A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis upon metabolic demands. Detection of these contact sites at the nanometer scale over time in living cells is challenging. We developed a tool kit for detecting contact sites based on fluorogen-activated bimolecular complementation at CONtact sites, FABCON, using a reversible, low-affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic regulation.

Mammary fat globules as a source of mRNA to model alterations in the expression of some milk component genes during lactation in bovines.

BACKGROUND: The milk's nutritional value is determined by its constituents, including fat, protein, carbohydrates, and minerals. The mammary gland's ability to produce milk is controlled by a complex network of genes. Thereby, the fat, protein, and lactose synthesis must be boost in milk to increase milk production efficiency. This can be accomplished by fusing genetic advancements with proper management practices. Therefore, this study aimed to investigate the association between the Lipoprotein lipase (LPL), kappa casein CSN3, and Glucose transporter 1 (GLUT1) genes expression levels and such milk components as fat, protein, and lactose in different dairy breeds during different stages of lactation. METHODS: To achieve such a purpose, 94 milk samples were collected (72 samples from 36 multiparous black-white and red-white Holstein-Friesian (HF) cows and 22 milk samples from 11 Egyptian buffaloes) during the early and peak lactation stages. The milk samples were utilized for milk analysis and genes expressions analyses using non- invasive approach in obtaining milk fat globules (MFGs) as a source of Ribonucleic acid (RNA). RESULTS: LPL and CSN3 genes expressions levels were found to be significantly higher in Egyptian buffalo than Holstein-Friesian (HF) cows as well as fat and protein percentages. On the other hand, GLUT1 gene expression level was shown to be significantly higher during peak lactation than early lactation. Moreover, lactose % showed a significant difference in peak lactation phase compared to early lactation phase. Also, fat and protein percentages were significantly higher in early lactation period than peak lactation period but lactose% showed the opposite pattern of Egyptian buffalo. CONCLUSION: Total RNA can be successfully obtained from MFGs. The results suggest that these genes play a role in glucose absorption and lactose synthesis in bovine mammary epithelial cells during lactation. Also, these results provide light on the differential expression of these genes among distinct Holstein-Friesian cow breeds and Egyptian buffalo subspecies throughout various lactation phases.

Species-specific histological characterizations of renal tubules and collecting ducts in the kidneys of cats and dogs.

The histomorphological features of normal kidneys in cats and dogs have been revealed despite the high susceptibility of cats to tubulointerstitial damage. Herein, the histological characteristics of the two species were compared. Cytoplasmic lipid droplets (LDs) were abundant in the proximal convoluted tubules (PCTs) of cats aged 23-27 months but scarce in dogs aged 24-27 months. LDs were rarely observed in the distal tubules (DTs) and collecting ducts (CDs) of either species, as visualized by the expression of Tamm-Horsfall protein 1, calbindin-D28K, and aquaporin 2. The occupational area ratio of proximal tubules (PTs) in the renal cortex was higher, but that of DTs or CDs was significantly lower in adult cats than in dogs. Single PT epithelial cells were larger, but PCT, DT, and CD lumens were significantly narrower in adult cats than in dogs. Unlike adults, young cats at 6 months exhibited significantly abundant cytoplasmic LDs in proximal straight tubules, indicating lipid metabolism-related development. Histochemistry of the 21 lectins also revealed variations in glycosylation across different renal tubules and CDs in both species. Sodium-glucose cotransporter 2 was expressed only in PTs, excluding the proximal straight tubules with few LDs in adult cats or the PCTs of young cats and adult dogs. These findings are crucial for understanding species-specific characteristics of renal histomorphology and pathogenesis.

Exploring selection signatures in the divergence and evolution of lipid droplet (LD) associated genes in major oilseed crops.

BACKGROUND: Oil bodies or lipid droplets (LDs) in the cytosol are the subcellular storage compartments of seeds and the sites of lipid metabolism providing energy to the germinating seeds. Major LD-associated proteins are lipoxygenases, phospholipaseD, oleosins, TAG-lipases, steroleosins, caleosins and SEIPINs; involved in facilitating germination and enhancing peroxidation resulting in off-flavours. However, how natural selection is balancing contradictory processes in lipid-rich seeds remains evasive. The present study was aimed at the prediction of selection signatures among orthologous clades in major oilseeds and the correlation of selection effect with gene expression. RESULTS: The LD-associated genes from the major oil-bearing crops were analyzed to predict natural selection signatures in phylogenetically close-knit ortholog clusters to understand adaptive evolution. Positive selection was the major force driving the evolution and diversification of orthologs in a lineage-specific manner. Significant positive selection effects were found in 94 genes particularly in oleosin and TAG-lipases, purifying with excess of non-synonymous substitution in 44 genes while 35 genes were neutral to selection effects. No significant selection impact was noticed in Brassicaceae as against LOX genes of oil palm. A heavy load of deleterious mutations affecting selection signatures was detected in T-lineage oleosins and LOX genes of Arachis hypogaea. The T-lineage oleosin genes were involved in mainly anther, tapetum and anther wall morphogenesis. In Ricinus communis and Sesamum indicum > 85% of PLD genes were under selection whereas selection pressures were low in Brassica juncea and Helianthus annuus. Steroleosin, caleosin and SEIPINs with large roles in lipid droplet organization expressed mostly in seeds and were under considerable positive selection pressures. Expression divergence was evident among paralogs and homeologs with one gene attaining functional superiority compared to the other. The LOX gene Glyma.13g347500 associated with off-flavor was not expressed during germination, rather its paralog Glyma.13g347600 showed expression in Glycine max. PLD-α genes were expressed on all the tissues except the seed,δ genes in seed and meristem while ß and γ genes expressed in the leaf. CONCLUSIONS: The genes involved in seed germination and lipid metabolism were under strong positive selection, although species differences were discernable. The present study identifies suitable candidate genes enhancing seed oil content and germination wherein directional selection can become more fruitful.

Scalable lipid droplet microarray fabrication, validation, and screening.

High throughput screening of small molecules and natural products is costly, requiring significant amounts of time, reagents, and operating space. Although microarrays have proven effective in the miniaturization of screening for certain biochemical assays, such as nucleic acid hybridization or antibody binding, they are not widely used for drug discovery in cell culture due to the need for cells to internalize lipophilic drug candidates. Lipid droplet microarrays are a promising solution to this problem as they are capable of delivering lipophilic drugs to cells at dosages comparable to solution delivery. However, the scalablility of the array fabrication, assay validation, and screening steps has limited the utility of this approach. Here we take several new steps to scale up the process for lipid droplet array fabrication, assay validation in cell culture, and drug screening. A nanointaglio printing process has been adapted for use with a printing press. The arrays are stabilized for immersion into aqueous solution using a vapor coating process. In addition to delivery of lipophilic compounds, we found that we are also able to encapsulate and deliver a water-soluble compound in this way. The arrays can be functionalized by extracellular matrix proteins such as collagen prior to cell culture as the mechanism for uptake is based on direct contact with the lipid delivery vehicles rather than diffusion of the drug out of the microarray spots. We demonstrate this method for delivery to 3 different cell types and the screening of 92 natural product extracts on a microarray covering an area of less than 0.1 cm2. The arrays are suitable for miniaturized screening, for instance in high biosafety level facilities where space is limited and for applications where cell numbers are limited, such as in functional precision medicine.

Infant formula supplemented with milk fat globule membrane compared with standard infant formula for the cognitive development of healthy term-born formula-fed infants: protocol for a randomised controlled trial.

INTRODUCTION: Milk fat globule membrane (MFGM) is a complex lipid-protein structure in mammalian milk and human milk that is largely absent from breastmilk substitutes. The objective of this trial is to investigate whether providing infant formula enriched with MFGM versus standard infant formula improves cognitive development at 12 months of age in exclusively formula-fed full-term infants. METHODS AND ANALYSIS: This is a randomised, controlled, clinician-blinded, researcher-blinded and participant-blinded trial of two parallel formula-fed groups and a breastfed reference group that were recruited in the suburban Adelaide (Australia) community by a single study centre (a medical research institute). Healthy, exclusively formula-fed, singleton, term-born infants under 8 weeks of age were randomised to either an MFGM-supplemented formula (intervention) or standard infant formula (control) from enrolment until 12 months of age. The reference group was not provided with formula. The primary outcome is the Cognitive Scale of the Bayley Scales of Infant Development, Fourth Edition (Bayley-IV) at 12 months. Secondary outcomes are the Bayley-IV Cognitive Scale at 24 months, other Bayley-IV domains (language, motor, emotional and behavioural development) at 12 and 24 months of age, infant attention at 4 and 9 months of age, parent-rated language at 12 and 24 months of age, parent-rated development at 6 and 18 months of age as well as growth, tolerance and safety of the study formula. To ensure at least 80% power to detect a 5-point difference in the mean Bayley-IV cognitive score, >200 infants were recruited in each group. ETHICS AND DISSEMINATION: The Women's and Children Health Network Human Research Ethics Committee reviewed and approved the study (HREC/19/WCHN/140). Caregivers gave written informed consent prior to enrolling in the trial. Findings of this study will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: ACTRN12620000552987; Australian and New Zealand Clinical Trial Registry: anzctr.org.au.

