Cell Membrane Function Research Articles

Sphingolipid Metabolism and Signaling in Lung Cancer: A Potential Therapeutic Target.

Sphingolipids are important bioactive lipids that not only play an important role in maintaining the barrier function and fluidity of cell membranes but also regulate multiple processes in cancer development by controlling multiple signaling pathways in the signal transduction network. Dysregulation of sphingolipid metabolism is thought to be one of the most important dysregulated pathways in lung cancer, the most prevalent type of cancer in terms of incidence and mortality worldwide. This article focuses on lung cancer, reviewing the important lipids in sphingolipid metabolism and the related enzymes in relation to lung cancer progression and their effects on the tumor microenvironment and discussing their roles in the diagnosis and treatment of lung cancer.

Integrated Analyses of DNA Methylation and Gene Expression of Rainbow Trout Muscle under Variable Ploidy and Muscle Atrophy Conditions.

Rainbow trout, Oncorhynchus mykiss, is an important cool, freshwater aquaculture species used as a model for biological research. However, its genome reference has not been annotated for epigenetic markers affecting various biological processes, including muscle growth/atrophy. Increased energetic demands during gonadogenesis/reproduction provoke muscle atrophy in rainbow trout. We described DNA methylation and its associated gene expression in atrophying muscle by comparing gravid, diploid females to sterile, triploid females. Methyl Mini-seq and RNA-Seq were simultaneously used to characterize genome-wide DNA methylation and its association with gene expression in rainbow trout muscle. Genome-wide enrichment in the number of CpGs, accompanied by depleted methylation levels, was noticed around the gene transcription start site (TSS). Hypermethylation of CpG sites within ±1 kb on both sides of TSS (promoter and gene body) was weakly/moderately associated with reduced gene expression. Conversely, hypermethylation of the CpG sites in downstream regions of the gene body +2 to +10 kb was weakly associated with increased gene expression. Unlike mammalian genomes, rainbow trout gene promotors are poor in CpG islands, at <1% compared to 60%. No signs of genome-wide, differentially methylated (DM) CpGs were observed due to the polyploidy effect; only 1206 CpGs (0.03%) were differentially methylated, and these were primarily associated with muscle atrophy. Twenty-eight genes exhibited differential gene expression consistent with methylation levels of 31 DM CpGs. These 31 DM CpGs represent potential epigenetic markers of muscle atrophy in rainbow trout. The DM CpG-harboring genes are involved in apoptosis, epigenetic regulation, autophagy, collagen metabolism, cell membrane functions, and Homeobox proteins. Our study also identified genes explaining higher water content and modulated glycolysis previously shown as characteristic biochemical signs of rainbow trout muscle atrophy associated with sexual maturation. This study characterized DNA methylation in the rainbow trout genome and its correlation with gene expression. This work also identified novel epigenetic markers associated with muscle atrophy in fish/lower vertebrates.

Oxidative Stress in the Oral Cavity before and After Prosthetic Treatment

BACKGROUND: Metal ions emitted from dental alloys may induce oxidative stress leading to numerous pathological changes. Lipid peroxidation may cause disturbance of structure and function of cell membranes, apoptosis, autophagy, and formation of potentially mutagenic compounds. Products of interaction between reactive oxygen species and biomolecules may be used for evaluation of oxidative stress level. AIM: The aim of this study was to evaluate the influence of the prosthetic dental treatment with metal ceramic restorations on the level of oxidative stress in the oral cavity. MATERIALS AND METHODS: Metal ceramic crowns with copings fabricated by direct metal laser sintering were produced for 35 patients. CoCr dental alloy EOS CobaltChrome SP2 (EOS) was used. Non-stimulated and stimulated saliva samples were collected from the patients before and after the prosthetic treatment. For evaluation of oxidative stress concentration of 8-isoPGF2-alpha was measured by liquid chromatography tandem mass spectrometry. For statistical processing, non-parametric Wilcoxon signed-rank test and Mann–Whitney test were applied. RESULTS: The concentration of isoprostane 8-isoPGF2-alpha in non-stimulated saliva was lower 2 h after fixing the crowns compared to the initial level and statistically significant difference was observed. On the 7th day the concentration of isoprostanes remained significantly lower than the initial one. No significant differences were found in isoprostane concentration in stimulated saliva before and after prosthetic treatment. CONCLUSION: Prosthetic dental treatment leads to decrease in oral oxidative stress.

