Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that controls blood phosphate levels by increasing renal phosphate excretion and reducing 1,25-dihydroxyvitamin D3 1,25(OH)2D production. ...Disorders of FGF23 homeostasis are associated with significant morbidity and mortality, but a fundamental understanding of what regulates FGF23 production is lacking. Because the kidney is the major end organ of FGF23 action, we hypothesized that it releases a factor that regulates FGF23 synthesis. Using aptamer-based proteomics and liquid chromatography-mass spectrometry-based (LC-MS-based) metabolomics, we profiled more than 1600 molecules in renal venous plasma obtained from human subjects. Renal vein glycerol-3-phosphate (G-3-P) had the strongest correlation with circulating FGF23. In mice, exogenous G-3-P stimulated bone and bone marrow FGF23 production through local G-3-P acyltransferase-mediated (GPAT-mediated) lysophosphatidic acid (LPA) synthesis. Further, the stimulatory effect of G-3-P and LPA on FGF23 required LPA receptor 1 (LPAR1). Acute kidney injury (AKI), which increases FGF23 levels, rapidly increased circulating G-3-P in humans and mice, and the effect of AKI on FGF23 was abrogated by GPAT inhibition or Lpar1 deletion. Together, our findings establish a role for kidney-derived G-3-P in mineral metabolism and outline potential targets to modulate FGF23 production during kidney injury.
Microorganism species can become pathogenic and cause bacterial infection, result from the imbalance in microbial ecosystem between host and microbe. Microorganisms emit secondary metabolites, known ...as volatile metabolites or organic compounds (VOCs) for various functions such as intra- or inter-species interactions, defense and attraction. Currently, VOCs are widely used as a biomarker for human diseases. This research is aimed to identify the relationship between microorganism species and volatile metabolite compound from the collected species and VOCs emitted organism data by using unsupervised machine learning approaches such as hierarchical clustering and graph-clustering method. Supervised machine learning methods also been used to classify the microorganism pathogenicity such as support vector machine (SVM) and random forest (RF). These data are collected from KNApSAcK and mVOC database where it provides most of the microorganism species and metabolites contents. From the collected data, there are in total of 1088 VOCs emitted by 517 microorganism species. As a result, the application of machine learning methods enable us to identify the relationship of species with their emitted VOCs and classify the microorganism species into their own pathogenicity.
CD14 is a multifunctional receptor expressed on many cell types and has been shown to mediate immune response resulting in the activation of an inflammatory cascade, with polymorphism of its promoter ...(rs2569190) found to be associated with susceptibility to several diseases. In malaria infection, the CD14 gene demonstrated a pathogenic profile in regulating experimental cerebral malaria, with reports of elevated levels of soluble CD14 in serum of patients but no definitive conclusion. We present a detailed analysis of genetic diversity of CD14 promoter gene (snp −159 C/T; rs2519190) polymorphism between a malaria-infected group and uninfected controls and its association with clinical parameters of disease. Genomic DNA samples obtained from 106 Plasmodium falciparum malaria–infected patients and 277 uninfected controls were elucidated with a polymerase chain reaction-restriction fragment length polymorphism (RFLP) assay. Our results show a significant diversity (P = 3.32E−06) in the genotypic frequency (3.8% versus 22.4%) of the rs2569190 mutant variant between the malaria-infected group and controls, respectively. The mutant allele had the lowest frequency among the malaria-infected group demonstrating its necessity for infection. Mean parasitemia (parasites/μL of blood) was significantly regulated based on CD14 polymorphic profile (19 855 versus 37 041 versus 49 396 for homozygote mutants, heterozygotes, and homozygote wild type, respectively). Interestingly, we found no association between CD14 genetic variants with fever, age of patients, or anemia. How this affects disease severity between subregional and continental groups deserves further clarification, including extending these studies in a larger group and among severe and asymptomatic patients with malaria.
Barth syndrome is an inherited X-linked disorder characterized by cardiomyopathy, skeletal muscle myopathy, and neutropenia. The syndrome arises because of inherited mutations in the gene TAZ, ...resulting in a loss of function of the protein tafazzin. Of note, a group of investigators recently described how tafazzin can regulate ‘stemness’ in models of acute myeloid leukemia (Cell Stem Cell, 2019).
Tafazzin is an enzyme that processes the final step of cardiolipin maturation, replacing saturated with unsaturated acyl chains. Cardiolipin is a 4-tailed phospholipid that is almost-exclusively found in the inner membrane of the mitochondria. The lack of tafazzin activity results in a cardiolipin pool that contains more highly saturated lipid tails and it is this lack of unsaturated cardiolipins that contributes to a disorganized inner mitochondrial membrane.
