Regulation Of Metabolic Pathways Research Articles

6494 GLP-1/GIP/Glucagon Triagonist Improves Glycemic Control via Gαq Signaling Pathway and TRPM5 Activation

Abstract Disclosure: N. Khajavi: None. P.C. Schreier: None. P. Beyerle: None. P.S. Reinach: None. A. Breit: None. I. Boekhoff: None. T.D. Müller: Grant Recipient; Self; Novo Nordisk. Speaker; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. T. Gudermann: None. Cutting-edge research on obesity and diabetes strives to pioneer innovative drug combinations targeting various dysfunctional metabolic regulatory pathways. The single-molecule triagonist has been developed to directly interact with the glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor (GIPR), and the glucagon receptor (GcgR). It provides a breakthrough in the treatment of obesity and type 2 diabetes because of greater efficacy compared to conventional single-agonist therapy. While the triagonist is already undergoing phase two clinical trials, the downstream mechanism requires further study. Previously, we showed that triagonist elicits transient rises in intracellular Ca2+ concentration that augment glucose-induced rises in insulin secretion in murine (MIN6) and human (1.1B4) pancreatic β cell lines. Although Gαs signaling is known as the main mediator of both GLP-1 and GIP signaling, our research indicates that the superior effect of the triagonist on insulin secretion is not solely dependent on the activation of the classical Gαs pathway and the exchange factor activated by cAMP (EPAC) signaling. Instead, we report that the triagonist enhances glucose-induced rises in insulin secretion via Gαq signaling and its downstream effector, the TRPM5 channel. This involvement is evident, as blocking Gαq signaling with a specific blocker, YM254890, reduced triagonist-induced rises in insulin secretion in mouse pancreatic islets. Similarly, blocking TRPM5 with the TPPO blocker attenuated triagonist-induced increases in insulin secretion and Ca2+ signaling in mouse pancreatic islets. Strikingly, both blockers had no remarkable impact on GLP-1- and GIP-induced rises in insulin secretion. Our in vivo analysis demonstrates that the triagonist improves glucose tolerance in diet-induced obese wild-type mice, but this effect was absent in TRPM5 knockout mice. Notably, the triagonist augments glucose-induced insulin secretion in isolated islets of wild-type mice more than treatment with GLP1 and GIP mono-agonists. Furthermore, this enhanced response is absent in TRPM5 knockout islets. Recent studies propose that persistent membrane depolarization of β cells leads to a switch from Gαs to Gαq signaling, which is a major amplifying pathway of insulin secretion under diabetic conditions. Our study shows that the enhancing effect of the triagonist, acting as a biased agonist, on insulin secretion is dependent on TRPM5 activation through the Gαq signaling pathway. Presentation: 6/2/2024

Pde3a and Pde3b regulation of murine pulmonary artery smooth muscle cell growth and metabolism.

A role for metabolically active adipose tissue in pulmonary hypertension (PH) pathogenesis is emerging. Alterations in cellular metabolism in metabolic syndrome are triggers of PH-related vascular dysfunction. Metabolic reprogramming in proliferative pulmonary vascular cells causes a metabolic switch from oxidative phosphorylation to glycolysis. PDE3A and PDE3B subtypes in the regulation of metabolism in pulmonary artery smooth muscle cells (PASMC) are poorly understood. We previously found that PDE3A modulates the cellular energy sensor, AMPK, in human PASMC. We demonstrate that global Pde3a knockout mice have right ventricular (RV) hypertrophy, elevated RV systolic pressures, and metabolic dysfunction with elevated serum free fatty acids (FFA). Therefore, we sought to delineate Pde3a/Pde3b regulation of metabolic pathways in PASMC. We found that PASMC Pde3a deficiency, and to a lesser extent Pde3b deficiency, downregulates AMPK, CREB and PPARγ, and upregulates pyruvate kinase dehydrogenase expression, suggesting decreased oxidative phosphorylation. Interestingly, siRNA Pde3a knockdown in adipocytes led to elevated FFA secretion. Furthermore, PASMC exposed to siPDE3A-transfected adipocyte media led to decreased α-SMA, AMPK and CREB phosphorylation, and greater viable cell numbers compared to controls under the same conditions. These data demonstrate that deficiencies of Pde3a and Pde3b alter pathways that affect cell growth and metabolism in PASMC.

