Sucrose Density Gradient Centrifugation Research Articles

Prevalence of serum antibodies to Toxoplasma gondii in the small Indian mongoose (Herpestes auropunctatus) on Amami-Oshima Island, Japan.

Prevalence of antibodies to Toxoplasm gondii was studied using the latex agglutination (LA) method, followed by sucrose density gradient centrifugation (SDGC) method on the small Indian mongoose (Herpestes auropunctatus), which inhabits Amami-Oshima Island. Of the 362 samples, 38 (10.5%) revealed positive. Single or double peaks in the 7–8 and/or 12–14 fraction to LA titer by SDGC indicated the early stage of T. gondii infection. It is suggested that domestic/feral cats play an important role for spreading this zoonotic pathogen to the mongoose as well as other species that are endemic to this island. Future studies are warranted to prevent the transmission of T. gondii among cats and wild animals in order to maintain the ecosystem health.

Dendrobium officinale polysaccharide ameliorates diabetic hepatic glucose metabolism via glucagon-mediated signaling pathways and modifying liver-glycogen structure.

Dendrobium officinale polysaccharide (DOP) is the main active ingredient of Dendrobium officinale Kimura & Migo, which is a precious traditional Chinese medicine and often used in treatment of hepatitis, diabetes, obesity and rheumatoid arthritis. DOP exhibits significant hypoglycemic activity, while its mechanism remains unclear. The present study aims to investigate the hypoglycemic mechanisms of DOP based on the glucagon-mediated signaling pathways and the liver glycogen structure, which catalyze hepatic glucose metabolism, and provide new knowledge about the antidiabetic mechanism of DOP and further evidence for its clinical use for diabetes. DOP were obtained from the dry stems of Dendrobium officinale by water extraction and alcohol precipitation method. T2DM mice model was established by high-fat diet combined with streptozotocin. Liver histopathological changes were observed by H&E and PAS straining. Pancreatic histology was studied by H&E staining and immunofluorescence analysis. The levels of glucagon and insulin were detected by Elisa Kit and the hepatic glycogen content was detected by GOPOD. The expressions of the hepatic glycogen-related metabolism enzymes, hepatic gluconeogenesis enzymes, and the related protein in cAMP-PKA and Akt/FoxO1 signaling pathways were detected by western blots. Liver glycogen was extracted from the liver tissues by sucrose density gradient centrifugation, and size exclusion chromatography (SEC) was used to analyze the structure of liver glycogen. DOP could significantly affect the glucagon-mediated signaling pathways, cAMP-PKA and Akt/FoxO1, to further promote hepatic glycogen synthesis, inhibit hepatic glycogen degradation and hepatic gluconeogenesis. Moreover, DOP could reverse the instability of the liver glycogen structure and thus probably suppressed glycogen degradation. Thus, DOP finally would ameliorate hepatic glucose metabolism via glucagon-mediated signaling pathways and modifying liver-glycogen structure in diabetic mice. The hypoglycemic mechanism of DOP might be associated with the regulation of glucagon-mediated hepatic glycogen metabolism and gluconeogenesis, and of liver glycogen structure, contributing to improved hepatic glucose metabolism in diabetic mice.

Neospora caninum antigens displaying virus-like particles as a bivalent vaccine candidate against neosporosis

Neospora caninum is a causative and transmissible agent of dog and bovine neosporosis. The resulting reproductive failures in infected cattle lead to significant economic losses worldwide. However, there is no satisfactory treatment or vaccine currently available to combat this pathogen. Thus, the development of appropriate vaccines to manage its infection and transmission is urgently needed. In this study, we expressed Rous sarcoma virus-like particles (RSV-LP) that displayed dual N. caninum antigens in silkworms. The antigen candidates are modified by adding a transmembrane domain of GP64 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) to the C-terminus of surface antigen 1 (NcSAG1) and SAG1-related sequence 2 (NcSRS2). The NcSRS2 alone or the NcSAG1/NcSRS2 bivalent form displaying RSV-LPs were purified using sucrose density gradient centrifugation. These purified VLPs were then used for immunizations in gerbils, Meriones unguiculatus, to evaluate the anti-N. caninum effects in vivo. The results demonstrated that antigens displaying RSV-LPs in immunized gerbils produced the antigen-specific antibody, leading to a relatively lower parasite load after infections of N. caninum. To the best of our knowledge, this is the first study to present an RSV-LP vaccine displaying bivalent antigens from neosporosis. Taken together, our strategy suggests that silkworm-expressed virus-like particles (VLPs) are promising bivalent vaccine candidates against N. caninum infections.

