scholarly journals The Ciliary Rootlet Maintains Long-Term Stability of Sensory Cilia

2005 ◽  
Vol 25 (10) ◽  
pp. 4129-4137 ◽  
Author(s):  
Jun Yang ◽  
Jiangang Gao ◽  
Michael Adamian ◽  
Xiao-Hong Wen ◽  
Basil Pawlyk ◽  
...  
Keyword(s):  
Cell Biology ◽  
Structural Integrity ◽  
In Vitro Assays ◽  
Ciliated Cells ◽  
Term Survival ◽  
Functional Deficits ◽  
Structural Support ◽  
Long Term Stability

ABSTRACT The striated ciliary rootlet is a prominent cytoskeleton originating from basal bodies of ciliated cells. Although a familiar structure in cell biology, its function has remained unresolved. In this study, we carried out targeted disruption in mice of the gene for rootletin, a component of the rootlet. In the mutant, ciliated cells are devoid of rootlets. Phototransduction and ciliary beating in sensory and motile cilia initially exhibit no apparent functional deficits. However, photoreceptors degenerate over time, and mutant lungs appear prone to pathological changes consistent with insufficient mucociliary clearance. Further analyses revealed a striking fragility at the ciliary base in photoreceptors lacking rootlets. In vitro assays suggest that the rootlet is among the least dynamic of all cytoskeletons and interacts with actin filaments. Thus, a primary function of the rootlet is to provide structural support for the cilium. Inasmuch as photoreceptors elaborate an exceptionally enlarged sensory cilium, they are especially dependent on the rootlet for structural integrity and long-term survival.

scholarly journals Pharmacologic marrow purging in murine T cell leukemia

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1656-1661 ◽  
Author(s):  
EA Copelan ◽  
SC Johnson ◽  
MR Grever ◽  
JF Sheridan ◽  
PJ Tutschka
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
In Vitro Assays ◽  
Cell Leukemia ◽  
Term Survival ◽  
Adenosine Deaminase Activity ◽  
Marrow Purging ◽  
Malignant T Cells

Abstract Deoxycoformycin in combination with deoxyadenosine was used to purge 6C3HED malignant T cells from murine marrow in vitro. Adenosine deaminase activity of 6C3HED cells was ablated by incubation with 10(- 6) mol/L deoxycoformycin (dCF). During a 12-hour incubation with 10(-6) mol/L dCF and 10(-4) mol/L deoxyadenosine, tumor cells sequentially accumulated dATP, became depleted of NAD followed by ATP, then died. More than 5 logs of 6C3HED cells were killed as measured by survival of mice injected with treated tumor cells. Identical incubation of 5 x 10(6) marrow cells did not interfere with rescue of syngeneic lethally irradiated mice. Long-term survival was demonstrated in 12 of 14 mice that received marrow that had been contaminated with 5% 6C3HED cells, incubated with deoxycoformycin and deoxyadenosine, then used to rescue lethally irradiated mice. This murine model provides information not available from in vitro assays and may be useful in the development of strategies to purge malignant T cells from marrow.

scholarly journals Stability Assessment of Four Chimeric Proteins for Human Chagas Disease Immunodiagnosis

Biosensors ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 289
Author(s):  
Paola Alejandra Fiorani Celedon ◽  
Leonardo Maia Leony ◽  
Ueriton Dias Oliveira ◽  
Natália Erdens Maron Freitas ◽  
Ângelo Antônio Oliveira Silva ◽  
...  
Keyword(s):  
Chagas Disease ◽  
Structural Integrity ◽  
Structural Characteristics ◽  
Control Group ◽  
Stability Tests ◽  
Adverse Conditions ◽  
Term Stability ◽  
Long Term Stability

The performance of an immunoassay relies on antigen-antibody interaction; hence, antigen chemical stability and structural integrity are paramount for an efficient assay. We conducted a functional, thermostability and long-term stability analysis of different chimeric antigens (IBMP), in order to assess effects of adverse conditions on four antigens employed in ELISA to diagnose Chagas disease. ELISA-based immunoassays have served as a model for biosensors development, as both assess molecular interactions. To evaluate thermostability, samples were heated and cooled to verify heat-induced denaturation reversibility. In relation to storage stability, the antigens were analyzed at 25 °C at different moments. Long-term stability tests were performed using eight sets of microplates sensitized. Antigens were structurally analyzed through circular dichroism (CD), dynamic light scattering, SDS-PAGE, and functionally evaluated by ELISA. Data suggest that IBMP antigens are stable, over adverse conditions and for over a year. Daily analysis revealed minor changes in the molecular structure. Functionally, IBMP-8.2 and IBMP-8.3 antigens showed reactivity towards anti-T. cruzi antibodies, even after 72 h at 25 °C. Long-term stability tests showed that all antigens were comparable to the control group and all antigens demonstrated stability for one year. Data suggest that the antigens maintained their function and structural characteristics even in adverse conditions, making them a sturdy and reliable candidate to be employed in future in vitro diagnostic tests applicable to different models of POC devices, such as modern biosensors in development.

