Spatial organization of putrescine synthesis in plants.

Three plant pathways for the synthesis of putrescine have been described to date. These are the synthesis of putrescine from ornithine, by ornithine decarboxylase (ODC); the synthesis of putrescine from arginine by arginine decarboxylase, agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (NLP1); and arginine decarboxylase and agmatinase. To address how these pathways are organized in plants, we have used transient expression analysis of these genes in the leaves of Nicotiana benthamiana. Brassicas do not have ODC, but the single ODC gene from rice and one of the soybean genes, were localized to the ER. Transient expression of the rice agmatinase gene showed that it was localized to the mitochondria. In A. thaliana there are five isoforms of AIH and three isoforms of NLP1. Stable GFP-tagged transformants of the longest isoforms of AIH and NLP1 showed that both proteins were localized to the ER, but in tissues with chloroplasts, the localization was concentrated to lamellae adjacent to chloroplasts. Transient expression analyses showed that four of the isoforms of AIH and all of the isoforms of NLP1 were localized to the ER. However, AIH.4 was localized to the chloroplast. Combining these results with other published data, reveal that putrescine synthesis is excluded from the cytoplasm and is spatially localized to the chloroplast, ER, and likely the mitochondria. Synthesis of putrescine in the ER may facilitate cell to cell transport via plasmodesmata, or secretion via vesicles. Differential expression of these pathways may enable putrescine-mediated activation of hormone-responsive genes.

ACOD1 mediates Staphylococcus aureus-induced inflammatory response via the TLR4/NF-κB signaling pathway.

Staphylococcus aureus (SA) is a common Gram-positive bacterium that activates inflammatory cells, expressing various cytokines and inducing an inflammatory response. Recent research revealed aconitate decarboxylase 1 (ACOD1) as a regulator of the immune response through various metabolic pathways, playing a dual role in the inflammatory response. However, the mechanism by which ACOD1 participates in the regulation of SA-induced inflammatory responses in macrophages remains unknown. Therefore, this study aims to investigate the function and underlying regulatory mechanisms of ACOD1 in SA-induced inflammatory response. This study reveals that SA induced a macrophage inflammatory response and upregulated ACOD1 expression. ACOD1 knockdown significantly inhibited SA-induced macrophage inflammatory response, attenuated SA-induced nuclear envelope wrinkling, and plasma membrane rupture, and suppressed the TLR4/NF-κB signaling pathway. Furthermore, ACOD1 knockdown reduced the inflammatory response and alleviated lung tissue injury and cellular damage, leading to decreased bacterial loads in the lungs of SA-infected mice. Collectively, these findings demonstrate that SA induces an inflammatory response in macrophages and increases ACOD1 expression. ACOD1 enhances SA-induced inflammatory responses via the TLR4/NF-κB signaling pathway. Our findings highlight the significant role of ACOD1 in mediating the inflammatory response in SA-infected macrophages and elucidate its molecular mechanism in regulating the SA-induced inflammatory response.

A monoallelic UXS1 variant associated with short-limbed short stature.

BACKGROUND: Serine residues in the protein backbone of heavily glycosylated proteoglycans are bound to glycosaminoglycans through a tetrasaccharide linker. UXS1 encodes UDP-glucuronate decarboxylase 1, which catalyzes synthesis of UDP-xylose, the donor of the first building block in the linker. Defects in other enzymes involved in formation of the tetrasaccharide linker cause so-called linkeropathies, characterized by short stature, radio-ulnar synostosis, decreased bone density, congenital contractures, dislocations, and more. METHODS: Whole exome sequencing was performed in a father and son who presented with a mild skeletal dysplasia, as well as the father's unaffected parents. Wild-type and mutant UXS1 were recombinantly expressed in Escherichia coli and purified. Enzyme activity was evaluated by LC-MS/MS. In vivo effects were studied using HeparinRed assay and metabolomics. RESULTS: The son had short long bones, normal epiphysis, and subtle metaphyseal changes especially in his legs. The likely pathogenic heterozygous variant NM_001253875.1(UXS1):c.557T>A p.(Ile186Asn) detected in the son was de novo in the father. Purified Ile186Asn-UXS1, in contrast to the wild-type, was not able to convert UDP-glucuronic acid to UDP-xylose. Plasma glycosaminoglycan levels were decreased in both son and father. CONCLUSION: This is the first report linking UXS1 to short-limbed short stature in humans.

