Putrescine Metabolism Research Articles

Transcriptomic analysis reveals the mechanism underlying salinity-induced morphological changes in Skeletonema subsalsum.

Diatom cell walls are diverse and unique, providing the basis for species identification and supporting the ecological and economic value of diatoms. However, these important structures sometimes change in response to environmental fluctuations, especially under salt adaptation. Although studies have shown that salinity induces morphological plasticity changes in diatom cell walls, most research has focused on physiological responses rather than molecular mechanisms. In this study, Skeletonema subsalsum was cultured under four salinity conditions (0, 3, 6, 12). Through morphological and physiological methods, we found that salinity increased the cell diameter, protrusion lengths, distance between adjacent cells (DBCs), and nanopore size, while reducing cell height and silicification degree. To further investigate the mechanism underlying morphological changes in S. subsalsum, complementary transcriptome analysis was performed. In total, 20,138 differentially expressed genes (DEGs) were identified among the four treatments. Among them, 231 DEGs were screened and found to be closely associated with morphological changes, of which 107 were downregulated and 124 were upregulated. The findings demonstrated that elevated salinity inhibited silicon transport and deposition via downregulating the expression of DEGs involved in functions including chitin metabolism, putrescine metabolism, and vesicle transport, resulting in reduced silicon content and cell height. Increased salinity promoted the expression of DEGs related to microtubules (MTs), actin, and ubiquitin, which synchronously induced morphological changes. These findings provide a more comprehensive understanding of the salt tolerance of algae and a foundation for future studies on cell wall morphogenesis.

Shotgun Proteomics Analysis, Functional Networks, and Peptide Biomarkers for Seafood-Originating Biogenic-Amine-Producing Bacteria.

Biogenic amine-producing bacteria are responsible for the production of basic nitrogenous compounds (histamine, cadaverine, tyramine, and putrescine) following the spoilage of food due to microorganisms. In this study, we adopted a shotgun proteomics strategy to characterize 15 foodborne strains of biogenic-amine-producing bacteria. A total of 10,673 peptide spectrum matches belonging to 4081 peptides and corresponding to 1811 proteins were identified. Relevant functional pathways were determined, and strains were differentiated into hierarchical clusters. An expected protein-protein interaction network was created (260 nodes/1973 interactions). Most of the determined proteins were associated with networks/pathways of energy, putrescine metabolism, and host-virus interaction. Additionally, 556 peptides were identified as virulence factors. Moreover, 77 species-specific peptide biomarkers corresponding to 64 different proteins were proposed to identify 10 bacterial species. This represents a major proteomic dataset of biogenic-amine-producing strains. These results may also be suitable for new treatments for food intoxication and for tracking microbial sources in foodstuffs.

Comparative genomics and transcriptomic response to root exudates of six rice root-associated Burkholderia sensu lato species

Beyond being a reliable nutrient provider, some bacteria will perceive the plant as a potential host and undertake root colonization leading to mutualistic or parasitic interactions. Bacteria of the Burkholderia and Paraburkholderia genera are frequently found in the rhizosphere of rice. While the latter are often described as plant growth promoting species, Burkholderia are often studied for their human opportunistic traits. Here, we used root exudate stimulation on three Burkholderia and three Paraburkholderia strains isolated from rice roots to characterize their preliminary adaptation to the rice host at the transcriptomic level. Instead of the awaited genus-dependent adaptation, we observed a strongly species-specific response for all tested strains. While all bacteria originate from the rice environment, there are great disparities in their levels of adaptation following the sensing of root exudates. We further report the shared major functions that were differentially regulated in this early step of bacterial adaptation to plant colonization, including amino acids and putrescine metabolism, the Entner-Doudoroff (ED) pathway as well as cyclic diguanylate monophosphate (c-di-GMP) cycling.

Genome collection of Shewanella spp. isolated from spoiled lamb.

