Metabolic modeling of single Th17 cells reveals regulators of autoimmunity.

Metabolism is a major regulator of immune cell function, but it remains difficult to study the metabolic status of individual cells. Here, we present Compass, an algorithm to characterize cellular metabolic states based on single-cell RNA sequencing and flux balance analysis. We applied Compass to associate metabolic states with T helper 17 (Th17) functional variability (pathogenic potential) and recovered a metabolic switch between glycolysis and fatty acid oxidation, akin to known Th17/regulatory T cell (Treg) differences, which we validated by metabolic assays. Compass also predicted that Th17 pathogenicity was associated with arginine and downstream polyamine metabolism. Indeed, polyamine-related enzyme expression was enhanced in pathogenic Th17 and suppressed in Treg cells. Chemical and genetic perturbation of polyamine metabolism inhibited Th17 cytokines, promoted Foxp3 expression, and remodeled the transcriptome and epigenome of Th17 cells toward a Treg-like state. In vivo perturbations of the polyamine pathway altered the phenotype of encephalitogenic T cells and attenuated tissue inflammation in CNS autoimmunity.

The telencephalon is a neuronal substrate for systemic inflammatory responses in teleosts via polyamine metabolism.

Systemic inflammation elicits sickness behaviors and fever by engaging a complex neuronal circuitry that begins in the preoptic area of the hypothalamus. Ectotherms such as teleost fish display sickness behaviors in response to infection or inflammation, seeking warmer temperatures to enhance survival via behavioral fever responses. To date, the hypothalamus is the only brain region implicated in sickness behaviors and behavioral fever in teleosts. Yet, the complexity of neurobehavioral manifestations underlying sickness responses in teleosts suggests engagement of higher processing areas of the brain. Using in vivo models of systemic inflammation in rainbow trout, we find canonical pyrogenic cytokine responses in the hypothalamus whereas in the telencephalon and the optic tectum il-1b and tnfa expression is decoupled from il-6 expression. Polyamine metabolism changes, characterized by accumulation of putrescine and decreases in spermine and spermidine, are recorded in the telencephalon but not hypothalamus upon systemic injection of bacteria. While systemic inflammation causes canonical behavioral fever in trout, blockade of bacterial polyamine metabolism prior to injection abrogates behavioral fever, polyamine responses, and telencephalic but not hypothalamic cytokine responses. Combined, our work identifies the telencephalon as a neuronal substrate for brain responses to systemic inflammation in teleosts and uncovers the role of polyamines as critical chemical mediators in sickness behaviors.

Increased hepatic putrescine levels as a new potential factor related to the progression of metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic liver condition that often progresses to more advanced stages, such as metabolic dysfunction-associated steatohepatitis (MASH). MASH is characterized by inflammation and hepatocellular ballooning, in addition to hepatic steatosis. Despite the relatively high incidence of MASH in the population and its potential detrimental effects on human health, this liver disease is still not fully understood from a pathophysiological perspective. Deregulation of polyamine levels has been detected in various pathological conditions, including neurodegenerative diseases, inflammation, and cancer. However, the role of the polyamine pathway in chronic liver disorders such as MASLD has not been explored. In this study, we measured the expression of liver ornithine decarboxylase (ODC1), the rate-limiting enzyme responsible for the production of putrescine, and the hepatic levels of putrescine, in a preclinical model of MASH as well as in liver biopsies of patients with obesity undergoing bariatric surgery. Our findings reveal that expression of ODC1 and the levels of putrescine, but not spermidine nor spermine, are elevated in hepatic tissue of both diet-induced MASH mice and patients with biopsy-proven MASH compared with control mice and patients without MASH, respectively. Furthermore, we found that the levels of putrescine were positively associated with higher aspartate aminotransferase concentrations in serum and an increased SAF score (steatosis, activity, fibrosis). Additionally, in in vitro assays using human HepG2 cells, we demonstrate that elevated levels of putrescine exacerbate the cellular response to palmitic acid, leading to decreased cell viability and increased release of CK-18. Our results support an association between the expression of ODC1 and the progression of MASLD, which could have translational relevance in understanding the onset of this disease. © 2024 The Pathological Society of Great Britain and Ireland.

