The OsTIL1 lipocalin protects cell membranes from reactive oxygen species damage and maintains the 18:3-containing glycerolipid biosynthesis under cold stress in rice.

Lipocalins constitute a conserved protein family that binds to and transports a variety of lipids while fatty acid desaturases (FADs) are required for maintaining the cell membrane fluidity under cold stress. Nevertheless, it remains unclear whether plant lipocalins promote FADs for the cell membrane integrity under cold stress. Here, we identified the role of OsTIL1 lipocalin in FADs-mediated glycerolipid remodeling under cold stress. Overexpression and CRISPR/Cas9 mediated gene edition experiments demonstrated that OsTIL1 positively regulated cold stress tolerance by protecting the cell membrane integrity from reactive oxygen species damage and enhancing the activities of peroxidase and ascorbate peroxidase, which was confirmed by combined cold stress with a membrane rigidifier dimethyl sulfoxide or a H2 O2 scavenger dimethyl thiourea. OsTIL1 overexpression induced higher 18:3 content, and higher 18:3/18:2 and (18:2 + 18:3)/18:1 ratios than the wild type under cold stress whereas the gene edition mutant showed the opposite. Furthermore, the lipidomic analysis showed that OsTIL1 overexpression led to higher contents of 18:3-mediated glycerolipids, including galactolipids (monoglactosyldiacylglycerol and digalactosyldiacylglycerol) and phospholipids (phosphatidyl glycerol, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and phosphatidyl inositol) under cold stress. RNA-seq and enzyme linked immunosorbent assay analyses indicated that OsTIL1 overexpression enhanced the transcription and enzyme abundance of four ω-3 FADs (OsFAD3-1/3-2, 7, and 8) under cold stress. These results reveal an important role of OsTIL1 in maintaining the cell membrane integrity from oxidative damage under cold stress, providing a good candidate gene for improving cold tolerance in rice.

DNA damage-regulated autophagy modulator 1 prevents glioblastoma cells proliferation by regulating lysosomal function and autophagic flux stability.

Glioblastoma (GBM) is the most aggressive and life-threatening brain tumor, characterized by its highly malignant and recurrent nature. DNA damage-regulated autophagy modulator 1 (DRAM-1) is a p53 target gene encoding a lysosomal protein that induces macro-autophagy and damage-induced programmed cell death in tumor growth. However, the precise mechanisms underlying how DRAM-1 affects tumor cell proliferation through regulation of lysosomal function and autophagic flux stability remain incompletely understood. We found that DRAM-1 expressions were evidently down-regulated in high-grade glioma and recurrent GBM tissues. The upregulation of DRAM-1 could increase mortality of primary cultured GBM cells. TEM analysis revealed an augmented accumulation of aberrant lysosomes in DRAM-1-overexpressing GBM cells. The assay for lysosomal pH and stability also demonstrated decreasing lysosomal membrane permeabilization (LMP) and impaired lysosomal acidity. Further research revealed the detrimental impact of lysosomal dysfunction, which impaired the autophagic flux stability and ultimately led to GBM cell death. Moreover, downregulation of mTOR phosphorylation was observed in GBM cells following upregulation of DRAM-1. In vivo and in vitro experiments additionally illustrated that the mTOR inhibitor rapamycin increased GBM cell mortality and exhibited an enhanced antitumor effect.

Stabilization of insect cell membranes and soluble enzymes by accumulated cryoprotectants during freezing stress.

Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.

