Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli

The global pool of all metabolites in a cell, or metabolome, is a reflection of all the metabolic functions of an organism under any particular growth condition. In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction. In the past, a variety of cell lysis methods were adopted for assays of individual metabolites or groups of intermediates in pathways. In this study, metabolites were extracted from Escherichia coli using six different commonly used procedures including acid or alkaline treatments, permeabilization by freezing with methanol, high-temperature extraction in the presence of ethanol or methanol, and by lysis with chloroform-methanol. Metabolites were extracted by the six methods from cells grown under identical conditions and labeled with [14C]glucose. The metabolomes were compared after 2-dimensional thin-layer chromatography of labeled compounds. For global analysis, extraction with cold (-40 degrees C) methanol showed the greatest promise, allowing simultaneous resolution of more than 95 metabolite spots. In contrast, 80 or less spots were obtained with other extraction methods. Extraction also influenced quantitative analysis of particular compounds. Metabolites such as adenosine exhibited up to 20-fold higher abundance after cold methanol extraction than after extraction with acid, alkali, or chloroform. The simplicity, rapidity, and universality of cold methanol extraction offer great promise if a single method of lysis is to be adopted in metabolome analysis.     

Plant Functional Traits and the Distribution of West African Rain Forest Trees along the Rainfall Gradient

Plant morphological and physiological traits affect the way plants tolerate environmental stresses and therefore play an important role in shaping species distribution patterns in relation to environmental gradients. Despite our growing knowledge on the role of functional traits in structuring plant communities, few studies have tested their importance at large scales in the wet tropics. Here, we describe the distribution patterns of the most important West African rain forest timber species along the regional rainfall gradient and relate them to their functional traits. We found that the distribution patterns of 25 out of the 31 studied species (80%) were significantly related to mean annual rainfall. Shade tolerance and drought resistance were identified as the main strategy axes of variation. Wood density and leaf deciduousness emerged as the best predictor traits of species position along the rainfall gradient, explaining respectively 32 and 15 percent of the variation. Species traits tended to show stronger relationships with estimated optimum annual rainfall for each species than to the extreme rainfall conditions where they occur. The significant role of rainfall in shaping timber species distribution and the strong relationships between species traits and rainfall indicate that changes in climate, especially declining rainfall, could have strong effects on species composition and abundance in these tropical forests.     

Antioxidants prevent oxidative DNA damage and cellular transformation elicited by the over-expression of c-MYC

Reactive oxygen species (ROS)-induced genomic damage may have important consequences in the initiation and progression of cancer. Deregulated expression of the proto-oncogene c-MYC is associated with intracellular oxidative stress and increased DNA damage. However, the protective role of antioxidants such as Vitamin C against MYC-induced genomic damage has not been fully investigated. In a variety of cell lines, we show that ectopic MYC over-expression results in the elevation of intracellular ROS levels and a concomitant increase in oxidative DNA damage, as assessed by levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in the genomic DNA. Loading cells with ascorbic acid (AA) relieved MYC-elicited intracellular oxidative stress and conferred genomic protection. A mitochondrially targeted Vitamin E analog, TPPB, also protected cells from MYC-elicited oxidative DNA damage, suggesting the involvement of mitochondria in increased ROS production. We found that deregulated MYC expression resulted in the attenuation of intracellular glutathione levels, which was reversed by loading cells with Vitamin C. Additionally, cells over-expressing MYC had elevated levels of intracellular superoxide, which was significantly quenched by Vitamin C or the selective superoxide quencher, Tiron. Consequently, Vitamin C and other antioxidants protected cells from MYC-induced cellular transformation. Our studies implicate a role for ROS, and superoxide in particular, in MYC-elicited oxidative DNA damage and cellular transformation, and point to a pharmacological role of antioxidants in cancer chemoprevention.     

Effect of different biosynthetic precursors on the production of nargenicin A1 from metabolically engineered Nocardia sp. CS682

Nargenicin A1 is a 28-membered polyketide macrolide, with antibacterial activity against methicillin-resistant Staphylococcus aureus, produced by Nocardia sp. CS682. In this study, the production of nargenicin A1 was improved by enhancing the supply of different biosynthetic precursors. In Nocardia sp. CS682 (KCTC11297BP), this improvement was ~4.62-fold with the supplementation of 30 mM methyl oleate, 4.25-fold with supplementation of 15 mM sodium propionate, and 2.81-fold with supplementation of 15 mM sodium acetate. In Nocardia sp. metK18 and Nocardia sp. CS682 expressing S-adenosylmethionine synthetase (MetK), the production of nargenicin A1 was improved by ~5.57-fold by supplementation with 30 mM methyl oleate, 5.01-fold by supplementation with 15 mM sodium propionate, and 3.64-fold by supplementation with 15 mM sodium acetate. Furthermore, supplementing the culture broth of Nocardia sp. ACC18 and Nocardia sp. CS682 expressing the acetyl-CoA carboxylase complex (AccA2 and AccBE) with 30 mM methyl oleate, 15 mM sodium propionate, or 15 mM sodium acetate resulted in ~6.99-, 6.46-, and 5.58-fold increases, respectively, in nargenicin A1 production. Our overall results showed that among the supplements, methyl oleate was the most effective precursor supporting the highest titers of nargenicin A1 in Nocardia sp. CS682, Nocardia sp. metK18, and Nocardia sp. ACC18.     

