Modifying the interfacial dynamics of oleosome (lipid droplet) membrane using curcumin.

Cells store energy in lipid droplets, known as oleosomes, which have a neutral lipid core surrounded by a dilatable membrane of phospholipids and proteins. Oleosomes can be loaded with therapeutic lipophilic cargos through their permeable membrane and used as carriers. However, the cargo can also adsorb between the phospholipids and affect the membrane properties. In the present work, we investigated the effect of adsorbed curcumin on the mechanical properties of oleosome membranes using dilatational interfacial rheology (LAOD). The oleosome membrane had a weak-stretchable behavior, while the adsorption of curcumin led to stronger in-plane interactions, which were dependent on curcumin concentration and indicated a glassy-like structure. Our findings showed that adsorbed curcumin molecules can enhance the molecular interactions on the oleosome membrane. This behavior suggests that oleosomes membranes can be modulated by loaded cargo. Understanding cargo and membrane interactions can help to design oleosome-based formulations with tailored mechanical properties for applications.

Micro-droplet printed ion-selective membrane sensors for in situ monitoring of marine heavy metal ions.

Fast, accurate, and reliable techniques for marine toxic heavy metal ions (HMI) detection are critical for the ecological environment and human health. One of the fatal drawbacks of traditional ion selective electrochemical sensors is that the modification of electrode cannot be accurately quantified, resulting in poor repeatability of the detection electrode and large error between the multi-electrode detection results. In order to tackle this challenge, this study presents ultra-fine micro-droplet printed electrodes for the in-situ detection of Cd2+, a carcinogenic and toxic HMI commonly found in the ocean. The ion selective membrane casting liquid was dispersed into tiny droplets with a diameter of micron through microfluidic technology, and the microdroplets were precisely arranged on the electrode surface. As a result, the modification error of electrode was reduced to pL level (accurate to 10 pL), which greatly improved the repeatability between electrodes prepared in different batches. The results of experiments with pure electrolyte, interference ions and artificial seawater indicated that the micro-droplet printed sensors possessed excellent properties of accuracy, precision, repeatability, and anti-interference. This novel micro-droplet printed sensor has the potential to capture an accurate picture of nearshore HMI in heterogeneous environments under shock conditions.

An intelligent NIR fluorescent dye with dual response to pH and viscosity for investigating the interactions between LDs and mitochondria.

The interaction between lipid droplets and mitochondria plays a pivotal role in biological processes including cellular stress, metabolic homeostasis, cellular autophagy and apoptosis. Deciphering the complex interplay between lipid droplets and mitochondria is essential for gaining insights into the fundamental workings of the cell and can have broad implications for the development of therapeutic strategies for various diseases, including metabolic disorders, neurodegenerative diseases, and cancer. In this study, we develop a pH and viscosity-responsive near-infrared (NIR) fluorescent probe PTOH to investigate the interaction between lipid droplets and mitochondria. This probe demonstrates a significant enhancement in fluorescence intensity at 470 nm when the pH increases, while under acidic conditions, its fluorescence intensity at 730 nm intensifies by a factor of 35 with rising system viscosity. Cell imaging experiments revealed that PTOH can effectively discriminate between normal and cancerous cells, as well as detect intracellular pH and viscosity alterations induced by drugs. Additionally, PTOH is utilized to visualize the interaction between lipid droplets and mitochondria and to differentiate between cellular autophagy and apoptosis phenomena, providing a valuable tool for elucidating the mechanisms underlying lipid droplet-mitochondria interactions and their associated diseases.

Analysis of lipid uptake, storage, and fatty acid oxidation by group 2 innate lymphoid cells.

Group 2 Innate Lymphoid Cells (ILC2) are critical drivers of both innate and adaptive type 2 immune responses, known to orchestrate processes involved in tissue restoration and wound healing. In addition, ILC2 have been implicated in chronic inflammatory barrier disorders in type 2 immunopathologies such as allergic rhinitis and asthma. ILC2 in the context of allergen-driven airway inflammation have recently been shown to influence local and systemic metabolism, as well as being rich in lipid-storing organelles called lipid droplets. However, mechanisms of ILC2 lipid anabolism and catabolism remain largely unknown and the impact of these metabolic processes in regulating ILC2 phenotypes and effector functions has not been extensively characterized. ILC2 phenotypes and effector functions are shaped by their metabolic status, and determining the metabolic requirements of ILC2 is critical in understanding their role in type 2 immune responses and their associated pathophysiology. We detail here a novel experimental method of implementing flow cytometry for large scale analysis of fatty acid uptake, storage of neutral lipids, and fatty acid oxidation in primary murine ILC2 with complementary morphological analysis of lipid storage using confocal microscopy. By combining flow cytometry and confocal microscopy, we can identify the metabolic lipid requirements for ILC2 functions as well as characterize the phenotype of lipid storage in ILC2. Linking lipid metabolism pathways to ILC2 phenotypes and effector functions is critical for the assessment of novel pharmaceutical strategies to regulate ILC2 functions in type 2 immunopathologies.

Neuroinvasive virus utilizes a lipid droplet surface protein, perilipin2, to restrict apoptosis by decreasing Bcl-2 ubiquitination.

Lipid droplets (LDs) can interact with other organelles to regulate cell death, and it has also been reported to play an important role in virus replication. However, the interplay among LDs, cell death, and viral replication remains unclear. Neuroinvasive viruses, such as Japanese encephalitis virus (JEV), rabies virus (RABV), and encephalomyocarditis virus (EMCV) still threaten global public health and raise intensive concerns. Here, we reveal that neuroinvasive virus infection enhances cellular triglyceride biosynthesis by upregulating the expression of diacylglycerol O-acyltransferase 2 (DGAT2) to promote LD formation and increase the expression of Perilipin 2 (PLIN2), an LD surface protein, which consequently facilitates neuroinvasive virus replication. Furthermore, PLIN2 could reduce mitochondrial damage and suppress apoptosis by restoring mitochondrial potential and interacting with anti-apoptotic protein Bcl-2, specifically the 136-209 amino acid region, to interrupt the BAX-Cytc-caspase-3 apoptotic pathway by decreasing the K48-linked ubiquitination of Bcl-2 at the 17th lysine. Together, we elucidate that neuroinvasive virus utilizes an LD surface protein to restrict the apoptosis of infected cells, providing a fresh insight into the pathogenesis and antiviral therapeutics development of neuroinvasive viruses. IMPORTANCE: The neuroinvasive virus is a kind of pathogen that is capable of infiltrating and infecting the central nervous system to potentially induce severe neurological damage and disorders, which pose a significant threat to public health. Here, we found that neuroinvasive viruses can utilize an LD surface protein PLIN2 to facilitate viral replication. Notably, PLIN2 could reduce mitochondrial damage and suppress apoptosis by restoring mitochondrial potential and interacting with anti-apoptotic protein Bcl-2, specifically the 136-209 amino acid region, to interrupt the BAX-Cytc-caspase-3 apoptotic pathway by decreasing the K48-linked ubiquitination of Bcl-2 at the 17th lysine. This study reveals a common strategy for neuroinvasive viruses to avoid apoptosis of infected cells by employing LDs, which extends the important role of LDs in viral pathogenesis and may inspire further research in this field.

Charge Modulation at the Liquid Crystal Droplet-Aqueous Interface Enables Ultrasensitive, Nonspecific Protein Detection.

Thermotropic nematic liquid crystals (LC) have been utilized to sense/detect various analytes such as polymers, surfactants, lipids, etc. However, their use for protein detection depends on pre-adsorbed molecules, co-nematogens, or biomolecular agents for specificity. This approach impedes the platform's sensitivity with a detection limit for the folded proteins generally reported in the micromolar concentration range. Here, this work provides fundamental insights into the type of molecular interactions and their modulation that can drive ultrasensitive protein detection at an LC microdroplet/aqueous interface formed without adding an auxiliary co-nematogen. Using ultraviolet (UV) light treated 4-cyano-4'-pentylbiphenyl (5CB) LC and a flow-focused microfluidic device, we prepared different populations of monodisperse and highly negatively charged microdroplets in water. Adding an aqueous solution of various model proteins (α-synuclein, α-chymotrypsin, myoglobin, or bovine serum albumin, BSA) with different secondary structures and surface charges triggers a rapid radial- to bipolar-defect transition in these microdroplets. Isothermal titration calorimetry measurement and molecular dynamic simulation studies attribute this to the dominant electrostatic force-mediated adsorption of proteins at the LC/aqueous interface. Further, bioconjugation-based variation of protein surface charge allows tuning their detection limit. These findings can provide crucial physical cues for designing responsive LC systems and establishing a foundation for developing versatile, molecularly tailored, and highly specific biomolecular detection platforms.

