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Related Articles Microfluidic Exosome Analysis toward Liquid Biopsy for Cancer. J Lab Autom. 2016 Aug;21(4):599-608 Authors: He M, Zeng Y Abstract Assessment of a tumor's molecular makeup using biofluid samples, known as liquid biopsy, is a prominent research topic in precision medicine for cancer, due to its noninvasive property allowing repeat sampling for monitoring molecular changes of tumors over time. Circulating exosomes recently have been recognized as promising tumor surrogates because they deliver enriched biomarkers, such as proteins, RNAs, and DNA. However, purification and characterization of these exosomes are technically challenging. Microfluidic lab-on-a-chip technology effectively addresses these challenges owing to its inherent advantages in integration and automation of multiple functional modules, enhancing sensing performance, and expediting analysis processes. In this article, we review the state-of-the-art development of microfluidic technologies for exosome isolation and molecular characterization with emphasis on their applications toward liquid biopsy-based analysis of cancer. Finally, we share our perspectives on current challenges and future directions of microfluidic exosome analysis. PMID: 27215792 [PubMed - indexed for MEDLINE]

Experimental design and reporting standards for metabolomics studies of mammalian cell lines. Cell Mol Life Sci. 2017 Jul 01;: Authors: Hayton S, Maker GL, Mullaney I, Trengove RD Abstract Metabolomics is an analytical technique that investigates the small biochemical molecules present within a biological sample isolated from a plant, animal, or cultured cells. It can be an extremely powerful tool in elucidating the specific metabolic changes within a biological system in response to an environmental challenge such as disease, infection, drugs, or toxins. A historically difficult step in the metabolomics pipeline is in data interpretation to a meaningful biological context, for such high-variability biological samples and in untargeted metabolomics studies that are hypothesis-generating by design. One way to achieve stronger biological context of metabolomic data is via the use of cultured cell models, particularly for mammalian biological systems. The benefits of in vitro metabolomics include a much greater control of external variables and no ethical concerns. The current concerns are with inconsistencies in experimental procedures and level of reporting standards between different studies. This review discusses some of these discrepancies between recent studies, such as metabolite extraction and data normalisation. The aim of this review is to highlight the importance of a standardised experimental approach to any cultured cell metabolomics study and suggests an example procedure fully inclusive of information that should be disclosed in regard to the cell type/s used and their culture conditions. Metabolomics of cultured cells has the potential to uncover previously unknown information about cell biology, functions and response mechanisms, and so the accurate biological interpretation of the data produced and its ability to be compared to other studies should be considered vitally important. PMID: 28669031 [PubMed - as supplied by publisher]

Dynamic changes of plasma metabolites in pigs with GalN-induced acute liver failure using GC-MS and UPLC-MS. Biomed Pharmacother. 2017 Jun 29;93:480-489 Authors: Chen E, Lu J, Chen D, Zhu D, Wang Y, Zhang Y, Zhou N, Wang J, Li J, Li L Abstract Metabolomics facilitates investigation of the mechanisms of disease and screening for biomarkers. Here, a gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)-based metabolomics approach was employed to identify plasma biomarkers of acute liver failure (ALF) in pigs. Blood was collected from pigs at 12h intervals during ALF. Hepatic injury was quantified by determining liver function and histopathology. Based on a multivariate data matrix and pattern recognition, two upregulated metabolites, namely, amino acids and conjugated bile acids, and two downregulated metabolites, lysophosphatidylcholines (LPCs) and phosphatidylcholines (PCs), were identified. All of these metabolites showed a strong relationship with the extent of liver injury. Amino acids were biomarkers of the severity of liver impairment, conjugated bile acids were predictive of early stage liver damage, and LPCs and PCs were related to the prognosis of liver injury. In conclusion, our results demonstrated the occurrence of marked metabolic disturbances during ALF and that integrated metabolomics analysis facilitates identification of biomarkers of disease. PMID: 28668767 [PubMed - as supplied by publisher]