PrG protects postovulatory oocytes aging in mice through the putrescine pathway.

Postovulatory aging of oocytes involves a series of deleterious molecular and cellular changes, which adversely affect oocyte maturation, fertilization, and early embryonic development. Petunidin-3-O-(6-O-pcoumaroyl)-rutinoside-5-O-glucoside (PrG), the main active ingredient of anthocyanin, exerts antioxidant effects. This study investigated whether PrG supplementation could delay postovulatory oocyte aging by alleviating oxidative stress. Our results showed that PrG supplementation decreased the number of abnormal morphology oocytes and improved the oxidative stress of aged oocytes by facilitating the reduction of the reactive oxygen species, the increase in glutathione content, and the recovery of expression of antioxidant-related gene expression. In addition, PrG treatment recovered mitochondrial dysfunction, including mitochondrial distribution, mitochondrial membrane potential and adenosine triphosphate in aged oocytes. PrG-treated oocytes returned to normal levels of cytoplasmic and mitochondrial calcium. Notably, PrG inhibited early apoptosis in aged oocytes. RNA-seq and qRT-PCR results revealed that PrG ameliorated oxidative stress injury in postovulatory aging oocytes of mice via the putrescine pathway. In conclusion, in vitro PrG supplementation is a potential therapy for delaying postovulatory oocyte aging.

Exogenous putrescine application imparts salt stress-induced oxidative stress tolerance via regulating antioxidant activity, potassium uptake, and abscisic acid to gibberellin ratio in Zinnia flowers.

This research was conducted to investigate the efficacy of putrescine (PUT) treatment (0, 1, 2, and 4 mM) on improving morphophysiological and biochemical characteristics of Zinnia elegans "State Fair" flowers under salt stress (0, 50, and 100 mM NaCl). The experiment was designed in a factorial setting under completely randomized design with 4 replications. The results showed that by increasing the salt stress intensity, the stress index (SSI) increased while morphological traits such as plant height decreased. PUT treatments effectively recovered the decrease in plant height and flower quality compared to the not-treated plants. Treatment by PUT 2 mM under 50 and 100 mM salt stress levels reduced the SSI by 28 and 35%, respectively, and increased plant height by 20 and 27% compared to untreated plants (PUT 0 mM). 2 mM PUT treatment also had the greatest effect on increasing fresh and dry biomass, number and surface area of leaves, flower diameter, internodal length, leaf relative water content, protein contents, total chlorophyll contents, carotenoids, leaf potassium (K+) content, and K+/Na+ ratio in treated plants compared to untreated control plants. The treatment of 2 mM PUT decreased the electrolyte leakage, leaf sodium (Na+) content, H2O2, malondialdehyde, and proline content. Furthermore, PUT treatments increased the activity of defense-related enzymes including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and phenylalanine ammonium lyase (PAL), and reduced the abscisic acid (ABA) content while increased the level of gibberellin (GA) content compared to untreated samples under all different levels of salinity stress. In this research, enhancing the plant's antioxidant system, increasing K+ absorption, K+/Na+ ratio, and reducing the ABA/GA ratio are likely the most important mechanisms of PUT treatment, which improved growth, and maintained the visual quality of zinnia flowers under salt stress conditions.

Exogenous putrescine plays a switch-like influence on the pH stress adaptability of biofilm-based activated sludge.