Improving organoleptic and antioxidant properties by inhibition of novel miRstv_7 to target key genes of steviol glycosides biosynthetic pathway in Stevia rebaudiana Bertoni.

Stevioside (5-10%) and rebaudioside-A (2-4%) are well-characterized diterpene glycosides found in leaves of Stevia rebaudiana known to have natural sweetening properties with zero glycaemic index. Stevioside has after-taste bitterness, whereas rebaudioside-A is sweet in taste. The ratio of rebaudioside-A to stevioside needs to be changed in order to increase the effectiveness and palatability of this natural sweetener. Plant-specific miRNAs play a significant role in the regulation of metabolic pathways for the biosynthesis of economically important secondary metabolites. In this study inhibition of miRNA through antisense technology was employed to antagonize the repressive action of miRstv_7 on its target mRNAs involved in the steviol glycosides (SGs) biosynthesis pathway. In transgenic plants expressing anti-miRstv_7, reduced expression level of endogenous miRstv_7 was observed than the non-transformed plants. As a result, enhanced expression of target genes, viz. KO (Kaurene oxidase), KAH (Kaurenoic acid-13-hydroxylase), and UGT76G1 (UDP-glycosyltransferase 76G1) led to a significant increase in the rebaudioside-A to stevioside ratio. Furthermore, metabolome analysis revealed a significant increase in total steviol glycosides content as well as total flavonoids content. Thus, our study can be utilized to generate more palatable varieties of Stevia with improved nutraceutical values including better organoleptic and antioxidant properties.

Evolution of the biochemistry underpinning purine alkaloid metabolism in plants.

Purine alkaloids are naturally occurring nitrogenous methylated derivatives of purine nucleotide degradation products, having essential roles in medicine, food and various other aspects of our daily lives. They are generated through convergent evolution in different plant species. The pivotal reaction steps within the purine alkaloid metabolic pathways have been largely elucidated, and the convergent evolution of purine alkaloids has been substantiated through bioinformatic, biochemical and other research perspectives within S-adenosyl-ʟ-methionine-dependent N-methyltransferases. Currently, the biological and ecological roles of purine alkaloids, further refinement of the purine alkaloid metabolic pathways and the investigation of purine alkaloid adaptive evolutionary mechanisms continue to attract widespread research interest. The exploration of the purine alkaloid metabolic pathways also enhances our comprehension of the biochemical mechanism, providing insights into inter-species interactions and adaptive evolution and offering potential value in drug development and agricultural applications. Here, we review the progress of research in the distribution, metabolic pathway elucidation and regulation, evolutionary mechanism and ecological roles of purine alkaloids in plants. The opportunities and challenges involved in elucidating the biochemical basis and evolutionary mechanisms of the purine alkaloid metabolic pathways, as well as other research aspects, are also discussed. This article is part of the theme issue 'The evolution of plant meta-bolism'.

Hepatic transcriptomic analysis reveals differential regulation of metabolic and immune pathways in three strains of chickens with distinct growth rates exposed to mixed parasite infections.

During parasite infections, the liver may prioritise immune-related pathways over its metabolic functions. Intestinal infections caused by Ascaridia galli and Heterakis gallinarum impair feed intake, nutrient absorption, and weight gain. Histomonas meleagridis, vectored by H. gallinarum, can also damage liver tissues, potentially impairing liver functions. This study examined the hepatic gene expression in three strains of chickens: Ross-308 (R), Lohmann Brown Plus (LB), and Lohmann Dual (LD), 2weeks after an experimental infection (n = 18) with both A. galli and H. gallinarum or kept as uninfected control (n = 12). Furthermore, H. gallinarum infection led to a co-infection with H. meleagridis. The mixed infections reduced feed intake and the average daily weight gain (P < 0.001). The infections also increased the plasma concentrations of alpha (1)-acid glycoprotein and the antibody titre against H. meleagridis (P = 0.049), with no strain differences (P > 0.05). For host molecular response, 1887 genes were differentially expressed in LD, while 275 and 25 genes were differentially expressed in R and LB, respectively. The up-regulated genes in R and LD were mostly related to inflammatory and adaptive immune responses, while down-regulated genes in LD were involved in metabolic pathways, including gluconeogenesis. Despite performance differences among the strains, worm burdens were similar, but hepatic molecular responses differed significantly. Moreover, there was an indication of a shift in hepatic functions towards immune-related pathways. We, therefore, conclude that the liver shifts its functions from metabolic to immune-related activities in chickens when challenged with mixed parasite species.