Exploring lipin1 as a promising therapeutic target for the treatment of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive and devastating muscle disease, resulting from the absence of dystrophin. This leads to cell membrane instability, susceptibility to contraction-induced muscle damage, subsequent muscle degeneration, and eventually disability and early death of patients. Currently, there is no cure for DMD. Our recent studies identified that lipin1 plays a critical role in maintaining myofiber stability and integrity. However, lipin1 gene expression levels are dramatically reduced in the skeletal muscles of DMD patients and mdx mice. METHODS: To identify whether increased lipin1 expression could prevent dystrophic pathology, we employed unique muscle-specific mdx:lipin1 transgenic (mdx:lipin1Tg/0) mice in which lipin1 was restored in the dystrophic muscle of mdx mice, intramuscular gene delivery, as well as cell culture system. RESULTS: We found that increased lipin1 expression suppressed muscle degeneration and inflammation, reduced fibrosis, strengthened membrane integrity, and resulted in improved muscle contractile and lengthening force, and muscle performance in mdx:lipin1Tg/0 compared to mdx mice. To confirm the role of lipin1 in dystrophic muscle, we then administered AAV1-lipin1 via intramuscular injection in mdx mice. Consistently, lipin1 restoration inhibited myofiber necroptosis and lessened muscle degeneration. Using a cell culture system, we further found that differentiated primary mdx myoblasts had elevated expression levels of necroptotic markers and medium creatine kinase (CK), which could be a result of sarcolemmal damage. Most importantly, increased lipin1 expression levels in differentiated myoblasts from mdx:lipin1Tg/0 mice substantially inhibited the elevation of necroptotic markers and medium CK levels. CONCLUSIONS: Overall, our data suggest that lipin1 is a promising therapeutic target for the treatment of dystrophic muscles.

Butyl isothiocyanate exhibits antifungal and anti-biofilm activity against Candida albicans by targeting cell membrane integrity, cell cycle progression and oxidative stress.

The prevalence of Candida albicans infection has increased during the past few years, which contributes to the need for new, effective treatments due to the increasing concerns regarding antifungal drug toxicity and multidrug resistance. Butyl isothiocyanate (butylITC) is a glucosinolate derivative, and has shown a significant antifungal effect contrary to Candida albicans. Additionally, how butylITC affects the virulence traits of C. albicans and molecular mode of actions are not well known. Present study shows that at 17.36 mM concentration butylITC inhibit planktonic growth. butylITC initially slowed the hyphal transition at 0.542 mM concentration. butylITC hampered biofilm development, and inhibits biofilm formation at 17.36 mM concentration which was analysed using metabolic assay (XTT assay) and Scanning Electron Microscopy (SEM). In addition, it was noted that butylITC inhibits ergosterol biosynthesis. The permeability of cell membranes was enhanced by butylITC treatment. Moreover, butylITC arrests cells at S-phase and induces intracellular Reactive Oxygen Species (ROS) accumulation in C. albicans. The results suggest that butylITC may have a dual mode of action, inhibit virulence factors and modulate cellular processes like inhibit ergosterol biosynthesis, cell cycle arrest, induces ROS production which leads to cell death in C. albicans.

Antifungal mechanism of rose, mustard, and their blended essential oils against Cladosporium allicinum isolated from Xinjiang naan and its storage application.

AIMS: To reveal the inhibition mechanism of rose, mustard, and blended essential oils against Cladosporium allicinum isolated from Xinjiang naan, and investigate the effect of the three essential oils on oxidative damage and energy metabolism. METHODS AND RESULTS: Rose and mustard essential oils significantly inhibited mycelial growth and spore viability in a dose-dependent relationship. After essential oil treatment, the cell membrane permeability was altered, and significant leakage of intracellular proteins and nucleic acids occurred. SEM observations further confirmed the disruption of cell structure. ROS, MDA, and SOD measurements indicated that essential oil treatment induced a redox imbalance in C. allicinum, leading to cell death. As for energy metabolism, essential oil treatment significantly reduced Na+K+-ATPase, Ca2+Mg2+-ATPase, MDH activity, and CA content, impairing metabolic functions. Finally, storage experiments showed that all three essential oils ensured better preservation of naan, with mustard essential oil having the best antifungal effect. CONCLUSIONS: Rose and mustard essential oils and their blends can inhibit C. allicinum at multiple targets and pathways, destroying cell morphological structure and disrupting metabolic processes.

From growth inhibition to ultrastructural changes: Toxicological assessment of lambda cyhalothrin and fosetyl aluminium against Bacillus subtilis and Pseudomonas aeruginosa.

