Dissecting the roles of dynamin and clathrin in platelet pinocytosis.

Platelets endocytose many molecules from their environment. However, this process of pinocytosis in platelets is poorly understood. Key endocytic regulators such as dynamin, clathrin, CDC42 and Arf6 are expressed in platelets but their roles in pinocytosis is not known. Stimulated platelets form two subpopulations of pro-aggregatory and procoagulant platelets. The effect of stimulation on pinocytosis is also poorly understood. In this study, washed human platelets were treated with a range of endocytosis inhibitors and stimulated using different activators. The rate of pinocytosis was assessed using pHrodo green, a pH-sensitive 10 kDa dextran. In unstimulated platelets, pHrodo fluorescence increased over time and accumulated as intracellular puncta indicating constituently active pinocytosis. Stimulated platelets (both pro-aggregatory and procoagulant) had an elevated pinocytosis rate compared to unstimulated platelets. Dynamin inhibition blocked pinocytosis in unstimulated, pro-aggregatory and procoagulant platelets indicating that most platelet pinocytosis is dynamin dependent. Although pinocytosis was clathrin-independent in unstimulated and procoagulant populations, clathrin partially contributed to pinocytosis in pro-aggregatory platelets.

Cytotoxicity of Quantum Dots in Receptor-Mediated Endocytic and Pinocytic Pathways in Yeast.

Despite the promising applications of the use of quantum dots (QDs) in the biomedical field, the long-lasting effects of QDs on the cell remain poorly understood. To comprehend the mechanisms underlying the toxic effects of QDs in yeast, we characterized defects associated with receptor-mediated endocytosis (RME) as well as pinocytosis using Saccharomyces cerevisiae as a model in the presence of cadmium selenide/zinc sulfide (CdSe/ZnS) QDs. Our findings revealed that QDs led to an inefficient RME at the early, intermediate, and late stages of endocytic patch maturation at the endocytic site, with the prolonged lifespan of GFP fused yeast fimbrin (Sac6-GFP), a late marker of endocytosis. The transit of FM1-43, a lipophilic dye from the plasma membrane to the vacuole, was severely retarded in the presence of QDs. Finally, QDs caused an accumulation of monomeric red fluorescent protein fused carbamoyl phosphate synthetase 1 (mRFP-Cps1), a vacuolar lumen marker in the vacuole. In summary, the present study provides novel insights into the possible impact of CdSe/ZnS QDs on the endocytic machinery, enabling a deeper comprehension of QD toxicity.

Macropinocytosis in Phagocyte Function and Immunity.

Phagocytes play critical roles in the maintenance of organismal homeostasis and immunity. Central to their role is their ability to take up and process exogenous material via the related processes of phagocytosis and macropinocytosis. The mechanisms and functions underlying macropinocytosis have remained severely understudied relative to phagocytosis. In recent years, however, there has been a renaissance in macropinocytosis research. Phagocytes can engage in various forms of macropinocytosis including an "induced" form and a "constitutive" form. This chapter, however, will focus on constitutive macropinocytosis and its role in the maintenance of immunity. Functions previously attributed to macropinocytosis, including antigen presentation and immune surveillance, will be revisited in light of recent revelations and emerging concepts will be highlighted.

Insights of Endocytosis Signaling in Health and Disease.

Endocytosis in mammalian cells is a fundamental cellular machinery that regulates vital physiological processes, such as the absorption of metabolites, release of neurotransmitters, uptake of hormone cellular defense, and delivery of biomolecules across the plasma membrane. A remarkable characteristic of the endocytic machinery is the sequential assembly of the complex proteins at the plasma membrane, followed by internalization and fusion of various biomolecules to different cellular compartments. In all eukaryotic cells, functional characterization of endocytic pathways is based on dynamics of the protein complex and signal transduction modules. To coordinate the assembly and functions of the numerous parts of the endocytic machinery, the endocytic proteins interact significantly within and between the modules. Clathrin-dependent and -independent endocytosis, caveolar pathway, and receptor mediated endocytosis have been attributed to a greater variety of physiological and pathophysiological roles such as, autophagy, metabolism, cell division, apoptosis, cellular defense, and intestinal permeabilization. Notably, any defect or alteration in the endocytic machinery results in the development of pathological consequences associated with human diseases such as cancer, cardiovascular diseases, neurological diseases, and inflammatory diseases. In this review, an in-depth endeavor has been made to illustrate the process of endocytosis, and associated mechanisms describing pathological manifestation associated with dysregulated endocytosis machinery.