Green synthesis of gold nanoclusters using seed aqueous extract of Cichorium intybus L. and their characterization

The present study reports the green synthesis of gold nanoparticles (AuNPs) by Cichorium intybus L. (chicory) aqueous seed extract and their total polyphenol content (TPC), ferric reducing antioxidant power, and photocatalytic activities as well as antiproliferative effect in C2C12 myotubes. The formation of AuNPs was achieved by reacting the extract with 1 mM final concentration of hydrochloroauric acid trihydrate (HAuCl4·3H2O) and confirmed by UV–visible spectroscopy. DLS particle size analyzer and TEM analysis showed mostly spherical crystals of around 1.7–3.2 nm (zeta potential, − 5.97) that converged to form a uniform collection of 20–40 nm clusters with irregular contour (zeta potential, − 19.7). In XRD analysis, AuNPs produced strong signal for gold; and the presence of extract components on AuNP surfaces was confirmed by FT-IR, ESD, and HPLC. TPC and the antioxidant activity were equivalent to about 1.2 mg of Gallic acid and 4.3 mM Fe2+ , per gram AuNP, respectively. The cytotoxic effect of AuNPs in C2C12 myotubes was examined by MTT assay in which higher concentrations of fraction “f1”, separated according to size by sucrose density gradient centrifugation, showed anti-proliferative activity in C2C12 myoblast cells. Despite their nontoxic behavior, AuNP nanoclusters possessed good antioxidant and photocatalytic activities.

Abstract 3640: Cancer cell membrane coated biomimetic nanoparticles as decoys for disrupting cancer cell-stromal cell interactions

Abstract Introduction: Biomimetic nanoparticles (NPs) combining synthetic and biological materials have flexibility and functionality for disruptive strategies.1 Stromal cells such as cancer associated fibroblasts (CAFs) mediate many of the aggressive characteristics of cancer and play a crucial role in proliferation, invasiveness, metastasis, and angiogenesis of cancer.2 NPs coated with cancer cell membrane fractions (CCMFs) inherit the repertoire of surface proteins from cancer cells, making them potentially useful as decoys to disrupt cancer cell-stromal cell interactions. Here, we coated cancer cell membranes onto poly(lactic-co-glycolic acid) (PLGA) NPs to form CCMF-PLGA NPs, and characterized their protein profile, size, purity, cellular internalization, and integrity. We investigated, for the first time, the ability of these “artificial cancer cells” to disrupt fibroblast-mediated migration and lung metastasis. Method: CCMFs were isolated upon cell homogenization, and sucrose density gradient centrifugation. PLGA NPs were prepared by nanoprecipitation. CCMFs and PLGA NPs were mixed and physically extruded through a porous membrane to obtain CCMF-PLGA NPs. The protein profile of CCMFs was analyzed by western blot with antibodies against cell fraction markers.. The experimental lung metastasis model was established by intravenously injecting MDA-MD-231 cells constitutively expressing luciferase (231-luc) through the tail vein of nude mice. The treated group was inoculated with the same number of 231-luc cells mixed with CCMFs-PLGA NPs and injected weekly with CCMF-PLGA NPs in the following weeks. Lung metastasis was monitored in vivo for three consecutive weeks by bioluminescence imaging (BLI). At the end of treatment, lungs were isolated and inflated. Metastatic nodules were imaged with BLI and examined by histology. Results: Plasma membrane purity was confirmed from western blot analysis that showed the significant enrichment of Na+/K+-ATPase, negligible amount of GPR78 or GAPDH in CCMFs. Confocal fluorescence microscopy and flow cytometry confirmed a “right-side” out orientation of CCMF-PLGA NPs and the integrity of membrane-associated proteins after membrane isolation and PLGA coating. When CCMFs or CCMF-PLGA NPs were added to HMFs in the transwell assay, fewer cancer cells migrated towards HMFs, identifying the unique ability of CCMF-PLGA NPs to disrupt HMF-cancer cell interactions. In the lung metastasis study, mice treated with CCMF-PLGA NPs had significantly less incidence and metastatic burden confirmed by BLI and histology. CCMF-PLGA NPs hold promise as decoys to disrupt cancer cell-stromal cell interactions.