In modern agricultural practices, agrochemicals and pesticides play an important role in protecting the crops from pests and elevating agricultural productivity. This strategic utilization is essential to meet global food demand due to the relentless growth of the world's population. However, the indiscriminate application of these substances may result in environmental hazards and directly affect the soil microorganisms and crop production. Considering this, an in vitro study was carried out to evaluate the pesticides' effects i.e. lambda cyhalothrin (insecticide) and fosetyl aluminum (fungicide) at lower, recommended, and higher doses on growth behavior, enzymatic profile, total soluble protein production, and lipid peroxidation of bacterial specimens (Pseudomonas aeruginosa and Bacillus subtilis). The experimental findings demonstrated a concentration-dependent decrease in growth of both tested bacteria, when exposed to fosetyl aluminium concentrations exceeding the recommended dose. This decline was statistically significant (p < 0.000). However, lambda cyhalothrin at three times of recommended dose induces 10% increase in growth of Pseudomonas aeruginosa (P. aeruginosa) and 76.8% decrease in growth of Bacillus subtilis (B. subtilis) respectively as compared to control. These results showed the stimulatory effect of lambda cyhalothrin on P. aeruginosa and inhibitory effect on B. subtilis. Pesticides induced notable alterations in biomarker enzymatic assays and other parameters related to oxidative stress among bacterial strains, resulting in increased oxidative stress and membrane permeability. Generally, the maximum toxicity of both (P. aeruginosa and B. subtilis) was shown by fosetyl aluminium, at three times of recommended dose. Fosetyl aluminium induced morphological changes like cellular cracking, reduced viability, aberrant margins and more damage in both bacterial strains as compared to lambda cyhalothrin when observed under scanning electron microscope (SEM). Conclusively the, present study provide an insights into a mechanistic approach of pyrethroid insecticide and phosphonite fungicide induced cellular toxicity towards bacteria.

Polyvinyl alcohol addition to freezing extender can improve the post-thaw quality, longevity and in vitro fertility of ram epididymal spermatozoa.

Recovering and cryopreserving epididymal spermatozoa are suitable methods for preserving the genetic potential of livestock and endangered species. Regarding encouraging reports on the use of polyvinyl alcohol (PVA) in cryopreserving various cell types, we conducted this study to examine the impact of PVA on the post-thaw quality, longevity, and in vitro fertility of ram epididymal sperm. In the first experiment, ram epididymal spermatozoa were frozen in extenders containing 6 % glycerol and 0, 0.5, 1, 2, 5, 10, or 15 mg/ml of PVA. Polyvinyl alcohol at concentrations of 0.5, 1, and 2 mg/ml improved the motility and functional membrane integrity (FMI) of the sperm compared with the control group (P < 0.05). In the second experiment, we investigated whether PVA could partially substitute glycerol in the freezing extender. PVA was added at 0, 0.5, 1, and 2 mg/ml to the extenders containing 1 % or 2 % glycerol. After thawing, the sperm motility parameters of the group containing 1 mg/ml PVA and 2 % glycerol were significantly higher than those of the un-supplemented groups (P < 0.05). In the third experiment, the effect of PVA on the post-thaw sperm longevity were examined. Sperm were frozen in 3 extenders: one containing 6 % glycerol and 1 mg/ml PVA (Gly6P1), another containing 2 % glycerol and 1 mg/ml PVA (Gly2P1), and a control extender with 6 % glycerol. After thawing, the quality of the sperm was evaluated. Sperm were then diluted in human tubal fluid (HTF) and incubated at 37 °C for 3 h. Afterwards, the quality of the sperm was evaluated once more. The presence of PVA in the freezing extender improved motility parameters and FMI. Additionally, PVA-containing groups had lower proportions of capacitated and acrosome reacted sperm compared with the control group (P < 0.05). The Gly6P1 group performed better than the other two groups (P < 0.05). In the fourth experiment, sperm from the Gly6P1 and Control groups were used in the IVF process immediately after thawing (T0) and after a 3-h incubation at 37 °C in HTF (T3). Cleavage, blastocyst and hatching rates in both groups were similar at T0, but they were lower in the Control group at T3 (P < 0.05). In conclusion, PVA as an additive to the freezing extender significantly improves post-thaw motility, viability, acrosome integrity, longevity, and fertile lifespan of ram epididymal spermatozoa.

