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Cell Systems May 2018The advent of mass cytometry increased the number of parameters measured at the single-cell level while decreasing the extent of crosstalk between channels relative to...
The advent of mass cytometry increased the number of parameters measured at the single-cell level while decreasing the extent of crosstalk between channels relative to dye-based flow cytometry. Although reduced, spillover still exists in mass cytometry data, and minimizing its effect requires considerable expert knowledge and substantial experimental effort. Here, we describe a novel bead-based compensation workflow and R-based software that estimates and corrects for interference between channels. We performed an in-depth characterization of the spillover properties in mass cytometry, including limitations defined by the linear range of the mass cytometer and the reproducibility of the spillover over time and across machines. We demonstrated the utility of our method in suspension and imaging mass cytometry. To conclude, our approach greatly simplifies the development of new antibody panels, increases flexibility for antibody-metal pairing, opens the way to using less pure isotopes, and improves overall data quality, thereby reducing the risk of reporting cell phenotype artifacts.
Topics: Antibodies; Breast Neoplasms; Female; Flow Cytometry; Humans; Image Cytometry; Immunophenotyping; Reproducibility of Results; Signal-To-Noise Ratio; Single-Cell Analysis; Software; Suspensions
PubMed: 29605184
DOI: 10.1016/j.cels.2018.02.010 -
Nature Communications Jul 2022Economic gold deposits result from a 100- to 10,000-fold enrichment in gold relative to crustal background. In hydrothermal systems, this enrichment is achieved through...
Economic gold deposits result from a 100- to 10,000-fold enrichment in gold relative to crustal background. In hydrothermal systems, this enrichment is achieved through the transport and accumulation of metals via deeply sourced fluids to a site of deposition. However, the generally low metal solubility of Au in aqueous solutions in orogenic systems requires additional processes in order to explain high-grade gold formation. Reports of Au nanoparticles in high-grade gold veins infer that their formation is linked to mineralisation. However, processes leading to nanoparticle nucleation and deposition remain poorly understood. Here we show that formation of metal nanoparticles (Au, AuAg, Cu, AgO) is one of the essential contributors to efficient and focused gold deposition. We report systematic and previously unrecognized metal nanoparticles preserved in amorphous silica and/or carbonic phases in five high-grade deposits. The association of metal, silica and carbonic phases helps to constrain the multiple reactive processes involved in Au, Cu and Ag metallogenesis and formation of high-grade gold mineralisation.
Topics: Calcinosis; Carbon; Gold; Humans; Metal Nanoparticles; Silicon Dioxide; Suspensions
PubMed: 35778393
DOI: 10.1038/s41467-022-31447-5 -
Particle and Fibre Toxicology Jan 2020The regulatory definition(s) of nanomaterials (NMs) frequently uses the term 'agglomerates and aggregates' (AA) despite the paucity of evidence that AA are significantly...
BACKGROUND
The regulatory definition(s) of nanomaterials (NMs) frequently uses the term 'agglomerates and aggregates' (AA) despite the paucity of evidence that AA are significantly relevant from a nanotoxicological perspective. This knowledge gap greatly affects the safety assessment and regulation of NMs, such as synthetic amorphous silica (SAS). SAS is used in a large panel of industrial applications. They are primarily produced as nano-sized particles (1-100 nm in diameter) and considered safe as they form large aggregates (> 100 nm) during the production process. So far, it is indeed believed that large aggregates represent a weaker hazard compared to their nano counterpart. Thus, we assessed the impact of SAS aggregation on in vitro cytotoxicity/biological activity to address the toxicological relevance of aggregates of different sizes.
RESULTS
We used a precipitated SAS dispersed by different methods, generating 4 ad-hoc suspensions with different aggregate size distributions. Their effect on cell metabolic activity, cell viability, epithelial barrier integrity, total glutathione content and, IL-8 and IL-6 secretion were investigated after 24 h exposure in human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic cells (THP-1). We observed that the de-aggregated suspension (DE-AGGR), predominantly composed of nano-sized aggregates, induced stronger effects in all the cell lines than the aggregated suspension (AGGR). We then compared DE-AGGR with 2 suspensions fractionated from AGGR: the precipitated fraction (PREC) and the supernatant fraction (SuperN). Very large aggregates in PREC were found to be the least cytotoxic/biologically active compared to other suspensions. SuperN, which contains aggregates larger in size (> 100 nm) than in DE-AGGR but smaller than PREC, exhibited similar activity as DE-AGGR.