Abstract 5804: The inhibition of PLA2G2A decreases K-ras-driven pancreatic cancer growth and migration through damaging cell membrane and mitochondria function

Abstract The activation of oncogenic K-ras by mutations may stimulate multiple cellular processes including lipids modification. However, the mechanisms by which K-ras affects lipid metabolism remain poorly understood. In this study, we found that PLA2G2A, an enzyme that hydrolyzes membrane phospholipids at sn-2 position to release polyunsaturated fatty acids (PUFA), was up-regulated by K-ras activation and its high expression was correlated with poor survival of patients with pancreatic ductal adenocarcinoma (PDAC). Abrogation of PLA2G2A by siRNA silencing or pharmacological inhibition significantly suppressed the proliferation of K-ras-driven cancer cells, as evidenced by inhibition of cell growth and loss of colony formation capacity. Suppression of PLA2G2A could also decrease K-ras-driven PDAC cell migration. Mechanistic study showed that PLA2G2A inhibition rendered cells vulnerability to lipid peroxidation, likely due to accumulation of excessive PUFA in the membranes. Such alterations resulted in membrane damage and mitochondrial dysfunction with elevated ROS, leading to a reduction in energy production and a decrease in acetyl-CoA, which further compromised de novo lipid biosynthesis. Our study suggests that PLA2G2A-mediated cell membrane remodeling plays an important role in cell survival and proliferation in during K-ras-driven cancer, and that PLA2G2 could be a potential therapeutic target for pancreatic cancer treatment. Citation Format: Mingquan zhang, Peng huang. The inhibition of PLA2G2A decreases K-ras-driven pancreatic cancer growth and migration through damaging cell membrane and mitochondria function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5804.

Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

Phosphatidylinositol 4-phosphate: a key determinant of plasma membrane identity and function in plants.

Phosphatidylinositol 4-phosphate (PI4P) is an anionic phospholipid which has been described as a master regulator of the Golgi apparatus in eukaryotic cells. However, recent evidence suggests that PI4P mainly accumulates at the plasma membrane in all plant cells analyzed so far. In addition, many functions that are typically attributed to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) in animal and yeast cells are also supported by PI4P in plants. For example, PI4P is the key anionic lipid that powers the strong electrostatic properties of the plasma membrane. Phosphatidylinositol 4-phosphate is also required for the establishment of stable membrane contacts between the endoplasmic reticulum and the plasma membrane, for exocytosis and to support signaling pathways. Thus, we propose that PI4P has a prominent role in specifying the identity of the plasma membrane and in supporting some of its key functions and should be considered a hallmark lipid of this compartment.

A novel strategy for improving watermelon resistance to cucumber green mottle mosaic virus by exogenous boron application.

The molecular mode controlling cucumber green mottle mosaic virus (CGMMV)‐induced watermelon blood flesh disease (WBFD) is largely unknown. In this study, we have found that application of exogenous boron suppressed CGMMV infection in watermelon fruit and alleviated WBFD symptoms. Our transcriptome analysis showed that the most up‐regulated differentially expressed genes (DEGs) were associated with polyamine and auxin biosynthesis, abscisic acid catabolism, defence‐related pathways, cell wall modification, and energy and secondary metabolism, while the down‐regulated DEGs were mostly involved in ethylene biosynthesis, cell wall catabolism, and plasma membrane functions. Our virus‐induced gene silencing results showed that silencing of SPDS expression in watermelon resulted in a higher putrescine content and an inhibited CGMMV infection correlating with no WBFD symptoms. SBT and TUBB1 were also required for CGMMV infection. In contrast, silencing of XTH23 and PE/PEI7 (low‐level lignin, cellulose and pectin) and ATPS1 (low‐level glutathione) promoted CGMMV accumulation. Furthermore, RAP2‐3, MYB6, WRKY12, H2A, and DnaJ11 are likely to participate in host antiviral resistance. In addition, a higher (spermidine + spermine):putrescine ratio, malondialdehyde content, and lactic acid content were responsible for fruit decay and acidification. Our results provide new knowledge on the roles of boron in watermelon resistance to CGMMV‐induced WBFD. This new knowledge can be used to design better control methods for CGMMV in the field and to breed CGMMV resistant watermelon and other cucurbit crops.

Shielding effect of melatonin improves seminal quality and oxidative stress indices during chilled storage of ram semen.

