Plasma Membrane Fraction Research Articles

The quick screening of binding compounds and proteins for drug discovery and pharmacological research

In drug discovery and pharmacological research, early identification of target molecules for compounds with pharmacological effects is crucial. However, this process often requires significant effort and can be rate-limiting, thereby slowing down research progress. This paper introduces a simplified and rapid method for quick screening of binding compounds or proteins. Utilizing Affinity Selection Mass Spectrometry (ASMS), this technique efficiently detects compound-target binding through size-exclusion chromatography and mass spectrometry. ASMS offers high sensitivity and specificity, making it ideal for accurate identification of binding interactions. We have further enhanced ASMS to handle membrane proteins without solubilization, creating Binder Selection Technology (BST). BST allows screening against both soluble and challenging membrane proteins such as GPCRs and SLC transporters. By using cell membrane fractions or organelle fractions with high target molecule expression, BST efficiently identifies potential binding compounds. This innovative method constructs a comprehensive database of binding compounds for various targets, facilitating rapid hypothesis testing and pharmacological evaluation. Additionally, BST screens 17,000 proteins, including membrane proteins, using wheat germ cell-free and animal cell expression systems. This approach allows exploration of binding interactions without labeling compounds or immobilizing proteins, preserving their native state. BST is powerful for identifying targets of compounds with known pharmacological effects but unknown targets in animal or cell-based assays. By utilizing BST, researchers can overcome bottlenecks in early drug discovery, significantly enhancing research speed and success rates. This method represents a major advancement, providing an efficient and effective way to identify and validate target molecules in drug discovery.

Optimization of Cell Membrane Purification for the Preparation and Characterization of Cell Membrane Liposomes.

Cell membrane nanoparticles have attracted increasing interest in nanomedicine because they allow to exploit the complexity of cell membrane interactions for drug delivery. Several methods are used to obtain plasma membrane to generate cell membrane nanoparticles. Here, an optimized method combining nitrogen cavitation in isotonic buffer and sucrose gradient fractionation is presented. The method allows to obtain cell membrane fractions of high purity from both suspension and adherent cells. Comparison with other common methods for membrane extraction, where mechanical lysis using cell homogenizers is performed in isotonic or hypotonic buffers, shows that the optimized procedure yields high purity membrane in a robust and reproducible way. Procedures to mix the purified membrane with synthetic lipids to obtain cell membrane liposomes (CMLs) are presented and indications on how to optimize these steps are provided. CMLs made using crude membrane isolates or the purified membrane fractions show different uptake by cells. The CMLs made with the optimized procedure and liposomes of the same composition but without cell membrane components are thoroughly characterized and compared for their size, zeta potential, bilayer and mechanical properties to confirm membrane protein inclusion in the CMLs. Cell uptake studies confirm that the inclusion of membrane components modifies liposome interactions with cells.

Abstract Tu077: Targeting β-adrenergic receptor resensitization attenuates cardiac dysfunction

β-adrenergic receptor (βAR) is a key regulator of cardiac function. Agonist activation of βAR leads to phosphorylation-mediated desensitization resulting in βAR trafficking to endosomes. Endosomal βARs undergo resensitization through protein phosphatase 2A (PP2A)-mediated dephosphorylation. We have shown that stimulation of βARs activates phosphoinositide 3-kinase γ (PI3Kγ) that phosphorylates endogenous inhibitor of PP2A (I2PP2A) promoting robust binding to PP2A resulting in PP2A inhibition. βAR dysfunction due to desensitization is considered as a classical hallmark of heart failure, while less is known about resensitization. Plasma membrane and endosomal fractions were isolated from non-failing and failing patient samples to determine whether βAR resensitization is altered in human heart failure. Endosomes from heart failure samples showed significant accumulation of phosphorylated β2ARs which was associated with reduced adenylyl cyclase and βAR-associated PP2A activity compared to non-failing showing that resensitization is inhibited in human heart failure. Since PP2A activity is inhibited by robust binding of I2PP2A to PP2A, we hypothesized that disrupting I2PP2A interaction with PP2A would unlock PP2A inhibition normalizing βAR resensitization. In silico modeling and biochemical studies mapped the PP2A interaction with I2PP2A to the C-terminal region of PP2A (PP2Act). Agonist isoproterenol (ISO) treatment of cells expressing PP2Act showed significant reduction in β2AR phosphorylation along with increased cAMP generation and normalized PP2A activity showing preserved βAR resensitization. While expression of PP2A mutant with deletion in the PP2Act (ΔPP2Act) did not preserve βAR resensitization. Challenging mice with cardiomyocyte-specific expression of PP2Act to ISO showed preserved cardiac function providing insights that targeting resensitization could potentially be a novel therapeutic strategy. Key words: Resensitization, I2PP2A, PP2A, Cardiac hypertrophy

