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The role of food constituents as pharmacological agents is an important consideration in health and obesity. Vitamin C acts as a small molecule antioxidant but is also a co-factor for numerous transition metal-dependent enzymes involved in healthy weight and energy metabolism. Vitamin C cannot be manufactured by humans and is mainly obtained from the dietary intake of fresh fruit and vegetables. There is great variability between different nutritional guidelines in the recommended daily allowance of vitamin C. Vitamin C deficiency results from an inadequate intake of vitamin C-containing foods and also increased utilization by oxidative and carbonyl stress. Risk factors for vitamin C deficiency include cigarette smoking, malnutrition, obesity, type 2 diabetes mellitus, age, race, sex, social isolation, major surgery, and Western-type diets. Despite the common belief that vitamin C deficiency is rare in affluent countries, surveys of large populations and specific patient groups suggest otherwise. Patients with obesity typically consume highly processed, energy-dense foods which contain inadequate micronutrients. As obesity increases, larger amounts of oral vitamin C are required to achieve adequate plasma and tissue concentrations, as compared to persons with a healthy weight. This is important in the control of oxidative stress and the maintenance of homeostasis and organ function. In this narrative review, the dosage, absorption, distribution, excretion, and catabolism of vitamin C are reviewed, together with the latest findings on vitamin C pharmacology in patients with obesity.

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Ammonium (NH4+) is essential to generate the nitrogenous building blocks of life. It gets assimilated via the canonical biosynthetic routes to glutamate and is further distributed throughout metabolism via a network of transaminases. To study the flexibility of this network, we constructed an Escherichia coli glutamate auxotrophic strain. This strain allowed us to systematically study which amino acids serve as amine sources. We found that several amino acids complemented the auxotrophy either by producing glutamate via transamination reactions or by their conversion to glutamate. In this network, we identified aspartate transaminase AspC as a major connector between many amino acids and glutamate. Additionally, we extended the transaminase network by the amino acids ß-alanine, alanine, glycine, and serine as new amine sources and identified d-amino acid dehydrogenase (DadA) as an intracellular amino acid sink removing substrates from transaminase reactions. Finally, ammonium assimilation routes producing aspartate or leucine were introduced. Our study reveals the high flexibility of the cellular amination network, both in terms of transaminase promiscuity and adaptability to new connections and ammonium entry points.

Nitrogen is an essential part of many of the cell's building blocks, including amino acids and nucleotides, which form proteins and DNA respectively. Therefore, nitrogen has to be available to cells so that they can survive and grow. In nature, some microorganisms convert the gaseous form of nitrogen into ammonium, which then acts as the nitrogen source of most organisms, including bacteria, plants and animals. Once cells take up ammonium, it is 'fixed' by becoming part of an amino acid called glutamate, which has a so-called 'amine group' that contains a nitrogen. Glutamate then becomes the central source for passing these amines on to other molecules, distributing nitrogen throughout the cell. This coupling between ammonium fixation and glutamate production evolved over millions of years and occurs in all organisms. However, the complete metabolic network that underlies the distribution of amines remains poorly understood despite decades of research. Furthermore, it is not clear whether ammonium can be fixed in a way that is independent of glutamate. To answer these questions, Schulz-Mirbach et al. used genetic engineering to create a strain of the bacterium E. coli that was unable to make glutamate. These mutant cells could only grow in the presence of certain amino acids, which acted as alternative amine sources. Schulz-Mirbach et al. found that enzymes called transaminases, and one called AspC in particular, were required for the cells to be able to produce glutamate using the amine groups from other amino acids. Notably, Schulz-Mirbach et al. showed that AspC, which had previously been shown to use an amino acid called aspartate as a source of amine groups, is indispensable if the cell is to use the amine groups from other amino acids ­ including histidine, tyrosine, phenylalanine, tryptophan, methionine, isoleucine and leucine. Schulz-Mirbach et al. also discovered that if they engineered the E. coli cells to produce transaminases from other species, the repertoire of molecules that the cells could use as the source of amines to generate glutamate increased. In a final set of experiments, Schulz-Mirbach et al. were able to engineer the cells to fix ammonium by producing aspartate and leucine, thus entirely bypassing the deleted routes of glutamate synthesis. These data suggest that fixing ammonium and distributing nitrogen in E. coli can be very flexible. The results from these experiments may shed light on how cells adapt when there is not a lot of ammonium available. Moreover, this study could advance efforts at metabolic engineering, for example, to create molecules through new pathways or to boost the production of amino acids needed for industrial purposes.