Molecular characterization of circulating strains of small ruminant lentiviruses in Brazil based on complete gag and pol genes

Small ruminant lentiviruses (SRLV) are widely distributed in the world and cause productive and economic losses. These pathogens have high genetic variability, which hinders diagnosis by molecular methods. Molecular characterization studies are good strategies for elaborating programs aimed to control or eradicate diseases. Hence, this study aimed to sequence, characterize and compare strains of SRLV, which were isolated from naturally infected goats from the states of Ceará, Rio Grande do Norte and Minas Gerais, Brazil, based on total nucleotide sequences of gag and pol genes. SRLV isolates from the microbiological collection of Sheep and Goat of Embrapa were cultured in goat synovial membrane. Supernatants containing cells were stored and viral particles were purified with sucrose density gradient centrifugation for RNA extraction. Then, sequencing in HiSeq 2500 platform (Illumina) was performed to obtain the total genome of the SRLV strains. The software MEGA version 7.0 was used for sequence editing and phylogeny analysis. A total of 17 isolates were multiplied in cellular culture and six were viable, which were two from Minas Gerais (MG), one from Rio Grande do Norte (RN) and three from Ceará (CE). Two sequences were obtained from the sequencing by synthesis, one from MG (BRMG CNPC) and one from RN (BRRN CNPC). From the total SRLV genome (9,200 bases), sample BRMG CNPC generated a consensus of 6,378 bases, approximately 69,34% of the viral genome and sample BRRN CNPC generated a consensus of 5,351 bases, which comprised 58.16% of the total genome. Samples in this study demonstrated closer phylogenetic relation with genotype B (B1 group). The remaining strains sequenced in Brazil are also present in the same group with the exception of three samples from genotype A. Immunodominant regions of gag gene and motifs of pol gene were similar to the standard CAEV Cork strain. For the first time, the complete gag and pol genes were sequenced in Brazil from the strain BRMG CNPC isolated in Minas Gerais state. In addition, 2490 bases from the total of 3316 bases were sequenced from the pol gene of the BRRN CNPC sample from Rio Grande do Norte.

VIPP1 rods engulf membranes containing phosphatidylinositol phosphates

In cyanobacteria and plants, VIPP1 plays crucial roles in the biogenesis and repair of thylakoid membrane protein complexes and in coping with chloroplast membrane stress. In chloroplasts, VIPP1 localizes in distinct patterns at or close to envelope and thylakoid membranes. In vitro, VIPP1 forms higher-order oligomers of >1 MDa that organize into rings and rods. However, it remains unknown how VIPP1 oligomerization is related to function. Using time-resolved fluorescence anisotropy and sucrose density gradient centrifugation, we show here that Chlamydomonas reinhardtii VIPP1 binds strongly to liposomal membranes containing phosphatidylinositol-4-phosphate (PI4P). Cryo-electron tomography reveals that VIPP1 oligomerizes into rods that can engulf liposomal membranes containing PI4P. These findings place VIPP1 into a group of membrane-shaping proteins including epsin and BAR domain proteins. Moreover, they point to a potential role of phosphatidylinositols in directing the shaping of chloroplast membranes.

Density Gradient Selection of Colloidal Silver Nanotriangles for Assembling Dye-Particle Plasmophores.

A simple method based on sucrose density gradient centrifugation is proposed here for the fractionation of colloidal silver nanotriangles. This method afforded particle fractions with surface plasmon resonances, spanning from red to infrared spectral ranges that could be used to tune optical properties for plasmonic applications. This feature was exemplified by selecting silver nanotriangle samples with spectral overlap with Atto-655 dye’s absorption and emission in order to assemble dye-particle plasmophores. The emission brightness of an individual plasmophore, as characterized by fluorescence correlation spectroscopy, is at least 1000-fold more intense than that of a single Atto-655 dye label, which renders them as promising platforms for the development of fluorescence-based nanosensors.