Exopolysaccharides from Rhizobium Tropici mitigate aluminum bioaccumulation and toxicity in earthworm Lumbricus Terrestris

Aluminum is one of the most abundant elements in earth’s crust and soils. Global soil acidification has occurred with industrialization and enhances Al solubility in acidic soils, resulting in Al phytoxicity and reducing crop production. Earthworms play an important role in nutrient recycling in soils and maintaining soil health. Rhizosphere microorganism excretes exopolysaccharides (EPS) into the rhizosphere. The objectives of this study is to examine effects of EPS from Rhizobium Tropici on aluminum uptake and ecotoxicity to earthworms in soils and litters. Overall this study provides the scientific understanding of the EPS in alleviation of Al toxicity to earthworms in soils. Results showed the clear mitigation of EPS in reducing earthworm mortality rate and increasing its reproductivity under Al stress. EPS significantly decreased Al uptake from soil and litters, and decreased Al in subcellular distribution (cell membrane, cytosol and organelle fractions) in earthworms from soils. Al in purges was found to be mainly present in the residual fraction, while EPS slightly increased Al in water soluble and exchangeable fractions in purges. Overall water soluble and organic matter bound Al in soils were correlated well with Al in earthworms, but Al in earthworms were controlled by total Al in purges. The current study concluded that earthworm Lumbricus Terrestre may be a potential bio-indicator of Al ecotoxicity in soils and EPS from Rhizobium Tropici effectively mitigated Al ecotoxicity in soils.

Complexity and modification of the bull sperm glycocalyx during epididymal maturation.

Mammalian spermatozoa have a surface covered with glycocalyx, consisting of heterogeneous glycoproteins and glycolipids. This complexity arises from diverse monosaccharides, distinct linkages, various isomeric glycans, branching levels, and saccharide sequences. The glycocalyx is synthesized by spermatozoa developing in the testis, and its subsequent alterations during their transit through the epididymis are a critical process for the sperm acquisition of fertilizing ability. In this study, we performed detailed analysis of the glycocalyx on the sperm surface of bull spermatozoa in relation to individual parts of the epididymis using a wide range (24) of lectins with specific carbohydrate binding preferences. Fluorescence analysis of intact sperm isolated from the bull epididymides was complemented by Western blot detection of protein extracts from the sperm plasma membrane fractions. Our experimental results revealed predominant sequential modification of bull sperm glycans with N-acetyllactosamine (LacNAc), followed by subsequent sialylation and fucosylation in a highly specific manner. Additionally, variations in the lectin detection on the sperm surface may indicate the acquisition or release of glycans or glycoproteins. Our study is the first to provide a complex analysis of the bull sperm glycocalyx modification during epididymal maturation.

High Potassium Suppresses Glycosuria and Gluconeogenesis in Tubule-specific mTORC2 Knockout Mice