The cytotoxic activities of the major diterpene extracted from Salvia multicaulis (Bardakosh) are mediated by the regulation of heat-shock response and fatty acid metabolism pathways in human leukemia cells

BackgroundLeukemia is one of the most lethal cancers worldwide and represents the sixth-leading cause of cancer deaths. The results of leukemia treatment have not been as positive as desired, and recurrence is common. PurposeThus, there is an urgent requirement for the development of new therapeutic drugs. Salvia multicaulis (Bardakosh) is a widespread species that contains multiple phytochemical components with anti-cancer activities. MethodsWe isolated and characterized the major diterpene candesalvone B methyl ester from S. multicaulis and investigated its action as a cytotoxic agent towards sensitive and drug-resistant leukemia cells by the resazurin reduction assay. Additionally, the targeted genes and the affected molecular mechanisms attributed to the potent cytotoxic activities were discovered by transcriptome-wide mRNA expression profiling. The targets predicted to be regulated by candesalvone B methyl ester in each cell line were confirmed by qRT-PCR, molecular docking, microscale thermophoresis, and western blotting. Moreover, cell cycle distribution and apoptosis were analyzed by flow cytometry. ResultsCandesalvone B methyl ester was cytotoxic with IC50 values of 20.95 ± 0.15 µM against CCRF-CEM cells and 4.13 ± 0.10 µM against multidrug-resistant CEM/ADR5000 leukemia cells. The pathway enrichment analysis disclosed that candesalvone B methyl ester could regulate the heat-shock response signaling pathway via targeting heat shock factor 1 (HSF1) in CCRF-CEM cells and ELOVL fatty acid elongase 5 (ELOVL5) controls the fatty acid metabolism pathway in CEM/ADR5000 cells. Microscale thermophoresis showed the binding of candesalvone B methyl ester with HSF1 and ELOVL5, confirming the results of molecular docking analysis. Down-regulation of both HSF1 and ELOVL5 by candesalvone B methyl ester as detected by both western blotting and RT-qPCR was related to the reversal of drug resistance in the leukemia cells. Furthermore, candesalvone B methyl ester increased the arrest in the sub-G1 phase of the cell cycle in a dose-dependent manner from 1.3 % to 32.3 % with concomitant induction of apoptosis up to 29.0 % in CCRF-CEM leukemic cells upon inhibition of HSF1. ConclusionCandesalvone B methyl ester isolated from S. multicaulis exerted cytotoxicity by affecting apoptosis, cell division, and modulation of expression levels of genes contributing to the heat stress signaling and fatty acid metabolism pathways that could relieve drug resistance of leukemia cells.

A complex and dynamic redox network regulates oxygen reduction at photosystem I in Arabidopsis.

Thiol-dependent redox regulation of enzyme activities plays a central role in regulating photosynthesis. Beside the regulation of metabolic pathways, alternative electron transport is subjected to thiol-dependent regulation. We investigated the regulation of O2 reduction at photosystem I. The level of O2 reduction in leaves and isolated thylakoid membranes depends on the photoperiod in which plants are grown. We used a set of Arabidopsis (Arabidopsis thaliana) mutant plants affected in the stromal, membrane and lumenal thiol network to study the redox protein partners involved in regulating O2 reduction. Light-dependent O2 reduction was determined in leaves and in thylakoids of plants grown in short day and long day conditions using a spin-trapping electron paramagnetic resonance (EPR) assay. In wild type samples from short day conditions, reactive oxygen species (ROS) generation was double that of samples from long day conditions, while this difference was abolished in several redoxin mutants. An in vitro reconstitution assay showed that thioredoxin m, NADPH-dependent reductase C and NADPH are required for high O2 reduction levels in thylakoids from plants grown in long day conditions. Using isolated photosystem I, we also showed that reduction of a photosystem I protein is responsible for the increase in O2 reduction. Furthermore, differences in the membrane localization of m-type thioredoxins and 2-Cys peroxiredoxin were detected between thylakoids of short day and long day plants. Overall, we propose a model of redox regulation of O2 reduction according to the reduction power of the stroma and the ability of different thiol-containing proteins to form a network of redox interactions.