FSH INCREASES LIPID DROPLET CONTENT BY REGULATING THE EXPRESSION OF GENES RELATED TO LIPID STORAGE IN RAT SERTOLI CELLS.

Sertoli cells (SCs) are essential for appropriate spermatogenesis. From a metabolic standpoint, they catabolize glucose and provide germ cells with lactate, which is their main energy source. SCs also oxidize fatty acids (FAs), which are stored as triacylglycerides (TAGs) within lipid droplets (LDs), to fulfill their own energy requirements. On the other hand, it has been demonstrated that FSH regulates some of SCs functions, but little is known about its effect on lipid metabolism. In the present study, we aimed to analyze FSH-mediated regulation of (1) lipid storage in LDs and (2) the expression of genes involved in FAs activation and TAG synthesis and storage in SCs. SCs obtained from 20-day-old rats were cultured for different incubation periods with FSH (100 ng/ml). It was observed that FSH increased LD content and TAG levels in SCs. There were also increments in the expression of Plin1, Fabp5, Acsl1, Acsl4, Gpat3, and Dgat1, which suggests that these proteins may mediate the increase in TAGs and LDs elicited by FSH. Regarding the signaling involved in FSH actions, it was observed that dbcAMP increased LD, and H89, a PKA inhibitor, inhibited FSH stimulus. Also, dbcAMP increased Plin2, Fabp5, Acsl1, Acsl4, and Dgat1 mRNA levels, confirming a role of the cAMP/PKA pathway in the regulation of lipid storage in SCs. Altogether, these results suggest that FSH, via the cAMP/PKA pathway, regulates lipid storage in SCs ensuring the availability of substrates to satisfy their energy requirements.

Facile construction of dual-response super-resolution probes for tracking organelles dynamics.

Super-resolution imaging techniques, such as structured illumination microscopy (SIM), have enabled researchers to obtain nanoscale organelle-level outputs in living systems, but they impose additional stringent requirements on fluorescence probes. However, high-performance, custom-designed SIM probes that can explain underlying biological processes remain unavailable. Herein, a customizable engineering toolkit is developed for the facile assembly of SIM probes suitable for subcellular component detection. This toolkit is used to customize a fluorescent molecule, CPC (coumarin-phenylhydrazine-carboxyl), capable of simultaneously monitoring peroxynitrite (ONOO-) and polarity distribution in mitochondria and lipid droplets (LDs), respectively, through functional ON-OFF mechanisms. The customized CPC molecule demonstrated excellent imaging capabilities under SIM, enabled the successful localization of multiple organelles, and reliably tracked the distribution of different components, thus facilitating the study of the interplay between organelles. Using CPC, the physical transition of intracellular LDs is demonstrated from heterogeneity to homogeneity. This was specifically observed during ferroptosis where the polarity of the LDs increased and their morphology became more contracted. Furthermore, the loss of LDs functionality could not counteract the accumulation of ONOO- within the mitochondria, leading to the decoupling of mitochondrial LDs during ferroptosis. These results confirmed the potential mechanism of LDs dysfunction and decoupling triggered via cumulative mitochondrial oxidative stress during ferroptosis. To summarize, this toolkit will be a powerful tool for examining subtle variations among components during the interplay between different organelles, thus offering novel avenues for understanding and treating related diseases.

Lipid droplet-associated proteins in alcohol-associated fatty liver disease: A proteomic approach.

