0000000000043619
AUTHOR
Varpu Marjomäki
Echovirus 1 internalization negatively regulates epidermal growth factor receptor downregulation
We have demonstrated previously that the human picornavirus Echovirus 1 (EV1) triggers an infectious internalization pathway that follows closely, but seems to stay separate, from the epidermal growth factor receptor (EGFR) pathway triggered by epidermal growth factor (EGF). Here, we confirmed by using live and confocal microscopy that EGFR and EV1 vesicles are following intimately each other but are distinct entities with different degradation kinetics. We show here that despite being sorted to different pathways and located in distinct endosomes, EV1 inhibits EGFR downregulation. Simultaneous treatment with EV1 and EGF led to an accumulation of EGFR in cytoplasmic endosomes, which was evi…
Slow infection due to lowering amount of intact versus empty particles is a characteristic feature of Coxsackievirus B5, dictated by the structural proteins
Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from a fast cytolysis causing echovirus 1 (EV1) with non-structural proteins from Coxsackievirus B5 (CVB5) showing persistent infection in RD cells. The results showed that the chimera behaved similar to the parental EV1 leading to efficient cytolysis in both …
Real-time Fluorescence Measurement of Enterovirus Uncoating
Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of the virus has previously been observed mainly by transmission electron microscopy using negative staining, cryo electron microscopy, X-ray crystallography or gradient separation (reviewed in Tuthill et al., 2010, Myllynen et al., 2016, Ruokolainen et al., 2019). However, monitoring of uncoating has been limited by the lack of methods detecting dynamic changes of the virions. Here, we present a real-time fluorescence based protocol, which detects the viral …
Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine
Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced pro…
Covalent and non-covalent coupling of a Au102 nanocluster with a fluorophore: energy transfer, quenching and intracellular pH sensing
Interactions between an atomically precise gold nanocluster Au102(p-MBA)44 (p-MBA = para mercaptobenzoic acid) and a fluorescent organic dye molecule (KU, azadioxatriangulenium) are studied. In solution, the constituents form spontaneously a weakly bound complex leading to quenching of fluorescence of the KU dye via energy transfer. The KU can be separated from the complex by lowering pH, leading to recovery of fluorescence, which forms a basis for an optical reversible pH sensor. However, the sensor is not a stable entity, which could be delivered inside cells. For this purpose, a covalently bound hybrid is synthesized by linking the KU dye to the ligand layer of the cluster via an ester b…
Advanced time-stamped total data acquisition control front-end for MeV ion beam microscopy and proton beam writing
Many ion-matter interactions exhibit [email protected] time dependences such as, fluorophore emission quenching and ion beam induced charge (IBIC). Conventional event-mode MeV ion microbeam data acquisition systems discard the time information. Here we describe a fast time-stamping data acquisition front-end based on the concurrent processing capabilities of a Field Programmable Gate Array (FPGA). The system is intended for MeV ion microscopy and MeV ion beam lithography. The speed of the system (>240,000 events s^-^1 for four analogue to digital converters (ADC)) is limited by the ADC throughput and data handling speed of the host computer.
Enhanced Gene Delivery by Avidin-Displaying Baculovirus
Flexible alteration of virus surface properties would be beneficial for enhanced and targeted gene delivery. A useful approach could be based on a high-affinity receptor–ligand pair, such as avidin and biotin. In this study, we have constructed an avidin-displaying baculovirus, Baavi. Avidin display was expected to enhance cell transduction due to the high positive charge of avidin in physiological pH and to provide a binding site for covering the virus with desired biotinylated ligands. Successful incorporation of avidin on the virus envelope was detected by immunoblotting and electron microscopy. Multiple biotin-binding sites per virus were detected with fluorescence-correlation spectrosc…
Early entry events in Echovirus 30 infection
Echovirus 30 (E30), a member of the enterovirus B species, is a major cause of viral meningitis, targeting children and adults alike. While it is a frequently isolated enterovirus and the cause of several outbreaks all over the world, surprisingly little is known regarding its entry and replication strategy within cells. In this study, we used E30 strain Bastianni (E30B) generated from an infectious cDNA clone in order to study early entry events during infection in human RD cells. E30B required the newly discovered Fc echovirus receptor (FcRn) for successful infection, but not the coxsackievirus and adenovirus receptor (CAR) or decay-accelerating factor (DAF), although an interaction with …
Hydrophobic pocket targeting probes for enteroviruses
Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron micros…
A comparative study of the effect of UV and formalin inactivation on the stability and immunogenicity of a Coxsackievirus B1 vaccine
Type B Coxsackieviruses (CVBs) belong to the enterovirus genus, and they cause both acute and chronic diseases in humans. CVB infections usually lead to flu-like symptoms but can also result in more serious diseases such as myocarditis, aseptic meningitis and life-threatening multi-organ infections in young infants. Thus, CVBs have long been considered as important targets of future vaccines. We have previously observed CVB1 capsid disintegration and virus concentration decrease with 12-day long formalin inactivation protocol. Here a scalable ion exchange chromatography purification method was developed, and purified CVB1 was inactivated with UV-C or formalin. Virus morphology and concentra…
Quantitative Analysis of Dynamic Association in Live Biological Fluorescent Samples
Determining vesicle localization and association in live microscopy may be challenging due to non-simultaneous imaging of rapidly moving objects with two excitation channels. Besides errors due to movement of objects, imaging may also introduce shifting between the image channels, and traditional colocalization methods cannot handle such situations. Our approach to quantifying the association between tagged proteins is to use an object-based method where the exact match of object locations is not assumed. Point-pattern matching provides a measure of correspondence between two point-sets under various changes between the sets. Thus, it can be used for robust quantitative analysis of vesicle …
Polyphenols Epigallocatechin Gallate and Resveratrol, and Polyphenol-Functionalized Nanoparticles Prevent Enterovirus Infection through Clustering and Stabilization of the Viruses
To efficiently lower virus infectivity and combat virus epidemics or pandemics, it is important to discover broadly acting antivirals. Here, we investigated two naturally occurring polyphenols, Epigallocatechin gallate (EGCG) and Resveratrol (RES), and polyphenol-functionalized nanoparticles for their antiviral efficacy. Concentrations in the low micromolar range permanently inhibited the infectivity of high doses of enteroviruses (107 PFU/mL). Sucrose gradient separation of radiolabeled viruses, dynamic light scattering, transmission electron microscopic imaging and an in-house developed real-time fluorescence assay revealed that polyphenols prevented infection mainly through clustering of…
Intracellular route of canine parvovirus entry.
