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Phosphorus-independent role of FGF23 in erythropoiesis and iron homeostasis.
Get PDFA number of studies have reported an association between phosphorus, red blood cell (RBC) production, and iron metabolism. However, it is difficult to distinguish whether the effect of phosphorus is direct or through the actions of FGF23, and it is not clear whether phosphorus is positively or negatively associated with RBC production. In the present study, we investigated the effects of a) increased phosphorus load and b) phosphorus deficiency on erythropoiesis and iron metabolism in association with FGF23. Mice were fed either a 1.2% or 1.65% phosphorus diet and compared to mice fed a control diet containing 0.6% of phosphorus. Moreover, we used two mouse models of hypophosphatemia-induced either by dietary intervention in the form of a low phosphorus (LP) diet (0.02% of Pi) or genetically in a mouse model of X-linked hypophosphatemia (XLH)-that had opposite FGF23 levels. Phosphorus supplementation appropriately increased FGF23 levels leading to excretion of excess phosphorus and normalization of serum phosphorus levels. We also found that a phosphorus-rich diet results in inflammation-induced hypoferremia associated with reduced iron export leading to tissue iron overload. Moreover, high phosphorus intake results in ineffective erythropoiesis caused by decreased production (decreased RBCs, hemoglobin, hematocrit, and erythroid progenitors in the bone marrow) and increased destruction of RBCs, leading to anemia despite increased EPO secretion. These complications occur through the actions of elevated FGF23 in the presence of normophosphatemia. Our data also show that LP diet induces a decrease in the serum concentrations of phosphorus and FGF23, resulting in increased RBC counts, hemoglobin concentration, and hematocrit compared to mice fed normal diet. Moreover, serum iron and transferrin saturation were increased and positively correlated with serum ferritin, liver ferritin protein and mRNA expression in mice fed LP diet. However, hyp mice, the murine model of XLH, exhibit hypophosphatemia and high serum FGF23 levels, along with low number of circulating RBCs, hemoglobin, and hematocrit compared to wild-type mice. In the bone marrow, hyp mice showed reduced number of erythroid progenitors and formed significantly less BFU-E colonies compared to control mice. Serum iron levels and transferrin saturation were also decreased in hyp mice in comparison to control mice. Taken together, our data show that FGF23 acts independent of phosphorus levels to regulate erythropoiesis and iron homeostasis
The primate gut microbiota contributes to interspecific differences in host metabolism.
Get PDFBecause large brains are energetically expensive, they are associated with metabolic traits that facilitate energy availability across vertebrates. However, the biological underpinnings driving these traits are not known. Given its role in regulating host metabolism in disease studies, we hypothesized that the gut microbiome contributes to variation in normal cross-vertebrate species differences in metabolism, including those associated with the brain\u27s energetic requirements. By inoculating germ-free mice with the gut microbiota (GM) of three primate species - two with relatively larger brains and one with a smaller brain - we demonstrated that the GM of larger-brained primates shifts host metabolism towards energy use and production, while that of smaller-brained primates stimulates energy storage in adipose tissues. Our findings establish a causal role of the GM in normal cross-host species differences in metabolism associated with relative brain size and suggest that the GM may have been an important facilitator of metabolic changes during human evolution that supported encephalization
STRprofiler: efficient comparisons of short tandem repeat profiles for biomedical model authentication.
Get PDFSUMMARY: Short tandem repeat (STR) profiling is commonly performed for authentication of biomedical models of human origin, yet no tools exist to easily compare sets of STR profiles to each other or an existing database in a high-throughput manner. Here, we present STRprofiler, a Python package, command line tool, and Shiny application providing methods for STR profile comparison and cross-contamination detection. STRprofiler can be run with custom databases or used to query against the Cellosaurus cell line database.
