Phosphatidylethanolamines link ferroptosis and autophagy during appressorium formation of rice blast fungus.

A cell death pathway, ferroptosis, occurs in conidial cells and is critical for formation and function of the infection structure, the appressorium, in the rice blast fungus Magnaporthe oryzae. In this study, we identified an orthologous lysophosphatidic acid acyltransferase (Lpaat) acting at upstream of phosphatidylethanolamines (PEs) biosynthesis and which is required for such fungal ferroptosis and pathogenicity. Two PE species, DOPE and SLPE, that depend on Lpaat function for production were sufficient for induction of lipid peroxidation and the consequent ferroptosis, thus positively regulating fungal pathogenicity. On the other hand, both DOPE and SLPE positively regulated autophagy. Loss of the LPAAT gene led to a decrease in the lipidated form of the autophagy protein Atg8, which is probably responsible for the autophagy defect of the lpaatΔ mutant. GFP-Lpaat was mostly localized on the membrane of lipid droplets (LDs) that were stained by the fluorescent dye monodansylpentane (MDH), suggesting that LDs serve as a source of lipids for membrane PE biosynthesis and probably as a membrane source of autophagosome. Overall, our results reveal novel intracellular membrane-bound organelle dynamics based on Lpaat-mediated lipid metabolism, providing a temporal and spatial link of ferroptosis and autophagy.

The Neuroprotective Flavonoids Sterubin and Fisetin Maintain Mitochondrial Health under Oxytotic/Ferroptotic Stress and Improve Bioenergetic Efficiency in HT22 Neuronal Cells.

The global increase in the aging population has led to a rise in many age-related diseases with continuing unmet therapeutic needs. Research into the molecular mechanisms underlying both aging and neurodegeneration has identified promising therapeutic targets, such as the oxytosis/ferroptosis cell death pathway, in which mitochondrial dysfunction plays a critical role. This study focused on sterubin and fisetin, two flavonoids from the natural pharmacopeia previously identified as strong inhibitors of the oxytosis/ferroptosis pathway. Here, we investigated the effects of the compounds on the mitochondrial physiology in HT22 hippocampal nerve cells under oxytotic/ferroptotic stress. We show that the compounds can restore mitochondrial homeostasis at the level of redox regulation, calcium uptake, biogenesis, fusion/fission dynamics, and modulation of respiration, leading to the enhancement of bioenergetic efficiency. However, mitochondria are not required for the neuroprotective effects of sterubin and fisetin, highlighting their diverse homeostatic impacts. Sterubin and fisetin, thus, provide opportunities to expand drug development strategies for anti-oxytotic/ferroptotic agents and offer new perspectives on the intricate interplay between mitochondrial function, cellular stress, and the pathophysiology of aging and age-related neurodegenerative disorders.

Fragment-based drug discovery and biological evaluation of novel cannabinol-based inhibitors of oxytosis/ferroptosis for neurological disorders.

The oxytosis/ferroptosis regulated cell death pathway is an emerging field of research owing to its pathophysiological relevance to a wide range of neurological disorders, including Alzheimer's and Parkinson's diseases and traumatic brain injury. Developing novel neurotherapeutics to inhibit oxytosis/ferroptosis offers exciting opportunities for the treatment of these and other neurological diseases. Previously, we discovered cannabinol (CBN) as a unique, potent inhibitor of oxytosis/ferroptosis by targeting mitochondria and modulating their function in neuronal cells. To further elucidate which key pharmacophores and chemical space are essential to the beneficial effects of CBN, we herein introduce a fragment-based drug discovery strategy in conjunction with cell-based phenotypic screens using oxytosis/ferroptosis to determine the structure-activity relationship of CBN. The resulting information led to the development of four new CBN analogs, CP1-CP4, that not only preserve the sub-micromolar potency of neuroprotection and mitochondria-modulating activities seen with CBN in neuronal cell models but also have better druglike properties. Moreover, compared to CBN, the analog CP1 shows improved in vivo efficacy in the Drosophila model of mild traumatic brain injury. Together these studies identify the key molecular scaffolds of cannabinoids that contribute to neuroprotection against oxytosis/ferroptosis. They also highlight the advantageous approach of combining in vitro cell-based assays and rapid in vivo studies using Drosophila models for evaluating new therapeutic compounds.