Background: Insulin promotes renal proximal tubule glucose reabsorption and suppresses gluconeogenesis (GNG). The kinase mTORC2 is critical for insulin signaling in multiple cell types. In the kidney tubules, mTORC2 knockout has recently been shown to cause glycosuria via reduced plasma membrane SGLT2 and SGLT1 as well as inappropriately increased renal GNG. Potassium (K+) also plays an important role in systemic glucose homeostasis. However, the overall importance of K+ for the regulation of renal glucose reabsorption and GNG is poorly understood. Methods: Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. To examine the role of dietary K+ on renal glucose homeostasis, TRKO mice and wild-type (WT) littermates were fed with either a normal 0.5% K+ diet or high 3% K+ diet while in balance cages. Kidney tissue was harvested after feeding and processed to study plasma membrane glucose transporters, gluconeogenic enzymes, and mTORC2-regulated targets by western blot and qPCR. Results: On a normal K+ diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane fractions showed decreased SGLT2 and SGLT1, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes. On a high K+ diet, glycosuria resolved and plasma membrane SGLT2 and SGLT1 were restored in TRKO mice. In addition, TRKO animals fed with a high K+ diet had suppressed gluconeogenic enzymes compared to WT mice. Regardless of dietary K+ content, TRKO animals had similar reductions in phosphorylation of mTORC2 substrates compared to WT controls. Conclusion: Renal tubule mTORC2 is critical for coordinated glucose reabsorption by SGLT2 and SGLT1 as well as suppression of renal GNG. High dietary K+ promotes sodium-glucose cotransport by restoring plasma membrane SGLT2 and SGLT1 in TRKO mice. High dietary K+ also prevents excessive GNG in TRKO mice. High K+ appears to regulate these processes independently of mTORC2 and its downstream substrates, such as Akt. High K+ could potentially provide an alternative signaling mechanism to regulate renal glucose reabsorption and GNG in states of insulin resistance. Grants from the NIH (T32, R01) and The James Hilton Manning and Emma Austin Manning Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2984: Prostate cancer associated glycosylated RNAs: Who are you and where are you hiding

Abstract Glycosylated RNA (glycoRNA) is an emerging field. Carbohydrate modifications are vital for biomolecule (proteins and lipids) stability, transport, and localization. Glycosylation influences cell functions like differentiation, apoptosis, and immune response, potentially impacting human disease. The Bertozzi lab initially discovered that RNAs can carry sugar modifications when they metabolically labeled mammalian cultured cells using bioorthogonal chemistry methods normally employed for glycosylated protein and lipid enrichment. Our lab wanted to investigate the translational potential of glycoRNAs and determine if these modifications could be developed as novel clinical targets for aggressive prostate cancer. (PCa). As advanced forms of PCa often lead to poor prognoses, it is imperative that better screening biomarkers and therapeutic treatment options are developed to increase patient survivorship. We predicted that glycoRNA expression would correlate with malignancy, metastatic status, and hormone sensitivity in prostate cells. Our lab used the sialic acid metabolic precursor Ac4ManNAz and click chemistry to label a panel of human prostate cancer cell lines representing the spectrum of prostate disease followed by rigorous RNA isolation procedures and northern blot analysis. We found that human prostate cell lines with no (RWPE-1) or low malignancy (LNCaP) expressed higher levels of glycoRNA belonging to the small <200 nt (but not large) noncoding RNA class compared to aggressive, metastatic PCa cell lines. Our glycosidase studies and lectin northern blot analysis indicated that RNA carries both N-linked and O-linked modifications. Based on these findings, we hypothesized that glycosylated RNA plays an important role in maintaining prostate homeostasis and in cancer protection. To better profile these glycoRNAs, we aimed to: 1) Test a wider range of metabolic labeling regents to characterize the glycoRNA populations using a panel of human prostate cell lines; 2) Perform cellular fractionation studies to determine localization of glycoRNA in prostate cells; and 3) Explore glycoRNAs in a genetic animal model. These studies focused on LNCaP PCa cell lines, which express high levels of glycoRNA. We found that small glycoRNA expression was noted when LNCaP cells were metabolically labeled with Ac4GalNAz (predominantly O-linked) and Ac4ManNAz (predominantly N-linked), verifying glycoRNA heterogeneity. Cellular fractionation studies indicated glycoRNA localization was enriched in the membrane fraction of LNCaP cells using these metabolic labels. Furthermore, we verified that C. elegans grown on Ac4GalNAz plates expressed small glycoRNA, indicating biological conservation. We are now characterizing these glycoRNAs using RNA sequencing and LC-MS/MS glycomics. Our findings will provide novel insights into cancer-associated glycoRNA, leading to improved clinical tools for PCa. Citation Format: Samantha McGuire, Esther Jones, Spencer Moen, Aurora Esquela-Kerscher. Prostate cancer associated glycosylated RNAs: Who are you and where are you hiding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2984.