AVAILABILITY AND IMPLEMENTATION: STRprofiler is freely available as a Python package with a rich CLI from PyPI https://pypi.org/project/strprofiler/ with source code available under the MIT license on GitHub https://github.com/j-andrews7/strprofiler and at https://zenodo.org/records/10989034. A web server hosting an example STRprofiler Shiny application backed by a database with data from the National Cancer Institute-funded PDXNet consortium and The Jackson Laboratory PDX program is available at https://sj-bakerlab.shinyapps.io/strprofiler/. Full documentation is available at https://strprofiler.readthedocs.io/en/latest/
Annotation of Metabolites in Stable Isotope Tracing Untargeted Metabolomics via Khipu-web.
No full textStable isotope tracing is a crucial technique for understanding the metabolic wiring of biological systems, determining metabolic flux through pathways of interest, and detecting novel metabolites and pathways. Despite the potential insights provided by this technique, its application remains limited to a small number of targeted molecules and pathways. Because previous software tools usually require chemical formulas to find relevant features, and the data are highly complex, especially in untargeted metabolomics and when the downstream reactions and metabolites are poorly characterized. We report here Khipu version 2 and its new user-friendly web application. New functions are added to enhance analyzing stable isotope tracing data including metrics that evaluate peak enrichment in labeled samples, scoring methods to facilitate robust detection of intensity patterns and integrated natural abundance correction. We demonstrate that this approach can be applied to untargeted metabolomics to systematically extract isotope-labeled compounds and annotate the unidentified metabolites
Assessing Anxiety Through Defecation Kinetics in BXD Mice
No full textWhile understanding anxiety in mice is crucial to behavioral research, methods of measuring anxiety have been notoriously difficult to understand. Defecation patterns of mice in open-field assays, measured by the number of fecal boli counts present at the end of the assay, have historically been used as one such anxiety measure. However, machine-learning-based analysis of open-field tests has opened the doors for higher-resolution quantification of defecation patterns. In this project, I analyzed anxiety measures across different tests and laboratories using correlation analysis, determining that many traditional anxiety measures are inconsistent. However, defecation remained consistent and proved to be highly genetically regulated. Using quantitative trait loci (QTL) and enrichment analysis, I determined that defecation kinetics are enriched for anxiety-related genes, phenotypes, and pathways, concluding that these novel kinetics measures are informative for future anxiety assays
Assessing the Incidence of Heart Failure with Preserved Ejection Fraction (HFpEF) within the Context of Aging
No full textHeart failure with preserved ejection (HFpEF)is an aging-associated disorder that is responsible for 50% of heart failure cases in humans and is a relatively common aging disorder that increases in severity and complication with age (Kobak et al. 2022). This project aims to discover if aged mice present with HFpEF phenotypes naturally as they age. Previous studies have induced HFpEF in mouse models using a combination of methods, most commonly an exclusively high fat diet and the use of a hypertensive drug N-nitro-l-arginine methyl ester (L-Name) (Schiattarella et al. 2019). The examination of aged mice, within this study, will allow for the investigation of HFpEF in a non-artificially induced state which allows for a more parallel model of the human experience. It was found that the aged mice did not exhibit some of the canonical HFpEF phenotypes, however the conclusions that can be drawn are limited due to the minimal sample size of n=4. Aged kidney samples showed a substantial increase in fibrotic content when compared to the heart, which is consistent with general phenotypes of aging. However, the mechanisms underlying this substantial deposition of fibrotic content within the kidney need to be further elucidated. Further investigation with a larger sample size to give more concrete results is recommended
Alzheimer\u27s age-dependent molecular signatures in individuals with the loss of function CETP I405V variant
