Cellular Membrane Integrity Research Articles

A Transcriptional Analysis Showing the Effects of GH12 Combined with Fluoride for Suppressing the Acidogenicity of Streptococcus mutans Biofilms.

The acidogenicity of Streptococcus mutans is important for caries development. The antimicrobial peptide GH12 can affect the integrity of cellular membranes and the virulence factors of S. mutans. Combining GH12 and NaF (GF) efficiently controlled the development of caries, but its mechanisms remained unrevealed. This research intended to verify the effects of GF on the acidogenicity of S. mutans biofilms and to reveal the mechanisms. Lactic acid production assays and pH monitoring assays were conducted to investigate the regulatory effects of the GF treatment on the acidogenicity of S. mutans biofilms. RNA sequencing and bioinformatics analyses were conducted to screen the transcriptional profile affected by the GF treatment. The results demonstrated the GF group had significantly less lactic acid and maintained the broth's pH values above 5.0 for longer times. Thereafter, GO/KEGG enrichment analyses and RT-qPCR validation revealed that the GF treatment mainly restrained the expression of genes related to the carbohydrates' internalization and metabolism. Compared with NaF, the GF treatment further downregulated the carbohydrates transportation genes. Moreover, compared with GH12, the GF treatment affected the membrane's integrity more significantly. Generally, GF treatment could arrest the acidogenicity of S. mutans biofilms, mainly through suppressing carbohydrates transportation and inhibiting overall metabolism.

Tea-based biochar-mediated changes in cation diffusion homeostasis in rice grown in heavy metal (loid) contaminated mining soil

Foreseeable future scenarios highlight the urgency of applying eco-safe avoidance methods or tolerance to heavy metal(loid) (HM) stress in agricultural production areas of contamination. The analyses show that the Ni, Mn, As, and Cr concentrations detected in the soils of the paddy fields in the Black Sea region vary between 123.60 and 263.30; 687–1271; 8.90–14.50; 162.00–340.00 mg kg−1 proving high accumulation of Ni, Mn, As, Cr in rice. Overconsumption of rice farmed extensively on these soils might also lead to human HM-related health problems. Therefore, in the current study, the approach of using tea-based biochar (BC) proven to have one of the most significant potentials as a soil amendment to reduce HM transmission to in-vitro-grown rice plants was investigated in the soil medium naturally contaminated with HMs. The tea-BC was produced from readily available local black tea waste of a conventional fermentation process and applied in the in-vitro experiments. Among the tested doses examined, 1% tea-BC showed a more positive effect on rice plant growth and development characterized by a better relative growth rate (59.7 and 84 mg g−1 d−1 for root and shoot tissues), photosynthetic pigment intactness (62.48 μg mL−1), cellular membrane integrity (93%), and relative water (96%) than the other rates (0% BC, 3%BC, 5%BC). The mRNA expression data highlights the probability of a cation diffusion facilitator (CDF) (OsMTP11) in concert with catalase isozyme (CATa) and dehydration-responsive element binding protein (DREB1a) linking the HM detoxification, oxidative defense, and dehydration pathways with the help of tea-BC. At the optimum concentration (1%BC), this approach might reduce HM accumulation levels of crops planted in HM-contaminated farmlands.

Arabidopsis flippase ALA3 is required for adjustment of early subcellular trafficking in plant response to osmotic stress.

To compensate for their sessile lifestyle, plants developed several responses to exogenous changes. One of the previously investigated and not yet fully understood adaptations occurs at the level of early subcellular trafficking, which needs to be rapidly adjusted to maintain cellular homeostasis and membrane integrity under osmotic stress conditions. To form a vesicle, the membrane needs to be deformed, which is ensured by multiple factors, including the activity of specific membrane proteins, such as flippases from the family of P4-ATPases. The membrane pumps actively translocate phospholipids from the exoplasmic/luminal to the cytoplasmic membrane leaflet to generate curvature, which might be coupled with recruitment of proteins involved in vesicle formation at specific sites of the donor membrane. We show that lack of the AMINOPHOSPHOLIPID ATPASE3 (ALA3) flippase activity caused defects at the plasma membrane and trans-Golgi network, resulting in altered endocytosis and secretion, processes relying on vesicle formation and movement. The mentioned cellular defects were translated into decreased intracellular trafficking flexibility failing to adjust the root growth on osmotic stress-eliciting media. In conclusion, we show that ALA3 cooperates with ARF-GEF BIG5/BEN1 and ARF1A1C/BEX1 in a similar regulatory pathway to vesicle formation, and together they are important for plant adaptation to osmotic stress.