Sperm characteristics of cryopreserved Prochilodus lineatus semen after adding cholesterol-loaded cyclodextrin.

The experiment evaluated the effect of adding cholesterol-loaded cyclodextrin (CLC) to Prochilodus lineatus fish (Curimata) semen on post-thaw sperm quality. Twelve adult fish were used for sperm collection after induced spermiation with carp pituitary gland. The semen was diluted and treated with CLC in concentrations of 0 (control), 0.5, 1.0, 2.0, 3.0, and 4.0 mg for 120 × 106 spermatozoa/ml, loaded in 0.5 ml straws, packaged and placed in dry vapor vessel cylinders for 24 h before being submerged in liquid nitrogen for storage. The samples were thawed in a water bath at 60 °C for 8 s, and the sperm parameters evaluated were motility, activation duration, longevity, plasma membrane integrity, and morphology. Data were tested for normal distribution and ANOVA, followed by Friedman test (P < 0.05). Spermatozoa treated with CLC displayed higher motility than the control (P < 0.05). The duration of sperm activation was longer in sperm treated with 0.5, 1.0, and 2.0 mg of CLC than in control (P < 0.05). The membrane integrity was higher in sperm treated with 0.5, 1.0, 2.0, and 3.0 mg of CLC than in control and four mg-treated samples (P < 0.05). The sperm longevity and morphology alterations did not differ between treatments (P > 0.05). Adding 0.5, 1.0, or 2.0 mg of CLC in Prochilodus lineatus semen before cryopreservation improves sperm motility and membrane integrity.

Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers.

The cryopreservation of endothelial cell monolayers is an important step that bridges the cryopreservation of cells in suspension to that of tissues. Previous studies have identified clear distinctions in freezing mechanisms between cells in suspension and in monolayers, as well as developed novel protocols for monolayer cryopreservation. Recently, our group has shown that human umbilical vein endothelial cell (HUVEC) and porcine corneal endothelial cell (PCEC) monolayers grown on Rinzl plastic substrate can be cryopreserved in 5% dimethyl sulfoxide, 6% hydroxyethyl starch, and 2% chondroitin sulfate, following a slow-cooling protocol (-1 °C/min) with rapid plunge into liquid nitrogen from -40 °C. However, membrane integrity assessments were done immediately post thaw, which may result in an overestimation of cell viability due to possible delayed injury responses. Here, we show that for the optimal protocol condition of plunge at the -40 °C interrupt temperature, HUVEC and PCEC monolayers exhibited no significant immediate post-thaw injuries nor delayed injury responses during the 24-h post-thaw overnight culture period. HUVEC monolayers experienced no significant impact to their natural growth rate during the post-thaw culture, while PCEC monolayers experienced significantly higher growth than the unfrozen controls. The difference in the low-temperature responses between HUVEC and PCEC monolayers was further shown under high temperature plunge conditions. At these suboptimal plunge temperatures, HUVEC monolayers exhibited moderate immediate membrane injury but a pronounced delayed injury response during the 24-h post-thaw culture, while PCEC monolayers showed significant immediate membrane injury but no additional delayed injury response during the same period. Therefore, we provide further validation of our group's previously designed endothelial monolayer cryopreservation protocol for HUVEC and PCEC monolayers, and we identify several cell-type-specific responses to the freezing process.

Effects of different cryopreservation parameters on the differences between trypan blue and fluorescent SYTO 13/GelRed assays.

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell-cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.

ROS mediated by TrPLD3 of Trichothecium roseum participated cell membrane integrity of apple fruit by influencing phosphatidic acid metabolism.

