Insights into the mechanism of action of pterostilbene against influenza A virus-induced acute lung injury.

BACKGROUND: Severe respiratory system illness caused by influenza A virus infection is associated with excessive inflammation and abnormal apoptosis in alveolar epithelial cells (AEC). However, there are limited therapeutic options for influenza-associated lung inflammation and apoptosis. Pterostilbene (PTE, trans-3,5-dimethoxy-4-hydroxystilbene) is a dimethylated analog of resveratrol that has been reported to limit influenza A virus infection by promoting antiviral innate immunity, but has not been studied for its protective effects on virus-associated inflammation and injury in AEC. PURPOSE: Our study aimed to investigate the protective effects and underlying mechanisms of PTE in modulating inflammation and apoptosis in AEC, as well as its effects on macrophage polarization during influenza virus infection. STUDY DESIGN AND METHODS: A murine model of influenza A virus-mediated acute lung injury was established by intranasal inoculation with 5LD50 of mouse-adapted H1N1 viruses. Hematoxylin and eosin staining, immunofluorescence, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, Luminex and flow cytometry were performed. RESULTS: PTE effectively mitigated lung histopathological changes and injury induced by H1N1 viruses in vivo. These beneficial effects of PTE were attributed to the suppression of inflammation and apoptosis in AEC, as well as the modulation of M1 macrophage polarization. Mechanistic investigations revealed that PTE activated the phosphorylated AMP-activated protein kinase alpha (P-AMPKα)/sirtui1 (Sirt1)/PPARγ coactivator 1-alpha (PGC1α) signal axis, leading to the inhibition of nuclear factor kappa-B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling induced by H1N1 viruses, thereby attenuating inflammation and apoptosis in AEC. PTE also forced activation of the P-AMPKα/Sirt1/PGC1α signal axis in RAW264.7 cells, counteracting the activation of phosphorylated signal transducer and activator of transcription 1 (P-STAT1) induced by H1N1 viruses and the augment of P-STAT1 activation in RAW264.7 cells with interferon-gamma (IFN-γ) pretreatment before viral infection, thereby reducing H1N1 virus-mediated M1 macrophage polarization as well as the enhancement of macrophages into M1 phenotypes elicited by IFN-γ pretreatment. Additionally, the promotion of the transition of macrophages towards the M2 phenotype by PTE was also related to activation of the P-AMPKα/Sirt1/PGC1α signal axis. Moreover, co-culturing non-infected AEC with H1N1 virus-infected RAW264.7 cells in the presence of PTE inhibited apoptosis and tight junction disruption, which was attributed to the suppression of pro-inflammatory mediators and pro-apoptotic factors in an AMPKα-dependent manner. CONCLUSION: In conclusion, our findings suggest that PTE may serve as a promising novel therapeutic option for treating influenza-associated lung injury. Its ability to suppress inflammation and apoptosis in AEC, modulate macrophage polarization, and preserve alveolar epithelial cell integrity highlights its potential as a therapeutic agent in influenza diseases.

Effects and mechanisms of glyphosate as phosphorus nutrient on element stoichiometry and metabolism in the diatom Phaeodactylum tricornutum.

The ability to utilize dissolved organic phosphorus (DOP) gives phytoplankton competitive advantages in P-limited environments. Our previous research indicates that the diatom Phaeodactylum tricornutum could grow on glyphosate, a DOP with carbon-phosphorus (C-P) bond and an herbicide, as sole P source. However, direct evidence and mechanism of glyphosate utilization are still lacking. In this study, using physiological and isotopic analysis, combined with transcriptomic profiling, we demonstrated the uptake of glyphosate by P. tricornutum and revealed the candidate responsible genes. Our data showed a low efficiency of glyphosate utilization by P. tricornutum, suggesting that glyphosate utilization costs energy and that the alga possessed an herbicide-resistant type of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase. Compared to the P-limited cultures, the glyphosate-grown P. tricornutum cells up-regulated genes involved in DNA replication, cell growth, transcription, translation, carbon metabolism, and many genes encoding antioxidants. Additionally, cellular C and silicon (Si) increased remarkably while cellular nitrogen (N) declined in the glyphosate-grown P. tricornutum, leading to higher Si:C and Si:N ratios, which corresponded to the up-regulation of genes involved in the C metabolism and Si uptake and the down-regulation of those encoding N uptake. This has the potential to enhance C and Si export to the deep sea when P is limited but phosphonate is available. In sum, our study documented how P. tricornutum could utilize the herbicide glyphosate as P nutrient and how glyphosate utilization may affect the element content and stoichiometry in this diatom, which have important ecological implications in the future ocean.IMPORTANCEGlyphosate is the most widely used herbicide in the world and could be utilized as phosphorus (P) source by some bacteria. Our study first revealed that glyphosate could be transported into Phaeodactylum tricornutum cells for utilization and identified putative genes responsible for glyphosate uptake. This uncovers an alternative strategy of phytoplankton to cope with P deficiency considering phosphonate accounts for about 25% of the total dissolved organic phosphorus (DOP) in the ocean. Additionally, accumulation of carbon (C) and silicon (Si), as well as elevation of Si:C ratio in P. tricornutum cells when grown on glyphosate indicates glyphosate as the source of P nutrient has the potential to result in more C and Si export into the deep ocean. This, along with the differential ability to utilize glyphosate among different species, glyphosate supply in dissolved inorganic phosphorus (DIP)-depleted ecosystems may cause changes in phytoplankton community structure. These insights have implications in evaluating the effects of human activities (use of Roundup) and climate change (potentially reducing DIP supply in sunlit layer) on phytoplankton in the future ocean.

