Biosynthetic Activity Research Articles

Increasing Energetic Demands on Photoreceptors in Diabetes Corrects Retinal Lipid Dysmetabolism and Reduces Subsequent Microvascular Damage

Mechanisms responsible for the pathogenesis of diabetic retinal disease remain incompletely understood, but they likely involve multiple cellular targets, including photoreceptors. Evidence suggests that dysregulated de novo lipogenesis in photoreceptors is a critical early target of diabetes. Following on this observation, the present study aimed to determine whether two interventions shown to improve diabetic retinopathy in mice-pharmacologic visual cycle inhibition and prolonged dark adaptation-reduce photoreceptor anabolic lipid metabolism. Elevated retinal lipid biosynthetic signaling was observed in two mouse models of diabetes, with both models showing reduced retinal AMP-activated kinase (AMPK) signaling, elevated acetyl CoA carboxylase (ACC) signaling, and increased activity of fatty acid synthase, which promotes lipotoxicity in photoreceptors. Although retinal AMPK-ACC axis signaling was dependent on systemic glucose fluctuations in healthy animals, mice with diabetes lacked such regulation. Visual cycle inhibition and prolonged dark adaptation reversed abnormal retinal AMPK-ACC signaling in mice with diabetes. Although visual cycle inhibition reduced the severity of diabetic retinopathy in control mice, as assessed by retinal capillary atrophy, this intervention was ineffective in fatty acid synthase gain-of-function mice. These results suggest that early diabetic retinopathy is characterized by glucose-driven elevations in retinal lipid biosynthetic activity, and that two interventions known to increase photoreceptor glucose demands alleviate disease by reversing these signals.

BAP (6-Benzylaminopurine) Seed-Priming Enhanced Growth, Antioxidant Accumulation and Anthocyanin Metabolism in Olive Sprouts

Given the importance of olive sprouts, it is crucial to explore their potential as an innovative source of bioactive and nutritive compounds through research. Here, we aimed at investigating the potential role of benzylaminopurine (BAP) in improving the tissue chemical composition and bioactivity of olive sprouts. To this end, seeds of two olive varieties (Olea europaea L. vr. Kroniaki and Coratina) were primed with BAP at 25 µM. A substantial enhancement was observed in biomass accumulation by 35% and 30% in Kroniaki and Coratina varieties, respectively. Likewise, the photosynthetic pigments (total chlorophyll, α- and β-carotene, lutein and β-cryptoxanthin) in both varieties were increased, mainly in Coratina. At primary metabolic level, BAP priming improved sprout lipid composition, particularly in Coratina variety. At antioxidant level, BAP priming improved lipid antioxidants (α-, β- and γ-tocopherols) and water-soluble antioxidants (phenols, flavonoids, ascorbate, glutathione and anthocyanins) in both olive varieties. At the anthocyanins level, their precursors (phenylalanine, cinnamic acid, coumaric acid and naringenin) and key biosynthetic enzyme activity (phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), coenzyme A ligase (4CL) and cinnamate 4-hydroxylase (C4H)) were improved in olive varieties, but to a greater extent in Coratina variety. Overall, the sprouts of BAP-primed olive seeds could potentially enhance their nutritional value, suggesting that the sprouts of BAP-primed olive seeds can be used as a food ingredient and additives.

Intermittent high voltage electrostatic field and static magnetic field assisted modified atmosphere packaging alleviate mildew of postharvest strawberries after simulated transportation by activating the phenylpropanoid pathway

The mildew is a typical symptom of strawberries during storage. The effectiveness of intermittent high voltage electrostatic field combined with static magnetic field (HVEF-SMF) technique in inhibiting the mildew of strawberries (before and after simulation of transport vibrations) was investigated. Intermittent HVEF, SMF and HVEF-SMF treatments inhibited spoilage fungal growth on the surface of strawberries by increasing the membrane permeability and leakage of intracellular materials of spoilage fungal. The HVEF-SMF alleviated mildew in strawberries, which probably via the increase of antifungal compounds (total phenolics and lignin), phenylpropanoid biosynthetic enzyme activities (Phenylalanine ammonia-lyase, 4-coumarate-CoA ligase) and pathogenesis-related proteins enzymes activities (chitinase and β-1,3-glucanase). Overall, HVEF-SMF contributed to alleviating the mildew and disease incidence of strawberries, improving the levels of antimicrobial activity, as well as extending their shelf life from 6 d to 12 d. Therefore, HVEF-SMF treatment is a promising technology for alleviating postharvest mildew in strawberries after transportation.

