Endocytosis Of Particles Research Articles

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

MicroRNA-mediated Regulation of LDL Receptor: Biological and Pharmacological Implications.

One of the main causes of atherosclerosis is a disruption in cellular cholesterol hemostasis. The low-density lipoprotein receptor (LDLR) is an important factor in maintaining cholesterol homeostasis by the receptor-mediated endocytosis of LDL particles. Defective hepatic LDLR activity and uptake of LDL particles lead to elevated blood levels of low-density lipoprotein cholesterol (LDL-C), which is associated with a higher risk of atherosclerotic cardiovascular disease. LDLR expression can be affected by microRNAs (miRNAs). Some miRNAs, like miR-148a, miR-185, miR-224, miR-520, miR-128-1, miR-27a/b, miR-130b, and miR-301 seem to be important post-transcriptional regulators of LDLR related genes. These findings indicate the critical role of miRNAs in regulating LDL metabolism. The aim of this review was to provide insight into the miRNAs involved in LDLR activity and their potential roles in the treatment of cardiovascular disease.

Virus-like particle endocytosis pathways and presentation of captured antigens

The proteins of many viruses can be assembled into strictly organized structures virus-like particles bearing antigens of the original viruses and may also be artificially decorated with antigens of other pathogens. These particles contain no viral genome and lack infectivity but can be highly immunogenic and therefore being actively used for vaccine development. Undoubtedly, while designing vaccines, it is necessary to take into account information about the interaction of vaccine components with immune system particularly antigen-presenting cells. This is especially important for virus-like particles because, like other nanometer-sized particles, they can enter antigen-presenting cells using various endocytosis pathways. The latter exploit multiple receptors, generate endocytic vesicles of different sizes, and, most importantly, are associated with varying fates of captured material. Here we review the mechanisms of phagocytosis, macropinocytosis, clathrin-mediated endocytosis, rapid endophilin-mediated endocytosis, and several endocytic pathways associated with lipid rafts. The data are presented on the relationship between various endocytic pathways and sorting of absorbed cargo in early endosomes as well as enzymatic degradation of the late endosomes contents. We also describe the mechanisms of distribution of absorbed antigens within antigen-presenting cells to be loaded onto the class I and II major histocompatibility complex molecules. The data are presented on the endocytosis of various viruses during cell infection, as well as a comparative analysis of the endocytosis pathways for virus-like particles and related viruses. It has been noted that virus-like particles, along with the absorption pathway specific for parent virus, can rely on additional endocytic pathways to be also artificially targeted at the selected endocytic receptor and relevant absorption pathway. It allows to select or design particles with optimal endocytosis and antigen presentation to induce a protective immune response upon vaccination. It should be assumed that most prophylactic vaccines require particles that are well engulfed by antigen presenting cells and direct material to endolysosomal degradation, or particles whose uptake directs material to both late and static early endosomes, making antigens available for direct and cross presentations. Finally, we discuss virus-like particles for the delivery of drugs or genetically engineered constructs, as well as optimal endocytic pathways that should protect the payload of these particles from endolysosomal degradation.

Nanomaterial Endocytosis: Quantification of Adsorption and Ingestion Mechanisms

The widespread use of nanomaterials in vaccines, therapeutics, and industrial applications creates an increasing demand for understanding their ingestion by living cells. Researchers in the field have called for a more robust understanding of physical/chemical particle–cell interactions and a means to determine the particles ingested per cell. Using superparamagnetic nanobeads, we measured the beads per cell and quantified the kinetics of the receptor-independent endocytosis of particles having seven surface chemistries. Poly(ethylene glycol) (PEG)-coated nanoparticles were ingested less effectively by cultured Chinese hamster ovary (CHO-K1) cells and more effectively by aminated nanoparticles than starch-coated particles. The cells ingested 2 to 4 × 105 of the most attractive particles. The interplay between Van der Waals and coulombic potentials was quantified on the basis of Derjaguin–Landau–Verwey–Overbeek (DLVO) theory modified to include hydration repulsion using physical parameters of the seven surface chemistries. Using dose–response curves for inhibitors of clathrin- or caveolae-dependent ingestion, we quantified how particle surface chemistry determines which endocytic pathway is used by the cell. Such characterization can be useful in predicting nanomaterial uptake in medical and toxicological applications and in the selection of particle surface chemistries for receptor-dependent endocytosis.

