scholarly journals Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632

2021 ◽  
Vol 22 (13) ◽  
pp. 6782
Author(s):  
Zixing Chen ◽  
Wenmeng He ◽  
Thomas Chun Ning Leung ◽  
Hau Yin Chung
Keyword(s):  
Calcium Imaging ◽  
Cultured Cells ◽  
Rho Kinase ◽  
Culture Method ◽  
Taste Bud ◽  
Label Free ◽  
Sprague Dawley ◽  
Rock Inhibitor ◽  
High Calcium

Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632–supplemented media were screened for the cultivation of RLKs isolated from Sprague–Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.

scholarly journals Pregnancy-associated adaptations in [Ca2+]i-dependent and Ca2+ sensitization mechanisms of venous contraction: implications in pregnancy-related venous disorders

2016 ◽  
Vol 310 (11) ◽  
pp. H1851-H1865 ◽  
Author(s):  
Yin Xia ◽  
Raouf A. Khalil
Keyword(s):  
Vena Cava ◽  
Rho Kinase ◽  
Sprague Dawley ◽  
Rock Inhibitor ◽  
Fura 2 ◽  
Arterial Circulation ◽  
Gf 109203X ◽  
Parallel Activation ◽  
Venous Disorders ◽  
In Pregnancy

Pregnancy is associated with significant adaptations in the maternal hemodynamics and arterial circulation, but the changes in the venous mechanisms during pregnancy are less clear. We hypothesized that pregnancy is associated with alterations in venous function, intracellular free Ca2+ concentration ([Ca2+]i), and Ca2+-dependent mechanisms of venous contraction. Circular segments of inferior vena cava (IVC) from virgin and late pregnant (Preg, day 19) Sprague-Dawley rats were suspended between two hooks, labeled with fura-2, and placed in a cuvet inside a spectrofluorometer for simultaneous measurement of contraction and [Ca2+]i (fura-2 340/380 ratio). KCl (96 mM), which stimulates Ca2+ influx, caused less contraction (35.6 ± 6.3 vs. 92.6 ± 19.9 mg/mg tissue) and smaller increases in [Ca2+]i (1.67 ± 0.12 vs. 2.19 ± 0.11) in Preg vs. virgin rat IVC. The α-adrenergic receptor agonist phenylephrine (Phe; 10−5 M) caused less contraction (23.8 ± 3.4 vs. 70.9 ± 12.9 mg/mg tissue) and comparable increases in [Ca2+]i (1.76 ± 0.10 vs. 1.89 ± 0.08) in Preg vs. virgin rat IVC. At increasing extracellular Ca2+ concentrations ([Ca2+]e) (0.1, 0.3, 0.6, 1, and 2.5 mM), KCl and Phe induced [Ca2+]e-contraction and [Ca2+]e-[Ca2+]i curves that were reduced in Preg vs. virgin IVC, supporting reduced Ca2+ entry mechanisms. The [Ca2+]e-contraction and [Ca2+]e-[Ca2+]i curves were used to construct the [Ca2+]i-contraction relationship. Despite reduced contraction and [Ca2+]i in Preg IVC, the Phe-induced [Ca2+]i-contraction relationship was greater than that of KCl and was enhanced in Preg vs. virgin IVC, suggesting parallel activation of Ca2+-sensitization pathways. The Ca2+ channel blocker diltiazem, protein kinase C (PKC) inhibitor GF-109203X, and Rho-kinase (ROCK) inhibitor Y27632 inhibited KCl- and Phe-induced contraction and abolished the shift in the Phe [Ca2+]i-contraction relationship in Preg IVC, suggesting an interplay between the decrease in Ca2+ influx and possible compensatory activation of PKC- and ROCK-mediated Ca2+-sensitization pathways. The reduced [Ca2+]i and [Ca2+]i-dependent contraction in Preg rat IVC, despite the parallel rescue activation of Ca2+-sensitization pathways, suggests that the observed reduction in [Ca2+]i-dependent contraction mechanisms is likely underestimated, and that the veins without the rescue Ca2+-sensitization pathways could be even more prone to dilation during pregnancy. These pregnancy-associated reductions in Ca2+ entry-dependent mechanisms of venous contraction, if occurring in human lower extremity veins and if not adequately compensated by Ca2+-sensitization pathways, may play a role in pregnancy-related venous disorders.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

scholarly journals Growth, lifetime, directional movement and myosin-dependent motility of mutant keratin granules in cultured cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. M. Lehmann ◽  
R. E. Leube ◽  
R. Windoffer
Keyword(s):  
Digital Image Analysis ◽  
Cultured Cells ◽  
Epidermolysis Bullosa Simplex ◽  
Continuous Growth ◽  
Relative Contribution ◽  
Complete Life Cycle ◽  
Directional Movement ◽  
Multiple Characteristics ◽  
Cell Centre