FACTORS ON THE BIOSYNTHESIS OF AMYLOLITIC ENZYMES OF STREPTOMYCETE ORIGIN

Modern enzyme biotechnology is a promising and rapidly developing field that requires the latest research on the conditions of enzyme biosynthesis. Optimizing the composition of the nutrient medium depending on the needs of microorganisms and physicochemical factors directly affect the increase in the efficiency of the biosynthesis of amylolytic enzymes, namely the biosynthetic capacity of the strain Streptomyces recifensis var. lyticus 2P-15. Modulation of the biosynthetic activity of strains producing amylolytic enzymes will allow to significantly increase their economic yield. Aim. The purpose of this work is to optimize the biosynthetic capacity of the strain Streptomyces recifensis var. lyticus 2P-15 in terms of the synthesis of amylolytic enzymes and the study of the dynamics of the influence of physical and chemical factors on optimization. Methods. The object of the study is the strain Streptomyces recifensis var. lyticus 2P-15, obtained by three-stage selection of the producer. The simplex method of selecting the composition of the environment was used for the research. The ratio of amylolytic activity to the level of biomass accumulation was taken as the biosynthetic capacity of the strain. A photocolometric method was used to determine amylolytic activity. The level of biomass accumulation was determined by the weight method. Results. It was established that as a result of optimizing the composition of the simplex nutrient medium by the method of mathematical modeling, the biosynthetic capacity increased by 3.63 compared to the control variant. It was also investigated that the optimal concentration of such a component of the nutrient medium as monosodium glutamate С5Н8NO4Na・H2O is 1.5%, which increases the amylolytic activity by 2.63 and increases the accumulation of biomass. Separately, it should be noted the obtained results of the study of the optimal concentrations of heavy metal ions added to the optimized version of the nutrient medium, which allow further research in this aspect to be continued and the use of Co, Mo, Cd ions in the composition of the nutrient medium. With the obtained results, there is an increase in amylolytic activity in the best response by 3.54. The obtained results have theoretical and practical significance for further research in the biotechnology of enzymes. Conclusions. The prospect of further research into the optimization of the biosynthesis of actinomycetes by the simplex method of other aspects of its regulation will be to increase the biosynthetic capacity of the studied strain, which will have a positive effect on the economic output of the production of amylolytic enzyme preparations by obtaining microbial synthesis.

Microbial synthesis of silver nanoparticles and their effect of wheat seeds’ germination

One of the little-studied and promising ways to increase the yield of crops is the use of nanoparticles, especially of the biological origin. Recently, research has been carried out in this direction, however, there is still very little information and it is unambiguous. In this regard, the purpose of this study was to obtain silver nanoparticles (AgNPs) with use of Pseudomonas stutzeri and Bacillus sp. bacteria, which expressed high biosynthetic activity, and to determine their effect on the germination of wheat seeds. The culture liquids containing AgNPs were used to treat wheat seeds (Unumli bugdoy variety). The treated seeds were placed in Petri dishes with moistened filter paper to germinate during 7 days. The received AgNPs were characterized by UV spectroscopy and AFM. It was determined that strains of Pseudomonas stutzeri and Bacillus sp. possess the ability to synthesize oval and spherical AgNPs ranging in size from 5 to 100 nm. It was revealed that the treatment of wheat seeds with a cultural liquid containing biogenic AgNPs stimulated seeds’ germination, and also had a stimulating effect on the growth of roots and stems of plants, the accumulation of the dry matter mass of the roots and the aerial part of the seedlings.

Metagenomic and metatranscriptomic exploration of the Egyptian Red Sea sponge Theonella sp. associated microbial community.