Macrophage-driven nutrient delivery to phagosomal Staphylococcus aureus supports bacterial growth.

Staphylococcus aureus is a notorious pathogen causing significant morbidity and mortality worldwide. The ability of S. aureus to survive and replicate within phagocytes such as macrophages represents an important facet of immune evasion and contributes to pathogenesis. The mechanisms by which S. aureus acquires nutrients within host cells to support growth remain poorly characterized. Here, we demonstrate that macrophages infected with S. aureus maintain their dynamic ruffling behavior and consume macromolecules from the extracellular milieu. To support the notion that fluid-phase uptake by macrophages can provide S. aureus with nutrients, we utilized the pharmacological inhibitors PIK-III and Dynasore to impair uptake of extracellular macromolecules. Inhibitor treatment also impaired S. aureus replication within macrophages. Finally, using a mutant of S. aureus that is defective in purine biosynthesis we show that intracellular growth is inhibited unless the macrophage culture medium is supplemented with the metabolite inosine monophosphate. This growth rescue can be impaired by inhibition of fluid-phase uptake. In summary, through consumption of the extracellular environment macrophages deliver nutrients to phagolysosomal S. aureus to promote bacterial growth.

Trypan Blue - Adapting a Dye Used for Labelling Dead Cells to Visualize Pinocytosis in Viable Cells.

BACKGROUND/AIMS: Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. METHODS: We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. RESULTS: We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. CONCLUSION: In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.

AMPKα promotes basal autophagy induction in Dictyostelium discoideum.

Autophagy is a degradation process, wherein long-lived proteins, damaged organelles, and protein aggregates are degraded to maintain cellular homeostasis. Upon starvation, 5'-AMP-activated protein kinase (AMPK) initiates autophagy. We show that ampkα- cells exhibit 50% reduction in pinocytosis and display defective phagocytosis. Re-expression of AMPKα in ampkα- cells co-localizes with red fluorescence protein-tagged bacteria. The ampkα- cells show reduced cell survival and autophagic flux under basal and starvation conditions. Co-immunoprecipitation studies show conservation of the AMPK-ATG1 axis in basal autophagy. Computational analyses suggest that the N-terminal region of DdATG1 is amenable for interaction with AMPK. Furthermore, ß-actin was found to be a novel interacting partner of AMPK, attributed to the alteration in macropinocytosis and phagocytosis in the absence of AMPK. Additionally, ampkα- cells exhibit enhanced poly-ubiquitinated protein levels and allied large ubiquitin-positive protein aggregates. Our findings suggest that AMPK provides links among pinocytosis, phagocytosis, autophagy, and is a requisite for basal autophagy in Dictyostelium.

Different rates of endocytic activity and vesicle transport from the apical and synaptic poles of the outer hair cell.

BACKGROUND: Intense endocytic activity at the apex of outer hair cells (OHCs)-the electromechanical cells of the cochlea-has been demonstrated using the vital plasma-membrane marker FM1-43 and confocal laser-scanning microscopy. Vesicular traffic toward the cell nucleus to distinct locations of the endoplasmic reticulum has also been shown. OBJECTIVE: The current study characterizes the dynamics of endocytic activity, as well as apicobasal and basoapical trafficking, using a local perfusion technique that we recently developed and published to visualize bidirectional trafficking in isolated bipolar cells. MATERIALS AND METHODS: The fluorescent plasma-membrane markers FM1-43 (10 µM) and FM4-64 (10 µM), together with a fluid-phase marker, Lucifer yellow (50 µM), were used to label endocytosed vesicles in isolated OHCs of the guinea pig cochlea. Targets of endocytosed vesicles were examined with a fluorescent marker of subsurface cisternae, DiOC6 (0.87 µM). Single- and two-photon confocal laser-scanning microscopy was used to visualize labeled vesicles. RESULTS: The plasma-membrane markers presented more intense vesicle internalization at the synaptic pole than at the apical pole of the OHC. Intracellular basoapical vesicle trafficking was faster than apicobasal trafficking. Vesicles endocytosed at the synaptic pole were transcytosed to the endoplasmic reticulum system. An intracellular Lucifer yellow signal was not detected. CONCLUSION: The larger endocytic fluorescent signals in the synaptic pole and the faster basoapical trafficking imply that membrane internalization and vesicle trafficking are more efficient at the synaptic pole than at the apical pole of the OHC.

