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Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3AG) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3AG variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.

Background:Congenital heart disease (CHD) is the most common anomaly at birth, with a prevalence of ≈1%. While infants born to mothers with diabetes or obesity have a 2- to 3-fold increased incidence of CHD, the cause of the increase is unknown. Damaging de novo variants (DNV) in coding regions are more common among patients with CHD, but genome-wide rates of coding and noncoding DNVs associated with these prenatal exposures have not been studied in patients with CHD.Methods:DNV frequencies were determined for 1812 patients with CHD who had whole-genome sequencing and prenatal history data available from the Pediatric Cardiac Genomics Consortium’s CHD GENES study (Genetic Network). The frequency of DNVs was compared between subgroups usingttest or linear model.Results:Among 1812 patients with CHD, the number of DNVs per patient was higher with maternal diabetes (76.5 versus 72.1,ttestP=3.03×10−11), but the difference was no longer significant after including parental ages in a linear model (paternal and maternal correctionP=0.42). No interaction was observed between diabetes risk and parental age (paternal and maternal interactionP=0.80 and 0.68, respectively). No difference was seen in DNV count per patient based on maternal obesity (72.0 versus 72.2 for maternal body mass index 30,ttestP=0.86).Conclusions:After accounting for parental age, the offspring of diabetic or obese mothers have no increase in DNVs compared with other children with CHD. These results emphasize the role for other mechanisms in the cause of CHD associated with these prenatal exposures.Registration:URL:https://clinicaltrials.gov; NCT01196182.

Objective The transcription factor Kruppel-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. Methods 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. Results 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. Conclusions 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 humans.

// Sarabjot Pabla 1 , * , Jonathan Andreas 1 , * , Felicia L. Lenzo 1 , Blake Burgher 1 , Jacob Hagen 1 , Vincent Giamo 1 , Mary K. Nesline 1 , Yirong Wang 1 , Mark Gardner 1 , Jeffrey M. Conroy 1 , 2 , Antonios Papanicolau-Sengos 1 , Carl Morrison 1 , 2 and Sean T. Glenn 1 , 2 , 3 1 OmniSeq Inc., Buffalo, NY 14203, USA 2 Center for Personalized Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA 3 Department of Molecular and Cellular Biology, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA * These authors contributed equally to this work Correspondence to: Sean T. Glenn, email: sean.glenn@omniseq.com Keywords: next-generation sequencing; NGS; microsatellite instability; MSI Abbreviations: NGS: next-generation sequencing Received: June 10, 2019 Accepted: July 29, 2019 Published: August 27, 2019 ABSTRACT Background We have developed and analytically validated a next-generation sequencing (NGS) assay to classify microsatellite instability (MSI) in formalin-fixed paraffin-embedded (FFPE) tumor specimens. Methodology The assay relies on DNA-seq evaluation of insertion/deletion (indel) variability at 29 highly informative genomic loci to estimate MSI status without the requirement for matched-normal tissue. The assay has a clinically relevant five-day turnaround time and can be conducted on as little as 20 ng genomic DNA with a batch size of up to forty samples in a single run. Results The MSI detection method was developed on a training set ( n = 94) consisting of 22 MSI-H, 24 MSS, and 47 matched normal samples and tested on an independent test set of 24 MSI-H and 24 MSS specimens. Assay performance with respect to accuracy, reproducibility, precision as well as control sample performance was estimated across a wide range of FFPE samples of multiple histologies to address pre-analytical variability (percent tumor nuclei), and analytical variability (batch size, run, day, operator). Analytical precision studies demonstrated that the assay is highly reproducible and accurate as compared to established gold standard PCR methodology and has been validated through NYS CLEP. Significance This assay provides clinicians with robust and reproducible NGS-based MSI testing without the need of matched normal tissue to inform clinical decision making for patients with solid tumors.

Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid β-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4. ispartof: FASEB JOURNAL vol:33 issue:3 pages:4355-4364 ispartof: location:United States status: published

Background Resistance to immune checkpoint inhibitors (ICIs) has been linked to local immunosuppression independent of major ICI targets (e.g., PD-1). Clinical experience with response prediction based on PD-L1 expression suggests that other factors influence sensitivity to ICIs in non-small cell lung cancer (NSCLC) patients. Methods Tumor specimens from 120 NSCLC patients from 10 institutions were evaluated for PD-L1 expression by immunohistochemistry, and global proliferative profile by targeted RNA-seq. Results Cell proliferation, derived from the mean expression of 10 proliferation-associated genes (namely BUB1, CCNB2, CDK1, CDKN3, FOXM1, KIAA0101, MAD2L1, MELK, MKI67, and TOP2A), was identified as a marker of response to ICIs in NSCLC. Poorly, moderately, and highly proliferative tumors were somewhat equally represented in NSCLC, with tumors with the highest PD-L1 expression being more frequently moderately proliferative as compared to lesser levels of PD-L1 expression. Proliferation status had an impact on survival in patients with both PD-L1 positive and negative tumors. There was a significant survival advantage for moderately proliferative tumors compared to their combined highly/poorly counterparts (p = 0.021). Moderately proliferative PD-L1 positive tumors had a median survival of 14.6 months that was almost twice that of PD-L1 negative highly/poorly proliferative at 7.6 months (p = 0.028). Median survival in moderately proliferative PD-L1 negative tumors at 12.6 months was comparable to that of highly/poorly proliferative PD-L1 positive tumors at 11.5 months, but in both instances less than that of moderately proliferative PD-L1 positive tumors. Similar to survival, proliferation status has impact on disease control (DC) in patients with both PD-L1 positive and negative tumors. Patients with moderately versus those with poorly or highly proliferative tumors have a superior DC rate when combined with any classification schema used to score PD-L1 as a positive result (i.e., TPS ≥ 50% or ≥ 1%), and best displayed by a DC rate for moderately proliferative tumors of no less than 40% for any classification of PD-L1 as a negative result. While there is an over representation of moderately proliferative tumors as PD-L1 expression increases this does not account for the improved survival or higher disease control rates seen in PD-L1 negative tumors. Conclusions Cell proliferation is potentially a new biomarker of response to ICIs in NSCLC and is applicable to PD-L1 negative tumors. Electronic supplementary material The online version of this article (10.1186/s40425-019-0506-3) contains supplementary material, which is available to authorized users.

Inbred mouse strains are a cornerstone of translational research but paradoxically many strains carry mild inborn errors of metabolism. For example, α-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 α-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.

Aims Mitochondrial fatty acid oxidation (FAO) is an important energy provider for cardiac work and changes in cardiac substrate preference are associated with different heart diseases. Carnitine palmitoyltransferase 1B (CPT1B) is thought to perform the rate limiting enzyme step in FAO and is inhibited by malonyl-CoA. The role of CPT1B in cardiac metabolism has been addressed by inhibiting or decreasing CPT1B protein or after modulation of tissue malonyl-CoA metabolism. We assessed the role of CPT1B malonyl-CoA sensitivity in cardiac metabolism. Methods and results We generated and characterized a knock in mouse model expressing the CPT1BE3A mutant enzyme, which has reduced sensitivity to malonyl-CoA. In isolated perfused hearts, FAO was 1.9-fold higher in Cpt1bE3A/E3A hearts compared with Cpt1bWT/WT hearts. Metabolomic, proteomic and transcriptomic analysis showed increased levels of malonylcarnitine, decreased concentration of CPT1B protein and a small but coordinated downregulation of the mRNA expression of genes involved in FAO in Cpt1bE3A/E3A hearts, all of which aim to limit FAO. In vivo assessment of cardiac function revealed only minor changes, cardiac hypertrophy was absent and histological analysis did not reveal fibrosis. Conclusions Malonyl-CoA-dependent inhibition of CPT1B plays a crucial role in regulating FAO rate in the heart. Chronic elevation of FAO has a relatively subtle impact on cardiac function at least under baseline conditions.