An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics strategy. J Pharm Biomed Anal. 2017 Jun 21;145:240-247 Authors: Sui Z, Li Q, Zhu L, Wang Z, Lv C, Liu R, Xu H, He B, Li Z, Bi K Abstract Aconiti kusnezoffii radix (AKR), the root of Aconitum kusnezoffii Reichb., is commonly used in the treatment of the rheumatoid arthritis. However, the clinical application is limited due to its potential toxicity. Therefore, to investigate the mechanism of its potential neurotoxicity and nephrotoxicity, a comprehensive metabolomics study combined with serum biochemistry and histopathology measurements was carried out. A UHPLC-Q-TOF mass spectrometry based metabolomics approach was applied to characterize the AKR toxicity, while the toxicity attenuation effects of Aconiti kusnezoffii radix cocta (AKRC) on Wistar rats were also investigated. Two chromatographic techniques involving reversed-phase chromatography and hydrophilic interaction chromatography were combined for the serum and urine detection, which balanced the integrity and selectivity of the two matrices. Principal component analysis was used to determine the groups, and principal component analysis discriminant analysis was carried out to confirm the important variables. Then, the developed integrative toxicity evaluation method was applied to assess the toxicity of AKR and the attenuation effect of AKRC. The highly sensitive and specific toxic biomarkers, which can provide practical bases were identified for the diagnosis of the neurotoxicity and nephrotoxicity induced by AKR. In all, a total of 19 putative biomarkers were characterized, and related metabolic pathways were identified. The study demonstrated that the established metabolomics strategy is a powerful approach for investigating the mechanisms of herbal toxicity and the attenuation effect of a processing method and would provide medical solutions for other toxic herbal medications and further clinical evidence on how AKR improves symptoms of rheumatoid arthritis patients. PMID: 28668652 [PubMed - as supplied by publisher]

GC-MS based metabolomics of CSF and blood serum: Metabolic phenotype for a rat model of cefoperazone-induced disulfiram-like reaction. Biochem Biophys Res Commun. 2017 Jun 28;: Authors: Liu L, Huang C, Bian Y, Miao L Abstract Cefoperazone are most popularly used in the treatment of complicated infections clinically. Concomitant ingestion of ethnaol and cefoperazone may cause a disulfiram-like reaction. However, very little is known about the possible interactions between cefoperazone treatment and an alcohol with regard to the induction of disulfiram-like reaction. Study of the metabolic impact of cotreatment with cefoperazone and alcohol on animals can facilitate the identification of markers relevant to disulfiram-like reaction. In this study, the serum and cerebrospinal fluid (CSF) metabolites from Sprague-Dawley rats were profiled using gas chromatography mass spectrometry. Serum levels of valine, leucine, glycine, palmitelaidic acid, and 2-hydroxyisobutyrate in combination application group were significantly higher than those in the control; while alanine and pyruvate deceased in cotreatment group. Most TCA intermediates, glutamate and aspartate had lower CSF level in combination application group, except citrate. In addition, most carbohydrates, ethylmalonate and N-acetylaspartate had higher level compared with control group. These results highlight concomitant ingestion of alcohol and cefoperazone generated disulfiram-like reaction by way of disrupting normal metabolic pathway. Cefoperazone magnifes ethanol-induced impairment of TCA cycle and aspartate metabolism, thereby affects energy metabolism and neural transmission. PMID: 28668395 [PubMed - as supplied by publisher]

