Computational Analysis of Plasma Lipidomics from Mice Fed Standard Chow and Ketogenic Diet

Dietary saturated fatty acids (SFAs) are upregulated in the blood circulation following digestion. A variety of circulating lipid species have been implicated in metabolic and inflammatory diseases; however, due to the extreme variability in serum or plasma lipid concentrations found in human studies, established reference ranges are still lacking, in addition to lipid specificity and diagnostic biomarkers. Mass spectrometry is widely used for identification of lipid species in the plasma, and there are many differences in sample extraction methods within the literature. We used ultra-high performance liquid chromatography (UPLC) coupled to a high-resolution hybrid triple quadrupole-time-of-flight (QToF) mass spectrometry (MS) to compare relative peak abundance of specific lipid species within the following lipid classes: free fatty acids (FFAs), triglycerides (TAGs), phosphatidylcholines (PCs), and sphingolipids (SGs), in the plasma of mice fed a standard chow (SC; low in SFAs) or ketogenic diet (KD; high in SFAs) for two weeks. In this protocol, we used Principal Component Analysis (PCA) and R to visualize how individual mice clustered together according to their diet, and we found that KD-fed mice displayed unique blood profiles for many lipid species identified within each lipid class compared to SC-fed mice. We conclude that two weeks of KD feeding is sufficient to significantly alter circulating lipids, with PCs being the most altered lipid class, followed by SGs, TAGs, and FFAs, including palmitic acid (PA) and PA-saturated lipids. This protocol is needed to advance knowledge on the impact that SFA-enriched diets have on concentrations of specific lipids in the blood that are known to be associated with metabolic and inflammatory diseases

Model Systems for the Study of Kidney Development: Use of the Pronephros in the Analysis of Organ Induction and Patterning

AbstractMost vertebrate organs, once formed, continue to perform the function for which they were generated until the death of the organism. The kidney is a notable exception to this rule. Vertebrates, even those that do not undergo metamorphosis, utilize a progression of more complex kidneys as they grow and develop. This is presumably due to the changing conditions to which the organism must respond to retain what Homer Smith referred to as our physiological freedom. To quote, “Recognizing that we have the kind of blood we have because we have the kind of kidneys we have, we must acknowledge that our kidneys constitute the major foundation of our physiological freedom. Only because they work the way they do has it become possible for us to have bones, muscles, glands, and brains. Superficially, it might be said that the function of the kidneys is to make urine; but in a more considered view one can say that the kidneys make the stuff of philosophy itself” (“From Fish to Philosopher,” Little, Brown and Co., Boston, 1953). Different kidneys are used to make the stuff of philosophy at different stages of development depending on the age and needs of the organism, rather than the usual approach of simply making embryonic organs larger as the animal grows. Although evolution has provided the higher vertebrates with complex adult kidneys, they continue to utilize simple kidneys in embryogenesis. In lower vertebrates with simple adult kidneys, even more simple versions are used during early developmental stages. In this review the anatomy, development, and gene expression patterns of the embryonic kidney, the pronephros, will be described and compared to the more complex kidney forms. Despite some differences in anatomy, similar developmental pathways seem to be responsible for the induction and the response to induction in both evanescent and permanent kidney forms. Gene expression patterns can, therefore, be added to the morphological and functional data indicating that all forms of the kidney are closely related structures. Given the similarities between the development of simple and complex kidneys, the embryonic kidneys may be an ideal model system in which to investigate the genesis of multicomponent organ systems

Developmental Basis of Pronephric Defects in Xenopus Body Plan Phenotypes

AbstractWe have used monoclonal antibodies that recognize the pronephric tubules or pronephric duct to explore the induction of the embryonic kidney in developing Xenopus embryos. Morphogenesis of the pronephros was examined in UV-ventralized and lithium-dorsalized embryos. We find that the pronephric tubules are present in all but the strongest UV-induced phenotypes, but absent from relatively moderate lithium phenotypes. Interestingly the pronephric duct, which develops from the ventroposterior portion of the pronephric anlage, is missing from more of the mild UV phenotypes than are pronephric tubules. The loss of the capacity to form pronephroi in UV-ventralized embryos is caused by the loss of tissues capable of inducing the pronephric mesoderm, as marginal zone explants from ventralized embryos are still competent to respond to pronephric-inductive signals. Explant recombination experiments indicate that the tissue responsible for both the loss of pronephroi in UV-ventralized embryos and the induction of pronephroi during normal development is the anterior somites. The absence of pronephroi in relatively mild lithium phenotypes has a developmental basis different from that of the UV phenotype, as explants from lithium-treated embryos are effective inducers of pronephroi in recombinants with competent mesoderm, even though they themselves do not form pronephroi in isolation. Together these data indicate that dorsal tissues, especially the anterior somites, are responsible for the establishment of the intermediate mesoderm and the induction of the embryonic kidneys and that even mild dorsalization destroys the capacity to form cells competent to receive this signal

Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized 129Xe NMR

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor–cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand

Disease heterogeneity of adult diabetes based on routine clinical parameters at diagnosis: Results from the German/Austrian DPV registry.

AIMS To cluster adults with diabetes using parameters from real-world clinical care at manifestation. MATERIALS AND METHODS We applied hierarchical clustering using Ward's method to 56,869 adults documented in the Prospective Diabetes Follow-up Registry (DPV). Clustering variables included age, sex, BMI, HbA1c, diabetic ketoacidosis (DKA), components of the metabolic syndrome (hypertension/dyslipidemia/hyperuricemia), and beta-cell antibody status. Time until use of oral antidiabetic drugs (OAD), use of insulin, chronic kidney disease (CKD), cardiovascular disease (CVD), retinopathy, or neuropathy were assessed using Kaplan Meier analysis and Cox regression models. RESULTS We identified eight clusters: Four clusters comprised early diabetes onset (median age between 40 and 50 years), but differed with regard to BMI, HbA1c, DKA and antibody positivity. Two clusters included adults with diabetes onset in their early 60s who met target HbA1c, but differed in BMI and sex distribution. Two clusters were characterized by late diabetes onset (median age 69 and 77 years) and relatively low BMI, but differences in HbA1c. Earlier insulin use was observed in adults with high HbA1c, and earlier OAD use was observed in those with high BMI. Time until CKD or CVD was shorter in those with late onset, whereas retinopathy occurred earlier in adults with late onset and high HbA1c, and in adults with early onset, but high HbA1c and high percentage of antibody positivity. CONCLUSIONS Adult diabetes is heterogeneous beyond classical type 1/type 2 diabetes, based on easily available parameters in clinical practice using an automated clustering algorithm which allows both continuous and binary variables. This article is protected by copyright. All rights reserved

Enriched Dietary Saturated Fatty Acids Induce Trained Immunity via Ceramide Production that Enhances Severity of Endotoxemia and clearance of infection

Trained immunity is an innate immune memory response that is induced by a primary inflammatory stimulus that sensitizes monocytes and macrophages to a secondary pathogenic challenge, reprogramming the host response to infection and inflammatory disease. Dietary fatty acids can act as inflammatory stimuli, but it is unknown if they can act as the primary stimuli to induce trained immunity. Here we find mice fed a diet enriched exclusively in saturated fatty acids (ketogenic diet; KD) confer a hyper-inflammatory response to systemic lipopolysaccharide (LPS) and increased mortality, independent of diet-induced microbiome and hyperglycemia. We find KD alters the composition of the hematopoietic stem cell compartment and enhances the response of bone marrow macrophages, monocytes, and splenocytes to secondary LPS challenge. Lipidomics identified enhanced free palmitic acid (PA) and PA-associated lipids in KD-fed mice serum. We found pre-treatment with physiologically relevant concentrations of PA induces a hyper-inflammatory response to LPS in macrophages, and this was dependent on the synthesis of ceramide. In vivo, we found systemic PA confers enhanced inflammation and mortality in response to systemic LPS, and this phenotype was not reversible for up to 7 days post-PA-exposure. Conversely, we find PA exposure enhanced clearance of Candida albicans in Rag1-/- mice. Lastly, we show that oleic acid, which depletes intracellular ceramide, reverses PA-induced hyper-inflammation in macrophages and enhanced mortality in response to LPS. These implicate enriched dietary SFAs, and specifically PA, in the induction of long-lived innate immune memory and highlight the plasticity of this innate immune reprogramming by dietary constituents

Визначення кількості рослинних антиоксидантів для захисту гірких хмелевих речовин від окисної деструкції

Досліджено кінетику окиснення гірких речовин водного розчину екстракту хмелю у прискорених умовах з різною концентрацією антиоксидантів із рослинної сировини. Визначено ефективну концентрацію антиоксидантів із кори дубу, трави звіробою та трави м'яти.Kinetics of bitter matters in aquatic solution of hope extract in speed-up terms with different concentration of antioxidants from the digister are investigated. Certainly effective concentration of antioxidants from the bark oak, st-john's-wort herbares and mint herbares are determinated