CONCLUSION
Overall, aggregation resulted in reduced toxicological activity of SAS. However, when comparing aggregates of different sizes, it appeared that aggregates > 100 nm were not necessarily less cytotoxic than their nano-sized counterparts. This study suggests that aggregates of SAS are toxicologically relevant for the definition of NMs.
Topics: Caco-2 Cells; Cell Culture Techniques; Cell Survival; Epithelial Cells; Glutathione; Humans; Nanoparticles; Particle Size; Silicon Dioxide; Surface Properties; Suspensions; THP-1 Cells
PubMed: 31900181
DOI: 10.1186/s12989-019-0331-3 -
Journal of Ocular Pharmacology and... 2022To compare ocular pharmacokinetics (PK), systemic absorption, and injectability of triamcinolone acetonide (TA) suprachoroidal (SC) and intravitreal (IVT) suspensions....
Suprachoroidal Injection of Triamcinolone Acetonide Suspension: Ocular Pharmacokinetics and Distribution in Rabbits Demonstrates High and Durable Levels in the Chorioretina.
To compare ocular pharmacokinetics (PK), systemic absorption, and injectability of triamcinolone acetonide (TA) suprachoroidal (SC) and intravitreal (IVT) suspensions. New Zealand White (NZW) rabbits received a bilateral injection of 4 mg TA injectable suspension, TRI (TRIESENCE ; Alcon) through either microneedle-based SC or standard IVT injection. Another group of NZW rabbits received a bilateral SC injection (4 mg) of either TRI or a proprietary TA suspension for SC use, CLS-TA (Clearside Biomedical). Blood and ocular tissues were analyzed for TA over 3 months. Separately, injectability of TRI and CLS-TA through a proprietary SC delivery system (SCS Microinjector; Clearside Biomedical) was compared using microinjector syringe-plunger glide force measurement. Suprachoroidal delivery of TRI, compared with IVT-TRI, resulted in ∼12-fold higher exposure in the retinal pigment epithelium-choroid-sclera, and comparable exposure in the retina. Conversely, SC-TRI, compared to IVT-TRI, resulted in 460-, 34-, and 22-fold lower exposure in the lens, iris-ciliary body, and vitreous humor, and negligible exposure in the aqueous humor. SC injection of either CLS-TA or TRI resulted in comparable and sustained ocular TA levels. Plasma TA levels were generally undetectable in both studies. A significantly greater and more variable glide force was measured for TRI vs. CLS-TA. Suprachoroidal delivery of TA enabled high and durable TA levels targeted to the chorioretina with limited anterior segment exposure. SC delivery of either CLS-TA or TRI resulted in comparable ocular PK profiles with low systemic exposure; however, CLS-TA required lower and less variable glide force than TRI, potentiating more consistent, controlled administration.
Topics: Animals; Choroid; Glucocorticoids; Rabbits; Retina; Suspensions; Triamcinolone Acetonide
PubMed: 35404132
DOI: 10.1089/jop.2021.0090 -
International Journal of Nanomedicine 2022Cyclosporin A (CsA) is a hydrophobic drug widely used as an immunosuppressant and anti-rejection drug in solid organ transplantation. On the market, there are two oral...
BACKGROUND
Cyclosporin A (CsA) is a hydrophobic drug widely used as an immunosuppressant and anti-rejection drug in solid organ transplantation. On the market, there are two oral CsA formulations available containing polyoxyethylene castor oil, which can cause serious allergic reactions and nephrotoxicity. In order to eliminate polyoxyethylene castor oil, CsA was formulated into a nanosuspension. This study aimed to design an oral cyclosporin A nanosuspensions (CsA-NSs) and investigate the effect of particle size on absorption of CsA-NSs.
METHODS
CsA-NSs were prepared using a wet bead milling method. Particle size, morphology and crystallinity state of CsA-NSs were characterized. The in vitro dissolution, the intestinal absorption properties and pharmacokinetic study of CsA-NSs were investigated.