BACKGROUND: The earliest manifestation of alcohol-associated liver disease (ALD) is steatosis characterized by deposition of fat in specialized organelles called lipid droplets (LDs). While alcohol administration causes a rise in LD numbers in the hepatocytes, little is known regarding their characteristics that allow their accumulation and size to increase. The aim of the present study is to gain insights into underlying pathophysiological mechanisms by investigating the ethanol-induced changes in hepatic LD proteome as a function of LD size. METHODS: Adult male Wistar rats (180-200 g BW) were fed with ethanol liquid diet for 6 weeks. At sacrifice, large-, medium-, and small-sized hepatic LD subpopulations (LD1, LD2, and LD3, respectively) were isolated and subjected to morphological and proteomic analyses. RESULTS: Morphological analysis of LD1-LD3 fractions of ethanol-fed rats clearly demonstrated that LD1 contained larger LDs compared with LD2 and LD3 fractions. Our preliminary results from principal component analysis showed that the proteome of different-sized hepatic LD fractions was distinctly different. Proteomic data analysis identified over 2000 proteins in each LD fraction with significant alterations in protein abundance among the three LD fractions. Among the altered proteins, several were related to fat metabolism, including synthesis, incorporation of fatty acid, and lipolysis. Ingenuity pathway analysis revealed increased fatty acid synthesis, fatty acid incorporation, LD fusion, and reduced lipolysis in LD1 compared to LD3. Overall, the proteomic findings indicate that the increased level of protein that facilitates fusion of LDs combined with an increased association of negative regulators of lipolysis dictates the generation of large-sized LDs during the development of alcohol-associated hepatic steatosis. CONCLUSION: Several significantly altered proteins were identified in different-sized LDs isolated from livers of ethanol-fed rats. Ethanol-induced increases in specific proteins that hinder LD lipid metabolism led to the accumulation and persistence of large-sized LDs in the liver.

The evolving landscape of ER-LD contact sites.

Lipid droplets (LDs) are evolutionarily conserved dynamic organelles that play an important role in cellular physiology. Growing evidence suggests that LD biogenesis occurs at discrete endoplasmic reticulum (ER) subdomains demarcated by the lipodystrophy protein, Seipin, lack of which impairs adipogenesis. However, the mechanisms of how these domains are selected is not completely known. These ER sites undergo ordered assembly of proteins and lipids to initiate LD biogenesis and facilitate establishment of ER-LD contact sites, a prerequisite for proper growth and maturation of droplets. LDs retain both physical and functional association with the ER throughout their lifecycle to facilitate bi-directional communication, such as exchange of proteins and lipids between the two organelles at these ER-LD contact sites. In recent years several molecular tethers have been identified that bridge ER and LDs together including few proteins that are found exclusively at these ER-LD contact interface. Here, we discuss recent advances in understanding the role of factors that ensure functionality of ER-LD contact site machinery for LD homeostasis.

Evolution and Screening of Trichoderma reesei Mutants for Secreted Protein Production at Elevated Temperature.

The filamentous fungus Trichoderma reesei is a mesophilic ascomycete commercially used to produce industrial enzymes for a variety of applications. Strain improvement efforts over many years have resulted in more productive hosts, but also in undesirable traits such as the need for lower temperatures to achieve maximum protein secretion rates. Lower fermentation temperatures increase the need for cooling resulting in higher manufacturing costs. We used a droplet-based evolution strategy to increase the protein secretion temperature of a highly productive T. reesei whole cellulase strain from 25°C to 28°C by first isolating an improved mutant and subsequently tracing the causative high temperature mutation to one gene designated gef1. An industrial host with a gef1 deletion was found to be capable of improved productivity at higher temperature under industrially relevant fermentation conditions.

Adsorption dynamics of heavy oil droplets on silica: Effect of asphaltene anionic carboxylic.

Understanding the adsorption behavior of asphaltene molecules on the surfaces of oil reservoir solids is essential for optimizing oil recovery processes. This study employed molecular dynamics simulations to investigate the adsorption behavior of oil droplets composed of charged and neutral asphaltenes on silica surfaces. The results revealed that oil droplet containing anionic asphaltene molecules were more likely to adsorb onto silica surfaces and exhibited greater resistance to detachment compared to oil droplet containing neutral asphaltene molecules. Specifically, anionic asphaltene molecules tended to accumulate at the oil-water-silica interface, whereas neutral asphaltene molecules primarily adsorbed near the oil-water interface. These findings provide valuable insights into the differing adsorption dynamics of charged and neutral asphaltene molecules on silica surfaces.

Endogenous Magnetic Lipid Droplet-Mediated Cascade-Targeted Sonodynamic Therapy as an Approach to Reversing Breast Cancer Multidrug Resistance.