StMAPK1 functions as a thermos-tolerant gene in regulating heat stress tolerance in potato (Solanum tuberosum).

Mitogen-activated protein kinases (MAPKs) have been reported to respond to various stimuli including heat stress. This research aimed to investigate whether StMAPK1 is implicated in the transduction of the heat stress signal to adapt heat stress as a thermos-tolerant gene. Potato plants were cultivated under mild (30°C) and acute (35°C) heat stress conditions to analyze mRNA expression of StMAPKs and physiological indicators. StMAPK1 was up-regulated and down-regulated by transfection. Subcellular localization of StMAPK1 protein was observed by fluorescence microscope. The transgenic potato plants were assayed for physiological indexes, photosynthesis, cellular membrane integrity, and heat stress response gene expression. Heat stress altered the expression prolife of StMAPKs. StMAPK1 overexpression changed the physiological characteristics and phenotypes of potato plants under heat stresses. StMAPK1 mediates photosynthesis and maintains membrane integrity of potato plants in response to heat stress. Stress response genes (StP5CS, StCAT, StSOD, and StPOD) in potato plants were altered by StMAPK1 dysregulation. mRNA expression of heat stress genes (StHSP90, StHSP70, StHSP20, and StHSFA3) was affected by StMAPK1. StMAPK1 overexpression increases the heat-tolerant capacity of potato plants at the morphological, physiological, molecular, and genetic levels.

Functional Role of Essential Oils as Antimicrobial and Antioxidat Agents in Food Industry: A Review

Essential oils (EOs) possess both antimicrobial and antioxidant activities in food systems. Variations in EOs effectiveness were dictated by their components, effective concentrations, intrinsic factors of food composition as well as extrinsic factors such as storage temperature. The antimicrobial and antioxidant activities of EOs are a result of the presence of phenolic components at high concentrations. EOs could have better effectiveness than single component because these constitutes could act additively or even synergistically in EOs. EOs have antimicrobial activity against wide range of microorganisms and their mode of action is related to disintegration of cellular membrane integrity followed by inactivation of other microbial cells components. The antioxidant mode of action for EOs is related to neutralization free radicals and peroxide decomposition in particularly when tested in meat, dairy, fruits and vegetables. The high effectiveness of EOs indicates that they could replace the synthetic food additives. This scientific review summarizes the most recent studies about effectiveness of EOs as antimicrobial and antioxidant agents to be used in food industry.

Phase angle as a prognostic indicator in critically ill children: A systematic review.

Phase angle (PhA) may reflect the integrity of cellular membranes, hydration status, and total body cell mass. Studies have shown that PhA can be used as a good predictor for evaluation of disease severity in critically ill adults. However, there is a lack of studies assessing the association between PhA and clinical outcomes in critically ill children. This systematic review described the association between PhA at pediatric intensive care unit (PICU) admission with clinical outcomes in critically ill children. The search was conducted using PubMed/Medline, Scopus, Web of Science, EMBASE and LILACS until July 22, 2022. Studies that evaluate the association between PhA at PICU admission in critically ill children and clinical outcomes were eligible. Data regarding population, study design, setting, bioelectrical impedance analysis (BIA) protocol used, PhA classification, and outcome analysis were extracted. Risk of bias was assessed by Newcastle-Ottawa Scale. Among the 4669 articles screened, five prospective studies were included. The studies have shown association between lower values of PhA at PICU admission with longer PICU and hospital length of stay, duration of mechanical ventilation, septic shock, and higher mortality risk. Small sample size, different clinical conditions, and methodological differences of the studies regarding BIA equipment and cutoffs of PhA were observed. Although the studies have limitations, the PhA has a potential role in predicting clinical outcomes in critically ill children. Larger studies with standardized PhA protocols and other relevant clinical outcomes are necessary.

