The plant-unique protein DRIF1 coordinates with sorting nexin 1 to regulate membrane protein homeostasis.

Membrane protein homeostasis is fine-tuned by the cellular pathways for vacuolar degradation and recycling, which ultimately facilitate plant growth and cell-environment interactions. The endosomal sorting complex required for transport (ESCRT) machinery plays important roles in regulating intraluminal vesicle (ILV) formation and membrane protein sorting to vacuoles. We previously showed that the plant-specific ESCRT component FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FREE1) performs multiple functions in plants, although the underlying mechanisms remain elusive. In this study, we performed a suppressor screen of the FREE1-RNAi mutant and identified and characterized 2 suppressor of free1 (sof) mutants in Arabidopsis (Arabidopsis thaliana). These mutants, sof10 and sof641, result in a premature stop codon or a missense mutation in AT5G10370, respectively. This gene was named DEAH and RING domain-containing protein as FREE1 suppressor 1 (DRIF1). DRIF1 has a homologous gene, DRIF2, in the Arabidopsis genome with 95% identity to DRIF1. The embryos of drif1 drif2 mutants arrested at the globular stage and formed enlarged multivesicular bodies (MVBs) with an increased number of ILVs. DRIF1 is a membrane-associated protein that coordinates with retromer component sorting nexin 1 to regulate PIN-FORMED2 recycling to the plasma membrane. Altogether, our data demonstrate that DRIF1 is a unique retromer interactor that orchestrates FREE1-mediated ILV formation of MVBs and vacuolar sorting of membrane proteins for degradation in plants.

Ufmylation reconciles salt stress-induced unfolded protein responses via ER-phagy in Arabidopsis.

In plants, the endomembrane system is tightly regulated in response to environmental stresses for maintaining cellular homeostasis. Autophagosomes, the double membrane organelles forming upon nutrient deprivation or stress induction, degrade bulky cytosolic materials for nutrient turnover. Though abiotic stresses have been reported to induce plant autophagy, few receptors or regulators for selective autophagy have been characterized for specific stresses. Here, we have applied immunoprecipitation followed by tandem mass spectrometry using the autophagosome marker protein ATG8 as bait and have identified the E3 ligase of the ufmylation system Ufl1 as a bona fide ATG8 interactor under salt stress. Notably, core components in the ufmylation cascade, Ufl1 and Ufm1, interact with the autophagy kinase complexes proteins ATG1 and ATG6. Cellular and genetic analysis showed that Ufl1 is important for endoplasmic reticulum (ER)-phagy under persisting salt stress. Loss-of-function mutants of Ufl1 display a salt stress hypersensitive phenotype and abnormal ER morphology. Prolonged ER stress responses are detected in ufl1 mutants that phenocopy the autophagy dysfunction atg5 mutants. Consistently, expression of ufmylation cascade components is up-regulated by salt stress. Taken together, our study demonstrates the role of ufmylation in regulating ER homeostasis under salt stress through ER-phagy.

The plant ESCRT component FREE1 regulates peroxisome-mediated turnover of lipid droplets in germinating Arabidopsis seedlings.

Lipid droplets (LDs) stored during seed development are mobilized and provide essential energy and lipids to support seedling growth upon germination. Triacylglycerols (TAGs) are the main neutral lipids stored in LDs. The lipase SUGAR DEPENDENT 1 (SDP1), which hydrolyzes TAGs in Arabidopsis thaliana, is localized on peroxisomes and traffics to the LD surface through peroxisomal extension, but the underlying mechanism remains elusive. Here, we report a previously unknown function of a plant-unique endosomal sorting complex required for transport (ESCRT) component FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) in regulating peroxisome/SDP1-mediated LD turnover in Arabidopsis. We showed that LD degradation was impaired in germinating free1 mutant; moreover, the tubulation of SDP1- or PEROXIN 11e (PEX11e)-marked peroxisomes and the migration of SDP1-positive peroxisomes to the LD surface were altered in the free1 mutant. Electron tomography analysis showed that peroxisomes failed to form tubules to engulf LDs in free1, unlike in the wild-type. FREE1 interacted directly with both PEX11e and SDP1, suggesting that these interactions may regulate peroxisomal extension and trafficking of the lipase SDP1 to LDs. Taken together, our results demonstrate a pivotal role for FREE1 in LD degradation in germinating seedlings via regulating peroxisomal tubulation and SDP1 targeting.

