Vesicle Formation Research Articles

Determination of Inhibitory Effect of PKM2 Enzyme and Antitumoral Activity of Novel Coumarin-Naphthoquinone Hybrids.

Oral squamous cell carcinoma (OSCC) represents the primary form of oral cancer, posing a significant global health threat. The existing chemotherapy options are accompanied by notable side effects impacting patient treatment adherence. Consequently, the exploration and development of novel substances with enhanced anticancer effects and fewer side effects have become pivotal in the realms of biological and chemical science. This work presents the pioneering examples of naphthoquinone-coumarin hybrids as a new category of highly effective cytotoxic substances targeting oral squamous cell carcinoma (OSCC). Given the significance of both naphthoquinones and coumarins as essential pharmacophores/ privileged structures in the quest for anticancer compounds, this study focused on the synthesis and evaluation of novel naphthoquinones/coumarin hybrids against oral squamous cell carcinoma. By several in vitro, in silico, and in vivo approaches, we demonstrated that compound 6e was highly cytotoxic against OSCC cells and several other cancer cell types and was more selective than current chemotherapeutic drugs (carboplatin) and the naphthoquinone lapachol. Furthermore, compound 6e was non-hemolytic and tolerated in vivo at 50 mg/kg with an LD50 of 62.5 mg/kg. Furthermore, compound 6e did not induce apoptosis and cell cycle arrest but led to intracellular vesicle formation with LC3 aggregation in autophagosomes, suggesting an autophagic cell death. Additionally, 6e had a high-affinity potential for PKM2 protein, higher than the known ligands, such as lapachol or shikonin, and was able to inhibit this enzyme activity in vitro. We assert that compound 6e shows promise as a potential lead for a novel chemotherapeutic drug targeting OSCC, with potential applicability to other cancer types.

Ultrastructure of the Bovine Testis in Cattle (Bos taurus): New View.

The purpose of this study was to analyze the ultrastructure of the testes of sexually immature calves and reproductive bulls of the Polish Holstein-Friesian Black-and-White breed. Utilizing TEM, this study identified three distinct stages of seminiferous tubule development in calves, characterized by varying shapes, distributions, and arrangements of individual cells. In immature animals, early developing spermatocytes, prespermatogonia, and pre-Sertoli cells were observed within the seminiferous tubules. In sexually mature bulls, all cells of the spermatogenic series were observed, situated on a thin, multilayered basal lamina, which forms characteristic undulations. An abundant smooth endoplasmic reticulum was observed in the cytoplasm of spermatogonia in both groups of animals, forming characteristic membranous swirls. In adult bulls, spermatogonia maintain contact with each other through numerous cytoplasmic bridges and cell connections, forming small spaces with visible microvilli between them. The ultrastructural analysis facilitated the identification of morphological changes occurring during the maturation of pre-Sertoli cells, transitioning from a large euchromatic nucleus to a nucleus in which the formation of characteristic vesicles and tubules could be observed. It should also be emphasized that two types of Sertoli cells, namely dark and light electron-dense cells, can be found in cattle. These cells differ from each other, indicating that they may perform different functions. The widespread recognition of the presence of two types of Sertoli cells in cattle will undoubtedly contribute to a better understanding of the processes occurring within the testes and provide a basis for further research in this area.

The lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

Cell-Impermeable Buffering Fluorogenic Probes for Live-Cell Super-Resolution Imaging of Plasma Membrane Morphology Dynamics.

Super-resolution fluorescence imaging has emerged as a potent tool for investigating the nanoscale structure and function of the plasma membrane (PM). Nevertheless, the challenge persists in achieving super-resolution imaging of PM dynamics due to limitations in probe photostability and issues with cell internalization staining. Herein, we report assembly-mediated buffering fluorogenic probes BMP-14 and BMP-16 exhibiting fast PM labeling and extended retention time (over 2 h) on PM. The incorporation of alkyl chains proves effective in promoting the aggregation of BMP-14 and BMP-16 into nonfluorescent nanoparticles to realize fluorogenicity and regulate the buffering capacity to rapidly replace photobleached probes ensuring stable long-term super-resolution imaging of PM. Utilizing these PM-buffering probes, we observed dynamic movements of PM filopodia and continuous shrinkage, leading to the formation of extracellular vesicles (EVs) using structured illumination microscopy (SIM). Furthermore, we discovered two distinct modes of EV fusion: one involving fusion through adjacent lipids and the other through filamentous lipid traction. The entire process of EV fusion outside the PM was dynamically tracked. Additionally, BMP-16 exhibited a unique capability of inducing single-molecule fluorescence blinking when used for cell membrane staining. This property makes BMP-16 suitable for the PAINT imaging of cell membranes.