Melatonin enhanced the heavy metal-stress tolerance of pepper by mitigating the oxidative damage and reducing the heavy metal accumulation

Heavy metals (HMs), like vanadium (V), chromium (Cr), cadmium (Cd), and nickel (Ni) toxicity due to anthropogenic, impair plant growth and yield, which is a challenging issue for agricultural production. Melatonin (ME) is a stress mitigating molecule, which alleviates HM-induced phytotoxicity, but the possible underlying mechanism of ME functions under HMs' phytotoxicity is still unclear. Current study uncovered key mechanisms for ME-mediated HMs-stress tolerance in pepper. HMs toxicity greatly reduced growth by impeding leaf photosynthesis, root architecture system, and nutrient uptake. Conversely, ME supplementation markedly enhanced growth attributes, mineral nutrient uptake, photosynthetic efficiency, as measured by chlorophyll content, gas exchange elements, chlorophyll photosynthesis genes' upregulation, and reduced HMs accumulation. ME treatment showed a significant decline in the leaf/root V, Cr, Ni, and Cd concentration which was about 38.1/33.2%, 38.5/25.9%, 34.8/24.9%, and 26.6/25.1%, respectively, when compared with respective HM treatment. Furthermore, ME remarkably reduced the ROS (reactive oxygen species) accumulation, and reinstated the integrity of cellular membrane via activating antioxidant enzymes (SOD, superoxide dismutase; CAT, catalase; APX, ascorbate peroxidase; GR, glutathione reductase; POD, peroxidase; GST, glutathione S-transferase; DHAR, dehydroascorbate reductase; MDHAR, monodehydroascorbate reductase) and as well as regulating ascorbate-glutathione (AsA-GSH) cycle. Importantly, oxidative damage showed efficient alleviations through upregulating the genes related to key defense such as SOD, CAT, POD, GR, GST, APX, GPX, DHAR, and MDHAR; along with the genes related to ME biosynthesis. ME supplementation also enhanced the level of proline and secondary metabolites, and their encoding genes expression, which may control excessive H2O2 (hydrogen peroxide) production. Finally, ME supplementation enhanced the HM stress tolerance of pepper seedlings.

Abstract 2764: Application of NanoBRET target engagement cellular assay for development of inhibitors against protein kinases, RAS, and epigenetic proteins in cancer therapy

Abstract NanoBRET target engagement (TE) is the first biophysical technique that broadly enables the quantitative determination of protein inhibitor occupancy in live cells, without disruption of cellular membrane integrity. This quantitative capability is achieved in live cells via BRET with an optimized set of cell-permeable tracers, allowing the measurement of compound binding to a selected cellular target protein. Our NanoBret assay is scaled to 384-well plate format and Z’ analysis demonstrated the assay to be High Throughput Screening compatible. Protein kinases, RAS, and epigenetic proteins are very important drug targets. NanoBRET TE cellular assay has enabled the development of live-cell quantitative compound binding assays for >360 individual wild type and mutant protein kinases including serine/threonine-specific kinases, receptor tyrosine kinases, and all 20 cyclin-dependent kinases (CDKs). RAS is a well-known oncogene that is frequently mutated in most lung, pancreatic and colorectal cancers and is associated with poor disease prognosis. The recent discovery of a unique switch II binding pocket and successful inhibition of the KRAS (G12C) mutant by covalent inhibitors have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Our data demonstrate that NanoBRET TE cellular assay can measure the apparent affinity of RAS inhibitors by competitive displacement of a NanoBRET RAS switch I/II pocket tracer, reversibly bound to the LgBiT- and SmBiT- KRAS, KRAS (G12C), KRAS (G12D), KRAS (G12V), KRAS (G13D), KRAS (Q61H), KRAS (Q61L), KRAS (Q61R), KRAS-G12A, KRAS-G12S, HRAS, HRAS-G12C, and HRAS-G12V fusion constructs co-transfected in live HEK293 cells. Our data also show NanoBRET cellular assay can be used to measure compound binding affinity to epigenetic drug target proteins such as BRD4, BRD4-BD1, BRD4-BD2, HDAC6, HDAC1, HDAC2, and HDAC3. Taken together, our results suggest NanoBRET TE cellular assay can serve as a great tool to facilitate new drug discovery against human cancers. Citation Format: Jianghong Wu, Yuan Wang, Shawn McGinley, Peter Gallagher, Li Liang, Alisha Di Lanni, Ashley Miles, Mohammad Haque, Yong Wan, Haiching Ma. Application of NanoBRET target engagement cellular assay for development of inhibitors against protein kinases, RAS, and epigenetic proteins in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2764.