Microbial community adaptability to pH stress plays a crucial role in biofilm formation. This study aims to investigate the regulatory mechanisms of exogenous putrescine on pH stress, as well as enhance understanding and application for the technical measures and molecular mechanisms of biofilm regulation. Findings demonstrated that exogenous putrescine acted as a switch-like distributor affecting microorganism pH stress, thus promoting biofilm formation under acid conditions while inhibiting it under alkaline conditions. As pH decreases, the protonation degree of putrescine increases, making putrescine more readily adsorbed. Protonated exogenous putrescine could increase cell membrane permeability, facilitating its entry into the cell. Subsequently, putrescine consumed intracellular H+ by enhancing the glutamate-based acid resistance strategy and the γ-aminobutyric acid metabolic pathway to reduce acid stress on cells. Furthermore, putrescine stimulated ATPase expression, allowing for better utilization of energy in H+ transmembrane transport and enhancing oxidative phosphorylation activity. However, putrescine protonation was limited under alkaline conditions, and the intracellular H+ consumption further exacerbated alkali stress and inhibits cellular metabolic activity. Exogenous putrescine promoted the proportion of fungi and acidophilic bacteria under acidic stress and alkaliphilic bacteria under alkali stress while having a limited impact on fungi in alkaline biofilms. Increasing Bdellovibrio under alkali conditions with putrescine further aggravated the biofilm decomposition. This research shed light on the unclear relationship between exogenous putrescine, environmental pH, and pH stress adaptability of biofilm. By judiciously employing putrescine, biofilm formation could be controlled to meet the needs of engineering applications with different characteristics.IMPORTANCEThe objective of this study is to unravel the regulatory mechanism by which exogenous putrescine influences biofilm pH stress adaptability and understand the role of environmental pH in this intricate process. Our findings revealed that exogenous putrescine functioned as a switch-like distributor affecting the pH stress adaptability of biofilm-based activated sludge, which promoted energy utilization for growth and reproduction processes under acidic conditions while limiting biofilm development to conserve energy under alkaline conditions. This study not only clarified the previously ambiguous relationship between exogenous putrescine, environmental pH, and biofilm pH stress adaptability but also offered fresh insights into enhancing biofilm stability within extreme environments. Through the modulation of energy utilization, exerting control over biofilm growth and achieving more effective engineering goals could be possible.

The loss-of-function of AtNATA2 enhances AtADC2-dependent putrescine biosynthesis and priming, improving growth and salinity tolerance in Arabidopsis.

Putrescine (Put) is a promising small molecule-based biostimulant to enhance plant growth and resilience, though its mode of action remains unclear. This study investigated the Put priming effect on Arabidopsis mutant lines (Atadc1, Atadc2, Atnata1, and Atnata2) under control conditions and salinity to understand its role in regulating plant growth. The Atadc2 mutant, characterized by reduced endogenous Put levels, showed insensitivity to Put priming without growth enhancement, which was linked to significant imbalances in nitrogen metabolism, including a high Gln/Glu ratio. Contrarily, the Atnata2 mutant exhibited significant growth improvement and upregulated AtADC2 expression, particularly under Put priming, highlighting these genes' involvement in regulating plant development. Put priming enhanced plant growth by inducing the accumulation of specific polyamines (free, acetylated, conjugated, or bound form) and improving light-harvesting efficiency, particularly in the Atnata2 line. Our findings suggest that AtNATA2 may negatively regulate Put synthesis and accumulation via AtADC2 in the chloroplast, impacting light harvesting in photosystem II (PSII). Furthermore, the Atadc2 mutant line exhibited upregulated AtADC1 but reduced AcPut levels, pointing to a cross-regulation among these genes. The regulation by AtNATA2 on AtADC2 and AtADC2 on AtADC1 could be crucial for plant growth and overall stress tolerance by interacting with polyamine catabolism, which shapes the plant metabolic profile under different growth conditions. Understanding the regulatory mechanisms involving crosstalk between AtADC and AtNATA genes in polyamine metabolism and the connection with certain SMBBs like Put can lead to more effective agricultural practices, improving plant growth, nitrogen uptake, and resilience under challenging conditions.

Bachmann-Bupp syndrome and treatment.

Bachmann-Bupp syndrome (BABS) is a neurodevelopmental disorder characterized by developmental delay, hypotonia, and varying forms of non-congenital alopecia. The condition is caused by 3'-end mutations of the ornithine decarboxylase 1 (ODC1) gene, which produce carboxy (C)-terminally truncated variants of ODC, a pyridoxal 5'-phosphate-dependent enzyme. C-terminal truncation of ODC prevents its ubiquitin-independent proteasomal degradation and leads to cellular accumulation of ODC enzyme that remains catalytically active. ODC is the first rate-limiting enzyme that converts ornithine to putrescine in the polyamine pathway. Polyamines (putrescine, spermidine, spermine) are aliphatic molecules found in all forms of life and are important during embryogenesis, organogenesis, and tumorigenesis. BABS is an ultra-rare condition with few reported cases, but it serves as a convincing example for drug repurposing therapy. α-Difluoromethylornithine (DFMO, also known as eflornithine) is an ODC inhibitor with a strong safety profile in pediatric use for neuroblastoma and other cancers as well as West African sleeping sickness (trypanosomiasis). Patients with BABS have been treated with DFMO and have shown improvement in hair growth, muscle tone, and development.