IRG1/itaconate alleviates acute liver injury in septic mice by suppressing NLRP3 expression and its mediated macrophage pyroptosis via regulation of the Nrf2 pathway.

Sepsis, a systemic inflammatory response triggered by infection, has a considerably high mortality rate. However, effective prevention and intervention measures against sepsis remain insufficient. Therefore, this study aimed to investigate the mechanisms underlying the protective properties of immune response gene-1 (IRG1) and 4-Octyl itaconate (OI) during acute liver damage in mice with sepsis. A sepsis mouse model was established to compare wild-type and IRG1-/- groups. The impact of IRG1/Itaconate on pro- and anti-inflammatory cytokines was evaluated using J774A.1 cells. IRG1/Itaconate substantially reduced pro-inflammatory cytokines and increased the release of anti-inflammatory cytokines. It reduced pathological damage to liver tissues, preserved normal liver function, decreased the release of reactive oxygen species (ROS) and LDH, and enhanced the GSH/GSSG ratio. Moreover, IRG1 and itaconic acid activated the Nrf2 signaling pathway, regulating the expression of its downstream antioxidative stress-related proteins. Additionally, they inhibited the activity of NLRP3 inflammatory vesicles to suppress the expression of macrophage-associated pyroptosis signaling molecules. Our findings demonstrate that IRG1/OI inhibits NLRP3 inflammatory vesicle activation and macrophage pyroptosis by modulating the Nrf2 signaling pathway, thereby attenuating acute liver injury in mice with sepsis. These findings could facilitate the clinical application of IRG1/Itaconate to prevent sepsis-induced acute liver injury.

Itaconate protects ferroptotic neurons by alkylating GPx4 post stroke.

Neuronal ferroptosis plays a key role in neurologic deficits post intracerebral hemorrhage (ICH). However, the endogenous regulation of rescuing ferroptotic neurons is largely unexplored. Here, we analyzed the integrated alteration of metabolomic landscape after ICH using LC-MS and MALDI-TOF/TOF MS, and demonstrated that aconitate decarboxylase 1 (Irg1) and its product itaconate, a derivative of the tricarboxylic acid cycle, were protectively upregulated. Deficiency of Irg1 or depletion of neuronal Irg1 in striatal neurons was shown to exaggerate neuronal loss and behavioral dysfunction in an ICH mouse model using transgenic mice. Administration of 4-Octyl itaconate (4-OI), a cell-permeable itaconate derivative, and neuronal Irg1 overexpression protected neurons in vivo. In addition, itaconate inhibited ferroptosis in cortical neurons derived from mouse and human induced pluripotent stem cells in vitro. Mechanistically, we demonstrated that itaconate alkylated glutathione peroxidase 4 (GPx4) on its cysteine 66 and the modification allosterically enhanced GPx4's enzymatic activity by using a bioorthogonal probe, itaconate-alkyne (ITalk), and a GPx4 activity assay using phosphatidylcholine hydroperoxide. Altogether, our research suggested that Irg1/itaconate-GPx4 axis may be a future therapeutic strategy for protecting neurons from ferroptosis post ICH.

Taurine Alleviates Experimental Colitis by Enhancing Intestinal Barrier Function and Inhibiting Inflammatory Response through TLR4/NF-κB Signaling.