Supplementation of antioxidant to semen extender maintains seminal quality by reducing oxidative stress during preservation time period. The main aim of this study was to investigate the effect of different concentrations of melatonin supplementation on liquid storage of Magra ram semen. This study was performed on adult Magra ram (n = 8), and seminal ejaculates (48) were collected and evaluated for various macroscopic and microscopic seminal quality parameters for further processing. After preliminary evaluation, ejaculates of each collection session were mixed and divided into four equal aliquots. All the aliquots were diluted (1:10) with Tris-citric fructose egg yolk extender contained sans melatonin served as control, whereas the other three aliquots were supplemented with 0.5, 1 and 2mM MLT which were grouped as MLT0.5, MLT1 and MLT2, respectively. Thereafter, the samples were stored at 4 ºC for 72h, and various seminal parameters (individual sperm progressive motility, viability, abnormalities, plasma membrane functionality) along with oxidative stress parameters (total antioxidant capacity (TAC), malondialdehyde (MDA)) were evaluated at 0, 24, 48 and 72h of preservation. The results indicated that the mean percent values for progressive sperm motility, viability, plasma membrane functionality and TAC were significantly higher (p < 0.05) in treated groups with the highest values in MLT1 group. Significantly lower (p < 0.05) percentage of total sperm abnormalities and MDA level was observed in treatment groups compared to control group. The results report that supplementation of 1mM melatonin efficiently maintains the seminal quality and ameliorates oxidative stress during preservation at 4 ºC to 72h.

The Influence of Rumen-Protected Choline and α-tocopherol Supplementation on Early Lactating Dairy Cows Metabolism.

It is well documented that choline is known as one of the essential ingredients of phospholipids. Choline acts as a determinative element for appropriate cell membrane functions. On the other hand α-tocopherol (Vit E) is a fat-soluble vitamin. This vitamin acts as a strong antioxidant in the living body's defense system against oxidative stress. Lipid peroxidation in peripartum and early lactating cows is significantly increased while the level of serum Vit E is decreases dramatically. These concomitant physiological changes demonstrate a higher level of oxidative stress subsequently leads to serious health issues in dairy cows. Therefore, the present research was designed to investigate the following items in dairy cattle: 1) evaluation of the possible changes in serum protein fractions, and 2) comparing the oxidative status of orally RPC and vitamin E supplementation in dairy cows in early lactation period. In the current study 30 early lactating primiparous and multiparous Holstein cows (body condition score (BCS)=2.51 ± 0.10) were used beginning five weeks postpartum. All the animals were randomly divided in to three groups (n=10) (number of lactation=2.61). The animals were randomly assigned to receive one of the following treatments. Group 1 served as control group were not received any supplement. The second group was supplemented with 90 g/d of RPC (Reashre Choline, Balchem, USA). The third group was administrated 4400 IU/d vitamin E (Roche, Vitamins Ltd; Switzerland). In the current study, serum protein electrophoresis showed four main fractions as follows: albumin, α-globulin, β-globulin, and γ-globulin. The recorded data showed that the percentages of albumin and γ-globulin fractions were higher in treated groups compared to the control group. In the animals supplementing with RPC and vitamin E the percentages of serum albumin increased to the value of 37. 70±1.63 and 38.21±1.28 respectively compare to the control group (34.69±1.21), which were significant (P<0.05).

Advancements in cell membrane camouflaged nanoparticles: A bioinspired platform for cancer therapy.

The idea of employing natural cell membranes as a coating medium for nanoparticles (NPs) endows man-made vectors with natural capabilities and benefits. In addition to retaining the physicochemical characteristics of the NPs, the biomimetic NPs also have the functionality of source cell membranes. It has emerged as a promising approach to enhancing the properties of NPs for drug delivery, immune evasion, imaging, cancer-targeting, and phototherapy sensitivity. Several studies have been reported with a multitude of approaches to reengineering the surface of NPs using biological membranes. Owing to their low immunogenicity and intriguing biomimetic properties, cell-membrane-based biohybrid delivery systems have recently gained a lot of interest as therapeutic delivery systems. This review summarises different kinds of biomimetic NPs reported so far, their fabrication aspects, and their application in the biomedical field. Finally, it briefs on the latest advances available in this biohybrid concept.

Effect of aqueous Nigella sativa extract on the functional parameters of post-thaw human spermatozoa during vitrification.