Marine sponges associated microorganisms are considered to be prolific source of bioactive natural products. Omics-based techniques such as metagenomics and metatranscriptomics have been used as effective tools to discover natural products. In this study, we used integrated metagenomic and metatranscriptomic analysis of three samples of the Egyptian Red Sea sponge Theonella sp. microbiome to obtain a complete picture of its biosynthetic activity to produce bioactive compounds. Our data revealed high biodiversity of the Egyptian sponge microbiota represented by 38 bacterial phyla with Candidate Phylum Poribacteria as the most abundant phyla with an average of 27.5% of the microbial community. The analysis also revealed high biosynthetic activity of the sponge microbiome through detecting different types of biosynthetic gene clusters (BGCs) with predicted antibacterial, cytotoxic and inhibitory bioactivity and the majority of these clusters were found to be actively transcribed. The complete BGCs of the cytotoxic theonellamide and misakinolide were detected and found to be actively transcribed. The majority of the detected BGCs were predicted to be novel as they did not show any similarity with any known cluster in the MIBiG database.

Metabolic Characteristics of Hairy Root Clones of Scutellaria pycnoclada and Scutellaria baicalensis

Scutellaria baicalensis is the most studied species of the genus, while Scutellaria pycnoclada is a poorly studied endemic species. Ten lines of the hairy roots of S. pycnoclada were obtained using Agrobacterium rhizogenes A4. The hairy root cultures of S. pycnoclada and the previously obtained roots of S. baicalensis were cultured on liquid and agar Gamborg media. A total of 14 flavonoids were detected via HPLC MS/MS in S. pycnoclada, and 17 were detected in S. baicalensis. Among them were flavones characteristic of both the roots and the aboveground parts of the plants. S. pycnoclada had a lower diversity of methylated flavones than S. baicalensis. Moreover, tenaxin I was absent in all S. pycnoclada lines on agar medium. HPLC analysis revealed that the flavone content in the different hairy root lines was 1.4–12.7 times higher on liquid medium than on agar medium. S. baicalensis and S. pycnoclada differed significantly in the ratio of the main flavones. In S. baicalensis, baicalin (7.83 mg/g DW) and wogonoside (6.29 mg/g DW) dominated when cultured on liquid medium, and wogonin (2.08 mg/g DW) dominated when cultured on solid medium. In S. pycnoclada, baicalin predominated (52–88% of the total content). S. pycnoclada is assumed to have a different set of O-methyltransferases and less biosynthetic enzyme activity than S. baicalensis.

Phytohormones affect the differentiation of human dermal fibroblasts via UPR activation

Normalization of secretory activity and the level of differentiation of mesenchymal cells, including fibroblasts, is the important biomedical problem. One of the plausible solutions to this problem is to affect the unfolded protein response (UPR) signaling cascade, which is activated during fibroblast differentiation. In the present study, the effect of phytohormones on the secretory activity and differentiation of cultured human dermal fibroblasts was investigated. By analyzing the expression level of genes encoding UPR markers in these cells, we found that phytohormone abscisic acid (ABA) upregulated the expression of GRP78 and ATF4 genes, while phytohormone gibberellic acid (GA) upregulated the expression of CHOP. Evaluation of the biosynthetic activity of fibroblasts showed that ABA elevated the level of secretion and synthesis of procollagen I and the level of fibronectin synthesis, as well as total production of collagen and non-collagen proteins of extracellular matrix. ABA also stimulated the synthesis of smooth muscle actin α (α-SMA) and increased the number of myofibroblasts in cell population. On the contrary, GA increased the level of fibronectin secretion, but decreased the level of procollagen I synthesis, and reduced the total production of collagen proteins of extracellular matrix. Concomitantly, in these conditions we observed the decreased level of α-SMA synthesis and the number of myofibroblasts in the cell population. Our results suggest that phytohormones are the modulators of the biosynthetic activity of fibroblasts and affect their differentiation status.