Utility of quantitative MRI metrics in brain ageing research.

The advent of new, advanced quantitative MRI metrics allows for in vivo evaluation of multiple biological processes highly relevant for ageing. The presented study combines several MRI parameters hypothesised to detect distinct biological characteristics as myelin density, cellularity, cellular membrane integrity and iron concentration. 116 healthy volunteers, continuously distributed over the whole adult age span, underwent a multi-modal MRI protocol acquisition. Scatterplots of individual MRI metrics revealed that certain MRI protocols offer much higher sensitivity to early adulthood changes while plateauing in higher age (e.g., global functional connectivity in cerebral cortex or orientation dispersion index in white matter), while other MRI metrics provided reverse ability-stable levels in young adulthood with sharp changes with rising age (e.g., T1ρ and T2ρ). Nonetheless, despite the previously published validations of specificity towards microstructural biology based on cytoarchitectonic maps in healthy population or alterations in certain pathologies, several metrics previously hypothesised to be selective to common measures failed to show similar scatterplot distributions, pointing to further confounding factors directly related to age. Furthermore, other metrics, previously shown to detect different biological characteristics, exhibited substantial intercorrelations, be it due to the nature of the MRI protocol itself or co-dependence of relevant biological microstructural processes. All in all, the presented study provides a unique basis for the design and choice of relevant MRI parameters depending on the age group of interest. Furthermore, it calls for caution in simplistic biological inferences in ageing based on one simple MRI metric, even though previously validated under other conditions. Complex multi-modal approaches combining several metrics to extract the shared subcomponent will be necessary to achieve the desired goal of histological MRI.

A bifunctional imidazolium-based cholesterol analog for the tracking of cellular cholesterol distributions and cholesterol-protein interactions.

Cholesterol is a sterol lipid found in all higher eukaryotic organisms. It is required to consolidate the basic structural integrity and dynamic principles of cellular membranes and participates in many essential cellular processes that range from signal transduction to membrane traffic and metabolism. Moreover, a growing number of clinically highly relevant diseases such as immunological disorders or cancer has been linked to changes or misfunctions in cholesterol homeostasis. Therefore, the development of molecular tools that help to further unravel the role of cholesterol in essential cellular processes is of high relevance. Herein, we report the synthesis and proof-of-concept of a novel bifunctional imidazolium-based cholesterol analog (X-CHIM) that we envision to serve as a broadly applicable tool for the simultaneous investigation of cellular cholesterol distributions as well as cholesterol-protein interactions.

Ursolic Acid Impairs Cellular Lipid Homeostasis and Lysosomal Membrane Integrity in Breast Carcinoma Cells.

Cancer is one of the leading causes of death worldwide, thus the search for new cancer therapies is of utmost importance. Ursolic acid is a naturally occurring pentacyclic triterpene with a wide range of pharmacological activities including anti-inflammatory and anti-neoplastic effects. The latter has been assigned to its ability to promote apoptosis and inhibit cancer cell proliferation by poorly defined mechanisms. In this report, we identify lysosomes as the essential targets of the anti-cancer activity of ursolic acid. The treatment of MCF7 breast cancer cells with ursolic acid elevates lysosomal pH, alters the cellular lipid profile, and causes lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol. Lysosomal membrane permeabilization precedes the essential hallmarks of apoptosis placing it as an initial event in the cascade of effects induced by ursolic acid. The disruption of the lysosomal function impairs the autophagic pathway and likely partakes in the mechanism by which ursolic acid kills cancer cells. Furthermore, we find that combining treatment with ursolic acid and cationic amphiphilic drugs can significantly enhance the degree of lysosomal membrane permeabilization and cell death in breast cancer cells.

