scholarly journals Development and test for long-term stability of a synthetic standard for a quantitative Cryptosporidium parvum LightCycler™ PCR assay

2005 ◽  
Vol 3 (1) ◽  
pp. 15-25 ◽  
Author(s):  
Renata Filkorn-Kaiser ◽  
Konrad Botzenhart ◽  
Albrecht Wiedenmann
Keyword(s):  
Cryptosporidium Parvum ◽  
Surrogate Marker ◽  
Target Sequence ◽  
Positive Trend ◽  
Long Term Stability ◽  
Synthetic Standard ◽  
One Year

A recently described quantitative rapid cycle real time PCR (LightCycler™) assay detects Cryptosporidium parvum after in vitro excystation, which is a surrogate marker for the viability of the organisms. In the original assay the quantification standard is a dilution series of C. parvum oocysts with a microscopically determined excystation rate. The need to keep suspensions of viable oocysts in stock and to continuously monitor their excystation rate, however, renders the assay impracticable for routine application. A synthetic standard was developed to replace the in vivo standard and was calibrated using oocysts with known excystation rates. The standard consists of a 486 bp DNA segment ranging from 229 bp upstream to 79 bp downstream of the actual PCR target site. Aliquots of the standard were frozen and stored at −20 °C and at −70 °C or lyophilised and stored at room temperature in the dark. For a period of one year samples preserved with each of the three methods were restored every four or five weeks. They were amplified in the LightCycler™ and the crossing points (CP) were monitored. No significant trend in the raw CP values could be observed for any of the three storage methods. However, when the methods were compared to each other by calculating the CP ratios (−20 °C/−70 °C; −20 °C/lyophilised; −70 °C/lyophilised) at the 10 monitoring dates, the CP ratios −20 °C/−70 °C and −20 °C/lyophilised showed a highly significant positive trend (p<0.0001) while the CP ratio −70 °C/lyophilised did not differ from the null hypothesis (p=0.53). It can be concluded that the latter two preservation methods are both appropriate, while storage at −20 °C is less advisable. Calculations based on the molecular weight of the standard and on the assumption of an average yield of three sporozoites per oocyst led to the conclusion that the target sequence is probably located on a double copy gene

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

Nontoxic Silicon Nanocrystals and Nanodiamonds as Substitution for Harmful Quantum Dots

2009 ◽  
Vol 1241 ◽  
Author(s):  
Anna Fucikova ◽  
Jan Valenta ◽  
Ivan Pelant ◽  
Vitezslav Brezina
Keyword(s):  
Quantum Dots ◽  
Chemical Composition ◽  
Silicon Nanocrystals ◽  
Nanocrystalline Silicon ◽  
Semiconductor Quantum Dots ◽  
In Vivo Studies ◽  
Long Term Stability

AbstractThe commercially available semiconductor quantum dots have been proven to be slightly to significantly toxic by recent publications depending on the chemical composition. We are developing new non-toxic fluorescent labels based on (i) nanocrystalline silicon, suitable for in vivo studies due to their biodegrability, and on (ii) nanodiamonds, intended mainly for in vitro use due to their long-term stability and nondegradilibity.

Robust oil-core nanocapsules with hyaluronate-based shells as promising nanovehicles for lipophilic compounds

Nanoscale ◽  
2017 ◽  
Vol 9 (47) ◽  
pp. 18867-18880 ◽  
Author(s):  
Joanna Szafraniec ◽  
Agnieszka Błażejczyk ◽  
Edyta Kus ◽  
Małgorzata Janik ◽  
Gabriela Zając ◽  
...  
Keyword(s):  
Lipophilic Compounds ◽  
Term Stability ◽  
Long Term Stability ◽  
Free Process

Biocompatible hyaluronate-based nanocapsules with liquid oil cores exhibiting long-term stability and tunable size were obtained in a versatile surfactant-free process and their biodistribution was studied in vivo and in vitro.

scholarly journals Comparison of the Efficacy of Two Novel Antitubercular Agents in Free and Liposome-Encapsulated Formulations

2021 ◽  
Vol 22 (5) ◽  
pp. 2457
Author(s):  
Nikoletta Kósa ◽  
Ádám Zolcsák ◽  
István Voszka ◽  
Gabriella Csík ◽  
Kata Horváti ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Drug Carrier ◽  
Antimycobacterial Activity ◽  
Size Exclusion ◽  
In Silico Docking ◽  
Term Stability ◽  
Long Term Stability