The diversity of the genus Shewanella and their roles across a variety of ecological niches is largely unknown highlighting the phylogenetic diversity of these bacteria. From a food safety perspective, Shewanella species have been recognized as causative spoilage agents of vacuum-packed meat products. However, the genetic basis and metabolic pathways for the spoilage mechanism are yet to be explored due to the unavailability of relevant Shewanella strains and genomic resources. In this study, whole-genome sequencing of 32 Shewanella strains isolated from vacuum-packaged refrigerated spoiled lamb was performed to examine their roles in meat spoilage. Phylogenomic reconstruction revealed their genomic diversity with 28 Shewanella spp. strains belonging to the same putative novel species, two Shewanella glacialipiscicola strains (SM77 and SM91), Shewanella xiamenensis NZRM825, and Shewanella putrefaciens DSM 50426 (ATCC 8072) isolated from butter. Genome-wide clustering of orthologous gene families revealed functional groupings within the major Shewanella cluster but also considerable plasticity across the different species. Pan-genome analysis revealed conserved occurrence of spoilage genes associated with sulfur and putrescine metabolism, while the complete set of trimethylamine metabolism genes was observed in only Shewanella sp. SM74, S. glacialipiscicola SM77 and SM91 strains. Through comparative genomics, some variations were also identified pertaining to genes associated with adaptation to environmental cues such as temperature, osmotic, salt, oxidative, antimicrobial peptide, and drug resistance stresses. Here we provide a reference collection of draft Shewanella genomes for subsequent species descriptions and future investigations into the molecular spoilage mechanisms for further applications in the meat industry.

Analysis of Growth Phases of Enterotoxigenic Escherichia coli Reveals a Distinct Transition Phase before Entry into Early Stationary Phase with Shifts in Tryptophan, Fucose, and Putrescine Metabolism and Degradation of Neurotransmitter Precursors.

ABSTRACTEnterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and adults in endemic areas. Gene regulation of ETEC during growth in vitro and in vivo needs to be further evaluated, and here we describe the full transcriptome and metabolome of ETEC during growth from mid-logarithmic growth to early stationary phase in rich medium (LB medium). We identified specific genes and pathways subjected to rapid transient alterations in gene expression and metabolite production during the transition from logarithmic to stationary growth. The transient phase was found to be different from the subsequent induction of early stationary phase-induced genes. The transient phase was characterized by the repression of genes and metabolites involved in organic substance transport. Genes involved in fucose and putrescine metabolism were upregulated, and genes involved in iron transport were repressed. Expression of toxins and colonization factors were not changed, suggesting retained virulence from mid-logarithmic to the start of the stationary phase. Metabolomic analyses showed that the transient phase was characterized by a drop of intracellular amino acids, e.g., l-tyrosine, l-tryptophan, l-phenylalanine, l-leucine, and l-glutamic acid, followed by increased levels at induction of stationary phase. A pathway enrichment analysis of the entire combined transcriptome and metabolome revealed that significant pathways during progression from logarithmic to early stationary phase are involved in the degradation of neurotransmitters aminobutyrate (GABA) and precursors of 5-hydroxytryptamine (serotonin). This work provides a comprehensive framework for further studies on transcriptional and metabolic regulation in pathogenic E. coli.IMPORTANCE We show that E. coli, exemplified by the pathogenic subspecies enterotoxigenic E. coli (ETEC), undergoes a stepwise transcriptional and metabolic transition into the stationary phase. At a specific entry point, E. coli induces activation and repression of specific pathways. This leads to a rapid decrease of intracellular levels of certain amino acids. The resulting metabolic activity leads to an intense but short peak of indole production, suggesting that this is the previously described “indole peak,” rapid decrease of intermediate molecules of bacterial neurotransmitters, increased putrescine and fucose uptake, increased glutathione levels, and decreased iron uptake. This specific transient shift in gene expression and metabolome is short-lived and disappears when bacteria enter the early stationary phase. We suggest that these changes mainly prepare bacteria for ceased growth, but based on the pathways involved, we could suggest that this transient phase substantially influences survival and virulence.