Whole-genome sequencing of Fusarium spp. causing sugarcane root rot on both chewing cane and sugar-making cane.

Previously we isolated three Fusarium strains (a F. sacchari strain namely GXUF-1, and another two F. commune strains namely GXUF-2 and GXUF-3), and we verified that GXUF-3 was able to cause sugarcane root rot to the chewing cane cultivar Badila. Considering that Fusarium spp. are a group of widely distributed fungal pathogens, we tested whether these three Fusarium isolates were able to cause root rot to Badila as well as sugar-making cane cultivar (Guitang42), using a suitable inoculation method established based on infection assays using Badila. We found that the three Fusarium strains were able to cause root rot symptoms to both Badila and Guitang42, to different extents. To better investigate the potential pathogenicity mechanisms, we performed Illumina high-throughput sequencing and analyzed the whole genomic sequence data of these three Fusarium strains. The results reveal that the assembly sizes of the three Fusarium strains were in a range of 44.7-48.2 Mb, with G + C contents of 48.0-48.5%, and 14,154-15,175 coding genes. The coding genes were annotated by multiple public databases, and potential pathogenic genes were predicted using proprietary databases (such as PHI, DFVF, CAZy, etc.). Furthermore, based on evolutionary analysis of the coding sequence, we found that contraction and expansion of gene families occurred in the three Fusarium strains. Overall, our results suggest a potential risk that the root rot disease may occur to the sugar-making canes although it was initially spotted from fruit cane, and provide clues to understand the pathogenic mechanisms of Fusarium spp. causing sugarcane root rot.

Polyamine signaling communications play a key role in regulating the pathogenicity of Dickeya fangzhongdai.

IMPORTANCE: Dickeya fangzhongdai is a newly identified plant bacterial pathogen with a wide host range. A clear understanding of the cell-to-cell communication systems that modulate the bacterial virulence is of key importance for elucidating its pathogenic mechanisms and for disease control. In this study, we present evidence that putrescine molecules from the pathogen and host plants play an essential role in regulating the bacterial virulence. The significance of this study is in (i) demonstrating that putrescine signaling system regulates D. fangzhongdai virulence mainly through modulating the bacterial motility and production of PCWD enzymes, (ii) outlining the signaling and regulatory mechanisms with which putrescine signaling system modulates the above virulence traits, and (iii) validating that D. fangzhongdai could use both arginine and ornithine pathways to synthesize putrescine signals. To our knowledge, this is the first report to show that putrescine signaling system plays a key role in modulating the pathogenicity of D. fangzhongdai.

Characterization of the Arn lipopolysaccharide modification system essential for zeamine resistance unveils its new roles in Dickeya oryzae physiology and virulence.

The zeamines produced by Dickeya oryzae are potent polyamine antibiotics and phytotoxins that are essential for bacterial virulence. We recently showed that the RND efflux pump DesABC in D. oryzae confers partial resistance to zeamines. To fully elucidate the bacterial self-protection mechanisms, in this study we used transposon mutagenesis to identify the genes encoding proteins involved in zeamine resistance in D. oryzae EC1. This led to the identification of a seven-gene operon, arnEC1 , that encodes enzyme homologues associated with lipopolysaccharide modification. Deletion of the arnEC1 genes in strain EC1 compromised its zeamine resistance 8- to 16-fold. Further deletion of the des gene in the arnEC1 mutant background reduced zeamine resistance to a level similar to that of the zeamine-sensitive Escherichia coli DH5α. Intriguingly, the arnEC1 mutants showed varied bacterial virulence on rice, potato, and Chinese cabbage. Further analyses demonstrated that ArnBCATEC1 are involved in maintenance of the bacterial nonmucoid morphotype by repressing the expression of capsular polysaccharide genes and that ArnBEC1 is a bacterial virulence determinant, influencing transcriptional expression of over 650 genes and playing a key role in modulating bacterial motility and virulence. Taken together, these findings decipher a novel zeamine resistance mechanism in D. oryzae and document new roles of the Arn enzymes in modulation of bacterial physiology and virulence.

