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PubMed1989

Ultrastructure, serogrouping and localization of surface antigens of Bacteroides intermedius.

The surface ultrastructure of 21 strains of Bacteroides intermedius was investigated by electron microscopy. Rat monoclonal antibodies (mAbs) were used to define serogroups and to detect the location of surface antigens. All 21 isolates had capsules as demonstrated by the use of wet and dry Indian ink stains. Negative staining of whole cells with 1% (w/v) methylamine tungstate showed that all 21 isolates carried clumped peritrichous fibrils with strain dependent morphology, density and length (less than or equal to 0.75 micron). Fibrils on 11 of 13 fresh clinical isolates were more conspicuously clumped and easily visible, whereas those on 6 of 8 laboratory strains were indistinct and were at the limits of the resolution of the negative staining technique. Staining with ruthenium red (RR), followed by thin sectioning, revealed a dense, amorphous RR staining layer (RRL), up to 24.8 +/- 3.0 nm thick, adjacent to the outer membrane on all of 15 strains examined. All isolates had a less dense RR staining matrix (RRM) extending away from the RRL. The structure of the RRM varied between strains. Four rat mAbs (37BI6.1, 38BI1, 39BI1.1 and 40BI3.2) were used to serogroup the 21 strains of B. intermedius. Immunonegative staining revealed that the mAbs were not directed against fibrilis. Antigens recognized by mAb 37BI6.1 and mAb 39BI1.1 were located on the surfaces of cells, beneath fibrils, and on extracellular vesicles. mAb 38BI1 recognized an antigen which was most accessible on lysed cells, and non-specific binding of mAb 40BI3.2 to grids prevented its localization on the cell surface.

Antigens, Bacterial↗

PubMed1991

The hemagglutinating adhesin HA-Ag2 of Bacteroides gingivalis is distinct from fimbrilin.

We carried out a series of immunoblots with antigenic preparations from the periodontal pathogen Bacteroides gingivalis using antisera of restricted specificity for the hemagglutinating adhesin HA-Ag2, and for the major structural subunit of the fimbriae (fimbrilin). We have been able to show that these 2 antigens are distinct. The fimbrilin subunit had an apparent molecular weight of 42 kDa in all of the bacterial preparations tested. HA-Ag2 occurred as a pair of bands at 43 and 49 kDa in outer membranes prepared as extracellular vesicles, and at 33 and 38 kDa in glass-bead-EDTA extracted antigens and in sheared-cell outer membranes prepared in the presence of EDTA. No HA-Ag2 was found in an enriched fimbrial preparation. The 2 antigens could thus be distinguished on the basis of their behaviour when subjected to different extraction techniques. The lower apparent molecular weight of HA-Ag2 (a pair of bands at 33 and 38 kDa) was invariably associated with the presence of EDTA in the buffers used to prepare the extracts, and the effect could be partially prevented by adding MgCl2 to the extraction buffer. The difference in apparent molecular weight of HA-Ag2 in the different extracts can thus be attributed either to an EDTA-sensitive tertiary conformation of its component polypeptides, or to an EDTA-sensitive linkage of each of these polypeptides to an unknown component of approximately 10 kDa.

Adhesins, Bacterial↗

PubMed1994

Selective growth inhibition of Porphyromonas gingivalis by bestatin.

Recent work in our laboratory indicates that selected protease/peptidase inhibitors interfere with the growth of Porphyromonas gingivalis. The aim of the present study was to further investigate the inhibitory effect of bestatin on the growth of P. gingivalis. Complete growth inhibition of P. gingivalis (11 strains) was observed when bestatin was incorporated at 2.5 micrograms ml-1 in a complex broth medium. Fifty percent inhibition was still obtained with bestatin at a final concentration of 0.5 microgram ml-1. The inhibitory effect of bestatin was highly specific as the growth of 20 different oral bacterial species, including Gram-positive and Gram-negative as well as saccharolytic and asaccharoltic bacteria, was not affected even at bestatin concentrations up to 50 micrograms ml-1. Bestatin did not significantly affect the viability of P. gingivalis indicating that it has a bacteriostatic rather than a bactericidal effect. Growth assays using other specific inhibitors suggested that the effect of bestatin on the growth of P. gingivalis was unlikely to be related to its aminopeptidase inhibitor activity. Cultivation of P. gingivalis with a subinhibitory concentration of bestatin did not modify the cell envelope protein profile, as determined by SDS-PAGE analysis, but significantly decreased the number of extracellular vesicles produced. The present study indicated that bestatin is a highly effective inhibitor of cell growth of P. gingivalis. Additional studies will indicate whether bestatin should be considered as a potential drug in the control of P. gingivalis, a suspected pathogen in adult chronic periodontitis.

