CYPOPATHIC EFFECT OF DIPHTHERIA PATHOGEN IN THE COMPOSITION OF BIOFILM

When the nasopharynx is colonized with toxigenic strains of the diphtheria pathogen, toxin is released, which contributes to the death of epithelial cells. But in bacterial carriers, the development of the clinical picture of the disease does not occur. This is due to the peculiarities of the state of their immune system, as well as the peculiarities of the production of diphtheria exotoxin by corynebacteria in the biofilm. Goal. Determining the nature of the cytopathic effect of C. diphtheriae as part of a biofilm in CHO-K1 cell culture. The planktonic and biofilm (120- and 720-hour) cultures of the strains were studied: C. diphtheriae gravis tox+ № 665, C. diphtheriae gravis tox+ № 6765, C. diphtheriae mitis tox+ № 269, C. diphtheriae gravis tox+ isolated from a patient with a diagnosis Localized oropharyngeal diphtheria C. diphtheriae gravis with a silent tox-gene. Biofilm (120- and 720-hour) cultures of diphtheria pathogen strains were obtained according to the Watnik method. The cytopathic effect of corynebacterial strains was studied on a CHO-K1 cell culture, taking into account in an inverted microscope. When studying the cytopathic effect of planktonic cultures of toxigenic strains of corynebacteria, it was found that the number of living CHO-K1 cells after 24 hours was insignificant (25.3±1.2%) and sharply decreased (2.5±0.5%) after 72 hours of cultivation. Under the influence of biofilm and, especially, 720-hour cultures, a different cytopathic effect dynamics was found: the number of living cells after 24 hours remained significant (82.5±2.2%), while at 72-hour it decreased to 25.0±3.0%. In the study of filtrates of planktonic and biofilm cultures of C. diphtheriae strain with a «silent» tox-gene, similar patterns were revealed. However, the number of live CHO-K1 cells when exposed to the filtrate of a 720-hour biofilm culture was significantly higher (p≤0.05) than when studying toxigenic strains of corynebacteria. Considering the nature of the cytopathic action, it was found that planktonic cultures of toxigenic strains of corynebacteria are characterized by a change in the cell monolayer, manifested by their thinning and elongation. The study of 720-hour biofilm cultures at 72-hour exposure revealed the appearance of a large number of rounded cells (63-69%). The cytopathic effect, formed under the influence of filtrates of planktonic and biofilm cultures of C. diphtheriae with a «silent» tox-gene, as well as strains of non-diphtheria corynebacteria, is characterized by rounding of cells and the formation of symplasts. In the biofilm, the intensity of the cytopathic effect of toxigenic C. diphtheriae strains and C. diphtheriae strain with a silent tox-gene decreased. CPD, manifested by thinning and lengthening of CHO-K1 cells, is associated with the action of diphtheria exotoxin, and rounding is associated with corynebacterial enzymes and, apparently, fragments of surface structures - adhesins. Decreased release of toxin and enzymes beyond the C. bihfilm matrix is a significant cause of the «asymptomatic» carriage of diphtheria.

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Real-time monitoring of liver fibrosis through embedded sensors in a microphysiological system

Nano Convergence ◽

10.1186/s40580-021-00253-y ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Hafiz Muhammad Umer Farooqi ◽

Bohye Kang ◽

Muhammad Asad Ullah Khalid ◽

Abdul Rahim Chethikkattuveli Salih ◽

Kinam Hyun ◽

...

Keyword(s):

Cell Culture ◽

Liver Fibrosis ◽

Real Time ◽

Hepatic Fibrosis ◽

Transforming Growth Factor ◽

Cell Monolayer ◽

Embedded Sensors ◽

Matrix Deposition ◽

Tgf Β1

AbstractHepatic fibrosis is a foreshadowing of future adverse events like liver cirrhosis, liver failure, and cancer. Hepatic stellate cell activation is the main event of liver fibrosis, which results in excessive extracellular matrix deposition and hepatic parenchyma's disintegration. Several biochemical and molecular assays have been introduced for in vitro study of the hepatic fibrosis progression. However, they do not forecast real-time events happening to the in vitro models. Trans-epithelial electrical resistance (TEER) is used in cell culture science to measure cell monolayer barrier integrity. Herein, we explored TEER measurement's utility for monitoring fibrosis development in a dynamic cell culture microphysiological system. Immortal HepG2 cells and fibroblasts were co-cultured, and transforming growth factor β1 (TGF-β1) was used as a fibrosis stimulus to create a liver fibrosis-on-chip model. A glass chip-based embedded TEER and reactive oxygen species (ROS) sensors were employed to gauge the effect of TGF-β1 within the microphysiological system, which promotes a positive feedback response in fibrosis development. Furthermore, albumin, Urea, CYP450 measurements, and immunofluorescent microscopy were performed to correlate the following data with embedded sensors responses. We found that chip embedded electrochemical sensors could be used as a potential substitute for conventional end-point assays for studying fibrosis in microphysiological systems.