MULTİPLE SKLEROZ HASTALARINDA APOE GEN EKSPRESYONUNUN BELİRLENMESİ

OBJECTIVE: Apolipoprotein E (ApoE) is a glycoprotein that functions as a ligand in receptor-mediated endocytosis of lipoprotein particles. There are studies showing the association of APOE with neurogenerative diseases such as Alzheimer's disease and multiple sclerosis (MS). The aim of this study was to investigate whether there is a relationship between APOE gene expression and MS.MATERIAL AND METHODS: To determine the effect of APOE gene expression on multiple sclerosis patients, we have included 35 MS patients and 20 healthy subjects into the study. The level of APOE mRNA expression was determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). For statistical analysis, t-test, chi-square test, Kruskal-Wallis test and Mann-Whitney U test were performed using SPSS package program and p <0.05 was considered as statistically significant.RESULTS: As a result, the comparison of ApoE gene expression in multiple sclerosis patients and healthy people using real-time PCR technique did not show any statistical significance between the groups (p=0.95).CONCLUSIONS: Our study does not support that ApoE gene expression may be related to MS. Further studies are needed to determine the role of ApoE in Multiple Sclerosis disease.

Adenovirus-Mimetic Nanoparticles: Sequential Ligand-Receptor Interplay as a Universal Tool for Enhanced In Vitro/In Vivo Cell Identification.

Viral infection patterns often rely on precisely coordinated sequences of distinct ligand-receptor interactions, leading in many cases to an outstanding target cell specificity. A successful mimicry of viral targeting strategies to create more site-specific nanoparticles (NPs) would therefore require particle-cell interactions to also be adequately controllable. In the present study, hetero-multivalent block-copolymer NPs present their attached ligands in a sterically controlled manner to create a sequential NP-cell interaction similar to the cell infiltration strategy of human adenovirus type 2. Targeting renal mesangial cells, particles therefore initially bind angiotensin II receptor type 1 (AT1r) on the cell surface via a structurally flexible AT1r antagonist. After a mandatory spatial approach, particle endocytosis is realized via binding of immobile αVβ3 integrins with a previously concealed secondary ligand, thereby creating a stepwise particle-cell interplay of primary NP attachment and subsequent uptake. Manufactured adenovirus-mimetic NPs show great avidity for both target motifs in vitro, leading to a substantial binding as well as subsequent cell uptake into target mesangial cells. Additionally, steric shielding of secondary ligand visibility leads to a highly controllable, sequential ligand-receptor interaction, whereby hetero-functional NPs activate mesangial cell surface integrins only after a successful prior binding to the AT1r. This stepwise cell identification significantly enhances mesangial cell specificity in co-culture assays with different off-target cells. Additionally, described NPs display excellent in vivo robustness by efficiently accumulating in the mesangium upon injection, thereby opening new paths for possible drug delivery applications.

Cell-in-Cell Structures in the Liver: A Tale of Four E's.

The liver is our largest internal organ and it plays major roles in drug detoxification and immunity, where the ingestion of extracellular material through phagocytosis is a critical pathway. Phagocytosis is the deliberate endocytosis of large particles, microbes, dead cells or cell debris and can lead to cell-in-cell structures. Various types of cell endocytosis have been recently described for hepatic epithelia (hepatocytes), which are non-professional phagocytes. Given that up to 80% of the liver comprises hepatocytes, the biological impact of cell-in-cell structures in the liver can have profound effects in liver regeneration, inflammation and cancer. This review brings together the latest reports on four types of endocytosis in the liver -efferocytosis, entosis, emperipolesis and enclysis, with a focus on hepatocyte biology.

Lipoprotein receptor signalling in atherosclerosis.