Advanced pathway engineering for phototrophic putrescine production

SummaryThe polyamine putrescine (1,4‐diaminobutane) contributes to cellular fitness in most organisms, where it is derived from the amino acids ornithine or arginine. In the chemical industry, putrescine serves as a versatile building block for polyamide synthesis. The green microalga Chlamydomonas reinhardtii accumulates relatively high putrescine amounts, which, together with recent advances in genetic engineering, enables the generation of a powerful green cell factory to promote sustainable biotechnology for base chemical production. Here, we report a systematic investigation of the native putrescine metabolism in C. reinhardtii, leading to the first CO2‐based bio‐production of putrescine, by employing modern synthetic biology and metabolic engineering strategies. A CRISPR/Cas9‐based knockout of key enzymes of the polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a gatekeeper for putrescine accumulation and demonstrated that the arginine decarboxylase (ADC) route is likely inactive and that amine oxidase 2 (AMX2) is mainly responsible for putrescine degradation in C. reinhardtii. A 4.5‐fold increase in cellular putrescine levels was achieved by engineered overexpression of potent candidate ornithine decarboxylases (ODCs). We identified unexpected substrate promiscuity in two bacterial ODCs, which exhibited co‐production of cadaverine and 4‐aminobutanol. Final pathway engineering included overexpression of recombinant arginases for improved substrate availability as well as functional knockout of putrescine degradation, which resulted in a 10‐fold increase in cellular putrescine titres and yielded 200 mg/L in phototrophic high cell density cultivations after 10 days.

Transcriptomic response of Anopheles gambiae sensu stricto mosquito larvae to Curry tree (Murraya koenigii) phytochemicals

BackgroundInsect growth regulators (IGRs) can control insect vector populations by disrupting growth and development in juvenile stages of the vectors. We previously identified and described the curry tree (Murraya koenigii (L.) Spreng) phytochemical leaf extract composition (neplanocin A, 3-(1-naphthyl)-l-alanine, lumiflavine, terezine C, agelaspongin and murrayazolinol), which disrupted growth and development in Anopheles gambiae sensu stricto mosquito larvae by inducing morphogenetic abnormalities, reducing locomotion and delaying pupation in the mosquito. Here, we attempted to establish the transcriptional process in the larvae that underpins these phenotypes in the mosquito.MethodsWe first exposed third-fourth instar larvae of the mosquito to the leaf extract and consequently the inherent phytochemicals (and corresponding non-exposed controls) in two independent biological replicates. We collected the larvae for our experiments sampled 24 h before peak pupation, which was 7 and 18 days post-exposure for controls and exposed larvae, respectively. The differences in duration to peak pupation were due to extract-induced growth delay in the larvae. The two study groups (exposed vs control) were consequently not age-matched. We then sequentially (i) isolated RNA (whole larvae) from each replicate treatment, (ii) sequenced the RNA on Illumina HiSeq platform, (iii) performed differential bioinformatics analyses between libraries (exposed vs control) and (iv) independently validated the transcriptome expression profiles through RT-qPCR.ResultsOur analyses revealed significant induction of transcripts predominantly associated with hard cuticular proteins, juvenile hormone esterases, immunity and detoxification in the larvae samples exposed to the extract relative to the non-exposed control samples. Our analysis also revealed alteration of pathways functionally associated with putrescine metabolism and structural constituents of the cuticle in the extract-exposed larvae relative to the non-exposed control, putatively linked to the exoskeleton and immune response in the larvae. The extract-exposed larvae also appeared to have suppressed pathways functionally associated with molting, cell division and growth in the larvae. However, given the age mismatch between the extract-exposed and non-exposed larvae, we can attribute the modulation of innate immune, detoxification, cuticular and associated transcripts and pathways we observed to effects of age differences among the larvae samples (exposed vs control) and to exposures of the larvae to the extract.ConclusionsThe exposure treatment appears to disrupt cuticular development, immune response and oxidative stress pathways in Anopheles gambiae s.s larvae. These pathways can potentially be targeted in development of more efficacious curry tree phytochemical-based IGRs against An. gambiae s.s mosquito larvae.Graphical

