scholarly journals Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
David Smith ◽  
Geetha Kannan ◽  
Isabelle Coppens ◽  
Fengrong Wang ◽  
Hoa Mai Nguyen ◽  
...  
Keyword(s):  
Animal Health ◽  
Genetic Ablation ◽  
Cellular Processes ◽  
Transmission Electron ◽  
Structural Abnormalities ◽  
Coccidian Parasites ◽  
Brain Cysts ◽  
Mitochondrial Networks

Many of the world's warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including an estimated one third of the global human population. The cellular processes that permit long-term persistence within the cyst are largely unknown for T. gondii and related coccidian parasites that impact human and animal health. Herein we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed numerous structural abnormalities occurring in ∆atg9 bradyzoites. Intriguingly, abnormal mitochondrial networks were observed in TgATG9-deficient bradyzoites, some of which contained numerous different cytoplasmic components and organelles. ∆atg9 bradyzoite fitness was drastically compromised in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.

scholarly journals Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts

2020 ◽  
Author(s):  
David Smith ◽  
Geetha Kannan ◽  
Isabelle Coppens ◽  
Fengrong Wang ◽  
Hoa Mai Nguyen ◽  
...  
Keyword(s):  
Cathepsin L ◽  
Animal Health ◽  
Genetic Ablation ◽  
Cellular Processes ◽  
Membrane Complex ◽  
Coccidian Parasites ◽  
Inner Membrane Complex ◽  
Brain Cysts

ABSTRACTMany of the world’s warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including up to an estimated one third of the global human population. The cellular processes that permit long-term parasite persistence within the cyst are largely unknown, not only for T. gondii but also for related coccidian parasites that impact human and animal health. A previous study revealed an accumulation of autophagic material in the lysosome-like Vacuolar Compartment (VAC) of chronic stage bradyzoites lacking functional cathepsin L protease (TgCPL) activity. Furthermore, it was shown that TgCPL knockout bradyzoites have compromised viability, indicating the turnover of autophagic material could be necessary for bradyzoite survival. However, the extent to which autophagy itself contributes to bradyzoite development and fitness remained unknown. Herein we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed structural abnormalities occurring in Δatg9 bradyzoites, including disorganization of the inner membrane complex and plasma membrane, the occurrence of multiple nuclei within a single bradyzoite cell, as well as various anomalies associated with the VAC. TgATG9-deficient bradyzoites accumulated significantly less undigested material in the VAC upon inhibition of TgCPL activity, suggesting that autophagy contributes material to the VAC for degradation. Intriguingly, abnormal mitochondria networks were observed in TgATG9-deficient bradyzoites. They were thin and elongated and often adopted a horseshoe conformation. Some abnormal mitochondrial structures were found to contain numerous different cytoplasmic components and organelles. Bradyzoite fitness was found to be drastically compromised, both in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.

scholarly journals Hedgehog pathway permissive conditions allow generation of immortal cell lines from granule cells derived from cancerous and non-cancerous cerebellum

Open Biology ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 180145 ◽  
Author(s):  
Constantin Heil
Keyword(s):  
Cell Lines ◽  
Hedgehog Pathway ◽  
Simple Method ◽  
Genetic Ablation ◽  
Neural Lineage ◽  
Shh Pathway ◽  
Pathway Activation