Using the yeast vacuole as a system to test the lipidic drivers of membrane heterogeneity in living cells.

The biophysical drivers of membrane lateral heterogeneity, often termed lipid rafts, have been largely explored using synthetic liposomes or mammalian plasma membrane-derived giant vesicles. Yeast vacuoles, an organelle comparable to mammalian lysosomes, is the only in vivo system that shows stable micrometer scale phase separation in unperturbed cells. The ease of manipulating lipid metabolism in yeast makes this a powerful system for identifying lipids involved in the onset of vacuole membrane heterogeneity. Vacuole domains are induced by stationary stage growth and nutritional starvation, during which they serve as a docking and internalization site for lipid droplet energy stores. Here we describe methods for characterizing vacuole phase separation, its physiological function, and its lipidic drivers. First, we detail methodologies for robustly inducing vacuole domain formation and quantitatively characterizing during live cell imaging experiments. Second, we detail a new protocol for biochemical isolation of stationary stage vacuoles, which allows for lipidomic dissection of membrane phase separation. Third, we describe biochemical techniques for analyzing lipid droplet internalization in vacuole domains. When combined with genetic or chemical perturbations to lipid metabolism, these methods allow for systematic dissection of lipid composition in the structure and function of ordered membrane domains in living cells.

Dietary intake of a MFGM/EV-rich concentrate promotes accretion of very long odd-chain sphingolipids and increases lipid metabolic turnover at the whole-body level.

Lipids from cow milk fat globule membranes (MFGMs) and extracellular vesicles (EVs) are considered beneficial for neurodevelopment, cognitive maintenance and human health in general. Nevertheless, it is largely unknown whether intake of infant formulas and medical nutrition products rich in these particles promote accretion of specific lipids and whether this affects metabolic homeostasis. To address this, we carried out a 16-week dietary intervention study where mice were supplemented with a MFGM/EV-rich concentrate, a control diet supplemented with a whey protein concentrate and devoid of milk lipids, or regular chow. Assessment of commonly used markers of metabolic health, including body weight, glucose intolerance and liver microanatomy, demonstrated no differences across the dietary regimes. In contrast, in-depth lipidomic analysis revealed accretion of milk-derived very long odd-chain sphingomyelins and ceramides in blood plasma and multiple tissues of mice fed the MFGM/EV diet. Furthermore, lipidomic flux analysis uncovered that mice fed the MFGM/EV diet have increased lipid metabolic turnover at the whole-body level. These findings help fill a long-lasting knowledge gap between the intake of MFGM/EV-containing foods and the health-promoting effects of their lipid constituents. In addition, the findings suggest that dietary sphingomyelins or ceramide-breakdown products with very long-chains can be used as structural components of cellular membranes, lipoprotein particles and signaling molecules that modulate metabolic homeostasis and health.

Effects of Milk Fat Globule Membrane Supplementation Following Exercise Training on Physical Performance in Healthy Young Adults: A Randomized Double-Blind, Placebo-Controlled Pilot Trial.

The purpose of this study was to examine whether 4 wk of daily ingestion of milk fat globule membrane (MFGM) combined with exercise training improves physical performance-muscle strength, agility and muscle power-in healthy young adults. The study was designed as a randomized, double-blind, and placebo-controlled trial. Twenty healthy young adults received either an MFGM powder containing 1.6 g of fat and 160 mg of sphingomyelin or an isocaloric placebo powder daily throughout 4 wk of power or agility training. Physical performance tests and body composition measurements were conducted before and after the 4-wk intervention. Ingestion of MFGM did not affect isometric or isokinetic muscle strength, but it was associated with a greater increase in vertical jump peak power compared with placebo. There were no significant changes in body weight or lean body mass during the intervention period in either group, and no significant differences between groups. We conclude that daily MFGM supplementation combined with exercise training has the potential to improve physical performance in young adults; however, further studies with larger sample sizes should be conducted to obtain more evidence supporting achievement of improved physical performance through MFGM supplementation.

