GABA and astrocytic cholesterol determine the lipid environment of GABA A R in cultured cortical neurons.

The γ-aminobutyric acid (GABA) type A receptor (GABA A R), a GABA activated pentameric chloride channel, mediates fast inhibitory neurotransmission in the brain. The lipid environment is critical for GABA A R function. How lipids regulate the channel in the cell membrane is not fully understood. Here we employed super resolution imaging of lipids to demonstrate that the agonist GABA induces a rapid and reversible membrane translocation of GABA A R to phosphatidylinositol 4,5-bisphosphate (PIP 2 ) clusters in mouse primary cortical neurons. This translocation relies on nanoscopic separation of PIP 2 clusters and lipid rafts (cholesterol-dependent ganglioside clusters). In a resting state, the GABA A R associates with lipid rafts and this colocalization is enhanced by uptake of astrocytic secretions. These astrocytic secretions enhance endocytosis and delay desensitization. Our findings suggest intercellular signaling from astrocytes regulates GABA A R location based on lipid uptake in neurons. The findings have implications for treating mood disorders associated with altered neural excitability.

An Experimental Investigation of the Precipitation Utilization of Plants in Arid Regions.

What represents a water source for the ecological restoration of a plant in an arid region is still up to debate. To address this issue, we conducted an in situ experiment in the Ulan Buh Desert of China, to study desert plants absorbing atmospheric water vapor. We selected Tamarisk, a common drought-salt-tolerant species in the desert, for ecological restoration as our research subject, used a newly designed lysimeter to monitor precipitation infiltration, and a sap flow system to track reverse sap flow that occurred in the shoot, branch, and stem during the precipitation event, and observed the precipitation redistribution process of the Tamarisk plot. The results showed that Tamarisk indeed directly absorbs precipitation water: when precipitation occurs, the main stem, lateral branch, and shoot all show the signs of reversed sap flow, and the reversed sap flow accounted for 21.5% of the annual sap flow in the shoot and branch, and 13.6% in the stem. The precipitation event in the desert was dominated by light precipitation events, which accounted for 81% of the annual precipitation events. It was found that light precipitation can be directly absorbed by the Tamarisk leaves, especially during nighttime or cloudy days. Even when the precipitation is absent, it was found that desert plants can still absorb water from the unsaturated atmospheric vapor; even the absorbed atmospheric water vapor was transported from the leaves to the stem, forming a reversed sap flow, as a reversed sap flow was observed when the atmospheric relative humidity reached 75%. This study indicated that the effect of light precipitation on desert plants was significant and should not be overlooked in terms of managing the ecological and hydrological systems in arid regions.

Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling.

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.

"Ouch!": you have just stabbed your little toe on the sharp corner of a coffee table. That painful sensation stems from nerve cells converting information about external forces into electric signals the brain can interpret. Increasingly, new evidence is suggesting that this process may be starting at fat-based structures within the membrane of these cells. The cell membrane is formed of two interconnected, flexible sheets of lipids in which embedded structures or molecules are free to move. This organisation allows the membrane to physically respond to external forces and, in turn, to set in motion chains of molecular events that help fine-tune how cells relay such information to the brain. For instance, an enzyme known as PLD2 is bound to lipid rafts ­ precisely arranged, rigid fatty 'clumps' in the membrane that are partly formed of cholesterol. PLD2 has also been shown to physically interact with and then activate the ion channel TREK-1, a membrane-based protein that helps to prevent nerve cells from relaying pain signals. However, the exact mechanism underpinning these interactions is difficult to study due to the nature and size of the molecules involved. To address this question, Petersen et al. combined a technology called super-resolution imaging with a new approach that allowed them to observe how membrane lipids respond to pressure and fluid shear. The experiments showed that mechanical forces disrupt the careful arrangement of lipid rafts, causing PLD2 and TREK-1 to be released. They can then move through the surrounding membrane where they reach a switch that turns on TREK-1. Further work revealed that the levels of cholesterol available to mouse cells directly influenced how the clumps could form and bind to PLD2, and in turn, dialled up and down the protective signal mediated by TREK-1. Overall, the study by Petersen et al. shows that the membrane of nerve cells can contain cholesterol-based 'fat sensors' that help to detect external forces and participate in pain regulation. By dissecting these processes, it may be possible to better understand and treat conditions such as diabetes and lupus, which are associated with both pain sensitivity and elevated levels of cholesterol in tissues.

Hypoxia Aggravates Neuron Ferroptosis in Early Brain Injury Following Subarachnoid Hemorrhage via NCOA4-Meditated Ferritinophagy.