Drought tolerant maize cultivar accumulates putrescine in roots

We compared putrescine metabolism in roots of tolerant maize cultivar (Karoon) to a susceptible cultivar (260) under drought stress. The experiment was conducted in growth chamber under controlled condition in 80 pots. Drought stress led to significant increase in root growth indices, H2O2 content and activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase). The qPCR analysis revealed that expression of maize polyamine oxidase (ZmPAO) genes including ZmPAO1, ZmPAO2, ZmPAO4 and ZmPAO6 as well as ornithine decarboxylase (ZmODC) significantly increased in root of both cultivars in response to drought. However, up-regulation of ZmPAO3 and ZmPAO5 was only observed in root of tolerant cultivar. Consistently, drought caused increased polyamine oxidase activity in the tolerant cultivar. Transcription of spermidine synthase (ZmSPDS) was down-regulated by drought stress. Transcript levels of ZmPOA, ZmODC and ZmSPDS were more affected by drought in tolerant cultivar compared to susceptible cultivar. Increase in putrescine content under drought stress was more prominent in roots of tolerant cultivar, compared to susceptible cultivar. We attribute the accumulation of putrescine to increased rate of putrescine synthesis, due to higher expression of ZmPAOs and ZmODC and decreased rate of its depletion for biosynthesis of higher polyamines due to down-regulation of ZmSPDS.

The effect of Poria cocos ethanol extract on the intestinal barrier function and intestinal microbiota in mice with breast cancer

Ethnopharmacological relevancePoria cocos Wolf has been used in traditional East-Asian medicine for centuries to effectively treat various gastrointestinal disorders such as diarrhea for its tonic, anti-fungal and anti-bacterial activities. Previous studies have revealed that the tumor development would induce intestinal microbiota dysbiosis and intestinal barrier dysfunction to the patients with breast cancer. Aim of studyTo investigate the effect and the mechanism of ethanol extract of Poria cocos (PC) on intestinal barrier function and intestinal microbiota in the mice with breast cancer. Materials and methodsThirty-six female BALB/c mice were randomly divided into four groups (the normal control, model, PC and positive control group). Intestinal histopathological was evaluated by H&E staining. The difference of the intestinal microbiota in each group was studied by 16S rDNA high-throughput sequencing. The level of plasma endotoxin, D -lactic acid (D-LA) and diamine oxidase (DAO) were measured by ELISA. The putrescine content in serum and urine were detected by HPLC. Expression of the tight junction (TJ) proteins, phosphorylated p38 MAPK and ERK1/2 were determined by western blotting. ResultsOur results showed that tumor development prominently induced the intestinal damage and microbiome dysbiosis in mice. PC prominently remit such histologic damage through enhancing the expression of TJ proteins and decreasing the levels of DAO, D-LA and endotoxin via upregulating the expression of phosphorylated ERK1/2 and p38 MAPK. Furthermore, PC increased the diversity of the intestinal microbiota and strikingly changed the structure and composition of the gut microbiota in the mice by increasing the beneficial bacteria Lactobacillus, Bifidobacterium, and decreasing the sulfate-reducing bacteria Desulfovibrio and inflammatory associated bacteria Mucispirillum, S24-7 and Staphylococcus. Moreover, PICRUSt analysis and the putrescine detection might indicate that PC might be involved in the putrescine metabolism in the mice. Correlation analysis indicated that Prevotella, Rikenellaceae and Bacteroidetes were significantly correlated with Claudin-8 and p38-MAPK expression (p < 0.05). ConclusionPC could improve the dysbacteriosis and repair the intestinal barrier function in the mice with breast cancer. This study provide more data to support the application of PC in breast cancer treatment.