Alkaline Phosphatase PhoD Mutation Induces Fatty Acid and Long-Chain Polyunsaturated Fatty Acid (LC-PUFA)-Bound Phospholipid Production in the Model Diatom Phaeodactylum tricornutum.

With rapid growth and high lipid contents, microalgae have become promising environmentally friendly candidates for renewable biodiesel and health supplements in our era of global warming and energy depletion. Various pathways have been explored to enhance algal lipid production, especially gene editing. Previously, we found that the functional loss of PhoD-type alkaline phosphatase (AP), a phosphorus-stress indicator in phytoplankton, could lead to increased lipid contents in the model diatom Phaeodactylum tricornutum, but how the AP mutation may change lipid composition remains unexplored. This study addresses the gap in the research and investigates the effects of PhoD-type AP mutation on the lipid composition and metabolic regulation in P. tricornutum using transcriptomic and lipidomic analyses. We observed significantly modified lipid composition and elevated production of fatty acids, lysophosphatidylcholine, lysophosphatidylethanolamine, ceramide, phosphatidylinositol bisphosphate, and monogalactosylmonoacylglycerol after PhoD_45757 mutation. Meanwhile, genes involved in fatty acid biosynthesis were upregulated in mutant cells. Moreover, the mutant exhibited increased contents of ω-3 long-chain polyunsaturated fatty acid (LC-PUFA)-bound phospholipids, indicating that PhoD_45757 mutation could improve the potential bioavailability of PUFAs. Our findings indicate that AP mutation could influence cellular lipid synthesis and probably redirect carbon toward lipid production and further demonstrate that AP mutation is a promising approach for the development of high-value microalgal strains for biomedical and other applications.

Phosphorus nutrition strategies in a Symbiodiniacean species: Implications in coral-alga symbiosis facing increasing phosphorus deficiency in future warmer oceans.

Coral reefs thrive in the oligotrophic ocean and rely on symbiotic algae to acquire nutrients. Global warming is projected to intensify surface ocean nutrient deficiency and anthropogenic discharge of wastes with high nitrogen (N): phosphorus (P) ratios can exacerbate P nutrient limitation. However, our understanding on how symbiotic algae cope with P deficiency is limited. Here, we investigated the responses of a coral symbiotic species of Symbiodiniaceae, Cladocopium goreaui, to P-limitation by examining its physiological performance and transcriptomic profile. Under P stress, C. goreaui exhibited decreases in algal growth, photosynthetic efficiency, and cellular P content but enhancement in carbon fixation, N assimilation, N:P ratio, and energy metabolism, with downregulated expression of carbohydrate exporter genes. Besides, C. goreaui showed flexible mechanisms of utilizing different dissolved organic phosphorus to relieve P deficiency. When provided glycerol phosphate, C. goreaui hydrolyzed it extracellularly to produce phosphate for uptake. When grown on phytate, in contrast, C. goreaui upregulated the endocytosis pathway while no dissolved inorganic phosphorus was released into the medium, suggesting that phytate was transported into the cell, potentially via the endocytosis pathway. This study sheds light on the survival strategies of C. goreaui and potential weakening of its role as an organic carbon supplier in P-limited environments, underscoring the importance of more systematic investigation on future projections of such effects.

Functional differentiation and complementation of alkaline phosphatases and choreography of DOP scavenging in a marine diatom.