Interaction of the Homer1 EVH1 domain and skeletal muscle ryanodine receptor

The skeletal muscle ryanodine receptor (RyR1) proteins are intracellular calcium (Ca2+) release channels on the membrane of the sarcoplasmic reticulum (SR) and required for skeletal muscle excitation-contraction coupling. Homer (Vesl) is a family of scaffolding proteins that modulate target proteins including RyRs (ryanodine receptors), mGluRs (group 1 metabotropic glutamate receptors) and IP3Rs (inositol-1,4,5-trisphosphate receptors) through a conserved EVH1 (Ena/VASP homology 1) domain. Here, we examined the interaction between Homer1 EVH1 domain and RyR1 by co-immunoprecipitation, continuous sucrose density-gradient centrifugation, and bio-layer interferometry binding assay at different Ca2+ concentrations. Our results show that there exists a high-affinity binding between the Homer1 EVH1 domain and RyR1, especially at 1 mM of Ca2+. Based on our data and the known structures of Homer1 EVH1 domain and RyR1, we found two consensus proline-rich sequences in the structure of RyR1, PPHHF and FLPPP, and proposed two corresponding binding models to show mechanisms of recognition different from those used by other proline-rich motifs. The side proline residues of two proline-rich motifs from RyR1 are away from the hydrophobic surface of Homer1 EVH1, rather than buried in this hydrophobic surface. Our results provide evidence that Homer1 regulates RyR1 by direct interaction.

Viroids as Companions of a Professional Career.

Since the early 1970s when “virus-like” agents were considered as the cause of two diseases (potato spindle tuber and citrus exocortis), their study and further characterization have been linked to the development and use of molecular biology tools. Sucrose density gradient centrifugation and polyacrylamide gel electrophoresis (PAGE) played a critical role in the pioneering studies of PSTVd and citrus exocortis viroid (CEVd). This was later modified by using other PAGEs (sequential PAGE, return PAGE, two-dimensional PAGE), and/or different staining methods (ethidium bromide, silver nitrate, etc.). Since then, disease-causing agents suspected to be viroids were usually subjected to a number of tests to define their: (i) Molecular nature (RNA or DNA; single stranded or double stranded; circular or linear RNA); (ii) molecular weight; (iii) secondary and tertiary structure. Further biological assays are also essential to establish the relationship of a viroid with plant disease and to fulfill Koch’s postulates.

CaMKII Modulates the Cardiac Transient Outward K+ Current through its Association with Kv4 Channels in Non-Caveolar Membrane Rafts.

To test whether the physiological regulation of the cardiac Kv4 channels by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is restricted to lipid rafts and whether the interactions observed in rat cardiomyocytes also occur in the human ventricle. Ventricular myocytes were freshly isolated from Sprague-Dawley rats. Ito was recorded by the whole-cell Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the proteins of interest was analysed by western blot. Immunogold staining and electron microscopy of heart vibrosections was performed to localize Kv4.2/Kv4.3 and CaMKII proteins. Protein-protein interactions were determined by co-immunoprecipitation experiments in rat and human ventricular mycoytes. Patch-Clamp recordings in control conditions and after lipid raft or caveolae disruption show that the CaMKII-Kv4 channel complex must associate in non-caveolar lipid rafts to be functional. Separation in density gradients, co-immunoprecipitation and electron microscopy show that there are two Kv4 channel populations: one located in caveolae, that is CaMKII independent, and another one located in planar membrane rafts, which is bound to CaMKII. CaMKII regulates only the Kv4 channel population located in non-caveolar lipid rafts. Thus, the regulation of cardiac Kv4 channels in rat and human ventricle depends on their subcellular localization.