Alterations of stool metabolome, phenome, and microbiome of the marine fish, red sea bream, Pagrus major, following exposure to phenanthrene: A non-invasive approach for exposure assessment

The present study aimed to assess the impact of phenanthrene (Phe) on fish health by addressing the alteration of fecal characteristics, in lieu of collecting biomarkers that often involves injurious or even fatal sampling of organisms. The marine fish red sea bream, Pagrus major, was exposed to Phe at a concentration of 18 μg/L for 16 days followed by depuration for 13 days. We collected feces from Phe-exposed or control (Phe-free) fish and then analyzed the fecal metabolite profile (metabolome), carbon utilization of microbiota (phenome), and bacterial 16s rRNA gene sequence (microbiome). Along with the increase in physiological stress markers (SOD and EROD) in serum and liver, we noted the possible role of intestine as a Phe reservoir. Furthermore, abnormal fecal appearance (green coloration) and remarkable changes in fecal characteristics were observed. These changes include alterations of cholesterol and putrescine metabolism and the enhanced utilization of putrescine as a carbon source. Phe also altered the microbial community, with an increase in Phe-degrading bacteria such as Pseudomonas. Interestingly, these enteric impairments were ameliorated by depuration. Taken together, our findings suggest that these alterations in feces were associated with adaptive responses to environmentally relevant Phe exposure scenarios, and that stool samples are potential candidates for exposure assessment in fish.

Exogenous allantoin improves the salt tolerance of sugar beet by increasing putrescine metabolism and antioxidant activities

Allantoin as a nitrogen metabolite can improve the salt tolerance in plants, but its mechanism of action remain elusive. Herein, the effects of pretreatment with exogenous allantoin in salt tolerance were investigated in sugar beet. The seedlings were subjected to salt stress (300 mM Na+) without or with different allantoin concentrations (0.01, 0.1, and 1 mM). The effects of allantoin on plant growth, homeostasis, oxidative damage, osmoregulation, and polyamine metabolism were studied. The results showed that salt stress inhibited the net photosynthetic rate and plant growth, and caused oxidative damage. However, these adverse effects were mitigated by exogenous allantoin in a dose-dependent manner, especially at 0.1 mM. Allantoin reduced the accumulation of ROS by increasing the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and AsA content. Under salt stress, allantoin reduced the root concentrations of free putrescine (Put) but increased the free spermine (Spm) in leaves and roots. Furthermore, allantoin decreased the Na+/K+ ratio and promoted the accumulation of betaine and soluble sugars in leaves and roots. Under salinity conditions, allantoin may enhance the antioxidant system and improve ion homeostasis by enhancing putrescine and/or spermine accumulation. In addition, Pearson's correlation and principal component analysis (PCA) established correlations between physiological parameters, and significant differences between different concentrations of allantoin were observed. In total, exogenous allantoin effectively reduced the oxidative damage and ion toxicity in sugar beet, caused by salinity, this finding would be helpful in improving salt tolerance in plant.

Inhibitors of tri- and tetra- polyamine oxidation, but not diamine oxidation, impair the initial stages of wound-induced suberization