Gram-negative bacteria resist antimicrobial agents by a DzrR-mediated envelope stress response.

BACKGROUND: Envelope stress responses (ESRs) are critical for adaptive resistance of Gram-negative bacteria to envelope-targeting antimicrobial agents. However, ESRs are poorly defined in a large number of well-known plant and human pathogens. Dickeya oryzae can withstand a high level of self-produced envelope-targeting antimicrobial agents zeamines through a zeamine-stimulated RND efflux pump DesABC. Here, we unraveled the mechanism of D. oryzae response to zeamines and determined the distribution and function of this novel ESR in a variety of important plant and human pathogens. RESULTS: In this study, we documented that a two-component system regulator DzrR of D. oryzae EC1 mediates ESR in the presence of envelope-targeting antimicrobial agents. DzrR was found modulating bacterial response and resistance to zeamines through inducing the expression of RND efflux pump DesABC, which is likely independent on DzrR phosphorylation. In addition, DzrR could also mediate bacterial responses to structurally divergent envelope-targeting antimicrobial agents, including chlorhexidine and chlorpromazine. Significantly, the DzrR-mediated response was independent on the five canonical ESRs. We further presented evidence that the DzrR-mediated response is conserved in the bacterial species of Dickeya, Ralstonia, and Burkholderia, showing that a distantly located DzrR homolog is the previously undetermined regulator of RND-8 efflux pump for chlorhexidine resistance in B. cenocepacia. CONCLUSIONS: Taken together, the findings from this study depict a new widely distributed Gram-negative ESR mechanism and present a valid target and useful clues to combat antimicrobial resistance.

Two-component system ArcBA modulates cell motility and biofilm formation in Dickeya oryzae.

Phytopathogen Dickeya oryzae is a causal agent of rice foot rot disease and the pathogen has an array of virulence factors, such as phytotoxin zeamines, plant cell wall degrading enzymes, cell motility, and biofilms, collectively contributing to the bacterial pathogenesis. In this study, through deletion analysis of predicted regulatory genes in D. oryzae EC1, we identified a two-component system associated with the regulation of bacterial virulence. The two-component system contains a histidine kinase ArcB and a response regulator ArcA, and deletion of their coding genes resulted in changed phenotypes in cell motility, biofilm formation, and bacterial virulence. Electrophoretic mobility shift assay revealed that ArcA bound to the promoters of the bcs operon and bssS, which respectively encode enzymes for the synthesis of celluloses and a biofilm formation regulatory protein. ArcA could also bind to the promoters of three virulence associated transcriptional regulatory genes, i.e., fis, slyA and ohrR. Surprisingly, although these three regulators were shown to modulate the production of cell wall degrading enzymes and zeamines, deletion of arcB and arcA did not seem to affect these phenotypes. Taken together, the findings from this study unveiled a new two-component system associated with the bacterial pathogenesis, which contributes to the virulence of D. oryzae mainly through its action on bacterial motility and biofilm formation.

Structural Requirements for the Neuroprotective and Anti-Inflammatory Activities of the Flavanone Sterubin.

Alzheimer's disease (AD) is the most frequent age-associated disease with no treatments that can prevent, delay, slow, or stop its progression. Thus, new approaches to drug development are needed. One promising approach is the use of phenotypic screening assays that can identify compounds that have therapeutic efficacy in target pathways relevant to aging and cognition, as well as AD pathology. Using this approach, we identified the flavanone sterubin, from Yerba santa (Eriodictyon californicum), as a potential drug candidate for the treatment of AD. Sterubin is highly protective against multiple initiators of cell death that activate distinct death pathways, potently induces the antioxidant transcription factor Nrf2, and has strong anti-inflammatory activity. Moreover, in a short-term model of AD, it was able to prevent decreases in short- and long-term memory. In order to better understand which key chemical functional groups are essential to the beneficial effects of sterubin, we compared the activity of sterubin to that of seven closely related flavonoids in our phenotypic screening assays. Surprisingly, only sterubin showed both potent neuroprotective activity against multiple insults as well as strong anti-inflammatory activity against several distinct inducers of inflammation. These effects correlated directly with the ability of sterubin to strongly induce Nrf2 in both nerve and microglial cells. Together, these results define the structural requirements underlying the neuroprotective and anti-inflammatory effects of sterubin and they provide the basis for future studies on new compounds based on sterubin.