Taurine (Tau) is a semiessential amino acid in mammals with preventive and therapeutic effects on several intestinal disorders. However, the exact function of taurine in ulcerative colitis (UC) is still largely unclear. In this study, we used two taurine-deficient mouse models (CSAD-/- and TauT-/- mice) to explore the influence of taurine on the progression of UC in both dextran sulfate sodium (DSS)-induced colitis and LPS-stimulated Caco-2 cells. We found that cysteine sulfinic acid decarboxylase (CSAD) and taurine transporter (TauT) expressions and taurine levels were markedly reduced in colonic tissues of mice treated with DSS. The CSAD and TauT knockouts exacerbated DSS-induced clinical symptoms and pathological damage and aggravated the intestinal barrier dysfunction and the colonic mucosal inflammatory response. Conversely, taurine pretreatment enhanced the intestinal barrier functions by increasing goblet cells and upregulating tight junction protein expression. Importantly, taurine bound with TLR4 and inhibited the TLR4/NF-κB pathway, ultimately reducing proinflammatory factors (TNF-α and IL-6) and oxidative stress. Our findings highlight the essential role of taurine in maintaining the intestinal barrier integrity and inhibiting intestinal inflammation, indicating that taurine is a promising supplement for colitis treatment.

Novel biallelic PISD missense variants cause spondyloepimetaphyseal dysplasia with disproportionate short stature and fragmented mitochondrial morphology.

Biallelic variants in PISD cause a phenotypic spectrum ranging from short stature with spondyloepimetaphyseal dysplasia (SEMD) to a multisystem disorder affecting eyes, ears, bones, and brain. PISD encodes the mitochondrial-localized enzyme phosphatidylserine decarboxylase. The PISD precursor is self-cleaved to generate a heteromeric mature enzyme that converts phosphatidylserine to the phospholipid phosphatidylethanolamine. We describe a 17-year-old male patient, born to unrelated healthy parents, with disproportionate short stature and SEMD, featuring platyspondyly, prominent epiphyses, and metaphyseal dysplasia. Trio genome sequencing revealed compound heterozygous PISD variants c.569C>T; p.(Ser190Leu) and c.799C>T; p.(His267Tyr) in the patient. Investigation of fibroblasts showed similar levels of the PISD precursor protein in both patient and control cells. However, patient cells had a significantly higher proportion of fragmented mitochondria compared to control cells cultured under basal condition and after treatment with 2-deoxyglucose that represses glycolysis and stimulates respiration. Structural data from the PISD orthologue in Escherichia coli suggest that the amino acid substitutions Ser190Leu and His267Tyr likely impair PISD's autoprocessing activity and/or phosphatidylethanolamine biosynthesis. Based on the data, we propose that the novel PISD p.(Ser190Leu) and p.(His267Tyr) variants likely act as hypomorphs and underlie the pure skeletal phenotype in the patient.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis.

BACKGROUND: UDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides. RESULTS: In this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark. CONCLUSION: Our findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

Identification and expression analysis of transcripts involved in taurine biosynthesis during early ontogeny of tropical gar Atractosteus tropicus.

In fishes, the availability of taurine is regulated during ontogenetic development, where its endogenous synthesis capacity is species dependent. Thus, different pathways and involved enzymes have been described: pathway I (cysteine sulfinate-dependent pathway), cysteine dioxygenase type 1 (cdo1) and cysteine sulfinic acid decarboxylase (csad); pathway II (cysteic acid pathway), cdo1 and glutamic acid decarboxylase (gad); and pathway III (cysteamine pathway), 2-aminoethanethiol dioxygenase (ado); whereas taurine transporter (taut) is responsible for taurine entry into cells on the cell membrane and the mitochondria. This study determined if the tropical gar (Atractosteus tropicus), an ancient holostean fish model, has the molecular mechanism to synthesize taurine through the identification and analysis expression of transcripts coding for proteins involved in its biosynthesis and transportation, at different embryo-larvae stages and in different organs of juveniles (31 dah). We observed a fluctuating expression of all transcripts involved in the three pathways at all analyzed stages. All transcripts are expressed during the beginning of larval development; however, ado and taut show a peak expression at 9 dah, and all transcripts but csad decreased at 23 dah, when the organism ended the larval period. Furthermore, at 31 dah, we observed taut expression in all examined organs. The transcripts involved in pathways I and III are expressed differently across all organs, whereas pathway II was only observed in the brain, eye, and skin. The results suggested that taurine biosynthesis in tropical gar is regulated during its early development before first feeding, and the pathway might also be organ-type dependent.

