The growing popularity of unpasteurized milk in the United States raises public health concerns. We estimated outbreak-related illnesses and hospitalizations caused by the consumption of cow’s milk and cheese contaminated with Shiga toxin–producing Escherichia coli, Salmonella spp., Listeria monocytogenes, and Campylobacter spp. using a model relying on publicly available outbreak data. In the United States, outbreaks associated with dairy consumption cause, on average, 760 illnesses/year and 22 hospitalizations/year, mostly from Salmonella spp. and Campylobacter spp. Unpasteurized milk, consumed by only 3.2% of the population, and cheese, consumed by only 1.6% of the population, caused 96% of illnesses caused by contaminated dairy products. Unpasteurized dairy products thus cause 840 (95% CrI 611–1,158) times more illnesses and 45 (95% CrI 34–59) times more hospitalizations than pasteurized products. As consumption of unpasteurized dairy products grows, illnesses will increase steadily; a doubling in the consumption of unpasteurized milk or cheese could increase outbreak-related illnesses by 96%.
The metabolism of tryptophan to nicotinamide adenine dinucleotide (NAD+) through the kynurenine pathway is increasingly linked to aging and age-associated disease. Kynurenine pathway enzymes and metabolites influence a range of molecular processes critical to healthy aging, including regulation of inflammatory and immune responses, cellular redox homeostasis, and energy production. Aberrant kynurenine metabolism is observed during normal aging and has been implicated in a range of age-associated pathologies, including chronic inflammation, atherosclerosis, neurodegeneration, and cancer. In previous work, we and others identified three genes-kynu-1, tdo-2, and acsd-1-encoding kynurenine pathway enzymes for which decreasing expression extends lifespan in invertebrate models. Here we report that knockdown of haao-1, a fourth kynurenine pathway gene encoding the enzyme 3-hydroxyanthranilic acid dioxygenase (HAAO), extends lifespan by ~30% and delays age-associated decline in health in Caenorhabditis elegans. This lifespan extension is mediated by increased physiological levels of the HAAO substrate 3-hydroxyanthranilic acid (3HAA). 3HAA increases resistance to oxidative stress during aging by directly degrading hydrogen peroxide and activating the Nrf2/SKN-1 oxidative stress response. Aging mice fed a diet supplemented with 3HAA are similarly long-lived. Our results identify HAAO and 3HAA as novel therapeutic targets for age-associated disease. This works provides a foundation for more detailed examination of the molecular mechanisms underlying the benefits of 3HAA, and how these mechanisms interact with other interventions both within and beyond the kynurenine pathway. We anticipate that these findings will bolster growing interest in developing pharmacological strategies to target tryptophan metabolism to improve health aging.
Caenorhabditis elegans are an important model system for host-microbe research due to the ability to rapidly quantify the influence of microbial exposure on whole-organism survival and rapidly quantify microbial load. To date, the majority of host-pathogen interaction studies rely on host group survival and cross-sectional examination of infection severity. Here we present a new system called Systematic Imaging of Caenorhabditis Killing Organisms (SICKO) capable of characterizing longitudinal interactions between host and pathogens in individual C. elegans, enabling researchers to capture dynamic changes in gut colonization between individuals and quantify the impact of bacterial colonization events on host survival. Using this system, we demonstrate that gut colonization by the strain of Escherichia coli used as a common laboratory food source dramatically impacts the lifespan of C. elegans. Additionally, we show that immunodeficient animals, lacking the pmk-1 gene, do not significantly alter the progression of bacterial infection, but rather suffer an increased rate of gut colony initiation. This new system provides a powerful tool into understanding underlying mechanisms of host-microbe interaction, opening a wide avenue for detailed research into therapies that combat pathogen induced illness, the benefits imparted by probiotic bacteria, and understanding the role of the microbiome in host health .
Introduction Mutant GOF p53 is an epigenetic regulator which promotes oncogenesis. Many cancers with a GOF mutation accumulate cholesterol and inhibition of cholesterol synthesis with statin treatment reverses some of the pro‐oncogenic properties of GOF p53. Our central hypothesis is that lipid raft signaling is altered by cholesterol accumulation in cells expressing a GOF p53. To address the role of cholesterol in promoting oncogenesis, we use the SW13 cell line bearing a GOF p53 (H193Y). This cell line has two epigenetically distinct cell subtypes that differentially express genes coding for GPI anchors, cholesterol biosynthesis, and sphingolipids, all of which are components of lipid rafts. In addition, the SW13 subtypes have different oncogenic profiles. The SW13(Vim−) subtype has an epithelial morphology and is highly proliferative. The SW13(Vim+) subtype has a mesenchymal like phenotype and a higher metastatic potential. When treated with histone deacetylase inhibitors (HDACi), SW13(Vim−) appear to adopt a SW13(Vim+) phenotype. Therefore, we treat with HDACi to control the subtype conversion and shRNA to knockdown GOF p53 expression. This experimental approach allows the composition of raft fraction to be correlated to GOF p53 expression and oncogenic properties of each SW13 subtype, with and without p53 expression. Methods Proliferation rate, MMP expression, and migration of SW13(Vim−), SW13(Vim+), shRNA SW13(Vim−), and shRNA SW13(Vim+) were quantitated by EdU assays and in situ zymography respectively. We have isolated lipid raft fractions by the detergent resistant enrichment technique from SW13 cells of each subtype, with and without GOF p53 expression knockdown by shRNA. Proteins were identified by mass spectrometry analysis. Fluorescent cholesterol assays and p53 western blots were used to quantify cholesterol levels and p53 protein expression, respectively. Results Data suggest that there is a shift from planar rafts in the SW13(Vim−) to caveolar rafts in the SW13(Vim+) line. Although the contribution of GOF p53 to this shift is under study, evidence suggests that raft composition is altered in the shRNA lines, in which GOF p53 has been knocked down by ~90%. In addition, p53 knockdown increases the proliferation rate in SW13(Vim−) (p = 0.01) but does not affect SW13(Vim+). In contrast, p53 knockdown decreases MMP activity in SW13(Vim+) (p = 0.05) but not SW13(Vim−). Conclusions Our data support the hypothesis that lipid raft signaling is significantly altered between the epigenetically distinct SW13 subtypes and that these differences may correlate to oncogenic potential. In addition, statin treatment, which reverses some of the pro oncogenic properties of GOF p53, may do so, in part, by altering raft composition and signaling.