Alpha-aminoadipic and alpha-ketoadipic aciduria is an autosomal recessive inborn error of lysine, hydroxylysine, and tryptophan degradation. To date, DHTKD1 mutations have been reported in two alpha-aminoadipic and alpha-ketoadipic aciduria patients. We have now sequenced DHTKD1 in nine patients diagnosed with alpha-aminoadipic and alpha-ketoadipic aciduria as well as one patient with isolated alpha-aminoadipic aciduria, and identified causal mutations in eight. We report nine novel mutations, including three missense mutations, two nonsense mutations, two splice donor mutations, one duplication, and one deletion and insertion. Two missense mutations, one of which was reported before, were observed in the majority of cases. The clinical presentation of this group of patients was inhomogeneous. Our results confirm that alpha-aminoadipic and alpha-ketoadipic aciduria is caused by mutations in DHTKD1, and further establish that DHTKD1 encodes the E1 subunit of the alpha-ketoadipic acid dehydrogenase complex.

In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which MTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of “master lysosomal regulators”. Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated. Here, we identify HEPES, a chemical buffering agent that is broadly applied in cell culture, as a potent inducer of lysosome biogenesis. Supplementation of HEPES to cell growth media is sufficient to decouple the MiT/TFE family members–TFEB, TFE3 and MITF–from regulatory mechanisms that control their cytosolic retention. Increased MiT/TFE nuclear import in turn drives the expression of a global network of lysosomal-autophagic and innate host-immune response genes, altering lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling. In addition, siRNA-mediated MiT/TFE knockdown effectively blunted HEPES-induced lysosome biogenesis and gene expression profiles. Mechanistically, we show that MiT/TFE activation in response to HEPES requires its macropinocytic ingestion and aberrant lysosomal storage/pH, but is independent of MTORC1 signaling. Altogether, our data underscore the cautionary use of chemical buffering agents in cell culture media due to their potentially confounding effects on experimental results.

Objective Cardiac hexokinase II (HKII) can translocate between cytosol and mitochondria and change its cellular expression with pathologies such as ischemia–reperfusion, diabetes and heart failure. The cardiac metabolic consequences of these changes are unknown. Here we measured energy substrate utilization in cytosol and mitochondria using stabile isotopes and oxygen consumption of the intact perfused heart for 1) an acute decrease in mitochondrial HKII (mtHKII), and 2) a chronic decrease in total cellular HKII. Methods/results We first examined effects of 200 nM TAT (Trans-Activator of Transcription)-HKII peptide treatment, which was previously shown to acutely decrease mtHKII by ~ 30%. In Langendorff-perfused hearts TAT-HKII resulted in a modest, but significant, increased oxygen consumption, while cardiac performance was unchanged. At the metabolic level, there was a nonsignificant (p = 0.076) ~ 40% decrease in glucose contribution to pyruvate and lactate formation through glycolysis and to mitochondrial citrate synthase flux (6.6 ± 1.1 vs. 11.2 ± 2.2%), and an 35% increase in tissue pyruvate (27 ± 2 vs. 20 ± 2 pmol/mg; p = 0.033). Secondly, we compared WT and HKII+/− hearts (50% chronic decrease in total HKII). RNA sequencing revealed no differential gene expression between WT and HKII+/− hearts indicating an absence of metabolic reprogramming at the transcriptional level. Langendorff-perfused hearts showed no significant differences in glycolysis (0.34 ± 0.03 μmol/min), glucose contribution to citrate synthase flux (35 ± 2.3%), palmitate contribution to citrate synthase flux (20 ± 1.1%), oxygen consumption or mechanical performance between WT and HKII+/− hearts. Conclusions These results indicate that acute albeit not chronic changes in mitochondrial HKII modestly affect cardiac oxygen consumption and energy substrate metabolism., final draft, peerReviewed