ABSTRACT The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18°C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membr…
Echovirus 1 entry into polarized Caco-2 cells depends on dynamin, cholesterol, and cellular factors associated with macropinocytosis
Enteroviruses invade their hosts by crossing the intestinal epithelium. We have examined the mechanism by which echovirus 1 (EV1) enters polarized intestinal epithelial cells (Caco-2). Virus binds to VLA-2 on the apical cell surface and moves rapidly to early endosomes. Using inhibitory drugs, dominant negative mutants, and small interfering RNAs (siRNAs) to block specific endocytic pathways, we found that virus entry requires dynamin GTPase and membrane cholesterol but is independent of both clathrin- and caveolin-mediated endocytosis. Instead, infection requires factors commonly associated with macropinocytosis, including amiloride-sensitive Na /H exchange, protein kinase C, and C-termina…
The Fibril-associated Collagen IX Provides a Novel Mechanism for Cell Adhesion to Cartilaginous Matrix
Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collage…
Structural characterization of site-modified nanocapsid with monodispersed gold clusters
AbstractHepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102pMBA44 (Au102) was an ideal conjugate given its small 2.5 nm s…
Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin, Cholesterol, and Cellular Factors Associated with Macropinocytosis
ABSTRACT Enteroviruses invade their hosts by crossing the intestinal epithelium. We have examined the mechanism by which echovirus 1 (EV1) enters polarized intestinal epithelial cells (Caco-2). Virus binds to VLA-2 on the apical cell surface and moves rapidly to early endosomes. Using inhibitory drugs, dominant negative mutants, and small interfering RNAs (siRNAs) to block specific endocytic pathways, we found that virus entry requires dynamin GTPase and membrane cholesterol but is independent of both clathrin- and caveolin-mediated endocytosis. Instead, infection requires factors commonly associated with macropinocytosis, including amiloride-sensitive Na + /H + exchange, protein kinase C, …
Expression and trafficking of fluorescent viral membrane proteins in baculovirus-transduced BHK cells
Baculovirus vectors show promise as a novel tool for gene delivery into mammalian cells and gene transfer with wild-type baculovirus has been demonstrated both in vitro and in vivo. To study expression and intracellular trafficking of foreign viral membrane proteins in baculovirus-transduced mammalian cells, the envelope proteins, E1 and E2, of rubella virus (RV) were chosen as a model. The enhanced green fluorescent protein (EGFP) and a red fluorescent protein (RFP) were fused to the C-terminus of E1 and E2, respectively. The proteins were cloned under a cytomegalovirus (CMV) promoter and expressed as fluorescent fusion proteins in baculovirus-transduced baby hamster kidney (BHK) cells. Ex…
Isoproterenol inhibits fluid-phase endocytosis from early to late endosomes
We have shown recently that isoproterenol affects both the cellular location and the morphology of late endosomes in a pH-dependent manner [Marjomäki et al., Eur. J. Cell Biol. 65, 1-13 (1994)]. In this study, using fluorescence and quantitative electron microscopy, we wanted to examine further what is the fate of internalized markers during their translocation from early to late endosomes under isoproterenol treatment. Fluorescein dextran internalized for 30 min (10-min pulse followed by a 20-min chase) showed accumulation in the cellular periphery during isoproterenol treatment in contrast to the control cells, which accumulated dextran in the perinuclear region. Quantitative electron mic…
Clustering induces a lateral redistribution of α2β1 integrin from membrane rafts to caveolae and subsequent protein kinase C-dependent internalization
Integrin alpha 2 beta 1 mediates the binding of several epithelial and mesenchymal cell types to collagen. The composition of the surrounding plasma membrane, especially caveolin-1- and cholesterol-containing membrane structures called caveolae, may be important to integrin signaling. On cell surface alpha 2 beta 1 integrin was located in the raft like membrane domain, rich in GPI-anchored proteins, rather than in caveolae. However, when antibodies were used to generate clusters of alpha 2 beta 1 integrin, they started to move laterally on cell surface along actin filaments. During the lateral movement small clusters fused together. Finally alpha 2 beta 1 integrin was found inside caveolae …
The closure of Pak1-dependent macropinosomes requires the phosphorylation of CtBP1/BARS
Membrane fission is an essential process in membrane trafficking and other cellular functions. While many fissioning and trafficking steps are mediated by the large GTPase dynamin, some fission events are dynamin independent and involve C-terminal-binding protein-1/brefeldinA-ADP ribosylated substrate (CtBP1/BARS). To gain an insight into the molecular mechanisms of CtBP1/BARS in fission, we have studied the role of this protein in macropinocytosis, a dynamin-independent endocytic pathway that can be synchronously activated by growth factors. Here, we show that upon activation of the epidermal growth factor receptor, CtBP1/BARS is (a) translocated to the macropinocytic cup and its surroundi…
Compensation of missing wedge effects with sequential statistical reconstruction in electron tomography.
Electron tomography (ET) of biological samples is used to study the organization and the structure of the whole cell and subcellular complexes in great detail. However, projections cannot be acquired over full tilt angle range with biological samples in electron microscopy. ET image reconstruction can be considered an ill-posed problem because of this missing information. This results in artifacts, seen as the loss of three-dimensional (3D) resolution in the reconstructed images. The goal of this study was to achieve isotropic resolution with a statistical reconstruction method, sequential maximum a posteriori expectation maximization (sMAP-EM), using no prior morphological knowledge about …
Novel activities of safe-in-human broad-spectrum antiviral agents
According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley feve…
Baculovirus capsid display: a novel tool for transduction imaging
Baculoviruses are enveloped insect viruses that can carry large quantities of foreign DNA in their genome. Baculoviruses have proved to be very promising gene therapy vectors but little is known about their transduction mechanisms in mammalian cells. We show in this study that Autographa californica multiple nuclear polyhedrosis virus capsid is compatible with the incorporation of desired proteins in large quantities. Fusions can be made to the N-terminus or C-terminus of the major capsid protein vp39 without compromising the viral titer or functionality. As an example of the baculovirus capsid display we show a tracking of the baculovirus transduction in mammalian cells by an enhanced gree…
Permeability changes of integrin-containing multivesicular structures triggered by picornavirus entry.
Cellular uptake of clustered α2β1-integrin induces the formation of membrane compartments that subsequently mature into a multivesicular body (MVB). Enhanced internalization mediated by clustered integrins was observed upon infection by the picornavirus echovirus 1 (EVI). We elucidated the structural features of virus-induced MVBs (vMVBs) in comparison to antibody-induced control MVBs (mock infection) by means of high-pressure cryo fixation of cells followed by immuno electron tomography during early entry of the virus. Three-dimensional tomograms revealed a marked increase in the size and complexity of these vMVBs and the intraluminal vesicles (ILVs) at 2 and 3.5 hours post infection (p.i.…
Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein
Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpa…
Subcellular localization of pentachlorophenol 4-monooxygenase in Sphingobium chlorophenolicum ATCC 39723.
Abstract We have studied the subcellular localization of pentachlorophenol 4-monooxygenase (PCP4MO) in Sphingobium chlorophenolicum ATCC 39723 during induction by pentachlorophenol (PCP). Using a monoclonal antibody CL6 specific to the native and recombinant PCP4MO, the enzyme was primarily found soluble as determined by immunoblot and ELISA analyses of cellular fractions. However, the enzyme was observed both in the soluble and membrane-bound forms during induction for 2–4 h, suggesting its translocation out from the cytoplasm. Electron microscopy confirmed that PCP4MO was predominantly present in the cytoplasm at 1 h, whereas at 4 h significant amount was detected also in the membrane and…
Reply to "Heparan Sulfate in Baculovirus Binding and Entry of Mammalian Cells"
(1), we investigated the interaction ofbaculovirus and mammalian cell surface heparan sulfate pro-teoglycans (HSPG). The data show that baculovirus requiresHSPG sulfation, particularly N- and 6-O-sulfation, to bind andtransduce mammalian cells. We also show that baculovirus asso-ciates specifically with syndecan-1 (SDC-1) but not with othersyndecans or glypicans.As discussed in the article, HS has previously been shown to beinvolved in glycoprotein 64 (gp64)-mediated baculovirus bindingonto mammalian cells. Heparin and heparinase I and II treatmentof cells have also been shown to prevent the virus binding (2, 3).The role of HS in baculovirus entry was further studied in ourarticle (1). Bindi…
Enzyme-assisted extraction of anthocyanins and other phenolic compounds from blackcurrant (Ribes nigrum L.) press cake: From processing to bioactivities
The effects of commercial enzymes (pectinases, cellulases, beta-1-3-glucanases, and pectin lyases) on the recovery of anthocyanins and polyphenols from blackcurrant press cake were studied considering two solid:solvent ratios (1:10 and 1:4 w/v). β-glucanase enabled the recovery of the highest total phenolic content – 1142 mg/100 g, and the extraction of anthocyanins was similar using all enzymes (∼400 mg/100 g). The use of cellulases and pectinases enhanced the extraction of antioxidants (DPPH − 1080 mg/100 g; CUPRAC – 3697 mg/100 g). The freeze-dried extracts presented antioxidant potential (CUPRAC, DPPH), which was associated with their biological effects in different systems: antiviral a…
Clathrin-independent entry of baculovirus triggers uptake of E. coli in non-phagocytic human cells
The prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus, an insect pathogen, holds great potential as a gene therapy vector. To develop transductional targeting and gene delivery by baculovirus, we focused on characterizing the nature and regulation of its uptake in human cancer cells. Baculovirus entered the cells along fluid-phase markers from the raft areas into smooth-surfaced vesicles devoid of clathrin. Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake. The internalization and nuclear uptake was, however, affected by mutants o…
Baculovirus entry into human hepatoma cells.
ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a prototype member of the Baculoviridae family, has gained increasing interest as a potential vector candidate for mammalian gene delivery applications. AcMNPV is known to enter both dividing and nondividing mammalian cell lines in vitro, but the mode and kinetics of entry as well as the intracellular transport of the virus in mammalian cells is poorly understood. The general objective of this study was to characterize the entry steps of AcMNPV- and green fluorescent protein-displaying recombinant baculoviruses in human hepatoma cells. The viruses were found to bind and transduce the cell line efficiently, and electron …
Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor–avidin fusion protein
The very high binding affinity of avidin to biotin is one of the highest to occur in nature. We constructed a fusion protein composed of avidin and the endocytotic LDL receptor in order to target biotinylated molecules to cells of the desired tissues. In addition to the native avidin, charge-mutated and nonglycosylated avidins were utilized as part of the fusion proteins, in order to modify its properties. All of the fusion protein versions retained the biotin-binding capacity. Although the specificity was not increased, however, fusion proteins composed of natural avidin and nonglycosylated avidin bound most efficiently to the biotinylated ligands. Fluorescence microscopy and atomic force …
Role of lipid rafts in virus infection
Rafts are domains of the plasma membrane, enriched in cholesterol and sphingolipids; they form a platform for signaling proteins and receptors. The lipid rafts are utilized in the replication cycle of numerous viruses. Internalization receptors of many viruses localize to rafts or are recruited there after virus binding. Arrays of signal transduction proteins found in rafts contribute to efficient trafficking and productive infection. Some viruses are dependent on raft domains for the biogenesis of their membranous replication structures. Finally, rafts are often important in virus assembly and budding. Subsequently, raft components in the viral envelope may be vital for the entry to a new…
Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy
AbstractFluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and pastimmunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7x10exp-7 cm(2)s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5x10exp-8 cm(2)s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a pastimmunity se…
Women With and Without Metabolic Disorder Differ in Their Gut Microbiota Composition
The aim of this study was to investigate whether overweight/obese women in metabolic disorder group (MDG, n = 27) differ in their gut microbiota composition from overweight/obese women in non-metabolic disorder group (NMDG, n = 47) and normal weight women group (NWG, n = 11). Gut microbiota was profiled from fecal samples by 16S rRNA fluorescence in situ hybridization and flow cytometry in 85 premenopausal women. Body composition was measured by bioimpedance, and dietary intakes were collected via food diaries. Standard procedures were used to assess plasma glucose, serum insulin, lipids, and inflammatory status. We found that the proportion of bacteria belonging to Eubacterium rectale-Clos…
BioImageXD: an open, general-purpose and high-throughput image-processing platform
BioImageXD puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXD has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes. We demonstrate its performance in a study of integrin clustering in response to selected inhibitors.
Endocytosis in skeletal muscle fibers.
Defining the organization of endocytic pathway in multinucleated skeletal myofibers is crucial to understand the routing of membrane proteins, such as receptors and glucose transporters, through this system. Here we analyzed the organization of the endocytic trafficking pathways in isolated rat myofibers. We found that sarcolemmal-coated pits and transferrin receptors were concentrated in the I band areas. Fluid phase markers were taken up into vesicles in the same areas along the whole length of the fibers and were then delivered into structures around and between the nuclei. These markers also accumulated beneath the neuromuscular and myotendinous junctions. The recycling compartment, lab…
Covalent and non-covalent coupling of a Au102 nanocluster with a fluorophore : energy transfer, quenching and intracellular pH sensing
Interactions between an atomically precise gold nanocluster Au102(p-MBA)44 (p-MBA = para mercaptobenzoic acid) and a fluorescent organic dye molecule (KU, azadioxatriangulenium) are studied. In solution, the constituents form spontaneously a weakly bound complex leading to quenching of fluorescence of the KU dye via energy transfer. The KU can be separated from the complex by lowering pH, leading to recovery of fluorescence, which forms a basis for an optical reversible pH sensor. However, the sensor is not a stable entity, which could be delivered inside cells. For this purpose, a covalently bound hybrid is synthesized by linking the KU dye to the ligand layer of the cluster via an ester b…
Coxsackievirus B3 VLPs purified by ion exchange chromatography elicit strong immune responses in mice
Coxsackievirus B3 (CVB3) is an important cause of acute and chronic viral myocarditis, and dilated cardiomyopathy (DCM). Although vaccination against CVB3 could significantly reduce the incidence of serious or fatal viral myocarditis and various other diseases associated with CVB3 infection, there is currently no vaccine or therapeutic reagent in clinical use. In this study, we contributed towards the development of a CVB3 vaccine by establishing an efficient and scalable ion exchange chromatography-based purification method for CVB3 virus and baculovirus-insect cell-expressed CVB3 virus-like particles (VLPs). This purification system is especially relevant for vaccine development and produ…
Field-Grown and In Vitro Propagated Round-Leaved Sundew (Drosera rotundifolia L.) Show Differences in Metabolic Profiles and Biological Activities
Drosera rotundifolia L. is a carnivorous plant used in traditional medicine for its therapeutic properties. Because of its small size, its collection in nature is laborious and different cultivation methods have been studied to ensure availability. However, only a few studies exist where the lab-grown sundew tissue and field-grown sundew would have been compared in their functionality or metabolic profiles. In this study, the antioxidant and antiviral activities of lab-grown and field-grown sundew extracts and their metabolic profiles are examined. The effect of drying methods on the chromatographic profile of the extracts is also shown. Antioxidant activity was significantly higher (5–6 ti…
Phosphomannosyl receptors of lysosomal enzymes of skeletal muscle in neuromuscular diseases.
The phosphomannosyl receptor system is responsible for both the receptor-mediated endocytosis and the intracellular transport of lysosomal enzymes. In the present study this receptor system was examined in affected muscles of patients with various neuromuscular diseases. The total activity of beta-N-acetyl-glucosaminidase, a marker enzyme of lysosomal hydrolases, was significantly elevated in the patients with myopathies (polymyositis and muscular dystrophies) but only slightly increased in those with neurogenic muscle atrophies (amyotrophic lateral sclerosis, polyneuropathy or other neurogenic muscle disease). The increase was most prominent in the group of polymyositis. The content of pho…
Assembly of citrate gold nanoparticles on hydrophilic monolayers
Abstract Self-assembled monolayers (SAMs) as model surfaces were linked onto planar gold films thorough lipoic acid or disulfide groups. The molecules used were polyethylene glycol (EG-S-S), N -[tris-(hydroxymethyl)methyl]acrylamide polymers with and without lipoic acid (Lipa-pTHMMAA and pTHMMAA) and a lipoic acid triazine derivative (Lipa-MF). All the layers, but Lipa-MF with a primary amino group were hydroxyl terminated. The layers were characterized by contact angle measurements and atomic force microscopy, AFM. Citrate stabilized nanoparticles, AuNPs in water and phosphate buffer were allowed to assemble on the layers for 10 min and the binding was followed in real-time with surface pl…
Toxicological and bioactivity evaluation of blackcurrant press cake, sea buckthorn leaves and bark from Scots pine and Norway spruce extracts under a green integrated approach
Aqueous extracts from blackcurrant press cake (BC), Norway spruce bark (NS), Scots pine bark (SP), and sea buckthorn leaves (SB) were obtained using maceration and pressurized hot water and tested for their bioactivities. Maceration provided the extraction of higher dry matter contents, including total phenolics (TPC), anthocyanins, and condensed tannins, which also impacted higher antioxidant activity. NS and SB extracts presented the highest mean values of TPC and antioxidant activity. Individually, NS extract presented high contents of proanthocyanidins, resveratrol, and some phenolic acids. In contrast, SB contained a high concentration of ellagitannins, ellagic acid, and quercetin, exp…