Multidrug resistance (MDR) has emerged as a major barrier to effective breast cancer treatment, contributing to high rates of chemotherapy failure and disease recurrence. There is thus a pressing need to overcome MDR and to facilitate the efficient and precise treatment of breast cancer in a targeted manner. In this study, endogenous functional lipid droplets (IR780@LDs-Fe3O4/OA) were developed and used to effectively overcome the limited diffusion distance of reactive oxygen species owing to their amenability to cascade-targeted delivery, thereby facilitating precise and effective sonodynamic therapy (SDT) for MDR breast cancer. Initially, IR780@LDs-Fe3O4/OA was efficiently enriched within tumor sites in a static magnetic field, achieving the visualization of tumor treatment. Subsequently, the cascade-targeted SDT combined with the Fenton effect induced lysosome membrane permeabilization and relieved lysosomal sequestration, thus elevating drug concentration at the target site. This treatment approach also suppressed ATP production, thereby inhibiting P-glycoprotein-mediated chemotherapeutic drug efflux. This cascade-targeted SDT strategy significantly increased the sensitivity of MDR cells to doxorubicin, increasing the IC50 value of doxorubicin by approximately 10-fold. Moreover, the cascade-targeted SDT also altered the gene expression profiles of MDR cells and suppressed the expression of MDR-related genes. In light of these promising results, the combination of cascade-targeted SDT and conventional chemotherapy holds great clinical promise as an effective treatment modality with excellent biocompatibility that can improve MDR breast cancer patient outcomes.

A dual-state emission luminogen for lipid droplet imaging and photodynamic therapy.

Organic luminogens with dual-state emission (DSE) have garnered widespread attention due to their versatility in the forms of both dilute solutions and solids. Despite the growing interest, most research on DSE focuses primarily on molecule design and photophysical investigation, with limited exploration of their practical applications. In this study, we introduce a novel fluorescent molecule, PCT, featuring a distinct D-π(A)-D' electronic structure. PCT exhibited efficient DSE properties, with high quantum yields in both dilute solutions (ΦTHF = 52.3 %) and solid-state (Φsolid = 74.6 %). Taking advantage of PCT's lipophilicity, we demonstrated its potential for targeted lipid droplet (LD) imaging in living cells and its utility in monitoring LD depletion during cellular starvation. To further enhance its applicability in photodynamic therapy (PDT), PCT was encapsulated within the amphiphilic triblock copolymer Pluronic F127, forming PCT@F127 nanoparticles with improved colloidal stability. These nanoparticles efficiently generated singlet oxygen (1O2) under white light irradiation, achieving a 1O2 quantum yield of 57.2 %. In vitro studies on MCF-7 cells revealed significant 1O2 generation and potent phototoxicity, leading to marked cell apoptosis and necrosis. These results underscore PCT's multifunctionality as a DSEgen, with promising applications in both bioimaging and PDT.

Alarmin-loaded extracellular lipid droplets induce airway neutrophil infiltration during type 2 inflammation.

Group 2 innate lymphoid cells (ILC2s) play a crucial role in allergic diseases by coordinating a complex network of various effector cell lineages involved in type 2 inflammation. However, their function in regulating airway neutrophil infiltration, a deleterious symptom of severe asthma, remains unknown. Here, we observed ILC2-dependent neutrophil accumulation in the bronchoalveolar lavage fluid (BALF) of allergic mouse models. Chromatography followed by proteomics analysis identified the alarmin high mobility group box-1 (HMGB1) in the supernatant of lung ILC2s initiated neutrophil chemotaxis. Genetic perturbation of Hmgb1 in ILC2s reduced BALF neutrophil numbers and alleviated airway inflammation. HMGB1 was loaded onto the membrane of lipid droplets (LDs) released from activated lung ILC2s. Genetic inhibition of LD accumulation in ILC2s significantly decreased extracellular HMGB1 abundance and BALF neutrophil infiltration. These findings unveil a previously uncharacterized extracellular LD-mediated immune signaling delivery pathway by which ILC2s regulate airway neutrophil infiltration during allergic inflammation.

Female-age-dependent changes in the lipid fingerprint of the mammalian oocytes.