Widely targeted metabolomics analysis reveals the mechanism of quality improvement of flue-cured tobacco.

Flue-curing of top leaves with stems is a widely applied curing technology. Owing to the presence of stems, the quality of flue-cured leaves was significantly improved. However, the contribution of stems to flue-cured leaves is still unknown. In this study, the differences in physicochemical properties and metabolomics data between separated leaves (stem(-)) and leaves with stems (stem(+)) were investigated. The metabolic profiling of stem(+) was significantly different from that of stem(-), with phytohormone indole-3-acetic acid (IAA) being one of the most differential metabolites. The presence of stems reduced the rate of water loss in leaves, which led to less ROS accumulation, higher antioxidant enzyme activities and a lower level of membrane lipid peroxidation in stem(+) than in stem(-). The presence of stems also helped maintain the cellular membrane integrity of leaf cells by preventing the accumulation of IAA in leaf cells. Better cellular membrane integrity during flue-curing means a lower risk of leaf browning. In addition, stem(+) had a lower starch content than stem(-) because of a higher level of amylase activity. In summary, these results indicated that the presence of stems caused metabolism changes in leaves, prevented flue-cured leaves from browning and enhanced starch degradation in leaves during flue-curing.

Single-walled carbon nanotube conjugated cytochrome c as exogenous nano catalytic medicine to combat intracellular oxidative stress.

Mitochondrial dysfunction has been reported to be one of the main causes of many diseases including cancer, type2 diabetes, neurodegenerative disorders, cardiac ischemia, sepsis, muscular dystrophy, etc. Under in vitro conditions, Cytochrome C (Cyt C) maintains mitochondrial homeostasis and stimulates apoptosis, along with being a key participant in the life-supporting function of ATP synthesis. Hence, the medicinal importance of Cyt C as catalytic defense is immensely important in various mitochondrial disorders. Here, we have developed a nanomaterial via electrostatically conjugating oxidized single-wall carbon nanotube with Cyt C (Cyt C@cSWCNT) for the exogenous delivery of Cyt C. The chemical and morphological characterization of the developed Cyt C@cSWCNT was done using UV-vis, FTIR, XPS, powder XRD, TGA/DSC, TEM, etc. The developed Cyt C@cSWCNT exhibited bifunctional catalase and peroxidase activity with Km (∼ 642.7 μM and 351.6 μM) and Vmax (∼0.33 μM/s and 2.62 μM/s) values, respectively. Also, through this conjugation Cyt C was found to retain its catalytic activity even at 60°C, excellent catalytic recyclability (at least up to 3 times), and wider pH activity (pH=3 to 9). Cyt C@cSWCNT was found to promote intracellular ROS quenching and maintain mitochondrial membrane potential and cellular membrane integrity via Na+/K+ ion homeostasis during the H2O2 stress. Overall the present strategy provides an alternative approach for the exogenous delivery of Cyt C which can be used as nano catalytic medicine.

Mass spectrometry-based approaches to explore metabolism regulating ferroptosis

Ferroptosis is a type of programmed cell death distinct from apoptosis or necroptosis. Ferroptosis is well characterized by an iron-dependent accumulation of lipid peroxides and disruption of cellular membrane integrity. Many metabolic alterations can prevent or accelerate ferroptosis induction. Recent advances in analytical techniques of mass spectrometry have allowed high-throughput analysis of metabolites known to be critical for understanding ferroptosis regulatory metabolism. In this review, we introduce mass spectrometry-based analytical methods contributing to recent discovery of various metabolic pathways regulating ferroptosis, focusing on cysteine metabolism, antioxidant metabolism, and poly-unsaturated fatty acid metabolism.