Targeting the sympathetic nervous system with the selective imidazoline receptor agonist moxonidine for the management of hypertension: an international position statement.

Hypertension is often linked with metabolic risk factors that share common pathophysiological pathways. Despite wide-spread availability of multiple drug classes, optimal blood pressure (BP) control remains challenging. Increased central sympathetic outflow is frequently neglected as a critical regulator of both circulatory and metabolic pathways and often remains unopposed therapeutically. Selective imidazoline receptor agonists (SIRAs) effectively reduce BP with a favorable side effect profile compared with older centrally acting antihypertensive drugs. Hard outcome data in hypertension, such as prevention of stroke, heart and kidney diseases, are not available with SIRAs. However, in direct comparisons, SIRAs were as effective as angiotensin-converting enzyme inhibitors, β-blockers, calcium channel blockers, and diuretics in lowering BP. Other beneficial effects on metabolic parameters in hypertensive patients with concomitant overweight and obesity have been documented with SIRAs. Here we review the existing evidence on the safety and efficacy of moxonidine, a widely available SIRA, compared with common antihypertensive agents and provide a consensus position statement based on inputs from 12 experts from Europe and Australia on SIRAs in hypertension management.

Development of a monoclonal antibody to study MARCH6, an E3 ligase that regulates proteins that control lipid homeostasis

Membrane-associated ring-CH-type finger 6 (MARCH6), also designated as TEB4 or RNF176, is an E3 ligase that is embedded in membranes of the endoplasmic reticulum where it ubiquitinates many substrate proteins to consign them to proteasome-mediated degradation. In recent years, MARCH6 has been identified as a key regulator of several metabolic pathways, including cholesterol and lipid droplet homeostasis, protein quality control, ferroptosis, and tumorigenesis. Despite its importance, there are currently no specific antibodies to detect and monitor MARCH6 levels in cultured cells and animals. Here, we address this deficiency by generating a monoclonal antibody that specifically detects MARCH6 in cultured cells of insect, mouse, hamster, and human origin, as well as in mouse tissues, with minimal cross-reactivity against other proteins. We then used this antibody to assess two properties of MARCH6. First, analysis of mouse tissues with this antibody revealed that the liver contained the highest levels of March6. Second, analysis of five different cell lines with this antibody showed that endogenous levels of MARCH6 are unchanged as the cellular content of cholesterol is varied. This reagent promises to be a useful tool in interrogating additional signaling roles of MARCH6.

Sex-specific molecular signature of mouse podocytes in homeostasis and in response to pharmacological challenge with rapamycin

BackgroundSex differences exist in the prevalence and progression of major glomerular diseases. Podocytes are the essential cell-type in the kidney which maintain the physiological blood-urine barrier, and pathological changes in podocyte homeostasis are critical accelerators of impairment of kidney function. However, sex-specific molecular signatures of podocytes under physiological and stress conditions remain unknown. This work aimed at identifying sexual dimorphic molecular signatures of podocytes under physiological condition and pharmacologically challenged homeostasis with mechanistic target of rapamycin (mTOR) inhibition. mTOR is a crucial regulator involved in a variety of physiological and pathological stress responses in the kidney and inhibition of this pathway may therefore serve as a general stress challenger to get fundamental insights into sex differences in podocytes.MethodsThe genomic ROSAmT/mG-NPHS2 Cre mouse model was used which allows obtaining highly pure podocyte fractions for cell-specific molecular analyses, and vehicle or pharmacologic treatment with the mTOR inhibitor rapamycin was performed for 3 weeks. Subsequently, deep RNA sequencing and proteomics were performed of the isolated podocytes to identify intrinsic sex differences. Studies were supplemented with metabolomics from kidney cortex tissues.ResultsAlthough kidney function and morphology remained normal in all experimental groups, RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways. Interestingly, rapamycin abolished prominent sex-specific clustering of podocyte gene expression and induced major changes only in male transcriptome. Several sex-biased transcription factors could be identified as possible upstream regulators of these sexually dimorphic responses. Concordant to transcriptomics, metabolomic changes were more prominent in males. Remarkably, high number of previously reported kidney disease genes showed intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition.ConclusionsOur results highlight remarkable intrinsic sex-differences and sex-specific response patterns towards pharmacological challenged podocyte homeostasis which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. This work provides rationale and an in-depth database for novel targets to be tested in specific kidney disease models to advance with sex-specific treatment strategies.