Molecular Insights into the Synergistic Effects of Putrescine and Ammonium on Dinoflagellates.

Ammonium and polyamines are essential nitrogen metabolites in all living organisms. Crosstalk between ammonium and polyamines through their metabolic pathways has been demonstrated in plants and animals, while no research has been directed to explore this relationship in algae or to investigate the underlying molecular mechanisms. Previous research demonstrated that high concentrations of ammonium and putrescine were among the active substances in bacteria-derived algicide targeting dinoflagellates, suggesting that the biochemical inter-connection and/or interaction of these nitrogen compounds play an essential role in controlling these ecologically important algal species. In this research, putrescine, ammonium, or a combination of putrescine and ammonium was added to cultures of three dinoflagellate species to explore their effects. The results demonstrated the dose-dependent and species-specific synergistic effects of putrescine and ammonium on these species. To further explore the molecular mechanisms behind the synergistic effects, transcriptome analysis was conducted on dinoflagellate Karlodinium veneficum treated with putrescine or ammonium vs. a combination of putrescine and ammonium. The results suggested that the synergistic effects of putrescine and ammonium disrupted polyamine homeostasis and reduced ammonium tolerance, which may have contributed to the cell death of K. veneficum. There was also transcriptomic evidence of damage to chloroplasts and impaired photosynthesis of K. veneficum. This research illustrates the molecular mechanisms underlying the synergistic effects of the major nitrogen metabolites, ammonium and putrescine, in dinoflagellates and provides direction for future studies on polyamine biology in algal species.

The Deficiency of Hypusinated eIF5A Decreases the Putrescine/Spermidine Ratio and Inhibits +1 Programmed Ribosomal Frameshifting during the Translation of Ty1 Retrotransposon in Saccharomyces cerevisiae.

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential in all eukaryotes. It is identified initially as an initiation factor and functions broadly in translation elongation and termination. The hypusination of eIF5A is specifically required for +1 PRF at the shifty site derived from the ornithine decarboxylase antizyme 1 (OAZ1) in Saccharomyces cerevisiae. However, whether the regulation of +1 PRF by yeast eIF5A is universal remains unknown. Here, we found that Sc-eIF5A depletion decreased the putrescine/spermidine ratio. The re-introduction of Sc-eIF5A in yeast eIF5A mutants recovered the putrescine/spermidine ratio. In addition, the Sc-eIF5A depletion decreases +1 PRF during the decoding of Ty1 retrotransposon mRNA, but has no effect on -1 PRF during the decoding of L-A virus mRNA. The re-introduction of Sc-eIF5A in yeast eIF5A mutants restored the +1 PRF rate of Ty1. The inhibition of the hypusine modification of yeast eIF5A by GC7 treatment or by mutating the hypusination site Lys to Arg caused decreases of +1 PRF rates in the Ty1 retrotransposon. Furthermore, mutational studies of the Ty1 frameshifting element support a model where the efficient removal of ribosomal subunits at the first Ty1 frame 0 stop codon is required for the frameshifting of trailing ribosomes. This dependency is likely due to the unique position of the frame 0 stop codon distance from the slippery sequence of Ty1. The results showed that eIF5A is a trans-regulator of +1 PRF for Ty1 retrotransposon and could function universally in yeast.

Influence of Some Heterocyclic, Cyclic, and Nitrogen-Containing Compounds on Oxidative Deamination of Polyamines in a Cell-Free Test System.