ObjectiveSperm vitrification leads to the production of reactive oxygen species (ROS) that can damage the functional parameters of sperm. The present study aimed to investigate the antioxidant effect of Nigella sativa extract on motility, plasma membrane function, mitochondrial membrane potential (MMP), DNA damage, and intracellular ROS production.MethodsA total of 20 sperm samples were used. Samples were divided into six experimental groups, including groups with aqueous extract from N. sativa seeds at concentrations of 1% to 6%, a cryopreserved control group, and a fresh control group.ResultsStatistical analysis showed significantly higher total sperm motility at concentrations of 3% to 6% than in the vitrified semen control group. Additionally, progressive motility and all motion characteristics at all concentrations were significantly higher than in the vitrified semen control group. The presence of N. sativa seed extract also improved the quality of the sperm parameters assayed in all experimental groups (1%–6%; intracellular ROS production, DNA damage, MMP, and sperm membrane function) compared to the control group.ConclusionHigher concentrations of N. sativa led to improvements in all sperm parameters and sperm quality. These findings indicate that N. sativa seed extract is effective for improving the quality of sperm after vitrification.

Acid adaptive response of Alicyclobacillus acidoterrestris: A strategy to survive lethal heat and acid stresses

Alicyclobacillus acidoterrestris causes the spoilage of pasteurized acidic fruit juice, seriouslydecreasing quality and posing a significant safety concern. We previously discovered that acid adaptation could induce stress adaptive responses of A. acidoterrestris, however, the underlying mechanisms of this induction have not been fully elucidated. In this work, the effects of acid adaptation (pH = 3.0, 1 h) on intracellular pH (pHi) and the morphophysiological properties of A. acidoterrestris under lethal heat and acid stresses were investigated, and gene expression profiles after acid adaptation were measured by transcriptomic analysis. The results showed that acid adaptation increased the pHi of A. acidoterrestris cells in response to lethal stresses, enhanced membrane integrity, decreased surface shrinkage and roughness, and altered the Fourier transform infrared spectra profiles. After acid adaptation of A. acidoterrestris, 517 differentially expressed genes (DEGs) were detected. Consistent with resistance phenotypes, DEGs included genes related to cell surface modification and pHi homeostasis. Specifically, the barrier function of cell membrane was strengthened during acid adaptation by increasing fatty acid (FA) chain length, promoting unsaturated FA biosynthesis, and maintaining balanced synthesis of zwitterionic and acidic phospholipids. To reduce excessive intracellular protons, cells upregulated glutamate decarboxylation, urease system, and branched-chain amino acid synthesis. Additionally, the nucleotide salvage pathway was activated, and homologous recombination, UvrD-mediated transcription-coupled, and ribonucleotide excision repair pathways were applied to repair DNA lesions. Sporulation metabolism was also induced. The findings of this study provide insight into the multiple layers of acid adaptive response strategies of A. acidoterrestris, with implications for the formulation of improved control measures in the fruit juice industry.

The importance of muscle mass in predicting intradialytic hypotension in patients undergoing maintenance hemodialysis.

BackgroundPatients undergoing hemodialysis are susceptible to sarcopenia. As intracellular reservoirs of water, skeletal muscles are important contributors to intradialytic hypotension. This study was designed to determine the role of skeletal muscle mass in intradialytic hypotension. MethodsIn a cross-sectional study, the body composition of 177 patients was measured immediately after hemodialysis using bioelectrical impedance analysis. The parameters measured were skeletal muscle mass, intracellular and extracellular water contents, total body water, and cell-membrane functionality (in phase angle at 50 kHz). Data from laboratory tests, chest radiography, measurements of handgrip strength and mid-arm circumference, and questionnaires were collected. The main outcome was intradialytic hypotension, defined as more than two episodes of hypotension (systolic blood pressure of <90 mmHg) with intervention over the 3 months following enrollment. Logistic regression models including each parameter related to sarcopenia were compared with a clinical model.ResultsPatients with a low ratio of skeletal muscle mass to dry body weight (SMM/WT) had a higher rate of intradialytic hypotension (40.7%). Most low-SMM/WT patients were female, obese, diabetic, and had a lower handgrip strength compared with the other patients. In the high-SMM/WT group, the risk of intradialytic hypotension was lower, with an odds ratio of 0.08 (95% confidence interval [CI], 0.02–0.28) and adjusted odds ratio of 0.06 (95% CI, 0.01–0.29).ConclusionMeasurement and maintenance of skeletal muscle can help prevent intradialytic hypotension in frail patients undergoing hemodialysis.