Stable and large-scale organic-inorganic halide perovskite nanocrystal/polymer nanofiber films prepared via a green in situ fiber spinning chemistry method.

Organic-inorganic halide perovskite nanocrystals (PNCs) have shown great advantages in recent years due to their tunable emission wavelengths, narrow full-width at half-maximum (FWHM) and high photoluminescence quantum yield (PLQY). However, PNCs still face the challenges of poor stability, difficulty in processing and generation of heavy metal wastes; therefore, it is necessary to develop a green synthetic method to prepare PNCs. Here, we present for the first time a facile fiber spinning chemistry (FSC) method for the rapid preparation of organic-inorganic halide PAN/MAPbX3 (MA = CH3NH3, X = Cl, Br and I) nanofiber films at room temperature. The FSC process utilizes spinning fibers as the reactor, and polymer solidification and the in situ generation of PNCs occur simultaneously with solvent evaporation during the spinning process. This method not only achieves a continuous large-scale preparation of PNC/polymer nanofiber films but also avoids the generation of heavy metal waste. The organic-inorganic halide PAN/MAPbX3 nanofiber films fabricated by FSC demonstrated tunable emission in the range of 464-612 nm and PLQY of up to 58%, and the fluorescence intensity remained essentially unchanged after 90 days of storage in the atmospheric environment. Interestingly, we successfully prepared high-efficiency white light-emitting diodes (WLEDs) and wide color gamut liquid crystal displays (LCDs) with a color gamut of 116.1% using PAN/MAPbBr3 nanofiber films as fluorescence conversion materials. This study provides a novel way to construct high-performance PNC/polymer fiber composites on a large scale.

Hfq Is a Critical Modulator of Pathogenicity of Dickeya oryzae in Rice Seeds and Potato Tubers.

The frequent outbreaks of soft-rot diseases caused by Dickeya oryzae have emerged as severe problems in plant production in recent years and urgently require the elucidation of the virulence mechanisms of D. oryzae. Here, we report that Hfq, a conserved RNA chaperone protein in bacteria, is involved in modulating a series of virulence-related traits and bacterial virulence in D. oryzae EC1. The findings show that the null mutation of the hfqEC1 gene totally abolished the production of zeamine phytotoxins and protease, significantly attenuated the production of two other types of cell wall degrading enzymes, i.e., pectate lyase and cellulase, as well as attenuating swarming motility, biofilm formation, the development of hypersensitive response to Nicotiana benthamiana, and bacterial infections in rice seeds and potato tubers. QRT-PCR analysis and promoter reporter assay further indicated that HfqEC1 regulates zeamine production via modulating the expression of the key zeamine biosynthesis (zms) cluster genes. Taken together, these findings highlight that the Hfq of D. oryzae is one of the key regulators in modulating the production of virulence determinants and bacterial virulence in rice seeds and potato tubers.

Nickel-Catalyzed 1,1-Dihydrophosphinylation of Nitriles with Phosphine Oxides.

Treatment of phosphine oxides with nitriles usually furnishes 1,2-dihydrophosphinylation products. Herein, we developed a nickel-catalyzed 1,1-dihydrophosphinylation of nitriles with phosphine oxides to access primary amines. This reaction proceeded smoothly under very mild conditions. A series of nitriles and phosphine oxides were compatible with this conversion, and the desired products were obtained in moderate to good yields.