We studied the effects of some nitrogen-containing, heterocyclic, and cyclic compounds on the rate of oxidative deamination of polyamines and putrescine in tissues with a high proliferation rate. For this purpose, the specific activities of the main enzymes of polyamine oxidative degradation - spermine oxidase (SMO), polyamine oxidase (PAO), and diamine oxidase (DAO) were determined using a cell-free test system from regenerating rat liver. The compounds methyl 2-(5-formylfuran-2-yl)benzoate and 2,7-bis-[2-(diethylamino)ethoxy]-9H-fluoren-9-one (and in the form of dihydrochloride) showed mainly activating effect on oxidative degradation of putrescine, spermidine, and spermine, which indirectly indicates their antiproliferative effect. Nitrogen-free compounds inhibited this process, thus exhibiting potentially carcinogenic properties. Correlations were calculated for activity of DAO, PAO, and SMO with 5 topological indices: Wiener (W), Rouvray (R), Balaban (J) in the Trinaistich modification, detour (Ip), and electropy (Ie). The highest dependence was noted for DAO and the Balaban index (R=-0.55), for PAO and the detour index (R=0.78), and for SMO and the electropy index (R=0.53). The remaining dependencies showed insignificant correlation strength.

Strikingly conserved gene expression changes of polyamine regulating enzymes among various forms of acute and chronic kidney injury.

The polyamines spermidine and spermine and their common precursor molecule putrescine are involved in tissue injury and repair. Here, we test the hypothesis that impaired polyamine homeostasis contributes to various kidney pathologies in mice during experimental models of ischemia-reperfusion, transplantation, rhabdomyolysis, cyclosporine treatment, arterial hypertension, diabetes, unilateral ureteral obstruction, high oxalate feeding, and adenine-induced injuries. We found a remarkably similar pattern in most kidney pathologies with reduced expression of enzymes involved in polyamine synthesis together with increased expression of polyamine degrading enzymes. Transcript levels of amine oxidase copper-containing 1 (Aoc1), an enzyme which catalyzes the breakdown of putrescine, were barely detectable by in situ mRNA hybridization in healthy kidneys. Aoc1 was highly expressed upon various experimental kidney injuries resulting in a significant reduction of kidney putrescine content. Kidney levels of spermine were also significantly reduced, whereas spermidine was increased in response to ischemia-reperfusion injury. Increased Aoc1 expression in injured kidneys was mainly accounted for by an Aoc1 isoform that harbors 22 additional amino acids at its N-terminus and shows increased secretion. Mice with germline deletion of Aoc1 and injured kidneys showed no decrease of kidney putrescine content; although they displayed no overt phenotype, they had fewer tubular casts upon ischemia-reperfusion injury. Hyperosmotic stress stimulated AOC1 expression at the transcriptional and post-transcription levels in metanephric explants and kidney cell lines. AOC1 expression was also significantly enhanced after kidney transplantation in humans. These data demonstrate that the kidneys respond to various forms of injury with down-regulation of polyamine synthesis and activation of the polyamine breakdown pathway. Thus, an imbalance in kidney polyamines may contribute to various etiologies of kidney injury.

Putrescine alleviates the oxidative damage of cumulus-oocyte complex via improving fatty acid oxidation.

BACKGROUND: Fatty acid oxidation of cumulus-oocyte complex (COC) provides sufficient energy for oocyte maturation. But, the relationship between fatty acid oxidation and oxidative stress in aging follicles, as well as the effect of putrescine, is still unclear. METHODS: The porcine COCs were randomly divided into four groups and cultured in in vitro maturation (IVM) medium with or without 1 mmol/L putrescine, with 50 µmol/L hydrogen peroxide (H2O2) or with 50 µmol/L H2O2 plus 1 mmol/L putrescine. Oocyte maturation was assessed by the first polar body extrusion. The expressions of genes involved in fatty acid oxidation were detected, and the mitochondrial function was analyzed by themembrane potential. RESULTS: The maturation rate of oocyte was significantly lower in the H2O2 group when compared with the control group (P＜0.001), and putrescine significantly increased this rate in the H2O2 plus putrescine group when compared with the H2O2 group (P＜0.001). The expressions of LKB1, STRAD, ACC2, AMPKα1 and AMPKα2 mRNAs in cumulus cells (CCs) were significantly downregulated by H2O2 treatment, and partly rescued by putrescine addition (P＜0.05-0.001). However, the changes of LKB1, STRAD, ACC2, AMPKα1 and AMPKα2 mRNAs in oocytes were inapparent. The mitochondrial membrane potential of CCs in the H2O2 group was significantly lower than that in the control group, while putrescine addition significantly increased the mitochondrial membrane potential (P＜0.001). CONCLUSION: The decrease of oocyte maturation due to oxidative stress is related with the decreased fatty acid oxidation, and putrescine may alleviate the COCs damage via improving fatty acid oxidation.