Cytotoxicity inhibition of catechol's type molecules by grafting on TiO2 and Fe2O3 nanoparticles surface

The potential toxicity deriving from the interaction between chemicals and manufactured nanoparticles (NPs) represents an emerging threat to the environment and human health. Several studies have focused on the risks and (eco)toxicity of manufactured NPs as a consequence of their extensive use in recent years, however, there is still a limited understanding of the combined effects caused by manufactured NPs in the presence of other environmental contaminants. This is particularly relevant to aquatic environments, where many types of pollutants are inevitably released and can be involved in many kinds of reactions. In this context, the interaction between catecholate type ligands and two different nanomaterials, namely TiO2 and Fe2O3 NPs, was investigated by performing cytotoxicity assays with the topminnow fish hepatoma cell line (PLHC-1) using: i) the original organic molecules, ii) pristine NPs alone, and iii) modified NPs obtained by grafting the ligands on the NPs surface. Cytotoxic effects were explored at three different levels, specifically on cellular metabolism, membrane integrity and lysosomal activity. The outcomes from these assays showed cytotoxicity only for the free catechol type ligands, while in general no significant decrease in cell viability was observed for pristine NPs, as well as for the modified NPs, regardless the initial cytotoxicity level of the organic ligands These results suggest that the binding of catechols on the NPs’ surface inhibited their cytotoxicity, indicating that TiO2 and Fe2O3 NPs may act as sorbents of these contaminants, thus reducing their possible detrimental effects.

Apoptosis Detection Assays.

Many apoptosis assays are available since there are many proteins regulated at multiple points and involved in apoptosis signaling cascade. To detect apoptosis accurately, two or more assays should be used since there are many overlapped features between apoptosis and necrosis. There are six major groups of available assays to detect apoptosis: membrane alteration, mitochondrial assays, cytomorphological alterations, DNA fragmentation, detection of caspase, cleaved substrate, inhibitors and regulators, and detection of apoptosis in whole mounts. Among those assay, early apoptosis could be detected through annexin V, which is based on the loss of the cellular membrane integrity. Also, there are many assays that can detect midphase of apoptosis using caspase activation and molecular processing including PARP degradation. Late phase of apoptosis could be detected with DNA fragmentation assays. Combinations of these assays allow us to identify the mechanisms of apoptosis induction after specific stimulus. This chapter will introduce three apoptosis detection assays including annexin assay, DNA/chromatin condensation assays, and TUNEL assay.

Nanoscale Delivery Systems of Lutein: An Updated Review from a Pharmaceutical Perspective.

Carotenoids are natural lipid-soluble pigments that produce yellow to red colors in plants as well as providing bright coloration in vegetables and fruits. Lutein belongs to the xanthophyll subgroup of the carotenoid family, which plays an essential role in photosynthesis and photoprotection in nature. In the human body, lutein, together with its isomer zeaxanthin and its metabolite meso-zeaxanthin, accumulates in the macula of the eye retina, which is responsible for central, high-resolution, and color vision. As a bioactive phytochemical, lutein has essential physiological functions, providing photoprotection against damaging blue light, along with the neutralization of oxidants and the preservation of the structural and functional integrity of cellular membranes. As a potent antioxidant and anti-inflammatory agent, lutein unfortunately has a low bioavailability because of its lipophilicity and a low stability as a result of its conjugated double bonds. In order to enhance lutein stability and bioavailability and achieve its controlled delivery to a target, nanoscale delivery systems, which have great potential for the delivery of bioactive compounds, are starting to be employed. The current review highlights the advantages and innovations associated with incorporating lutein within promising nanoscale delivery systems, such as liposomes, nanoemulsions, polymer nanoparticles, and polymer–lipid hybrid nanoparticles, as well as their unique physiochemical properties.

Amphotericin B and 5-flucytosine as fungicides against Penicillium italicum for citrus fruit rot

The expansion of the global population and improved living standards has created an increasing demand for fresh fruit. The combination of agricultural and medical development for new fungicides is one of the most effective approaches for keeping fruit fresh. Both amphotericin B and 5-flucytosine are quite safe and have a wide spectrum of antifungal activity. For the purpose of investigating their potential applications for preserving fruit freshness, we evaluated their antifungal efficiencies against Penicillium italicum in vitro and in vivo. The results showed that amphotericin B and 5-flucytosine significantly inhibited spore germination and the mycelial growth of P. italicum. The 5-flucytosine showed much better bioactivity than the well-known agricultural fungicide prochloraz, both in vitro and in vivo. Furthermore, crystal violet staining, propidium iodide dyeing, ultrastructural observation and the ergosterol content indicated that amphotericin B and 5-flucytosine destroyed the cellular ultrastructure and membrane integrity. In addition, nucleic acids (OD260) and proteins (OD280) escaped from P. italicum cells in a dose-dependent manner. The results of this study indicated that amphotericin B and 5-flucytosine have good potential as antifungal preservatives for postharvest citrus fruit due to their excellent antifungal effects, and that the primary mode of action was the destruction of the membrane integrity and permeability of P. italicum.