Abstract C022: Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in pancreatic ductal adenocarcinoma

Abstract Alteration of essential metabolic pathways is a major mechanism by which oncogenic KRAS promotes tumor development and growth in pancreatic ductal adenocarcinoma (PDAC). KRAS- driven PDAC is dependent on nutrient scavenging pathways, including macropinocytosis and autophagy to fuel the metabolic demand of rapid proliferation. Thus, these metabolic processes are attractive targets for the development of treatments for PDAC. Acute KRAS loss results in downregulation of macropinocytosis in PDAC. Additionally, our lab demonstrated that KRAS loss or inhibition of ERK MAPK signaling decreased glucose consumption and glycolysis but increased autophagy, thereby enhancing dependency on autophagy for survival and growth. Accordingly, dual ERK MAPK and autophagy inhibition (via chloroquine) synergistically enhanced anti-tumor efficacy in PDAC. However, early clinical data has demonstrated that resistance to this treatment arises over time through unknown mechanisms. We observed that both autophagy induction and macropinocytosis downregulation are transient following RAS, MEK, or ERK inhibition—with autophagic and macropinocytic activity returning to or surpassing basal levels after within two weeks of treatment. Furthermore, we found that prolonged pharmacological inhibition of RAS, MEK, or ERK pathway consistently resulted in significant upregulation of macropinocytosis. We propose that prolonged MAPK inhibition upregulates macropinocytosis over time, consequently abrogating dependency on autophagy and therefore reducing sensitivity to autophagy inhibition. Furthermore, we found that chloroquine, which is commonly thought of as a lysosomotropic drug, does not prevent degradation of proteins engulfed via macropinocytosis. Together our data suggests that upregulation of macropinocytosis may mediate resistance to the combination of ERK MAPK inhibition and chloroquine. With the recent advance in development RAS inhibitors, the resistance mechanisms by which PDAC can overcome RAS inhibitor treatment is a major focus of the field. Given the dependence of PDAC on macropinocytic uptake for tumorigenic growth, it is imperative to understand how long-term treatment with RAS inhibitors regulate macropinocytosis. We demonstrated that PDAC cells with acquired resistance to RAS inhibition exhibit a 2- to 10-fold increase in macropinocytic uptake. We found that upregulated macropinocytosis following long-term RAS or MAPK inhibition is accompanied by increased albumin uptake and sensitivity to albumin-bound paclitaxel (nab-paclitaxel), a therapeutic that is included in the current standard of care for PDAC patients. These results suggest that RAS inhibitor pretreatment or concurrent treatment with nab-paclitaxel may represent a strategy to improve this current PDAC treatment. We conclude that a more complete understanding of the regulation of essential metabolic pathways following both acute and sustained RAS inhibition will better inform future RAS inhibitor combination therapies. Citation Format: Ryan Robb, Sarah Ackermann, Khalilah Taylor, Runying Yang. Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C022.

Post-Translational Modifications to Cysteine Residues in Plant Proteins and Their Impact on the Regulation of Metabolism and Signal Transduction.