The use of metabolomic quantitative trait locus mapping and osmotic adjustment traits for the improvement of crop yields under environmental stresses. Semin Cell Dev Biol. 2017 Jun 28;: Authors: Adbelrahman M, Burritt DJ, Tran LP Abstract The sustainable production of food to feed an increasing world population is a major challenge for plant scientists, especially due to the unpredictable and dynamic nature of global climatic conditions. Heat waves, drought, increased soil salinity, unseasonal cold and flooding are all becoming more common climate-related causes of stress for crop plants, and are already affecting yields and the geographical distributions of optimal growing regions for many crops. Therefore, the development and application of multi-faceted strategies, including sustainable agricultural practices and the development and cultivation of intraspecific hybrids containing genetic traits associated with abiotic stress tolerance from wild relatives, will either alone or together be essential to sustainably grow high-yielding crops under increasingly stressful environmental conditions. The development of abiotic stress-resilient crops requires an in-depth knowledge of plant development and of the biological processes that enable plants to survive in stressful environments, and this knowledge can be obtained from "omic" studies, such as bioinformatics, genomics, transcriptomics, proteomics and metabolomics. The plant metabolome can provide a snapshot of the physiological and biochemical status of a plant cell under normal or stressful conditions, and thus it is closely related to the plant phenotypes. Analysis of the metabolomes of plants growing under stressful conditions can be used to identify stress resistance-associated metabolites, or biomarkers, which can then be used by plant breeders as selective markers to help identify the phenotypes, resulted from the complex interactions between genotype and environment, associated with stress-tolerant crop plants. Osmotic adjustment is an important metabolic adaptation mechanism which helps plants survive abiotic stress and can support higher crop yield under stressful environmental conditions. This review highlights the recent advances in our understanding of the functions of abiotic stress-responsive metabolites, with an emphasis on the use of metabolomic quantitative trait locus mapping and osmotic adjustment agronomic traits, for the improvement of crop yields under environmental stresses. PMID: 28668354 [PubMed - as supplied by publisher]

Unravelling HIV-1 Latency, One Cell at a Time. Trends Microbiol. 2017 Jun 28;: Authors: Kok YL, Ciuffi A, Metzner KJ Abstract A single virus is capable of infecting and replicating in a single cell. Recent advances across single-cell omics technologies - genomics, epigenomics, transcriptomics, epitranscriptomics, proteomics, and metabolomics - will offer unprecedented opportunities to gain more insights into the various aspects of the life cycle of viruses and their impact on the host cell. Here, using the human immunodeficiency virus type 1 (HIV-1) as an example, we summarize the current knowledge and the future potential of single-cell omics in the investigation of an important aspect of the life cycle of HIV-1 that represents a major hurdle in achieving viral eradication, HIV-1 latency. PMID: 28668335 [PubMed - as supplied by publisher]

Valproic acid alters the content and function of the cell-free DNA released by hepatocellular carcinoma (HepG2) cells in vitro. Biochimie. 2017 Jun 28;: Authors: Aucamp J, Van Dyk HC, Bronkhorst AJ, Pretorius PJ Abstract BACKGROUND: It has long been believed that cell-free DNA (cfDNA) actively released into circulation can serve as intercellular messengers, and their involvement in processes such as the bystander effect strongly support this. However, this intercellular messaging function of cfDNA may have clinical implications that have not yet been considered. METHODS: CfDNA was isolated from the growth medium of HepG2 cells treated with valproic acid (VPA). This cfDNA was then administered to untreated cells and cellular metabolic activity was measured. RESULTS: VPA altered the characteristics of cfDNA released by treated HepG2 cells in vitro. When administered to untreated cells, the cfDNA from cells treated with VPA resulted in the dose-dependent induction of glycolytic activity within 36 min of administration, but little to no alterations in oxidative phosphorylation. The glycolytic activity lasted for 4-6 h, whereas changes in subsequent cfDNA release and characteristics were found to remain persistent after two 24 h treatments. Fragmented genomic DNA from VPA-treated cells did not induce the effects observed for cfDNA obtained VPA-treated cells. CONCLUSIONS: It is possible for cfDNA to, under in vitro conditions, transfer pharmaceutically-induced effects to untreated recipient cells. Further investigation regarding this occurrence under in vivo conditions is, therefore, strongly encouraged. GENERAL SIGNIFICANCE: The intercellular messaging functions of cfDNA present in donated biological fluids has potential clinical implications that require urgent attention. These implications may, however, also have potential as new forms of treatment that can circumvent pharmacological barriers. PMID: 28668269 [PubMed - as supplied by publisher]