Emerging roles of calpain proteolytic systems in macrophage cholesterol handling.

Calpains are Ca2+-dependent intracellular proteases that play central roles in the post-translational processing of functional proteins. In mammals, calpain proteolytic systems comprise the endogenous inhibitor calpastatin as well as 15 homologues of the catalytic subunits and two homologues of the regulatory subunits. Recent pharmacological and gene targeting studies in experimental animal models have revealed the contribution of conventional calpains, which consist of the calpain-1 and -2 isozymes, to atherosclerotic diseases. During atherogenesis, conventional calpains facilitate the CD36-dependent uptake of oxidized low-density lipoprotein (LDL), and block cholesterol efflux through ATP-binding cassette transporters in lesional macrophages, allowing the expansion of lipid-enriched atherosclerotic plaques. In addition, calpain-6, an unconventional non-proteolytic calpain, in macrophages reportedly potentiates pinocytotic uptake of native LDL, and attenuates the efferocytic clearance of apoptotic and necrotic cell corpses from the lesions. Herein, we discuss the recent progress that has been made in our understanding of how calpain contributes to atherosclerosis, in particular focusing on macrophage cholesterol handling.

Small molecule pinocytosis and clathrin-dependent endocytosis at the intestinal brush border: Two separate pathways into the enterocyte.

Pinocytosis at the small intestinal brush border was studied in postweaned porcine cultured mucosal explants, using the fluorescent polar probes Alexa hydrazide (AH, MW 570), Texas red dextran (TRD, MW ~ 3000), and Cascade blue dextran (CBD, MW ~ 10,000). Within 1 h, AH appeared in a string of subapical punctae in enterocytes, indicative of an ongoing constitutive pinocytosis. By comparison, TRD was taken up less efficiently into the same compartment, and no intracellular labeling of CBD was detectable, indicating that only small molecules are pinocytosed from the postweaned gut lumen. AH remained in the terminal web region in EEA-1-positive endosomes ("TWEEs") for at least 2 h, implying that the pinocytic uptake does not proceed towards a transcytic pathway. Like AH, cholera toxin B subunit (CTB) was readily internalized, but the two probes appeared in completely non-overlapping subapical compartments, indicating the existence of two different uptake mechanisms operating simultaneously at the brush border. CTB is internalized by clathrin-dependent receptor mediated endocytosis, but surprisingly the toxin also caused a rapid disappearance from the apical cell surface of two major brush border enzymes, alkaline phosphatase and aminopeptidase N, demonstrating the disruptive effect of this pathway. By immunofluorescence, caveolin-1 was hardly detectable in enterocytes, arguing against a caveolae-mediated uptake of AH, whereas the pinocytosis/phagocytosis inhibitors dimethyl amiloride and cytochalasin D both arrested AH uptake. We propose that the constitutive pinocytic mechanism visualized by AH contributes to maintenance of membrane homeostasis and to enrich the contents of lipid raft constituents at the brush border.

Measurement of Aortic Cell Fluid-Phase Pinocytosis in vivo by Flow Cytometry.

OBJECTIVE: Fluid-phase pinocytosis is a receptor-independent mechanism of endocytosis that occurs in all mammalian cells and may be a mechanism for the uptake of LDL by macrophages. As there are currently no methods for the measurement of fluid-phase pinocytosis by individual aortic cells in vivo, we sought to identify a suitable method. METHODS: ApoE-/- mice were retro-orbitally injected with AngioSPARK fluorescent nanoparticles specifically designed to not interact with cells. After 24 h, mice were sacrificed, and the aortas were isolated and then digested to analyze aortic cell uptake of AngioSPARK by flow cytometry. RESULTS: CD11b-expressing aortic macrophages from mice injected with AngioSPARK showed high levels of fluid-phase pinocytosis compared to aortic cells not expressing CD11b (4,393.7 vs. 408.3 mean fluorescence intensity [MFI], respectively). CONCLUSION: This new technique allows for the measurement of fluid-phase pinocytosis by aortic cells in vivo, making it possible to examine the cell-signaling molecules and drugs that affect this process. Published by S. Karger AG, Basel.