Trichothecium roseum is a typical necrotrophic fungal pathogen that not only bring about postharvest disease, but contribute to trichothecenes contamination in fruit and vegetables. Phospholipase D (PLD), as an important membrane lipid degrading enzyme, can produce phosphatidic acid (PA) by hydrolyzing phosphatidylcholine (PC) and phosphatidylinositol (PI). PA can promote the production of reactive oxygen species (ROS) by activating the activity of NADPH oxidase (NOX), thereby increasing the pathogenicity to fruit. However, the ROS mediated by TrPLD3 how to influence T. roseum infection to fruit by modulating phosphatidic acid metabolism, which has not been reported. In this study, the knockout mutant and complement strain of TrPLD3 were constructed through homologous recombination, TrPLD3 was tested for its effect on the colony growth and pathogenicity of T. roseum. The experimental results showed that the knockout of TrPLD3 inhibited the colony growth of T. roseum, altered the mycelial morphology, completely inhibited the sporulation, and reduced the accumulation of T-2 toxin. Moreover, the knockout of TrPLD3 significantly decreased pathogenicity of T. roseum on apple fruit. Compared to inoculated apple fruit with the wide type (WT), the production of ROS in apple infected with ΔTrPLD3 was slowed down, the relative expression and enzymatic activity of NOX, and PA content decreased, and the enzymatic activity and gene expression of superoxide dismutase (SOD) increased. In addition, PLD, lipoxygenase (LOX) and lipase activities were considerably decreased in apple fruit infected with ΔTrPLD3, the changes of membrane lipid components were slowed down, the decrease of unsaturated fatty acid content was alleviated, and the accumulation of saturated fatty acid content was reduced, thereby maintaining the cell membrane integrity of the inoculated apple fruit. We speculated that the decreased PA accumulation in ΔTrPLD3-inoculated apple fruit further weakened the interaction between PA and NOX on fruit, resulting in the reduction of ROS accumulation of fruits, which decreased the damage to the cell membrane and maintained the cell membrane integrity, thus reducing the pathogenicity to apple. Therefore, TrPLD3-mediated ROS plays a critical regulatory role in reducing the pathogenicity of T. roseum on apple fruit by influencing phosphatidic acid metabolism.

Antifungal Mechanism of Ruta graveolens Essential Oil: A Colombian Traditional Alternative against Anthracnose Caused by Colletotrichum gloeosporioides.

Here, we report for the first time on the mechanisms of action of the essential oil of Ruta graveolens (REO) against the plant pathogen Colletotrichum gloeosporioides. In particular, the presence of REO drastically affected the morphology of hyphae by inducing changes in the cytoplasmic membrane, such as depolarization and changes in the fatty acid profile where straight-chain fatty acids (SCFAs) increased by up to 92.1%. In addition, REO induced changes in fungal metabolism and triggered apoptosis-like responses to cell death, such as DNA fragmentation and the accumulation of reactive oxygen species (ROS). The production of essential enzymes involved in fungal metabolism, such as acid phosphatase, ß-galactosidase, ß-glucosidase, and N-acetyl-ß-glucosaminidase, was significantly reduced in the presence of REO. In addition, C. gloeosporioides activated naphthol-As-BI phosphohydrolase as a mechanism of response to REO stress. The data obtained here have shown that the essential oil of Ruta graveolens has a strong antifungal effect on C. gloeosporioides. Therefore, it has the potential to be used as a surface disinfectant and as a viable replacement for fungicides commonly used to treat anthracnose in the postharvest testing phase.

Engineering the synthesis of unsaturated fatty acids by introducing desaturase improved the stress tolerance of yeast.