Abstract 379: Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells

Abstract NanoBRET࣪ target engagement (TE) is the first biophysical technique that broadly enables the quantitative determination of protein inhibitor occupancy in live cells, without disruption of cellular membrane integrity. This quantitative capability is achieved in live cells via BRET with an optimized set of cell-permeable tracers, allow the measurement of compound binding to a selected cellular target protein. RAS is a well-known oncogene that is frequently mutated in most lung, pancreatic and colorectal cancers and is associated with poor disease prognosis. Mutated RAS is locked in the activated GTP bound state and facilitates enhanced RAS signaling in cancer cells. While being a desirable target, the absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful. The recent discovery of a unique switch II binding pocket and successful inhibition of the KRAS (G12C) mutant by covalent inhibitors have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Here, we performed NanoBRET࣪ TE cellular assay with RAS inhibitors against transfected RAS and its mutants. Our data demonstrate that NanoBRET࣪ TE cellular assay can measure the apparent affinity of RAS inhibitors by competitive displacement of a NanoBRET࣪ RAS switch I/II pocket tracer, reversibly bound to the LgBiT- and SmBiT- KRAS, KRAS (G12C), KRAS (G12D), KRAS (G12V), KRAS (G13D), KRAS (Q61H), KRAS (Q61L), KRAS (Q61R), or HRAS fusion constructs co-transfected in live HEK293 cells. Z factor analysis indicates the assay is High Throughput Screening (HTS) compatible and reproducible. In addition, we are able to confirm the signaling inhibition of downstream phospho-ERK1/2 by KRAS (G12C)-specific inhibitors AMG 510 (Sotorasib), ARS-1620, MRTX849, and MRTX1257 in MIA PaCa-2 pancreas and SW837 colon cancer cells bearing KRAS (G12C) mutation by Western blot assay. Our results suggest NanoBRET࣪ TE cellular assay can serve as a great tool to facilitate RAS pathway drug discovery against human cancers. Citation Format: Jianghong Wu, Shawn McGinley, Yuan Wang, Peter Gallagher, Haiching Ma. Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells [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 379.

Exploring the integrity of cellular membrane and resistance to digestive juices of dehydrated lactic acid bacteria as influenced by drying kinetics

The survival of probiotics after spray drying is largely related to drying kinetics experienced by cells in atomized droplets. In this study, the effect of drying kinetics on the viability, integrity of cellular membrane, and digestive stability of Lactobacillus rhamnosus GG and Lactococcus lactis subsp. cremoris was quantitatively examined, using the single droplet drying technique with reconstituted skimmed milk as protectant. Distinct inactivation histories of the strains were observed at 70 °C, with droplet sizes of 1 and 2 μL and air flow velocities of 0.45 and 1 m/s. The two strains showed similar inactivation profiles under the same condition. The order of inactivation rate and the degree of damages on the cellular membrane of dehydrated cells followed the order of drying rate, which was the highest for 1 µL at 1 m/s, followed by 1 µL at 0.45 m/s and 2 µL at 1 m/s, while 2 µL at 0.45 m/s showed the slowest inactivation. For individual drying process, the percentage of live cells with intact membrane was constantly higher than cell survival ratio. Dehydrated cells showed increased viability after in situ gastric digestion for 30 min, which could be due to the resistance of milk proteins to simulated gastric fluid and the rich nutrients of the protectant. Bacterial viability declined again after subsequent in situ intestinal digestion. The findings demonstrated the sensitivity of lactic acid bacteria toward minor changes in drying kinetics. Optimizing drying kinetics may effectively increase the survival and lower the cellular injury of spray-dried probiotics.