The Effects of N-Glycosylation on the Expression and Transport Activity of OATP1A2 and OATP2B1

Organic anion transporting polypeptide (OATP)1A2 and OATP2B1 have potential N-glycosylation sites, but their influence remains unclear. This study aimed to identify the N-glycosylation sites of OATP1A2/2B1 and investigate their impact on the expression and function of OATP1A2/2B1.Human embryonic kidney cells expressing OATP1A2 or OATP2B1 (HEK293-OATP1A2/2B1) were exposed to tunicamycin, an N-glycosylation inhibitor, and a plasma membrane fraction (PMF) Western blot assay and an estrone 3-sulfate (E3S) uptake study were conducted. HEK293-OATP1A2/OATP2B1 cell lines with mutation(s) at potential N-glycosylation sites were established, and the Western blotting and uptake study were repeated.Tunicamycin reduced the PMF levels and E3S uptake of OATP1A2/OATP2B1. The Asn124Gln, Asn135Gln, and Asn492Gln mutations in OATP1A2 and Asn176Gln and Asn538Gln mutations in OATP2B1 reduced the molecular weights of the OATP molecules and their PMF levels. The PMF levels of OATP1A2 Asn124/135Gln, OATP1A2 Asn124/135/492Gln, and OATP2B1 Asn176/538Gln were further reduced. The maximum transport velocities of OATP1A2 Asn124Gln, OATP1A2 Asn135Gln, and OATP2B1 Asn176/538Gln were markedly reduced to 10 %, 4 %, and 10 % of the wild-type level, respectively.In conclusion, the N-glycans at Asn124 and Asn135 of OATP1A2 and those at Asn176 and Asn538 of OATP2B1 are essential for the plasma membrane expression of these molecules and also affect their transport function.

MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C.

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.

Mycobacterium tuberculosis protein PPE15 (Rv1039c) possesses eukaryote-like SH3 domain that interferes with NADPH Oxidase assembly and Reactive Oxygen Species production

Inhibition of Reactive Oxygen Species (ROS) is one of the strategies that Mycobacterium tuberculosis (Mtb) employs as its defence mechanism. In this study, the role of PPE15 (Rv1039c), a late-stage protein, has been investigated in modulating the cellular ROS. We discovered PPE15 to be a secretory protein that downregulates ROS generation in THP1 macrophages. Our in-silico analysis revealed the presence of a eukaryote-like SH3 (SH3e) domain in PPE15. The predicted SH3e-domain of PPE15 was found to interact with cytosolic components of NADPH Oxidase (NOX), p67phox and p47phox through molecular docking. In-vitro experiments using THP1 macrophages showed a diminished NADP/NADPH ratio, indicating reduced NOX activity. We also observed increased levels of p67phox and p47phox in the cytoplasmic fraction of PPE15 treated macrophages as compared to the plasma membrane fraction. To understand the role of the SH3e-domain in ROS modulation, this domain was deleted from the full-length PPE15 (PPE15-/-SH3). We observed an increase in cellular ROS and NADP/NADPH ratio in response to PPE15-/-SH3 protein. The interaction of PPE15-/-SH3 with p67phox or p47phox was also reduced in the cytoplasm, indicating migration of NOX subunits to the plasma membrane. Additionally, M. smegmatis expressing PPE15 was observed to be resistant to oxidative stress with significant intracellular survival in THP1 macrophages as compared to M. smegmatis expressing PPE15-/-SH3. These observations suggest that the SH3e-domain of PPE15 interferes with ROS generation by sequestering NOX components that inhibit NOX assembly at the cell membrane. Therefore, PPE15 acts like a molecular mimic of SH3-domain carrying eukaryotic proteins that can be employed by Mtb at late stages of infection for its survival. These findings give us new insights about the pathogen evading strategy of Mtb which may help in improving the therapeutics for TB treatment.

GRP78 Contributes to the Beneficial Effects of SGLT2 Inhibitor on Proximal Tubular Cells in DKD.