Triacontanol delivery by nano star shaped polymer promoted growth in maize

Plant Growth Regulators (PGRs) are functional compounds known for enhancing plant growth and development. However, their environmental impact is a concern due to poor water solubility and the need for substantial organic solvents. Recently, nano-delivery systems have emerged as a solution, offering a broad range of applications for small molecule compounds. This study introduces a nano-delivery system for Triacontanol (TA), utilizing a star polymer (SPc), aimed at promoting maize growth and improving physiological indicators. The system forms nearly spherical nanoparticles through TA's hydroxyl group and SPc's tertiary amine group. The TA/SPc nano-complex notably outperforms separate TA or SPc treatments in maize, increasing biomass, chlorophyll content, and nutrient absorption. It elevates chlorophyll content by 16.4%, 10.0%, and 6.2% over water, TA, and SPc treatments, respectively, and boosts potassium and nitrate ion uptake by up to 2 and 1.6 times compared to TA alone, leading to enhanced plant height and leaf growth. qRT-PCR analysis further demonstrated that the nano-complex enhanced cellular uptake through the endocytosis pathway by up-regulating endocytosis-related gene expression. The employment of TEM to observe vesicle formation during the internalization of maize leaves furnishes corroborative evidence for the participation of the endocytosis pathway in this process. This research confirms that SPc is an effective carrier for TA, significantly enhancing biological activity and reducing TA dosage requirements.

Molecular characterization and expression profile of estrogen receptor subtypes in female hilsa (Tenualosa ilisha)

Hilsa (Tenualosa ilisha), a highly prized edible fish, is consumed by over 250 million people worldwide. Estrogens are essential hormones required during reproduction and bind with estrogen receptors in target organs for biological activity. The two unique subtypes of the estrogen receptor found in fish are alpha (α) and beta (β) and exhibit distinctive roles in reproduction. The present study aimed to understand the breeding physiology of hilsa during the seasonal gonadal cycles through GSI seasonal variation, histological study, and molecular identification, characterization, and transcriptional modification of estrogen receptors in hilsa. Monthly GSI analysis from females showed that during September, the GSI value was maximum (22.01 ± 0.68), followed by the May GSI value (18.78 ± 0.97). Histological observation showed the formation of asynchronous gametes during the development of ovaries. The histological analysis revealed the formation of developing oocytes, nucleus, presence of primary oogonia, secondary oogonia, chromatin nucleolar oocytes, and early perinucleolar oocytes in the ovary (January–early July). Progressively, formations of the yolk vesicles, yolk granule stage, premature stage, and mature stage with post-ovulatory follicles were also identified. Linear observation was recorded during the monsoon season from July to November. Furthermore, partial length cDNAs for estrogen receptors were characterized, and their mRNA expression patterns demonstrate that ER-α expression significantly increased in September, followed by April and August in the ovary and liver tissue. Both the liver and ovary showed the highest mRNA expression of ER-β in September. The study revealed that ER-α expression was higher in the ovary as compared with liver tissue. Furthermore, we introduce three-dimensional (3D) models depicting the hilsa estrogen receptors in complex with estradiol (E2), constructed through homology modeling. This investigation contributes valuable insights into the molecular characteristics of estrogen receptors in this teleost fish. Our present work provided preliminary evidence of estrogen receptors during reproduction in hilsa.

A New Class of Polyion Complex Vesicles (PIC-somes) to Improve Antimicrobial Activity of Tobramycin in Pseudomonas Aeruginosa Biofilms.