Computational and experimental characterizations of the spatiotemporal activity and functional role of TGF-β in the synovial joint

TGF-β is a prominent anabolic signaling molecule associated with synovial joint health. Recent work has uncovered mechanochemical mechanisms that activate the latent form of TGF-β (LTGF-β) in the synovial joint—synovial fluid (SF) shearing or cartilage compression—pointing to mechanobiological phenomena, whereby enhanced TGF-β activity occurs during joint stimulation. Here, we implement computational and experimental models to better understand the role of mechanochemical-activated TGF-β (aTGF-β) in regulating the functional biosynthetic activities of synovial joint tissues. Reaction-diffusion models describe the pronounced role of extracellular chemical reactions—load-induced activation, reversible ECM-binding, and cell-mediated internalization—in modulating the spatiotemporal distribution of aTGF-β in joint tissues. Of note, aTGF-β from SF shearing predominantly acts on cells in peripheral tissue regions (superficial zone [SZ] chondrocytes and synoviocytes) and aTGF-β from cartilage compression acts on chondrocytes through all cartilage layers. Further, ECM reversible binding sites in cartilage act to modulate the temporal delivery of aTGF-β to cells, creating a dynamic where short durations of joint activity give rise to extended periods of aTGF-β exposure at moderated doses. Ex vivo tissue models were subsequently utilized to characterize the influence of physiologic aTGF-β activity regimens in regulating functional biosynthetic activities. Physiologic exposure regimens of aTGF-β in SF induce strong 4-fold to 9-fold enhancements in the secretion rate of the synovial biolubricant, PRG4, from SZ cartilage and synovium explants. Further, aTGF-β inhibition in cartilage over 1-month culture leads to a pronounced loss of GAG content (30–35% decrease) and tissue softening (60–65% EY reduction). Overall, this work advances a novel perspective on the regulation of TGF-β in the synovial joint and its role in maintaining synovial joint health.

Phytohormones Affect Differentiation Status of Human Skin Fibroblasts via UPR Activation.

Normalization of secretory activity and differentiation status of mesenchymal cells, including fibroblasts, is an important biomedical problem. One of the possible solutions is modulation of unfolded protein response (UPR) activated during fibroblast differentiation. Here, we investigated the effect of phytohormones on the secretory activity and differentiation of cultured human skin fibroblasts. Based on the analysis of expression of genes encoding UPR markers, abscisic acid (ABA) upregulated expression of the GRP78 and ATF4 genes, while gibberellic acid (GA) upregulated expression of CHOP. Evaluation of the biosynthetic activity of fibroblasts showed that ABA promoted secretion and synthesis of procollagen I and synthesis of fibronectin, as well as the total production of collagen and non-collagen proteins of the extracellular matrix (ECM). ABA also stimulated the synthesis of smooth muscle actin α (α-SMA), which is the marker of myofibroblasts, and increased the number of myofibroblasts in the cell population. On the contrary, GA increased the level of fibronectin secretion, but reduced procollagen I synthesis and the total production of the ECM collagen proteins. GA downregulated the synthesis of α-SMA and decreased the number of myofibroblasts in the cell population. Our results suggest that phytohormones modulate the biosynthetic activity of fibroblasts and affect their differentiation status.

Stochastic scanning events on the GCN4 mRNA 5' untranslated region generate cell-to-cell heterogeneity in the yeast nutritional stress response.

Gene expression stochasticity is inherent in the functional properties and evolution of biological systems, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. In a distinct form of non-transcriptional noise, we find that interactions of the yeast translation machinery with the GCN4 mRNA 5'UTR, which underpins starvation-induced regulation of this transcriptional activator gene, manifest stochastic variation across cellular populations. We use flow cytometry, fluorescence-activated cell sorting and microfluidics coupled to fluorescence microscopy to characterize the cell-to-cell heterogeneity of GCN4-5'UTR-mediated translation initiation. GCN4-5'UTR-mediated translation is generally not de-repressed under non-starvation conditions; however, a sub-population of cells consistently manifests a stochastically enhanced GCN4 translation (SETGCN4) state that depends on the integrity of the GCN4 uORFs. This sub-population is eliminated upon deletion of the Gcn2 kinase that phosphorylates eIF2α under nutrient-limitation conditions, or upon mutation to Ala of the Gcn2 kinase target site, eIF2α-Ser51. SETGCN4 cells isolated using cell sorting spontaneously regenerate the full bimodal population distribution upon further growth. Analysis of ADE8::ymRuby3/ GCN4::yEGFP cells reveals enhanced Gcn4-activated biosynthetic pathway activity in SETGCN4 cells under non-starvation conditions. Computational modeling interprets our experimental observations in terms of a novel translational noise mechanism underpinned by natural variations in Gcn2 kinase activity.