The mechanisms regulating, and modulating potato wound-healing processes are of great importance in reducing tuber infections, reducing shrinkage and maintaining quality and nutritional value for growers and consumers. Wound-induced changes in tuber polyamine metabolism have been linked to the modulation of wound healing (WH) and in possibly providing the crucial amount of H2O2 required for suberization processes. In this investigation we determined the effect of inhibition of specific steps within the pathway of polyamine metabolism on polyamine content and the initial accumulation of suberin polyphenolics (SPP) during WH. The accumulation of SPP represents a critical part of the beginning or inchoate phase of tuber WH during closing-layer formation because it serves as a barrier to bacterial infection and is a requisite for the accumulation of suberin polyaliphatics which provide the barrier to fungal infection. Results showed that the inhibitor treatments that caused changes in polyamine content generally did not influence wound-induced accumulation of SPP. Such lack of correlation was found for inhibitors involved in metabolism and oxidation of putrescine (arginine decarboxylase, ornithine decarboxylase, and diamine oxidase). However, accumulation of SPP was dramatically reduced by treatment with guazatine, a potent inhibitor of polyamine oxidase (PAO), and methylglyoxal-bis(guanylhydrazone), a putative inhibitor of S-adenosylmethione decarboxylase which may also cross-react to inhibit PAO. The mode of action of these inhibitors is presumed to be blockage of essential H2O2 production within the WH cell wall. These results are of great importance in understanding the mechanisms modulating WH and ultimately controlling related infections and associated postharvest losses.

Complete genome sequence provides insights into the quorum sensing-related spoilage potential of Shewanella baltica 128 isolated from spoiled shrimp

Shewanella baltica 128 is a specific spoilage organism (SSO) isolated from the refrigerated shrimp that results in shrimp spoilage. This study reported the complete genome sequencing of this strain, with the primary annotations associated with amino acid transport and metabolism (8.66%), indicating that S. baltica 128 has good potential for degrading proteins. In vitro experiments revealed Shewanella baltica 128 could adapt to the stress conditions by regulating its growth and biofilm formation. Genes that related to the spoilage-related metabolic pathways, including trimethylamine metabolism (torT), sulfur metabolism (cysM), putrescine metabolism (speC), biofilm formation (rpoS) and serine protease production (degS), were identified. Genes (LuxS, pfs, LuxR and qseC) that related to the specific QS system were also identified. Complete genome sequence of S. baltica 128 provide insights into the QS-related spoilage potential, which might provide novel information for the development of new approaches for spoilage detection and prevention based on QS target.

Nicotine Biosynthesis inNicotiana: A Metabolic Overview

Alkaloids are important compounds found in Nicotiana plants, essential in plant defense against herbivores. The main alkaloid of Nicotiana tabacum, nicotine, is produced in roots and translocated to the leaves. Nicotine is formed by a pyrrolidine and a pyridine ring in a process involving several enzymes. The pyridine ring of nicotine is derived from nicotinic acid, whereas the pyrrolidine ring originates from polyamine putrescine metabolism. After synthesis in root cortical cells, a set of transporters is known to transport nicotine upward to the aerial part and store it in leaf vacuoles. Moreover, nicotine can be metabolized in leaves, giving rise to nornicotine through the N-demethylation process. Some Nicotiana wild species produce acyltransferase enzymes, which allow the plant to make N-acyl-nornicotine, an alkaloid with more potent insecticidal properties than nicotine. However, although we can find a wealth of information about the alkaloid production in Nicotiana spp., our understanding about nicotine biosynthesis, transport, and metabolism is still incomplete. This review will summarize these pathways on the basis on recent literature, as well as highlighting questions that need further investigation.

The Genome of the Toluene-Degrading Pseudomonas veronii Strain 1YdBTEX2 and Its Differential Gene Expression in Contaminated Sand.

The natural restoration of soils polluted by aromatic hydrocarbons such as benzene, toluene, ethylbenzene and m- and p-xylene (BTEX) may be accelerated by inoculation of specific biodegraders (bioaugmentation). Bioaugmentation mainly involves introducing bacteria that deploy their metabolic properties and adaptation potential to survive and propagate in the contaminated environment by degrading the pollutant. In order to better understand the adaptive response of cells during a transition to contaminated material, we analyzed here the genome and short-term (1 h) changes in genome-wide gene expression of the BTEX-degrading bacterium Pseudomonas veronii 1YdBTEX2 in non-sterile soil and liquid medium, both in presence or absence of toluene. We obtained a gapless genome sequence of P. veronii 1YdBTEX2 covering three individual replicons with a total size of 8 Mb, two of which are largely unrelated to current known bacterial replicons. One-hour exposure to toluene, both in soil and liquid, triggered massive transcription (up to 208-fold induction) of multiple gene clusters, such as toluene degradation pathway(s), chemotaxis and toluene efflux pumps. This clearly underlines their key role in the adaptive response to toluene. In comparison to liquid medium, cells in soil drastically changed expression of genes involved in membrane functioning (e.g., lipid composition, lipid metabolism, cell fatty acid synthesis), osmotic stress response (e.g., polyamine or trehalose synthesis, uptake of potassium) and putrescine metabolism, highlighting the immediate response mechanisms of P. veronii 1YdBTEX2 for successful establishment in polluted soil.