BACKGROUND: Yeast is often used to build cell factories to produce various chemicals or nutrient substances, which means the yeast has to encounter stressful environments. Previous research reported that unsaturated fatty acids were closely related to yeast stress resistance. Engineering unsaturated fatty acids may be a viable strategy for enhancing the stress resistance of cells. RESULTS: In this study, two desaturase genes, OLE1 and FAD2 from Z. rouxii, were overexpressed in S. cerevisiae to determine how unsaturated fatty acids affect cellular stress tolerance of cells. After cloning and plasmid recombination, the recombinant S. cerevisiae cells were constructed. Analysis of membrane fatty acid contents revealed that the recombinant S. cerevisiae with overexpression of OLE1 and FAD2 genes contained higher levels of fatty acids C16:1 (2.77 times), C18:1 (1.51 times) and C18:2 (4.15 times) than the wild-type S. cerevisiae pY15TEF1. In addition, recombinant S. cerevisiae cells were more resistant to multiple stresses, and exhibited improved membrane functionality, including membrane fluidity and integrity. CONCLUSION: These findings demonstrated that strengthening the expression of desaturases was beneficial to stress tolerance. Overall, this study may provide a suitable means to build a cell factory of industrial yeast cells with high tolerance during biological manufacturing. © 2023 Society of Chemical Industry.

Lipin1 plays complementary roles in myofibre stability and regeneration in dystrophic muscles.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by dystrophin mutations, leading to the loss of sarcolemmal integrity, and resulting in progressive myofibre necrosis and impaired muscle function. Our previous studies suggest that lipin1 is important for skeletal muscle regeneration and myofibre integrity. Additionally, we discovered that mRNA expression levels of lipin1 were significantly reduced in skeletal muscle of DMD patients and the mdx mouse model. To understand the role of lipin1 in dystrophic muscle, we generated dystrophin/lipin1 double knockout (DKO) mice, and compared the limb muscle pathology and function of wild-type B10, muscle-specific lipin1 deficient (lipin1Myf5cKO ), mdx and DKO mice. We found that further knockout of lipin1 in dystrophic muscle exhibited a more severe phenotype characterized by increased necroptosis, fibrosis and exacerbated membrane damage in DKO compared to mdx mice. In barium chloride-induced muscle injury, both lipin1Myf5cKO and DKO showed prolonged regeneration at day 14 post-injection, suggesting that lipin1 is critical for muscle regeneration. In situ contractile function assays showed that lipin1 deficiency in dystrophic muscle led to reduced specific force production. Using a cell culture system, we found that lipin1 deficiency led to elevated expression levels of necroptotic markers and medium creatine kinase, which could be a result of sarcolemmal damage. Most importantly, restoration of lipin1 inhibited the elevation of necroptotic markers in differentiated primary lipin1-deficient myoblasts. Overall, our data suggests that lipin1 plays complementary roles in myofibre stability and muscle function in dystrophic muscles, and overexpression of lipin1 may serve as a potential therapeutic strategy for dystrophic muscles. KEY POINTS: We identified that lipin1 mRNA expression levels are significantly reduced in skeletal muscles of Duchenne muscular dystrophy patients and mdx mice. We found that further depletion of lipin1 in skeletal muscles of mdx mice induces more severe dystrophic phenotypes, including enhanced myofibre sarcolemma damage, muscle necroptosis, inflammation, fibrosis and reduced specific force production. Lipin1 deficiency leads to elevated expression levels of necroptotic markers, whereas restoration of lipin1 inhibits their expression. Our results suggest that lipin1 is functionally complementary to dystrophin in muscle membrane integrity and muscle regeneration.

Hydrophobic Residues at the Intracellular Domain of the M2 Protein Play an Important Role in Budding and Membrane Integrity of Influenza Virus.