scholarly journals Pharmacologic marrow purging in murine T cell leukemia

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1656-1661
Author(s):  
EA Copelan ◽  
SC Johnson ◽  
MR Grever ◽  
JF Sheridan ◽  
PJ Tutschka
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
In Vitro Assays ◽  
Cell Leukemia ◽  
Term Survival ◽  
Adenosine Deaminase Activity ◽  
Marrow Purging ◽  
Malignant T Cells

Deoxycoformycin in combination with deoxyadenosine was used to purge 6C3HED malignant T cells from murine marrow in vitro. Adenosine deaminase activity of 6C3HED cells was ablated by incubation with 10(- 6) mol/L deoxycoformycin (dCF). During a 12-hour incubation with 10(-6) mol/L dCF and 10(-4) mol/L deoxyadenosine, tumor cells sequentially accumulated dATP, became depleted of NAD followed by ATP, then died. More than 5 logs of 6C3HED cells were killed as measured by survival of mice injected with treated tumor cells. Identical incubation of 5 x 10(6) marrow cells did not interfere with rescue of syngeneic lethally irradiated mice. Long-term survival was demonstrated in 12 of 14 mice that received marrow that had been contaminated with 5% 6C3HED cells, incubated with deoxycoformycin and deoxyadenosine, then used to rescue lethally irradiated mice. This murine model provides information not available from in vitro assays and may be useful in the development of strategies to purge malignant T cells from marrow.

Human thymic epithelial cells maintain long-term survival of clonogenic myeloid and erythroid progenitor cells in vitro

2000 ◽  
Vol 111 (1) ◽  
pp. 363-370 ◽  
Author(s):  
Katsuto Takenaka ◽  
Mine Harada ◽  
Tomoaki Fujisaki ◽  
Koji Nagafuji ◽  
Shinichi Mizuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Progenitor Cells ◽  
Thymic Epithelial Cells ◽  
Erythroid Progenitor ◽  
Term Survival ◽  
Long Term Survival ◽  
Erythroid Progenitor Cells

THE ROLE OF POLYETHYLENIMINE IN ENHANCING PERFORMANCE OF GLUTAMATE BIOSENSORS

2018 ◽  
Vol 8 (3) ◽  
pp. 36-41
Author(s):  
Diep Do Thi Hong ◽  
Duong Le Phuoc ◽  
Hoai Nguyen Thi ◽  
Serra Pier Andrea ◽  
Rocchitta Gaia
Keyword(s):  
First Generation ◽  
Good Reproducibility ◽  
Complex Matrices ◽  
Long Term Stability ◽  
In Vitro Characterization ◽  
Glutamate Oxidase

Background: The first biosensor was constructed more than fifty years ago. It was composed of the biorecognition element and transducer. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples Glutamate is important biochemicals involved in energetic metabolism and neurotransmission. Therefore, biosensors requires the development a new approach exhibiting high sensibility, good reproducibility and longterm stability. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples. The aims of this work: To find out which concentration of polyethylenimine (PEI) exhibiting the most high sensibility, good reproducibility and long-term stability. Methods: We designed and developed glutamate biosensor using different concentration of PEI ranging from 0% to 5% at Day 1 and Day 8. Results: After Glutamate biosensors in-vitro characterization, several PEI concentrations, ranging from 0.5% to 1% seem to be the best in terms of VMAX, the KM; while PEI content ranging from 0.5% to 1% resulted stable, PEI 1% displayed an excellent stability. Conclusions: In the result, PEI 1% perfomed high sensibility, good stability and blocking interference. Furthermore, we expect to develop and characterize an implantable biosensor capable of detecting glutamate, glucose in vivo. Key words: Glutamate biosensors, PEi (Polyethylenimine) enhances glutamate oxidase, glutamate oxidase biosensors

Cellular and immunological basis of the host-parasite relationship during infection withNeospora caninum

Parasitology ◽  
2006 ◽  
Vol 133 (3) ◽  
pp. 261-278 ◽  
Author(s):  
A. HEMPHILL ◽  
N. VONLAUFEN ◽  
A. NAGULESWARAN
Keyword(s):  
Host Cell ◽  
Cell Biology ◽  
Neospora Caninum ◽  
Host Cells ◽  
Term Survival ◽  
Prevention Of Infection ◽  
Parasite Relationship ◽  
Potential Applications