Alteration of ADP-ribosylation in aging rat brain astrocytes

DNA damage and the enzyme poly (ADP-ribose) polymerase (PARP) associated with the pathogenesis of numerous age-related neurodegenerative disorders. Astrocytes play crucial roles in both support metabolic functions and cell viability of the brain. PARP regulates DNA damage and repair in the brain cells. In this study PARP activity and DNA strand break were investigated in the astrocytes isolated from young and aged rat brain. Three and 30-month-old rats were killed by decapitation and brains were removed onto an ice cooled glass plate. Astrocytes were isolated by sucrose density gradient centrifugation and glutamine synthetase (GS) served as a marker of the astrocytes lineage. The specific activity of PARP was assayed in permeabilized cells by measuring the incorporation of the ADPribose moiety of [3H]NAD into the nuclear acceptor proteins. The rate of DNA strand breaks was determined using a fluorescent dye and monitored spectrofluorimetry. An increase (about 75%) in the PARP activity was observed in the whole homogenates of aged rats, whereas this rise was more pronounced (about 360%) when the reaction was measured in the purified astrocyte preparations. The amount of DNA strand breaks was also higher in the astrocytes isolated from the aged brain as compared to that of young levels. The close relationship between the level of DNA strand breaks and PARP activity in the astrocytes suggest that these cells are susceptible to the metabolic alterations in aging. It is concluded that the astrocytes PARP might be considered as a therapeutic target for combating age related neurodegenerative disorders.

Exosomes from nicotine-stimulated macrophages accelerate atherosclerosis through miR-21-3p/PTEN-mediated VSMC migration and proliferation.

Rationale: During the development of atherosclerosis, macrophages secrete exosomes that regulate vascular smooth muscle cells (VSMCs); however, whether nicotine, a major constituent of cigarettes, can modulate this communication in the context of atherogenesis remains to be further studied. In this study, we hypothesized that nicotine induces macrophages to secrete atherogenic exosomes containing microRNAs (miRNAs) to mediate cell-to-cell crosstalk and encourage proatherogenic phenotypes of VSMCs.Methods: In an in vivo study, nicotine was administered subcutaneously to 8-week-old male ApoE-/- mice fed a high-fat diet (HFD) for 12 weeks. Oil red O and hematoxylin and eosin (HE) were used to stain atherosclerotic lesions. Lesion macrophages, VSMCs and exosomes were stained for CD68, α-smooth muscle actin (α-SMA) and CD9, and plaque exosomes were observed by transmission electron microscopy (TEM). Exosomes derived from control macrophages (M-Exos) and from nicotine-treated macrophages (NM-Exos) were isolated by ultracentrifugation, purified by sucrose density gradient centrifugation and characterized based on specific morphology and surface markers. The IVIS® Spectrum in vivo imaging system showed the biodistribution of NM-Exos and M-Exos in circulation. Chitosan hydrogel-incorporated exosomes were applied to simulate exosome secretion in situ. Scratch wound assay, transwell assay and EdU staining were conducted to assess the effects of NM-Exos on the migration and proliferation of mouse VSMCs. RNA-seq was performed to determine the miRNA profiles of M-Exos and NM-Exos. Quantitative real-time PCR (qRT-PCR) analysis was conducted to detect the expression levels of miRNAs and mRNAs. The roles of the candidate miRNA and its target gene were assessed using specific RNA inhibitors, siRNAs and miRNA mimics. Western blotting was used to detect candidate protein expression levels. A dual-luciferase reporting system was utilized to confirm the binding of a specific miRNA to its target gene.Results: Nicotine induced atherosclerotic lesion progression and resulted in plaque exosome retention in vivo. The biodistribution of NM-Exos showed that plaque-resident exosomes might be secreted in situ. VSMCs cocultured in vitro with nicotine-stimulated macrophages presented an increased capacity for migration and proliferation, which was exosome-dependent. In addition, isolated NM-Exos helped promote VSMC migration and proliferation. miRNA profiling showed that miR-21-3p was enriched in NM-Exos, and this miRNA was shown to play a key role in regulating NM-Exos-induced effects by directly targeting phosphatase and tension homologue (PTEN).Conclusion: Exosomal miR-21-3p from nicotine-treated macrophages may accelerate the development of atherosclerosis by increasing VSMC migration and proliferation through its target PTEN.

Separation and Optimisation of a Sucrose Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells (PBMC)

One step centrifugation procedure used commonly for separation of blood cells is the ficoll gradient centrifugation. In this method, after centrifugation, the peripheral blood mononuclear cells (PBMCs) are located on the top of the separation fluid, whereas other blood cells erythrocytes and granulocytes sediment to the bottom. In the present study 75% of lymphocyte suspension could be separated by using a one-step density gradient centrifugation of sodium heparin blood with Sucrose. Sucrose was diluted into different concentrations using miliQ water (10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%,). 4 mL of diluted blood was layered on 4 mL of each sucrose solution and centrifuged for 45 minutes at 1000 rpm. Clear separation of PBMCs could be observed in solution with 40% sucrose. The separated PBMCs were analysed in haeme analyser which showed 75% lymphocytes, 23% monocytes and 2% of other cells.