Metabolic profiling of the traditional Chinese medicine formulation Yu Ping Feng San for the identification of constituents relevant for effects on expression of TNF-α, IFN-γ, IL-1β and IL-4 in U937 cells. J Pharm Biomed Anal. 2017 Jun 15;145:219-229 Authors: Stefanie N, Marlene M, Huiqin Z, Yong L, Xiaojuan H, Danping F, Aiping L, Kate Y, Giorgis I, Rudolf B Abstract Yu Ping Feng San (YPFS) is a classical TCM formulation which has been traditionally used for treatment of immune system related diseases such as chronic bronchitis, allergic rhinitis and asthma. The formula is a mixture of Radix Saposhnikoviae (Fangfeng), Radix Astragali (Huangqi), and Rhizoma Atractylodis macrocephalae (Baizhu). TLC- and LC-DAD-ESI-MS/MS methods have been developed for the analysis of the metabolic profiles of the single herbs and of the formula. Decoctions and ASE extracts were analyzed in order to trace components of the individual herbs in YPFS. Nine constituents of Radix Saposhnikoviae, ten constituents of Radix Astragali and five constituents of Rhizoma Atractylodis macrocephalae have been assigned in the chemical profiles of the formula, which now allow the standardisation of YPFS. The pharmacological testing showed that all extracts significantly inhibited expression of TNF-α, IFN-γ, and IL-1β in U937 cells, while the inhibition of IL-4 was consistently low. Compared to conventional analyses which are focused on a limited set of compounds, metabolomics approaches, together with novel data processing tools, enable a more holistic comparison of the herbal extracts. In order to identify the constituents which are relevant for the immunomodulatory effects of the formula, metabolomics studies (PCA, OPLS-DA) have been performed using UPLC/QTOF MS data. PMID: 28667937 [PubMed - as supplied by publisher]

Metabolomics analysis of TiO2 nanoparticles induced toxicological effects on rice (Oryza sativa L.). Environ Pollut. 2017 Jun 28;230:302-310 Authors: Wu B, Zhu L, Le XC Abstract The wide occurrence and high environmental concentration of titanium dioxide nanoparticles (nano-TiO2) have raised concerns about their potential toxic effects on crops. In this study, we employed a GC-MS-based metabolomic approach to investigate the potential toxicity of nano-TiO2 on hydroponically-cultured rice (Oryza sativa L.) after exposed to 0, 100, 250 or 500 mg/L of nano-TiO2 for fourteen days. Results showed that the biomass of rice was significantly decreased and the antioxidant defense system was significantly disturbed after exposure to nano-TiO2. One hundred and five identified metabolites showed significant difference compared to the control, among which the concentrations of glucose-6-phosphate, glucose-1-phosphate, succinic and isocitric acid were increased most, while the concentrations of sucrose, isomaltulose, and glyoxylic acid were decreased most. Basic energy-generating ways including tricarboxylic acid cycle and the pentose phosphate pathway, were elevated significantly while the carbohydrate synthesis metabolism including starch and sucrose metabolism, and glyoxylate and dicarboxylate metabolism were inhibited. However, the biosynthetic formation of most of the identified fatty acids, amino acids and secondary metabolites which correlated to crop quality, were increased. The results suggest that the metabolism of rice plants is distinctly disturbed after exposure to nano-TiO2, and nano-TiO2 would have a mixed effect on the yield and quality of rice. PMID: 28667911 [PubMed - as supplied by publisher]

38 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2017/07/01PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