The founding member of the lipoprotein receptor family, low-density lipoprotein receptor (LDLR) plays a major role in the atherogenesis through the receptor-mediated endocytosis of LDL particles and regulation of cholesterol homeostasis. Since the discovery of the LDLR, many other structurally and functionally related receptors have been identified, which include low-density lipoprotein receptor-related protein (LRP)1, LRP5, LRP6, very low-density lipoprotein receptor, and apolipoprotein E receptor 2. The scavenger receptor family members, on the other hand, constitute a family of pattern recognition proteins that are structurally diverse and recognize a wide array of ligands, including oxidized LDL. Among these are cluster of differentiation 36, scavenger receptor class B type I and lectin-like oxidized low-density lipoprotein receptor-1. In addition to the initially assigned role as a mediator of the uptake of macromolecules into the cell, a large number of studies in cultured cells and in in vivo animal models have revealed that these lipoprotein receptors participate in signal transduction to modulate cellular functions. This review highlights the signalling pathways by which these receptors influence the process of atherosclerosis development, focusing on their roles in the vascular cells, such as macrophages, endothelial cells, smooth muscle cells, and platelets. Human genetics of the receptors is also discussed to further provide the relevance to cardiovascular disease risks in humans. Further knowledge of the vascular biology of the lipoprotein receptors and their ligands will potentially enhance our ability to harness the mechanism to develop novel prophylactic and therapeutic strategies against cardiovascular diseases.

Quasi-ultrafine particles promote cell metastasis via HMGB1-mediated cancer cell adhesion

With increasingly severe air pollution, the aggravated health risks of particulate matter, especially ultrafine particles, are emerging as an urgent and sensitive topic. Considering the heterogeneity and complexity of ultrafine particles, there is insufficient evidence about their toxic effects and possible molecular mechanisms. To address this question, we analyzed the emission characteristics of quasi-ultrafine particles collected during winter in a typical coal-burning city, Taiyuan, and confirmed their contribution to lung cancer cell adhesion and metastasis. For the specific mechanism, we revealed that the endocytosis of quasi-ultrafine particles stimulated the release of HMGB1, induced NFκB-facilitated proinflammatory cytokine production through the interaction of HMGB1 with RAGE, and resulted in cancer-endothelial cell adhesion. These findings remind us of the potential effects of anthropogenic quasi-ultrafine particle pollution and provide a theoretical reference for the mitigation of tumorigenesis in a severe particulate matter contaminated environment.

Transfection Efficiency Evaluation and Endocytosis Exploration of Different Polymer Condensed Agents.

DNA condensed agents can improve the transfection efficiency of the cationic liposome delivery system. However, various condensed agents have distinct transfection efficiency and cellular cytotoxicity. The object of this study was to screen the optimal agents with the high transfection efficiency and low cytotoxicity from four polymer compressive materials, polyethylenimine (PEI), chitosan, poly-l-lysine (PLL), and spermidine. DNA was precompressed with these four agents and then combined to cationic liposomes. Subsequently, the entrapment and transfection efficiency of the obtained complexes were investigated. Finally, the particle sizes, cytotoxicity, and endocytosis fashion of these copolymers (Lipo-PEI, Lipo-chitosan, Lipo-PLL, and Lipo-spermidine) were examined. It was found that these four copolymers had significantly lower cytotoxicity and higher transfection efficiency (45.5%, 42.4%, 36.8%, and 47.4%, respectively) than those in the control groups. The transfection efficiency of Lipo-PEI and Lipo-spermidine copolymers were better than the other two copolymers. In 293T cells, nystatin significantly inhibited the transfection efficiency of Lipo-PEI-DNA and Lipo-spermidine-DNA (51.88% and 46.05%, respectively), which suggest that the endocytosis pathway of Lipo-spermidine and Lipo-PEI copolymers was probably caveolin dependent. Our study indicated that these dual-degradable copolymers especially liposome-spermidine copolymer could be used as the potential biocompatible gene delivery carriers.

Human Cytomegalovirus Cell Tropism and Host Cell Receptors.