The shear rate promotes pinocytosis of extracellular dextran in platelets.

BACKGROUND: Several conventional studies focused on platelet pinocytosis for possible utilization as drug delivery systems. Although platelet pinocytosis is important in such utilization, the impact of the shear rate on pinocytosis is unclear. OBJECTIVE: Our objective was to investigate the relationship between shear rate and platelet pinocytosis in vitro. In addition, this study addressed the change in platelet aggregation reactivity with adenosine diphosphate (ADP) stimulation after pinocytosis. METHOD: Porcine platelet-rich plasma was mixed with fluorescein isothiocyanate (FITC)-conjugated dextran and incubated for 15 min under shear conditions of 0, 500, and 1500 s-1. After incubation, confocal microscopic scanning and three-dimensional rendering were performed to confirm the internalization of FITC-dextran into platelets. The amount of FITC-dextran accumulated via platelet pinocytosis was compared using flow cytometry at each shear rate. In addition, light transmission aggregometry by ADP stimulation was applied to platelets after pinocytosis. RESULTS: The amount of intracellular FITC-dextran increased with higher shear rates. Platelets with increased amounts of intracellular FITC-dextran did not show changes in the aggregation reactivity to ADP. CONCLUSIONS: A higher shear rate promotes platelet pinocytosis, but enhanced pinocytosis does not affect aggregation sensitivity, which is stimulated by ADP.

Using Ion Substitution and Fluid Indicators to Monitor Macropinosome Dynamics in Live Cells.

All forms of endocytosis involve the incidental uptake of fluid (pinocytosis). Macropinocytosis is a specialized type of endocytosis that results in the bulk ingestion of extracellular fluid via large (>0.2 µm) vacuoles called macropinosomes. The process is a means of immune surveillance, a point of entry for intracellular pathogens, and a source of nutrients for proliferating cancer cells. Macropinocytosis has also recently emerged as a tractable system that can be experimentally exploited to understand fluid handling in the endocytic pathway. In this chapter, we describe how stimulating macropinocytosis in the presence of extracellular fluids of a defined ionic composition can be combined with high-resolution microscopy to understand the role of ion transport in controlling membrane traffic.

Mechanisms of Nanoparticle Transport across Intestinal Tissue: An Oral Delivery Perspective.

Oral drug administration has been a popular choice due to patient compliance and limited clinical resources. Orally delivered drugs must circumvent the harsh gastrointestinal (GI) environment to effectively enter the systemic circulation. The GI tract has a number of structural and physiological barriers that limit drug bioavailability including mucus, the tightly regulated epithelial layer, immune cells, and associated vasculature. Nanoparticles have been used to enhance oral bioavailability of drugs, as they can act as a shield to the harsh GI environment and prevent early degradation while also increasing uptake and transport of drugs across the intestinal epithelium. Evidence suggests that different nanoparticle formulations may be transported via different intracellular mechanisms to cross the intestinal epithelium. Despite the existence of a significant body of work on intestinal transport of nanoparticles, many key questions remain: What causes the poor bioavailability of the oral drugs? What factors contribute to the ability of a nanoparticle to cross different intestinal barriers? Do nanoparticle properties such as size and charge influence the type of endocytic pathways taken? In this Review, we summarize the different components of intestinal barriers and the types of nanoparticles developed for oral delivery. In particular, we focus on the various intracellular pathways used in nanoparticle internalization and nanoparticle or cargo translocation across the epithelium. Understanding the gut barrier, nanoparticle characteristics, and transport pathways may lead to the development of more therapeutically useful nanoparticles as drug carriers.

Daily Consumption of Lemon and Ginger Herbal Infusion Caused Tumor Regression and Activation of the Immune System in a Mouse Model of Breast Cancer.