The occurrence of early brain injury (EBI) significantly contributes to the unfavorable prognosis observed in patients with subarachnoid hemorrhage (SAH). During the process of EBI, a substantial quantity of iron permeates into the subarachnoid space and brain tissue, thereby raising concerns regarding its metabolism. To investigate the role and metabolic processes of excessive iron in neurons, we established both in vivo and in vitro models of SAH. We substantiated that ferritinophagy participates in iron metabolism disorders and promotes neuronal ferroptosis using an in vivo model, as detected by key proteins such as ferritin heavy chain 1, glutathione peroxidase 4, autophagy related 5, nuclear receptor coactivator 4 (NCOA4), LC3B, and electron microscopy results. By interfering with NCOA4 expression in vitro and in vivo, we confirmed the pivotal role of elevated NCOA4 levels in ferritinophagy during EBI. Additionally, our in vitro experiments demonstrated that the addition of oxyhemoglobin alone did not result in a significant upregulation of NCOA4 expression. However, simultaneous addition of oxyhemoglobin and hypoxia exposure provoked a marked increase in NCOA4 expression and heightened ferritinophagy in HT22 cells. Using YC-1 to inhibit hypoxia signaling in in vitro and in vitro models effectively attenuated neuronal ferroptosis. Collectively, we found that the hypoxic microenvironment during the process of EBI exaggerates iron metabolism abnormalities, leading to poor prognoses in SAH. The findings also offer a novel and potentially effective foundation for the treatment of SAH, with the aim of alleviating hypoxia.

Effects of Ecological Restoration and Climate Change on Herbaceous and Arboreal Phenology.

With global climate change, changes in vegetation phenology have become increasingly evident. Horqin Sandy Land is located near the eastern part of the West Liaohe River. It is the largest sandy land in China and its ecological environment is fragile. Investigating the changes in vegetation phenology in these sandy areas and determining the relationship between vegetation phenology and meteorological factors are of great importance for predicting the impacts of future climate change and understanding the response mechanisms of ecosystems. In this study, we used the time series of the Normalized Difference Vegetation Index (NDVI) from 2000 to 2021 and extracted the vegetation phenology in the Horqin Sandy Land using high-order curve fitting methods, including the start date of the growing season (SOS), the end date of the growing season (EOS), and the length of the growing season (LOS). We analyzed their temporal variation and used partial correlation analysis to determine their relationship with meteorological factors (temperature and precipitation). In addition, we compared the phenology and microclimate of forest and grassland within the study area. In the Horqin Sandy Land, the vegetation SOS was concentrated between the 115th and 150th day, the EOS was concentrated between the 260th and 305th day, and the LOS ranged from 125 to 190 days. Over the past 22 years, the SOS, EOS, and LOS of vegetation in the Horqin Sandy Land showed trends of delay, shift, and extension, with rates of change of 0.82 d/10a, 5.82 d/10a, and 5.00 d/10a, respectively. The start date of the growing season in the Horqin Sandy Land was mainly influenced by precipitation in April of the current year, while the end date was mainly influenced by precipitation in August of the current year. Overall, the SOS in the forested areas of the Horqin Sandy Land was slightly later than in the grasslands, but the EOS in the forested areas was significantly later than in the grasslands, resulting in a longer LOS in the forests. In addition, annual precipitation and the rate of precipitation increase were higher in the forested areas than in the grasslands, but soil temperature was higher in the grasslands than in the forests. Vegetation phenology in the Horqin Sandy Land has undergone significant changes, mainly manifested in the delayed end date of the growing season, the extended length of the growing season, and the differences between forest and grassland. This indicates that climate change has indeed affected phenological changes and provides a theoretical basis for subsequent ecological restoration and desertification prevention efforts in the region.

A Novel Low Molecule Peptides-calcium Chelate from Silkworm Pupae Protein Hydrolysate: Preparation, Antioxidant Activity, and Bioavailability.