Many chemometricians today are dependent on having access to Matlab for development of algorithms and analysis of data sets. This article describes some of the potential dangers with such a dependency and how it may affect the field of chemometrics as a whole. Instead of using a proprietary scripting language, it is here suggested that the field should find a solution based on free or open source software. The most realistic open source alternative to Matlab today is Octave which covers most of the Matlab syntax and data structures. However, until now it has been lacking an interactive graphics system like Matlab's Handle Graphics (HG) which is very important to chemometricians for viewing data and inspecting results from analyses. To rectify this situation we are developing the Oplot graphics system which is compatible with the HG syntax. The main structure of Oplot is described and how it compares with HG. Not all HG functionality is currently implemented, however the most common functions and objects are available such as figure, axes, line, image, text, plot, subplot, set, get, clf, hold on/off, grid on/off, labels and title. Ensuring that Octave/Oplot is as compatible with Matlab/HG as possible, will make it easier for chemometricians to make a switch. We argue that such a switch to an open source solution is necessary to ensure long term stability and control over our future scientific and technological developments.

Novel, single nucleotide mutations were identified in the mitochondrial methionyl amino-acyl-tRNA synthetase gene (MARS2) via whole exome sequencing in two affected siblings with developmental delay, poor growth, and sensorineural hearing loss. We show that compound heterozygous mutations c.550C>T:p.Gln184* and c.424C>T:p.Arg142Trp in MARS2 lead to decreased MARS2 protein levels in patient lymphoblasts. Analysis of respiratory complex (RC) enzyme activities in patient fibroblasts revealed decreased Complex I and IV activities. Immunoblotting of patient fibroblast and lymphoblast samples revealed reduced protein levels of NDUFB8 and COXII, representing Complex I and IV respectively. Additionally, overexpression of wild-type MARS2 in patient fibroblasts increased NDUFB8 and COXII protein levels. These findings suggest that recessive single nucleotide mutations in MARS2 are causative for a new mitochondrial translation deficiency disorder with a primary phenotype including developmental delay and hypotonia. Identification of additional patients with single nucleotide mutations in MARS2 is necessary to determine if pectus carinatum is also a consistent feature of this syndrome.

Abstract During the studies of ice loads on an Arctic Floater in ice, a series of numerical simulations were carried out in an advanced ship simulator with advanced ice module. The operation of the Arctic Floater will periodically be supported by an ice management system involving also physical ice management. This paper starts with introduction to the theoretical background for the Ice model, wherein the numerical model enables modelling of the arctic floater the mooring and different managed ice conditions. The managed ice is created as convex ice floes with comparable size ice floes used in model scale tests. A number of simulations were compared with and validated against results from identical set up in ice tank tests. Based on a set of assumptions from these simulations, a series of situations with varying ice drift direction, ice drift speed, floe thickness, domain size and ice floe coverage made the input parameters to the numerical model. Ice loading on the Arctic Floater is governed by ice breaking loads and ice transport around the hull. During the numerical simulations, the movement of the Arctic Floater was studied, the forces were calculated and influence of boundary conditions was investigated. The numerical simulations gave the possibility to compare ice floe interaction with and without boundary influence of an ice tank set up. Simulations was also used to show what would happen if the Arctic Floater is hit by a large unbreakable ice floe. The results presented in this paper show good correlation with model scale results for the Arctic Floater in drifting managed ice. Results from additional simulations gave useful input for predicting ice forces and system behaviour in ice conditions beyond what is possible in an ice tank test set-up.

Numerous studies have implicated impaired early growth (low birth weight [LBW]) as a risk factor for the development of obesity (1) and type 2 diabetes (2). Obesity, particularly abdominal obesity, is associated with an increased risk of insulin resistance and type 2 diabetes, and although the mechanistic basis is not fully known, several characteristics of the metabolically active visceral fat depot, including ready release of free fatty acids and adipokines into the portal circulation, have been proposed as potential mediators of whole-body insulin resistance. In contrast, leg fat appears to be protective against the development of glucose intolerance (3). Little is known about time of onset of fat accumulation and the regional distribution of fat tissue in LBW subjects. Previous studies have used indirect methods such as BMI, waist and hip circumference, and waist-to-hip ratio to assess adiposity and body fat distribution. The limitation of those measurements was recently demonstrated (4). We determined whole-body fat content and regional fat distribution by anthropometrical measurements (BMI and waist and hip circumference) and dual-energy X-ray absorptiometry (DEXA) (Norland XR-26 …