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With the emergence of high-throughput methods in plant biology, the importance of long-term projects characterized by incremental advances involving multiple laboratories can sometimes be overlooked. Here, I highlight my 40-year effort to isolate and characterize the most common class of mutants encountered in Arabidopsis (Arabidopsis thaliana): those defective in embryo development. I present an updated dataset of 510 EMBRYO-DEFECTIVE (EMB) genes identified throughout the Arabidopsis community; include important details on 2200 emb mutants and 241 pigment-defective embryo (pde) mutants analyzed in my laboratory; provide curated datasets with key features and publication links for each EMB gene identified; revisit past estimates of 500-1000 total EMB genes in Arabidopsis; document 83 double mutant combinations reported to disrupt embryo development; emphasize the importance of following established nomenclature guidelines and acknowledging allele history in research publications; and consider how best to extend community-based curation and screening efforts to approach saturation for this diverse class of mutants in the future. Continued advances in identifying EMB genes and characterizing their loss-of-function mutant alleles are needed to understand genotype-to-phenotype relationships in Arabidopsis on a broad scale, and to document the contributions of large numbers of essential genes to plant growth and development.

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The bacteriophage lambda replication initiation protein P exhibits a toxic effect on its Escherichia coli (E. coli) host, likely due to the formation of a dead-end P-DnaB complex, sequestering the replicative DnaB helicase from further activity. Intracellular expression of P triggers SOS-independent cellular filamentation and rapidly cures resident ColE1 plasmids. The toxicity of P is suppressed by alleles of P or dnaB. We asked whether P buildup within a cell can influence E. coli replication fidelity. The influence of P expression from a defective prophage, or when cloned and expressed from a plasmid was examined by screening for auxotrophic mutants, or by selection for rifampicin resistant (Rif(R)) cells acquiring mutations within the rpoB gene encoding the ß-subunit of RNA polymerase (RNAP), nine of which proved unique. Using fluctuation assays, we show that the intracellular expression of P evokes a mutator effect. Most of the Rif(R) mutants remained P(S) and localized to the Rif binding pocket in RNAP, but a subset acquired a P(R) phenotype, lost sensitivity to ColE1 plasmid curing, and localized outside of the pocket. One P(R) mutation was identical to rpo*Q148P, which alleviates the UV-sensitivity of ruv strains defective in the migration and resolution of Holliday junctions and destabilizes stalled RNAP elongation complexes. The results suggest that P-DnaB sequestration is mutagenic and supports an earlier observation that P can interact with RNAP.

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Enrichment of proteins with isotopes such as (2)H, (15)N, and (13)C is commonly carried out in magnetic resonance and vibrational spectroscopic characterization of protein structures, mechanisms, and dynamics. Although uniform isotopic labeling of proteins is straightforward, efficient labeling of proteins with only a selected set of amino acid types is often challenging. A number of approaches have been described in the literature for amino acid-selective isotope labeling of proteins, each with its own limitations. Since Escherichia coli represents the most cost-effective and widely used host for heterologous production of foreign proteins, an efficient method to express proteins selectively labeled with isotopes would be highly valuable for these studies. However, an obvious drawback is misincorporation and dilution of input isotope labels to unwanted amino acid types due to metabolic scrambling in vivo. To overcome this problem, we have generated E. coli auxotroph strains that are compatible with the widely used T7 RNA polymerase overexpression systems and that minimize metabolic scrambling. We present several examples of selective amino acid isotope labeling of simple and complex proteins with bound cofactors, as an initial guide for practical applications of these E. coli strains.

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INTRODUCTION: For at least two centuries, there have been reports that cancer patients infected with various bacteria had what appeared to be spontaneous remission. In the late nineteenth and early twentieth centuries, W.B. Coley, of what is now the Memorial Sloan-Kettering Cancer Center, pioneered bacterial therapy of cancer in the clinic with considerable success. After Coley died in 1936, bacterial therapy of cancer started to go out of favor. In the current twenty-first century, there is great resurgent interest in developing bacterial therapy for treating cancer using either obligate or facultative anaerobic bacteria. There is also controversy about which bacteria are optimum for cancer treatment and whether bacteria should be used as tumor-targeting vectors, immune stimulators, or for direct tumor killing. AREAS COVERED: This review covers various types of bacteria currently used for tumor targeting. It also covers methods used to develop maximal tumor targeting and minimally attenuated bacteria, which grow in viable as well as necrotic areas of tumors and directly kill cancer cells. EXPERT OPINION: The current paradigm of cancer chemotherapy lacks sufficient efficacy, and a new paradigm is needed. Bacterial therapy is a candidate for a 'new' approach to cancer treatment. In the authors' opinion, the current revival of bacterial therapy of cancer is one of the most promising approaches to treatment.