21 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2017/06/30PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer. Nature. 2017 Jun 28;: Authors: Zabala-Letona A, Arruabarrena-Aristorena A, Martín-Martín N, Fernandez-Ruiz S, Sutherland JD, Clasquin M, Tomas-Cortazar J, Jimenez J, Torres I, Quang P, Ximenez-Embun P, Bago R, Ugalde-Olano A, Loizaga-Iriarte A, Lacasa-Viscasillas I, Unda M, Torrano V, Cabrera D, van Liempd SM, Cendon Y, Castro E, Murray S, Revandkar A, Alimonti A, Zhang Y, Barnett A, Lein G, Pirman D, Cortazar AR, Arreal L, Prudkin L, Astobiza I, Valcarcel-Jimenez L, Zuñiga-García P, Fernandez-Dominguez I, Piva M, Caro-Maldonado A, Sánchez-Mosquera P, Castillo-Martín M, Serra V, Beraza N, Gentilella A, Thomas G, Azkargorta M, Elortza F, Farràs R, Olmos D, Efeyan A, Anguita J, Muñoz J, Falcón-Pérez JM, Barrio R, Macarulla T, Mato JM, Martinez-Chantar ML, Cordon-Cardo C, Aransay AM, Marks K, Baselga J, Tabernero J, Nuciforo P, Manning BD, Marjon K, Carracedo A Abstract Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program. PMID: 28658205 [PubMed - as supplied by publisher]

The past decade in bench research into pulmonary infectious diseases: What do clinicians need to know? Respirology. 2017 Jun 28;: Authors: Finch S, Keir HR, Dicker AJ, Chalmers JD Abstract Respiratory infections are primarily treated with antibiotics, drugs that are mostly inexpensive and have been widely available since the 1940s and 1950s. Nevertheless, despite antibiotics, the burden of disease in pneumonia, bronchiectasis, cystic fibrosis, COPD and rare respiratory infections remains exceptionally high. There is an urgent need for translational studies to develop new treatments or new biomarkers to improve outcomes in these conditions. The 'translational gaps' between bench science and clinical practice are particularly challenging in respiratory infections. This is partly due to the poor representativeness of animal models of infection to human disease, and a long-term lack of investment into pulmonary infection research. The revolution in genomics and other omics technologies, however, is beginning to unlock clinically important information about the host response to infection, the behaviour of bacterial communities and the development of new antibiotics. It is not possible to review the extensive progress made in the last decade into the pathophysiology of the different respiratory infections and so here, we focus on major technologies that are now changing respiratory infection research, specifically bacterial whole-genome sequencing, the microbiota, personalized medicine with omics technologies, new antibiotic development and host inflammatory cell biology. PMID: 28657170 [PubMed - as supplied by publisher]

Evaluation of microextraction by packed sorbent, liquid-liquid microextraction and derivatization pretreatment of diet-derived phenolic acids in plasma by gas chromatography with triple quadrupole mass spectrometry. J Sep Sci. 2017 Jun 28;: Authors: Bustamante L, Cárdenas D, von Baer D, Pastene E, Duran-Sandoval D, Vergara C, Mardones C Abstract Miniaturized sample pretreatments for the analysis of phenolic metabolites in plasma, involving protein precipitation, enzymatic deconjugation, extraction procedures and different derivatization reactions were systematically evaluated. The analyses were conducted by gas chromatography with mass spectrometry for the evaluation of 40 diet-derived phenolic compounds. An enzyme purification was necessary for the phenolic deconjugation before extraction. Trimethylsilanization reagent and two different tetrabutylammonium salts for derivatization reactions were compared. The optimum reaction conditions were 50 μL of trimethylsilanization reagent at 90°C for 30 min, while tetrabutylammonium salts were associated with loss of sensitivity due to rapid activation of the inert gas chromatograph liner. Phenolic acids extractions from plasma were optimized, optimal microextraction by packed sorbent performance was achieved using an octadecylsilyl packed bed and better recoveries for less polar compounds, such as methoxylated derivatives, were observed. Despite the low recovery for many analytes, repeatability using an automated extraction procedure in the gas chromatograph inlet was 2.5%. Instead, using liquid-liquid microextraction, better recoveries (80-110%) for all analytes were observed at the expense of repeatability (3.8-18.4%). The phenolic compounds in gerbil plasma samples, collected before and 4 h after the administration of a calafate extract, were analyzed with the optimized methodology. This article is protected by copyright. All rights reserved. PMID: 28657140 [PubMed - as supplied by publisher]