Formation, Degradation, and Detoxification of Putrescine by Foodborne Bacteria: A Review

AbstractBiogenic amines (BAs) represent a considerable toxicological risk in some food products. Putrescine is one of the most common BAs in food. Its increased occurrence in food may lead to alimentary poisoning, due to enhancement of the toxic effects of other BAs, and also to lower quality of food, this amine is potentially carcinogenic. Increased occurrence of putrescine in food is mainly due to the bacterial metabolism of the Gram‐negative as well as Gram‐positive bacteria present. The bacterial metabolism of putrescine is very specific due to its complexity (in comparison with the metabolism of other BAs). There are 3 distinct known pathways leading toward the formation of putrescine, in some splices involving up to 6 different enzymes. The existence of more metabolic pathways and the possibility of their simultaneous use by different bacteria complicate the specification of the best conditions for food production and storage, which could lead to a lower content of putrescine. This review provides a summary of the existing knowledge about putrescine production and detection (mainly detection of specific genes for different enzymes using polymerase chain reaction) in both starter and contaminating microorganisms. Thus, this comprehensive review gives a useful overview for further research.

Establishment of quantitative severity evaluation model for spinal cord injury by metabolomic fingerprinting.

Spinal cord injury (SCI) is a devastating event with a limited hope for recovery and represents an enormous public health issue. It is crucial to understand the disturbances in the metabolic network after SCI to identify injury mechanisms and opportunities for treatment intervention. Through plasma 1H-nuclear magnetic resonance (NMR) screening, we identified 15 metabolites that made up an “Eigen-metabolome” capable of distinguishing rats with severe SCI from healthy control rats. Forty enzymes regulated these 15 metabolites in the metabolic network. We also found that 16 metabolites regulated by 130 enzymes in the metabolic network impacted neurobehavioral recovery. Using the Eigen-metabolome, we established a linear discrimination model to cluster rats with severe and mild SCI and control rats into separate groups and identify the interactive relationships between metabolic biomarkers in the global metabolic network. We identified 10 clusters in the global metabolic network and defined them as distinct metabolic disturbance domains of SCI. Metabolic paths such as retinal, glycerophospholipid, arachidonic acid metabolism; NAD–NADPH conversion process, tyrosine metabolism, and cadaverine and putrescine metabolism were included. In summary, we presented a novel interdisciplinary method that integrates metabolomics and global metabolic network analysis to visualize metabolic network disturbances after SCI. Our study demonstrated the systems biological study paradigm that integration of 1H-NMR, metabolomics, and global metabolic network analysis is useful to visualize complex metabolic disturbances after severe SCI. Furthermore, our findings may provide a new quantitative injury severity evaluation model for clinical use.