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The concept of auxotrophic complementation has been proposed as an approach to identify genes in essential metabolic pathways in Drosophila melanogaster. However, it has achieved limited success to date, possibly due to the low probability of finding mutations fit with the chemically defined profile. Instead of using the chemically defined culture media lacking specific nutrients, we used bare minimum culture medium, i.e., 4% sucrose, for adult Drosophila. We identified a nutritional conditional lethal mutant and localized a c.95C > A mutation in the Drosophila pyridoxine 5'-phosphate oxidase gene [dPNPO or sugarlethal (sgll)] using meiotic recombination mapping, deficiency mapping, and whole genome sequencing. PNPO converts dietary vitamin B6 such as pyridoxine to its active form pyridoxal 5'-phosphate (PLP). The missense mutation (sgll(95)) results in the substitution of alanine to aspartate (p.Ala32Asp). The sgll(95) flies survive well on complete medium but all die within 6 d on 4% sucrose only diet, which can be rescued by pyridoxine or PLP supplement, suggesting that the mutation does not cause the complete loss of PNPO activity. The sgll knockdown further confirms its function as the Drosophila PNPO. Because better tools for positional cloning and cheaper whole genome sequencing have made the identification of point mutations much easier than before, alleviating the necessity to pinpoint specific metabolic pathways before gene identification, we propose that nutritional conditional screens based on bare minimum growth media like ours represent promising approaches for discovering important genes and mutations in metabolic pathways, thereby accelerating the establishment of in vivo models that recapitulate human metabolic diseases.

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pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae.

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Toxoplasma gondii is an intracellular parasite that has evolved to actively control its invaded host cells. Toxoplasma triggers then actively regulates host innate interleukin-12 (IL-12) and interferon-γ (IFN-γ) responses that elicit T cell control of infection. A live, nonreplicating avirulent uracil auxotroph vaccine strain (cps) of Toxoplasma triggers novel innate immune responses that stimulate amplified CD8(+) T cell responses and life-long immunity in vaccinated mice. Here, we review recent reports showing that intratumoral treatment with cps activated immune-mediated regression of established solid tumors in mice. We speculate that a better understanding of host-parasite interaction at the molecular level and applying improved genetic models based on Δku80 Toxoplasma strains will stimulate development of highly effective immunotherapeutic cancer vaccine strategies using engineered uracil auxotrophs.

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Among the various types of mutations studied in rhizobia, the auxotrophic mutations (which confer on the mutants the inability to synthesize certain essential substances such as amino acids, vitamins and nucleic acids), are the most favoured ones as these can be used as suitable markers for genetic analysis. An important property of rhizobia is their effectiveness i.e. their ability to fix atmospheric nitrogen into ammonia within the nodule. Special interest in this category of mutations by rhizobial geneticists is due to the fact that there is a strong correlation between the metabolic defects and the ineffectiveness (Nod(-) and/or Fix(-)) of the rhizobial strains. Auxotrophic mutants of various species of rhizobia with defects in the synthesis of nucleic bases, vitamins and amino acids have been obtained by mutagenising with physical, chemical and Tn5 mutagens. These mutants have been used in mapping studies as well as in establishing a correlation between its metabolic requirement and symbiotic relationship with the host plant. The present review deals with the isolation of auxotrophs, and their genetic, biochemical and symbiotic characterization. The review also encompasses the studies on the elucidation of biosynthetic pathways of nutritional substances in rhizobia.

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Protoplasts of the wild type strain of Pleurotus osteatus were mutagenized with UV light, and 3,000 colonies were examined for abnormal mycelial and fruiting phenotypes. Forty one strains displayed variant phenotypes in mycelia and fruiting processes. The variant phenotypes were classified into 6 groups: (1) auxotrophic strains, which are incapable of growing on minimal media and can only grow when provided with their specific requirements; (2) abnormal vegetative strains, which grow very slowly on minimal and complete media; (3) primordiumless strains, which fail to develop to the formation of primordia; (4) maturationless strains, which form primordia, but do not form mature fruiting bodies; (5) specifically colored strains, which have Specific bluish grey or bluish white pileus; (6) poorly spored strains, which fail to produce basidiospore or which produce few spores. These variant strains may be useful in genetic breeding programs and for the studies of fungal development and genetics.

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