Cys is one of the least abundant amino acids in proteins. However, it is often highly conserved and is usually found in important structural and functional regions of proteins. Its unique chemical properties allow it to undergo several post-translational modifications, many of which are mediated by reactive oxygen, nitrogen, sulfur, or carbonyl species. Thus, in addition to their role in catalysis, protein stability, and metal binding, Cys residues are crucial for the redox regulation of metabolism and signal transduction. In this review, we discuss Cys post-translational modifications (PTMs) and their role in plant metabolism and signal transduction. These modifications include the oxidation of the thiol group (S-sulfenylation, S-sulfinylation and S-sulfonylation), the formation of disulfide bridges, S-glutathionylation, persulfidation, S-cyanylation S-nitrosation, S-carbonylation, S-acylation, prenylation, CoAlation, and the formation of thiohemiacetal. For each of these PTMs, we discuss the origin of the modifier, the mechanisms involved in PTM, and their reversibility. Examples of the involvement of Cys PTMs in the modulation of protein structure, function, stability, and localization are presented to highlight their importance in the regulation of plant metabolic and signaling pathways.

The Efficacy of Vitamins in the Prevention and Treatment of Cardiovascular Disease.

Vitamins are known to affect the regulation of several biochemical and metabolic pathways that influence cellular function. Adequate amounts of both hydrophilic and lipophilic vitamins are required for maintaining normal cardiac and vascular function, but their deficiencies can contribute to cardiovascular abnormalities. In this regard, a deficiency in the lipophilic vitamins, such as vitamins A, D, and E, as well as in the hydrophilic vitamins, such as vitamin C and B, has been associated with suboptimal cardiovascular function, whereas additional intakes have been suggested to reduce the risk of atherosclerosis, hypertension, ischemic heart disease, arrhythmias, and heart failure. Here, we have attempted to describe the association between low vitamin status and cardiovascular disease, and to offer a discussion on the efficacy of vitamins. While there are inconsistencies in the impact of a deficiency in vitamins on the development of cardiovascular disease and the benefits associated with supplementation, this review proposes that specific vitamins may contribute to the prevention of cardiovascular disease in individuals at risk rather than serve as an adjunct therapy.

Interplanting potato with grapes improved yield and soil nutrients by optimizing the interactions of soil microorganisms and metabolites.

Interplanting crops is the best method to grow crops synergistically for better utilization of land and agro-resources. Grape (Vitis vinifera) and potato (Solanum tuberosum L.) have highly efficient agricultural planting systems in China, however, how soil physicochemical properties and soil microbial communities and metabolites affect the output of grape-potato interplanting remained unknown. In this study, we employed three planting patterns (CK: grape monocropping; YY: grape interplanted with potato (variety 'Favorita'); LS: grape interplanted with potato (variety 'Longshu7')) at two experimental sites i.e., the Huizhou (2022) site and the Qingyuan site (2023). The grape variety for all planting patterns was 'Sunshine Rose'. Soil samples (top 0-20 cm) at both sites were collected to observe the diversity of bacterial communities and soil metabolites. Our findings revealed that, compared with monocropping, the interplanted systems resulted in higher concentrations of total nitrogen, available phosphorus, and available potassium and enhanced the activities of acid phosphatase, urease, and protease. The potato root exudates also altered the relative abundance of Bacillus, Kaistobacter, and Streptomyces in the rhizosphere. Among the soil metabolites, lipids and organic acids showed the most significant changes. Notably, 13-L-hydroperoxylinoleic acid is the key differentially abundant metabolite involved in the regulation of linoleic acid metabolism pathways. The association analyses of the metabolome, microbiome, and soil physicochemical properties revealed that the interactions of microbes and metabolites resulted in differences in the soil nutrient content, whereas the interactions of 13-L-hydroperoxylinoleic acid and Firmicutes improved the soil nutrient levels and bacterial composition in the interplanting systems. In summary, our findings demonstrated that intercropping grapes with potato 'Favorita' was better with respect to improving soil nutrients, soil enzyme activity, the diversity of soil bacteria, and soil metabolites without causing adverse impacts on grape yield. Overall, this study explained the physiological mechanisms by which soil microorganisms and metabolites promote potato growth in grape interplanting and provided new perspectives for the utilization of soil resources in vineyards.

Multiple Mechanisms of Action of Sulfodyne®, a Natural Antioxidant, against Pathogenic Effects of SARS-CoV-2 Infection.