Molecular identification of a laccase that catalyzes the oxidative coupling of a hydroxycinnamic acid amide for hordatine biosynthesis in barley.

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound accumulated at high levels in young barley (Hordeum vulgare) seedlings. The enzyme responsible for the oxidative dimerization of pCA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H2 O2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes (HvLAC1 and HvLAC2) that are highly expressed at the seedling stage and may encode enzymes that oxidize pCA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis-related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo (Phyllostachys nigra) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N. benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo-configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of pCA during HA biosynthesis.

Photosynthetic capacity in seagrass seeds and early-stage seedlings of Zostera marina.

In many terrestrial seeds, photosynthetic activity supplies O2 to the developing plant embryo to sustain aerobic metabolism and enhance biosynthetic activity. However, whether seagrass seeds possess similar photosynthetic capacity to alleviate intra-seed hypoxic stress conditions is unknown. We used a novel combination of microscale variable chlorophyll fluorescence imaging, a custom-made O2 optode microrespirometry system and planar optode O2 imaging, to determine the O2 microenvironment and photosynthetic activity in developing seeds and seedlings of seagrass (Zostera marina). Developing, sheath-covered seeds exhibited high O2 concentrations in the photosynthetic active seed sheath and low O2 concentrations in the centre of the seed at the position of the embryo. In light, photosynthesis in the seed sheath increased O2 availability in central parts of the seed enabling enhanced respiratory energy generation for biosynthetic activity. Early-stage seedlings also displayed photosynthetic capacity in hypocotyl and cotyledonary tissues, which may be beneficial for seedling establishment. Sheath O2 production is important for alleviating intra-seed hypoxic stress, which might increase endosperm storage activity, improving the conditions for successful seed maturation and germination.

PpMYB44 positively affects salicylic acid biosynthesis in Pichia guilliermondii-induced peach fruit resistance against Rhizopus stolonifer

Antagonistic yeast induces postharvest disease resistance and delays fruit decay. Salicylic acid (SA), a signal molecule, is required for activating induced disease resistance in various harvested fruit. However, there is limited information about the transcriptional regulatory mechanism of SA biosynthesis in response to Rhizopus stolonifer in peaches. Our results showed that Pichia guilliermondii application promoted SA accumulation accompanied by increasing SA biosynthetic enzyme activity of PAL and ICS and upregulating transcripts of SA synthesis-related genes and defense genes, including PpPAL1, PpPAL2, PpICS1, PpICS2, PpCM1, PpCM2, PpCM3, PpBA2H, PpSARD1, PpCBP60g, PpPR1, PpPR2, and PpPR5. In addition, an R2R3 MYB transcription factor, PpMYB44, was induced in P. guilliermondii-peach fruit in response to fungal disease, and PpMYB44 was identified to mediate SA synthesis by binding to the promoters of PpPAL1 and PpSARD1. Furthermore, the transient overexpression of PpMYB44 in peach fruit reduced disease incidence and severity, upregulated the expression of SA synthesis-related genes (PpPAL1 and PpSARD1) and enhanced SA accumulation. Silencing of PpMYB44 in peach fruit increased disease incidence and severity, downregulated the transcript levels of PpPAL1 and PpSARD1, and decreased SA accumulation. Thus, the study indicates that PpMYB44 regulates yeast-induced disease resistance by increasing the SA content in peaches.

A cytokine-induced spheroid-based in vitro model for studying osteoarthritis pathogenesis.