Protocol for observing lipid droplet dynamics in chicken cone cells.

Chicken cone cells are an excellent model for studying the regulation of lipid droplet dynamics. Here, we present a protocol for studying cone cell lipid droplets from in vivo and ex vitro cultured retinas of chicken embryos. We describe steps for dissecting chicken retinas, electroporating retinas, culturing retinas ex vivo and in vitro, and staining lipid droplets with neutral lipid dye. This protocol is also applicable to investigating other organelles in retinas. For complete details on the use and execution of this protocol, please refer to Pan et al.1.

Regulating donor configuration to develop AIE-active type I photosensitizers for lipid droplet imaging and high-performance photodynamic therapy under hypoxia.

Type I photodynamic therapy is considered to be a more promising cancer treatment than type II photodynamic therapy due to its non-oxygen-dependent characteristics. In this work, three D-A structure N,N'-dihydrophenazine (DHP)-based photosensitizers DP-CNPY, SMP-CNPY and DMP-CNPY were designed and synthesized by introducing different numbers of methyl groups in the backbone neighbor of DHP as the donor and combined with the typical strong electron acceptor 2-(pyridin-4-yl)acetonitrile. Among the three photosensitizers, SMP-CNPY with one methyl modification showed the best type I ROS (O2-˙, ˙OH) generation capacity and AIE performance. By encapsulation, SMP-CNPY was fabricated into nanoparticles, and SMP-CNPY NPs exhibited lipid droplet targeting ability with near-infrared (NIR) emission. Cell experiments have proved that SMP-CNPY NPs can effectively kill different kinds of cancer cells under normal oxygen conditions. Even under hypoxic and extreme hypoxic conditions, SMP-CNPY NPs can still produce ROS and kill cancer cells. This work holds significant potential in the field of type I AIE-active photosensitizers and provides a new strategy for overcoming the hypoxic dilemma in the malignant tumor microenvironment.

PGC-1α repression dysregulates lipid metabolism and induces lipid droplet accumulation in retinal pigment epithelium.

Drusen, the yellow deposits under the retina, are composed of lipids and proteins, and represent a hallmark of age-related macular degeneration (AMD). Lipid droplets are also reported in the retinal pigment epithelium (RPE) from AMD donor eyes. However, the mechanisms underlying these disease phenotypes remain elusive. Previously, we showed that Pgc-1α repression, combined with a high-fat diet (HFD), induce drastic AMD-like phenotypes in mice. We also reported increased PGC-1α acetylation and subsequent deactivation in the RPE derived from AMD donor eyes. Here, through a series of in vivo and in vitro experiments, we sought to investigate the molecular mechanisms by which PGC-1α repression could influence RPE and retinal function. We show that PGC-1α plays an important role in RPE and retinal lipid metabolism and function. In mice, repression of Pgc-1α alone induced RPE and retinal degeneration and drusen-like deposits. In vitro inhibition of PGC1A by CRISPR-Cas9 gene editing in human RPE (ARPE19- PGC1A KO) affected the expression of genes responsible for lipid metabolism, fatty acid ß-oxidation (FAO), fatty acid transport, low-density lipoprotein (LDL) uptake, cholesterol esterification, cholesterol biosynthesis, and cholesterol efflux. Moreover, inhibition of PGC1A in RPE cells caused lipid droplet accumulation and lipid peroxidation. ARPE19-PGC1A KO cells also showed reduced mitochondrial biosynthesis, impaired mitochondrial dynamics and activity, reduced antioxidant enzymes, decreased mitochondrial membrane potential, loss of cardiolipin, and increased susceptibility to oxidative stress. Our data demonstrate the crucial role of PGC-1α in regulating lipid metabolism. They provide new insights into the mechanisms involved in lipid and drusen accumulation in the RPE and retina during aging and AMD, which may pave the way for developing novel therapeutic strategies targeting PGC-1α.

Compartmentalised enzyme-induced phase transformations in self-assembling lipid systems.

