Vitamins B9 (folate) and B12 are essential water-soluble vitamins that play a crucial role in the maintenance of one-carbon metabolism: a set of interconnected biochemical pathways driven by folate ...and methionine to generate methyl groups for use in DNA synthesis, amino acid homeostasis, antioxidant generation, and epigenetic regulation. Dietary deficiencies in B9 and B12, or genetic polymorphisms that influence the activity of enzymes involved in the folate or methionine cycles, are known to cause developmental defects, impair cognitive function, or block normal blood production. Nutritional deficiencies have historically been treated with dietary supplementation or high-dose parenteral administration that can reverse symptoms in the majority of cases. Elevated levels of these vitamins have more recently been shown to correlate with immune dysfunction, cancer, and increased mortality. Therapies that specifically target one-carbon metabolism are therefore currently being explored for the treatment of immune disorders and cancer. In this review, we will highlight recent studies aimed at elucidating the role of folate, B12, and methionine in one-carbon metabolism during normal cellular processes and in the context of disease progression.
The formation, inheritance, and removal of DNA methylation in the genome of mammalian cells is directly regulated by two families of enzymes–DNA methyltransferases (DNMTs) and Ten-Eleven ...Translocation proteins (TETs). DNMTs generate and maintain the inheritance of 5-methylcytosine (5mC), which is the substrate targeted by the TET enzymes for conversion to 5-hydroxymethylcytosine (5hmC) and its downstream oxidized derivatives. The activity of DNMT and TET is dependent on the availability of micronutrients and metabolite co-factors, including essential vitamins, amino acids, and trace metals, highlighting how DNA methylation levels can be directly enhanced, suppressed, or remodeled via metabolic and nutritional perturbations. Dynamic changes in DNA methylation are required during embryonic development, lineage specification, and maintenance of somatic cell function that can be fine-tuned based on the influence of essential micronutrients. As we age, DNA methylation and hydroxymethylation levels drift in patterning, leading to epigenetic dysregulation and genomic instability that underlies the formation and progression of multiple diseases including cancer. Understanding how DNA methylation can be regulated by micronutrients will have important implications for the maintenance of normal tissue function upon aging, and in the prevention and treatment of diseases for improved health and lifespan.
A cost‐effective way of undertaking comprehensive, continental‐scale, assessments of ecological condition is needed to support large‐scale conservation planning, monitoring, reporting, and ...decision‐making. Currently, cross‐jurisdictional inconsistency in assessment methods limits the capacity to scale‐up monitoring. Here we present a novel way to build a coherent continent‐wide site‐level ecological condition dataset, using cross‐calibration methods to integrate assessments from many observers. We focus on the use of condition assessments from individual expert observers, a currently untapped resource. Our approach has two components: (1) a simple online tool that captures expert assessments at specific locations; (2) a process of calibrating and rescaling disparate expert evaluations that can be applied to the data to provide a consistent dataset for use in conservation assessments. We describe a pilot study, involving 28 experts, who contributed 314 individual site condition assessments across a wide range of ecosystems and regions throughout continental Australia. A correction factor for each expert was used to rescale the contributed site condition assessment scores, based on a set of 77 photographic images, each scored for their condition by multiple experts, using a linear mixed model. Our approach shows strong promise for delivering the volumes of data required to develop continental‐scale reference libraries of site condition assessments. Although developed from expert elicitation, the approach could also be used to harmonize the collation of existing condition datasets. The process we demonstrate can also facilitate online citizen scientists to make site condition assessments that can be cross‐calibrated using contributed images.
To assess the feasibility of embolization of aortic side branches and its impact on the incidence of type II endoleak after endovascular aneurysm repair.
Endovascular aneurysm repair was performed in ...74 patients. Aortic side branch vessels were evaluated on the preoperative angiogram and computed tomography (CT) and, where embolization of lumbar and inferior mesenteric vessels was considered technically possible, this was attempted prior to endovascular repair. Follow-up CT was used to assess the presence of type II endoleak.
Seventy-two patients were followed up for longer than 1 month. Embolization was attempted in 25 cases, successfully in 10, with partial success in 11, and failure in four. Twenty patients with successful or partly successful preoperative embolization were discharged and followed-up. Four (20%) had demonstrable type II endoleak during follow-up, with two of these persisting at latest follow-up. Of 43 patients without previous embolization, there were 10 (23.3%) type II endoleaks during the follow-up period, four of these persisting. In cases with type II endoleak, mean sac diameter change was -0.5 mm in the cases with previous embolization and +3.1 mm without. The mean period to onset of type II endoleak was 6.9 months without, and 15.3 months with, previous embolization.