Arabidopsis ORP2A mediates ER-autophagosomal membrane contact sites and regulates PI3P in plant autophagy.

Autophagy is an intracellular degradation system for cytoplasmic constituents which is mediated by the formation of a double-membrane organelle termed the autophagosome and its subsequent fusion with the lysosome/vacuole. The formation of the autophagosome requires membrane from the endoplasmic reticulum (ER) and is tightly regulated by a series of autophagy-related (ATG) proteins and lipids. However, how the ER contacts autophagosomes and regulates autophagy remain elusive in plants. In this study, we identified and demonstrated the roles of Arabidopsis oxysterol-binding protein-related protein 2A (ORP2A) in mediating ER-autophagosomal membrane contacts and autophagosome biogenesis. We showed that ORP2A localizes to both ER-plasma membrane contact sites (EPCSs) and autophagosomes, and that ORP2A interacts with both the ER-localized VAMP-associated protein (VAP) 27-1 and ATG8e on the autophagosomes to mediate the membrane contact sites (MCSs). In ORP2A artificial microRNA knockdown (KD) plants, seedlings display retarded growth and impaired autophagy levels. Both ATG1a and ATG8e accumulated and associated with the ER membrane in ORP2A KD lines. Moreover, ORP2A binds multiple phospholipids and shows colocalization with phosphatidylinositol 3-phosphate (PI3P) in vivo. Taken together, ORP2A mediates ER-autophagosomal MCSs and regulates autophagy through PI3P redistribution.

Efficacy and safety of Mahuang Fuzi and Shenzhuo Decoction for treatment of primary membranous nephropathy: a multicenter prospective trial.

BACKGROUND: This study aims to undertake a comprehensive assessment of the effectiveness and safety profile of Mahuang Fuzi and Shenzhuo Decoction (MFSD) in the management of primary membranous nephropathy (PMN), within the context of a prospective clinical investigation. METHODS: A multicenter, open-label clinical trial was executed on patients diagnosed with PMN. These individuals were subjected to MFSD therapy for a duration of at least 24 months, with primary outcome of clinical remission rates. The Cox regression analysis was employed to discern the pertinent risk factors exerting influence on the efficacy of MFSD treatment, with scrupulous monitoring of any adverse events. RESULTS: The study comprised 198 participants in total. Following 24 months of treatment, the remission rate was 58.6% (116/198). Among the subgroup of 130 participants subjected to a 36-month follow-up, the remission rate reached 70% (91/130). Subgroup analysis revealed that neither a history of immunosuppressive therapy (HIST) nor an age threshold of ≥60 years exhibited a statistically significant impact on the remission rate at the 24-month mark (p > .05). Multivariate Cox regression analyses elucidated HIST, nephrotic syndrome, or mass proteinuria, and a high-risk classification as noteworthy risk factors in the context of MFSD treatment. Remarkably, no fatalities resulting from side effects were documented throughout the study's duration. CONCLUSIONS: This trial establishes the efficacy of MFSD as a treatment modality for membranous nephropathy. MFSD demonstrates a favorable side effect profile, and remission rates are consistent across patients, irrespective of HIST and age categories.

Correlation of vacuole morphology with stomatal lineage development by whole-cell electron tomography.