Facing phosphate deficiency, phytoplankton use alkaline phosphatase (AP) to scavenge dissolved organophosphate (DOP). AP is a multitype (e.g., PhoA, PhoD) family of hydrolases and is known as a promiscuous enzyme with broad DOP substrate compatibility. Yet, whether the multiple types differentiate on substrates and collaborate to provide physiological flexibility remain elusive. Here we identify PhoA and PhoDs and document the functional differentiation between PhoA and a PhoD (PhoD_45757) in Phaeodactylum tricornutum. CRISPR/Cas9-based mutations and physiological analyses reveal that (1) PhoA is a secreted enzyme and contributes the majority of total AP activity whereas PhoD_45757 is intracellular and contributes a minor fraction of the total AP activity, (2) AP gene expression compensates for each other after one is disrupted, (3) the DOP→PhoA→phosphate_uptake and the DOP_uptake→PhoD→phosphate pathways function interchangeably for some DOP substrates. These findings shed light on the underpinning of AP's multiformity and have important implications in phytoplankton phosphorus-nutrient niche differentiation, physiological plasticity, and competitive strategy.

Phytate as a Phosphorus Nutrient with Impacts on Iron Stress-Related Gene Expression for Phytoplankton: Insights from the Diatom Phaeodactylum tricornutum.

Phytoplankton have evolved a capability to acquire phosphorus (P) from dissolved organic phosphorus (DOP) since the preferred form, dissolved inorganic phosphate (DIP, or Pi), is often limited in parts of the ocean. Phytic acid (PA) is abundantly synthesized in plants and rich in excreta of animals, potentially enriching the DOP pool in coastal oceans. However, whether and how PA can be used by phytoplankton are poorly understood. Here, we investigated PA utilization and underlying metabolic pathways in the diatom model Phaeodactylum tricornutum. The physiological results showed that P. tricornutum could utilize PA as a sole source of P nutrient to support growth. Meanwhile, the replacement of PA for DIP also caused changes in multiple cellular processes, such as inositol phosphate metabolism, photosynthesis, and signal transduction. These results suggest that PA is bioavailable to P. tricornutum and can directly participate in the metabolic pathways of PA-grown cells. However, our data showed that the utilization of PA was markedly less efficient than that of DIP, and PA-grown cells exhibited P and iron (Fe) nutrient stress signals. Implicated in these findings is the potential of complicated responses of phytoplankton to an ambient DOP species, which calls for more systematic investigation. IMPORTANCE PA is abundant in plants and cannot be digested by nonruminant animals. Hence, it is potentially a significant component of the DOP pool in coastal waters. Despite this potential importance, there is little information about its bioavailability to phytoplankton as a source of P nutrient and the molecular mechanisms involved. In this study, we found that part of PA could be utilized by the diatom P. tricornutum to support growth, and another portion of PA can act as a substrate directly participating in various metabolism pathways and cellular processes. However, our physiological and transcriptomic data show that PA-grown cells still exhibited signs of P stress and potential Fe stress. These results have significant implications in phytoplankton P nutrient ecology and provide a novel insight into multifaceted impacts of DOP utilization on phytoplankton nutrition and metabolism.

SPX-related genes regulate phosphorus homeostasis in the marine phytoplankton, Phaeodactylum tricornutum.

Phosphorus (P) is an essential nutrient for marine phytoplankton. Maintaining intracellular P homeostasis against environmental P variability is critical for phytoplankton, but how they achieve this is poorly understood. Here we identify a SPX gene and investigate its role in Phaeodactylum tricornutum. SPX knockout led to significant increases in the expression of phosphate transporters, alkaline phosphatases (the P acquisition machinery) and phospholipid hydrolases (a mechanism to reduce P demand). These demonstrate that SPX is a negative regulator of both P uptake and P-stress responses. Furthermore, we show that SPX regulation of P uptake and metabolism involves a phosphate starvation response regulator (PHR) as an intermediate. Additionally, we find the SPX related genes exist and operate across the phytoplankton phylogenetic spectrum and in the global oceans, indicating its universal importance in marine phytoplankton. This study lays a foundation for better understanding phytoplankton adaptation to P variability in the future changing oceans.

Roles of Alkaline Phosphatase PhoA in Algal Metabolic Regulation under Phosphorus-replete Conditions.

Alkaline phosphatase (AP) in plants and algae is known to hydrolyze dissolved organophosphate (DOP) in order to obtain phosphorus when the preferred dissolved inorganic phosphorus (DIP) is present in limited supply. By conducting comparative analyses of physiologies and transcriptomes on a mutant of PhoA type AP (mPhoA) and wild type (WT) of the marine diatom Phaeodactylum tricornutum CCAP 1055/1 under P-replete and P-depleted conditions, we document other roles of this gene than DOP scavenging. PhoA mutation created by CRISPR/Cas9 diminished its DOP hydrolase activity but led to significant increases in cellular contents of pigment, carbon, and lipids, photosynthetic rate, growth rate, and the transcriptional levels of their corresponding metabolic pathways. All the results in concert indicate that besides P-nutrient scavenging under DIP deficiency, AP also functions, under the P-replete condition, to constrain pigment biosynthesis, photosynthesis, fatty acid biosynthesis, and cell division. These functions have important implications in maintaining metabolic homeostasis and preventing premature cell division.