Analysis of gene expression related to polyamine concentration and dimorphism induced in ornithine decarboxylase (odc) and spermidine synthase (spd) Ustilago maydis mutants.

Polyamines are ubiquitous small organic cations, and their roles as regulators of several cellular processes are widely recognized. They are implicated in the key stages of the fungal life cycle. Ustilago maydis is a phytopathogenic fungus, the causal agent of common smut of maize and a model system to understand dimorphism and virulence. U. maydis grows in yeast form at pH 7 and it can develop its mycelial form in vitro at pH 3. Δodc mutants that are unable to synthesize polyamines, grow as yeast at pH 3 with a low putrescine concentration, and to complete its dimorphic transition high putrescine concentration is require. Δspd mutants require spermidine to grow and cannot form mycelium at pH 3. In this work, the increased expression of the mating genes, mfa1 and mfa2, on Δodc mutants, was related to high putrescine concentration. Global gene expression analysis comparisons of Δodc and Δspd U. maydis mutants indicated that 2,959 genes were differentially expressed in the presence of exogenous putrescine at pH 7 and 475 genes at pH 3. While, in Δspd mutant, the expression of 1,426 genes was affected by exogenous spermine concentration at pH 7 and 11 genes at pH 3. Additionally, we identified 28 transcriptional modules with correlated expression during seven tested conditions: mutant genotype, morphology (yeast, and mycelium), pH, and putrescine or spermidine concentration. Furthermore, significant differences in transcript levels were noted for genes in modules relating to pH and genotype genes involved in ribosome biogenesis, mitochondrial oxidative phosphorylation, N-glycan synthesis, and Glycosylphosphatidylinositol (GPI)-anchor. In summary, our results offer a valuable tool for the identification of potential factors involved in phenomena related to polyamines and dimorphism.

SnRK2.4-mediated phosphorylation of ABF2 regulates ARGININE DECARBOXYLASE expression and putrescine accumulation under drought stress.

Arginine decarboxylase (ADC)-mediated putrescine (Put) biosynthesis plays an important role in plant abiotic stress response. SNF1-related protein kinases 2s (SnRK2s) and abscisic acid (ABA)-response element (ABRE)-binding factors (ABFs), are core components of the ABA signaling pathway involved in drought stress response. We previously reported that ADC of Poncirus trifoliata (PtrADC) functions in drought tolerance. However, whether and how SnRK2 and ABF regulate PtrADC to modulate putrescine accumulation under drought stress remains largely unclear. Herein, we employed a set of physiological, biochemical, and molecular approaches to reveal that a protein complex composed of PtrSnRK2.4 and PtrABF2 modulates putrescine biosynthesis and drought tolerance by directly regulating PtrADC. PtrABF2 was upregulated by dehydration in an ABA-dependent manner. PtrABF2 activated PtrADC expression by directly and specifically binding to the ABRE core sequence within its promoter and positively regulated drought tolerance via modulating putrescine accumulation. PtrSnRK2.4 interacts with and phosphorylates PtrABF2 at Ser93. PtrSnRK2.4-mediated PtrABF2 phosphorylation is essential for the transcriptional regulation of PtrADC. Besides, PtrSnRK2.4 was shown to play a positive role in drought tolerance by facilitating putrescine synthesis. Taken together, this study sheds new light on the regulatory module SnRK2.4-ABF2-ADC responsible for fine-tuning putrescine accumulation under drought stress, which advances our understanding on transcriptional regulation of putrescine synthesis.