Hepatitis B Surface Antigen With N-terminal Addition of mCherry Can Assemble Into Functional Subviral Particles

Aim: To label HBsAg with the mCherry protein without impairing its functionality. Materials & methods: A vector expressing mCherry–HBsAg fusion protein was constructed and transfected into Huh7 cell lines. The expression, secretion and subcellular localization of HBsAg was detected by western blotting, ELISA and immunofluorescence staining, respectively. Then the assembly of subviral particles was evaluated by sucrose density gradient centrifugation, dot blotting and electron microscopic assay. Results: mCherry–HBsAg fusion protein can be expressed and secreted in a similar manner to HBsAg. More importantly, mCherry–HBsAg fusion protein can self-assemble into spherical subviral-like particles. Conclusion: mCherry could be introduced into HBsAg without affecting its biological characters including expression, secretion and assembly.

Kinetic characterization of the gill (Na+, K+)-ATPase in a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na+, K+)-ATPase with Mr of ≈110 kDa is present that hydrolyzes ATP with VM = 130.3 ± 4.8 nmol Pi min-1 mg protein-1 and K0.5 = 0.065 ± 0.00162 mmol L-1, exhibiting site-site interactions. Stimulation by Na+ (VM = 127.5 ± 5.3 nmol Pi min-1 mg protein-1, K0.5 = 5.3 ± 0.42 mmol L-1), Mg2+ (VM = 130.6 ± 6.8 nmol Pi min-1 mg protein-1, K0.5 = 0.33 ± 0.042 mmol L-1), K+ (VM = 126.7 ± 7.7 nmol Pi min-1 mg protein-1, K0.5 = 0.65 ± 0.0079 mmol L-1) and NH4+ (VM = 134.5 ± 8.6 nmol Pi min-1 mg protein-1, K0.5 = 1.28 ± 0.44 mmol L-1) also obeys cooperative kinetics. Ouabain (KI = 0.18 ± 0.058 mmol L-1) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na+, K+)-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats.

Torquetenovirus detection in exosomes enriched vesicles circulating in human plasma samples

BackgroundTorquetenovirus (TTV) belongs to Anelloviridae family, infects nearly all people indefinitely without causing overt disease establishing a fine and successful interaction with the host. Increasing evidence have shown some human viruses exploit extracellular vesicles thereby helping viral persistence in the host. Here, the presence of TTV in extracellular vesicles circulating in human plasma was investigated.MethodsTTV DNA was quantified in plasma-derived exosomes from 122 samples collected from 97 diseased patients and 25 healthy donors. Exosomes enriched vesicles (EEVs) were extracted from plasma and characterized by Nanoparticle tracking analysis, by western blot for presence of tetraspanin CD63, CD81 and annexin II protein and, finally, by electron microscopy (EM). Presence and quantitation of TTV DNA were assessed with an universal single step real-time TaqMan PCR assay.ResultsPreliminary investigation showed that the human plasma extracted extracellular vesicles exhibited a main size of 70 nm, had concentration of 2.5 × 109/ml, and scored positive for tetraspanin CD63, CD81 and annexin II, typical characteristic of the exosomes vesicles. EEVs extracted from pooled plasma with TTV DNA viremia of 9.7 × 104 copies/ml showed to contain 6.3 × 102 TTV copies/ml, corresponding to 0.65% of total viral load. Important, TTV yield changed significantly following freezing/thawing, detergents and DNAse treatment of plasma before EEVs extraction. EEVs purified by sucrose-density gradient centrifugation and analysis of gradient fraction positive for exosomes marker CD63 harbored 102 TTV copies/ml. Moreover, EM evidenced the presence of TTV-like particles in EEVs. Successive investigation of plasma EEVs from 122 subjects (37 HIV-positive, 20 HCV infected, 20 HBV infected, 20 kidney transplant recipients, and 25 healthy) reported TTV DNA detection in 42 (34%) of the viremic samples (37 were from diseased patients and 5 from healthy people) at a mean level of 4.8 × 103 copies/ml. The examination of EEVs selected samples reported the presence of TTV genogroup 1, 3, 4 and 5, with genogroup 3 highly observed.ConclusionsCollectively, although these observations should be confirmed by further studies, circulation of TTV particles in EEVs opens new avenues and mechanistic insights on the molecular strategies adopted by anelloviruses to persist in the host.