M2 protein of influenza virus plays an important role in virus budding, including membrane scission and vRNP packaging. Three hydrophobic amino acids (91F, 92V, and 94I) at the intracellular domain of the M2 protein constitute a hydrophobic motif, also known as the LC3-interacting region (LIR), whereas the role of this motif remains largely unclear. To explore the role of the 91-94 hydrophobic motif for influenza virus, all three hydrophobic amino acids were mutated to either hydrophilic S or hydrophobic A, resulting in two mutant viruses (WSN-M2/SSS and WSN-M2/AAA) in the background of WSN/H1N1. The results showed that the budding ability of the M2/SSS protein was inhibited and the bilayer membrane integrity of the WSN-M2/SSS virion was impaired based on transmission electron microscopy (TEM), which in turn abolished the resistance to trypsin treatment. Moreover, the mutant WSN-M2/SSS was dramatically attenuated in mice. In contrast, the AAA mutations did not have a significant effect on the budding of the M2 proteins or the bilayer membrane integrity of the viruses, and the mutant WSN-M2/AAA was still lethal to mice. In addition, although the 91-94 motif is an LIR, knocking out of the LC3 protein of A549 cells did not significantly affect the membrane integrity of the influenza viruses propagated on the LC3KO cells, which suggested that the 91-94 hydrophobic motif affected the viral membrane integrity and budding is independent of the LC3 protein. Overall, the hydrophobicity of the 91-94 motif is crucial for the budding of M2, bilayer membrane integrity, and pathogenicity of the influenza viruses. IMPORTANCE M2 plays a crucial role in the influenza virus life cycle. However, the function of the C-terminal intracellular domain of M2 protein remains largely unclear. In this study, we explored the function of the 91-94 hydrophobic motif of M2 protein. The results showed that the reduction of the hydrophobicity of the 91-94 motif significantly affected the budding ability of the M2 protein and impaired the bilayer membrane integrity of the mutant virus. The mouse study showed that the reduction of the hydrophobicity of the 91-94 motif significantly attenuated the mutant virus. All of the results indicated that the hydrophobicity of the 91-94 motif of the M2 protein plays an important role in budding, membrane integrity, and pathogenicity of influenza virus. Our study offers insights into the mechanism of influenza virus morphogenesis, particularly into the roles of the 91-94 hydrophobic motif of M2 in virion assembly and the pathogenicity of the influenza viruses.

Membrane component ergosterol builds a platform for promoting effector secretion and virulence in Magnaporthe oryzae.

The plasma membrane (PM) functions as a physical border between the extracellular and cytoplasmic environments that contribute to the interaction between host plants and pathogenic fungi. As a specific sterol constituent in the cell membrane, ergosterol plays a significant role in fungal development. However, the role of ergosterol in the infection of the rice blast fungus Magnaporthe oryzae remains unclear. In this study, we found that a sterol reductase, MoErg4, is involved in ergosterol biosynthesis and the regulation of plasma membrane integrity in M. oryzae. We found that defects in ergosterol biosynthesis disrupt lipid raft formation in the PM and cause an abnormal distribution of the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein MoSso1, inhibiting its interaction with the v-SNARE protein MoSnc1. In addition, we found that MoSso1-MoSnc1 interaction is important for biotrophic interface complex development and cytoplasmic effector protein secretion. Our findings suggested that ergosterol-enriched lipid rafts constitute a platform for interactions among various SNARE proteins that are required for the development and pathogenicity of M. oryzae.

Cryopreservation of human cerebral microvascular endothelial cells with glycerol.

The cryopreservation of human cerebral microvascular endothelial cells (hCMEC) has facilitated their commercial availability for research studying the blood-brain barrier. The currently employed cryopreservation protocol uses 10% dimethyl sulfoxide (Me2SO) in cell medium, or 5% Me2SO in 95% fetal bovine serum (FBS) as cryoprotective agents (CPAs). However, Me2SO is toxic to cells and FBS is animal-derived and not chemically defined, so reducing the concentrations of these components is desirable. Recently, we showed that cryopreserving hCMEC in cell medium with 5% Me2SO and 6% hydroxyethyl starch (HES) results in over 90% post-thaw cell viability. This previous work was performed using an interrupted slow cooling (graded freezing) approach followed by SYTO13/GelRed staining to assay for membrane integrity. In this paper, we repeated graded freezing of hCMEC in cell medium containing 5% Me2SO and 6% HES, but this time using Calcein AM/propidium iodide staining to ensure that the stain is an equivalent alternative to SYTO13/GelRed for assessment of cell viability, and that results are comparable to those previously published. Next, using graded freezing experiments and Calcein AM/propidium iodide staining, we examined the effectiveness of non-toxic glycerol as a CPA at different concentrations, loading times, and cooling rates. The cryobiological response of hCMEC was used to develop a protocol that optimizes both the permeating and non-permeating capabilities of glycerol. HCMEC in cell medium loaded with 10% glycerol for 1 h at room temperature, ice nucleated at -5 °C and held for 3 min, and then cooled at -1 °C/min to -30 °C before plunging into liquid nitrogen had post-thaw viability of 87.7% ± 1.8%. Matrigel tube formation assay and immunocytochemical staining of junction protein ZO-1 were carried out on post-thaw hCMEC to ensure that the cryopreserved cells were viable and functional, in addition to being membrane-intact.