Spermidine Is an Intercellular Signal Modulating T3SS Expression in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a vital opportunistic human bacterial pathogen that causes acute and chronic infections. In this study, we set to determine whether the endogenous spermidine biosynthesis plays a role in regulation of type III secretion system (T3SS). The results showed that deletion of speA and speC, which encode putrescine biosynthesis, did not seem to affect cellular spermidine level and the T3SS gene expression. In contrast, mutation of speD and speE encoding spermidine biosynthesis led to significantly decreased spermidine production and expression of T3SS genes. We also showed that endogenous spermidine could auto-induce the transcriptional expression of speE and its full functionality required the transporter SpuDEFGH. Cytotoxicity analysis showed that mutants ΔspeE and ΔspuE were substantially attenuated in virulence compared with their wild-type strain PAO1. Our data imply a possibility that spermidine biosynthesis in P. aeruginosa may not use putrescine as a substrate, and that spermidine signaling pathway may interact with other two T3SS regulatory mechanisms in certain degree, i.e., cAMP-Vfr and GacS/GacA signaling systems. Taken together, these results specify the role of endogenous spermidine in regulation of T3SS in P. aeruginosa and provide useful clues for design and development antimicrobial therapies. IMPORTANCE Type III secretion system (T3SS) is one of the pivotal virulence factors of Pseudomonas aeruginosa responsible for evading phagocytosis, and secreting and translocating effectors into host cells. Previous studies underline the complicated and elaborate regulatory mechanisms of T3SS for the accurate, fast, and malicious pathogenicity of P. aeruginosa. Among these regulatory mechanisms, our previous study indicated that the spermidine from the host was vital to the host-pathogen interaction. However, the role of endogenous spermidine synthesized by P. aeruginosa on the regulation of T3SS expression is largely unknown. Here we reveal the role and regulatory network of endogenous spermidine synthesis in regulation of T3SS and bacterial virulence, showing that the spermidine is an important interspecies signal for modulating the virulence of P. aeruginosa through regulating T3SS expression.

Fibrous Nanoreactors from Microfluidic Blow Spinning for Mass Production of Highly Stable Ligand-Free Perovskite Quantum Dots.

Nano-/micro-reactors have emerged as a powerful platform for chemical synthesis. Here, we develop fiber-spinning chemistry (FSC) based on a microfluidic blow spinning (MBS) technique, allowing the availability of nanoreactors for chemical synthesis with scale-up capacities. Proof-of-concept experiments focus on the utilization of MBS-derived fibrous nanoreactors for large-scale production of ligand-free perovskite quantum dots (PQDs) in one step. Typically, methylammonium lead halide (MAPbX3 , X=Cl, Br, and I) PQDs in situ synthesized at large scale inside polyacrylonitrile (PAN) nanofiber films (size 120 cm ×30 cm per hour), exhibit high photoluminescence (PL) quantum yield (QY) of 71 %, tunable emissive peaks (448-600 nm), and superb PL stability. The PQDs/polymer nanofiber films are potentially useful for CO2 conversion, wide-color-gamut displays and light-emitting diode (LED) devices. These findings may guide the development of nano-/micro-reactor technology for scale-up production of nanomaterials with various potential applications.

Cyclic di-GMP modulates sessile-motile phenotypes and virulence in Dickeya oryzae via two PilZ domain receptors.

Dickeya oryzae is a bacterial pathogen causing the severe rice stem rot disease in China and other rice-growing countries. We showed recently that the universal bacterial second messenger c-di-GMP plays an important role in modulation of bacterial motility and pathogenicity, but the mechanism of regulation remains unknown. In this study, bioinformatics analysis of the D. oryzae EC1 genome led to the identification of two proteins, YcgR and BcsA, both of which contain a conserved c-di-GMP receptor domain, known as the PilZ-domain. By deleting all the genes encoding c-di-GMP-degrading enzymes in D. oryzae EC1, the resultant mutant 7ΔPDE with high c-di-GMP levels became nonmotile, formed hyperbiofilm, and lost the ability to colonize and invade rice seeds. These phenotypes were partially reversed by deletion of ycgR in the mutant 7ΔPDE, whereas deletion of bcsA only reversed the hyperbiofilm phenotype of mutant 7ΔPDE. Significantly, double deletion of ycgR and bcsA in mutant 7ΔPDE rescued its motility, biofilm formation, and virulence to levels of wild-type EC1. In vitro biochemical experiments and in vivo phenotypic assays further validated that YcgR and BcsA proteins are the receptors for c-di-GMP, which together play a critical role in regulating the c-di-GMP-associated functionality. The findings from this study fill a gap in our understanding of how c-di-GMP modulates bacterial motility and biofilm formation, and provide useful clues for further elucidation of sophisticated virulence regulatory mechanisms in this important plant pathogen.