Anti-Bacterial Agents↗

PubMed1995

Immunological characterization and localization of a Porphyromonas gingivalis BApNA-hydrolyzing protease possessing hemagglutinating activity.

A monoclonal antibody (mAb-PC) was produced against a BA pNA-hydrolyzing protease possessing hemagglutinating activity (Pase-C) from Porphyromonas gingivalis. Other P. gingivalis BA pNA-hydrolyzing enzymes (Pase-B and Pase-S) did not react with this antibody. By ELISA or SDS-PAGE and Western immunoblotting analysis, mAb-PC recognized all P. gingivalis and P. endodontalis strains tested but did not recognize other members of the Porphyromonas genus nor other putative periodontopathogenic organisms. Pase-C, extracellular vesicles (ECV) and human strains of P. gingivalis showed two major immunoreactive bands (44 kDa and 40 kDa), whereas a different pattern was obtained with animal strains of P. gingivalis. Biotinylarginyl chloromethane, an irreversible inhibitor of trypsin-like proteases, did not affect the reactivity of Pase-C with mAb-PC on immunoblot. By reversed-phase electronmicroscopy following immunogold labeling, the antibody was shown to bind to the cell surface of P. gingivalis. mAb-PC inhibited the hemagglutinating activity of both P. gingivalis cells and ECV whereas a monoclonal antibody against LPS of P. gingivalis did not. These results suggest that Pase-C is located on the cell surface of P. gingivalis and may participate in erythrocyte binding.

Animals↗

PubMed1999

Cellular localization of a Hsp90 homologue in Porphyromonas gingivalis.

We previously reported an association between elevated serum antibody titers to the 90-kDa human heat shock protein (Hsp90), periodontal health and colonization by Porphyromonas gingivalis. In this study, we examined the cellular localization of the Hsp90 homologue of P. gingivalis. Cultures of P. gingivalis were heat-stressed (45 degrees C) and examined for localization of the Hsp90 homologue. Heat stress induced a 4-5-fold increase in anti-Hsp90 antibody reactivity over that of the unstressed controls. Western blot analysis revealed two bands (44 and 68 kDa) that reacted with anti-Hsp90 antibodies. The 68-kDa band was heat-inducible, while the 44-kDa band was not. Immunogold staining revealed that the Hsp90 homologue localized principally to the membrane and extracellular vesicles. Subcellular fractionation confirmed that the Hsp90 homologue was primarily membrane-associated.

Blotting, Western↗

PubMed2006

Effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of Bacteroides fragilis.

The effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of species of the Bacteroides fragilis group isolated from children with diarrhea were determined. The minimal inhibitory and subinhibitory concentrations for clindamycin were determined. Minimal inhibitory concentration values ranging from 0.25 to 512 microg mL(-1) were observed. Cultures grown with clindamycin were used to determine the macroscopic morphological characteristics, cellular viability, ultrastructural characteristics and DNA integrity. Clindamycin did not alter colonial morphology, but after 6 h elongated cells were observed. Also, extracellular vesicles and electron-lucent areas inside the cytoplasm were observed. Bacteria treated with clindamycin also showed fragmentation of DNA as determined by electrophoresis. The alterations produced by clindamycin might be indicative of a possible modification of the structures involved in bacterial pathogenesis.

Anti-Bacterial Agents↗

PubMed1989

Surface properties and ultrastructure of Porphyromonas gingivalis W50 and pleiotropic mutants.

Cell surface ultrastructure and other surface properties of Porphyromonas gingivalis strain W50 and pleiotropic mutants W50/BP1 (brown), and W50/BE1 (beige) were studied. The percentage hydrophobicity of strains W50, W50/BP1, W50/BR1, and W50/BE1 gradually decreased from 24 to 9. Ruthenium red stained cells studied by transmission electron microscopy revealed a layer of extracellular polymeric material of varying thickness depending on the strain. The layer was thickest in W50/BP1 (15-20 nm), strains W50 and W50/BR1 both had a layer of 12-15 nm, while strain W50/BE1 completely lacked this layer. The results clearly showed that the hydrophobicity of P. gingivalis was related not only to the thickness of the layer but also to other factors like the composition of the capsular material, such that only strain W50/BE1, for example, showed no haemagglutinating activity. The surface properties of the pleiotropic mutants appeared to be stable characteristics as cells grown on either solid or in liquid media gave comparable results. The loss of virulence of the beige strain (W50/BE1) is probably partly due to the alteration of these surface properties. Both virulent and avirulent strains, however, possessed extracellular vesicles.