Cerebellar granule cell progenitors (GCPs) undergo proliferation in the post-natal cerebellum that is dependent on sonic hedgehog (SHH) signalling. Deregulated SHH signalling leads to type 2 medulloblastoma (MB). In this work, a novel cell culture protocol is described, which is suitable for the establishment and long-term maintenance of GCP-derived cells. This method is first applied to SHH pathway active MB cells from Atoh1 -cre; Ptch1 FL/FL tumours, which leads to the generation of neurosphere-like cell lines expressing GCP markers and an active SHH signalling pathway. These cells also show high sensitivity to the Smoothened inhibitor vismodegib, therefore recapitulating the SHH pathway requirement for survival shown by type 2 MB. Analysis of culture supplements reveals that bFGF and fetal bovine serum act as inhibitors of the SHH pathway and therefore preclude generation of cell lines that are relevant to the study of the SHH pathway. Consequently, these insights are transferred from the context of MB to non-transformed, post-natal day 7 cerebellum-derived cellular explants. In contrast to other, previously used methods, these GCP cultures proliferate indefinitely and depend on SHH pathway activation, either by means of the small molecule SAG or through genetic ablation of Ptch1 . This culture method therefore leads to the generation of immortal neurosphere-like cell lines, that are named murine SAG-dependent spheres (mSS). Despite long-term culture, mSS cells remain dependent on continuous stimulation of the SHH pathway. Further, mSS cells maintain their lineage after extensive periods in vitro, as demonstrated by their differentiation towards the neural lineage. Herein a simple method for the generation of immortal cell lines from murine cerebella is defined. These lines can be maintained indefinitely through hedgehog pathway activation and maintain the GCP lineage.

scholarly journals Dynamic Nature of Host-Pathogen Interactions inMycobacterium marinum Granulomas

2001 ◽  
Vol 69 (12) ◽  
pp. 7820-7831 ◽  
Author(s):  
Donna M. Bouley ◽  
Nafisa Ghori ◽  
K. Lynne Mercer ◽  
Stanley Falkow ◽  
Lalita Ramakrishnan
Keyword(s):  
Cell Activation ◽  
Morphological Changes ◽  
Tuberculous Infection ◽  
Dynamic Nature ◽  
Leopard Frogs ◽  
Transmission Electron ◽  
Phosphatase Cytochemistry ◽  
Phagocytic Killing

ABSTRACT Mycobacterium marinum causes long-term subclinical granulomatous infection in immunocompetent leopard frogs (Rana pipiens). These granulomas, organized collections of activated macrophages, share many morphological features with persistent human tuberculous infection. We examined organs of frogs with chronicM. marinum infection using transmission electron microscopy in conjunction with immunohistochemistry and acid phosphatase cytochemistry to better define the bacterium-host interplay during persistent infection. Bacteria were always found within macrophage phagosomes. These phagosomes were often fused to lysosomes, in sharp contrast to those formed during in vitro infection of J774 macrophage-like cells by M. marinum. The infected macrophages in frog granulomas showed various levels of activation, as evidenced by morphological changes, including epithelioid transformation, recent phagocytic events, phagolysosomal fusion, and disintegration of bacteria. Our results demonstrate that even long-term granulomas are dynamic environments with regard to the level of host cell activation and bacterial turnover and suggest a continuum between constantly replicating bacteria and phagocytic killing that maintains relatively constant bacterial numbers despite an established immune response. Infection with a mutant bacterial strain with a reduced capacity for intracellular replication shifted the balance, leading to a greatly reduced bacterial burden and inflammatory foci that differed from typical granulomas.

Glucose Dialysate Induces Mitochondrial DNA Damage in Peritoneal Mesothelial Cells

2002 ◽  
Vol 22 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Yoshitaka Ishibashi ◽  
Tokuichiro Sugimoto ◽  
Yasuko Ichikawa ◽  
Akira Akatsuka ◽  
Toshio Miyata ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mitochondrial Dna ◽  
Mitochondrial Membrane ◽  
Atp Content ◽  
Tissue Samples ◽  
Peritoneal Mesothelial Cells ◽  
Mitochondrial Dna Damage ◽  
Transmission Electron