BACKGROUND: The antioxidant properties of active peptides from silkworm pupae protein hydrolysate are of interest, and it serves as a novel source of calcium supplement. METHODS: Optimize the preparation parameters of silkworm pupae bioactive peptide-calcium chelate, and investigate the mechanism and bioavailability of silkworm pupae active peptide as a transport carrier to promote calcium ion absorption using simulated gastrointestinal digestion and Caco-2 monolayer cell model. RESULTS: The optimal process parameters for preparing peptide calcium chelate were the peptide calcium mass ratio of 3:1, pH of 6.7, a temperature of 35.6°C, and time of 32.8 min by Box-Behnken design, and the calciumchelating rate reached 84.67%. The DPPH radical scavenging activity of silkworm pupae protein hydrolysatecalcium chelate was 79.36 ± 4.31%, significantly higher than silkworm pupae protein hydrolysate (61.00 ± 9.56%). Fourier transform infrared spectroscopy shows that the COO-, N-H, C-H, and C-O groups participated in the formation of silkworm pupae protein hydrolysate-calcium chelate. The particle size of the silkworm pupae protein hydrolysate-calcium chelate was 970.75 ± 30.12 nm, which was significantly higher than that of silkworm pupae protein hydrolysate (253.14 ± 5.72 nm). The silkworm pupae protein hydrolysate-calcium chelate showed a calcium dissolution rate of 71.01 ± 1.91% in the simulated intestinal phase, significantly higher than that of CaCl2 (59.34 ± 1.24%). In the Caco-2 cell monolayers, the silkworm pupae protein hydrolysatecalcium chelate was more favorable for calcium transport. CONCLUSION: A novel silkworm pupa protein hydrolysate-calcium chelate with high antioxidant activity was successfully prepared to improve the bioavailability of calcium.

The role of high cholesterol in SARS-CoV-2 infectivity.

Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2. The virus binds to angiotensinogen converting enzyme 2 (ACE2), which mediates viral entry into mammalian cells. COVID-19 is notably severe in the elderly and in those with underlying chronic conditions. The cause of selective severity is not well understood. Here we show cholesterol and the signaling lipid phosphatidyl-inositol 4,5 bisphosphate (PIP2) regulate viral infectivity through the localization of ACE2's into nanoscopic (<200 nm) lipid clusters. Uptake of cholesterol into cell membranes (a condition common to chronic disease) causes ACE2 to move from PIP2 lipids to endocytic ganglioside (GM1) lipids, where the virus is optimally located for viral entry. In mice, age and high-fat diet increase lung tissue cholesterol by up to 40%. And in smokers with chronic disease, cholesterol is elevated 2-fold, a magnitude of change that dramatically increases infectivity of virus in cell culture. We conclude increasing the ACE2 location near endocytic lipids increases viral infectivity and may help explain the selective severity of COVID-19 in aged and diseased populations.

MitoStores: a place for precursors to ride out the storm.

The fate of unimported mitochondrial precursors has been increasingly studied in recent years, mostly focusing on protein degradation. In this issue of the EMBO journal, Krämer et al discovered MitoStores, a new protective mechanism that temporarily stores mitochondrial proteins in cytosolic deposits.

Getting in on the action: New tools to see SARS-CoV-2 infect a cell.

In this issue of Cell Chemical Biology, Miao et al. develop probes for live cell tracking of SARS-CoV-2. The probes reveal the endocytic pathway for viral entry. Unexpectedly, the antiviral compound BafA1 traps the virus on the cell surface, highlighting the power of super-resolution imaging in live cells.

Cholesterol Regulation of Membrane Proteins Revealed by Two-Color Super-Resolution Imaging.

Cholesterol and phosphatidyl inositol 4,5-bisphosphate (PIP2) are hydrophobic molecules that regulate protein function in the plasma membrane of all cells. In this review, we discuss how changes in cholesterol concentration cause nanoscopic (<200 nm) movements of membrane proteins to regulate their function. Cholesterol is known to cluster many membrane proteins (often palmitoylated proteins) with long-chain saturated lipids. Although PIP2 is better known for gating ion channels, in this review, we will discuss a second independent function as a regulator of nanoscopic protein movement that opposes cholesterol clustering. The understanding of the movement of proteins between nanoscopic lipid domains emerged largely through the recent advent of super-resolution imaging and the establishment of two-color techniques to label lipids separate from proteins. We discuss the labeling techniques for imaging, their strengths and weakness, and how they are used to reveal novel mechanisms for an ion channel, transporter, and enzyme function. Among the mechanisms, we describe substrate and ligand presentation and their ability to activate enzymes, gate channels, and transporters rapidly and potently. Finally, we define cholesterol-regulated proteins (CRP) and discuss the role of PIP2 in opposing the regulation of cholesterol, as seen through super-resolution imaging.

Hydroxychloroquine blocks SARS-CoV-2 entry into the endocytic pathway in mammalian cell culture.