RESULTS
CsA-NSs with sizes of 280 nm, 522 nm and 2967 nm were prepared. The shape of CsA-NSs with smaller size was similar to that of spheres. The crystallinity of CsA in nanocrystals was reduced. The dissolution rate of CsA-NSs (280 nm) was greater than that of CsA-NSs (522 nm) and CsA-NSs (2967 nm). CsA-NSs (280 nm) showed higher absorption rate constants ( ) and effective permeability coefficients ( ) of different intestinal segments compared with that of CsA-NSs (522 nm) and CsA-NSs (2967 nm). AUC of 280 nm CsA-NSs was about 1.12-fold of that of 522 nm CsA-NSs, and about 1.51-fold of that of 2967 nm CsA-NSs. In particular, the particle size of CsA-NSs was nanoscale, and their bioavailability was bioequivalent with marked self-microemulsion (Sandimmun Neoral).
CONCLUSION
It is feasible to prepare CsA-NSs. The dissolution rate, gastrointestinal transport properties and the oral absorption of CsA-NSs were promoted by reducing size. Considering the cost, efficiency and energy consumption, there should be an optimal particle size range in industrial production.
Topics: Administration, Oral; Biological Availability; Cyclosporine; Particle Size; Suspensions
PubMed: 35469173
DOI: 10.2147/IJN.S357541 -
Human Reproduction Update Nov 2016Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex... (Review)
Review
BACKGROUND
Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful.
OBJECTIVE AND RATIONALE
This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed.
SEARCH METHODS
An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy.
OUTCOMES
The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the human is lacking. In addition, few to no data are available on the safety aspects inherent to offspring generation with gametes derived from frozen-thawed TT or TCSs. Moreover, clarification is needed on whether it is better to cryopreserve TT or TCS.
WIDER IMPLICATIONS
Fertility restoration techniques are very promising and expected to be implemented in the clinic in the near future. However, inter-center variability needs to be overcome and the gametes produced for reproduction purposes need to be subjected to safety studies. With the perspective of a future clinical application, there is a dire need to optimize and standardize cryopreservation and safety testing before using frozen-thawed TT of TCSs for fertility restoration.
Topics: Animals; Cryopreservation; Fertility Preservation; Humans; Male; Models, Animal; Sexual Maturation; Spermatogenesis; Spermatozoa; Suspensions
PubMed: 27566839
DOI: 10.1093/humupd/dmw029 -
Food Research International (Ottawa,... Nov 2022The polymeric suspension of chitosan (Ch) has been an effective media for the extraction of total phenolic compounds (TPC) from the acerola by-product. It facilitates...
The polymeric suspension of chitosan (Ch) has been an effective media for the extraction of total phenolic compounds (TPC) from the acerola by-product. It facilitates the subsequent production of nanoparticles loaded with the phenolics (Np-TPC) by ionic gelation. However, neither the effects of Ch concentration on encapsulation efficiency (EE%) of TPC nor which compounds are extracted in its media are known, being it the first objective of this study. The second objective was to analyze the stability of the Np-TPC under accelerated conditions and its release profile at pHs 3.0 and 7.0. The results showed that Ch does not affect the extraction of TPC. However, the EE increased from 35.0 to 48.1 % with the increase of Ch concentration (0.4 to 1.0 %). LC/ESI-QTOF MS analysis showed that phenolic acids and flavonoids are extracted in 0.8 % Ch medium. After encapsulation, microscopy images revealed particle sizes ranging between 110 and 150 nm. Additionally, the presence of phenolics did not change the stability of the particles under accelerated conditions and the actives were fully released into the released medium for 10 h. The Np-TPC suspension appears to be useful for the production of edible antioxidant coatings to preserve fruits/vegetables, with potential application as carrier of other food ingredients.
Topics: Antioxidants; Ascorbic Acid; Chitosan; Flavonoids; Food Ingredients; Malpighiaceae; Phenols; Rutin; Suspensions
PubMed: 36192901
DOI: 10.1016/j.foodres.2022.111855 -
Soft Matter May 2021Neonatal respiratory distress syndrome (NRDS) is treated by intratracheal delivery of suspensions of animal-derived lung surfactant in saline. Lung surfactants are...