Few therapeutic options are available to treat COVID-19. The KEAP1/NRF2 pathway, the major redox-responsive pathway, has emerged as a potential therapeutic target for COVID-19 as it regulates redox homeostasis and inflammation that are altered during SARS-CoV-2 infection. Here, we characterized the effects of NRF2-agonist Sulfodyne®, a stabilized natural Sulforaphane, in cellular and animal models of SARS-CoV-2 infection. In pulmonary or colonic epithelial cell lines, Sulfodyne® elicited a more efficient inhibition of SARS-CoV-2 replication than NRF2-agonists DMF and CDDO. This antiviral activity was not dependent on NRF2 but was associated with the regulation of several metabolic pathways, including the inhibition of ER stress and mTOR signaling, which are activated during SARS-CoV-2 infection. Sulfodyne® also decreased SARS-CoV-2 mediated inflammatory responses by inhibiting the delayed induction of IFNB1 and type I IFN-stimulated genes in infected epithelial cell lines and by reducing the activation of human by-stander monocytes recruited after SARS-CoV-2 infection. In K18-hACE2 mice infected with SARS-CoV-2, Sulfodyne® treatment reduced both early lung viral load and disease severity by fine-tuning IFN-beta levels. Altogether, these results provide evidence for multiple mechanisms that underlie the antiviral and anti-inflammatory activities of Sulfodyne® and pinpoint Sulfodyne® as a potent therapeutic agent against pathogenic effects of SARS-CoV-2 infection.

Sex-dependent regulation of retinal pigment epithelium and retinal function by Pgc-1α.

Age-related macular degeneration (AMD) is a major cause of blindness that affects people over 60. While aging is the prominent factor in AMD, studies have reported a higher prevalence of AMD in women compared to age-matched men. Higher levels of the innate immune response's effector proteins complement factor B and factor I were also found in females compared to males in intermediate AMD. However, the mechanisms underlying these differences remain elusive. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is a key regulator of mitochondrial biogenesis and metabolic pathways. Previously, we showed that Pgc-1α repression and high-fat diet induce drastic AMD-like phenotypes in mice. Our recent data revealed that Pgc-1α repression alone can also induce retinal pigment epithelium (RPE) and retinal dysfunction in mice, and its inhibition in vitro results in lipid droplet accumulation in human RPE. Whether sex is a contributing factor in these phenotypes remains to be elucidated. Using electroretinography, we demonstrate that sex could influence RPE function during aging independent of Pgc-1α in wild-type (WT) mice. We further show that Pgc-1α repression exacerbates RPE and retinal dysfunction in females compared to aged-match male mice. Gene expression analyses revealed that Pgc-1α differentially regulates genes related to antioxidant enzymes and mitochondrial dynamics in males and females. RPE flat mounts immunolabeled with TOMM20 and DRP1 indicated a sex-dependent role for Pgc-1α in regulating mitochondrial fission. Analyses of mitochondrial network morphology suggested sex-dependent effects of Pgc-1α repression on mitochondrial dynamics. Together, our study demonstrates that inhibition of Pgc-1α induces a sex-dependent decline in RPE and retinal function in mice. These observations on the sex-dependent regulation of RPE and retinal function could offer novel insights into targeted therapeutic approaches for age-related RPE and retinal degeneration.

Molecular mechanisms of natural antifreeze phenomena and their application in cryopreservation.

Cryopreservation presents a critical challenge due to cryo-damage, such as crystallization and osmotic imbalances that compromise the integrity of biological tissues and cells. In contrast, various organisms in nature exhibit remarkable freezing tolerance, leveraging complex molecular mechanisms to survive extreme cold. This review explores the adaptive strategies of freeze-tolerant species, including the regulation of specific genes, proteins, and metabolic pathways, to enhance survival in low-temperature environments. We then discuss recent advancements in cryopreservation technologies that aim to mimic these natural phenomena to preserve cellular and tissue integrity. Special focus is given to the roles of glucose metabolism, microRNA expression, and cryoprotective protein modulation in improving cryopreservation outcomes. The insights gained from studying natural antifreeze mechanisms offer promising directions for advancing cryopreservation techniques, with potential applications in medical, agricultural, and conservation fields. Future research should aim to further elucidate these molecular mechanisms to develop more effective and reliable cryopreservation methods.