The Role of AMP-activated Protein Kinase in Oxytosis/Ferroptosis: Protector or Potentiator?

Significance: Evidence for a role for the oxytosis/ferroptosis regulated cell death pathway in aging and neurodegenerative diseases has been growing over the past few years. Because of this, there is an increasing necessity to identify endogenous signaling pathways that can be modulated to protect cells from this form of cell death. Recent Advances: Recently, several studies have identified a protective role for the AMP-activated protein kinase (AMPK)/acetyl CoA carboxylase 1 (ACC1) pathway in oxytosis/ferroptosis. However, there are also a number of studies suggesting that this pathway contributes to cell death initiated by various inducers of oxytosis/ferroptosis. Critical Issues: The goals of this review are to provide an overview and analysis of the published studies and highlight specific areas where more research is needed. Future Directions: Much remains to be learned about AMPK signaling in oxytosis/ferroptosis, especially the conditions where it is protective. Furthermore, the role of AMPK signaling in the brain and especially the aging brain needs further investigation.

The Alzheimer's disease drug candidate J147 decreases blood plasma fatty acid levels via modulation of AMPK/ACC1 signaling in the liver.

J147 is a novel drug candidate developed to treat neurological dysfunction. Numerous studies have demonstrated the beneficial effects of J147 in cellular and animal models of disease which has led to the transitioning of the compound into human clinical trials. However, no biomarkers for its target engagement have been identified. Here, we determined if specific metabolites in the plasma could be indicative of J147's activity in vivo. Plasma lipidomics data from three independent rodent studies were assessed along with liver lipidomics data from one of the studies. J147 consistently reduced plasma free fatty acid (FFA) levels across the independent studies. Decreased FFA levels were also found in the livers of J147-treated mice that correlated well with those in the plasma. These changes in the liver were associated with activation of the AMP-activated protein kinase/acetyl-CoA carboxylase 1 signaling pathway. A reduction in FFA levels by J147 was confirmed in HepG2 cells, where activation of the AMPK/ACC1 pathway was seen along with increases in acetyl-CoA and ATP levels which correlated with enhanced cellular bioenergetics. Our data show that J147 targets liver cells to activate the AMPK/ACC1 signaling pathway and preserve energy at the expense of inhibiting FFA synthesis.

The GacA-GacS Type Two-Component System Modulates the Pathogenicity of Dickeya oryzae EC1 Mainly by Regulating the Production of Zeamines.

The GacS-GacA type two-component system (TCS) positively regulates pathogenicity-related phenotypes in many plant pathogens. In addition, Dickeya oryzae EC1, the causative agent of soft rot disease, produces antibiotic-like toxins called zeamines as one of the major virulence factors that inhibit the germination of rice seeds. The present study identified a GacS-GacA type TCS, named TzpS-TzpA, that positively controls the virulence of EC1, mainly by regulating production of the toxin zeamines. RNA-seq analysis of strain EC1 and its tzpA mutant showed that the TCS regulated a wide range of virulence genes, especially those encoding zeamines. Protein-protein interaction was detected between TzpS and TzpA through the bacterial two-hybrid system and pull-down assay. In trans expression of tzpA failed to rescue the defective phenotypes in both the ΔtzpS and ΔtzpSΔtzpA mutants. Furthermore, TzpA controls target gene expression by direct binding to DNA promoters that contain a Gac-box motif, including a regulatory RNA rsmB and the vfm quorum-sensing system regulator vfmE. These findings therefore suggested that the EC1 TzpS-TzpA TCS system mediates the pathogenicity of Dickeya oryzae EC1 mainly by regulating the production of zeamines.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.