Tuberculosis is one of the top ten causes of death worldwide, and due to the appearance of drug-resistant strains, the development of new antituberculotic agents is a pressing challenge. Employing an in silico docking method, two coumaran (2,3-dihydrobenzofuran) derivatives—TB501 and TB515—were determined, with promising in vitro antimycobacterial activity. To enhance their effectiveness and reduce their cytotoxicity, we used liposomal drug carrier systems. Two types of small unilamellar vesicles (SUV) were prepared: multicomponent pH-sensitive stealth liposome (SUVmixed) and monocomponent conventional liposome. The long-term stability of our vesicles was obtained by the examination of particle size distribution with dynamic light scattering. Encapsulation efficiency (EE) of the two drugs was determined from absorption spectra before and after size exclusion chromatography. Cellular uptake and cytotoxicity were determined on human MonoMac-6 cells by flow cytometry. The antitubercular effect was characterized by the enumeration of colony-forming units on Mycobacterium tuberculosis H37Rv infected MonoMac-6 cultures. We found that SUVmixed + TB515 has the best long-term stability. TB515 has much higher EE in both types of SUVs. Cellular uptake for native TB501 is extremely low, but if it is encapsulated in SUVmixed it appreciably increases; in the case of TB515, quasi total uptake is accessible. It is concluded that SUVmixed + TB501 seems to be the most efficacious antitubercular formulation given the presented experiments; to find the most promising antituberculotic formulation for therapy further in vivo investigations are needed.

Polydopamine nanoparticles' activity and long-term stability should be fully studied for gene therapy applications

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Structural Control ◽  
Thrombus Formation ◽  
Contact Process ◽  
Protein Corona ◽  
Structure Property ◽  
Term Stability ◽  
Long Term Stability

Many substantial hurdles must be solved for in vivo or in vitro clinical translation of the Polydopamine (PDA)-based nanomaterials. Excessive accumulation of residual unreacted DA and specific metabolites (DA or other small molecules) of PDA in vivo may trigger a possible syndrome of dopamine dysregulation characterized by addictive behaviour, as DA may act as an endogenous neurotoxin when its vesicular sequestration is dysregulated. PDA nanoparticles' activity and long-term stability should be fully studied for in vivo applications aside from probable toxicity. According to the findings, PDA's strong reactivity with numerous functional groups (catechol, quinone, and amine) is comparatively favorable, but in mild circumstances it may have negative effects on the organism owing to direct alcohol interactions. More crucially, the charged, moist adhesive PDA has a high affinity for protein attachment, which might be a major defect in the blood contact process. Direct blood contact with these PDA-based nanomaterials with high specific surface area would result in fast protein adsorption, the establishment of a "protein corona" within minutes, and increased thrombus formation risk. In vitro applications, on the other hand, can prevent the threat of detrimental cell or tissue effects. New rules, theories and processes on structure property performance relationships may be developed by researching the in vivo bioapplications of the above-mentioned PDA nanoarchitectures, possibly leading to fundamental and useful insights into in-vitro material translations. Despite the fact that major impediments to structural control persist, it is predicted that in the future, electron coupling will bring new answers to challenges of improved illness diagnosis and therapy.

scholarly journals Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

2017 ◽  
Vol 85 (3) ◽  
Author(s):  
Maria A DeCicco RePass ◽  
Ying Chen ◽  
Yinan Lin ◽  
Wenda Zhou ◽  
David L. Kaplan ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Drug Targets ◽  
Diarrheal Disease ◽  
Three Dimensional ◽  
Content Type ◽  
Tissue Model ◽  
Culture Models

ABSTRACT Cryptosporidium spp. are apicomplexan parasites of global importance that cause human diarrheal disease. In vitro culture models that may be used to study this parasite and that have physiological relevance to in vivo infection remain suboptimal. Thus, the pathogenesis of cryptosporidiosis remains poorly characterized, and interventions for the disease are limited. In this study, we evaluated the potential of a novel bioengineered three-dimensional (3D) human intestinal tissue model (which we developed previously) to support long-term infection by Cryptosporidium parvum. Infection was assessed by immunofluorescence assays and confocal and scanning electron microscopy and quantified by quantitative reverse transcription-PCR. We found that C. parvum infected and developed in this tissue model for at least 17 days, the extent of the study time used in the present study. Contents from infected scaffolds could be transferred to fresh scaffolds to establish new infections for at least three rounds. Asexual and sexual stages and the formation of new oocysts were observed during the course of infection. Additionally, we observed ablation, blunting, or distortion of microvilli in infected epithelial cells. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium infection will be a useful tool for the study of host-parasite interactions, identification of putative drug targets, screening of potential interventions, and propagation of genetically modified parasites.