Hydroxychloroquine (HCQ), a drug used to treat lupus and malaria, was proposed as a treatment for SARS-coronavirus-2 (SARS-CoV-2) infection, albeit with controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ's mechanism of actions in vitro is needed. Recently, anesthetics were shown to disrupt ordered clusters of monosialotetrahexosylganglioside1 (GM1) lipid. These same lipid clusters recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to endocytic lipids, away from phosphatidylinositol 4,5 bisphosphate (PIP2) clusters. Here we employed super-resolution imaging of cultured mammalian cells (VeroE6, A549, H1793, and HEK293T) to show HCQ directly perturbs clustering of ACE2 receptor with both endocytic lipids and PIP2 clusters. In elevated (high) cholesterol, HCQ moves ACE2 nanoscopic distances away from endocytic lipids. In cells with resting (low) cholesterol, ACE2 primarily associates with PIP2 clusters, and HCQ moves ACE2 away from PIP2 clusters-erythromycin has a similar effect. We conclude HCQ inhibits viral entry through two distinct mechanisms in high and low tissue cholesterol and does so prior to inhibiting cathepsin-L. HCQ clinical trials and animal studies will need to account for tissue cholesterol levels when evaluating dosing and efficacy.

Disrupted and Elevated Circadian Secretion of Glucagon-Like Peptide-1 in a Murine Model of Type 2 Diabetes.

Metabolism and circadian rhythms are intimately linked, with circadian glucagon-like peptide-1 (GLP-1) secretion by the intestinal L-cell entraining rhythmic insulin release. GLP-1 secretion has been explored in the context of obesogenic diets, but never in a rodent model of type 2 diabetes (T2D). There is also considerable disagreement regarding GLP-1 levels in human T2D. Furthermore, recent evidence has demonstrated decreased expression of the ß-cell exocytotic protein secretagogin (SCGN) in T2D. To extend these findings to the L-cell, we administered oral glucose tolerance tests at 6 time points in 4-hour intervals to the high-fat diet/streptozotocin (HFD-STZ) mouse model of T2D. This revealed a 10-fold increase in peak GLP-1 secretion with a phase shift of the peak from the normal feeding period into the fasting-phase. This was accompanied by impairments in the rhythms of glucose, glucagon, mucosal clock genes (Arntl and Cry2), and Scgn. Immunostaining revealed that L-cell GLP-1 intensity was increased in the HFD-STZ model, as was the proportion of L-cells that expressed SCGN; however, this was not found in L-cells from humans with T2D, which exhibited decreased GLP-1 staining but maintained their SCGN expression. Gcg expression in isolated L-cells was increased along with pathways relating to GLP-1 secretion and electron transport chain activity in the HFD-STZ condition. Further investigation into the mechanisms responsible for this increase in GLP-1 secretion may give insights into therapies directed toward upregulating endogenous GLP-1 secretion.

A neural circuit integrates pharyngeal sensation to control feeding.

Swallowing is an essential step of eating and drinking. However, how the quality of a food bolus is sensed by pharyngeal neurons is largely unknown. Here we find that mechanical receptors along the Drosophila pharynx are required for control of meal size, especially for food of high viscosity. The mechanical force exerted by the bolus passing across the pharynx is detected by neurons expressing the mechanotransduction channel NOMPC (no mechanoreceptor potential C) and is relayed, together with gustatory information, to IN1 neurons in the subesophageal zone (SEZ) of the brain. IN1 (ingestion neurons) neurons act directly upstream of a group of peptidergic neurons that encode satiety. Prolonged activation of IN1 neurons suppresses feeding. IN1 neurons receive inhibition from DSOG1 (descending subesophageal neurons) neurons, a group of GABAergic neurons that non-selectively suppress feeding. Our results reveal the function of pharyngeal mechanoreceptors and their downstream neural circuits in the control of food ingestion.

The role of high cholesterol in age-related COVID19 lethality.

Coronavirus disease 2019 (COVID19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan, China in 2019. The disease is notably severe in elderly and those with underlying chronic conditions. A molecular mechanism that explains why the elderly are vulnerable and why children are resistant is largely unknown. Here we show loading cells with cholesterol from blood serum using the cholesterol transport protein apolipoprotein E (apoE) enhances the entry of pseudotyped SARS-CoV-2 and the infectivity of the virion. Super resolution imaging of the SARS-CoV-2 entry point with high cholesterol shows almost twice the total number of endocytic entry points. Cholesterol concomitantly traffics angiotensinogen converting enzyme (ACE2) to the endocytic entry site where SARS-CoV-2 presumably docks to efficiently exploit entry into the cell. Furthermore, in cells producing virus, cholesterol optimally positions furin for priming SARS-CoV-2, producing a more infectious virion with improved binding to the ACE2 receptor. In vivo, age and high fat diet induces cholesterol loading by up to 40% and trafficking of ACE2 to endocytic entry sites in lung tissue from mice. We propose a component of COVID19 severity based on tissue cholesterol level and the sensitivity of ACE2 and furin to cholesterol. Molecules that reduce cholesterol or disrupt ACE2 localization with viral entry points or furin localization in the producer cells, may reduce the severity of COVID19 in obese patients.