The importance of putrescine in the human body

Putrescine plays a very important role in the regulation of division, differentiation and maturation of cells as well as apoptosis. As the polycationic molecule it stabilizes the structure of DNA and participates in the functioning of cell membranes. It is able to interact with series of ion channels and has affinity for many receptors. The article presents the participation of putrescine in the metabolism of iron and mechanism of its transport across biological membranes. Especially important for the homeostasis of putrescine has ornithine decarboxylase and availability of its substrate--ornithine. Affecting to this enzyme is the simplest and widely used method of controlling the concentration of putrescine. For this purpose its inhibitor-eflornithine is applied. There was also a number of other enzymes involved in the metabolism of putrescine that was presented. Current information about the clinical relevance of putrescine in infertility, embryonic development, hirsutism, epilepsy, Alzheimer's disease, Parkinson's disease, prevention of metastases and hemostasis was also described. These processes were presented, in which putrescine plays a major role and focused on the latest reports. Attention was drawn to the situations where it has beneficial effects and those in which it is the cause of the pathology. Some of the cited reports are in phase of speculation on the possible use of it, but a significant part is already confirmed and used in clinical practice. The facts presented in this article show how great is the meaning of putrescine and how important role this simple specimen plays in the metabolic processes of living organisms.

Novel touchdown-PCR method for the detection of putrescine producing Gram-negative bacteria in food products

Formation of biogenic amines may occur in food due to metabolic activities of contaminating Gram-negative bacteria. Putrescine is assumed to be the major biogenic amine associated with microbial food spoilage. Gram-negative bacteria can form putrescine by three metabolic pathways that can include eight different enzymes. The objective of this study was to design new sets of primers able to detect all important enzymes involved in the production of putrescine by Gram-negative bacteria. Seven new sets of consensual primers based on gene sequences of different bacteria were designed and used for detection of the speA, adiA, adi, speB, aguA, speC, and speF genes. A newly developed touchdown polymerase chain reaction (PCR) method using these primers was successfully applied on several putrescine-producers. Selected PCR products were sequenced and high similarity of their sequences (99–91%) with known sequences of the corresponding genes confirmed high specificity of the developed sets of primers. Furthermore, all the investigated bacteria produced both putrescine and agmatine, an intermediate of putrescine production, which was confirmed by chemical analysis. The developed new touchdown PCR method could easily be used to detect potential foodborne Gram-negative producers of putrescine. The newly developed sets of primers could also be useful in further research on putrescine metabolism in contaminating microbiota.

Effects of glucose and insulin on HepG2‐C3A cell metabolism

HepG2, hepatocellular carcinoma cells, are used in drug toxicity studies and have also been explored for bioartificial livers. For these applications, the cells are under variable levels of nutrients and hormones, the effects of which on metabolism are poorly understood. In this study, HepG2-C3A cells were cultured under varying levels of glucose (high, low, and glucose-free) and insulin (without and with physiological levels of insulin) for 5 days. Cell growth was found to be comparable between high and low glucose media and lowest for glucose-free medium. Several features of central metabolism were affected profoundly by the medium glucose levels. Glucose consumption was greater for low glucose medium compared to high glucose medium, consistent with known glucose feedback regulation mechanisms. Urea productivity was highest in glucose-free medium. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high and low glucose media. Using a metabolic network flexibility analysis (MNFA) framework with stoichiometric and thermodynamic constraints, intracellular fluxes under varying levels of glucose and insulin were evaluated. The analysis indicates that urea production in HepG2-C3A cells arises via the arginase II pathway rather than from ammonia detoxification. Further, involvement of the putrescine metabolism with glutamine metabolism caused higher urea production in glucose-free medium consistent with higher glutamine uptake. MNFA indicated that in high and low glucose media, glycolysis, glutaminolysis, and oxidative phosphorylation were the main sources of energy (NADH, NADPH, and ATP). In the glucose-free medium, due to very low glycolytic flux, higher malate to pyruvate glutaminolytic flux and TCA cycle contributed more significantly to energy metabolism. The presence of insulin lowered glycerol uptake and corresponding fluxes involved in lipid metabolism for all glucose levels but otherwise exerted negligible effect on metabolism. HepG2-C3A cells thus show distinct differences from primary hepatocytes in terms of energy metabolism and urea production. This knowledge can be used to design media supplements and metabolically engineer cells to restore necessary hepatic functions to HepG2-C3A cells for a range of applications.