STUDY QUESTION: Can oocyte functionality be assessed by observing changes in their intracytoplasmic lipid droplets (LDs) profiles? SUMMARY ANSWER: Lipid profile changes can reliably be detected in human oocytes; lipid changes are linked with maternal age and impaired developmental competence in a mouse model. WHAT IS KNOWN ALREADY: In all cellular components, lipid damage is the earliest manifestation of oxidative stress (OS), which leads to a cascade of negative consequences for organelles and DNA. Lipid damage is marked by the accumulation of LDs. We hypothesized that impaired oocyte functionality resulting from aging and associated OS could be assessed by changes in LDs profile, hereafter called lipid fingerprint (LF). STUDY DESIGN, SIZE, DURATION: To investigate if it is possible to detect differences in oocyte LF, we subjected human GV-stage oocytes to spectroscopic examinations. For this, a total of 48 oocytes derived from 26 young healthy women (under 33 years of age) with no history of infertility, enrolled in an oocyte donation program, were analyzed. Furthermore, 30 GV human oocytes from 12 women were analyzed by transmission electron microscopy (TEM). To evaluate the effect of oocytes' lipid profile changes on embryo development, a total of 52 C57BL/6 wild-type mice and 125 Gnpat+/- mice were also used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were assessed by label-free cell imaging via coherent anti-Stokes Raman spectroscopy (CARS). Further confirmation of LF changes was conducted using spontaneous Raman followed by Fourier transform infrared (FTIR) spectroscopies and TEM. Additionally, to evaluate whether LF changes are associated with developmental competence, mouse oocytes and blastocysts were evaluated using TEM and the lipid dyes BODIPY and Nile Red. Mouse embryonic exosomes were evaluated using flow cytometry, FTIR and FT-Raman spectroscopies. MAIN RESULTS AND THE ROLE OF CHANCE: Here we demonstrated progressive changes in the LF of oocytes associated with the woman's age consisting of increased LDs size, area, and number. LF variations in oocytes were detectable also within individual donors. This finding makes LF assessment a promising tool to grade oocytes of the same patient, based on their quality. We next demonstrated age-associated changes in oocytes reflected by lipid peroxidation and composition changes; the accumulation of carotenoids; and alterations of structural properties of lipid bilayers. Finally, using a mouse model, we showed that LF changes in oocytes are negatively associated with the secretion of embryonic exosomes prior to implantation. Deficient exosome secretion disrupts communication between the embryo and the uterus and thus may explain recurrent implantation failures in advanced-age patients. LIMITATIONS, REASONS FOR CAUTION: Due to differences in lipid content between different species' oocytes, the developmental impact of lipid oxidation and consequent LF changes may differ across mammalian oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Our findings open the possibility to develop an innovative tool for oocyte assessment and highlight likely functional connections between oocyte LDs and embryonic exosome secretion. By recognizing the role of oocyte LF in shaping the embryo's ability to implant, our original work points to future directions of research relevant to developmental biology and reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by National Science Centre of Poland, Grants: 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547 (to G.E.P.); 2022/44/C/NZ4/00076 (to M.F.H.) and 2019/35/N/NZ3/03213 (to L.G.). M.F.H. is a National Agency for Academic Exchange (NAWA) fellow (GA ULM/2019/1/00097/U/00001). K.F. is a Diamond Grant fellow (Ministry of Education and Science GA 0175/DIA/2019/28). The open-access publication of this article was funded by the Priority Research Area BioS under the program "Excellence Initiative - Research University" at the Jagiellonian University in Krakow. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

A cell-free bacteriophage synthesis system for directed evolution.

Efficient phage production has always been an urgent need in fields such as drug discovery, disease treatment, and gene evolution. To meet this demand, we constructed a robust cell-free synthesis system for generating M13 phage by simplifying its genome, enabling a three-times faster efficiency compared with the traditional method in vivo. We further developed a cell-free directed evolution system in droplets, comprising a modified helper plasmid (ΔPS-ΔgIII-ΔgVI) and the simplified M13 genome-carrying gene mutation library. This system was greatly improved when coupled with fluorescence-activated droplet sorting (FADS). We successfully evolved the T7 RNA polymerase (RNAP), achieving a twofold higher activity to read through the T7 terminator. Moreover, we evolved the tryptophan tRNA into a suppressor tRNA with an eightfold increase in activity to read through the stop codon UAG.

High-fat and high-sucrose diets induce an experimental rabbit model for age-related macular degeneration (AMD).