The Mediterranean diet includes the consumption of various fruits and vegetables. Lemon and ginger are highly popular in Mediterranean cuisine. The current study aims to evaluate both anticancer and immunomodulatory activities of lemon and ginger combination. The antiproliferative activities of the combination were tested against different cancer cell lines using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. The degree of apoptosis induction and vascular endothelial growth factor expression were detected using ELISA. Balb/C mice were inoculated with the EMT6/P breast cancer cells and received combination water extract orally for 14 days. The effect of the water extract on splenocytes proliferation was measured using the mitogen proliferation assay. Macrophage function was evaluated using the nitro blue tetrazolium assay and pinocytosis was assessed using the neutral red method. Gas chromatography coupled to the tandem mass spectrometry was used to determine the composition of the combination. The lemon and ginger combination showed significant apoptosis induction and angiogenesis suppression effects. Fifty percent of the mice taking this combination did not develop tumors with a percentage of tumor reduction of 32.8%. This combination showed a potent effect in stimulating pinocytosis. Alpha-pinene and α-terpineol were detected in high percentages in the combination water extract. The lemon and ginger combination represents promising options to develop anticancer infusions for augmenting conventional anticancer therapies. Further testing is required to understand the exact molecular mechanisms of this combination.

Antimicrobial peptide nisin induces spherical distribution of macropinocytosis-like cytokeratin 5 and cytokeratin 17 following immediate derangement of the cell membrane.

The anti-aging effects of Lactococcus lactis are extensively investigated. Nisin is an antimicrobial peptide produced by L. lactis subsp. lactis. We previously reported that 24-hour nisin treatment disturbs the intermediate filament distribution in human keratinocytes. Additionally, we showed that the ring-like distribution of the intermediate filament proteins, cytokeratin (CK) 5 and CK17 is a marker of nisin action. However, two questions remained unanswered: 1) What do the CK5 and CK17 ring-like distributions indicate? 2) Is nisin ineffective under the experimental conditions wherein CK5 and CK17 do not exhibit a ring-like distribution? Super resolution microscopy revealed that nisin treatment altered CK5 and CK17 distribution, making them spherical rather than ring-like, along with actin incorporation. This spherical distribution was not induced by the suppression of endocytosis. The possibility of a macropinocytosis-like phenomenon was indicated, because the spherical distribution was >1 µm in diameter and the spherical distribution was suppressed by macropinocytosis inhibiting conditions, such as the inclusion of an actin polymerization inhibitor and cell migration. Even when the spherical distribution of CK5 and CK17 was not induced, nisin induced derangement of the cell membrane. Nisin treatment for 30 minutes deranged the regular arrangement of the lipid layer (flip-flop); the transmembrane structure of the CK5-desmosome or CK17-desmosome protein complex was disturbed. To the best of our knowledge, this is the first study to report that CK5 and CK17 in a spherical distribution could be involved in a macropinosome-like structure, under certain conditions of nisin action in keratinocytes.

Ultrastructural changes in mitochondria during oogenesis in two phylogenetically close fish species.

The ultrastructure of oocyte mitochondria and their contribution to the endogenous autosynthesis of the yolk was investigated in two clupeoid species, the Mediterranean sardine, Sardina pilchardus, and the European anchovy, Engraulis encrasicolus. The structure and abundance of mitochondria differ in secondary growth oocytes of the two species, whereas they are similar in chromatin nucleolus and primary growth oocytes. Sardine oocytes show a higher percentage of mitochondria in the cytoplasm as they develop. However, the individual size of each mitochondrion decreases, becoming smaller than those observed in anchovy oocytes. The volume fraction of cristae in mitochondria of sardine oocytes gradually increased throughout the oocyte developmental phases up to the early secondary growth phase and then slightly decreased during the mid-secondary growth phase. In the cytoplasm of early secondary growth oocytes of anchovy, the percentage of mitochondria is larger than in mid-secondary growth oocytes. As oocytes develop, the size of mitochondria diminishes as well. In contrast to the volume fraction of cristae in mitochondria of sardine oocytes, the volume fraction of cristae in anchovy was decreased in early secondary growth oocytes and then it was increased during the mid-secondary growth phase. As a result, based on both cytoplasmic dynamics of each species and mitochondrial alterations, it was assumed that mitochondria in sardine play a role in the formation of yolk granules, whereas mitochondria in anchovy play a role in the lipid synthesis pathway. Both species showed exogenous heterosynthesis of yolk, through the process of pinocytosis in the zona radiata of oocytes.

Elucidation of iron homeostasis in Acanthamoeba castellanii.