Piriformospora indica recruits host-derived putrescine for growth promotion in plants.

Growth promotion induced by the endosymbiont Piriformospora indica has been observed in various plants; however, except growth phytohormones, specific functional metabolites involved in P. indica-mediated growth promotion are unknown. Here, we used a gas chromatography-mass spectrometry-based untargeted metabolite analysis to identify tomato (Solanum lycopersicum) metabolites whose levels were altered during P. indica-mediated growth promotion. Metabolomic multivariate analysis revealed several primary metabolites with altered levels, with putrescine (Put) induced most significantly in roots during the interaction. Further, our results indicated that P. indica modulates the arginine decarboxylase (ADC)-mediated Put biosynthesis pathway via induction of SlADC1 in tomato. Piriformospora indica did not promote growth in Sladc1-(virus-induced gene silencing of SlADC1) lines of tomato and showed less colonization. Furthermore, using LC-MS/MS we showed that Put promoted growth by elevation of auxin (indole-3-acetic acid) and gibberellin (GA4 and GA7) levels in tomato. In Arabidopsis (Arabidopsis thaliana) adc knockout mutants, P. indica colonization also decreased and showed no plant growth promotion, and this response was rescued upon exogenous application of Put. Put is also important for hyphal growth of P. indica, indicating that it is co-adapted by both host and microbe. Taken together, we conclude that Put is an essential metabolite and its biosynthesis in plants is crucial for P. indica-mediated plant growth promotion and fungal growth.

Enhanced cell growth, production, and mAb quality produced in Chinese hamster ovary-K1 cells by supplementing polyamine in the media.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. KEY POINTS: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing.

Evaluation of the stability of polyamines in human serum at different storage temperatures using capillary electrophoresis with indirect ultraviolet detection.

Polyamines are low molecular weight compounds that are present in all living organisms. They are related to the pathological processes, and have been studied as biomarkers for tumor progression, being analyzed in patients' biological fluids. However, polyamines can undergo degradation in serum samples, depending on storage conditions, which impairs their quantification in these matrices. In this work, capillary electrophoresis using indirect ultraviolet detection has been developed and applied to evaluate the stability of polyamines [cadaverine (Cad), putrescine (Put), spermine (Spm), and spermidine (Spd)] in human serum at different storage temperatures. By using this method, Cad, Put, Spm, and Spd were separated in less than 4 min. The range of the correlation coefficients was 0.993-0.998. The corresponding limits of detection and quantification were as follows (in mg L-1 ): Spm: 0.209 and 0.697; Spd: 0.165 and 0.549; Put: 0.189 and 0.632; Cad: 0.125 and 0.417. Besides, the coefficient of variation was lower than 1% for all analytes and the recovery was 92%-110%. The method was successfully applied for polyamines spiked in human serum samples from healthy people. The results showed that the degradation of polyamines was lower in samples stored in a freezer (-20°C).

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.

Dynamics of Polyamines, Proline, and Ethylene Metabolism under Increasing Cold in Winter Oilseed Rape.

Cold stress is among the most important environmental factors reducing the yield of crops. The present study aimed to investigate the impact of increasing cold stress conditions on winter oilseed rape polyamines, proline, and ethylene metabolism in acclimated and non-acclimated winter oilseed rape. This study was carried out under controlled conditions in the laboratory. The winter oilseed rape hybrid 'Visby' was used in the experiment. Acclimated and non-acclimated plants were subjected to a two-day-long increasing cold (from -1 °C to -3 °C) treatment. HPTLC, RT-qPCR, spectral analysis, and gas chromatography methods were used to analyse the levels of polyamines, gene expression, proline, and ethylene, respectively. This study showed a decrease in putrescine, spermidine, and spermine content during cold acclimation and a decrease in putrescine and spermidine levels at sub-zero temperatures. There were intensive changes in ADC2 gene expression, proline, and ethylene levels in non-acclimated plants: a substantial increase after exposure to -1 °C temperature and a sharp decrease after exposure to -3 °C temperature. The changes in these parameters were lower or absent in acclimated plants. The phenomena observed in this study add new insights to the knowledge about the plant stress response and suggest questions to be answered in the future.