In the 1970s–1980s, a striking increase in the number of disseminated human cytomegalovirus (HCMV) infections occurred in immunosuppressed patient populations. Autopsy findings documented the in vivo disseminated infection (besides fibroblasts) of epithelial cells, endothelial cells, and polymorphonuclear leukocytes. As a result, multiple diagnostic assays, such as quantification of HCMV antigenemia (pp65), viremia (infectious virus), and DNAemia (HCMV DNA) in patient blood, were developed. In vitro experiments showed that only low passage or endothelial cell-passaged clinical isolates, and not laboratory-adapted strains, could reproduce both HCMV leuko- and endothelial cell-tropism, which were found through genetic analysis to require the three viral genes UL128, UL130, and UL131 of the HCMV UL128 locus (UL128L). Products of this locus, together with gH/gL, were shown to form the gH/gL/pUL128L pentamer complex (PC) required for infection of epithelial cells/endothelial cells, whereas gH/gL and gO form the gH/gL/gO trimer complex (TC) required for infection of all cell types. In 2016, following previous work, a receptor for the TC that mediates entry into fibroblasts was identified as PDGFRα, while in 2018, a receptor for the PC that mediates entry into endothelial/epithelial cells was identified as neuropilin2 (Nrp2). Furthermore, the olfactory receptor family member OR14I1 was recently identified as a possible additional receptor for the PC in epithelial cells. Thus, current data support two models of viral entry: (i) in fibroblasts, following interaction of PDGFRα with TC, the latter activates gB to fuse the virus envelope with the cell membrane, whereas (ii) in epithelial cells/endothelial cells, interaction of Nrp2 (and OR14I1) with PC promotes endocytosis of virus particles, followed by gB activation by gH/gL/gO (or gH/gL) and final low-pH entry into the cell.

Cholesterol and phosphatidylserine are engaged in adenoviral dodecahedron endocytosis

Adenoviral dodecahedron is a virus-like particle composed of twelve penton base proteins, derived from the capsid of human adenovirus type 3. Due to the high cell penetration capacity, it was used as a vector for protein, peptide and drug delivery. Two receptors are known to be involved in the endocytic dodecahedron uptake, namely αv integrins and heparan sulfate proteoglycans. Since it has been observed, that dodecahedron efficiently penetrates a wide range of cancer cells, it suggests that other cellular compounds may play a role in the particle endocytosis. To shed some light onto the interactions with membrane lipids and their potential role in dodecahedron entry, we performed a series of experiments including biochemical assays, fluorescence confocal imaging of giant unilamellar vesicles and surface plasmon resonance, which indicated specific preference of the particle to anionic phosphatidylserine. Experiments performed on cholesterol-depleted epithelial cells showed that cholesterol is essential in the endocytic uptake, however a direct interaction was not observed. We believe that the results will allow to better understand the role of lipids in dodecahedron entry and to design more specific dodecahedron-based vectors for drug delivery to cancer cells.

(Invited) Length-Dependent Intracellular Bundling of Single-Wall Carbon Nanotubes Influences Retention

Single-walled carbon nanotubes (SWCNTs) are increasingly being investigated for biomedical imaging, sensing, and drug delivery. Cell types, cellular entry mechanisms, and SWCNT lengths dictate SWCNT uptake, subsequent intracellular trafficking, and retention. Specialized immune cells known as macrophages are capable of two size-dependent entry mechanisms: endocytosis of small particles (diameter of 200 nm) and phagocytosis of large particles (diameter 4 500 nm). In comparison, fibroblasts uptake particles predominantly through endocytosis. We report dependence of cellular processing including uptake, subcellular distribution, and retention on the SWCNT length and immune cell-specific processes. We chose SWCNTs of three different average lengths: 50 nm (ultrashort, US), 150 nm (short) and 500 nm (long) to encompass two different entry mechanisms, and noncovalently dispersed them in water, cell culture media, and phosphate buffer (pH 5) with bovine serum albumin, which maintains the SWCNT optical properties and promotes their cellular uptake. Using confocal Raman imaging and spectroscopy, we quantified cellular uptake, tracked the intracellular dispersion state (i.e., individualized versus bundled), and monitored recovery as a function of SWCNT lengths in macrophages. Cellular uptake of SWCNTs increases with decreasing SWCNT length. Interestingly, short-SWCNTs become highly bundled in concentrated phase dense regions of macrophages after uptake and most of these SWCNTs are retained for at least 24 h. On the other hand, both US- and long-SWCNTs remain largely individualized after uptake into macrophages and are lost over a similar elapsed time. After uptake into fibroblasts, however, short-SWCNTs remain individualized and are exocytosed over 24 h. We hypothesize that aggregation of SWCNTs within macrophages but not fibroblasts may facilitate the retention of SWCNTs within the former cell type. Furthermore, the differential length dependent cellular processing suggests potential applications of macrophages as live cell carriers of SWCNTs into tumors and regions of inflammation for therapy and imaging.