No full textAlzheimer’s disease (AD) risk genes have been associated with lipid biology, including the strongest late-onset AD (LOAD) risk allele, APOE4. Research indicates that other lipid associated genes such as Cholesteryl Ester Transfer Protein (CETP) are also associated with AD risk; the CETP variant I405V (rs5882) is associated with greater longevity and reduced cognitive decline in centenarians. Other studies suggest that CETP*I405V may play pathogenic role in AD, however genetic studies indicate that the protective effect of CETP*I405V is dependent on the presence of APOE4 and has an age-specific effect in female individuals. Our goal was to determine the influence of aging on the transcriptomic signatures of individuals with CETP*I405V to better understand how lipid metabolism contributes to LOAD pathology. We achieved this by using two independent datasets to identify transcriptional signatures altered in CETP*I405V carriers and perform correlation analysis and enrichment analysis to determine enrichment of known AD transcriptomic signatures and gene ontology pathways with CETP*I405V carriers. The results indicate that there are transcriptional signatures that change in CETP*I405V carriers with age and are associated with LOAD pathways. Anti-correlation of CETP*I405V transcriptional signatures with AD transcriptional signatures in immune response and mitochondrial metabolism gene modules in older individuals suggest this variant may provide protection from AD through these pathways, however, correlation with AD transcriptional signatures in neuronal and glial pathways suggests that CETP*I405V may also contribute to AD pathogenic mechanisms through these specific biological processes
Tumor Immune Microenvironment in Humanized NSG-SGM3 Mice with Breast Cancer Undergo HER2 Chimeric Antigen Receptor T Cell Therapy
No full textBreast cancer (BC) is one of the leading causes of death in women and current treatment options are not always the most effective or efficient when treating this disease. Chimeric antigen receptor (CAR) T cell therapy has therapeutic potential but faces challenges when controlling solid breast cancer tumor growth due to tumor immune microenvironment (TIME). Thus, our collaborators have designed novel CAR T cells simultaneously targeting tumor receptor HER2 and the myeloid cell receptor TNF-related apoptosis-inducing ligand receptor 2 (TR2). Here, we performed multicolor immunofluorescence staining on tumors from hNSG-SGM3 mice that had received different CAR T cell constructs. We found that by targeting HER2 and TR2, there was an increase in the number of T cells and a decrease of myeloid cells intratumorally. Many of the T cells expressed PD1, which could indicate recognition of target antigen. However, tumor volume was not controlled by this therapy which could be due to exhaustion and further analysis could be done to determine this. Thus, this study gave us a closer look at the interactions within the tumor immune microenvironment and our model will allow us to design a more effective CAR-T cell treatment in further studies
Chapter 18 - Genetic modification of mice using CRISPR-Cas9: Best practices and practical concepts explained
No full textThe development of precision targetable nucleases has led to a massive acceleration in creating ge- netically modified mice and other species [1–6]. This began more than a decade ago with Zinc Finger Nucleases (ZFNs) [7], followed by TALENs (Transcription Activator-Like Effector Nucleases) [8–10], and then CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats with CRISPR- associated effector protein 9) [11,12]. At present, RNA-guided CRISPR-Cas9 is the most affordable and straightforward to design, construct, and implement. This accessibility, combined with its gener- ally high degree of targeting efficiency, has pushed CRISPR-Cas9 to the forefront of gene-editing methods. Regardless of its relative simplicity, the complexity of the resulting nuclease-derived genetic modifications, including the modified organism’s phenotype, should not be underestimated [13–15].
Herein, we outline our experience using CRISPR-Cas9 to precisely and directly engineer mouse zygotes, focusing on the general methodology and screening used to characterize the resulting alleles. These screening strategies are simple, straightforward, and reproducible. While the focus of this chap- ter is on CRISPR-modified alleles generated in mice, these screening regimes can be applied to other organisms and to the characterization of genetic modifications resulting from ZFNs, TALENs, or any other gene-editing technology
Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C\u3eT variant and correction with moderate dose folic acid supplementation.
Get PDF5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C\u3eT) results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2 mg folic acid/kg diet) and assess the effects of folic acid supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing and whole-genome bisulfite sequencing (WGBS). Reproductive parameters and locus-specific imprinted gene methylation were unaffected by genotype or diet. Using WGBS, sperm from 677TT mice had 360 differentially methylated tiles as compared to 677CC mice, predominantly hypomethylation (60% of tiles). Folic acid supplementation mostly caused hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic