Mild inborn errors of metabolism in commonly used inbred mouse strains.

Inbred mouse strains are a cornerstone of translational research but paradoxically many strains carry mild inborn errors of metabolism. For example, alpha-aminoadipic acidemia and branched-chain ketoacid dehydrogenase deficiency are known in C57BL/6J mice. Using RNA sequencing, we now reveal the causal variants in Dhtkd1 and Bckdhb, and the molecular mechanism underlying these metabolic defects. C57BL/6J mice have decreased Dhtkd1 mRNA expression due to a solitary long terminal repeat (LTR) in intron 4 of Dhtkd1. This LTR harbors an alternate splice donor site leading to a partial splicing defect and as a consequence decreased total and functional Dhtkd1 mRNA, decreased DHTKD1 protein and alpha-aminoadipic acidemia. Similarly, C57BL/6J mice have decreased Bckdhb mRNA expression due to an LTR retrotransposon in intron 1 of Bckdhb. This transposable element encodes an alternative exon 1 causing aberrant splicing, decreased total and functional Bckdhb mRNA and decreased BCKDHB protein. Using a targeted metabolomics screen, we also reveal elevated plasma C5-carnitine in 129 substrains. This biochemical phenotype resembles isovaleric acidemia and is caused by an exonic splice mutation in Ivd leading to partial skipping of exon 10 and IVD protein deficiency. In summary, this study identifies three causal variants underlying mild inborn errors of metabolism in commonly used inbred mouse strains

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

spores germinate in the presence of a single germinant, inosine, yet with a significant lag period. spores. spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination

Germline deletion of Krüppel-like factor 14 does not increase risk of diet induced metabolic syndrome in male C57BL/6 mice

The transcription factor Krüppel-like factor 14 (KLF14) has been associated with type 2 diabetes and high-density lipoprotein-cholesterol (HDL-C) through genome-wide association studies. The mechanistic underpinnings of KLF14's control of metabolic processes remain largely unknown. We studied the physiological roles of KLF14 in a knockout (KO) mouse model. Male whole body Klf14 KO mice were fed a chow or high fat diet (HFD) and diet induced phenotypes were analyzed. Additionally, tissue-specific expression of Klf14 was determined using RT-PCR, RNA sequencing, immunoblotting and whole mount lacZ staining. Finally, the consequences of KLF14 loss-of-function were studied using RNA sequencing in tissues with relatively high Klf14 expression levels. KLF14 loss-of-function did not affect HFD-induced weight gain or insulin resistance. Fasting plasma concentrations of glucose, insulin, cholesterol, HDL-C and ApoA-I were also comparable between Klf14(+/+) and Klf14(-/-) mice on chow and HFD. We found that in mice expression of Klf14 was the highest in the anterior pituitary (adenohypophysis), lower but detectable in white adipose tissue and undetectable in liver. Loss of KLF14 function impacted on the pituitary transcriptome with extracellular matrix organization as the primary affected pathway and a predicted link to glucocorticoid receptor signaling. Whole body loss of KLF14 function in male mice does not result in metabolic abnormalities as assessed under chow and HFD conditions. Mostly likely there is redundancy for the role of KLF14 in the mouse and a diverging function in human

Characterization and structure of the human lysine-2-oxoglutarate reductase domain, a novel therapeutic target for treatment of glutaric aciduria type 1

In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyses the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine-2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for inborn errors of metabolism such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR and show that it is activated by alkylation of cysteine 414 by N-ethylmaleimide. We also provide evidence that AASS is rate-limiting upon high lysine exposure of mice. Finally, we present the crystal structure of the human LOR domain. Our combined work should enable future efforts to identify inhibitors of this novel drug target

Mild inborn errors of metabolism in commonly used inbred mouse strains

Inbred mouse strains are a cornerstone of translational research but paradoxically many strains carry mild inborn errors of metabolism. For example, alpha-aminoadipic acidemia and branched-chain ketoacid dehydrogenase deficiency are known in C57BL/6J mice. Using RNA sequencing, we now reveal the causal variants in Dhtkd1 and Bckdhb, and the molecular mechanism underlying these metabolic defects. C57BL/6J mice have decreased Dhtkd1 mRNA expression due to a solitary long terminal repeat (LTR) in intron 4 of Dhtkd1. This LTR harbors an alternate splice donor site leading to a partial splicing defect and as a consequence decreased total and functional Dhtkd1 mRNA, decreased DHTKD1 protein and alpha-aminoadipic acidemia. Similarly, C57BL/6J mice have decreased Bckdhb mRNA expression due to an LTR retrotransposon in intron 1 of Bckdhb. This transposable element encodes an alternative exon 1 causing aberrant splicing, decreased total and functional Bckdhb mRNA and decreased BCKDHB protein. Using a targeted metabolomics screen, we also reveal elevated plasma C5-camitine in 129 substrains. This biochemical phenotype resembles isovaleric acidemia and is caused by an exonic splice mutation in Ivd leading to partial skipping of exon 10 and ND protein deficiency. In summary, this study identifies three causal variants underlying mild inborn errors of metabolism in commonly used inbred mouse strains

Enhanced germination rate of B. cereus spores germinated in conditioned supernatants.

<p>Wild-type <i>B. cereus</i> spores were germinated with a 0.2 mM inosine solution (•) or in conditioned supernatants containing 0.2 mM inosine (▪). <i>B. cereus</i> spores were also germinated with 0.2 mM inosine and 20 µM alanine (X).</p

<i>B. cereus</i> spore germination at low spore concentrations.

<p>(A) <i>B. cereus</i> spores were diluted in 10 or 4000 ml (OD₅₈₀ of 1 or 0.0025, respectively) of germination buffer supplemented with 0.2 mM inosine and 0 or 40 µM alanine. Thirty min post-inosine exposure, spores were collected by centrifugation, and pellets were treated with malachite green to stain resting spores and safranin-O to stain germinated cells. Samples were placed under a microscope and a field selected at random. (B) <i>B. cereus</i> spores were diluted in germination buffer (OD₅₈₀ of 1 to 0.0025) in the presence of 0.2 mM inosine. Stained samples were placed under a light microscope and the amount of resting spores and germinated cells were counted on three different fields selected at random. The percentage of germinated spores was plotted against the initial spore optical density.</p

7-AMC adducts detected in <i>wt B. cereus</i> conditioned supernatants.

<p>Wild type <i>B. cereus</i> 569 spores were resuspended in 200 µl TMB buffer to OD₅₈₀ = 1. Spores were treated with 0.2 mM inosine and supernatants were collected 30 min post-inosine addition. Collected supernatants were labeled with 7-AMC. 7-AMC adducts sere separated by RP-HPLC and identified by mass spectrometry. Concentrations were calculated by fluorescence spectroscopy.</p