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Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.36.8.2178-2182.1998 ◽

1998 ◽

Vol 36 (8) ◽

pp. 2178-2182 ◽

Cited By ~ 237

Author(s):

Haru Kato ◽

Naoki Kato ◽

Kunitomo Watanabe ◽

Naoichi Iwai ◽

Haruhi Nakamura ◽

...

Keyword(s):

Cell Culture ◽

Clostridium Difficile ◽

Pcr Amplification ◽

Enzyme Linked Immunosorbent Assay ◽

Pcr Assay ◽

Toxin A ◽

Toxin B ◽

Cell Monolayers ◽

Toxigenic Strains ◽

Cell Culture Assay

Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A−, toxin B−) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A−, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A−, toxin B− strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A−, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A−, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A−, toxin B+ strains from toxin A+, toxin B+ and toxin A−, toxin B− strains and will contribute to elucidation of the precise role of toxin A−, toxin B+ strains in intestinal diseases.

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Glass beads load macromolecules into living cells

Journal of Cell Science ◽

10.1242/jcs.88.5.669 ◽

1987 ◽

Vol 88 (5) ◽

pp. 669-678

Author(s):

P.L. McNeil ◽

E. Warder

Keyword(s):

Culture Medium ◽

Small Volume ◽

Cultured Cells ◽

Cell Monolayer ◽

Cell Loss ◽

Living Cells ◽

Glass Beads ◽

Cell Type ◽

Large Numbers ◽

Micron Diameter

We describe and characterize an exceptionally rapid and simple new technique for loading large numbers of cultured cells with large macromolecules. The culture medium of the cell monolayer is replaced by a small volume of the macromolecule to be loaded. Glass beads (75–500 micron diameter) are then sprinkled onto the cells, the cells are washed free of beads and exogenous macromolecules, and ‘bead-loading’ is completed. The conditions for bead-loading can readily be modified to accommodate cell type and loading objectives: for example, the amount of loading per cell increases if bead size is increased or if beads are agitated after sprinkling onto the monolayer, but at the expense of increased cell loss. As many as 97% of a population of bovine aortic endothelial (BAE) cells were loaded with a 10,000 Mr dextran; and 79% with a 150,000 Mr dextran using bead-loading. Various cell lines have been loaded using glass beads. Moreover, bead-loading has the advantage of producing loaded cells that remain adherent and well-spread, thus minimizing recovery time and permitting immediate microscopic examination.

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Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenicEscherichia colito bladder epithelial cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801564115 ◽

2018 ◽

Vol 115 (40) ◽

pp. 10106-10111 ◽

Cited By ~ 16

Author(s):

Emily C. Hollenbeck ◽

Alexandra Antonoplis ◽

Chew Chai ◽

Wiriya Thongsomboon ◽

Gerald G. Fuller ◽

...

Keyword(s):

Cell Surface ◽

Bacterial Cell ◽

Cell Monolayer ◽

Surface Structures ◽

Bacterial Cell Surface ◽

Cell Surface Structures ◽

Bladder Cell ◽

Type 1 Pili ◽

Tract Infections

UropathogenicEscherichia coli(UPEC) are the major causative agents of urinary tract infections, employing numerous molecular strategies to contribute to adhesion, colonization, and persistence in the bladder niche. Identifying strategies to prevent adhesion and colonization is a promising approach to inhibit bacterial pathogenesis and to help preserve the efficacy of available antibiotics. This approach requires an improved understanding of the molecular determinants of adhesion to the bladder urothelium. We designed experiments using a custom-built live cell monolayer rheometer (LCMR) to quantitatively measure individual and combined contributions of bacterial cell surface structures [type 1 pili, curli, and phosphoethanolamine (pEtN) cellulose] to bladder cell adhesion. Using the UPEC strain UTI89, isogenic mutants, and controlled conditions for the differential production of cell surface structures, we discovered that curli can promote stronger adhesive interactions with bladder cells than type 1 pili. Moreover, the coproduction of curli and pEtN cellulose enhanced adhesion. The LCMR enables the evaluation of adhesion under high-shear conditions to reveal this role for pEtN cellulose which escaped detection using conventional tissue culture adhesion assays. Together with complementary biochemical experiments, the results support a model wherein cellulose serves a mortar-like function to promote curli association with and around the bacterial cell surface, resulting in increased bacterial adhesion strength at the bladder cell surface.

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Associated Anisotropy of intrinsic NAD(P)H for monitoring changes in the metabolic activities of breast cancer cells in three-dimensional collagen matrix

Physical Chemistry Chemical Physics ◽

10.1039/d0cp06635d ◽

2021 ◽

Author(s):

Anh Cong ◽

Rafaela M. L. Pimenta ◽

Jon Holy ◽

Ahmed A Heikal

Keyword(s):

Breast Cancer ◽

Cell Culture ◽

Breast Cancer Cells ◽

Monolayer Culture ◽

Three Dimensional ◽

Collagen Matrix ◽

Living Cells ◽

Two Dimensional ◽

Metabolic Activities

The majority of in vitro studies of living cells are routinely conducted in a two-dimensional (2D) monolayer culture. Recent studies, however, suggest that 2D cell culture promotes specific types of...