Neofunctionalization of S-adenosylmethionine decarboxylase into pyruvoyl-dependent L-ornithine and L-arginine decarboxylases is widespread in bacteria and archaea.

S-adenosylmethionine decarboxylase (AdoMetDC/SpeD) is a key polyamine biosynthetic enzyme required for conversion of putrescine to spermidine. Autocatalytic self-processing of the AdoMetDC/SpeD proenzyme generates a pyruvoyl cofactor from an internal serine. Recently, we discovered that diverse bacteriophages encode AdoMetDC/SpeD homologs that lack AdoMetDC activity and instead decarboxylate L-ornithine or L-arginine. We reasoned that neofunctionalized AdoMetDC/SpeD homologs were unlikely to have emerged in bacteriophages and were probably acquired from ancestral bacterial hosts. To test this hypothesis, we sought to identify candidate AdoMetDC/SpeD homologs encoding L-ornithine and L-arginine decarboxylases in bacteria and archaea. We searched for the anomalous presence of AdoMetDC/SpeD homologs in the absence of its obligatory partner enzyme spermidine synthase, or the presence of two AdoMetDC/SpeD homologs encoded in the same genome. Biochemical characterization of candidate neofunctionalized genes confirmed lack of AdoMetDC activity, and functional presence of L-ornithine or L-arginine decarboxylase activity in proteins from phyla Actinomycetota, Armatimonadota, Planctomycetota, Melainabacteria, Perigrinibacteria, Atribacteria, Chloroflexota, Sumerlaeota, Omnitrophota, Lentisphaerota, and Euryarchaeota, the bacterial candidate phyla radiation and DPANN archaea, and the δ-Proteobacteria class. Phylogenetic analysis indicated that L-arginine decarboxylases emerged at least three times from AdoMetDC/SpeD, whereas L-ornithine decarboxylases arose only once, potentially from the AdoMetDC/SpeD-derived L-arginine decarboxylases, revealing unsuspected polyamine metabolic plasticity. Horizontal transfer of the neofunctionalized genes appears to be the more prevalent mode of dissemination. We identified fusion proteins of bona fide AdoMetDC/SpeD with homologous L-ornithine decarboxylases that possess two, unprecedented internal protein-derived pyruvoyl cofactors. These fusion proteins suggest a plausible model for the evolution of the eukaryotic AdoMetDC.

Testosterone enhances taurine synthesis by upregulating androgen receptor and cysteine sulfinic acid decarboxylase expressions in male mouse liver.