Given the lack of in vitro models faithfully reproducing the osteoarthritis (OA) disease on-set, this work aimed at manufacturing a reliable and predictive in vitro cytokine-based Articular Cartilage (AC) model to study OA progression. Cell spheroids of primary human fetal chondrocytes (FCs) and h-TERT mesenchymal stem cells differentiated chondrocytes (Y201-C) were analysed in terms of growth kinetics, cells proliferation and apoptosis over 10 days of culture, in healthy condition or in presence of cytokines (interleukin-1ß, -6 and TNF-α). Then, the spheroids were assembled into chondrospheres using a bottom-up strategy, to obtain an in vitro cytokines-induced OA model. The resulting chondrospheres were evaluated for gene expression and anabolic ECM proteins. Compared to the healthy environment, the simulated OA environment induced chondrocyte hyperproliferation and apoptotic pathway, decreased expression of anabolic ECM proteins, and diminished biosynthetic activity, resembling features of early-stage OA. These characteristics were observed for both Y201-C and HC at high and low concentrations of cytokines. Both HC and Y201-C demonstrated the suitability for the manufacturing of a scaffold-free in vitro OA model to facilitate studies into OA pathogenesis and therapeutic strategies. Our approach provides a faithful reproduction of early-stage osteoarthritis, demonstrating the ability of obtaining different disease severity by tuning the concentration of OA-related cytokines. Given the advantages in easy access and more reproducible performance, Y201-C may represent a more favourable source of chondrocytes for establishing more standardized protocols to obtain OA models.

Intercellular contacts affect secretion and biosynthesis of pancreatic islet cells.

Cell protein biosynthesis is regulated by different factors, but implication of intercellular contacts on alpha and beta cell protein biosyntheses activity has not been yet investigated. Islet cell biosynthetic activity is essential in regulating not only the hormonal reserve within cells but also in renewing all the proteins involved in the control of secretion. Here we aimed to assess whether intercellular interactions affected similarly secretion and protein biosynthesis of rat alpha and beta cells. Insulin and glucagon secretion were analyzed by ELISA or reverse hemolytic plaque assay, and protein biosynthesis evaluated at single cell level using bioorthogonal noncanonical amino acid tagging. Regarding beta cells, we showed a positive correlation between insulin secretion and protein biosynthesis. We also observed that homologous contacts increased both activities at low or moderate glucose concentrations. By contrast, at high glucose concentration, homologous contacts increased insulin secretion and not protein biosynthesis. In addition, heterogeneous contacts between beta and alpha cells had no impact on insulin secretion and protein biosynthesis. Regarding alpha cells, we showed that when they were in contact with beta cells, they increased their glucagon secretion in response to a drop of glucose concentration, but, on the other hand, they decreased their protein biosynthesis under any glucose concentrations. Altogether, these results emphasize the role of intercellular contacts on the function of islet cells, showing that intercellular contacts increased protein biosynthesis in beta cells, except at high glucose, and decreased protein biosynthesis in alpha cells even when glucagon secretion is stimulated.

Arabidopsis nicotianamine synthases comprise a common core-NAS domain fused to a variable autoinhibitory C terminus

Nicotianamine synthase (NAS) catalyzes the biosynthesis of the low-molecular-mass metal chelator nicotianamine (NA) from the 2-aminobutyrate moieties of three SAM molecules. NA has central roles in metal nutrition and metal homeostasis of flowering plants. The enzymatic function of NAS remains poorly understood. Crystal structures are available for archaeal and bacterial NAS-like proteins that carry out simpler aminobutanoyl transferase reactions. Here, we report amino acids essential for the activity of AtNAS1 based on structural modeling and site-directed mutagenesis. Using a newly developed enzyme-coupled continuous activity assay, we compare differing NAS proteins identified through multiple sequence alignments and phylogenetic analyses. In most NAS of dicotyledonous and monocotyledonous plants (class Ia and Ib), the core-NAS domain is fused to a variable C-terminal domain. Compared to fungal and moss NAS that comprise merely a core-NAS domain (class III), NA biosynthetic activities of the four paralogous Arabidopsis thaliana NAS proteins were far lower. C-terminally trimmed core-AtNAS variants exhibited strongly elevated activities. Of 320 amino acids of AtNAS1, twelve, 287-TRGCMFMPCNCS-298, accounted for the autoinhibitory effect of the C terminus, of which approximately one-third was attributed to N296 within a CNCS motif that is fully conserved in Arabidopsis. No detectable NA biosynthesis was mediated by two representative plant NAS proteins that naturally lack the C-terminal domain, class Ia Arabidopsis halleri NAS5 and Medicago truncatula NAS2 of class II which is found in dicots and diverged early during the evolution of flowering plants. Next, we will address a possible posttranslational release of autoinhibition in class I NAS proteins.