Transcriptome Analysis of Multiple Plant Parts in the Woody Oil Tree Camellia drupifera Loureiro

Camellia drupifera is mainly used in forestry for its high-value industrial products; however, limited information is available on its transcriptome. This study aimed to construct a full-length transcriptome sequence based on the PacBio sequencing platform for various plant parts of C. drupifera, including flower buds, leaves, leaf buds, branches, the pericarp, and seed kernels. The transcriptomes were annotated with 23,207 genes, with 58 subgroups in the GO classification. The KEGG database revealed 10,407 genes involved in the metabolic pathway analysis, with 68,192 coding sequences, 3352 TF families, 48,541 SSRs, 1421 IncRNAs, and 2625 variable shears predicted. The transcriptomes of different parts were analyzed and compared. The majority of differentially expressed genes (DEGs) were found between the pericarp and seed kernels, followed by leaves and the pericarp with 5662 DEGs, and flower buds and leaf buds with 1616 DEGs. GO and KEGG enrichment analyses showed that KEGG differential genes were significant in microbial metabolism, carbon metabolism, and other functions. The data annotation and analysis of the full-length transcriptome and the comparative analysis between different plant parts provided a theoretical basis for studying gene function, metabolic pathway regulation, and gene expression analysis in KEGG.

Comparative genomics reveals the molecular mechanisms of two drought-tolerant forages adapted to extreme environments in the northwest of China

Carex breviculmis, is a perennial herb with effective resistance of salt and drought stress in alkaline habitats and is widely used for forage production and turf management. We present its 469 Mb chromosome-level genome with 45,262 annotated protein-coding genes. The genome had 50.15 % repetitive sequences, mainly driven by TEs insertion in intronic regions. Phylogenetic analysis reveals C. breviculmis divergence from C. littledalei at 6.61 Mya, shared a WGD event with Ananas comosus about 103.12 Mya. Tandem duplicate genes rapidly expanded in sugar metabolism, amino acid synthesis, and phenylpropanoid biosynthesis. We identified 125/128 positively selected genes and 277/309 rapidly evolving genes in C. breviculmis and Achnatherum splendens, respectively, and analysis found that they were co-enriched in pathways like amino acid biosynthesis and fructose and mannose metabolism. In addition, a total of 51 convergent evolutionary genes were identified by two method, including stress-responsive genes like ACBP4, CIPK1, HMA5, OTC, SMT3, SPP2, VDE, VFB1, and VLN2. Finally, we reconstructed the sucrose metabolic pathways in C. breviculmis and A. splendens, and found that the major structural genes responsible for the regulation of sucrose metabolic pathways were significantly amplified. This study lays the foundation for future research on their environmental adaptations, and potential genetic breeding.

Application of genetic code expansion to regulate the synthesis of poly(lactate-co-3-hydroxybutyrate) in Escherichia coli

Genetic codon expansion has the potential to introduce a variety of unnatural amino acids to specific sites within target proteins. In this study, genetic codon expansion was employed to regulate the enzyme expression in metabolic pathways. Firstly, a purple protein from Actinia tenebrosa was selected as the candidate to be engineered. Bringing in UAG stop codon caused premature termination of translation, while expressing orthogonal aminoacyl-tRNA synthetase and tRNA from Methanococcus jannaschii restored translation at UAG site. However, leakage expression was observed without addition of unnatural amino acids, still it can be decreased by increasing numbers of UAG mutations. Subsequently, poly(lactate-co-3-hydroxyburyrate) [P(LA-3HB)] biosynthesis pathway was constructed in Escherichia coli, and propionyl-CoA transferase was mutated to harboring one or two more stop codons. With genetic codon expansion tools, the function of propionyl-CoA transferase was restored, promoting cells to synthesize P(LA-3HB) copolymer. Moreover, the lactate monomer content was regulated ranging from 0 to 33.42 mol% by altering the addition time of inducers. Finally, the strain accumulated 27.09 g/L P(25.1 mol% LA-3HB) in 5-L bioreactor cultivation. This is the first report on metabolic engineering of polyhydroxyalkanoate biosynthesis through genetic codon expansion and would provide helpful strategies to achieve dynamic regulation of multiple metabolic pathways.