Unexpected subcellular distribution of a specific isoform of the Coxsackie and adenovirus receptor, CAR-SIV, in human pancreatic beta cells
Aims/hypothesis: The Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the princi…
Coxsackievirus A9 Infects Cells via Nonacidic Multivesicular Bodies
ABSTRACT Coxsackievirus A9 (CVA9) is a member of the human enterovirus B species in the Enterovirus genus of the family Picornaviridae . According to earlier studies, CVA9 binds to αVβ3 and αVβ6 integrins on the cell surface and utilizes β2-microglobulin, dynamin, and Arf6 for internalization. However, the structures utilized by the virus for internalization and uncoating are less well understood. We show here, based on electron microscopy, that CVA9 is found in multivesicular structures 2 h postinfection (p.i.). A neutral red labeling assay revealed that uncoating occurs mainly around 2 h p.i., while double-stranded RNA is found in the cytoplasm after 3 h p.i. The biogenesis of multivesicu…
Enteroviruses and coronaviruses: similarities and therapeutic targets
ABSTRACT Introduction: Enteroviruses are common viruses causing a huge number of acute and chronic infections and producing towering economic costs. Similarly, coronaviruses cause seasonal mild infections, epidemics, and even pandemics and can lead to severe respiratory symptoms. It is important to develop broadly acting antiviral molecules to efficiently tackle the infections caused by thes. Areas covered: This review illuminates the differences and similarities between enteroviruses and coronaviruses and examines the most appealing therapeutic targets to combat both virus groups. Publications of both virus groups and deposited structures discovered through PubMed to March 2021 for viral p…
Structural Studies Reveal that Endosomal Cations Promote Formation of Infectious Coxsackievirus A9 A-Particles, Facilitating RNA and VP4 Release
Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N termini, and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here, we show that fatty acid-free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results showed that CVA9 treatment with albumin or endosomal ions generated a heterogeneous population of virions, which could b…
Salix spp. Bark Hot Water Extracts Show Antiviral, Antibacterial, and Antioxidant Activities—The Bioactive Properties of 16 Clones
Funding Information: This work was supported by Business Finland corona-co-creation funding for the project Antiviral Fibers?pilot with extracts from Finnish forests (grant: 40699/31/2020). This study was also funded by the Natural Resources Institute Finland?s strategic research funding to the projects ?More, faster, higher quality: potential of short-rotation aspen and willow biomass for novel products in bioeconomy? (AspenWill) and ?Added value potential of new and under-utilized fibre sources in Finnish value networks of green bioeconomy: prefeasibility, prototyping, and market acceptance? (VALUEPOT). In addition, Academy of Finland has supported this study via the project ?Antivirals f…
The impact of the receptor binding profiles of the vascular endothelial growth factors on their angiogenic features
Abstract Background Vascular endothelial growth factors (VEGFs) are potential therapeutic agents for treatment of ischemic diseases. Their angiogenic effects are mainly mediated through VEGF receptor 2 (VEGFR2). Methods Receptor binding, signaling, and biological efficacy of several VEGFR2 ligands were compared to determine their characteristics regarding angiogenic activity and vascular permeability. Results Tested VEGFR2 ligands induced receptor tyrosine phosphorylation with different efficacy depending on their binding affinities. However, the tyrosine phosphorylation pattern and the activation of the major downstream signaling pathways were comparable. The maximal angiogenic effect stim…
A Common Receptor Found for Echoviruses.
Echoviruses are amongst the most common causative agents of aseptic meningitis worldwide and are particularly devastating in the neonatal population, where they are associated with severe hepatitis, neurological disease, including meningitis and encephalitis, and even death. Here, we identify the neonatal Fc receptor (FcRn) as a pan-echovirus receptor. We show that loss of expression of FcRn or its binding partner beta 2 microglobulin (β2M) renders cells resistant to infection by a panel of echoviruses at the stage of virus attachment, and that a blocking antibody to β2M inhibits echovirus infection in cell lines and in primary human intestinal epithelial cells. We also show that expression…
Antiviral Mechanisms of N-Phenyl Benzamides on Coxsackie Virus A9
Enteroviruses are one of the most abundant groups of viruses infecting humans, and yet there are no approved antivirals against them. To find effective antiviral compounds against enterovirus B group viruses, an in-house chemical library was screened. The most effective compounds against Coxsackieviruses B3 (CVB3) and A9 (CVA9) were CL212 and CL213, two N-phenyl benzamides. Both compounds were more effective against CVA9 and CL213 gave a better EC50 value of 1 µM with high a specificity index of 140. Both drugs were most effective when incubated directly with viruses suggesting that they mainly bound to the virions. A real-time uncoating assay showed that the compounds stabilized the virion…
Internalization of coxsackievirus A9 is mediated by {beta}2-microglobulin, dynamin, and Arf6 but not by caveolin-1 or clathrin.
ABSTRACT Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae . It has been shown to utilize αV integrins, particularly αVβ6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin β6 subunit inhibited virus proliferation, confirming that αVβ6 mediates the CAV9 infection. However, siRNAs against integrin-linked signaling molecules, such as Src, Fyn, R…
Internalization of Echovirus 1 in Caveolae
ABSTRACT Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor α2β1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, α2β1 integrin, and …
Antiviral Agents From Fungi : Diversity, Mechanisms and Potential Applications
Viral infections are amongst the most common diseases affecting people worldwide. New viruses emerge all the time and presently we have limited number of vaccines and only few antivirals to combat viral diseases. Fungi represent a vast source of bioactive molecules, which could potentially be used as antivirals in the future. Here, we have summarized the current knowledge of fungi as producers of antiviral compounds and discuss their potential applications. In particular, we have investigated how the antiviral action has been assessed and what is known about the molecular mechanisms and actual targets. Furthermore, we highlight the importance of accurate fungal species identification on ant…
6- O - and N -Sulfated Syndecan-1 Promotes Baculovirus Binding and Entry into Mammalian Cells
ABSTRACT Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our da…
Viral entry, lipid rafts and caveosomes.
Lipid rafts and caveolae are detergent-insoluble plasma membrane microdomains, involved in cellular endocytic processes and signalling. Several viruses, including a human pathogen, echovirus 1, and an extensively studied simian virus 40 utilize these domains for internalization into the host cells. Interaction of viruses with receptors on the cell surface triggers specific conformational changes of the virus particle and can give rise to signalling events, which determine the mechanisms of virus entry. After internalization via cell surface lipid rafts or caveolae, virus-containing vesicles can fuse with caveosomes, pre-existing cytoplasmic organelles, or dock on other intracellular organel…
BioImageXD - Free Microscopy Image Processing Software
Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008
Infectious Entry Pathway of Enterovirus B Species
Enterovirus B species (EV-B) are responsible for a vast number of mild and serious acute infections. They are also suspected of remaining in the body, where they cause persistent infections contributing to chronic diseases such as type I diabetes. Recent studies of the infectious entry pathway of these viruses revealed remarkable similarities, including non-clathrin entry of large endosomes originating from the plasma membrane invaginations. Many cellular factors regulating the efficient entry have recently been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1), actin, Na/H exchanger, phospholipace C (PLC) and protein kinase Cα (PKCα). Another…
Autophagy, cathepsin L transport, and acidification in cultured rat fibroblasts.