Physiological and transcriptomic analysis of postharvest Jiashi melon at different storage temperatures

Low temperature storage prolongs the postharvest life of various fresh fruit and vegetables. However, tropical fruit may suffer from cold stress during storage. Here, Jiashi melons was stored at 3 °C, and 0.5 °C for a period of 36 days, with storage at 21 °C serving as a control. Rotting was delayed at the lower temperatures compared to the control. Storage at 21 °C resulted in more severe weight loss and a high rotting rate. By day 36, the weight loss rate as a result of moisture reduction reached 11% in the control, but only 8% at the lower temperatures. Chilling injury was observed at both low temperatures over time, but its onset was later for storage at 3 °C. The fruit stored at 3 °C also scored better for rotting rate, weight loss rate, and a chilling injury index than fruit stored at 0.5 °C. Storage at 3 °C was associated with higher activity of peroxidase (POD), catalase (CAT), fatty acid desaturase (FAD), and adenosine triphosphatase (ATPase). The transcriptomes of the fruit were sequenced for the time points 0 d, 12 d and 24 d at all three storage temperatures. This identified a total of 415 co-expressed differential genes that are mainly involved in regulation of transcription, signal transduction, response to stress, lipid metabolism and other processes. The findings were corroborated by targeted RT-qPCR analysis. The expression levels of differentially expressed genes (DEGs) indicated that storage at 3 °C induced overexpression of the early cold-responsive genes ICE1 and calcium-dependent protein kinase (CDPK). Extended storage at 3 °C reduced the damage of low-temperature induced oxidative stress and altered lipid metabolism that otherwise would negatively affect cell membrane functions, so that cell homeostasis was maintained for longer. This was enabled by increased expression of MYB, auxin response factor, heat shock protein (HSPs), FAD synthetase and ATPase. This study provides insights in the molecular responses of Jiashi melon towards cold stress, and provides a theoretical basis for the development of new storage and preservation technologies.

ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF Impatiens balsamina (L.): LITERATURE REVIEW ARTICLE

The human body contains a diverse range of microorganisms, including bacteria and fungi. The most common bacteria and fungi caused skin pathogens are Staphylococcus aureus, Propionibacterium acnes, Staphylococcus epidermis and Candida albicans. Many studies have found that Impatiens balsamina (L.) contains antibacterial and antifungal activities. Acne and Candidiasis are diseases that often arise and can interfere with comfort for sufferers. Usually, acne is caused by the bacteria Propionibacterium acnes, Staphylococcus epidermis and Staphylococcus aureus, and Candidiasis caused by the Candida albicans. One of the plants that is widely used as an antiacne and antifungal treatment is the Impatiens balsamina (L.). Impatiens balsamina (L.) has antiacne and antifungal activities. The purpose of the review is to find out if the Impatiens balsamina (L.) has activity as an antiacne, antifungal and can identify secondary metabolites that can inhibit the emergence of bacteria in acne and candidiasis in various journals. The method used is the Literature Review Article (LRA) method. A search through Google Scholar and Pubmed found 12 articles that Impatiens balsamina (L.) contains secondary metabolites, namely flavonoids and saponins. The mechanism of action of flavonoids is by inhibiting the function of cell membranes and energy metabolism of bacteria, while the mechanism of action of saponins as antifungals is by damaging cell membranes, causing cell leakage which ultimately leads to cell death. From the two mechanisms, these secondary metabolites can have antiacne and antifungal activities.

Use of a Short, In‐Class, Free Response Activity to Assess Student Understanding of Core Concepts of the Cell Membrane in an Undergraduate Physiology Course