Background It is known that peritoneal mesothelial cells (PMCs) are denuded in patients undergoing long-term continuous ambulatory peritoneal dialysis (CAPD); the mechanism of damage is not well known. A high quantity of glucose loaded onto PMCs in these patients may generate toxic radicals during the mitochondrial metabolism, leading to mitochondrial DNA damage that accumulates due to the incomplete repair system of this DNA. Objective To study damage to the PMCs of long-term CAPD patients, and to examine whether glucose overload accelerates this damage in vitro. Design Descriptive clinical and in vitro study. Participants Stable CAPD patients and nonuremic patients undergoing elective abdominal surgery. Methods ( 1 ) Clinical Samples: 13 peritoneal tissue samples from CAPD patients and 5 omental tissue samples from patients with normal renal function were investigated. PMCs in dialysate effluent were collected from another 13 stable CAPD patients. ( 2 ) In Vitro Samples: Primary cultured PMCs were incubated for up to 144 hours in medium containing one of the following: 5.6 mmol/L glucose (control), 56 mmol/L glucose (G), 222 mmol/L glucose (high G), or 222 mmol/L mannitol (high M; osmolar control for high G). The tissues and cells of clinical and in vitro samples were stained for light and immunoelectron microscopy with anti–8-hydroxy-2'-deoxyguanosine (anti–8-OH-dG) antibody, a marker of oxidative DNA damage. In vitro cells were also studied using transmission electron microscopy. Cellular ATP content, mitochondrial membrane potential, and intracellular generation of reactive oxygen species (ROS) were analyzed by luciferase–luciferin system, or by flow cytometry using rhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Results Biopsy specimens showed strong cytoplasmic staining with 8-OH-dG in patients on long-term CAPD, but only faint staining in patients with end-stage renal disease before the initiation of CAPD, and no staining in patients with normal renal function. Dialysate effluent showed strong granular staining with 8-OH-dG in most PMCs in all long-term CAPD patients, but only faint and focal staining in patients at the start and after 3 – 5 months of CAPD. In vitro experiments also showed strong granular staining by 8-OH-dG in most PMCs cultured in high G, weak staining in G and high M, and no staining in the control. Immunoelectron microscopy revealed the localization of 8-OH-dG to mitochondria. Transmission electron microscopy showed swelling of mitochondria, with decreased cristae, in PMCs cultured in high G. However, only partial expansion of mitochondria was seen in G and high M, and no changes were seen in the control. Cellular ATP content and mitochondrial membrane potential were reduced early, followed by an increase when cultured in high G. Intracellular ROS production was also increased in PMCs cultured in high G and high M. Conclusions These data suggest that high-glucose peritoneal dialysate may promote oxidative mitochondrial DNA damage in PMCs in CAPD patients.

Involvement of extracellular calcium in gastric stimulation

1981 ◽  
Vol 241 (5) ◽  
pp. G365-G375
Author(s):  
C. D. Logsdon ◽  
T. E. Machen
Keyword(s):  
Morphological Changes ◽  
Phosphodiesterase Inhibitor ◽  
Ringer Solution ◽  
Tetraacetic Acid ◽  
Dibutyryl Camp ◽  
Long Term Effects ◽  
Gastric Stimulation ◽  
Transmission Electron

We have tested whether external Ca2+ is required for either initiation or maintenance of secretory parameters, including membrane elaboration of oxyntic cells, in frog gastric mucosa. Ca2+ was removed from in vitro mucosal preparations [by washing repeatedly in Ca2+-free Ringer solution and adding 0.1 mM ethylene glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid to the serosal solution] either before (i.e., resting tissues) or after addition of stimulants. Electrophysiology [transepithelial potential difference (PD) and resistance], morphology (morphometric analysis of transmission electron micrographs), and transport (H+ secretion) were monitored. La3+ (1 mM) was added to the mucosal solution to help maintain resistance and PD. La3+ decreased tissue shunt conductance during Ca2+-free conditions, as evidenced by a decreased mucosal-serosal flux of 22Na+, presumably by preserving tight-junction integrity. Secretion was elicited by histamine alone or in combination with dibutyryl cAMP and isobutylmethylxanthine (a phosphodiesterase inhibitor). External Ca2+ is not required for the initiation of H+ secretion or the accompanying morphological changes when the combined stimulants are used, whereas H+ secretion and the morphological change showed some Ca2+ dependency when histamine alone was used. Thus, histamine-elicited secretion seems to be more sensitive to Ca2+ removal than that brought about by the combined stimulants. Long-term effects of Ca2+-free solutions on resistance, PD, and H+ secretion can largely be explained by disruptive effects on tight junctions.