Beneficial effects of SGLT2 inhibitors on kidney function are well-known; however, their molecular mechanisms are not fully understood. We focused on 78 kDa glucose-regulated protein (GRP78) and its interaction with SGLT2 and Integrin ß1 beyond the chaperone property of GRP78. In STZinduced diabetic mouse kidneys, GRP78, SGLT2, and Integrin ß1 increased in the plasma membrane fraction, while they were suppressed by canagliflozin. The altered subcellular localization of GRP78/Integrin ß1 in STZ mice promoted epithelial mesenchymal transition (EMT) and fibrosis, which were mitigated by canagliflozin. High glucose conditions reduced intracellular GRP78, increased its secretion, and caused EMT-like changes in cultured HK2 cells, which were again inhibited by canagliflozin. Urinary GRP78 increased in STZ mice, and in vitro experiments with recombinant GRP78 suggested that inflammation spread to surrounding tubular cells and canagliflozin reversed this effect. Under normal glucose culture, canagliflozin maintained SERCA activity, promoted endoplasmic reticulum (ER) robustness, reduced ER stress response impairment, and protected proximal tubular cells. In conclusion, canagliflozin restored subcellular localization of GRP78, SGLT2 and Integrin ß1 and inhibited EMT and fibrosis in DKD. In non-diabetic CKD, canagliflozin promoted ER robustness by maintaining SERCA activity and preventing ER stress response failure, and contribute to tubular protection.

Compartmentalization of galactan biosynthesis in mycobacteria

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[14C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.

Eliminating Lipopolysaccharide (LPS) Using Lactic Acid Bacteria (LAB).

Gram-negative bacteria such as Escherichia coli, among intestinal bacteria, have lipopolysaccharide (LPS), which induces inflammation of human intestines. However, lactic acid bacteria (LAB) can improve human intraintestinal conditions. One reason is that ingestion of LAB prevents bacterial diarrhea. This chapter describes a method of LPS elimination using lactic acid bacteria (LAB). First, the LPS concentration is assayed using an LPS assay kit with the limulus cascade reaction made by limulus amebocyte lysate. Some LABs, four bacillus strains and one coccus strain, have LPS-elimination activity. Particularly, the coccus strain Pediococcus pentosaceus eliminates LPS to 43%. The cells fractionate and eliminate four fractions: the extracellular fraction, cell membrane fraction, cytoplasm fraction, and cell wall fraction. Only the cell wall digesting fraction eliminates LPS to 45%. Results confirm that the LAB eliminates all LPS having O-antigen under a low-sugar medium condition at temperatures of 15-30°C. This method can be used for assay of LPS elimination by LABs exactly and easily for the probiotics field.

Oligomerization and aggregation of NAP-22 with several metal ions

Metal ions participate in various biochemical processes such as electron transport chain, gene transcription, and enzymatic reactions. Furthermore, the aggregation promoting effect of several metal ions on neuronal proteins such as prion, tau, Aβ peptide, and α-synuclein, has been reported. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the detergent-resistant membrane microdomain fraction of the neuronal cell membrane. Previously, we showed oligomer formation of NAP-22 in the presence of several phospholipids and fatty acids. In this study, we found the aggregation of NAP-22 by FeCl2, FeCl3, and AlCl3 using native-PAGE. Oligomer or aggregate formation of NAP-22 by ZnCl2 or CuSO4 was shown with SDS-PAGE after cross-linking with glutaraldehyde. Morphological analysis with electron microscopy revealed the formation of large aggregates composed of small annular oligomers in the presence of FeCl3, AlCl3, or CuSO4. In case of FeCl2 or ZnCl2, instead of large aggregates, scattered annular and globular oligomers were observed. Interestingly, metal ion induced aggregation of NAP-22 was inhibited by several coenzymes such as NADP+, NADPH, or thiamine pyrophosphate. Since NAP-22 is highly expressed in the presynaptic region of the synapse, this result suggests the participation of metal ions not only on the protein and membrane dynamics at the presynaptic region, but also on the metabolic regulation though the interaction with coenzymes.