Neospora caninumis an apicomplexan parasite that is closely related toToxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast toT. gondii, N. caninumrepresents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused byN. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cellsin vitroandin vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite orvice versa). Thirdly, by analogy withT. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features ofN. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

scholarly journals Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures

2020 ◽  
Author(s):  
Ameen A. Salahudeen ◽  
Shannon S. Choi ◽  
Arjun Rustagi ◽  
Junjie Zhu ◽  
Sean M. de la O ◽  
...  
Keyword(s):  
Lung Disease ◽  
Single Cell ◽  
Basal Cell ◽  
Human Lung ◽  
Basal Cells ◽  
Ciliated Cells ◽  
Club Cells ◽  
Distal Lung

ABSTRACTThe distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 “apical-out” organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

scholarly journals An updated protocol based on CLSI document C37 for preparation of off-the-clot serum from individual units for use alone or to prepare commutable pooled serum reference materials

2020 ◽  
Vol 58 (3) ◽  
pp. 368-374 ◽  
Author(s):  
Uliana Danilenko ◽  
Hubert W. Vesper ◽  
Gary L. Myers ◽  
Patric A. Clapshaw ◽  
Johanna E. Camara ◽  
...  
Keyword(s):  
Human Serum ◽  
Reference Materials ◽  
Frozen Storage ◽  
Metrological Traceability ◽  
Medical Laboratory ◽  
Serum Samples ◽  
Long Term Stability ◽  
Individual Human

AbstractManufacturers of in vitro diagnostic medical devices, clinical laboratories, research laboratories and calibration laboratories require commutable reference materials that can be used in the calibration hierarchies of medical laboratory measurement procedures used for human specimens to establish metrological traceability to higher order reference systems. Commutable materials are also useful in external quality assessment surveys. In order to achieve these goals, matrix-based reference materials with long-term stability, appropriate measurand concentrations and commutability with individual human specimens are required. The Clinical and Laboratory Standards Institute (CLSI) guideline C37-A (now archived) provided guidance to prepare commutable pooled serum reference materials for use in the calibration hierarchies of cholesterol measurement procedures. Experience using the C37-A guideline has identified a number of technical enhancements as well as applications to measurands other than cholesterol. This experience is incorporated into this updated protocol to ensure the procedure will continue to meet the needs of the medical laboratory. The updated protocol describes a procedure for preparing frozen human serum units or pools with minimal matrix alterations that are likely to be commutable with individual human serum samples. The protocol provides step-by-step guidance for the planning phase, collection of individual serum units, processing the units, qualifying the units for use in a pool and frozen storage of aliquots of pooled sera to manufacture frozen serum pools. Guidance on how to perform quality control of the final product and suggestions on documentation are also provided.

scholarly journals Long-Term Stability of a RAFT-Modified Bulk-Fill Resin-Composite under Clinically Relevant versus ISO-Curing Conditions

Materials ◽  
2020 ◽  
Vol 13 (23) ◽  
pp. 5350
Author(s):  
Niklas Graf ◽  
Nicoleta Ilie
Keyword(s):  
Fracture Mechanism ◽  
Resin Composite ◽  
Bending Test ◽  
Depth Sensing ◽  
Matrix Formulation ◽  
Curing Conditions ◽  
Term Stability ◽  
Long Term Stability

The addition of RAFT (reversible addition-fragmentation chain transfer) agents to the matrix formulation of a bulk-fill resin composite can significantly decrease the required curing time down to a minimum of 3 s. Evaluating the long term-stability of this resin composite in relation to varied curing conditions in an in-vitro environment was this study’s goal. Specimens were produced according to either an ISO or one of two clinical curing protocols and underwent a maximum of three successive aging procedures. After each one of the aging procedures, 30 specimens for each curing condition were extracted for a three-point bending test. Fragments were then stereo-microscopically characterized according to their fracture mechanism. Weibull analysis was used to quantify the reliability of each aging and curing combination. Selected fragments (n = 12) underwent further testing via depth-sensing indentation. Mechanical values for either standardized or clinical curing were mostly comparable. However, changes in fracture mechanism and Weibull modulus were observed after each aging procedure. The final procedure exposed significant differences in the mechanical values due to curing conditions. Curing conditions with increased radiant exposure seemingly result in a higher crosslink in the polymer-matrix, thus increasing resistance to aging. Yet, the clinical curing conditions still resulted in acceptable mechanical values, proving the effectiveness of RAFT-polymerization.

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