Pseudomonas aeruginosa (PA) is a major healthcare concern due to its tolerance to antibiotics when enclosed in biofilms. Tobramycin (Tob), an effective cationic aminoglycoside antibiotic against planktonic PA, loses potency within PA biofilms due to hindered diffusion caused by interactions with anionic biofilm components. Loading Tob into nano-carriers can enhance its biofilm efficacy by shielding its charge. Polyion complex vesicles (PIC-somes) are promising nano-carriers for charged drugs, allowing higher drug loadings than liposomes and polymersomes. In this study, a new class of nano-sized PIC-somes, formed by Tob-diblock copolymer complexation is presented. This approach replaces conventional linear PEG with brush-like poly[ethylene glycol (methyl ether methacrylate)] (PEGMA) in the shell-forming block, distinguishing it from past methods. Tob paired with a block copolymer containing hydrophilic PEGMA induces micelle formation (PIC-micelles), while incorporating hydrophobic pyridyldisulfide ethyl methacrylate (PDSMA) monomer into PEGMA chains reduces shell hydrophilicity, leads to the formation of vesicles (PIC-somes). PDSMA unit incorporation enables unprecedented dynamic disulfide bond-based shell cross-linking, significantly enhancing stability under saline conditions. Neither PIC-somes nor PIC-micelles show any relevant cytotoxicity on A549, Calu-3, and dTHP-1 cells. Tob's antimicrobial efficacy against planktonic PA remains unaffected after encapsulation into PIC-somes and PIC-micelles, but its potency within PA biofilms significantly increases.

Fluidic shear stress alters clathrin dynamics and vesicle formation in endothelial cells

Endothelial cells (ECs) experience a variety of highly dynamic mechanical stresses. Among others, cyclic stretch and increased plasma membrane tension inhibit clathrin-mediated endocytosis (CME) in non-ECs. It remains elusive how ECs maintain CME in these biophysically unfavorable conditions. Previously, we have used simultaneous two-wavelength axial ratiometry (STAR) microscopy to show that endocytic dynamics are similar between statically cultured human umbilical vein endothelial cells (HUVECs) and fibroblast-like Cos-7 cells. Here, we asked whether biophysical stresses generated by blood flow influence CME. We used our data processing platform—DrSTAR—to examine if clathrin dynamics are altered in HUVECs after experiencing fluidic shear stress (FSS). We found that HUVECs cultivated under a physiological level of FSS had increased clathrin dynamics compared with static controls. FSS increased both clathrin-coated vesicle formation and nonproductive flat clathrin lattices by 2.3-fold and 1.9-fold, respectively. The curvature-positive events had significantly delayed curvature initiation relative to clathrin recruitment in flow-stimulated cells, highlighting a shift toward flat-to-curved clathrin transitions in vesicle formation. Overall, our findings indicate that clathrin dynamics and clathrin-coated vesicle formation can be modulated by the local physiological environment and represent an important regulatory mechanism.

The negative mycorrhizal growth response of host plants to Acaulospora cf. morrowiae irrespective of soil P availability

Acaulospora is one of the dominant genera of AMF associated with waterlogged vegetation, and its dominance was also revealed in the roots of a lowland rice variety indigenous to Southern Thailand. In a preliminary finding, the isolated Acaulospora showed growth suppression of lowland japonica Nipponbare rice, even in high phosphorus (P) soil. The isolated Acaulospora was identified to species level and assigned as Acaulospora cf. morrowiae Phattalung 1. The mycorrhizal growth response (MGR) of an upland indica rice and maize to A. cf. morrowiae was further tested under low and high P conditions. AMF colonization rates were high, over 70%, in both cases, with perfect arbuscule and vesicle formation; however, growth depression of the host plants was markedly observed. Reduced nutrient accumulation in both shoots and roots of host plants was noted, as depicted by nutrient profiling. To further substantiate the negative MGR to inoculation of A. cf. morrowiae, a standardized in vivo bioassay was performed using maize seedlings in a sand and perlite mixture, ensuring low P with 20 mg kg−1 P in the form of insoluble CaH2PO4. Shoot and root growth of maize seedlings were reduced at 23.7 and 36.1%, respectively, by the inoculation of AMF. The nutrient-parasitic nature of this AMF results from unproportioned drainage of photosynthates, indicating an unbalanced primary/nutrient trade-off system between symbionts. The overall associative merits or demerits of A. cf. morrowiae cannot be ruled out without thorough investigations considering several ecological perspectives and its fitter survival and dominance in waterlogged soils.

Identification and characterization of the COPII vesicle-forming GTPase Sar1 in Chlamydomonas.