Adaptation of the maize seedling seminal roots to srought: Essential role of plasma membrane H+-ATPases activity

To understand how maize plants adapt to drought, this study examines the role of plasma membrane proton pumps in root growth. This study delves into the physiological mechanisms through which maize plants respond to drought conditions, with a particular emphasis on elucidating the crucial role played by plasma membrane proton pumps in facilitating adaptive changes in root growth. Our results underscore the indispensable nature of these pumps in orchestrating precise modulation of root growth patterns during drought stress, highlighting their profound significance in stress responses. Additionally, the study reveals that osmotic stress alters lipid profiles in the plasma membrane, potentially impacting its functioning and the activity of membrane proteins. To understand the role of plasma membrane (PM) H<sup>+</sup>-ATPases in the adaptative response to osmotic stress and in the regulation of root growth in maize, we studied the gene expression and enzyme activity of PM H<sup>+</sup>-ATPases, as well as the changes in plant biomass and total root growth, in the seedlings of two maize cultivars: the drought-tolerant Calo cultivar and the drought-sensitive Abelardo. The seedlings were exposed to simulated drought for 24 h (treatment with 20% PEG). The enzyme activity and gene expression of the <i>MHA4</i> H<sup>+</sup>-ATPase increased in the Calo variety but declined in Abelardo plants treated with PEG. The growth of roots in Abelardo plants exposed to 24 h of PEG treatment was reduced to almost 50% of the control. Conversely, for the Calo cultivar, there was no remarkable morpho-physiological difference between the roots of stressed and non-stressed plants. Therefore, the activity of the PM H<sup>+</sup>-ATPase seems to be an important factor for proper root growth during the adaptation of maize to drought. In addition, osmotic stress also induced changes in the levels of saturated polyisoprenoid alcohols in the plasma membrane fraction of maize roots. The increased levels of this class of lipids might modulate the physico-chemical properties of the PM lipid bilayer and thus affect its functioning and modify the activity of membrane proteins, such as PM H<sup>+</sup>-ATPases.

Practical Effect of Cervical Softening Decoction Combined with Delivery Ball Approach in Promoting Vaginal Delivery in Primiparous Women

In Traditional Chinese Medicine, Cervical Softening Decoction is derived from a modified “Wan Bing Hui Chun” formulation and consists of natural herbal ingredients such as Angelica sinensis, Ligusticum chuanxiong, Leonurus heterophyllus, and Cortex Daphnes. The decoction regulates Qi, promotes blood circulation, and facilitates fetus descent. Genkwanin is an active compound in L. heterophyllus and previous studies identified this compound in ethanolic extracts from rat uterine smooth muscle cell membrane fractions. To further understand the pharmacological activities of Cervical Softening Decoction, we examined genkwanin effects on isolated rat uterine contractions and protein expression in rat tissue with postpartum hemorrhage complicated by multiple organ dysfunction syndrome (MODS). We also explored the practical effects of Cervical Softening Decoction combined with a birthing ball approach on promoting natural delivery in primiparous women. First, an isolated rat uterine smooth muscle contraction model was generated and three groups established: oxytocin group (0.002 U/mL), conventional genkwanin dose group (3 μg/mL), and high genkwanin dose group (6.0 μg/mL). Changes in uterine smooth muscle contraction and relaxation amplitudes were recorded before and after administration. Second, 50 rats were used to establish the following groups: (1) control group, (2) postpartum hemorrhage with MODS model group (MODS group), and (3) a postpartum hemorrhage with MODS treated with genkwanin group (genkwanin group). Western blotting was used to detect and compare tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 protein expression levels in lung tissue from groups. Finally, 84 primiparous women admitted to our hospital between January 2020 and December 2021 were selected and divided into two groups based on their obstetric interventions. The control group (n = 42) received routine obstetric intervention, while the observation group (n = 42) received Cervical Softening Decoction and delivery ball interventions. Delivery conditions were compared across groups. In basic studies, genkwanin doses significantly increased contraction and relaxation values in isolated rat uterine smooth muscle (P <0.05). Moreover, the genkwanin contractile effects at conventional and high doses were comparable with oxytocin. TNF-α and IL-6 protein expression in MODS group lung tissue was significantly higher when compared with the control group, while expression in the genkwanin group was significantly reduced when compared with the MODS group (P <0.05). In clinical studies, the observation group showed significantly higher natural delivery rates and Labor Agentry Scale (LAS) scores when compared with the control group. In contrast, cesarean section rates, first and second stage labor duration, total labor duration, and Facial Pain Scale (FPS) scores were significantly lower in the observation group when compared with the control group (P <0.05). In basic studies, genkwanin elicited significant contraction effects on isolated rat uterine smooth muscle and effectively inhibited inflammatory responses in rats with postpartum hemorrhage complicated by MODS. In clinical studies, combined Cervical Softening Decoction and delivery ball generated promising practical effects in promoting vaginal delivery in primiparous women. The intervention significantly increased the probability of a natural delivery, shortened labor duration, relieved maternal pain, and enhanced maternal control during delivery.