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A patient with respiratory toxigenic diphtheria in Greece after more than 30 years

Epidemiology and Infection ◽

10.1017/s0950268820002605 ◽

2020 ◽

Vol 148 ◽

Author(s):

T. Georgakopoulou ◽

K. Tryfinopoulou ◽

A. Doudoulakakis ◽

F. Nikolaou ◽

I. Magaziotou ◽

...

Keyword(s):

Health Management ◽

Asymptomatic Carrier ◽

Fatal Outcome ◽

Asymptomatic Carriage ◽

Laboratory Confirmation ◽

Toxigenic Strains ◽

Susceptible Populations ◽

High Level ◽

Close Contacts

Abstract The introduction of treatment and systematic vaccination has significantly reduced diphtheria mortality; however, toxigenic strains continue to circulate worldwide. The emergence of an indigenous diphtheria case with fatal outcome in Greece, after 30 years, raised challenges for laboratory confirmation, clinical and public health management. Toxigenic Corynebacterium diphtheriae was isolated from an incompletely vaccinated 8-year-old boy with underlying conditions. The child passed away due to respiratory distress syndrome, before the administration of diphtheria antitoxin (DAT). All close contacts in family, school and hospital settings were investigated. Pharyngeal swabs were obtained to determine asymptomatic carriage. Chemoprophylaxis was given for 7 days to all close contacts and a booster dose to those incompletely vaccinated. Testing revealed a classmate, belonging to a subpopulation group (Roma), and incompletely vaccinated, as an asymptomatic carrier with an indistinguishable toxigenic strain (same novel multilocus sequence type, designated ST698). This case highlights the role of asymptomatic carriage, as the entry of toxigenic strains into susceptible populations can put individuals and their environment at risk. Maintenance of high-level epidemiological and microbiological surveillance, implementation of systematic vaccination in children and adults with primary and booster doses, availability of a DAT stockpile, and allowing timely administration are the cornerstone to prevent similar incidents in the future.

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Experiments in Nanomechanical Properties of Live Osteoblast Cells and Cell–Biomaterial Interface

Journal of Nanotechnology in Engineering and Medicine ◽

10.1115/1.4005666 ◽

2011 ◽

Vol 2 (4) ◽

Cited By ~ 8

Author(s):

Rohit Khanna ◽

Kalpana S. Katti ◽

Dinesh R. Katti

Keyword(s):

Cell Culture ◽

Culture Medium ◽

Cell Structure ◽

Living Cells ◽

Actin Stress Fiber ◽

Cell Culture Medium ◽

Nanomechanical Properties ◽

Cell Substrate ◽

Significant Difference

Characterizing the mechanical characteristics of living cells and cell–biomaterial composite is an important area of research in bone tissue engineering. In this work, an in situ displacement-controlled nanoindentation technique (using Hysitron Triboscope) is developed to perform nanomechanical characterization of living cells (human osteoblasts) and cell–substrate constructs under physiological conditions (cell culture medium; 37 °C). In situ elastic moduli (E) of adsorbed proteins on tissue culture polystyrene (TCPS) under cell culture media were found to be ∼4 GPa as revealed by modulus mapping experiments. The TCPS substrates soaked in cell culture medium showed significant difference in surface nanomechanical properties (up to depths of ∼12 nm) as compared to properties obtained from deeper indentations. Atomic force microscopy (AFM) revealed the cytoskeleton structures such as actin stress fiber networks on flat cells which are believed to impart the structural integrity to cell structure. Load-deformation response of cell was found to be purely elastic in nature, i.e., cell recovers its shape on unloading as indicated by linear loading and unloading curves obtained at 1000 nm indentation depth. The elastic response of cells is obtained during initial cell adhesion (ECell, 1 h, 1000 nm = 4.4–12.4 MPa), cell division (ECell, 2 days, 1000 nm = 1.3–3.0 MPa), and cell spreading (ECell, 2 days, 1000 nm = 6.9–11.6 MPa). Composite nanomechanical responses of cell–TCPS constructs were obtained by indentation at depths of 2000 nm and 3000 nm on cell-seeded TCPS. Elastic properties of cell–substrate composites were mostly dominated by stiff TCPS (EBulk = 5 GPa) lying underneath the cell.

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Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220954388 ◽

2020 ◽

pp. 247255522095438

Author(s):

Emmanuelle Soleilhac ◽

Marjorie Comte ◽

Anaelle da Costa ◽

Caroline Barette ◽

Christèle Picoli ◽

...

Keyword(s):

Cell Culture ◽

Fluorescent Protein ◽

Transmembrane Proteins ◽

Living Cells ◽

Brain Disorders ◽

Iodide Uptake ◽

Physiological Conditions ◽

Central Nervous System Disorders ◽

Approved Drugs ◽

New Treatments

In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFPQL, by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

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