Eukaryotic cells are highly compartmentalized, requiring elaborate transport mechanisms to facilitate the movement of proteins between membrane-bound compartments. Most proteins synthesized in the endoplasmic reticulum (ER) are transported to the Golgi apparatus through COPII-mediated vesicular trafficking. Sar1, a small GTPase that facilitates the formation of COPII vesicles, plays a critical role in the early steps of this protein secretory pathway. Sar1 was characterized in yeast, animals and plants, but no Sar1 homolog has been identified and functionally analyzed in algae. Here we identified a putative Sar1 homolog (CrSar1) in the model green alga Chlamydomonas reinhardtii through amino acid sequence similarity. We employed site-directed mutagenesis to generate a dominant-negative mutant of CrSar1 (CrSar1DN). Using protein secretion assays, we demonstrate the inhibitory effect of CrSar1DN on protein secretion. However, different from previously studied organisms, ectopic expression of CrSar1DN did not result in collapse of the ER-Golgi interface in Chlamydomonas. Nonetheless, our data suggest a largely conserved role of CrSar1 in the ER-to-Golgi protein secretory pathway in green algae.

Effect of leaflet asymmetry on the stretching elasticity of lipid bilayers with phosphatidic acid

The asymmetry of membranes has a significant impact on their biophysical characteristics and behavior. This study investigates the composition and mechanical properties of symmetric and asymmetric membranes in giant unilamellar vesicles (GUVs) made of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidic acid (POPA). A combination of fluorescence quantification, zeta potential measurements, micropipette aspiration, and bilayer molecular dynamics simulations are used to characterize these membranes. The outer leaflet composition in vesicles is found consistent across the two preparation methods we employed, namely electroformation and inverted emulsion transfer. However, characterizing the inner leaflet poses challenges. Micropipette aspiration of GUVs show that oil residues do not substantially alter membrane elasticity, but simulations reveal increased membrane thickness and decreased interleaflet coupling in the presence of oil. Asymmetric membranes with a POPC:POPA mixture in the outer leaflet and POPC in the inner leaflet display similar stretching elasticity values to symmetric POPC:POPA membranes, suggesting potential POPA insertion into the inner leaflet during vesicle formation and suppressed asymmetry. The inverse compositional asymmetry, with POPC in the outer leaflet and POPC:POPA in the inner one yield less stretchable membranes with higher compressibility modulus compared with their symmetric counterparts. Challenges in achieving and predicting compositional correspondence highlight the limitations of phase-transfer-based methods. In addition, caution is advised when using fluorescently labeled lipids (even at low fractions of 0.5 mol %), as unexpected gel-like domains in symmetric POPC:POPA membranes were observed only with a specific type of labeled DOPE (dioleoylphosphatidylethanolamine) and the same fraction of unlabeled DOPE. The latter suggest that such domain formation may result from interactions between lipids and membrane fluorescent probes. Overall, this study underscores the complexity of factors influencing GUV membrane asymmetry, emphasizing the need for further research and improvement of characterization techniques.

Inappropriate use of antibiotic enhances antibiotic resistance dissemination in ESBL-EC: Role of ydcz in outer membrane vesicles biogenesis and protein transport

Extended-spectrumβ-lactam producing Escherichia coli (ESBL-EC) readily colonizes live poultry and serves as a major source of contamination in retail chicken meat, posing significant threats to public health. This study aims to investigate the impact of inappropriate antibiotic use on the dissemination and exacerbation of antibiotic resistance in ESBL-EC and explore the underlying molecular mechanisms. Through experimental analysis, we propose a hypothesis that inappropriate antibiotic use may exacerbate resistance by affecting vesicle formation and protein secretion. Experimental results demonstrate that under the influence of amoxicillin, the concentration of proteins secreted in outer membrane vehicles (OMVs) by ESBL-EC significantly increases, along with a significant upregulation in the expression of the CTX-M-55-type Extended-spectrum beta-lactamase (CTX-M-55). Proteomic analysis and differential gene knockout experiments identified the key protein YdcZ, associated with OMVs formation and protein transportation in ESBL-EC under amoxicillin treatment. Further investigations reveal direct interactions between YdcZ and other proteins (YdiH and BssR). Upon ydcz gene knockout, a significant decrease in protein concentration within OMVs is observed, accompanied by a noticeable reduction in protection against sensitive bacteria. These findings suggest a critical role of YdcZ in regulating the process of protein transportation to OMVs in ESBL-EC under the influence of amoxicillin. In summary, our research uncovers the significant role of inappropriate antibiotic use in promoting the secretion of OMVs by ESBL-EC, aiding the survival of antibiotic-sensitive bacteria in the vicinity of infection sites. These findings provide new insights into the mechanisms underlying antibiotic-induced bacterial resistance dissemination and offer novel avenues for exploring prevention and control strategies against bacterial resistance propagation.