Hydroxychloroquine: mechanism of action inhibiting SARS-CoV2 entry.

Hydroxychloroquine (HCQ) has been proposed in the treatment of SARS-coronavirus 2 (SARS-CoV-2) infection, albeit with much controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ's mechanism of actions in vitro is needed to resolve these conflicts. Recently, anesthetics were shown to disrupt ordered monosialotetrahexosylganglioside1 (GM1) lipid rafts. These same lipid rafts recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to an endocytic entry point, away from phosphatidylinositol 4,5 bisphosphate (PIP2) domains. Here we employed super resolution imaging of cultured mammalian cells to show HCQ directly perturbs GM1 lipid rafts and inhibits the ability of ACE2 receptor to associate with the endocytic pathway. HCQ also disrupts PIP2 domains and their ability to cluster and sequester ACE2. Similarly, the antibiotic erythromycin inhibits viral entry and both HCQ and erythromycin decrease the antimicrobial host defense peptide amyloid beta in cultured cells. We conclude HCQ is an anesthetic-like compound that disrupts GM1 lipid rafts similar to anesthetics. The disruption likely decreases viral clustering at both endocytic and putative PIP2 entry points.

Indole-3-acetic acid has long-term effects on long non-coding RNA gene methylation and growth in Populus tomentosa.

DNA methylation and long non-coding RNAs (lncRNAs) regulate plant growth and development, but their relationship and effect on responses to the auxin phytohormone indole-3-acetic acid (IAA) remain largely unknown, particularly in woody plants such as poplar (Populus tomentosa). Following treatment of 1-year-old clonal plants with 100 µM IAA, key poplar lncRNA genes showed changes in methylation, but whole-genome methylation levels showed no significant change. Moreover, 100 µM IAA inhibited growth of the 1-year-old poplar clones, possibly through the suppression of photosynthesis. This inhibition had a long-term effect, persisting at 1 month after removal of the exogenous IAA. Transcriptome analysis identified two candidate lncRNA genes that show changes in expression following IAA treatment, TCONS_00003480 and TCONS_00004832. TCONS_00003480 contains the same microRNA target sites of ptc-miR6464 as the 4-coumarate: CoA ligase 2 transcript, which encodes a lignin biosynthesis enzyme. And TCONS_00004832 shares the same target sites of ptc-miR6437a with the Photosystem II reaction center protein D and Cytochrome C Oxidase 17 transcripts, which are related to photosynthesis. The two lncRNAs as the mimics to corresponding target genes of miRNAs to prevent them from degrading. Examination of lncRNA gene expression and methylation revealed a negative relationship (r = - 0.29, P < 0.05); moreover, hypermethylation of the two candidate lncRNA genes remained 1 month after IAA treatment, suggesting that changes in methylation might be involved in the long-term effects of plant hormones. Therefore, our study reveals a long-term effect of IAA on the growth of P. tomentosa, possibly via methylation-mediated epigenetic changes in lncRNA gene expression and the interaction with corresponding miRNAs, leading to regulation of genes related to photosynthesis and growth.

Inhibition of glioma growth by a GOLPH3 siRNA-loaded cationic liposomes.

PURPOSE: GOLPH3 has been shown to be involved in glioma proliferation. In this study, we aimed to demonstrate that GOLPH3 can serve as a target for glioma gene therapy. METHODS: During the experiment, cationic liposomes with angiopep-2 (A2-CL) were used to deliver siGOLPH3 crossing the blood-brain barrier and reaching the glioma. RESULTS: At the cellular level, the A2-CL/siGOLPH3 could silence GOLPH3 and then effectively inhibited the proliferation of cells. In vivo experiments, using U87-GFP-Luci-bearing BALB/c mouse models, we demonstrated that A2-CL could deliver GOLPH3-siRNA specifically to glioma and effectively inhibit glioma growth. CONCLUSIONS: This study shows that GOLPH3 has great potential as a target for the gene therapy of glioma and is of great value in precise medical applications.