Nano-sized CA125 antigen glycocamouflage: Mucin - Extracellular vesicles alliance to watch?

Mucin 16 (MUC16) is a transmembrane type mucin and its released extracellular portion is designated as CA125 antigen. It is considered to be part of a supramolecular glycoprotein complex having a complicated epitope map and extreme structural heterogeneity.Starting from the initial transmembrane localization of MUC16/CA125 antigen and its alternative routes of release by shedding or putative secretion, CA125 antigen from human amniotic fluid soluble and extracellular vesicles (EVs)-containing fractions were characterized aiming at the possible glycosylation-associated mode of distribution as a factor contributing to the reported conflicting structural data.Ultracentrifugation, sucrose density gradient centrifugation, ion-exchange chromatography and TEM were used for analysis.The results indicated that the smeared abundantly glycosylated high molecular mass CA125-immunoreactive species, which follow the wheat germ agglutinin-binding pattern, were shared across amniotic fluid soluble and particulate fractions. A lower molecular mass glycoprotein-like CA125-immunoreactive species which follows the peanut agglutinin-binding pattern and was specifically associated with the EVs-enriched fraction was observed.CA125 presentation in the particulate amniotic fluid fraction was found to be shaped by a complex interactome partially involving lactose-sensitive galectin-3 binding. The MUC16 - EVs alliance as well as heterogeneous mucin/macromolecular complexes, at membranes or extracellularly, may represent cryptic pools of distinct CA125 species.

SIMPLE binds specifically to PI4P through SIMPLE-like domain and participates in protein trafficking in the trans-Golgi network and/or recycling endosomes.

Small integral membrane protein of the lysosome/late endosome (SIMPLE) is a 161-amino acid cellular protein that contains a characteristic C-terminal domain known as the SIMPLE-like domain (SLD), which is well conserved among species. Several studies have demonstrated that SIMPLE localizes to the trans-Golgi network (TGN), early endosomes, lysosomes, multivesicular bodies, aggresomes and the plasma membrane. However, the amino acid regions responsible for its subcellular localization have not yet been identified. The SLD resembles the FYVE domain, which binds phosphatidylinositol (3)-phosphate (PI3P) and determines the subcellular localization of FYVE domain-containing proteins. In the present study, we have found that SIMPLE binds specifically to PI4P through its SLD. SIMPLE co-localized with PI4P and Rab11, a marker for recycling endosomes (REs, organelles enriched in PI4P) in both the IMS32 mouse Schwann cell line and Hela cells. Sucrose density-gradient centrifugation revealed that SIMPLE co-fractionated with syntaxin-6 (a TGN marker) and Rab11. We have also found that SIMPLE knockdown impeded recycling of transferrin and of transferrin receptor. Our overall results indicate that SIMPLE may regulate protein trafficking physiologically by localizing to the TGN and/or REs by binding PI4P.

Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V

Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46.0% (w/v) or 31.5% (w/v) based on weight. After 0.7% (w/v) deoxycholic acid, 0.4% (w/v) decyl maltoside, and 7.2% (w/v) potassium chloride were added to the mitochondrial membranes, those membranes were solubilized. At a membrane concentration of 46%, complex V was selectively solubilized, whereas at a concentration of 31.5% (w/v), complexes I and III were solubilized. Two steps—sucrose density gradient centrifugation and anion-exchange chromatography on a POROS HQ 20 μm column—enabled selective purification of samples that retained their structure and functions. These two steps enabled complexes I, III, and V to be purified in two days with a high yield. Complexes I, III, and V were stabilized with n-decyl-β-D-maltoside. A total of 200 mg–300 mg of those complexes from one bovine heart (1.1 kg muscle) was purified with good reproducibility, and the complexes retained the same functions they possessed while in mitochondrial membranes.