Taurine is an end-product of cysteine metabolism, whereas cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSAD) are key enzymes regulating taurine synthesis. Sex steroids, including estrogens and androgens, are associated with liver physiopathological processes; however, we still do not know whether taurine and sex steroids interact in regulating liver physiology and hepatic diseases, and whether there are sex differences, although our recent study shows that the estrogen is involved in regulating taurine synthesis in mouse liver. The present study was thus proposed to identify whether 17-ß-estradiol and testosterone (T) play their roles by regulating CDO and CSAD expression and taurine synthesis in male mouse liver. Our results demonstrated that testosterone did not have a significant influence on CDO expression but significantly enhanced CSAD, androgen receptor (AR) expressions, and taurine levels in mouse liver, cultured hepatocytes, and HepG2 cells, whereas these effects were abrogated by AR antagonist flutamide. Furthermore, our results showed that testosterone increased CSAD-promoter-luciferase activity through the direct interaction of the AR DNA binding domain with the CSAD promoter. These findings first demonstrate that testosterone acts as an important factor to regulate sulfur amino acid metabolism and taurine synthesis through AR/CSAD signaling pathway. In addition, the in vivo and in vitro experiments showed that 17-ß-estradiol has no significant effects on liver CSAD expression and taurine synthesis in male mice and suggest that the effects of sex steroids on the taurine synthesis in mouse liver have sex differences. These results are crucial for understanding the physiological functions of taurine/androgen and their interacting mechanisms in the liver.NEW & NOTEWORTHY This study demonstrates that testosterone functions to enhance taurine synthesis by interacting with androgen receptor and binding to cysteine sulfinate decarboxylase (CSAD) promoter zone. Whereas estrogen has no significant effects either on liver CSAD expression or taurine synthesis in male mice and suggests that the effects of sex steroids on taurine synthesis in the liver have gender differences. These new findings are the potential for establishing effective protective and therapeutic strategies for liver diseases.

Pathogenic variants of the coenzyme A biosynthesis-associated enzyme phosphopantothenoylcysteine decarboxylase cause autosomal-recessive dilated cardiomyopathy.

Coenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1-4]), 4'-phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC 4.1.1.36) catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency problems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDC.

Neural Stem Cells Overexpressing Arginine Decarboxylase Improve Functional Recovery from Spinal Cord Injury in a Mouse Model.

Current therapeutic strategies for spinal cord injury (SCI) cannot fully facilitate neural regeneration or improve function. Arginine decarboxylase (ADC) synthesizes agmatine, an endogenous primary amine with neuroprotective effects. Transfection of human ADC (hADC) gene exerts protective effects after injury in murine brain-derived neural precursor cells (mNPCs). Following from these findings, we investigated the effects of hADC-mNPC transplantation in SCI model mice. Mice with experimentally damaged spinal cords were divided into three groups, separately transplanted with fluorescently labeled (1) control mNPCs, (2) retroviral vector (pLXSN)-infected mNPCs (pLXSN-mNPCs), and (3) hADC-mNPCs. Behavioral comparisons between groups were conducted weekly up to 6 weeks after SCI, and urine volume was measured up to 2 weeks after SCI. A subset of animals was euthanized each week after cell transplantation for molecular and histological analyses. The transplantation groups experienced significantly improved behavioral function, with the best recovery occurring in hADC-mNPC mice. Transplanting hADC-mNPCs improved neurological outcomes, induced oligodendrocyte differentiation and remyelination, increased neural lineage differentiation, and decreased glial scar formation. Moreover, locomotor and bladder function were both rehabilitated. These beneficial effects are likely related to differential BMP-2/4/7 expression in neuronal cells, providing an empirical basis for gene therapy as a curative SCI treatment option.

Unraveling the genetics of polyamine metabolism in barley for senescence-related crop improvement.

We explored the polyamine (PA) metabolic pathway genes in barley (Hv) to understand plant development and stress adaptation in Gramineae crops with emphasis on leaf senescence. Bioinformatics and functional genomics tools were utilized for genome-wide identification, comprehensive gene features, evolution, development and stress effects on the expression of the polyamine metabolic pathway gene families (PMGs). Three S-adenosylmethionine decarboxylases (HvSAMDCs), two ornithine decarboxylases (HvODCs), one arginine decarboxylase (HvADC), one spermidine synthase (HvSPDS), two spermine synthases (HvSPMSs), five copper amine oxidases (HvCuAOs) and seven polyamine oxidases (HvPAOs) members of PMGs were identified and characterized in barley. All the HvPMG genes were found to be distributed on all chromosomes of barley. The phylogenetic and comparative assessment revealed that PA metabolic pathway is highly conserved in plants and the prediction of nine H. vulgare miRNAs (hvu-miR) target sites, 18 protein-protein interactions and 961 putative CREs in the promoter region were discerned. Gene expression of HvSAMDC3, HvCuAO7, HvPAO4 and HvSPMS1 was apparent at every developmental stage. SPDS/SPMS gene family was found to be the most responsive to induced leaf senescence. This study provides a reference for the functional investigation of the molecular mechanism(s) that regulate polyamine metabolism in plants as a tool for future breeding decision management systems.