Metabolism of tryptophan by the kynurenine pathway is increasingly linked to aging. Kynurenine pathway enzymes and metabolites influence a range of molecular processes critical to healthy aging, including regulation of inflammatory and immune responses, cellular redox homeostasis, and energy production. Aberrant kynurenine metabolism occurs during normal aging and is implicated in many age-associated pathologies including chronic inflammation, atherosclerosis, neurodegeneration, and cancer. We and others previously identified three kynurenine pathway genes—kynu-1, tdo-2, and acsd-1—for which decreasing expression extends lifespan in invertebrates. More recently we discovered that knockdown of haao-1, a fourth kynurenine pathway gene encoding the enzyme 3-hydroxyanthranilic acid dioxygenase (HAAO), extends lifespan by ~30% and delays age-associated health decline in Caenorhabditis elegans. Lifespan extension is mediated by increased physiological levels of the HAAO substrate 3-hydroxyanthranilic acid (3HAA). Aging mice fed a diet supplemented with 3HAA are similarly long-lived. The mechanism of action liking 3HAA to aging is complex and partially overlaps with multiple pathways previously implicated in aging. We recently identified activation of the Nrf2/SKN-1 oxidative stress response and alterations to iron homeostasis as key players in the benefits 3HAA. Ongoing work explores the relationship between 3HAA, Nrf2/SKN-1, and iron in C. elegans and mammalian aging, age-associated immune decline, and cancer. This works provides a foundation for detailed examination of the molecular mechanisms underlying the benefits of 3HAA, and how these mechanisms interact with other anti-aging interventions. We anticipate that these findings will bolster growing interest in developing pharmacological strategies to target tryptophan metabolism to improve health aging.
Cancer cells have elevated energy demands to sustain continuous growth and other malignant processes and undergo extensive metabolic reprogramming to meet these demands. One element of this reprogramming in many cancer subtypes is elevated synthesis of nicotinamide adenine dinucleotide (NAD+), a critical co-enzyme that supports energy production through both glycolysis and the TCA cycle. The kynurenine metabolic pathway is the evolutionarily conserved means by which cells produce NAD+ de novo from tryptophan. NAD+ levels drop with age, a contributing factor to many forms of age-related disease. While interventions that increase NAD+ have been shown to extend lifespan, previous work from our lab demonstrates that knockdown of several kynurenine pathway enzymes, thus decreasing de novo NAD+ production, results in increased longevity of Caenorhabditis elegans by 20-30%. To address this apparent contradiction, we propose that kynurenine pathway inhibition may produce metabolic feedback that results in upregulation of NAD+ recycling. Eukaryotic cells recycle NAD+ from nicotinamide (NAM) through one of two pathways: the Salvage pathway in mammalian cells and the Preiss-Handler pathway in C. elegans and related invertebrates species. We are using tools in C. elegans and human cell culture to examine the interaction between kynurenine/de novo NAD+ synthesis and NAD+ recycling through Salvage and Preiss-Handler. In particular, we are interested in how combining interventions between these pathways will influence activity throughout the NAD+ metabolic networks (measured via mass spectrometry), physiological phenotypes, and transcriptomic changes (via RNA sequence data) involved in aging and age-associated disease.
Aging is characterized by a progressive decline in the normal physiological functions of an organism, ultimately leading to mortality. Metabolic changes throughout the aging process disrupt the balance and homeostasis of the cell. The kynurenine metabolic pathway is the sole de novo biosynthetic pathway for producing NAD+ from ingested tryptophan. Altered kynurenine pathway activity is associated with both aging and a variety of age-associated diseases, and kynurenine-based interventions can extend lifespan in Caenorhabditis elegans. Our laboratory recently demonstrated knockdown of the kynurenine pathway enzymes kynureninase (KYNU) or 3-hydroxyanthranilic acid dioxygenase (HAAO) increases lifespan by 20-30% in C elegans. However, the mechanism of how these interventions may modulate response against different stressors during the aging process has yet to be explored. Fluorescent reporter strains show the stress-responsive transcription factors skn-1 (ortholog of NRF2/NFE2L2; oxidative stress response) and hif-1 (ortholog of HIF1A; hypoxic stress response) to be highly upregulated when the kynurenine pathway is inhibited. We also demonstrated the increase expression of gst-4 and gcs-1 (transcriptional targets skn-1), which are involved in production of the antioxidant glutathione (GSH), as well as upregulation of cysl-2 (transcriptional target of hif-1), a regulator of cysteine biosynthesis from serine. We hypothesize that lifespan extension resulting from kynurenine pathway inhibition is mediated, at least in part, by upregulation of these transcription factors, providing elevated defense against oxidative stress and hypoxic stress.
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