Bacteroides↗

PubMed2026

Defective EV-mediated transport of SHH alters neural fate specification in EPM1 epilepsy.

The extracellular milieu, including extracellular vesicles (EVs), plays a pivotal role in brain development. In this study, we sought to elucidate the pathogenesis of progressive myoclonus epilepsy type 1 (EPM1), a disease caused by mutations in the CSTB gene, using cerebral organoids (COs) derived from patient cells. The results demonstrate that EPM1 COs display increased electrophysiological activity and disrupted excitatory/inhibitory (E/I) balance. Single-cell RNA sequencing analysis of ventral EPM1-COs revealed an abnormal specification of progenitor fate, with a shift toward dorsal neuron identities. We demonstrated that this misspecification is driven by a functional alteration of the ventral signaling niche, resulting from impaired EV dynamics and altered protein cargo. Mechanistically, we identified Sonic Hedgehog (SHH) as a direct physical interactor of CSTB and demonstrated that CSTB deficiency leads to reduced SHH content and secretion. Our findings establish CSTB as a safeguard of ventral patterning and identify the CSTB-SHH-EV axis as a potential therapeutic target for mitigating the E/I imbalance associated with EPM1.

Hedgehog Proteins↗

PubMed2025

Stereo-cell: Spatial enhanced-resolution single-cell sequencing with high-density DNA nanoball-patterned arrays.

Single-cell sequencing technologies have advanced our understanding of cellular heterogeneity and biological complexity. However, existing methods face limitations in throughput, capture uniformity, cell size flexibility, and technical extensibility. We present Stereo-cell, a spatial enhanced-resolution single-cell sequencing platform based on high-density DNA nanoball (DNB)-patterned arrays, which enables scalable and unbiased cell capture at a wide input range and supports high-fidelity transcriptome profiling. Stereo-cell further allows integration with imaging-based modalities and multiomics strategies, including immunofluorescence and epitope profiling. This platform is also compatible with profiling extracellular vesicles, microstructures, and large cells, whereas its spatial resolution facilitates in situ analysis of cell-cell interactions, cellular microenvironments, and subcellular transcript localization. Together, Stereo-cell provides a flexible framework for expanding single-cell research applications.

Animals↗

PubMed1987

Properties of Desulfovibrio carbinolicus sp. nov. and Other Sulfate-Reducing Bacteria Isolated from an Anaerobic-Purification Plant.

Several sulfate-reducing microorganisms were isolated from an anaerobic-purification plant. Four strains were classified as Desulfovibrio desulfuricans, Desulfovibrio sapovorans, Desulfobulbus propionicus, and Desulfovibrio sp. The D. sapovorans strain contained poly-beta-hydroxybutyrate granules and seemed to form extracellular vesicles. A fifth isolate, Desulfovibrio sp. strain EDK82, was a gram-negative, non-spore-forming, nonmotile, curved organism. It was able to oxidize several substrates, including methanol. Sulfate, sulfite, thiosulfate, and sulfur were utilized as electron acceptors. Pyruvate, fumarate, malate, and glycerol could be fermented. Because strain EDK82 could not be ascribed to any of the existing species, a new species, Desulfovibrio carbinolicus, is proposed. The doubling times of the isolates were determined on several substrates. Molecular hydrogen, lactate, propionate, and ethanol yielded the shortest doubling times (3.0 to 6.3 h). Due to the presence of support material in an anaerobic filter system, these species were able to convert sulfate to sulfide very effectively at a hydraulic retention time as short as 0.5 h.

Journal Article↗

PubMed2004

Ultrastructural alterations of Erwinia carotovora subsp. atroseptica caused by treatment with aluminum chloride and sodium metabisulfite.

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO2 with little damage to the cellular membrane.

Aluminum Chloride↗

PubMed1995

Characterization of the human immunoglobulin G Fc-binding activity in Prevotella intermedia.