Biosynthesis of rubradirin as an ansamycin antibiotic from Streptomyces achromogenes var. rubradiris NRRL3061

The four overlapping cosmids from the rubradirin producer, Streptomyces achromogenes var rubradiris NRRL 3061, have 58 ORFs within a 105.6 kb fragment. These ORFs harbored essential genes responsible for the formation and attachment of four distinct moieties, along with the genes associated with regulatory, resistance, and transport functions. The PKS (rubA) and glycosyltransferase (rubG2) genes were disrupted in order to demonstrate a complete elimination of rubradirin production. The rubradirin biosynthetic pathway was proposed based on the putative functions of the gene products, the functional identification of sugar genes, and the mutant strains. The GeneBank accession number for the sequence reported in this paper is AJ871581.     

Effect of <Emphasis Type="Italic">Liriomyza huidobrensis</Emphasis> (Diptera: Agromyzidae) density on foliar leaf damage and yield loss in potato

Liriomyza huidobrensis (Blanchard) is a key pest of potatoes worldwide and an emerging pest of potatoes in Korea. To understand the nature of the damage caused by this pest and the potential for yield loss, potato (Solanum tuberosum), cv. Superior was exposed to different pest infestation levels (0, 2, 5, 10, 20, and 50 individuals/cage) for 27 days and the relationship between pest density and crop damage assessment was determined using both laboratory cage studies and field cages. Leaf stippling, number of mines per leaf, % leaf area mined, and yield loss varied significantly with infestation density level in both the laboratory and field. The greatest leaf area mined and yield loss were associated with the 20- and 50-individual infestation rates in both the laboratory and field. There was a significant relationship between female choice (as shown by stippling, caused by adult feeding and oviposition) and larval performance (as reflected by the number of mines, damaged leaf area, and yield loss). It is important to understand the nature of the damage caused by L. huidobrensis and to determine its economic threshold for potato in order to optimize the management of this leafminer and minimize management costs.     

MUC1-C influences cell survival in lung adenocarcinoma Calu-3 cells after SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces coronavirus disease 2019 (COVID-19) and may increase the risk of adverse outcomes in lung cancer patients. In this study, we investigated the expression and function of mucin 1 (MUC1) after SARS-CoV-2 infection in the lung epithelial cancer cell line Calu-3. MUC1 is a major constituent of the mucus layer in the respiratory tract and contributes to pathogen defense. SARS-CoV-2 infection induced MUC1 C-terminal subunit (MUC1-C) expression in a STAT3 activation-dependent manner. Inhibition of MUC1-C signaling increased apoptosis-related protein levels and reduced proliferation-related protein levels; however, SARS-CoV-2 replication was not affected. Together, these results suggest that increased MUC1-C expression in response to SARS-CoV-2 infection may trigger the growth of lung cancer cells, and COVID-19 may be a risk factor for lung cancer patients.     

Evaluating the influence of quality control decisions and software algorithms on SNP calling for the affymetrix 6.0 SNP array platform

Objective: Our goal was to evaluate the influence of quality control (QC) decisions using two genotype calling algorithms, CRLMM and Birdseed, designed for the Affymetrix SNP Array 6.0. Methods: Various QC options were tried using the two algorithms and comparisons were made on subject and call rate and on association results using two data sets. Results: For Birdseed, we recommend using the contrast QC instead of QC call rate for sample QC. For CRLMM, we recommend using the signal-to-noise rate ≥4 for sample QC and a posterior probability of 90% for genotype accuracy. For both algorithms, we recommend calling the genotype separately for each plate, and dropping SNPs with a lower call rate (<95%) before evaluating samples with lower call rates. To investigate whether the genotype calls from the two algorithms impacted the genome-wide association results, we performed association analysis using data from the GENOA cohort; we observed that the number of significant SNPs were similar using either CRLMM or Birdseed. Conclusions: Using our suggested workflow both algorithms performed similarly; however, fewer samples were removed and CRLMM took half the time to run our 854 study samples (4.2 h) compared to Birdseed (8.4 h).