HYPOTHESIS: Understanding the digestion of lipid-based pharmaceutical formulations and food systems is necessary for optimising drug and nutrient delivery and has been extensively studied in bulk emulsion systems using the pH-stat method [1]. However, this approach is not suitable for investigation of individual lipid droplets, in particular the interface where the lipase acts. Microfluidic approaches to study digestion at lipid-water interfaces using droplet trapping have been proposed, however the aqueous phase in that case washes over the interface presenting uncertainty over the stoichiometry of interactions [2]. The internal interface of a Janus-like droplet, containing distinct aqueous and lipid compartments, mimics the interface of a lipid droplet in aqueous solution with controlled stoichiometry [3]. Hence, it was hypothesised that the internal interface of Janus droplets can offer a precise way to study the enzymatic digestion of lipids formulations. EXPERIMENTS: Using microfluidic methods, Janus-like droplets were formed by coalescing emulsion droplets containing lipid formulation and pancreatic lipase. Polarised light microscopy (PLM) and in-situ small-angle X-ray scattering (SAXS) were used to investigate the droplets. FINDINGS: PLM revealed the growth of an aligned inverse hexagonal phase (H2), and with SAXS showed that this phase transformation and alignment resulted from enzymatic digestion. A subsequent partial transformation from H2 to inverse bicontinuous cubic phase occurred when simulated intestinal fluid was used instead of Tris buffer. Suggesting that phospholipids and bile salts could diffuse across the internal interface to locally affect their surroundings.

Qualitative and quantitative analysis of lipid droplets in mature 3T3-L1 adipocytes using oil red O.

By differentiating into mature adipocytes, 3T3-L1 cells can be utilized as a model cell line to investigate (pre)adipocyte function in vitro. Here, we present a protocol for combining qualitative and quantitative analysis of lipid droplets in mature 3T3-L1 adipocytes using oil red O. We describe steps to differentiate 3T3-L1 preadipocytes to adipocytes and give detailed procedures to determine total lipid amount as well as lipid droplet size and number using microscopic devices and an ImageJ macro. For complete details on the use and execution of this protocol, please refer to Kaczmarek et al.1.

Multiscale approach to the characterization of the interfacial properties of micellar casein and whey protein blends and their effects on recombined dairy creams.

In this study, whipped cream with blends of micellar casein (MCN) and whey protein (WPI) in different ratios were prepared to investigate the role of protein interfacial behavior in determining foam properties at multiple scales, using theoretical modeling, and microscopic and macroscopic analysis. Fluid force microscopy has been used for the first time as a more realistic and direct means of analyzing interfaces properties in multiphase systems. The adsorption kinetics showed that the interfacial permeability constant of WPI (4.24 × 10-4 s-1) was significantly higher than that of the MCN (2.97 × 10-4 s-1), and the WPI interfacial layer had a higher modulus of elasticity (71.38 mN/m) than that of the MCN (47.89 mN/m). This model was validated via the mechanical analysis of the fat globules in real emulsions. The WPI-stabilized fat globule was found to have a higher Young's modulus (219.67 Pa), which contributes to the integrity of its fat globule morphology. As the ratio of MCN was increased in the sample, however, both the interfacial modulus and Young's modulus decreased. Moreover, the rate of partial coalescence was found to increase, a phenomenon that decreased the stability of the emulsion and increased the rate of aeration. The mechanical analysis also revealed a higher level of adhesion between MCN-stabilized fat globule (25.16 nN), which increased fat globule aggregation and emulsion viscosity, while improving thixotropic recovery. The synergistic effect of the blended MCN and WPI provided the highest overrun, at 194.53 %. These studies elucidate the role of the interfacial behavior of proteins in determining the quality of whipped cream and provide ideas for the application of proteins in multiphase systems.

Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.

A fluorescence probe for imaging lipid droplet and visualization of diabetes-related polarity variations.

Diabetes mellitus is a disorder that affects lipid metabolism. Abnormalities in the lipid droplets (LDs) can lead to disturbances in lipid metabolism, which is a significant feature of diabetic patients. Nevertheless, the correlation between diabetes and the polarity of LDs has received little attention in the scientific literature. In order to detect LDs polarity changes in diabetes illness models, we created a new fluorescence probe LD-DCM. This probe has a stable structure, high selectivity, and minimal cytotoxicity. The probe formed a typical D-π-A molecular configuration with triphenylamine (TPA) and dicyanomethylene-4H-pyran (DCM) as electron donor and acceptor parts. The LD-DCM molecule has an immense solvatochromic effect (λem = 544-624 nm), fluorescence enhancement of around 150 times, and a high sensitivity to polarity changes within the linear range of Δf = 0.28 to 0.32, all due to its distinctive intramolecular charge transfer effect (ICT). In addition, LD-DCM was able to monitor the accumulation of LDs and the reduction of LDs polarity in living cells when stimulated by oleic acid, lipopolysaccharide, and high glucose. More importantly, LD-DCM has also been used effectively to detect polarity differences in organs from diabetic, drug-treated, and normal mice. The results showed that the liver polarity of diabetic mice was lower than that of normal mice, while the liver polarity of drug-treated mice was higher than that of diabetic mice. We believe that LD-DCM has the potential to serve as an efficient instrument for the diagnosis of disorders that are associated with the polarity of LDs.