A Novel Injectable Collagen Matrix: In Vitro Characterization and In Vivo Evaluation

2000 ◽  
Vol 122 (3) ◽  
pp. 231-235 ◽  
Author(s):  
Damien Laude ◽  
Kevin Odlum ◽  
Stewart Rudnicki ◽  
Nathaniel Bachrach
Keyword(s):  
Collagen Matrix ◽  
Porous Matrix ◽  
In Vivo Evaluation ◽  
Long Term Stability ◽  
In Vitro Characterization ◽  
Essential Properties ◽  
Novel Material

We present here a unique engineered collagen formulation that is injectable and compacts into a porous viscoelastic solid after implantation, achieving completely focal application without cross-linking. This implant provides a cohesive continuously porous matrix, as demonstrated by permeability and compression experiments. Those experiments also provide initial mechanical characterization of the material and establish the ability to modify these essential properties by design. Further, the short-term compaction and long-term stability of the implant in vivo in terms of both physical and histological responses are assessed in an animal model to demonstrate the mechanism of action and long-term persistence of this novel material. [S0148-0731(00)00403-9]

Magnetic Microparticles for Endovascular Aneurysm Treatment: In Vitro and In Vivo Experimental Results

Neurosurgery ◽  
2011 ◽  
Vol 68 (5) ◽  
pp. 1388-1398 ◽  
Author(s):  
Johanna Oechtering ◽  
Peter J. Kirkpatrick ◽  
Alexander G. K. Ludolph ◽  
Franz J. Hans ◽  
Bernd Sellhaus ◽  
...  
Keyword(s):  
Magnetic Field ◽  
External Magnetic Field ◽  
Observational Period ◽  
Magnetic Microparticles ◽  
Term Stability ◽  
Long Term Stability ◽  
Aneurysm Model

AbstractOBJECTIVE:Endovascular treatment of intracranial aneurysms employing endosaccular coiling can be associated with aneurysm perforation, coil herniation or incomplete obliteration fueling the interest to investigate novel endovascular techniques. We aimed to test a novel embolization material in experimental aneurysms in vitro and in vivo whereby intra-arterially administered magnetic microparticles (MMPs) are navigated into the lumen of vascular aneurysms with assistance from an external magnetic field.METHODS:MMPs are core-shell particles suspended in saline that have a shell made of a polymeric material and a core made of magnetite (Fe3O4). They have a diameter of 1.4 μm. During MMP administration via a microcatheter, a magnetic field was applied externally to direct the particles with the use of a solid-state neodymium magnet. Experiments were performed in a perfused silicone vessel and aneurysm model to evaluate application techniques and fluid dynamics and in the elastase aneurysm model in rabbits to evaluate in vivo compatibility, including multiorgan histological examinations and long-term stability of aneurysm embolization.RESULTS:It was possible to steer and hold the MMPs within the aneurismal cavity where they occluded the lumen progressively. After removal of the external magnetic field, the results remained stable in vivo for the remainder of the observational period (30 minutes); after a 12-week observational period, recanalization of the aneurysm occurred.CONCLUSION:MMPs can be magnetically directed into aneurysms, allowing short-term obliteration. Although the method has yet to show reliable long-term stability, these experiments provide proof of concept, encouraging further investigation of intravascular magnetic compounds.

scholarly journals Defining Stage-Specific Activity of Potent New Inhibitors of Cryptosporidium parvum Growth In Vitro

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Lisa J. Funkhouser-Jones ◽  
Soumya Ravindran ◽  
L. David Sibley
Keyword(s):  
Life Cycle ◽  
Cryptosporidium Parvum ◽  
Specific Activity ◽  
Trna Synthetase ◽  
Mechanisms Of Action ◽  
Cryptosporidium Hominis ◽  
Content Type

ABSTRACT Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro. These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity. IMPORTANCE Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.

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