Related Articles Metabolomics Identifies Metabolic Markers of Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes. Theranostics. 2017;7(7):2078-2091 Authors: Bhute VJ, Bao X, Dunn KK, Knutson KR, McCurry EC, Jin G, Lee WH, Lewis S, Ikeda A, Palecek SP Abstract Cardiovascular disease is a leading cause of death worldwide. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold immense clinical potential and recent studies have enabled generation of virtually pure hPSC-CMs with high efficiency in chemically defined and xeno-free conditions. Despite these advances, hPSC-CMs exhibit an immature phenotype and are arrhythmogenic in vivo, necessitating development of strategies to mature these cells. hPSC-CMs undergo significant metabolic alterations during differentiation and maturation. A detailed analysis of the metabolic changes accompanying maturation of hPSC-CMs may prove useful in identifying new strategies to expedite hPSC-CM maturation and also may provide biomarkers for testing or validating hPSC-CM maturation. In this study we identified global metabolic changes which take place during long-term culture and maturation of hPSC-CMs derived from three different hPSC lines. We have identified several metabolic pathways, including phospholipid metabolism and pantothenate and Coenzyme A metabolism, which showed significant enrichment upon maturation in addition to fatty acid oxidation and metabolism. We also identified increases in glycerophosphocholine and the glycerophosphocholine:phosphocholine ratio as potential metabolic biomarkers of maturation. These biomarkers were also affected in a similar manner during murine heart development in vivo. These results support that hPSC-CM maturation is associated with extensive metabolic changes in metabolic network utilization and understanding the roles of these metabolic changes has the potential to develop novel approaches to monitor and expedite hPSC-CM maturation. PMID: 28656061 [PubMed - in process]

Related Articles Cell-free DNA in a three-dimensional spheroid cell culture model: A preliminary study. Int J Biochem Cell Biol. 2017 Jun 24;: Authors: Aucamp J, Calitz C, Bronkhorst AJ, Wrzesinski K, Hamman S, Gouws C, Pretorius PJ Abstract BACKGROUND: Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo state, may be of significant benefit for cfDNA research. METHODS: CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose consumption and was related to isolated cfDNA characteristics. RESULTS: Changes in spheroid growth and stability were effectively mirrored by cfDNA characteristics. CfDNA characteristics correlated with that of previous two-dimensional (2D) cell culture and human plasma research. CONCLUSIONS: 3D spheroid cultures can serve as effective, simplified in vivo-simulating "closed-circuit" models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. BIOLOGICAL SIGNIFICANCE: 3D cell cultures can be used to translate "closed-circuit" in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields. PMID: 28655575 [PubMed - as supplied by publisher]