Adsorption and Aggregation Behaviors of Oleyl Alcohol-Based Extended Surfactant and Its Mixtures.

An oleyl alcohol-based extended surfactant, sodium oleyl polyethylene oxide-polypropylene oxide sulfate (OE3P3S), was synthesized and identified using FT-IR and 1H NMR. The adsorption and aggregation behaviors of OE3P3S and its mixture with cationic surfactant alkyltrimethylammoniumbromide (ATAB) were investigated under different molar ratios. The static surface tension analysis indicated that the critical micellization concentration (cmc) and the critical surface tension (γcmc) of OE3P3S were 0.72 mmol/L, and 36.16 mN/m, respectively. The cmc and γcmc values of the binary system were much lower than that of the individual component. And the cmc values of OE3P3S/ATAB = 6:4 mixtures decreased with an increase in the chain length of the cationic surfactant in the binary system. It was found from the dynamic surface tension that there was a slower diffusion rate in the binary system compared to the pure surfactant, and the adsorption processes for OE3P3S/ATAB = 6:4 were mixed diffusion-kinetic adsorption mechanisms. With a combination of DLS data and TEM measurements, formations of vesicles in OE3P3S/ATAB = 6:4 solutions appeared to occur at a concentration of 0.05 mmol/L. By studying the formation of liquid crystal structures in an emulsion prepared with OE3P3S as the surfactant, it was found that the oil-in-water emulsion is birefringent with a Maltese cross texture, and the rheological properties revealed its predominant viscoelastic behavior and shear thinning properties.

Ampholytic Peptides Consisting of an Alternating Lysine/Glutamic Acid Sequence for the Simultaneous Formation of Polyion Complex Vesicles.

Nanoarchitectures such as micelles and vesicles that self-assemble via electrostatic interactions between their charged polymeric components have been widely used as material delivery platforms. In this work, ampholytic peptides with a sequence of alternating lysine and glutamic acid residues were designed and synthesized via chemoenzymatic polymerization. This alternating sequence was achieved by trypsin-catalyzed polymerization of a dipeptide monomer. Due to the electrostatic interaction between the anionic and cationic residues, the prepared ampholytic peptides spontaneously formed nanosized assemblies with a size of 100-200 nm in water. Modification with tetra(ethylene glycol) (TEG) at the N-terminus of these ampholytic alternating peptides resulted in the formation of stable nanosized assemblies, while peptides consisting of random sequences of lysine and glutamic acid formed large aggregates with deteriorated stability even with TEG modification. Morphological observations using a field-emission scanning electron microscope and an atomic force microscope revealed that the obtained assemblies were spherical and hollow, indicating the spontaneous formation of vesicles from the TEG-modified ampholytic alternating peptides. These vesicles were able to encapsulate a model fluorescent protein within their hollow structures without structural collapse causing loss of fluorescence, demonstrating the potential of these nanocarriers for use in material delivery systems.

Sar1A overexpression in Chinese hamster ovary cells and its effects on antibody productivity and secretion

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.

Controlling the formation of ionic complex vesicles through double-tailed surfactants.