Neonatal respiratory distress syndrome (NRDS) is treated by intratracheal delivery of suspensions of animal-derived lung surfactant in saline. Lung surfactants are extracted via organic solvents from animal lung lavage, followed by solvent removal and surfactant re-hydration to form multi-bilayer particles suspended in saline. Following intra-tracheal administration, the surfactant suspension spreads throughout the lungs by surface tension gradient induced flow; the spreading rate is limited by suspension viscoelasticity. Here we examine the rheology of three clinical lung surfactant suspensions: Survanta (bovine lung), Curosurf (porcine lung), and Infasurf (calf lung). These surfactants have widely different rheological properties that depend on the lipid composition and bilayer organization. The steady shear viscosity is related to the bilayer particle volume fraction as for a suspension of hard spheres, but the lipid volume fraction is not simply related to the mass loading. Optical and electron microscopy and small angle X-ray scattering show that the viscosity variation is due to the temperature and composition dependent bilayer aggregate shapes and internal particle organization. Survanta forms crystalline bilayers at 37 °C, resulting in high aspect ratio asymmetric particles. Infasurf forms aggregates of unilamellar vesicles containing water pockets, while Curosurf forms onion-like multi-layered liposomes. While the mass loading of the three clinical surfactants is different, the different bilayer organization causes the particle volume fractions to be similar. Adding polyethylene glycol dehydrates and partially flocculates the bilayer aggregates in all suspensions, leading to smaller particle volume fractions and a reduced suspension viscosity even though the solvent viscosity increases almost six-fold.
Topics: Animals; Cattle; Lung; Pulmonary Surfactants; Surface-Active Agents; Suspensions; Swine; Viscosity
PubMed: 33929473
DOI: 10.1039/d1sm00337b -
Computational Intelligence and... 2021Intelligent methods and algorithms have promoted the development of the intelligent transportation system in many ways. In the rail transportation, the vertical...
Intelligent methods and algorithms have promoted the development of the intelligent transportation system in many ways. In the rail transportation, the vertical performance of a high-speed train suspension system has a great impact on the riding comfort of the train. Based on the intelligent optimization method of particle swarm optimization (PSO) algorithm, different inerter-spring-damper (ISD) suspension layouts are proposed for better riding comfort. A 10-degree-of-freedom (10-DOF) vertical dynamic model of a high-speed train is established, and the new suspension layouts are applied to the primary and secondary suspension of the train at the same time. Optimizations are carried out for the suspension parameters of the high-speed train. Performances of different suspension layouts at different running speeds are analysed and compared. The best layout for suspension is concluded. What is more, the virtual prototype simulation and analysis of a high-speed train with consideration of nonlinear inerters are carried out. Friction of a rack-pinion inerter is simulated in the virtual prototype simulation. And the influence of nonlinearity is discussed compared with the ideal suspensions. All the results can represent a guidance for future train suspension design and help the intelligent rail transportation system to be more comfortable.
Topics: Algorithms; Computer Simulation; Suspensions; Transportation
PubMed: 34721561
DOI: 10.1155/2021/1526792 -
Methods in Molecular Biology (Clifton,... 2022Membrane proteins are favored drug targets and antibody therapeutics represent the fastest-growing category of pharmaceuticals. However, there remains a need for rapid...
Membrane proteins are favored drug targets and antibody therapeutics represent the fastest-growing category of pharmaceuticals. However, there remains a need for rapid and effective approaches for the discovery of antibodies that recognize membrane proteins to develop a robust clinical pipeline for targeted therapeutics. The challenges associated with recombinant expression of membrane proteins make whole cell screening techniques desirable, as these strategies allow presentation of the target membrane proteins in their native conformations. Here, we describe a workflow that employs both adherent cell-based and suspension cell-based whole cell panning methodologies to enrich for specific binders within a yeast-displayed antibody library. The first round of selection consists of an adherent cell-based approach, wherein a diverse library is panned over target-expressing mammalian cell monolayers in order to debulk the naïve library. Subsequent rounds involve the use of suspension cell-based approaches, facilitated with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS), to achieve further library enrichment. Finally, we describe a high-throughput approach to screen target binding and specificity of individual clones isolated from selection campaigns.
Topics: Animals; Antibodies; Flow Cytometry; Immunologic Tests; Mammals; Membrane Proteins; Peptide Library; Suspensions
PubMed: 35482192
DOI: 10.1007/978-1-0716-2285-8_11