Effects of Atherogenic Factors on Endothelial Cells: Bioinformatics Analysis of Differentially Expressed Genes and Signaling Pathways.

(1) Background: Atherosclerosis is a serious medical condition associated with high morbidity and mortality rates. It develops over many years as a complex chain of events in the vascular wall involving various cells and is influenced by many factors of clinical interest. (2) Methods: In this study, we performed a bioinformatic analysis of Gene Expression Omnibus (GEO) datasets to investigate the gene ontology of differentially expressed genes (DEGs) in endothelial cells exposed to atherogenic factors such as tobacco smoking, oscillatory shear, and oxidized low-density lipoproteins (oxLDL). DEGs were identified using the limma R package, and gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction (PPI) network analysis were performed. (3) Results: We studied biological processes and signaling pathways involving DEGs in endothelial cells under the influence of atherogenic factors. GO enrichment analysis demonstrated that the DEGs were mainly involved in cytokine-mediated signaling pathway, innate immune response, lipid biosynthetic process, 5-lipoxygenase activity, and nitric-oxide synthase activity. KEGG pathway enrichment analysis showed that common pathways included tumor necrosis factor signaling pathway, NF-κB signaling pathway, NOD-like receptor signaling pathway, lipid and atherosclerosis, lipoprotein particle binding, and apoptosis. (4) Conclusions: Atherogenic factors such as smoking, impaired flow, and oxLDL contribute to impaired innate immune response, metabolism, and apoptosis in endothelial cells, potentially leading to the development of atherosclerosis.

Bursts in biosynthetic gene cluster transcription are accompanied by surges of natural compound production in the myxobacterium Sorangium sp.

A better understanding of the genetic regulation of the biosynthesis of microbial compounds could accelerate the discovery of new biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and non-ribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these bursts in BGC transcriptional activity were associated with surges in the net production rates of known natural compounds, indicating that their biosynthesis was critically regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provide unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under nutrient-limited conditions. The close association observed between BGC transcription and compound production warrants additional efforts to develop genetic engineering tools for boosting compound yields from myxobacterial producer strains.

Hepatic mitochondrial NAD + transporter SLC25A47 activates AMPKα mediating lipid metabolism and tumorigenesis.

SLC25A47 was initially identified as a mitochondrial HCC-downregulated carrier protein, but its physiological functions and transport substrates are unknown. We aimed to investigate the physiological role of SLC25A47 in hepatic metabolism. In the treatment of hepatocytes with metformin, we found that metformin can transcriptionally activate the expression of Slc25a47 , which is required for AMP-activated protein kinase α (AMPKα) phosphorylation. Slc25a47 -deficient mice had increased hepatic lipid content, triglycerides, and cholesterol levels, and we found that Slc25a47 deficiency suppressed AMPKα phosphorylation and led to an increased accumulation of nuclear SREBPs, with elevated fatty acid and cholesterol biosynthetic activities. Conversely, when Slc25a47 was overexpressed in mouse liver, AMPKα was activated and resulted in the inhibition of lipogenesis. Moreover, using a diethylnitrosamine-induced mouse HCC model, we found that the deletion of Slc25a47 promoted HCC tumorigenesis and development through the activated mammalian target of rapamycin cascade. Employing homology modeling of SLC25A47 and virtual screening of the human metabolome database, we demonstrated that NAD + was an endogenous substrate for SLC25A47, and the activity of NAD + -dependent sirtuin 3 declined in Slc25a47 -deficient mice, followed by inactivation of AMPKα. Our findings reveal that SLC25A47, a hepatocyte-specific mitochondrial NAD + transporter, is one of the pharmacological targets of metformin and regulates lipid homeostasis through AMPKα, and may serve as a potential drug target for treating NAFLD and HCC.