Although the cohort size is below a level that would confer significance, the trend of these findings is such as to suggest a lack of influence of aortic side branch embolization on the incidence of type II endoleak during the follow-up period.
Abstract
TET2 loss-of-function mutations induce a pre-malignant state known as clonal hematopoiesis of indeterminate potential (CHIP). CHIP occurs in approximately 10% of people over 65 years of age ...and confers a 10-fold greater risk of developing hematological malignancy. Several environmental factors, including radiation, sleep deprivation, atherosclerosis, and diet, have been associated with the expansion of pre-malignant clones in CHIP patients. Tet2-deficiency in mice has also been shown to trigger a pro-inflammatory state with increased intestinal permeability and accelerated myeloid expansion. Gut microbes exert an influence on host disease progression through the synthesis of many compounds including short-chain fatty acids (SCFAs), which promote gut barrier integrity. Dietary levels of one-carbon metabolites and cofactors, including methionine and folate, have been found to alter gut microbial composition in disease-free adults and influence SCFA production in the gut in murine models. Given the connection between diet, SCFAs and gut permeability, we sought to determine the impact of dietary one-carbon metabolites on gut microbial composition and function in a murine model of pre-malignant hematopoiesis. We performed competitive bone marrow transplantation assays in mice fed diets with altered one-carbon metabolite supplementation, such as high and low folate or methionine. We found that differential supplementation with these one-carbon metabolites did not influence the competitiveness of Tet2-deficient hematopoietic cells, however, increased dietary methionine promoted a myeloid lineage differentiation bias and an elevation in circulating pro-inflammatory cytokines. After 8 months of dietary treatment, fecal samples were collected, and shotgun sequencing was performed to examine the role of one-carbon metabolite levels on gut microbial diversity. Alterations in dietary methionine and folate caused significant changes to gut microbial composition in Tet2-deficient mice. High folate or methionine supplementation led to a decrease in the ratio of Firmicutes to Bacteroidetes and high folate supplementation was also associated with decreased alpha diversity and a decrease in SCFA-producing bacteria in the gut. These studies highlight the potential influence of dietary one-carbon metabolites on the microbiome and inflammatory microenvironment of pre-malignant hematopoiesis.
Citation Format: Peter Lyon, Praveen Singh, Byron Fang, Victoria Strippoli, Sabita Roy, Luisa Cimmino. The influence of dietary one-carbon metabolites on gut dysbiosis during pre-malignant hematopoiesis abstract. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A44.
Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or ...stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.
TET2 haploinsufficiency is a driving event in myeloid cancers and associated with a worse prognosis in patients with AML. Enhancing residual TET2 activity using vitamin C increases oxidized ...5-methylcytosine (oxi-mC) formation and promotes active DNA demethylation via base-excision repair (BER) that slows leukemia progression. We utilized genetic and compound library screening approaches to identify rational combination treatment strategies to improve the use of vitamin C as an adjuvant therapy for AML. In addition to increasing the efficacy of multiple FDA approved drugs, vitamin C treatment with poly-ADP-ribose polymerase inhibitors (PARPi) elicited a strong synergistic effect at blocking AML self-renewal in murine and human models. AML cells treated with a combination of vitamin C and PARPi in vitro led to reduced replating capacity in colony-forming assays, reduced viability in liquid culture, and increased survival upon treatment in vivo. These phenotypes were associated with increased p21 expression, cell-cycle stalling in S-phase and differentiation of AML cells toward a more mature myeloid phenotype. Furthermore, we show that vitamin C-mediated TET activation combined with PARPi causes an enrichment of chromatin-bound PARP1 protein specifically at 5-formylcytosine (5fC) oxi-mCs, and an enrichment for yH2AX at these sites in addition to its accumulation during mid-S phase. This work provides the first proof of PARP1 as a direct reader of 5fC, and enrichment of 5fC at sites marked by yH2AX. The generation of BER-inducing oxi-mCs imparts a novel therapeutic potential of PARPi, which are currently in clinical trial to determine their therapeutic efficacy for AML. Given the majority of AML subtypes maintain residual TET2 expression, vitamin C could elicit broad efficacy as a PARPi therapeutic adjuvant to improve treatment outcome.
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