Stomatal movement is essential for plants to optimize transpiration and therefore photosynthesis. Rapid changes in the stomatal aperture are accompanied by adjustment of vacuole volume and morphology in guard cells (GCs). In Arabidopsis (Arabidopsis thaliana) leaf epidermis, stomatal development undergoes a cell-fate transition including four stomatal lineage cells: meristemoid, guard mother cell, young GC, and GC. Little is known about the mechanism underlying vacuole dynamics and vacuole formation during stomatal development. Here, we utilized whole-cell electron tomography (ET) analysis to elucidate vacuole morphology, formation, and development in different stages of stomatal lineage cells at nanometer resolution. The whole-cell ET models demonstrated that large vacuoles were generated from small vacuole stepwise fusion/maturation along stomatal development stages. Further ET analyses verified the existence of swollen intraluminal vesicles inside distinct vacuoles at certain developmental stages of stomatal lineage cells, implying a role of multivesicular body fusion in stomatal vacuole formation. Collectively, our findings demonstrate a mechanism mediating vacuole formation in Arabidopsis stomatal development and may shed light on the role of vacuoles in stomatal movement.

Prenatal ultrasound manifestations of partial urorectal septum malformation sequence from the first trimester to postnatal: a case report.

BACKGROUND: Urorectal septum malformation sequence (URSMS) is characterized by incomplete partitioning of the genital, rectal, and urinary tracts, resulting in a severe form of anorectal malformation. The partial URSMS, also known as the persistent cloaca, represents a milder variant where a single perineal opening serves as a passage for the urinary, gastrointestinal, and reproductive tracts. CASE PRESENTATION: We present a rare case of partial URSMS accompanied by duplicated vagina and uterus, hydronephrosis, ascites, and anal atresia. CONCLUSIONS: This case report describes the sonographic findings at different stages of pregnancy and their changes throughout gestation.

RST1 Is a FREE1 Suppressor That Negatively Regulates Vacuolar Trafficking in Arabidopsis.

FYVE domain protein required for endosomal sorting1 (FREE1), a plant-specific endosomal sorting complex required for transport-I component, is essential for the biogenesis of multivesicular bodies (MVBs), vacuolar degradation of membrane protein, cargo vacuolar sorting, autophagic degradation, and vacuole biogenesis in Arabidopsis (Arabidopsis thaliana). Here, we report the characterization of RESURRECTION1 (RST1) as a suppressor of free1 that, when mutated as a null mutant, restores the normal MVB and vacuole formation of a FREE1-RNAi knockdown line and consequently allows survival. RST1 encodes an evolutionarily conserved multicellular organism-specific protein, which contains two Domain of Unknown Function 3730 domains, showing no similarity to known proteins, and predominantly localizes in the cytosol. The depletion of FREE1 causes substantial accumulation of RST1, and transgenic Arabidopsis plants overexpressing RST1 display retarded seedling growth with dilated MVBs, and inhibition of endocytosed FM4-64 dye to the tonoplast, suggesting that RST1 has a negative role in vacuolar transport. Consistently, enhanced endocytic degradation of membrane vacuolar cargoes occurs in the rst1 mutant. Further transcriptomic comparison of rst1 with free1 revealed a negative association between gene expression profiles, demonstrating that FREE1 and RST1 have antagonistic functions. Thus, RST1 is a negative regulator controlling membrane protein homeostasis and FREE1-mediated functions in plants.

[Diagnosis boundary values of metabolic syndrome obesity index for children and adolescents].

OBJECTIVE: To determine the distribution characteristics of waist circumference (WC), waist height ratio (WHtR) of 6-18 years olds in Guangzhou, and to put forward the WC and WHtR appropriate boundary values for 6-18 years olds on the basis of cardiovascular disease (CVD) risk factor assessment. METHODS: We analyzed the height, weight, WC and its metabolic indication data (blood pressure, fasting blood glucose, and blood lipids) of 15 000 children in Guangzhou, aged 6-18, with the receiver-operating characteristic curve (ROC), and explored the best value point of WC and WHtR for the prediction of cardiovascular diseases. RESULTS: When the WC percent reached P85, and WHtR reached 0.48, the cardiovascular risk factors of fasting blood-glucose, blood pressure, and blood fat were significantly higher. CONCLUSION: The 85th percentile value of WC and 0.48 of WHtR are the appropriate boundary values in increasing the cardiovascular disease risk factors in Chinese children and teenagers. WC and WHtR as a relatively simple inspection method, can well predict cardiovascular diseases, and be used in the conventional measuring items among students.