Many pathogenic bacteria possess cell surface receptors which can bind immunoglobulins via the Fc portion. The aim of this study was to characterize the human immunoglobulin G (IgG) Fc-binding activity of Prevotella intermedia, a suspected etiologic agent of adult chronic periodontitis. The Fc-binding activity of P. intermedia on whole cells and on extracellular vesicles was demonstrated. Incubation of P. intermedia cells in the presence of Zwittergent 3-14 allowed complete solubilization of the Fc receptor from the cell surface. This cell envelope extract was thus used to characterize the Fc-binding activity. A microtiter plate assay using alkaline phosphatase-labeled Fc fragments showed that preincubation of the cell envelope extract with human IgG, human IgG Fc fragments, or human serum completely inhibited the Fc-binding activity. Partial inhibition was obtained with human IgG F(ab')2 fragments, whereas no inhibition occurred following preincubation with human IgA, carbohydrates, and selected proteins. Preincubation of the cell envelope extract with IgG from a variety of animals demonstrated that rabbit, mouse, rat, goat, and sheep IgG did not inhibit Fc-binding activity, whereas cow, pig, and dog IgG partially inhibited Fc-binding activity. A strong inhibition comparable to that obtained with human IgG was noted with monkey IgG. The Fc receptor of P. intermedia is thus different from the six types previously reported in other nonoral bacteria. Polyacrylamide gel electrophoresis and Western blotting (immunoblotting) analysis of the cell envelope extract revealed a major band with a molecular mass of approximately 65 kDa which reacted with peroxidase-labeled human IgG Fe fragments. Transmission electron microscopy showed a uniform distribution of the Fc receptor on the bacterial surface, as revealed by gold labeling. The Fc-binding activity demonstrated in this study may act as an additional virulence factor for P. intermedia by reducing IgG reactions with the bacterial cell.

Bacterial Proteins↗

PubMed1990

Surface-active novel glycolipid and linked 3-hydroxy fatty acids produced by Serratia rubidaea.

A Serratia rubidaea isolate with wetting activity when grown at 30 but not 37 degrees C was examined for the production of specific lipids. Two novel lipids (rubiwettins R1 and RG1) were isolated and shown to be able to lower the surface tension of saline to 26 mN/m. These lipids were located in extracellular vesicles found in a 30 degrees C culture of S. rubidaea. Chemical structures of these biosurfactants were determined by degradation product analyses, infrared spectroscopy, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Rubiwettin R1 was proposed to be a mixture of 3-(3'-hydroxytetradecanoyloxy)decanoate, 3-(3'-hydroxyhexadecenoyloxy)decanoate, and minor molecular isomers. The structure of rubiwettin RG1 was proposed to be beta-D-glucopyranosyl 3-(3'-hydroxytetradecanoyloxy)decanoate. The importance of such surface-active exolipids in bacterial occupancy on surfaces was suggested.

Chromatography, Thin Layer↗

PubMed2026

Secreted protein circuits in the gastrointestinal tumour microenvironment: determinants of immunotherapy response and resistance.

Immune checkpoint blockade has transformed treatment in selected gastrointestinal (GI) cancers, yet primary resistance, incomplete responses and acquired resistance remain common. This heterogeneity is not explained by tumour-cell genomics alone; extracellular signalling programmes within the tumour microenvironment can determine immune recruitment, access and adaptation to therapy. The tumour secretome-including cytokines, chemokines, growth factors, complement components, matricellular proteins, soluble checkpoint molecules and extracellular-vesicle-associated cargo-regulates immune-cell recruitment, exclusion, suppression, tertiary lymphoid structure formation and exhaustion across anatomical and molecular contexts. Across gastric and esophageal cancers, colorectal cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma and biliary tract cancers, recurrent suppressive circuits include TGF-β, VEGF, CXCL12-CXCR4, CXCL8/IL-8-CXCR1/2, CCL2-CCR2, CSF1-CSF1R, IL-6-family cytokines, SPP1/osteopontin, periostin, galectins, DKK1, MIF, complement and soluble or vesicular PD-L1. Conversely, CXCL9/10/11-CXCR3 signalling and CXCL13-associated tertiary lymphoid structures characterise immune-permissive states that can support checkpoint responsiveness. We organise these circuits into four overlapping functional modules-myeloid-enriched, fibroblast-driven exclusion, angiogenic-immunosuppressive and immune-permissive-and apply a four-level evidence hierarchy that separates clinical validation from mechanistic inference. Clinically useful secretome biomarkers will therefore need to integrate cellular source, spatial localisation, receptor context, temporal dynamics and linkage to actionable immune-state transitions.

Humans↗

PubMed1990

Invasive growth of Actinobacillus actinomycetemcomitans on solid medium (TSBV).

When grown on agar, most Actinobacillus actinomycetemcomitans form circular and convex colonies with an internal star-shaped morphology. Such colonies adhere firmly to the agar, and when removed, a star-shaped imprint similar to that of the intact colony remains. This study was undertaken to determine the nature of this in vitro growth. Stereo-microscopy and scanning and transmission electron microscopy showed that the star-shaped imprint of A. actinomycetemcomitans colonies reflected pseudopod-like extensions of the bacterial colony which penetrated deep into the TSBV agar. The center of the colonies consisted primarily of ghost-like cells, while the dense border of each colony, including the pseudopods, expressed characteristics of vital cells. The latter were embedded in vast amounts of extracellular vesicles, and the outer aspect of the pseudopods was lined with a border of such vesicles.