The Tumor Necrosis Factor Alpha and Interleukin 6 Auto-paracrine Signaling Loop Controls Mycobacterium avium Infection via Induction of IRF1/IRG1 in Human Primary Macrophages.

Macrophages sense and respond to pathogens by induction of antimicrobial and inflammatory programs to alert other immune cells and eliminate the infectious threat. We have previously identified the transcription factor IRF1 to be consistently activated in macrophages during Mycobacterium avium infection, but its precise role during infection is not clear. Here, we show that tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) autocrine/paracrine signaling contributes to controlling the intracellular growth of M. avium in human primary macrophages through activation of IRF1 nuclear translocation and expression of IRG1, a mitochondrial enzyme that produces the antimicrobial metabolite itaconate. Small interfering RNA (siRNA)-mediated knockdown of IRF1 or IRG1 increased the mycobacterial load, whereas exogenously provided itaconate was bacteriostatic at high concentrations. While the overall level of endogenous itaconate was low in M. avium-infected macrophages, the repositioning of mitochondria to M. avium phagosomes suggests a mechanism by which itaconate can be delivered directly to M. avium phagosomes in sufficient quantities to inhibit growth. Using mRNA hybridization, we further show that uninfected bystander cells actively contribute to the resolution of infection by producing IL-6 and TNF-α, which, via paracrine signaling, activate IRF1/IRG1 and strengthen the antimicrobial activity of infected macrophages. This mechanism contributes to the understanding of why patients on anti-inflammatory treatment, e.g., with tocilizumab or infliximab, can be more susceptible to mycobacterial disease. IMPORTANCE The prevalence of lung diseases caused by nontuberculous mycobacteria, such as Mycobacterium avium, is increasing in countries where tuberculosis is not endemic, most likely because of an aging population that is immunocompromised from underlying disease or immunosuppressive therapy. Our study contributes to the understanding of mycobacterial survival and killing in human macrophages and, more broadly, to the impact of immunometabolism during infection. We show evidence of an antimicrobial program in human primary macrophages where activation of the transcription factor IRF1 and expression of the mitochondrial enzyme IRG1 restrict the intracellular growth of M. avium, possibly by directed delivery of itaconate to M. avium phagosomes. The study also sheds light on why patients on immunosuppressive therapy are more susceptible to mycobacterial infections, since TNF-α and IL-6 contribute to driving the described antimycobacterial program.

Genomic analysis of the polyamine biosynthesis pathway in duckweed Spirodela polyrhiza L.: presence of the arginine decarboxylase pathway, absence of the ornithine decarboxylase pathway, and response to abiotic stresses.