Students in an upper division, stand‐alone animal physiology course at Missouri Western State University are required to have completed a freshman‐level introductory cell biology course and a sophomore‐level genetics course as prerequisites. These prerequisite courses include discussion of the eukaryotic cell membrane and basic properties and functions of the membrane. However, many students remember the basic information about membrane structure but have difficulty relating that structure to membrane function and related topics in our physiology course, including vesicular transport, primary and secondary active transport, osmosis, and current flow across the membrane. To gain a better understanding of what students recall about the cell membrane before covering membrane structure and function in class, students were given an open‐ended prompt, “What do you know about the structure and function of the animal cell membrane?” The students were allowed to work in small groups or alone, and could generate their response as drawings, written statements, or both. They were given 10 minutes to construct a response. These student responses were analyzed using the conceptual framework for the “cell membrane” core concept recently published by Michael and Modell (Adv Physiol Educ 2019). This activity was repeated 2 weeks after finishing discussing of the cell membrane. Prior to covering this content in class, 100% (19 out of 19) submissions included a drawing or description of the cell membrane as a phospholipid bilayer, and 84% of submissions included integral and peripheral membrane proteins. Similar percentages of students included these concepts in the post‐coverage assessment. However, other components of the “cell membrane” conceptual framework were included much less frequently or not at all prior to covering the content in class, but improved dramatically afterwards. In the pre‐assessment, only 11% of responses included solute concentration gradients or selective permeability, while 70% of responses in the post‐assessment included this concepts. In the pre‐assessment less than 10% mentioned vesicular transport, receptors, or channel proteins; after covering the content in class, these topics were included by 15%, 45%, and 35% of student responses, respectively. This quick activity provides valuable information about what students recall from prior coursework and what concepts need to be revisited before moving into more detailed discussion of cell membrane function. When used in addition to objective exam or assignment questions, this free‐response activity can also provide a complementary view of student understanding of core concepts of the cell membrane.

Regulation of PIP2 homeostasis at ER‐plasma membrane contacts by Nir proteins

Phosphatidylinositol 4,5‐bisphosphate (PIP2) in the plasma membrane is a critical lipid molecule that regulates nearly all functional processes occurring at the cell surface. Additionally, PIP2 is hydrolyzed following receptor‐induced activation of phospholipase C to generate second messengers for signal transduction. It is crucial to quickly replenish PIP2 in the plasma membrane following receptor‐induced hydrolysis to sustain signaling outputs and to maintain PIP2‐dependent cellular functions. Replenishment of plasma membrane PIP2 in receptor‐stimulated cells involves transport of phosphatidylinositol (PI) from the endoplasmic reticulum (ER) to the plasma membrane for PIP2 re‐synthesis. ER‐plasma membrane contacts are subcellular loci characterized by the close apposition of the ER to the plasma membrane. Recent findings revealed that the PI transfer proteins, Nir2 and Nir3, dynamically localize at ER‐plasma membrane contacts to mediate replenishment of PIP2 hydrolyzed following receptor stimulation. Deficiency in Nir2 and Nir3 suppressed replenishment of PIP2 in the plasma membrane and Ca2+ signaling in receptor‐stimulated cells. A third mammalian Nir protein, Nir1, has been linked to several human diseases including retinal dystrophy. Unlike Nir2 and Nir3, Nir1 lacks a PI transfer protein domain and readily localizes at ER‐plasma membrane contacts in resting cells. The presence of Nir1 promotes Nir2 localization at ER‐plasma membrane contacts to mediate PIP2 replenishment following receptor stimulation. Together, Nir proteins maintain plasma membrane PIP2 homeostasis at ER‐plasma membrane contacts to support cell signaling and plasma membrane functions in receptor‐stimulated cells.

Abstract 238: Serum Opacity Factor Normalizes Dysfunctional High Density Lipoproteins In Srbi -/- Female Mice And Rescues Fertility

Although plasma HDL-C levels negatively correlate with atherosclerotic cardiovascular disease (ACVD), attempts to reduce ACVD risk by raising plasma HDL have disappointed. Thus, hypotheses about salutary HDL effects have shifted from higher-is-better to function-is-more-important. The SRBI-/- mouse is an extreme model of HDL dsyfunctionality; compared to WT mice, SRB1-/- mice have higher plasma HDL levels and an HDL surface that is free cholesterol (FC)-rich (60 vs. 15 mol%). This would be expected to increase HDL-FC bioavailability to cytotoxic levels. HDL dysfunctionality among SRB1-/- mice is associated with multiple metabolic abnormalities—impaired cell membrane structure and function and atherosusceptibility, despite having high plasma HDL-C levels, and infertility among female SRB1-/- mice. Dietary probucol, an HDL-lowering drug, partially reverses infertility in SR-B1-/- mice. The mechanisms underlying ovaritoxicity is not known and interventions that fully reverse this state has not been identified. We hypothesized that a bacterial protein, serum opacity factor (SOF), which acts on HDL and lowers cholesterol in WT mice by 43% in 3 hours, would normalize HDL functionality and rescue infertility in SRBI-/- female mice. SOF DNA was cloned into a TBG-AAV8 plasmid and virus produced by UPENN Vector Lab. The AAV-SOF was delivered by IP injection leading to the endogenous expression of SOF. Probucol-fed and AAV-GFP mice were used as controls. SOF expression drastically decreased plasma cholesterol and normalized the size of HDL in SRBI-/- females comparable to that of WT mice. Fertility was restored in female mice injected with SOF while GFP controls remained infertile. Compared to Probucol-fed mice, AAV-SOF females had a higher percentage of fertile females and shorter time to first litter drop. We concluded that SOF performed equally or better than probucol in rescuing fertility and normalizing HDL.