RIP1 kinase inactivation protects against LPS-induced acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome (ARDS) is characterized by lung tissue oedema and inflammatory cell infiltration, with limited therapeutic interventions available. Receptor-interacting protein kinase 1 (RIPK1), a critical regulator of cell death and inflammation implicated in many diseases, is not fully understood in the context of ARDS. In this study, we employed RIP1 kinase-inactivated (Rip1K45A/K45A) mice and two distinct RIPK1 inhibitors to investigate the contributions of RIP1 kinase activity in lipopolysaccharide (LPS)-induced ARDS pathology. Our results indicated that RIPK1 kinase inactivation, achieved through both genetic and chemical approaches, significantly attenuated LPS-induced ARDS pathology, as demonstrated by reduced polymorphonuclear neutrophil percentage (PMN%) in alveolar lavage fluid, expression of inflammatory and fibrosis-related factors in lung tissues, as well as histological examination. Results by tunnel staining and qRT-PCR analysis indicated that RIPK1 kinase activity played a role in regulating cell apoptosis and inflammation induced by LPS administration in lung tissue. In summary, employing both pharmacological and genetic approaches, this study demonstrated that targeted RIPK1 kinase inactivation attenuates the pathological phenotype induced by LPS inhalation in an ARDS mouse model. This study enhances our understanding of the therapeutic potential of RIPK1 kinase modulation in ARDS, providing insights for the pathogenesis of ARDS.

Realization of Fine-Tuning the Lattice Thermal Conductivity and Anharmonicity in Layered Semiconductors via Entropy Engineering.

Entropy engineering is widely proven to be effective in achieving ultra-low thermal conductivity for well-performed thermoelectric and heat management applications. However, no strong correlation between entropy and lattice thermal conductivity is found until now, and the fine-tuning of thermal conductivity continuously via entropy-engineering in a wide entropy range is still lacking. Here, a series of high-entropy layered semiconductors, Ni1- x(Fe0.25Co0.25Mn0.25Zn0.25)xPS3, where 0 ≤ x < 1, with low mass/size disorder is designed. High-purity samples with mixing configuration entropy of metal atomic site in a wide range of 0-1.61R are achieved. Umklapp phonon-phonon scattering is found to be the dominating phonon scattering mechanism, as revealed by the linear T-1 dependence of thermal conductivity. Meanwhile, fine tuning of the lattice thermal conductivity via continuous entropy engineering at metal atomic sites is achieved, in an almost linear dependence in middle-/high- entropy range. Moreover, the slope of the κ - T-1 curve reduces with the increase in entropy, and a linear response of the reduced Grüneisen parameter is revealed. This work provides an entropy engineering strategy by choosing multiple metal elements with low mass/size disorder to achieve the fine tuning of the lattice thermal conductivity and the anharmonic effect.

Fluorescent Fusion Protein Expression in Plant Cells.

Fluorescent proteins (FPs) revolutionized the cell biology research by visualizing the dynamics of cellular events. In fusion with the targeted proteins, the FPs can be utilized to monitor the protein dynamics and localization in cells. Recently, FPs have been used as reporters for live cell imaging to study the protein localization or organelles dynamics in plants, allowing cell biologists to explore the plant cell function by obtaining tremendous details of cell structures and functions in combination with confocal imaging. To facilitate the usage of fluorescent proteins for protein localization and dynamic analysis in plant cell biology research, here we describe the updated protocol of Agrobacterium-mediated transformation of Arabidopsis thaliana using fluorescent proteins to generate the stable expression transgenic plants for protein trafficking and localization study. We further use the GFP-tagged SDP1 (sugar-dependent protein) lipase, mCherry-tagged peroxisome marker, and BODYPY or Nile Red (lipid droplet staining dye) as examples to introduce the method for the protein localization analysis in plants.

Decoding plant specialized metabolism: new mechanistic insights.