Acanthamoeba castellanii is a ubiquitously distributed amoeba that can be found in soil, dust, natural and tap water, air conditioners, hospitals, contact lenses and other environments. It is an amphizoic organism that can cause granulomatous amoebic encephalitis, an infrequent fatal disease of the central nervous system, and amoebic keratitis, a severe corneal infection that can lead to blindness. These diseases are extremely hard to treat; therefore, a more comprehensive understanding of this pathogen's metabolism is essential for revealing potential therapeutic targets. To propagate successfully in human tissues, the parasites must resist the iron depletion caused by nutritional immunity. The aim of our study is to elucidate the mechanisms underlying iron homeostasis in A. castellanii. Using a comparative whole-cell proteomic analysis of cells grown under different degrees of iron availability, we identified the primary proteins involved in Acanthamoeba iron acquisition. Our results suggest a two-step reductive mechanism of iron acquisition by a ferric reductase from the STEAP family and a divalent metal transporter from the NRAMP family. Both proteins are localized to the membranes of acidified digestive vacuoles where endocytosed medium and bacteria are trafficked. The expression levels of these proteins are significantly higher under iron-limited conditions, which allows Acanthamoeba to increase the efficiency of iron uptake despite the observed reduced pinocytosis rate. We propose that excessive iron gained while grown under iron-rich conditions is removed from the cytosol into the vacuoles by an iron transporter homologous to VIT/Ccc1 proteins. Additionally, we identified a novel protein that may participate in iron uptake regulation, the overexpression of which leads to increased iron acquisition.

Entanglement of polymer chains in hypertonic medium enhances the delivery of DNA and other biomacromolecules into cells.

HYPOTHESIS: Most experimental procedures applied in modern biology involve cargo delivering into cells. One of the ways to cargo introduction is osmotic-mediated intracellular vesicle swelling. However, its widespread use was hindered due to cargo size (<10 nm) and cell-type-related restrictions. We addressed the issue of the composition of colloidal loading solution to enhance the efficiency of cellular delivery. EXPERIMENTS: We examined the effectiveness of colloidal loading solutions of varied compositions, including various types and sizes of polymers building osmotic pressure. We used confocal imaging coupled with fluorescence correlation spectroscopy to evaluate the introduction of polymers, proteins, nanoparticles, and DNA plasmids (cargos of sizes 1-175 nm) to cells representing eight cell lines: cancer, normal, epithelial, and mesenchymal ones. FINDINGS: We found that cellular delivery effectiveness strongly correlates with the size and concentration of osmotic pressure building polymers and not with the high value of the osmotic pressure itself. We show that polymer solutions at the entangled regime of concentrations enhance the delivery of large biomacromolecules even of size 200 nm (DNA plasmids) into cells, including MDA-MB-231 cells - so far resistant to the osmotic procedure. We show that the colloid loading medium based on entangled polymer chains is a versatile cargo delivery tool for molecular biology.

Pendimethalin-based herbicide impairs cellular immune response and haemocyte morphology in a beneficial ground beetle.

Herbicides have become the most commonly applied agrochemicals in agroecosystems. Thus, basic knowledge of their physiological effects on insects is needed, especially for understanding their impact on beneficial insect species. In this study, we evaluated the effect of a pendimethalin-based herbicide (PND) on the cellular immune response of the carabid beetle Harpalus (Pseudoophonus) rufipes (De Geer 1774) (Coleoptera, Carabidae), acting as biocontrol agent in agroecosystems. Total and differential haemocyte counts and phagocytosis assay, performed by injecting in vivo carboxylate-modified polystyrene latex beads, were measured in beetles exposed to a recommended field dose (4L per ha) of PND to evaluate the exposure effects over the time. The pattern of haemocyte subpopulations and the decrease of the phagocytic index after the exposure to PND suggested a lowering of P. rufipes ability to face an infection performing a cell-mediated response. PND was also found to cause cytotoxic effects on the haemocyte ultrastructure. Ultrastructural alterations such as irregular shape, large vacuolization of the cytoplasm, and condensation of marginated chromatin were recorded from 2d of exposure. The loss of RER, Golgi apparatus, mitochondria integrity and the swelling of the outer nuclear membrane found in some haemocytes suggested an interference of PND with the membrane permeability. Results indicated that the exposure to PND impairs the distribution, morphology and physiological functions of haemocytes causing a decrease of P. rufipes immunocompetence. Moreover, the sensitivity to herbicide exposure makes this species a suitable model and a useful bioindicator for monitoring exposure effects on non-target species. This study provides useful information to protect and preserve biodiversity of insects in agroecosystems.