Deciphering the effect of salinity and boron stress on broccoli plants reveals that membranes phytosterols and PIP aquaporins facilitate stress adaptation

Abiotic stresses, such as salinity and boron toxicity/deficiency, are prevalent in arid and semi-arid regions where broccoli is largely cultivated. This study aimed to investigate the physiological response of broccoli leaves to these stresses, focusing on parameters such as growth, relative water content, stomatal conductance, and mineral concentration after 15 days of treatment application. The effects of individual and combined stresses of salinity and boron (deficiency and toxicity) were examined. Additionally, the study explored the molecular aspects of PIP aquaporins in relation to their presence in the plasma membrane and their interaction with the lipid environment. The results showed that the combined stress of salinity and boron deficiency resulted in a significant reduction in plant biomass, suggesting a specific adaptation to this stress combination. Changes in stomatal conductance and mineral nutrient levels indicated that the adaptation mechanisms were associated with water and boron concentration in the leaves. The expression patterns of PIP aquaporins varied among the different stress treatments, either individually or in combination. Furthermore, the presence of aquaporins in the plasma membrane and microsomal fraction highlighted the potential regulatory roles of trafficking along with the membrane composition, particularly the concentration of phytosterols. The results underscore the importance of water transport by aquaporins and their interaction with the sterol composition in the membranes, in facilitating salinity-boron stress adaptation mechanisms.

Novel Cancer Immunotherapy Using Cell-Derived Membrane Vesicles Orchestrates Multimodal Antitumor Immunity

Cancer immunotherapy has emerged as a promising therapy for hematological malignancies. Although multiple immunotherapeutics are currently available in the clinic such as chimeric antigen receptor-engineered T-cell therapy (CAR-T cell therapy) and bispecific T-cell engager (BiTE), their long-term efficacy is still insufficient in most of the cases. Simultaneous inputs of multiple signals may provide more optimal T-cell activation and overcome the immunosuppressive tumor microenvironment (TME), which results in a durable therapeutic effect. However, integrating multifaceted immunomodulatory signals into a single drug or cell is technically difficult. To overcome these limitations, we developed nanosized cell-derived membrane vesicles (MVs) that orchestrate multimodal antigen-specific antitumor immunity in endogenous T cells. We isolated MVs with 100-150 nm in size and intact membrane-bound proteins from the HLA-negative leukemia cell line K562 by mechanical homogenization followed by isolation of the plasma membrane fraction using magnetic beads and ultracentrifugation. MVs can be loaded with a desired combination of immunomodulatory molecules on the surface by preparing cells stably transduced with those proteins. First, K562 cells were genetically engineered with a membrane-bound form of the anti-CD3/CD19 BiTE and homogenized to form BiTE-expressing MVs. When co-cultured with T cells and CD19-positive leukemia cell line NALM-6, MVs loaded with anti-CD3/CD19 BiTE induced potent cytotoxic activity in nonspecific T cells in a dose-dependent manner. To provide optimal T-cell activation, we further loaded MVs with a variety of immunostimulatory molecules including costimulatory ligands (CD80 and 4-1BBL) and cytokines (IL-7 and IL-15) on the surface. The generated antitumor MVs simultaneously induced multiple immunostimulatory signals in T cells, resulting in superior proliferation, cytokine production, and cytolytic functions compared with MVs loaded with BiTE alone. Intriguingly, MVs activated T cells only in the presence of target tumor cells, which is considered to provide a safety advantage in clinical settings. Changing the antitumor mAb sequences of the BiTE enabled to generate MVs against any type of antigen including B cell maturation antigen (BCMA), EGFR, and mesothelin. To test functions of our MVs in vivo, NSG mice were transplanted with NALM-6 and then infused with human peripheral blood T cells. While infusion of T cells alone did not contribute to controlling leukemia progression, infusion of anti-CD19 MVs with CD80, 4-1BBL, IL-7, and Il-15 induced potent antitumor response. Repeated infusion of MVs further augmented their therapeutic efficacy. To overcome the immunosuppressive TME, we additionally loaded MVs with immune checkpoint inhibitors (anti-PD-1 and anti-CTLA-4 antibody), TGF-b receptors to trap TGF-b, and inflammatory cytokines (IL-12 and IL-18) that activate both T cells and myeloid cells in the TME. Immunocompetent Balb/c mice were subcutaneously inoculated with colon carcinoma cell line CT26 ectopically expressed with CD19 and treated with MVs targeting CD19. The injected MVs converted tumor-associated M2-type macrophages into an inflammatory phenotype and promoted CD8+ T cell infiltration within the TME, resulting in better control of tumor progression in in vivo solid tumor mouse models. K562 cells are highly sensitive to NK cells because they endogenously express several ligands for NK cell activation receptors such as MIC-A and MIC-B, and lack the expression of HLA class I that represses NK cell activation. K562-derived MVs induced potent cytotoxic activity in NK cells against NK-cell-resistant myeloma cell line MM1S in an antigen-specific manner. In summary, this novel technology enables to deliver desired combinations of antitumor immune signals to not only T cells but also NK cells and other types of immune cells, which can transmit superior therapeutic efficacy against the tumor cells that are resistant to the present immunotherapeutics.