Actinobacillus↗

PubMed1989

The effects of occlusive dressings on inflammation and granulation tissue formation in excised wounds in rats.

The effects on the healing of full-thickness excisional wounds treated with either of two occlusive dressings (Mezinc or Duoderm) were compared with the effects of gauze soaked in saline. The wounds were made on 86 rats and were examined clinically, histologically and biochemically four, eight and twelve days after wounding. Four days postoperatively the Duoderm-treated wounds differed significantly from the other two groups. Clinically, an adherent discolored gelatinous mass remained after removal of the firm part of the Duoderm dressing. Histologically it corresponded to a superficial exudate containing polymorphonuclear leukocytes (PMNs), macrophages and condensed foreign material. There was also a more extensive inflammatory reaction in the underlying tissues compared with gauze or Mezinc treatment and debris was seen in vesicles extracellularly and in foamy macrophages. Foamy macrophages were only seen in the Duoderm-treated wounds. These macrophages were mainly confined to the granulation tissue, which was about twice as thick as in the other two treatment groups twelve days after excision.

Animals↗

PubMed2026

Single-section multiplex spatial proteomics of immune microenvironments in kidney transplantation.

Characterizing kidney disease is challenged by marked cellular heterogeneity and limited tissue availability from renal biopsies. Conventional diagnostic workflows rely on multiple serial sections for parallel staining, increasing tissue consumption, sampling bias, and loss of spatial information, thereby constraining molecular characterization within intact tissue architecture. High-plex spatial proteomics may overcome these limitations by enabling comprehensive molecular profiling on a single section. Here, we present and evaluate a high-plex cyclic immunofluorescence imaging workflow (MACSima™, Miltenyi Biotec) applied to kidney transplant biopsies, including BK virus nephropathy (BKVN) and focal segmental glomerulosclerosis (FSGS), to characterize spatial immune organization with a focus on complement system components. Feasibility and subcellular resolution were first assessed in a lupus nephritis section, demonstrating compatibility with diagnostic immune panels and preservation of tissue morphology. A 48-marker multiplex panel interrogating immunity, oxidative stress, senescence, and fibrosis was then applied to BKVN samples, including paired pre- and post-treatment biopsies, revealing distinct proteomic patterns and dynamic changes following therapy. In FSGS, a glomerulus-focused panel identified spatially resolved innate and adaptive immune signatures, including complement-related patterns supporting exploratory analysis of glomerular immune architecture. Structural, nuclear, membrane, and phosphorylated signaling markers enabled precise delineation of renal compartments and assessment of cellular states such as proliferation, DNA damage, and pathway activation. The workflow also supported detection of extracellular vesicles in cultured renal cells, highlighting its versatility. Overall, this approach provides a robust, tissue-sparing platform for integrated spatial and molecular profiling of renal biopsies, reducing sampling bias while enabling discovery-level phenotyping from a single section. This unified strategy is particularly suited to kidney transplantation, where diagnosis, therapeutic decision-making, and longitudinal monitoring are closely interconnected.

Kidney Transplantation↗

PubMed2022

An Exosomal miRNA Biomarker for the Detection of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains a difficult tumor to diagnose and treat. To date, PDAC lacks routine screening with no markers available for early detection. Exosomes are 40-150 nm-sized extracellular vesicles that contain DNA, RNA, and proteins. These exosomes are released by all cell types into circulation and thus can be harvested from patient body fluids, thereby facilitating a non-invasive method for PDAC detection. A bioinformatics analysis was conducted utilizing publicly available miRNA pancreatic cancer expression and genome databases. Through this analysis, we identified 18 miRNA with strong potential for PDAC detection. From this analysis, 10 (MIR31, MIR93, MIR133A1, MIR210, MIR330, MIR339, MIR425, MIR429, MIR1208, and MIR3620) were chosen due to high copy number variation as well as their potential to differentiate patients with chronic pancreatitis, neoplasms, and PDAC. These 10 were examined for their mature miRNA expression patterns, giving rise to 18 mature miRs for further analysis. Exosomal RNA from cell culture media was analyzed via RTqPCR and seven mature miRs exhibited statistical significance (miR-31-5p, miR-31-3p, miR-210-3p, miR-339-5p, miR-425-5p, miR-425-3p, and miR-429). These identified biomarkers can potentially be used for early detection of PDAC.

Humans↗