The link between tafazzin-deficiency and myopathy is generally explained by the dependence of muscle cells on mitochondrial function as well as oxidative respiration. The components of the electron transport chain are co-localized with cardiolipin in the inner mitochondrial membrane, and it is felt that their appropriate organization within the membrane lipid bilayer is dependent on the presence of mature cardiolipin which is lacking in those individuals with Barth syndrome.
The link between tafazzin-deficiency and neutropenia is less clear. Neutrophils are terminally-differentiated effector cells of the innate immune system. They are critical for protection against bacterial and fungal pathogens and patients without sufficient neutrophils are among the most immunocompromised and at risk of lethal infection. Neutrophils have few mitochondria at baseline and are generally believed to rely primarily on glycolysis for energy production. It is not known if the mechanism of neutropenia in Barth syndrome is due to a lack of production or due to increased clearance (e.g. more prone to apoptosis).
We undertook the study of tafazzin-deficient neutrophils to try to elucidate the mechanism of neutropenia in patients with Barth syndrome. We took advantage of an existing tafazzin-knockout mouse and a system of conditional immortalization of granulocyte-monocyte progenitors (GMP) using the ER-Hoxb8 system pioneered in our laboratory. This ER-Hoxb8 system allows for the unlimited ex vivo expansion of myeloid progenitors in the presence of estradiol and active Hoxb8. Once estradiol is removed from culture media, the Hoxb8 protein is inactive and the cells undergo normal, synchronous and terminal neutrophilic differentiation. In this manner, we were able to generate tafazzin-wild-type and knockout GMP lines from murine fetal liver cells.
Analysis of the myeloid progenitor compartment in fetal liver cells (d14.5-d16.5) showed no difference between wild-type and knockout mice, arguing against a developmental defect (E15 results shown in PANEL A). Furthermore, the tafazzin-deficient ER-Hoxb8 GMPs and neutrophils were remarkably normal when tested across a variety of assays including phagocytosis, cytokine production and ROS generation (ROS by H2DCFDA shown in PANEL B).
We hypothesized that the unpredictable neutropenia in patients with Barth Syndrome might be due to an increased proclivity to apoptosis because of the mitochondrial membrane defect. Indeed, the tafazzin-deficient GMPs showed an increased sensitivity to Bcl2-inhibition following treatment with ABT199 (PANEL C).
Two lines of evidence have suggested that the increased tendency towards apoptosis may be due to endoplasmic-reticulum (ER) stress. (1) Transmission electron microscopy demonstrated ‘swollen’ ER in the tafazzin-deficient cells (not shown) and (2) a comparison of gene expression patterns demonstrated an increased expression of ATF4 and CHOP (DDIT3) in the tafazzin-deficient cells (PANEL D).
We are now focused on validating these findings and in establishing models to confirm the ER-stress phenotype in vivo in the TAZ-knockout mouse model as well as primary samples from patients with Barth Syndrome.
We hope that this line of work will confirm the mechanism of neutropenia and shed light on potential targets for therapeutic intervention. In addition, this very rare disorder has provided insight into a previously-unexpected link between neutrophil survival and the membrane integrity of the inner mitochondrial membrane.
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Sykes:Clear Creek Bio: Equity Ownership, Other: Co-Founder.
Abstract
CD14 is a multifunctional receptor expressed on many cell types, and has been shown to mediate immune response resulting in the activation of an inflammatory cascade. Genetic polymorphisms ...of the CD14 promoter (rs2569190) has been found to be associated with susceptibility to several diseases. In malaria infection, CD14 gene demonstrates a pathogenic profile in regulating experimental cerebral malaria, with reports of elevated levels of soluble CD14 in serum of malaria patients, with no definitive conclusions. We present a detailed analysis of genetic diversity of CD14 promoter gene polymorphism between a malaria-infected group and uninfected controls, and its association with clinical parameters of disease. Genomic DNA samples obtained from 107 Plasmodium falciparum-infected (microscopy and PCR-confirmed) patients and 380 uninfected controls were subjected to a PCR-RFLP analysis. Our results show a significant diversity (p=3.32E-06) in the genotypic frequency (3.8% versus 22.4%) of the rs2569190 mutant variant between infected and uninfected controls respectively, with the mutant allele less frequent in infected group. Mean parasitemia (parasites/μl of blood) was significantly regulated based on CD14 genetic profile (19,855 vs 37,041 vs 49,396 for mutants, heterozygotes and wild type alleles respectively), but showed no association with other markers of disease severity (fever, age of patients or anemia). Our results show the mutant variant pushed Th1 cell differentiation resulting in interferon-γ response leading to reduced parasitemia. How this affects disease severity between sub-regional or continental malaria groups deserves elucidation, including extending this results among cerebral malaria patients.