MAIN CONCLUSION: Identification of the polyamine biosynthetic pathway genes in duckweed S. polyrhiza reveals presence of prokaryotic as well as land plant-type ADC pathway but absence of ODC encoding genes. Their differential gene expression and transcript abundance is shown modulated by exogenous methyl jasmonate, salinity, and acidic pH. Genetic components encoding for polyamine (PA) biosynthetic pathway are known in several land plant species; however, little is known about them in aquatic plants. We utilized recently sequenced three duckweed (Spirodela polyrhiza) genome assemblies to map PA biosynthetic pathway genes in S. polyrhiza. PA biosynthesis in most higher plants except for Arabidopsis involves two pathways, via arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). ADC-mediated PA biosynthetic pathway genes, namely, one arginase (SpARG1), two arginine decarboxylases (SpADC1, SpADC2), one agmatine iminohydrolase/deiminase (SpAIH), one N-carbamoyl putrescine amidase (SpCPA), three S-adenosylmethionine decarboxylases (SpSAMDc1, 2, 3), one spermidine synthase (SpSPDS1) and one spermine synthase (SpSPMS1) in S. polyrhiza genome were identified here. However, no locus was found for ODC pathway genes in this duckweed. Hidden Markov Model protein domain analysis established that SpADC1 is a prokaryotic/biodegradative type ADC and its molecular phylogenic classification fell in a separate prokaryotic origin ADC clade with SpADC2 as a biosynthetic type of arginine decarboxylase. However, thermospermine synthase (t-SPMS)/Aculis5 genes were not found present. Instead, one of the annotated SPDS may also function as SPMS, since it was found associated with the SPMS phylogenetic clade along with known SPMS genes. Moreover, we demonstrate that S. polyrhiza PA biosynthetic gene transcripts are differentially expressed in response to unfavorable conditions, such as exogenously added salt, methyl jasmonate, or acidic pH environment as well as in extreme temperature regimes. Thus, S. polyrhiza genome encodes for complete polyamine biosynthesis pathway and the genes are transcriptionally active in response to changing environmental conditions suggesting an important role of polyamines in this aquatic plant.

Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity.

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

Regulating COX10-AS1 / miR-142-5p / PAICS axis inhibits the proliferation of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the main causes of death in the world. To improve the diagnostic level and find new biological targets，GSE datasets were selected from GEO databaseto analyze the differential expression genes and construct ceRNA network. Cell apoptosis detection showed that both the early and late apoptosis rates were increased after inhibition of COX10-AS1. Glycolysis cell-based assay also found that the content of L-lactate decreased significantly after using miR-142-5p mimics but increased after using si-COX10-AS1. Dual-luciferase reporter analysis showed that the luciferase activity of PAICS-WT reporter vector was inhibited by miR-142-5p mimics, but there was no significant change in PAICS-MUT reporter vector after transfection of miR-142-5p mimics. And overexpression of miR-142-5p reduced the level of PAICS, but inhibition of miR-142-5p expression increased the expression of PAICS. After using COX10-AS1, the expression of PAICS inhibited by miR-142-5p was restored. Through bioinformatics analysis, we constructed the COX10-AS1/miR-142-5p/PAICS axis, which is a ceRNA regulatory network. We confirmed that COX10-AS1 down-expression can restore the inhibitory effect of miR-142-5p on PAICS, promote the apoptosis of NSCLC cells, and inhibit the proliferation of NSCLC cells. This process may be mediated by the activation of glycolysis pathway. The glycolysis-related gene PAICS may be a new and significant target for the regulation of the development of NSCLC.

Ist2 recruits the lipid transporters Osh6/7 to ER-PM contacts to maintain phospholipid metabolism.

ER-plasma membrane (PM) contacts are proposed to be held together by distinct families of tethering proteins, which in yeast include the VAP homologues Scs2/22, the extended-synaptotagmin homologues Tcb1/2/3, and the TMEM16 homologue Ist2. It is unclear whether these tethers act redundantly or whether individual tethers have specific functions at contacts. Here, we show that Ist2 directly recruits the phosphatidylserine (PS) transport proteins and ORP family members Osh6 and Osh7 to ER-PM contacts through a binding site located in Ist2's disordered C-terminal tethering region. This interaction is required for phosphatidylethanolamine (PE) production by the PS decarboxylase Psd2, whereby PS transported from the ER to the PM by Osh6/7 is endocytosed to the site of Psd2 in endosomes/Golgi/vacuoles. This role for Ist2 and Osh6/7 in nonvesicular PS transport is specific, as other tethers/transport proteins do not compensate. Thus, we identify a molecular link between the ORP and TMEM16 families and a role for endocytosis of PS in PE synthesis.