scholarly journals Genomic Instability Is an Early Event in Aluminium-Induced Tumorigenesis

2020 ◽  
Vol 21 (23) ◽  
pp. 9332
Author(s):  
Stefano J. Mandriota ◽  
Mirna Tenan ◽  
Adeline Nicolle ◽  
Julia D. Jankowska ◽  
Paolo Ferrari ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Genomic Instability ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Aluminium Chloride ◽  
Early Event ◽  
Environmental Carcinogens ◽  
Structural Abnormalities

Genomic instability is generally considered as a hallmark of tumorigenesis and a prerequisite condition for malignant transformation. Aluminium salts are suspected environmental carcinogens that transform mammary epithelial cells in vitro through unknown mechanisms. We report here that long-term culture in the presence of aluminium chloride (AlCl3) enables HC11 normal mouse mammary epithelial cells to form tumours and metastases when injected into the syngeneic and immunocompetent BALB/cByJ strain. We demonstrate that AlCl3 rapidly increases chromosomal structural abnormalities in mammary epithelial cells, while we failed to detect direct modulation of specific mRNA pathways. Our observations provide evidence that clastogenic activity—a well-recognized inducer of genomic instability—might account in part for the transforming abilities of aluminium in mammary epithelial cells.

scholarly journals FORMULATION AND EVALUATION OF SUBMICRON EMULSION CONTAINING ENTRAPPED FLUOROQUINOLONE FOR OCULAR DELIVERY

2018 ◽  
Vol 11 (7) ◽  
pp. 431 ◽  
Author(s):  
Durga Pandey ◽  
Deepti Jain
Keyword(s):  
Drug Release ◽  
Penetrating Trauma ◽  
Spherical Shape ◽  
High Energy ◽  
In Vitro Drug Release ◽  
Transmission Electron ◽  
Sustain Release ◽  
Submicron Emulsion

Objective: The objective of the present work was to develop and characterize the submicron emulsion bearing antimicrobial drug sparfloxacin for improvement of ocular activity by improved retention in eyes. The developed delivery system was results with prolonged drug release as compared to the conventional dosage form.Methods: SE prepared by high energy emulsification and sonication to obtain uniform globule size.Results: Average internal droplets size of the optimized formulation was 0.278±0.6 μm, pH of the optimized formulation was 6.9±0.6 (average of three determinations), and viscosity 2.9±0.5 cps suitable for ocular use. Entrapment of SF was 63±3.4%. Stable under accelerated and long-term at 4°C and 37°C. No major changes reported on pH and viscosity of optimized formulations. In vitro, drug release pattern showed sustain release of SF, a cumulative percent release of SF was found 87.8±1.7% within 24 h. Transmission electron microscopy showed spherical shape and size within 1 μm.Conclusion: Designed formulation can be a good candidate for ocular drug delivery for severe ocular infections where frequent dosing required for conditions such as endophthalmitis, corneal ulcer, and penetrating trauma. 

scholarly journals Preventing Microbial Contamination during Long-Term In Vitro Culture of Human Granulosa-Lutein Cells: An Ultrastructural Analysis

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
C. O. Campos ◽  
M. P. Bernuci ◽  
A. A. Vireque ◽  
J. R. Campos ◽  
M. F. Silva-de-Sá ◽  
...  
Keyword(s):  
Bacterial Contamination ◽  
Microbial Contamination ◽  
High Dose ◽  
Transmission Electron ◽  
Cellular Debris ◽  
Reproductive Techniques ◽  
And Function ◽  
Cell Plating