AbstractIntermediate filament polypeptides (IFPs) are prominent components of cytoplasmic aggregates, which are pathognomonic for multiple diseases. Recent observations in cultured cells suggest that they are dynamic and subject to regulated turnover. The emerging concept is that multiple factors contribute to motility and turnover of IFP-containing aggregates. To understand their relative contribution, quantitative tools are needed. The current study addresses this need using epithelial cells producing mutant keratin IFPs that have been identified as the cause of the hereditary blister-forming skin disease epidermolysis bullosa simplex. Digital image analysis of individual granules allowed mapping of their complete life cycle, with information on multiple characteristics at any given time-point. The deduced signet features revealed rapid granule fusion and directed transport from the periphery towards the cell centre, and a limited, ~ 30 min lifetime with a slow, continuous growth phase followed by fast disassembly. As paradigmatic proof-of-principle, we demonstrate that inhibition of myosin II selectively reduces granule movement, linking keratin granule motility to retrograde cortical acto-myosin flow. The newly developed methods and established parameters will help in the characterization of known and the identification of novel regulators of IFP-containing aggregates.

scholarly journals Potential use of molecular and structural characterization of the gut bacterial community for postmortem interval estimation in Sprague Dawley rats

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Huan Li ◽  
Siruo Zhang ◽  
Ruina Liu ◽  
Lu Yuan ◽  
Di Wu ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Interval Estimation ◽  
The Body ◽  
Taxon Richness ◽  
Time Since Death ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Level Data ◽  
Species Taxon

AbstractOnce the body dies, the indigenous microbes of the host begin to break down the body from the inside and play a key role thereafter. This study aimed to investigate the probable shift in the composition of the rectal microbiota at different time intervals up to 15 days after death and to explore bacterial taxa important for estimating the time since death. At the phylum level, Proteobacteria and Firmicutes showed major shifts when checked at 11 different intervals and emerged at most of the postmortem intervals. At the species level, Enterococcus faecalis and Proteus mirabilis showed a downward and upward trend, respectively, after day 5 postmortem. The phylum-, family-, genus-, and species-taxon richness decreased initially and then increased considerably. The turning point occurred on day 9, when the genus, rather than the phylum, family, or species, provided the most information for estimating the time since death. We constructed a prediction model using genus-level data from high-throughput sequencing, and seven bacterial taxa, namely, Enterococcus, Proteus, Lactobacillus, unidentified Clostridiales, Vagococcus, unidentified Corynebacteriaceae, and unidentified Enterobacteriaceae, were included in this model. The abovementioned bacteria showed potential for estimating the shortest time since death.

scholarly journals Characterization of Liposomes Using Quantitative Phase Microscopy (QPM)

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 590
Author(s):  
Jennifer Cauzzo ◽  
Nikhil Jayakumar ◽  
Balpreet Singh Ahluwalia ◽  
Azeem Ahmad ◽  
Nataša Škalko-Basnet
Keyword(s):  
Rapid Development ◽  
Fluorescent Labeling ◽  
Label Free ◽  
Batch Mode ◽  
Full Field ◽  
Quantitative Phase ◽  
Phase Microscopy ◽  
Quantitative Phase Microscopy ◽  
Tracking Ability