Adults diagnosed with acute myeloid leukemia have a poor prognosis, with only 30% surviving at five years. Despite advances in our understanding of the molecular underpinnings of AML, the ...chemotherapy backbone used to treat most patients (combination cytarabine and anthracycline) remains unchanged since 1973.
With the goal of identifying a new differentiation therapy, we previously performed a small molecule phenotypic screen to find compounds that triggered myeloid maturation. Unexpectedly, our most active hits were inhibitors of the enzyme dihydroorotate dehydrogenase (DHODH). The DHODH-inhibitor brequinar demonstrated dramatic activity in vitro and in vivo (Figure 1A), leading to differentiation (Figure 1B) and prolonged survival across multiple AML models when compared to standard induction chemotherapy (iCT) (Figure 1C). Brequinar treatment also depleted leukemia initiating cells in vivo as measured by secondary transplant analysis (Figure 1D).
Cancer therapy requires a ‘therapeutic window’ such that the treatment must target and kill malignant cells before killing normal cells and causing unacceptable side effects. An essential and ubiquitously expressed enzyme, DHODH is not typical among most cancer drug targets and is not known to be mutated or overexpressed in malignant cells. Yet empirically, a DHODH-inhibitor therapeutic window exists.
The competitive transplantation of hematopoietic stem cells following 36-days of brequinar treatment (at the same dose that is anti-leukemic and pro-differentiation) does not lead to a competitive disadvantage or deleterious differentiation of normal stem cells (Figure 1E). The basis of this therapeutic window in the case of DHODH inhibitors is not known.
Here we address the question: how are malignant cells metabolically programmed such that they are preferentially sensitive to pyrimidine starvation following inhibition of DHODH? Uridine is involved in myriad cellular processes beyond DNA and RNA synthesis, implicating many metabolic pathways (Figure 1F). We are taking an approach of (1) measuring metabolite levels and (2) metabolic pathway flux, as well as (3) using small molecule and genetic perturbations (shRNA) to unravel the differences in pyrimidine metabolism between normal and malignant cells.
The mechanism through which treatment with brequinar and inhibition of pyrimidine synthesis results in myeloid differentiation is also not known. Our preliminary data suggest that brequinar treatment not only leads to a rapid depletion of pyrimidines and downstream metabolites, but also to changes in TCA cycle metabolites including succinate and fumarate. Given the importance of these metabolites in the regulation of the TET-family of proteins, we measured changes in 5-hydroxy-methylcytosine as a surrogate for TET-enzyme activity following treatment with brequinar (Figure 1G). An increase in global 5hmC, suggesting an increase in TET-enzyme activity, paralleled the cellular differentiation over the first 72-hours. This is an intriguing association given the importance of TET2 in myeloid differentiation and the observation that inhibition of TET2 contributes to myeloid differentiation arrest.
As of summer 2018, two small molecule DHODH inhibitors are in early stage human clinical trials with two more inhibitors poised for 2019. A careful understanding of these metabolic pathways is important to inform the clinical experience of this class of anti-metabolite therapeutics. Understanding the basis of this therapeutic window will be critical to the safe and effective use of DHODH inhibitors in patients with advanced hematologic malignancies.
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Vander Heiden:Agios: Membership on an entity's Board of Directors or advisory committees; Aeglea: Membership on an entity's Board of Directors or advisory committees. Sykes:Clear Creek Bio: Equity Ownership, Other: Co-founder.
Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. ...Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.
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•Results of screens to identify drivers of hematopoietic differentiation•Perturbing nucleotide metabolism and DNA replication promotes differentiation•Replication stress activates primed regulatory loci to induce lineage programs•Canonical signaling and DNA damage responses to replication stress are not required
Do, Hsu et al. find that in non-transformed and transformed hematopoietic systems, perturbing nucleotide metabolism and DNA replication drives cells toward a more differentiated state and that replication stress activates primed chromatin to induce lineage-specifying programs, even when oncogenic differentiation blockades remain intact.