The mechanisms of enzyme delivery to and acidification of early autophagic vacuoles in cultured fibroblasts were elucidated by cryoimmunoelectron microscopic methods. The cation-independent mannose-6-phosphate receptor (MPR) was used as a marker of the pre-lysosomal compartment, and cathepsin L and an acidotropic amine (3-(2,4-dinitroanilino)-3'-amino-N-methyl-dipropylamine (DAMP), a cytochemical probe for low-pH organelles) as markers of both pre-lysosomal and lysosomal compartments. In addition, cationized ferritin was used as an endocytic marker. In ultrastructural double labeling experiments, the bulk of all the antigens was found in vesicles containing tightly packed membrane material…
Microbial strategies to exploit host cells
The European Research Conference (EURESCO) on Spatial and Temporal Dynamics of the Endomembrane System was held in sunny San Feliu de Guixols, Spain, between 16 and 21 October 2004. The conference was organized by D. Holden and H. Stenmark ![][1] By bringing together scientists from the fields of microbiology and cell biology, the European Research Conference (EURESCO) on ‘Spatial and Temporal Dynamics of the Endomembrane System’ combined the best of both worlds at the intersection where intracellular pathogens and host cells meet. The mixture of studies, which focused on the molecular mechanisms behind endocytic and secretory transport, and the pathogenic microbes that exploit these pathwa…
Echovirus 1 infection depends on biogenesis of novel multivesicular bodies
Summary Non-enveloped picornavirus echovirus 1 (EV1) clusters its receptor α2β1 integrin and causes their internalization and accumulation in α2β1 integrin enriched multivesicular bodies (α2-MVBs). Our results here show that these α2-MVBs are distinct from acidic late endosomes/lysosomes by several criteria: (i) live intra-endosomal pH measurements show that α2-MVBs are not acidic, (ii) they are not positive for the late endosomal marker LBPA or Dil-LDL internalized to lysosomes, and (iii) simultaneous stimulation of epidermal growth factor receptor (EGFR) and α2β1 integrin clustering leads to their accumulation in separate endosomes. EGFR showed downregulation between 15 min and 2 h, where…
Development of functionalized SYBR green II related cyanine dyes for viral RNA detection
Abstract Fluorescent probes for sensing nucleic acids have found widespread use in the field of cell and molecular biology. However, probes combined with potential for post-synthetic conjugation, e.g. for intra-endosomal measurements of RNA, are unavailable. Herein we developed cyanine dyes that can be conjugated to viral capsid or other targets. First, we solved the crystal structure of SYBR Green II. The structural elucidation of this commonly used RNA probe provided the basis for synthesizing similar molecules with much desired function for post-synthetic conjugation. To address this need, cyanine dyes were prepared using an alternative synthesis protocol. All studied compounds showed co…
Modification of face masks with zeolite imidazolate framework-8
The worldwide spread of the SARS-CoV-2 virus has continued to accelerate, putting a considerable burden on public health, safety, and the global economy. Taking into consideration that the main route of virus transmission is via respiratory particles, the face mask represents a simple and efficient barrier between potentially infected and healthy individuals, thus reducing transmissibility per contact by reducing transmission of infected respiratory particles. However, long-term usage of a face mask leads to the accumulation of significant amounts of different pathogens and viruses onto the surface of the mask and can result in dangerous bacterial and viral co-infections. Zeolite imidazolat…
Serological Follow-Up Study Indicates High Seasonal Coronavirus Infection and Reinfection Rates in Early Childhood
Seasonal human coronaviruses (HCoVs) cause respiratory infections, especially in children. Currently, the knowledge on early childhood seasonal coronavirus infections and the duration of antibody levels following the first infections is limited. Here we analyzed serological follow-up samples to estimate the rate of primary infection and reinfection(s) caused by seasonal coronaviruses in early childhood. Serum specimens were collected from 140 children at ages of 13, 24, and 36 months (1, 2, and 3 years), and IgG antibody levels against recombinant HCoV nucleoproteins (N) were measured by enzyme immunoassay (EIA). Altogether, 84% (118/140) of the children were seropositive for at least one s…
Additives for vaccine storage to improve thermal stability of adenoviruses from hours to months
Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10−8–10−6 M) or polyethylene glycol (PEG, molecular weight ∼8,000 Da, 10−7–10−4 M) increase the half-life of a green fluorescent protein expressing adenovirus from ∼48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of…
Lack of R-Ras Leads to Increased Vascular Permeability in Ischemic Retinopathy
PURPOSE. The role of R-Ras in retinal angiogenesis and vascular permeability was evaluated in an oxygen-induced retinopathy (OIR) model using R-Ras knockout (KO) mice and in human diabetic neovascular membranes. METHODS. Mice deficient for R-Ras and their wild-type (WT) littermates were subjected to 75% oxygen from postnatal day 7 (P7) to P12 and then returned to room air. At P17 retinal vascularization was examined from whole mounts, and retinal vascular permeability was studied using Miles assay. Real-time RT-PCR, Western blotting, and immunohistochemistry were used to assess the expression of R-Ras in retina during development or in the OIR model. The degree of pericyte coverage and vasc…
Single-cell analysis of population context advances RNAi screening at multiple levels
Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image‐based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single‐cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome‐wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell‐based screens at this depth reveals widespread RNAi‐induce…
Distinctive Evasion Mechanisms to Allow Persistence of Borrelia burgdorferi in Different Human Cell Lines
Lyme borreliosis is a multisystemic disease caused by the pleomorphic bacteria of the Borrelia burgdorferi sensu lato complex. The exact mechanisms for the infection to progress into a prolonged sequelae of the disease are currently unknown, although immune evasion and persistence of the bacteria in the host are thought to be major contributors. The current study investigated B. burgdorferi infection processes in two human cell lines, both non-immune and non-phagocytic, to further understand the mechanisms of infection of this bacterium. By utilizing light, confocal, helium ion, and transmission electron microscopy, borrelial infection of chondrosarcoma (SW1353) and dermal fibroblast (BJ) c…
Human enterovirus group B viruses rely on vimentin dynamics for efficient processing of viral nonstructural proteins
We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with IDPN did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the non-structural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, thus leading to prolonged cell survival. Furthermore, the loc…
Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel α2β1 Integrin Internalization Pathway
We have previously shown that soluble collagen and a human pathogen, echovirus 1 (EV1) cluster α2β1 integrin on the plasma membrane and cause their internalization into cytoplasmic endosomes. Here we show that cholesterol plays a major role not only in the uptake of α2β1 integrin and its ligands but also in the formation of α2 integrin-specific multivesicular bodies (α2-MVBs) and virus infection. EV1 infection and α2β1 integrin internalization were totally halted by low amounts of the cholesterol-aggregating drugs filipin or nystatin. Inhibition of cholesterol synthesis and accumulation of lanosterol after ketoconazole treatment inhibited uptake of collagen, virus and clustered integrin, an…
Cloning and characterization of Scavidin, a fusion protein for the targeted delivery of biotinylated molecules.
We have constructed a novel fusion protein "Scavidin" consisting of the macrophage scavenger receptor class A and avidin. The Scavidin fusion protein is transported to plasma membranes where the avidin portion of the fusion protein binds biotin with high affinity and forms the basis for the targeted delivery of biotinylated molecules. Subcellular fractionation analysis, immunostaining, and electron microscopy demonstrated endosomal localization of the fusion protein. According to pulse-labeling and cross-linking studies Scavidin is found as monomers (55 kDa), dimers, and multimers, of which the 220-kDa form was the most abundant. The biotin binding capacity and active endocytosis of the bio…
Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1▿
ABSTRACT Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. …
Effects of free fatty acids, lysophosphatides and phospholipase treatment on lipid peroxidation of myocardial homogenates and membrane fractions
The effects of various free fatty acids, lysophosphatides and phospholipase treatments on the enzymatic and the non-enzymatic lipid peroxidation capacities in the heart homogenates and subcellular fractions were studied. The results showed a dose related inhibition of both the enzymatic and non-enzymatic lipid peroxidation with free fatty acids. A significant inhibition occurred as early as at the concentration of 25–50 μM of several fatty acids both in homogenates and in organelle fractions. In general, the inhibition was greatest with cis-unsaturated, long-chain fatty acids. The inhibition was also induced by the pretreatment of the homogenates with phospholipase A2 but not with phospholi…
From the forest to the plate – Hemicelluloses, galactoglucomannan, glucuronoxylan, and phenolic-rich extracts from unconventional sources as functional food ingredients
This study aimed to characterise pressurised hot water (PHW) extracts from nonconventional sources of functional carbohydrates and phenolic compounds in terms of antioxidant capacity, antiviral activity, toxicity, and human erythrocytes’ protection antidiabetic potential. PHW extracts of Norway spruce bark (E1 + E2) and Birch sawdust (E3 + E4) contained mostly galactoglucomannan and glucuronoxylan. In contrast, samples E5 to E9 PHW extracted from Norway spruce, and Scots pine bark are rich sources of phenolic compounds. Overall, phenolic-rich extracts presented the highest inhibition of α-amylase and α-glucosidase and protection against stable non-enveloped enteroviruses. Additionally, all …
Therapeutic targets for enterovirus infections
Enteroviruses are among the most common viruses causing a huge number of acute and chronic infections leading to high economic costs. Novel nontoxic antivirals that reduce the virus load in acutely infected individuals and from various surfaces are needed to efficiently combat these viruses.This review summarizes the recent findings of compounds and tools targeting the enteroviruses and host cell molecules that are crucial for virus infection. In addition, the review states the modern methods to find new targets and tools that help to understand the mechanisms of action.High-throughput molecular screens have revealed important aspects of virus life cycle in host cells and, concomitantly, so…
Cell Susceptibility to Baculovirus Transduction and Echovirus Infection Is Modified by Protein Kinase C Phosphorylation and Vimentin Organization
ABSTRACT Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and it…
Structural and functional analysis of integrin alpha2I domain interaction with echovirus 1.