Selective Enrichment of Cell Surface Proteins Using ProMTag to Detect O‐glycosylation‐Dependent Changes

O‐linked glycosylation, which is regulated by the enzyme GalNAc transferase, plays a vital role in cellular function by regulating membrane protein composition and sorting. Changes in O‐linked glycosylation are very common in diseases such as cancers, diabetes, and Alzheimer’s. It is important to study the effects of changes in O‐link glycosylation on cell membrane composition and function to understand how these changes affect the onset, phenotype, and progression of these diseases. However, membrane proteins are notoriously challenging to analyze at the proteomic level due to their hydrophobicity and the inability to separate the membrane proteins from other proteins in the cell.Here we introduce a new sample preparation workflow that enriches membrane proteins for mass spectrometry analysis to understand changes in protein composition of the plasma membrane as the result of changes in O‐linked glycosylation. To do this, we used the bifunctional tag ProMTag to label and capture plasma membrane proteins via their extracellular domains in cells treated with GalNAc Transferase inhibitors. One end of the ProMTag forms a reversible, covalent bond with protein the other end of the ProMTag is capable of forming an irreversible, covalent bond with a solid bead support via the click chemistry pair of methyltetrazine and trans‐Cyclooctene (TCO). The extracellular domains of proteins from intact wild type HEK cells and HEK cells treated with GalNAc Transferase inhibitors were ProMTagged, captured, cleaned up, and eluted for comparison by mass spectrometry (MS) analysis. MS confirmed enrichment of plasma membrane proteins after sample preparation and protein changes in the plasma membrane as a result of GalNAc Transferase inhibition were characterized. This work establishes a method for enrichment of plasma membrane proteins and reveals how changes in O‐linked glycosylation via GalNAc Transferase inhibition leads to changes in plasma membrane protein composition due to altered membrane protein stability and sorting.

Spatiotemporal Dynamics and Mechanisms of Actin Cytoskeletal Re-modeling in Cells Perforated by Ultrasound-Driven Microbubbles.

To develop new strategies for improving the efficacy and biosafety of sonoporation-based macromolecule delivery, it is essential to understand the mechanisms underlying plasma membrane re-sealing and function recovery of the cells perforated by ultrasound-driven microbubbles. However, we lack a clear understanding of the spatiotemporal dynamics of the disrupted actin cytoskeleton and its role in the re-sealing of sonoporated cells. Here we used a customized experimental setup for single-pulse ultrasound (133.33-µs duration and 0.70-MPa peak negative pressure) exposure to microbubbles and for real-time recording of single-cell (human umbilical vein endothelial cell) responses by laser confocal microscopic imaging. We found that in reversibly sonoporated cells, the locally disrupted actin cytoskeleton, which was spatially correlated with the perforated plasma membrane, underwent three successive phases (expansion; contraction and re-sealing; and recovery) to re-model and that each phase of the disrupted actin cytoskeleton was approximately synchronized with that of the perforated plasma membrane. Moreover, compared with the closing time of the perforated plasma membrane, the same time was used for the re-sealing of the actin cytoskeleton in mildly sonoporated cells and a longer time was required in moderately sonoporated cells. Further, the generation, directional migration, accumulation and re-polymerization of globular actin polymers during the three phases drove the re-modeling of the actin cytoskeleton. However, in irreversibly sonoporated cells, the actin cytoskeleton, which underwent expansion and no contraction, was progressively de-polymerized and could not be re-sealed. Finally, we found that intracellular calcium transients were essential for the recruitment of globular actin and the re-modeling of the actin cytoskeleton. These results provide new insight into the role of actin cytoskeleton dynamics in the re-sealing of sonoporated cells and serve to guide the design of new strategies for sonoporation-based delivery.