EFR3A: a new raft domain organizing protein?

BackgroundMembrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft domain formation and regulation, including in cells with low expression of MPP1.MethodsWe sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and mass spectrometry technique. The results were further confirmed via immunoblotting and via co-immunoprecipitation. In order to study the effect of the candidate protein on the physicochemical properties of the plasma membrane, the gene was knocked down via siRNA, and fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy was employed.ResultsEFR3A was identified as a candidate protein that interacts with flotillin-2. Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergent-resistant membrane fraction of HeLa cells, and its presence was sensitive to the removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles derived from knocked down cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor and phospholipase C gamma phosphorylation and affect epidermal growth factor-dependent cytosolic Ca2+ concentration.ConclusionsAltogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which might be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling which points to the possible role in human health, in particular human cancer biology.

Targeted siRNA Delivery by Bioinspired Cancer Cell Membrane-Coated Nanoparticles with Enhanced Anti-Cancer Immunity.

Cell-membrane nanocarriers are usually constructed by modifying the nanoparticle surface with cell membrane extracts, which has a direct benefit in endowing targeting capacity to nanocarriers based on their original cell types. However, delivering nucleic acid cargos by cell membrane-based nanoparticles is difficult owing to the strong negative charge of the cell membrane fraction. In this study, we developed a cancer cell membrane-based drug delivery system, the cMDS, for efficient siRNA delivery. Meanwhile, the cancer-specific immune response stimulated by the gene vector itself could offer synergistic anti-cancer ability. The cMDS was prepared by ultrasound, and its transfection efficiency and anti-cancer ability were examined using cultures of CT26 cells. MTT and red blood cell hemolysis tests were performed to assess the safety of cMDS, while its targeted gene delivery and strong immune stimulation were investigated in a subcutaneous tumor model. Moreover, the detailed anti-cancer immune stimulation mechanisms of cMDS are uncovered by protein chip analysis. The cMDS was spherical core-shell structure. It showed high transfection efficiency and anti-cancer ability in vitro. In animal experiments, intravenously administered cMDS/siStat3 complex efficiently suppress the growth of colon cancer. Moreover, the result of protein chip analysis suggested that cMDS affect the migration and chemotaxis of immune cells. The cMDS shows obvious tumor tissue-specific accumulation properties and strong immune stimulation ability. It is an advanced targeted gene delivery system with potent immunotherapeutic properties.