Integrins are cell surface receptors for several microbial pathogens including echovirus 1 (EV1), a picornavirus. Cryo-electron microscopy revealed that the functional domain (alpha(2)I) of human alpha(2)beta(1) integrin binds to a surface depression on the EV1 capsid. This three-dimensional structure of EV1 bound to alpha(2)I domain provides the first structural details of an integrin interacting with a picornavirus. The model indicates that alpha(2)beta(1) integrin cannot simultaneously bind both EV1 and the physiological ligand collagen. Compared with collagen binding to the alpha(2)I domain, the virus binds with a 10-fold higher affinity but in vitro uncoating of EV1 was not observed as…
Development of the Jyväskylä microbeam facility
Abstract A new microbeam facility is being constructed at the 1.7 MV Pelletron Accelerator in Jyvaskyla. The facility is designed for easy upgrading and incorporates a number of innovative features. Initially, it is based on a Heidelberg doublet with a design capability of a 3 × 5 μm beamspot at PIXE intensities and later upgraded to nanobeam performance. A thermal-expansion compensated rigid frame mounted on a mechanically isolated floor section is used to support the ion optical components. A compact-post focusing electrostatic deflector is used for high linearity beam scanning. This together with a novel time-stamped data collection (TDC) allows dynamic effects in IBIC, fluorescence blea…
Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues
The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging &minus
Echovirus 1 internalization negatively regulates epidermal growth factor receptor downregulation
We have demonstrated previously that the human picornavirus Echovirus 1 (EV1) triggers an infectious internalization pathway that follows closely, but seems to stay separate, from the epidermal growth factor receptor (EGFR) pathway triggered by epidermal growth factor (EGF). Here, we confirmed by using live and confocal microscopy that EGFR and EV1 vesicles are following intimately each other but are distinct entities with different degradation kinetics. We show here that despite being sorted to different pathways and located in distinct endosomes, EV1 inhibits EGFR downregulation. Simultaneous treatment with EV1 and EGF led to an accumulation of EGFR in cytoplasmic endosomes, which was evi…
Site-specific targeting of enterovirus capsid by functionalized monodisperse gold nanoclusters
Development of precise protocols for accurate site-specific conjugation of monodisperse inorganic nanoparticles to biological material is one of the challenges in contemporary bionanoscience and nanomedicine. We report here a successful site-specific covalent conjugation of functionalized atomically monodisperse gold clusters with 1.5-nm metal cores to viral surfaces. Water-soluble Au102(para-mercaptobenzoic acid)44 clusters, functionalized by maleimide linkers to target cysteines of viral capsid proteins, were synthesized and conjugated to enteroviruses echovirus 1 and coxsackievirus B3. Quantitative analysis of transmission electron microscopy images and the known virus structures showed …
A Raft-derived, Pak1-regulated Entry Participates in α2β1 Integrin-dependent Sorting to Caveosomes
We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor alpha2beta1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. alpha2beta1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered alpha2beta1 integrin does not enter…
Ellagitannin-rich cloudberry inhibits hepatocyte growth factorinduced cell migration and phosphatidylinositol 3-kinase/AKT activation in colon carcinoma cells and tumors in Min mice
// Anne-Maria Pajari 1, 2 , Essi Paivarinta 1 , Lassi Paavolainen 3 , Elina Vaara 1 , Tuuli Koivumaki 4 , Ritu Garg 5 , Anu Heiman-Lindh 1 , Marja Mutanen 1 , Varpu Marjomaki 3 , Anne J. Ridley 2, 5 1 Department of Food and Environmental Sciences, Division of Nutrition, University of Helsinki, Helsinki, Finland 2 University College London, Ludwig Institute for Cancer Research, London, UK 3 Department of Biological and Environmental Science / Nanoscience Center, University of Jyvaskyla, Jyvaskyla, Finland 4 Department of Food and Environmental Sciences, Division of Food Chemistry, University of Helsinki, Helsinki, Finland 5 Randall Division of Cell & Molecular Biophysics, King’s College Lond…
Optimized production and purification of Coxsackievirus B1 vaccine and its preclinical evaluation in a mouse model.
Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus. Safety…
Calpains promote α2β1 integrin turnover in nonrecycling integrin pathway
A novel virus- and integrin clustering–specific pathway diverts integrin from its normal endo/exocytic traffic to a nonrecycling degradative endosomal route. Clustering of α2β1 integrin causes redistribution of the integrin to perinuclear endosomes, leading to enhanced integrin turnover promoted by calpains.
Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes
Summary In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation o…
Automatic detection and analysis of cell motility in phase-contrast time-lapse images using a combination of maximally stable extremal regions and Kalman filter approaches
Phase-contrast illumination is simple and most commonly used microscopic method to observe nonstained living cells. Automatic cell segmentation and motion analysis provide tools to analyze single cell motility in large cell populations. However, the challenge is to find a sophisticated method that is sufficiently accurate to generate reliable results, robust to function under the wide range of illumination conditions encountered in phase-contrast microscopy, and also computationally light for efficient analysis of large number of cells and image frames. To develop better automatic tools for analysis of low magnification phase-contrast images in time-lapse cell migration movies, we investiga…
Development of micro-contact printing of osteosarcoma cells using MeV ion beam lithography
Abstract For investigation of spatial effects in signalling between cells and also signal substances that trigger cell proliferation and behaviour we are developing a micro Contact Printing process ( μ CP ) . In order to allow printing of cells stamps with high aspect ratio are required and these have been fabricated using Programmed Proximity Aperture Lithography (PPAL) with 3 MeV 4 He 2 + ions to produce PMMA masters for casting the stamps in PDMS. A simple printing device was developed and the first results using this to print human osteosarcoma cells is demonstrated.
Echovirus 1 Endocytosis into Caveosomes Requires Lipid Rafts, Dynamin II, and Signaling EventsV⃞
Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the α2β1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection…
A Novel Open and Infectious Form of Echovirus 1.
ABSTRACT One of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on previous studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N terminus of VP1 and to have reduced infectivity. Here, in addition to the native and intact particle type, we have identified another type of infectious echovirus 1 (E1) particle population during infection. Our results show that E1 is slightly altered during entry, which leads to the broadening of the major virion peak in the sucrose gradient. In contrast, CsCl gradient separation revealed that in addit…
Raman Spectroscopic Signatures of Echovirus 1 Uncoating
ABSTRACT In recent decades, Raman spectroscopy has entered the biological and medical fields. It enables nondestructive analysis of structural details at the molecular level and has been used to study viruses and their constituents. Here, we used Raman spectroscopy to study echovirus 1 (EV1), a small, nonenveloped human pathogen, in two different uncoating states induced by heat treatments. Raman signals of capsid proteins and RNA genome were observed from the intact virus, the uncoating intermediate, and disrupted virions. Transmission electron microscopy data revealed general structural changes between the studied particles. Compared to spectral characteristics of proteins in the intact v…
Production of biologically active recombinant avidin in baculovirus-infected insect cells
Abstract An efficient lepidopteran insect cell system was established for the expression of a recombinant form of chicken egg-white avidin. The gene product was obtained in both secreted and intracellular forms, and biologically active recombinant avidin was isolated using affinity chromatography on an iminobiotin–agarose column. Similar to the known quaternary structure of the native egg-white protein, the purified recombinant protein was glycosylated and assembled mainly into tetramers. Like native avidin, the recombinant tetramer also exhibited a high level of thermostability, and was further stabilized upon binding biotin. The biotin-binding and structural properties of the recombinant …
Vemurafenib Inhibits Acute and Chronic Enterovirus Infection by Affecting Cellular Kinase Phosphatidylinositol 4-Kinase Type IIIβ
Enteroviruses are one of the most abundant viruses causing mild to serious acute infections in humans and also contributing to chronic diseases like type 1 diabetes. Presently, there are no approved antiviral drugs against enteroviruses. Here, we studied the potency of vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, as an antiviral against enteroviruses. We showed that vemurafenib prevented enterovirus translation and replication at low micromolar dosage in an RAF/MEK/ERK-independent manner. Vemurafenib was effective against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Fo…
A novel rat CVB1-VP1 monoclonal antibody 3A6 detects a broad range of enteroviruses
AbstractEnteroviruses (EVs) are common RNA viruses that cause diseases ranging from rash to paralytic poliomyelitis. For example, EV-A and EV-C viruses cause hand-foot and mouth disease and EV-B viruses cause encephalitis and myocarditis, which can result in severe morbidity and mortality. While new vaccines and treatments for EVs are under development, methods for studying and diagnosing EV infections are still limited and therefore new diagnostic tools are required. Our aim was to produce and characterize new antibodies that work in multiple applications and detect EVs in tissues and in vitro. Rats were immunized with Coxsackievirus B1 capsid protein VP1 and hybridomas were produced. Hybr…
Molecular mechanism of α2β1 integrin interaction with human echovirus 1
Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did …
Lysosomal trafficking in rat cardiac myocytes.