Effects of scavenger receptors-1 class A stimulation on macrophage morphology and highly modified advanced glycation end product-protein phagocytosis

Advanced glycation end-products (AGEs), which comprise non-enzymatically glycosylated proteins, lipids, and nucleic acid amino groups, play an important role in several diseases and aging processes including angiopathy, renal failure, diabetic complications, and neurodegenerative diseases. Among AGE-associated phenotypes, toxic AGEs, glyceraldehyde-derived AGE-2, and glycolaldehyde-derived AGE-3 are involved in the pathogenesis of diabetic complications. In addition, macrophages are reported to remove extracellular AGEs from tissues via scavenger receptors, leading to the progression of atherosclerosis. In the present study, we found that AGE-2 and AGE-3 enhanced their own endocytic uptake by RAW264.7 mouse macrophage-like cells in a concentration-dependent manner. Furthermore, we demonstrated, for the first time, the morphology of phagocytic macrophages and the endocytosis of AGE particles. The toxic AGEs induced the expression of a scavenger receptor, CD204/scavenger receptors-1 class A (SR-A). Notably, an antibody against CD204 significantly prevented toxic AGE uptake. Moreover, an SR-A antagonistic ligand, fucoidan, also attenuated the AGE-2- and AGE-3-evoked uptake in a concentration-dependent manner. These results indicated that SR-A stimulation, at least in part, plays a role in AGE uptake.

The Role of Platelet Syntaxins in Endocytosis

In healthy patients, platelets serve as the first line of defense in forming a hemostatic plug when damage to the vasculature is detected. Hemostasis is a balancing act, however, where hyperactive platelets can cause unwanted blood clots which may lead to heart attack or stroke, while hypoactive platelets can cause excessive bleeding. For this reason, examining the proteins critical to platelet function and understanding the bleeding phenotype when these proteins are knocked out can provide potential drug targets and help explain and diagnose platelet disorders. In addition to their role in hemostasis, platelets act as vascular “sentries,” patrolling the vasculature for damage and endocytosing small molecules and viral particles they encounter [1]. While the exact purpose of platelet endocytosis of viral particles is unknown, studies have shown that AIDS patients have increased chances of experiencing a thrombotic event [2], which implies that the uptake of the HIV virus by platelets could be implicated in this disparity, perhaps by altering platelet function. Platelets contain SNARE (soluble N‐ethylmaleimide sensitive fusion protein attachment protein receptor) proteins which are known to control trafficking in most cells [3] but this trafficking role has not been established in platelets, making them an attractive target of study. To this end, we created Syntaxin 2, Syntaxin 4 (two t‐SNARE proteins) and double knock out mice to study the endocytic capabilities of their platelets. Platelets from the Syntaxin 4 and double knock out mice showed a defect in the uptake of total fibrinogen while Syntaxin 4 knockouts did not show a defect in uptake of transferrin.Support or Funding InformationThis work is supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL56652 and HL138179), and a Veterans Affairs Merit Award to SWW.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping the microscale origins of magnetic resonance image contrast with subcellular diamond magnetometry

Magnetic resonance imaging (MRI) is a widely used biomedical imaging modality that derives much of its contrast from microscale magnetic field patterns in tissues. However, the connection between these patterns and the appearance of macroscale MR images has not been the subject of direct experimental study due to a lack of methods to map microscopic fields in biological samples. Here, we optically probe magnetic fields in mammalian cells and tissues with submicron resolution and nanotesla sensitivity using nitrogen-vacancy diamond magnetometry, and combine these measurements with simulations of nuclear spin precession to predict the corresponding MRI contrast. We demonstrate the utility of this technology in an in vitro model of macrophage iron uptake and histological samples from a mouse model of hepatic iron overload. In addition, we follow magnetic particle endocytosis in live cells. This approach bridges a fundamental gap between an MRI voxel and its microscopic constituents.