Multifunctional lipid droplet probes for observing the polarity change of ferroptosis, inflammation, fatty liver and evaluating the efficacy of drugs.

BACKGROUND: Lipid droplets (LDs) polarity is intricately linked to diverse biological processes and diseases. The visualization of LDs-polarity is of vital importance but challenging due to the lack of high-specificity, high-sensitivity and large-Stokes shift probes for real-time tracking LDs-polarity in biological systems. RESULTS: Four D-π-A based fluorescent probes (TPA-TCF1-TPA-TCF4) have been developed by combining tricyanofuran (an electron acceptor, A) and triphenylamine (an electron donor, D) derivatives with different terminal groups. Among them, TPA-TCF1 and TPA-TCF4 exhibit excellent polar sensitivity, large Stokes shift (≥182 nm in H2O), and efficient LDs targeting ability. In particular, TPA-TCF4 is capable of monitoring the change of LDs-polarity during ferroptosis, inflammation, apoptosis of cancer cell, and fatty liver. SIGNIFICANCE: All these features render TPA-TCF4 a versatile tool for pharmacodynamic evaluation of anti-cancer drugs, in-depth understanding of the biological effect of LDs on ferroptosis, and medical diagnosis of LDs-polarity related diseases.

Dynamic label-free analysis of SARS-CoV-2 infection reveals virus-induced subcellular remodeling.

Assessing the impact of SARS-CoV-2 on organelle dynamics allows a better understanding of the mechanisms of viral replication. We combine label-free holotomographic microscopy with Artificial Intelligence to visualize and quantify the subcellular changes triggered by SARS-CoV-2 infection. We study the dynamics of shape, position and dry mass of nucleoli, nuclei, lipid droplets and mitochondria within hundreds of single cells from early infection to syncytia formation and death. SARS-CoV-2 infection enlarges nucleoli, perturbs lipid droplets, changes mitochondrial shape and dry mass, and separates lipid droplets from mitochondria. We then used Bayesian network modeling on organelle dry mass states to define organelle cross-regulation networks and report modifications of organelle cross-regulation that are triggered by infection and syncytia formation. Our work highlights the subcellular remodeling induced by SARS-CoV-2 infection and provides an Artificial Intelligence-enhanced, label-free methodology to study in real-time the dynamics of cell populations and their content.

Polarity-Driven Fluorescence Monitoring of Lipid Droplet Dynamics in Dry Eye Disease.

The emergence of lipid droplets (LDs) has been recognized as cellular markers of ocular surface hyperosmosis, which is recognized as a fundamental mechanism driving dry eye disease (DED), while their dynamics during DED progression and therapy remains unlocked. For this purpose, an LD-specific fluorescent probe P1 is presented in this work that exhibits highly selective and sensitive emission enhancement in response to a decreased ambient polarity (Δf) from 0.209 to 0.021. The hydrophobic nature of P1 enables specific staining of LDs, facilitating visualization of changes in polarity within these cellular structures. Utilizing P1, we observe a decrease in polarity accompanied by an increase in the size and number of LDs in hyperosmotic human corneal epithelial cells (HCECs). Furthermore, interplays between LDs and cellular organelles such as mitochondria and the Golgi apparatus are visualized, suggesting the underlying pathogenesis in DED. Notably, the variations of LDs are observed after the inhibition of ferroptosis or activation of autophagy in hyperosmotic HCECs, implying the great potential of LDs as indicators for the design and efficacy evaluation of DED drugs regarding ferroptosis or autophagy as targets. Finally, LDs are confirmed to be overproduced in corneal tissues from DED mice, and the application of clinical eye drops effectively impedes these changes. This detailed exploration underscores the significant roles of LDs as an indicator for the deep insight into DED advancement and therapy.