Secondary metabolite (SM) production provides biotic and abiotic stress resistance and enables plants to adapt to the environment. Biosynthesis of these metabolites involves a complex interplay between transcription factors (TFs) and regulatory elements, with emerging evidence suggesting an integral role for chromatin dynamics. Here we review key TFs and epigenetic regulators that govern SM biosynthesis in different contexts. We summarize relevant emerging technologies and results from the model species arabidopsis (Arabidopsis thaliana) and outline aspects of regulation that may also function in food, feed, fiber, oil, or industrial crop plants. Finally, we highlight how effective translation of fundamental knowledge from model to non-model species can benefit understanding of SM production in a variety of ecological, agricultural, and pharmaceutical contexts.

Progranulin released from microglial lysosomes reduces neuronal ferroptosis after cerebral ischemia in mice.

The cellular redox state is essential for inhibiting ferroptosis. Progranulin (PGRN) plays an important role in maintaining the cellular redox state after ischemic brain injury. However, the effect of PGRN on ferroptosis and its underlying mechanism after cerebral ischemia remains unclear. This study assesses whether PGRN affects ferroptosis and explores its mechanism of action on ferroptosis after cerebral ischemia. We found endogenous PGRN expression in microglia increased on day 3 after ischemia. In addition, PGRN agonists chloroquine and trehalose upregulated PGRN expression, reduced brain infarct volume, and improved neurobehavioral outcomes after cerebral ischemia compared to controls (p < 0.05). Moreover, PGRN upregulation attenuated ferroptosis by decreasing malondialdehyde and increasing Gpx4, Nrf2, and Slc7a11 expression and glutathione content (p < 0.05). Furthermore, chloroquine induced microglial lysosome PGRN release, which was associated with increased neuron survival. Our results indicate that PGRN derived from microglial lysosomes effectively inhibits ferroptosis during ischemic brain injury, identifying it as a promising target for ischemic stroke therapy.

The plant unique ESCRT component FREE1 regulates autophagosome closure.

The energy sensor AMP-activated protein kinase (AMPK) can activate autophagy when cellular energy production becomes compromised. However, the degree to which nutrient sensing impinges on the autophagosome closure remains unknown. Here, we provide the mechanism underlying a plant unique protein FREE1, upon autophagy-induced SnRK1α1-mediated phosphorylation, functions as a linkage between ATG conjugation system and ESCRT machinery to regulate the autophagosome closure upon nutrient deprivation. Using high-resolution microscopy, 3D-electron tomography, and protease protection assay, we showed that unclosed autophagosomes accumulated in free1 mutants. Proteomic, cellular and biochemical analysis revealed the mechanistic connection between FREE1 and the ATG conjugation system/ESCRT-III complex in regulating autophagosome closure. Mass spectrometry analysis showed that the evolutionary conserved plant energy sensor SnRK1α1 phosphorylates FREE1 and recruits it to the autophagosomes to promote closure. Mutagenesis of the phosphorylation site on FREE1 caused the autophagosome closure failure. Our findings unveil how cellular energy sensing pathways regulate autophagosome closure to maintain cellular homeostasis.

[The quality standard study on Hugan qingzhi tablets].

OBJECTIVE: To develop a method of quality control for Hugan qingzhi tablets. METHODS: Fructus Crataegi, Rhizoma Alismatis and Radix Notoginseng were identified by TLC. HPLC was used for the determination of ursolic acid in Hugan qingzhi tablets. RESULTS: The chromatographic spots were identified without the interference of negative control. Ursolic acid had a good linearity over the concentration range of 40-200 microg/mL (r = 1.000). The average recoveries was 99.05% with relatively standard deviations of 1.3%. CONCLUSION: This method is reliable, accurate and specific and can be used for the quality control of Hugan qingzhi tablets.

BK Channel-Mediated Microglial Phagocytosis Alleviates Neurological Deficit After Ischemic Stroke.