Related Articles A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in Aspergillus fumigatus. MBio. 2017 Feb 07;8(1): Authors: Shwab EK, Juvvadi PR, Waitt G, Soderblom EJ, Moseley MA, Nicely NI, Asfaw YG, Steinbach WJ Abstract Invasive aspergillosis (IA), caused by the filamentous fungal pathogen Aspergillus fumigatus, is a major cause of death among immunocompromised patients. The cyclic AMP/protein kinase A (PKA) signaling pathway is essential for hyphal growth and virulence of A. fumigatus, but the mechanism of regulation of PKA remains largely unknown. Here, we discovered a novel mechanism for the regulation of PKA activity in A. fumigatus via phosphorylation of key residues within the major catalytic subunit, PkaC1. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of PkaC1 at four sites (S175, T331, T333, and T337) with implications for important and diverse roles in the regulation of A. fumigatus PKA. While the phosphorylation at one of the residues (T333) is conserved in other species, the identification of three other residues represents previously unknown PKA phosphoregulation in A. fumigatus Site-directed mutagenesis of the phosphorylated residues to mimic or prevent phosphorylation revealed dramatic effects on kinase activity, growth, conidiation, cell wall stress response, and virulence in both invertebrate and murine infection models. Three-dimensional structural modeling of A. fumigatus PkaC1 substantiated the positive or negative regulatory roles for specific residues. Suppression of PKA activity also led to downregulation of PkaC1 protein levels in an apparent novel negative-feedback mechanism. Taken together, we propose a model in which PkaC1 phosphorylation both positively and negatively modulates its activity. These findings pave the way for future discovery of fungus-specific aspects of this key signaling network. IMPORTANCE: Our understanding of signal transduction networks in pathogenic fungi is limited, despite the increase in invasive fungal infections and rising mortality rates in the immunosuppressed patient population. Because PKA is known to be essential for hyphal growth and virulence of A. fumigatus, we sought to identify fungus-specific regulatory mechanisms governing PKA activity. In this study, we identify, for the first time, a novel mechanism for the regulation of PKA signaling in which differential phosphorylation of the PkaC1 catalytic subunit can lead to either positive or negative regulation of activity. Furthermore, we show that inactivation of PKA signaling leads to downregulation of catalytic subunit protein levels in a negative-feedback mechanism distinct from expression patterns previously reported in the yeasts. Our findings represent a divergence in the regulation of PKA signaling in A. fumigatus, which could potentially be exploited as a target and also open the avenue for discovery of fungus-specific downstream effectors of PKA. PMID: 28174315 [PubMed - indexed for MEDLINE]

Metabolite Measurement: Pitfalls to Avoid and Practices to Follow. Annu Rev Biochem. 2017 Jun 20;86:277-304 Authors: Lu W, Su X, Klein MS, Lewis IA, Fiehn O, Rabinowitz JD Abstract Metabolites are the small biological molecules involved in energy conversion and biosynthesis. Studying metabolism is inherently challenging due to metabolites' reactivity, structural diversity, and broad concentration range. Herein, we review the common pitfalls encountered in metabolomics and provide concrete guidelines for obtaining accurate metabolite measurements, focusing on water-soluble primary metabolites. We show how seemingly straightforward sample preparation methods can introduce systematic errors (e.g., owing to interconversion among metabolites) and how proper selection of quenching solvent (e.g., acidic acetonitrile:methanol:water) can mitigate such problems. We discuss the specific strengths, pitfalls, and best practices for each common analytical platform: liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and enzyme assays. Together this information provides a pragmatic knowledge base for carrying out biologically informative metabolite measurements. PMID: 28654323 [PubMed - in process]

Replication Study: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Elife. 2017 Jun 27;6: Authors: Showalter MR, Hatakeyama J, Cajka T, VanderVorst K, Carraway KL, Fiehn O, Reproducibility Project: Cancer Biology Abstract In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fiehn et al., 2016), that described how we intended to replicate selected experiments from the paper "The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate" (Ward et al., 2010). Here, we report the results of those experiments. We found that cells expressing R172K mutant IDH2 did not display isocitrate-dependent NADPH production above vector control levels, in contrast to the increased production observed with wild-type IDH2. Conversely, expression of R172K mutant IDH2 resulted in increased alpha-ketoglutarate-dependent consumption of NADPH compared to wild-type IDH2 or vector control. These results are similar to those reported in the original study (Figure 2; Ward et al., 2010). Further, expression of R172K mutant IDH2 resulted in increased 2HG levels within cells compared to the background levels observed in wild-type IDH2 and vector control, similar to the original study (Figure 3D; Ward et al., 2010). In primary human AML samples, the 2HG levels observed in samples with mutant IDH1 or IDH2 status were higher than those observed in samples without an IDH mutation, similar to what was observed in the original study (Figure 5C; Ward et al., 2010). Finally, we report meta-analyses for each result. PMID: 28653623 [PubMed - in process]