Age-related macular degeneration (AMD) is a leading cause of blindness. Metabolic disorders and diets are risk factors. We compared lipid profiles and retinal phenotypes with long-term feeding of four diets in male Chinchilla rabbits. Animals were fed with a normal diet (ND), high-fat (HFD), high-sucrose (HSD), or high-fat and high-sucrose diet (HFSD) for six months. The eyes were examined using multimodal imaging modalities and electroretinogram (ERG). Retinal sections were analyzed using H&E staining, toluidine blue staining, immunostaining, and transmission electron microscopy. Lipids and complement C3 in serum or aqueous humour were measured. RNA sequencing was performed to evaluate the retinal transcriptomes. HFD and HSD had minor effects on lipid profiles but synergistically induced severe dyslipidemia. All diets did not cause obesity. HFSD feeding induced retinal lesions like reticular pseudo-drusen (RPD) and pigmentary abnormalities. The RPD-like lesions were mainly lipid droplets around RPE cells. HFSD induced elevated ocular C3 levels and reduced retinal vessel density. In conclusion, HFD and HSD can synergistically induce normal-weight dyslipidemia and RPD-like retinal lesions. HFSD-fed male Chinchilla rabbits are a good model of early AMD.

Altered fatty acid metabolism rewires cholangiocarcinoma stemness features.

Background & Aims: Among the reprogrammed metabolic pathways described in cancer stem cells, aberrant lipid metabolism has recently drawn increasing attention. Our study explored the contribution of fatty acids (FA) in the regulation of stem-like features in intrahepatic cholangiocarcinoma (iCCA). Methods: We previously identified a functional stem-like subset in human iCCA by using a three-dimensional sphere (SPH) model in comparison to parental cells grown as monolayers (MON). In this study, quantification of intracellular free FA and lipidomic analysis (triacylglycerol [TAG] composition, de novo synthesis products) was performed by Liquid chromatography-mass spectrometry (LC-MS); quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS), respectively, in both SPH and MON cultures. Results: Stem-like SPH showed a superior content of free FA (citric, palmitic, stearic, and oleic acids) and unsaturated TAG. Molecularly, SPH showed upregulation of key metabolic enzymes involved in de novo FA biosynthesis (AceCS1, ACLY, ACAC, FASN, ACSL1) and the mTOR signalling pathway. In patients with iCCA (n = 68), tissue expression of FASN, a key gene involved in FA synthesis, correlated with 5-year overall survival. Interference with FASN activity in SPH cells through both specific gene silencing (siRNA) or pharmacological inhibition (orlistat) decreased sphere-forming ability and expression of stem-like markers. In a murine xenograft model obtained by injection of iCCA-SPH cells, FASN inhibition by orlistat or injection of FASN-silenced cells significantly reduced tumour growth and expression of stem-like genes. Conclusion: Altered FA metabolism contributes to the maintenance of a stem-like phenotype in iCCA. FASN inhibition may represent a new approach to interfere with the progression of this deadly disease. Impact and implications: Recent evidence indicates that metabolic disorders correlate with an increased susceptibility to intrahepatic cholangiocarcinoma (iCCA). Our investigation emphasises the pivotal involvement of lipid metabolism in the tumour stem cell biology of iCCA, facilitated by the upregulation of crucial enzymes and the mTOR signalling pathway. From a clinical perspective, this underscores the dual role of FASN as both a prognostic indicator and a therapeutic target, suggesting that FASN inhibitors could enhance patient outcomes by diminishing stemness and tumour aggressiveness. These findings pave the way for novel therapeutic strategies for iCCA and shed light on its relationship with metabolic disorders such as diabetes, obesity, metabolic syndrome, and metabolic dysfunction-associated steatotic liver disease.

Observation of Brownian elastohydrodynamic forces acting on confined soft colloids.

Confined motions in complex environments are ubiquitous in microbiology. These situations invariably involve the intricate coupling between fluid flow, soft boundaries, surface forces, and fluctuations. In the present study, such a coupling is investigated using a method combining holographic microscopy and advanced statistical inference. Specifically, the Brownian motion of soft micrometric oil droplets near rigid walls is quantitatively analyzed. All the key statistical observables are reconstructed with high precision, allowing for nanoscale resolution of local mobilities and femtonewton inference of conservative or nonconservative forces. Strikingly, the analysis reveals the existence of a novel, transient, but large, soft Brownian force. The latter might be of crucial importance for microbiological and nanophysical transport, target finding, or chemical reactions in crowded environments, and hence the whole life machinery.