Light scattering based analyses of the effects of bovine serum proteins on interactions of magnetite spherical particles with cells

Information concerns endocytosis and autophagic effects of magnetite particles is crucial for understanding the particle-cell interactions. In this work, we investigated the effects of bovine serum proteins on the endocytosis of magnetite spherical particles (MSPs). Autophagic effects of MSPs in breast cancer cells were studied. Light scattering based flow cytometry and microscopy were used for evaluating the uptake potential of MSPs by cells and the cellular autophagosome accumulation. Results showed bovine serum proteins significantly reduced the endocytosis of MSPs by decreasing their adsorption to cell membranes. Additionally, serum proteins had influences on the endocytic mechanisms of MSPs. Autophagosome accumulation could be caused by the internalized MSPs rather than the particles associated with cell membrane. Above fundamental findings promote our understandings upon the interactions of MSPs with cells. Light scattering based methods were proved to be simple and effective. The present work may promote their application in studies upon endocytosis of metallic particles in the future.

Complement Opsonization Promotes Herpes Simplex Virus 2 Infection of Human Dendritic Cells.

ABSTRACTHerpes simplex virus 2 (HSV-2) is one of the most common sexually transmitted infections globally, with a very high prevalence in many countries. During HSV-2 infection, viral particles become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of immune responses. In genital mucosa, the primary target cells for HSV-2 infection are epithelial cells, but resident immune cells, such as dendritic cells (DCs), are also infected. DCs are the activators of the ensuing immune responses directed against HSV-2, and the aim of this study was to examine the effects opsonization of HSV-2, either with complement alone or with complement and antibodies, had on the infection of immature DCs and their ability to mount inflammatory and antiviral responses. Complement opsonization of HSV-2 enhanced both the direct infection of immature DCs and their production of new infectious viral particles. The enhanced infection required activation of the complement cascade and functional complement receptor 3. Furthermore, HSV-2 infection of DCs required endocytosis of viral particles and their delivery into an acid endosomal compartment. The presence of complement in combination with HSV-1- or HSV-2-specific antibodies more or less abolished HSV-2 infection of DCs. Our results clearly demonstrate the importance of studying HSV-2 infection under conditions that ensue in vivo, i.e., conditions under which the virions are covered in complement fragments and complement fragments and antibodies, as these shape the infection and the subsequent immune response and need to be further elucidated.IMPORTANCE During HSV-2 infection, viral particles should become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of the immune responses. The dendritic cells are activators of the immune responses directed against HSV-2, and the aim of this study was to examine the effects of complement alone or complement and antibodies on HSV-2 infection of dendritic cells and their ability to mount inflammatory and antiviral responses. Our results demonstrate that the presence of antibodies and complement in the genital environment can influence HSV-2 infection under in vitro conditions that reflect the in vivo situation. We believe that our findings are highly relevant for the understanding of HSV-2 pathogenesis.

Evaluation of endocytosis of silica particles used in biodegradable implants in the brain

Silica nanoparticles embedded in a biodegradable scaffold have been proposed to offer several advantages when used in laser-tissue-soldering of blood vessels in the brain. During degradation, these nanoparticles are likely to be released into the surrounding brain tissue. The aim of this study was to investigate possible cellular uptake mechanism(s) of the two silica nanoparticle types in microglial cells as well as their effect on autophagy and inflammatory cytokines. The nanoparticle uptake was analysed quantitatively using high-content analysis. Nanoparticle incubation did not modulate cytokine secretion and autophagy at any time point investigated. The nanoparticles were taken up by the microglia cells in a time- and particle-dependent manner. The maximal uptake was reached after 4hours and the nanoparticles were found in the endoplasmic reticulum and lysosomes. Macropinocytosis and phagocytosis were predominantly responsible for the uptake, whereas clathrin- and caveolin-independent endocytosis were involved to a minor extent.

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment.

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.