Antibacterial Activity of Juglone Revealed in a Wound Model of Staphylococcus aureus Infection.

A serious problem currently facing the field of wound healing is bacterial infection, especially Staphylococcus aureus (S. aureus) infection. Although the application of antibiotics has achieved good effects, their irregular use has resulted in the emergence of drug-resistant strains. It is thus the purpose of this study to analyze whether the naturally extracted phenolic compound, juglone, can inhibit S. aureus in wound infection. The results show that the minimum inhibitory concentration (MIC) of juglone against S. aureus was 1000 µg/mL. Juglone inhibited the growth of S. aureus by inhibiting membrane integrity and causing protein leakage. At sub-inhibitory concentrations, juglone inhibited biofilm formation, the expression of α-hemolysin, the hemolytic activity, and the production of proteases and lipases of S. aureus. When applied to infected wounds in Kunming mice, juglone (50 µL juglone with a concentration of 1000 µg/mL) significantly inhibited the number of S. aureus and had a significant inhibitory effect on the expression of inflammatory mediators (TNF-α, IL-6 and IL-1ß). Moreover, the juglone-treated group promoted wound healing. At the same time, in animal toxicity experiments, juglone had no obvious toxic effects on the main tissues and organs of mice, indicating that juglone has good biocompatibility and has the potential to be used in the treatment of wounds infected with S. aureus.

Functional Characterization of Lobularia maritima LmTrxh2 Gene Involved in Cold Tolerance in Tobacco through Alleviation of ROS Damage to the Plasma Membrane.

Cold stress is a key environmental factor affecting plant growth and development, crop productivity, and geographic distribution. Thioredoxins (Trxs) are small proteins that are ubiquitously expressed in all organisms and implicated in several cellular processes, including redox reactions. However, their role in the regulation of cold stress in the halophyte plant Lobularia maritima remains unknown. We recently showed that overexpression of LmTrxh2, which is the gene that encodes the h-type Trx protein previously isolated from L. maritima, led to an enhanced tolerance to salt and osmotic stress in transgenic tobacco. This study functionally characterized the LmTrxh2 gene via its overexpression in tobacco and explored its cold tolerance mechanisms. Results of the RT-qPCR and western blot analyses indicated differential temporal and spatial regulation of LmTrxh2 in L. maritima under cold stress at 4 °C. LmTrxh2 overexpression enhanced the cold tolerance of transgenic tobacco, as evidenced by increased germination rate, fresh weight and catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities; reduced malondialdehyde levels, membrane leakage, superoxide anion (O2-), and hydrogen peroxide (H2O2) levels; and higher retention of chlorophyll than in non-transgenic plants (NT). Furthermore, the transcript levels of reactive oxygen species (ROS)-related genes (NtSOD and NtCAT1), stress-responsive late embryogenis abundant protein 5 (NtLEA5), early response to dehydration 10C (NtERD10C), DRE-binding proteins 1A (NtDREB1A), and cold-responsive (COR) genes (NtCOR15A, NtCOR47, and NtKIN1) were upregulated in transgenic lines compared with those in NT plants under cold stress, indicating that LmTrxh2 conferred cold stress tolerance by enhancing the ROS scavenging ability of plants, thus enabling them to maintain membrane integrity. These results suggest that LmTrxh2 promotes cold tolerance in tobacco and provide new insight into the improvement of cold-stress resistance to cold stress in non-halophyte plants and crops.