Liposomes are often used in cosmetics since they are naturally derived and have excellent texture enhancing capabilities. However, when preparing them by using phospholipids with unsaturated acyl groups, they easily suffer from oxidative degradation. Accordingly, hydrogenated phospholipids are preferred, however, it is difficult to prepare stable liposomes due to its high gel-liquid crystalline phase transition temperature. On the other hand, although dialkyl dimethyl ammonium type cationic surfactants are widely known to form vesicles, they have rarely been used for skincare products except for water-in-oil type emulsion creams stabilized by organically modified clay minerals. We decided to overcome all of the problems above through ionic complex vesicles formed by double-tailed cationic and anionic surfactants. Distearyl dimethyl ammonium chloride (DSAC) and sodium dilauramidoglutamide lysine (DLGL) were selected as cationic and anionic surfactants, respectively. Differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering (SWAXS) measurements were performed to confirm the DSAC/DLGL/water ternary phase diagram. Newly developed ionic complex vesicle formation was confirmed by cryogenic transmission electron microscopy (cryo-TEM). The adsorbed cosmetic film structure on the skin invivo was evaluated through the polarized infrared external reflection (PIR-ER). Finally, a cosmetic lotion formula was developed and the vesicle size was determined by dynamic light scattering (DLS). DSC and SWAXS data indicated that stable vesicles could be obtained at a molar ratio of DLGL to DSAC = 6:4. At this molar ratio, multi lamellar vesicles with diameters less than 100 nm were observed through cryo-TEM. PIR-ER data revealed that the developed vesicles formed a highly perpendicular orientation to the human skin surface. We have succeeded in formulating a cosmetic lotion containing developed vesicles with a mean diameter of 63.2 nm, which was stable over 1 month at 0, 37, and 50°C. Our newly developed vesicles can be easily obtained through a coagulation process. Also, the adsorbed film structure supported by PIR-ER experiments implies that the developed lotion has an excellent texture that is the same as cosmetic lotions containing liposomes. Therefore, it's possible that this ionic complex vesicle could take the place of liposomes.

#1841 Trimethylamine N-oxide (TMAO) induces the formation of endothelial extracellular vesicles

Abstract Background and Aims Endothelial dysfunction caused by the accumulation of uremic toxins is an important contributor to the development of cardiovascular disease in patients with chronic kidney disease (CKD). The uremic toxin trimethylamine n-oxide (TMAO) is associated with oxidative stress, vascular calcification, and inflammation, triggering the release of several proinflammatory molecules and Extracellular Vesicles (EVs). Clinical studies have shown that patients with CKD have high levels of circulating EVs, however, their role is still poorly understood. In this dysfunctional scenario, EVs can be used as damage biomarkers for CKD, as they carry information from their cells of origin. Here, we aim to elucidate the formation of EVs in endothelial cells exposed to TMAO. Method Human endothelial cells (EA.hy926, ATCC CRL-2922) were exposed for 24 h with TMAO at uremic concentrations (7.49 mg/L). EVs were isolated by differential ultracentrifugation. Protein concentration was analyzed by Pierce™ BCA Protein Assay Kit, and particle size and particle concentration were analyzed by NanoParticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and Scanning Electron Microscopy (SEM). EVs were stained with calcein and their internalization in endothelial cells was analyzed with Fluorescence Confocal Microscopy. Results TMAO treatment at uremic concentration induced the formation of EVs with a size of 202.8 ± 3.1 nm (image 1B) and 1.09 × 109 ± 2.68 × 107 particles/mL and a total protein concentration of 88.57 µg/mL. The morphological features of EVs are shown in images 1C-1F. TMAO-induced EVs were incorporated by endothelial cells, being dispersed in the cytoplasm or perinuclear regions (images 1G and end 1H). Conclusion TMAO was able to induce the formation of EVs in endothelial cells, with characteristics morphological features. When compared to control EVs (image 1A), TMAO-EVs have a more heterogeneous size, which indicate a change in the size pattern, and thus the nature of the EVs induced by TMAO. TMAO-EVs were able to be internalized by human endothelial cells. This uptake can be closely related to the effects of EVs in endothelial cells, but more experiments need to be performed. Taken together, these data could contribute to understanding how the formation and uptake of TMAO-EVs by the endothelium occurs, relating their effects to the cardiovascular complications of CKD.

Prominin 1 and Tweety Homology 1 both induce extracellular vesicle formation.