Purpose. To investigate whether the addition of antibiotic/antimycotic during human granulosa-lutein cells (GLCs) isolation and cell-plating procedures prevents microbial contamination after 144 h of culture and also evaluate the effects of contamination on GLCs ultrastructure and steroid secretion. Methods. GLCs obtained from five women submitted to assisted reproductive techniques (ARTs) were isolated with PBS supplemented with antibiotic/antimycotic or PBS nonsupplemented and cultured for 144 h. GLCs were evaluated by transmission electron microscopy (TEM), and estradiol (E2) and progesterone (P4) secretion was assayed by chemiluminescence. Results. Although no contaminating microorganisms were identified by light microscopy, TEM analyses revealed several bacterial colonies in culture dishes of GLCs isolated with only PBS. Bacterial contamination disrupted the adherence of the GLCs to the culture plate interfering with monolayer formation affecting the growth pattern of GLCs. Various cellular debris and bacteria were observed, and no organelles were found in the cytoplasm of infected cells. While bacterial contamination decreased estradiol media levels, it increased progesterone, as compared with noncontaminated group. Conclusion. Taken together, our data showed that the addition of a high dose of antibiotic/antimycotic during the isolation and cell-plating procedures prevents microbial contamination of long-term GLCs culture as its effects on cells growth and function in vitro.

Maytansine-Induced Mitotic Arrest in Human Lymphoblasts

1977 ◽  
Vol 35 ◽  
pp. 460-461
Author(s):  
Tai-Te Chao ◽  
John Sullivan ◽  
Awtar Krishan
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transmission Electron Microscopy ◽  
Tubulin Polymerization ◽  
Vinca Alkaloids ◽  
Antimitotic Activity ◽  
Transmission Electron ◽  
Flow Microfluorometry ◽  
Brain Tubulin

Maytansine, a novel ansa macrolide (1), has potent anti-tumor and antimitotic activity (2, 3). It blocks cell cycle traverse in mitosis with resultant accumulation of metaphase cells (4). Inhibition of brain tubulin polymerization in vitro by maytansine has also been reported (3). The C-mitotic effect of this drug is similar to that of the well known Vinca- alkaloids, vinblastine and vincristine. This study was carried out to examine the effects of maytansine on the cell cycle traverse and the fine struc- I ture of human lymphoblasts.Log-phase cultures of CCRF-CEM human lymphoblasts were exposed to maytansine concentrations from 10-6 M to 10-10 M for 18 hrs. Aliquots of cells were removed for cell cycle analysis by flow microfluorometry (FMF) (5) and also processed for transmission electron microscopy (TEM). FMF analysis of cells treated with 10-8 M maytansine showed a reduction in the number of G1 cells and a corresponding build-up of cells with G2/M DNA content.

EELS characterization of “Smut” layer films on steel surface prepared for chrome plating by anodic etching

1995 ◽  
Vol 53 ◽  
pp. 538-539
Author(s):  
E Y. Wang ◽  
J. T. Cherian ◽  
A. Madsen ◽  
R. M. Fisher
Keyword(s):  
Steel Surface ◽  
Surface Preparation ◽  
Treatment Method ◽  
Chrome Plating ◽  
Transmission Electron ◽  
Alloy Steels ◽  
Pre Treatment ◽  
Hard Chrome ◽  
Electron Energy Loss

Many steel parts are electro-plated with chromium to protect them against corrosion and to improve their wear-resistance. Good adhesion of the chrome plate to the steel surface, which is essential for long term durability of the part, is extremely dependent on surface preparation prior to plating. Recently, McDonnell Douglas developed a new pre-treatment method for chrome plating in which the steel is anodically etched in a sulfuric acid and hydrofluoric acid solution. On carbon steel surfaces, this anodic pre-treatment produces a dark, loosely adhering material that is commonly called the “smut” layer. On stainless steels and nickel alloys, the surface is only darkened by the anodic pre-treatment and little residue is produced. Anodic pre-treatment prior to hard chrome plating results in much better adherence to both carbon and alloy steels.We have characterized the anodic pre-treated steel surface and the resulting “smut” layer using various techniques including electron spectroscopy for chemical analysis (ESCA) on bulk samples and transmission electron microscopy (TEM) and electron energy-loss spectroscopy (EELS) on stripped films.

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