The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms of system’s instability, machine sensitivity, and loss of tracking ability, among others. In this study, we explore some of the downsides of batch-mode analyses and fluorescent labeling, while introducing quantitative phase microscopy (QPM) as a label-free complimentary characterization technique. Liposomes were used as a model nanocarrier for their therapeutic relevance and structural versatility. A successful immobilization of liposomes in a non-dried setup allowed for static imaging conditions in an off-axis phase microscope. Image reconstruction was then performed with a phase-shifting algorithm providing high spatial resolution. Our results show the potential of QPM to localize subdiffraction-limited liposomes, estimate their size, and track their integrity over time. Moreover, QPM full-field-of-view images enable the estimation of a single-particle-based size distribution, providing an alternative to the batch mode approach. QPM thus overcomes some of the drawbacks of the conventional methods, serving as a relevant complimentary technique in the characterization of nanosystems.

scholarly journals Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 750
Author(s):  
Pasquale Marrazzo ◽  
Valeria Pizzuti ◽  
Silvia Zia ◽  
Azzurra Sargenti ◽  
Daniele Gazzola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Defense Mechanisms ◽  
Live Cells ◽  
Label Free ◽  
Bacterial Diseases ◽  
Prospective Application ◽  
Therapy Strategies ◽  
Stromal Stem Cells ◽  
Different Sources

Antibiotic resistance is creating enormous attention on the development of new antibiotic-free therapy strategies for bacterial diseases. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials and included in several cell-therapy protocols. Together with the well-known immunomodulatory and regenerative potential of the MSC secretome, these cells have shown direct and indirect anti-bacterial effects. However, the low reproducibility and standardization of MSCs from different sources are the current limitations prior to the purification of cell-free secreted antimicrobial peptides and exosomes. In order to improve MSC characterization, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. We discuss the potential of emerging microfluidic technologies providing new insights into density, shape, and size of live cells, starting from heterogeneous or 3D cultured samples. The prospective application of these technologies to studying MSC populations may contribute to developing new biopharmaceutical strategies with a view to naturally overcoming bacterial defense mechanisms.

scholarly journals Mineralogical and Microstructure Analysis for Characterization and Provenance of Ceramic Artifacts from Late Helladic Kastrouli Settlement, Delphi (Central Greece)

Geosciences ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 36
Author(s):  
Vayia Xanthopoulou ◽  
Ioannis Iliopoulos ◽  
Ioannis Liritzis
Keyword(s):  
Raw Materials ◽  
Analytical Techniques ◽  
Sem Analysis ◽  
Petrographic Analysis ◽  
Ceramic Assemblage ◽  
Thin Sections ◽  
Central Greece ◽  
High Calcium ◽  
Local Geology

The present study deals with the characterization of a ceramic assemblage from the Late Mycenaean (Late Helladic III) settlement of Kastrouli, at Desfina near Delphi, Central Greece using various analytical techniques. Kastrouli is located in a strategic position supervising the Mesokampos plateau and the entire peninsula and is related to other nearby coeval settlements. In total 40 ceramic sherds and 8 clay raw materials were analyzed through mineralogical, petrographic and microstructural techniques. Experimental briquettes (DS) made from clayey raw materials collected in the vicinity of Kastrouli, were fired under temperatures (900 and 1050 °C) in oxidizing conditions for comparison with the ancient ceramics. The petrographic analysis performed on thin sections prepared from the sherds has permitted the identification of six main fabric groups and a couple of loners. The aplastic inclusions recognized in all fabric groups but one confirmed the local provenance since they are related to the local geology. Fresh fractures of representative sherds were further examined under a scanning electron microscope (SEM/EDS) helping us to classify them into calcareous (CaO > 6%) and non-calcareous (CaO < 6%) samples (low and high calcium was noted in earlier pXRF data). Here, the ceramic sherds with broad calcium separation are explored on a one-to-one comparison on the basis of detailed mineralogical microstructure. Moreover, their microstructure was studied, aiming to estimate their vitrification stage. The mineralogy of all studied samples was determined by means of X-ray powder diffraction (XRPD), permitting us to test the validity of the firing temperatures revealed by the SEM analysis. The results obtained through the various analytical techniques employed are jointly assessed in order to reveal potters’ technological choices.

scholarly journals Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 586
Author(s):  
Liam Cole ◽  
Diogo Fernandes ◽  
Maryam T. Hussain ◽  
Michael Kaszuba ◽  
John Stenson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Viral Vector ◽  
Structural Characteristics ◽  
Genetic Material ◽  
Label Free ◽  
Gene Therapies ◽  
Multiple Challenges

Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

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