Prominin-1 (Prom1) is a five-transmembrane-pass integral membrane protein that associates with curved regions of the plasma membrane. Prom1 interacts with membrane cholesterol and actively remodels the plasma membrane. Membrane bending activity is particularly evident in photoreceptors, where Prom1 loss-of-function mutations cause failure of outer segment homeostasis, leading to cone-rod retinal dystrophy (CRRD). The Tweety Homology (Ttyh) protein family has been proposed to be homologous to Prominin, but it is not known whether Ttyh proteins have an analogous membrane-bending function. Here, we characterize the membrane-bending activity of human Prom1 and Ttyh1 in native bilayer membranes. We find that Prom1 and Ttyh1 both induce formation of extracellular vesicles (EVs) in cultured mammalian cells and that the EVs produced are physically similar. Ttyh1 is more abundant in EV membranes than Prom1 and produces EVs with membranes that are more tubulated than Prom1 EVs. We further show that Prom1 interacts more stably with membrane cholesterol than Ttyh1 and that this may contribute to membrane bending inhibition in Prom1 EVs. Intriguingly, a loss-of-function mutation in Prom1 associated with CRRD induces particularly stable cholesterol binding. These experiments provide mechanistic insight into Prominin function in CRRD and suggest that Prom and Ttyh belong to a single family of functionally related membrane-bending, EV-generating proteins.

Lysis-Free Isolation and Direct Amplification of Pathogenic Bacterial DNA Using Diatom Frustules.

DNA has been implicated as an important biomarker for the diagnosis of bacterial infections. Herein, we developed a streamlined methodology that uses diatom frustules (DFs) to liberate and capture bacterial DNA and allows direct downstream amplification tests without any lysis, washing, or elution steps. Unlike most conventional DNA isolation methods that rely on cell lysis to release bacterial DNA, DFs can trigger the oxidative stress response of bacterial cells to promote bacterial membrane vesicle formation and DNA release by generating reactive oxygen species in aqueous solutions. Due to the hierarchical porous structure, DFs provided high DNA capture efficiency exceeding 80% over a wide range of DNA amounts from 10 pg to 10 ng, making only 10 μg DFs sufficient for each test. Since laborious liquid handling steps are not required, the entire DNA sample preparation process using DFs can be completed within 3 min. The diagnostic use of this DF-based methodology was illustrated, which showed that the DNA of the pathogenic bacteria in serum samples was isolated by DFs and directly detected using polymerase chain reaction (PCR) at concentrations as low as 102 CFU/mL, outperforming the most used approaches based on solid-phase DNA extraction. Furthermore, most of the bacterial cells were still alive after DNA isolation using DFs, providing the possibility of recycling samples for storage and further diagnosis. The proposed DF-based methodology is anticipated to simplify bacterial infection diagnosis and be broadly applied to various medical diagnoses and biological research.

Autophagy favors survival of corpora lutea during the long-lasting pregnancy of the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

The corpus luteum (CL) is a transient endocrine gland that plays a crucial role in establishing and maintaining pregnancy. Although autophagy and apoptosis have been suggested as cooperative mechanisms, their interaction within the CL of pregnant mammals has not been thoroughly investigated. To understand the collaborative function of autophagy and apoptosis in the CL, we analyzed both mechanisms during pregnancy in the South American plains vizcacha, Lagostomus maximus. This rodent undergoes a decline in progesterone levels during mid-gestation, a reactivation of the hypothalamus-hypophysis-gonadal axis, and the incorporation of new functional secondary CL. Our analysis of autophagy markers BECLIN 1 (BECN1), SEQUESTOSOME1 (SQSTM1), Microtubule-associated protein light chain 3 (LC3B), and lysosomal-associated membrane protein 1 (LAMP1) and anti- and pro-apoptotic markers BCL2 and ACTIVE CASPASE 3 (A-C3) revealed interactive behaviors between both processes. Healthy primary and secondary CL exhibited positive expression of BECN1, SQSTM1, LC3B, and LAMP1, while regressed CL displayed enhanced expression of these autophagy markers along with nuclear A-C3. Transmission electron microscopy revealed a significant formation of autophagic vesicles in regressed CL during full-term pregnancy, whereas healthy CL exhibited a low number of autophagy vesicles. The co-localization between LC3B and SQSTM1 and LC3B with LAMP1 was observed in both healthy and regressed CL during pregnancy, while co-localization of BECN1 and BCL2 was only detected in healthy CL. LC3B and ACTIVE CASPASE 3 co-localization were detected in a subset of luteal cells within the regressing CL. We propose that autophagy could act as a survival mechanism in the CL, allowing the pregnancy to progress until full-term, while also serving as a mechanism to eliminate remnants of regressed CL, thereby providing the necessary space for subsequent follicular maturation.