Microglial phagocytosis benefits neurological recovery after stroke. Large-conductance Ca2+-activated K+ currents are expressed in activated microglia, and BK channel knockout aggravates cerebral ischemic injury. However, the effect of BK channels on microglial phagocytosis after ischemic stroke remains unknown. Here, we explored whether BK channel activation is beneficial for neurological outcomes through microglial phagocytosis after ischemic stroke. ICR mice after transient middle cerebral artery occlusion (tMCAO) were treated with dimethyl sulfoxide (DMSO), BK channel activator NS19504, and inhibitor Paxilline. The results showed a decrease in BK channel expression after tMCAO. BK channel activator NS19504 alleviates neurological deficit after experimental modeling of tMCAO in mice compared to the control. Furthermore, we treated primary microglia with DMSO, NS19504, and Paxilline after oxygen glucose deprivation (OGD). NS19504 promoted primary microglial phagocytosing fluorescent beads and neuronal debris, which reduced neuronal apoptosis after stroke. These effects could be reversed by BK channel inhibitor Paxilline. Finally, NS19504 increased relative phosphorylated extracellular signal-regulated kinase 1/2 expression compared to the Paxilline group at the third day after stroke. Our findings indicate that microglial BK channels are a potential target for acute stage of ischemic stroke therapy.

M2 microglial small extracellular vesicles reduce glial scar formation via the miR-124/STAT3 pathway after ischemic stroke in mice.

Rationale: Glial scars present a major obstacle for neuronal regeneration after stroke. Thus, approaches to promote their degradation and inhibit their formation are beneficial for stroke recovery. The interaction of microglia and astrocytes is known to be involved in glial scar formation after stroke; however, how microglia affect glial scar formation remains unclear. Methods: Mice were treated daily with M2 microglial small extracellular vesicles through tail intravenous injections from day 1 to day 7 after middle cerebral artery occlusion. Glial scar, infarct volume, neurological score were detected after ischemia. microRNA and related protein were examined in peri-infarct areas of the brain following ischemia. Results: M2 microglial small extracellular vesicles reduced glial scar formation and promoted recovery after stroke and were enriched in miR-124. Furthermore, M2 microglial small extracellular vesicle treatment decreased the expression of the astrocyte proliferation gene signal transducer and activator of transcription 3, one of the targets of miR-124, and glial fibrillary acidic protein and inhibited astrocyte proliferation both in vitro and in vivo. It also decreased Notch 1 expression and increased Sox2 expression in astrocytes, which suggested that astrocytes had transformed into neuronal progenitor cells. Finally, miR-124 knockdown in M2 microglial small extracellular vesicles blocked their effects on glial scars and stroke recovery. Conclusions: Our results showed, for the first time, that microglia regulate glial scar formation via small extracellular vesicles, indicating that M2 microglial small extracellular vesicles could represent a new therapeutic approach for stroke.

P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis.

INTRODUCTION: Clearance of damaged cells and debris is beneficial for the functional recovery after ischemic brain injury. However, the specific phagocytic receptor that mediates microglial phagocytosis after ischemic stroke is unknown. AIM: To investigate whether P2Y6 receptor-mediated microglial phagocytosis is beneficial for the debris clearance and functional recovery after ischemic stroke. RESULTS: The expression of the P2Y6 receptor in microglia increased within 3 days after transient middle cerebral artery occlusion. Inhibition of microglial phagocytosis by the selective inhibitor MRS2578 enlarged the brain atrophy and edema volume after ischemic stroke, subsequently aggravated neurological function as measured by modified neurological severity scores and Grid walking test. MRS2578 treatment had no effect on the expression of IL-1α, IL-1ß, IL-6, IL-10, TNF-α, TGF-ß, and MPO after ischemic stroke. Finally, we found that the expression of myosin light chain kinase decreased after microglial phagocytosis inhibition in the ischemic mouse brain, which suggested that myosin light chain kinase was involved in P2Y6 receptor-mediated phagocytosis. CONCLUSION: Our results indicate that P2Y6 receptor-mediated microglial phagocytosis plays a beneficial role during the acute stage of ischemic stroke, which can be a therapeutic target for ischemic stroke.

Observation of NMR signals from proteins introduced into living mammalian cells by reversible membrane permeabilization using a pore-forming toxin, streptolysin O.

We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.