Abstract only
A major hallmark of infectious diseases is inflammation, particularly significant in endemic countries, where low levels of antigenic stimulation that is insufficient to cause disease ...are significant contributors to chronic inflammation, disrupting immunological balance. Interleukin (IL)‐10 is a cytokine produced by many cell types, stimulating the differentiation of innate and adaptive immunity cells and the control of inflammatory processes. We hypothesize that the diversity in inflammatory response is most importantly dependent on host genetic pathways, with this inter‐individual diversity more pronounced in individuals from Africa, thereby possessing cytokine gene variants abnormally affecting immune response. To this end, we examined the interethnic frequency of cytokine IL‐10 (rs1800872) promoter gene polymorphisms in three, disparate, well‐characterized population groups as well as malaria‐infected group and determined its association with disease. Genomic DNA samples were obtained from 189, 189 and 159 Africans, African Americans and Caucasians respectively. In addition, 105
Plasmodium falciparum
malaria‐infected patients from south‐west Nigeria were included, with comparative analysis among and between groups carried out with a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay. There was a statistically significant (
p
=1.07E‐03) interethnic diversity in the genotypic frequency of IL‐10 (rs1800872) mutant variant between population groups (20.1% versus 14.8% versus 6.3% for Africans, African Americans and Caucasians respectively). A similar interethnic observation was made for allelic frequency of IL‐10 polymorphism (
p
=4.35E‐08). This significant interethnic diversity clarifies the heterogeneous distribution of this polymorphism between groups and its essentiality in differential regulation of disease susceptibility or outcome. Interestingly, we found no significant difference in either genotypic (27.6% versus 20.1%;
p
=0.14) or allelic frequency (46.7% versus 43.9%;
p
=0.5) of IL‐10 (rs1800872) mutant variant between malaria‐infected patients and controls from West Africa. The lack of a significant difference in the malaria‐infected group is not surprising since malaria infection will drive T cell differentiation into Th1 cells, rather than Th2 cells, for which IL‐10 cytokine anti‐inflammatory properties would inhibit protective response. The possibility that the reported elevated levels of serum IL‐10 in malaria infection and consequent disease severity is regulated by host polymorphic variants deserves further investigation. This confirms our hypothesis that elucidating interethnic host genetic variations before initiating population‐stratified association studies are imperative. In addition, this host genetic variations are potential contributors to individual or sub‐continental differences in disease susceptibility and prevalence.
Support or Funding Information
Funding provided by College of Health Sciences and Technology Faculty Development and Laboratory Support Fund, Rochester Institute of Technology
Susceptibility to malaria infection has been associated with host genetic polymorphisms that differs between groups. We hypothesize that Toll-interacting proteins (
TOLLIP
), vitamin D receptor (VDR) ...and tumor necrosis factor-α (TNF) genes are significant contributors to susceptibility and disease severity in
Plasmodium falciparum (Pf)
infection. Our aim is to explore the genomic diversity and haplotype frequency of these genes, as well as extrapolate possible association with markers of severity, between malaria-infected and healthy controls. Genomic DNA samples extracted from the blood of 107 malaria-infected patients and 190 uninfected controls were analyzed, with no difference in genotypic or allelic frequencies of TOLLIP and VDR polymorphisms. However, a significant difference in the genotypic (
p
= 2.20E-16) and allelic frequencies (
p
= 2.20E-16) of the TNF-α (snp rs1800629) polymorphism was found. The preponderance of the mutant variant among the malaria-infected show a possible impaired capacity to mount an effective immune response, potentially confirmed by our association results. This result calls for analysis of clearly delineated uncomplicated versus severe disease groups, including serum assays, providing a basis to conclude that susceptibility to malaria infection and potential contribution to disease severity is significantly associated with polymorphisms of the tumor necrosis factor-α but not
TOLLIP
or VDR genes.
Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy, and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a ...transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knockout (KO) animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN-KO hematopoietic stem cells. We also used the ER-Hoxb8 system (estrogen receptor fused to Hoxb8) of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN-KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress-mediated and non-ER stress-mediated triggers of apoptosis. Although the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some patients with Barth syndrome remains to be clarified, our studies and new model system set a foundation for further investigation.
•ER-Hoxb8 conditional immortalization provides a system for the study of TAFAZZIN deficiency in myeloid progenitors and mature neutrophils.•Barth syndrome (ie, TAFAZZIN-deficient) murine neutrophils may be more sensitive to apoptosis, possibly because of increased ER stress.
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