By immunolabeling of cryosections, we have characterized in rat cardiac myocytes the cation-independent mannose-6-phosphate receptor (MPR), a lysosomal membrane glycoprotein, lgp120, and a lysosomal enzyme, MEP (homologous to cathepsin L). Most of the MPR label was located in large membrane-filled structures (MPR structures) in large clusters of mitochondria adjacent to but distinct from the Golgi complex. Lpg120 and MEP showed typical lysosomal localization throughout the cell, often associated with regions that appeared to contain autophagosome-like structures. In addition, MEP and lgp120 co-localized within MPR structures. MEP and MPR were localized inside the lumen of MPR structures. M…
Cannabinoid receptor 1 and acute resistance exercise – In vivo and in vitro studies in human skeletal muscle
Abstract Aim This study aimed to determine whether Cannabinoid receptor 1 (CB1) is involved in mammalian target of rapamycin (mTOR) signaling and skeletal muscle protein synthesis. Methods This study used human vastus lateralis skeletal muscle biopsies obtained before and after a resistance exercise (RE) bout in young men (n = 18). The signaling mechanisms were studied in vitro in human myotubes. Protein expression was determined by Western blot and confocal microscopy, and gene expression by quantitative PCR. Protein synthesis was measured in vitro using puromycin-based SuNSET technique. Results In human skeletal muscle, an anabolic stimulus in the form of RE down-regulated CB1 expression.…
Human parvovirus B19 induced apoptotic bodies contain altered self-antigens that are phagocytosed by antigen presenting cells.
Human parvovirus B19 (B19V) from the erythrovirus genus is known to be a pathogenic virus in humans. Prevalence of B19V infection has been reported worldwide in all seasons, with a high incidence in the spring. B19V is responsible for erythema infectiosum (fifth disease) commonly seen in children. Its other clinical presentations include arthralgia, arthritis, transient aplastic crisis, chronic anemia, congenital anemia, and hydrops fetalis. In addition, B19V infection has been reported to trigger autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. However, the mechanisms of B19V participation in autoimmunity are not fully understood. B19V induced chronic dise…
Polymorphic and solvate structures of ethyl ester and carboxylic acid derivatives of WIN 61893 analogue and their stability in solution
3-Ethyl ester- (1) and 3-carboxylic acid-isoxazole (2) derivatives of an antiviral drug analogue WIN 61893 were synthesized and characterized by X-ray crystallography and NMR spectroscopy. Crystallization experiments afforded two polymorphic structures for the ethyl ester derivative and two solvate structures for the carboxylic acid derivative based on their ability to form intermolecular hydrogen bonding interactions with the solvent molecules. The conformations of the derivatives depended greatly on the orientation of the planar isoxazole and phenyl-oxadiazole ring systems with respect to one another and were found to take up perpendicular, linear or tilted conformations. The carboxylic a…
Slow Infection due to Lowering the Amount of Intact versus Empty Particles Is a Characteristic Feature of Coxsackievirus B5 Dictated by the Structural Proteins
Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from fast-cytolysis-causing echovirus 1 (EV1) with nonstructural proteins from coxsackievirus B5 (CVB5), which shows persistent infection in RD cells. The results showed that the chimera behaved similarly to parental EV1, leading to efficient cytolysis in both…
Entry of Human Parechovirus 1
ABSTRACT Human parechovirus 1 (HPEV-1) is a prototype member of parechoviruses, a recently established picornavirus genus. Although there is preliminary evidence that HPEV-1 recognizes α V integrins as cellular receptors, our understanding of early events during HPEV-1 infection is still very limited. The aim of this study was to clarify the entry mechanisms of HPEV-1, including the attachment of the virus onto the host cell surface and subsequent internalization. In blocking experiments with monoclonal antibodies against different receptor candidates, antibodies against α V and β 3 integrin subunits, in particular in combination, appeared to be the most efficient ones in preventing the HPE…
Recombinant avidin and avidin-fusion proteins.
Both chicken egg-white avidin and its bacterial relative streptavidin are well known for their extraordinary high affinity with biotin (Kd approximately 10(-15) M). They are widely used as tools in a number of affinity-based separations, in diagnostic assays and in a variety of other applications. These methods have collectively become known as (strept)avidin-biotin technology. Biotin can easily and effectively be attached to different molecules, termed binders and probes, without destroying their biological activity. The exceptional stability of the avidin-biotin complex and the wide range of commercially available reagents explain the popularity of this system. In order by genetic enginee…
Capillary enlargement, not sprouting angiogenesis, determines beneficial therapeutic effects and side effects of angiogenic gene therapy.
Aims Currently, it is still unclear which mechanisms drive metabolic benefits after angiogenic gene therapy. The side-effect profile of efficient angiogenic gene therapy is also currently incompletely understood. In this study, the effects of increasing doses of adenoviral (Ad) vascular endothelial growth factor-A (VEGF-A) were evaluated on vascular growth, metabolic benefits, and systemic side effects. Methods and results Adenoviral vascular endothelial growth factor-A or AdLacZ control was injected intramuscularly (109–1011 vp/mL) or intra-arterially (5 × 1011 vp/mL) into rabbit ( n = 102) hindlimb muscles and examined 6 or 14 days later. Blood flow, tissue oedema, metabolic benefits, and…
Characteristics of lysosomal phosphomannosyl-enzyme receptors in the rat heart
The receptor system recognizing mannose 6-phosphate groups of lysosomal enzymes has been characterized, e.g. in fibroblasts and liver cells. The purpose of this study was to demonstrate the presence of a phosphomannosyl receptor system in rat heart muscle. The characterization of receptors was accomplished with beta-N-acetylglucosaminidase (beta-GA) secreted by rat embryo fibroblasts after ammonium chloride stimulation. The receptor binding of ligand enzymes was saturated by adding increasing concentrations of beta-GA and the binding increased linearly when the content of membrane protein was increased. The binding of beta-GA was inhibited by mannose and glucose phosphates, especially manno…
Vascular endothelial growth factor-A and platelet-derived growth factor-B combination gene therapy prolongs angiogenic effects via recruitment of interstitial mononuclear cells and paracrine effects rather than improved pericyte coverage of angiogenic vessels
Vessel stabilization and the inhibition of side effects such as tissue edema are essential in angiogenic gene therapy. Thus, combination gene transfers stimulating both endothelial cell and pericyte proliferation have become of interest. However, there is currently little data to support combination gene transfer in large animal models. In this study, we evaluated the potential advantages of such a strategy by combining the transfer of adenoviral (Ad) vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B into rabbit hindlimb skeletal muscle. AdLacZ alone or in combination with AdVEGF-A were used as controls. Contrast-enhanced ultrasound, modified Miles assa…
At reduced temperature, endocytic membrane traffic is blocked in multivesicular carrier endosomes in rat cardiac myocytes.
Temperatures around 20 degrees C are known to block degradation of endocytosed material by preventing its transport to lysosomes, accordingly reduced temperature has been widely used to define endosomes. Newer studies have revealed that the low temperature block is proximal to perinuclear late endosomes, but it is not clear whether the block is already in early endosomes, or whether the traffic proceeds to multivesicular carrier endosomes which mediate transport from early to late compartments. We have now focused on this problem using rat cardiac myocytes. First, cell fractionation on Percoll gradients showed that at reduced temperatures (22 degrees C and 26 degrees C), with prolonged chas…
Application independent greedy particle tracking method for 3D fluorescence microscopy image series
Single-particle tracking is computationally a challenging problem, and usually solved with local methods. Local methods suffer from defects in the image data or in the detection of particles, such as temporal disappearing of particles. A particle tracking method has to provide a solution also to real disappearing and appearing of particles as a result of merging and splitting. Here, we present an efficient, greedy algorithm as a solution to the particle tracking problem. This improved local method is application independent, as it has high configurability of the function used to solve particle correspondence. To demonstrate the accuracy of the method, we apply it to real microscopy image da…
6-O sulfated and N-sulfated Syndecan-1 promotes baculovirus binding and entry into mammalian cells.
Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show t…
Competitive binding of Rab21 and p120RasGAP to integrins regulates receptor traffic and migration
P120RasGAP competes with Rab21 for binding to the cytoplasmic domain of integrin α-subunits, thereby promoting receptor escape from early endosomes and recycling to the plasma membrane.
Efficient gene therapy based targeting system for the treatment of inoperable tumors
Background A considerable percentage of tumors are not amenable to surgery. We have designed a simple and powerful targeting system that offers an alternative option for the multi-component pre-targeting strategies used clinically. This targeting system can be used for any type of solid tumors independent of the tumor type, thereby omitting the need to engineer unique antibodies for each specific application or tumour type. In the present study, we show the expression of a chimeric fusion protein, which contains the low-density lipoprotein receptor transmembrane domains and avidin, after local gene transfer and its ability to bind biotinylated compounds in vivo. Methods Semliki Forest virus…
Single-cell analysis of population context advances RNAi screening at multiple levels
Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image‐based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single‐cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome‐wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell‐based screens at this depth reveals widespread RNAi‐induce…
Collagenase-3 (MMP-13) Enhances Remodeling of Three-Dimensional Collagen and Promotes Survival of Human Skin Fibroblasts
Collagenase-3 (MMP-13) is a matrix metalloproteinase capable of cleaving a multitude of extracellular matrix proteins in addition to fibrillar collagens. Human MMP-13 is expressed by fibroblasts in chronic cutaneous ulcers, but not in normally healing adult skin wounds. However, MMP-13 is produced by fibroblasts in adult gingival and in fetal skin wounds characterized by rapid collagen remodeling and scarless healing. Here, we have examined the role of human MMP-13 in remodeling of three-dimensional (3D) collagenous matrix by primary adult human skin fibroblasts. The high level of human MMP-13 expression by fibroblasts achieved by adenoviral gene delivery resulted in potent enhancement of r…
Calpains promote α2β1 integrin turnover in non-recycling integrin pathway.
Collagen receptor integrins recycle between the plasma membrane and endosomes and facilitate formation and turnover of focal adhesions. In contrast, clustering of α2β1 integrin with antibodies or the human pathogen echovirus 1 (EV1) causes redistribution of α2 integrin to perinuclear multivesicular bodies, α2-MVBs. We show here that the internalized clustered α2 integrin remains in α2-MVBs and is not recycled back to the plasma membrane. Instead, receptor clustering and internalization lead to an accelerated down-regulation of α2β1 integrin compared to the slow turnover of unclustered α2 integrin. EV1 infection or integrin degradation is not associated with proteasomal or autophagosomal pro…
Ion-induced fluorescence imaging of endosomes
Abstract Imaging laboratories at Jyvaskyla and Singapore are collaborating on the development of fluorescence imaging of cytoplasmic endosomes using a combination of proton induced fluorescence (PIF) with direct Scanning Transmission Ion Microscopy (direct-STIM) for sub-cellular structural imaging. A549 lung carcinoma cells were cultivated and stained for epidermal growth factor receptor (EGFR) and receptor α2β1 integrin. In this paper, we demonstrate that cells can be imaged at sub-150 nm resolution using the PIF technique. In addition, the same target cell was imaged at 50 and 25 nm resolution by using proton and He-STIM, respectively. The combination of both techniques offer a powerful t…
Extracellular Albumin and Endosomal Ions Prime Enterovirus Particles for Uncoating That Can Be Prevented by Fatty Acid Saturation
ABSTRACT There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37°C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectiv…
ADC measurements in diffuse large B-cell lymphoma and follicular lymphoma: a DWI and cellularity study.
Diffusion-weighted magnetic resonance imaging (DW-MRI) allows quantifying the random motion of water molecules in tissue by means of apparent diffusion coefficient (ADC) measurements. The aim of the study was to determine whether ADC measurements allow discrimination of diffuse large B-cell lymphoma (DLBCL) from follicular lymphoma (FL), and to examine the relationship between cellularity and ADC value of the tumor using DWI.Thirty-two patients with histologically proven non-Hodgkin lymphoma (21 with DLBCL and 11 with FL, 17 males and 15 females, mean age 62±13 years) underwent conventional MRI and DWI examination before treatment. The ADC values of DLBCL were compared to those of FL. The A…
Regulation of kynurenine biosynthesis during influenza virus infection.
Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus–host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T-cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine-2,3-dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furt…
Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel alpha2beta1 Integrin Internalization Pathway
We have previously shown that soluble collagen and a human pathogen, echovirus 1 (EV1) cluster α2β1 integrin on the plasma membrane and cause their internalization into cytoplasmic endosomes. Here we show that cholesterol plays a major role not only in the uptake of α2β1 integrin and its ligands but also in the formation of α2 integrin-specific multivesicular bodies (α2-MVBs) and virus infection. EV1 infection and α2β1 integrin internalization were totally halted by low amounts of the cholesterol-aggregating drugs filipin or nystatin. Inhibition of cholesterol synthesis and accumulation of lanosterol after ketoconazole treatment inhibited uptake of collagen, virus and clustered integrin, an…
Formalin treatment increases the stability and immunogenicity of coxsackievirus B1 VLP vaccine
Type B Coxsackieviruses (CVBs) are a common cause of acute and chronic myocarditis, dilated cardiomyopathy and aseptic meningitis. However, no CVB-vaccines are available for human use. We have previously produced virus-like particles (VLPs) for CVB3 with a baculovirus-insect cell production system. Here we have explored the potential of a VLP-based vaccine targeting CVB1 and describe the production of CVB1-VLPs with a scalable VLP purification method. The developed purification method consisting of tangential flow filtration and ion exchange chromatography is compatible with industrial scale production. CVB1-VLP vaccine was treated with UV-C or formalin to study whether stability and immuno…
Nanomolar pyrophosphate detection in water and in a self-assembled hydrogel of a simple terpyridine-Zn2+ complex.
A simple terpyridine-Zn(II) complex is shown to act as an efficient and highly selective fluorescent sensor for pyrophosphate in water at physiological pH. The sensor complex showed an unprecedented fluorescence response (∼500 fold increase) and a record nanomolar sensitivity (detectable fluorescent response at 20 nM and LOD ∼ 0.8 nM). It has successfully been used to stain and record confocal fluorescence microscopy images of HeLa cells. Moreover, the complex was found to self-assemble into a hydrogel which was subsequently used to coat disposable paper strips for easy, low-cost detection of pyrophosphate.
CCDC 999257: Experimental Crystal Structure Determination
Related Article: Kirsi Salorinne, Tanja Lahtinen, Varpu Marjomäki, Hannu Häkkinen|2014|CrystEngComm|16|9001|doi:10.1039/C4CE01152J
CCDC 999254: Experimental Crystal Structure Determination
Related Article: Kirsi Salorinne, Tanja Lahtinen, Varpu Marjomäki, Hannu Häkkinen|2014|CrystEngComm|16|9001|doi:10.1039/C4CE01152J
CCDC 977466: Experimental Crystal Structure Determination
Related Article: Sandip Bhowmik, Biswa Nath Ghosh, Varpu Marjomäki, and Kari Rissanen|2014|J.Am.Chem.Soc.|136|5543|doi:10.1021/ja4128949
CCDC 999255: Experimental Crystal Structure Determination
Related Article: Kirsi Salorinne, Tanja Lahtinen, Varpu Marjomäki, Hannu Häkkinen|2014|CrystEngComm|16|9001|doi:10.1039/C4CE01152J
CCDC 999256: Experimental Crystal Structure Determination
Related Article: Kirsi Salorinne, Tanja Lahtinen, Varpu Marjomäki, Hannu Häkkinen|2014|CrystEngComm|16|9001|doi:10.1039/C4CE01152J