Collagen immobilization on ultra-thin nanofiber membrane to promote in vitro endothelial monolayer formation

The endothelialization on the poly (ε-caprolactone) nanofiber has been limited due to its low hydrophilicity. The aim of this study was to immobilize collagen on an ultra-thin poly (ε-caprolactone) nanofiber membrane without altering the nanofiber structure and maintaining the endothelial cell homeostasis on it. We immobilized collagen on the poly (ε-caprolactone) nanofiber using hydrolysis by NaOH treatment and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo- N-hydroxysulfosuccinimide reaction as a cost-effective and stable approach. NaOH was first applied to render the poly (ε-caprolactone) nanofiber hydrophilic. Subsequently, collagen was immobilized on the surface of the poly (ε-caprolactone) nanofibers using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo- N-hydroxysulfosuccinimide. Scanning electron microscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and fluorescence microscopy were used to verify stable collagen immobilization on the surface of the poly (ε-caprolactone) nanofibers and the maintenance of the original structure of poly (ε-caprolactone) nanofibers. Furthermore, human endothelial cells were cultured on the collagen-immobilized poly (ε-caprolactone) nanofiber membrane and expressed tight junction proteins with the increase in transendothelial electrical resistance, which demonstrated the maintenance of the endothelial cell homeostasis on the collagen-immobilized-poly (ε-caprolactone) nanofiber membrane. Thus, we expected that this process would be promising for maintaining cell homeostasis on the ultra-thin poly (ε-caprolactone) nanofiber scaffolds.

Download Full-text

Maytansine-Induced Mitotic Arrest in Human Lymphoblasts

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079747 ◽

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.

Download Full-text

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100116620 ◽

1975 ◽

Vol 33 ◽

pp. 600-601

Author(s):

John C. Garancis ◽

Robert O. Hussa ◽

Michael T. Story ◽

Donald Yorde ◽

Roland A. Pattillo

Keyword(s):

Electron Microscopy ◽

Cellular Proliferation ◽

Morphological Changes ◽

Culture Fluid ◽

Cellular Functions ◽

Human Trophoblast ◽

Transmission Electron ◽

Marked Activity ◽

Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

Download Full-text

Dietary Nanoparticles Interact with Gluten Peptides and Alter the Intestinal Homeostasis Increasing the Risk of Celiac Disease

International Journal of Molecular Sciences ◽

10.3390/ijms22116102 ◽

2021 ◽

Vol 22 (11) ◽

pp. 6102

Author(s):

Clara Mancuso ◽

Francesca Re ◽

Ilaria Rivolta ◽

Luca Elli ◽

Elisa Gnodi ◽

...

Keyword(s):

Celiac Disease ◽

Metallic Nanoparticles ◽

Synergistic Effects ◽

Food Sector ◽

Intestinal Homeostasis ◽

Gluten Peptides ◽

Transmission Electron ◽

Duodenal Biopsies ◽

Junction Proteins

The introduction of metallic nanoparticles (mNPs) into the diet is a matter of concern for human health. In particular, their effect on the gastrointestinal tract may potentially lead to the increased passage of gluten peptides and the activation of the immune response. In consequence, dietary mNPs could play a role in the increasing worldwide celiac disease (CeD) incidence. We evaluated the potential synergistic effects that peptic-tryptic-digested gliadin (PT) and the most-used food mNPs may induce on the intestinal mucosa. PT interaction with mNPs and their consequent aggregation was detected by transmission electron microscopy (TEM) analyses and UV–Vis spectra. In vitro experiments on Caco-2 cells proved the synergistic cytotoxic effect of PT and mNPs, as well as alterations in the monolayer integrity and tight junction proteins. Exposure of duodenal biopsies to gliadin plus mNPs triggered cytokine production, but only in CeD biopsies. These results suggest that mNPs used in the food sector may alter intestinal homeostasis, thus representing an additional environmental risk factor for the development of CeD.

Download Full-text

Fabrication of a liquid cell for in situ transmission electron microscopy

Microscopy ◽

10.1093/jmicro/dfaa076 ◽

2020 ◽

Author(s):

Xiaoguang Li ◽

Kazutaka Mitsuishi ◽

Masaki Takeguchi

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Cost Effective ◽

Production Method ◽

Microscopy Imaging ◽

Transmission Electron ◽

In Situ Electron Microscopy ◽

Liquid Cell ◽

Si Wafer

Abstract Liquid cell transmission electron microscopy (LCTEM) enables imaging of dynamic processes in liquid with high spatial and temporal resolution. The widely used liquid cell (LC) consists of two stacking microchips with a thin wet sample sandwiched between them. The vertically overlapped electron-transparent membrane windows on the microchips provide passage for the electron beam. However, microchips with imprecise dimensions usually cause poor alignment of the windows and difficulty in acquiring high-quality images. In this study, we developed a new and efficient microchip fabrication process for LCTEM with a large viewing area (180 µm × 40 µm) and evaluated the resultant LC. The new positioning reference marks on the surface of the Si wafer dramatically improve the precision of dicing the wafer, making it possible to accurately align the windows on two stacking microchips. The precise alignment led to a liquid thickness of 125.6 nm close to the edge of the viewing area. The performance of our LC was demonstrated by in situ transmission electron microscopy imaging of the dynamic motions of 2-nm Pt particles. This versatile and cost-effective microchip production method can be used to fabricate other types of microchips for in situ electron microscopy.

Download Full-text

Ethosomes and Transethosomes for Mangiferin Transdermal Delivery

Antioxidants ◽

10.3390/antiox10050768 ◽

2021 ◽

Vol 10 (5) ◽

pp. 768

Author(s):

Maddalena Sguizzato ◽

Francesca Ferrara ◽

Supandeep Singh Hallan ◽

Anna Baldisserotto ◽

Markus Drechsler ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Radical Scavenging ◽

Human Keratinocytes ◽

Antioxidant Response ◽

Correlation Spectroscopy ◽

Inflammatory Status ◽

Transmission Electron ◽

Anti Inflammatory

Mangiferin is a natural glucosyl xanthone with antioxidant and anti-inflammatory activity, making it suitable for protection against cutaneous diseases. In this study ethosomes and transethosomes were designed as topical delivery systems for mangiferin. A preformulation study was conducted using different surfactants in association with phosphatidylcholine. Vesicle dimensional distribution was monitored by photon correlation spectroscopy, while antioxidant capacity and cytotoxicity were respectively assessed by free radical scavenging analysis and MTT on HaCaT keratinocytes. Selected nanosystems were further investigated by cryogenic transmission electron microscopy, while mangiferin entrapment capacity was evaluated by ultracentrifugation and HPLC. The diffusion kinetics of mangiferin from ethosomes and transethosomes evaluated by Franz cell was faster in the case of transethosomes. The suitability of mangiferin-containing nanovesicles in the treatment of skin disorders related to pollutants was investigated, evaluating, in vitro, the antioxidant and anti-inflammatory effect of ethosomes and transethosomes on human keratinocytes exposed to cigarette smoke as an oxidative and inflammatory challenger. The ability to induce an antioxidant response (HO-1) and anti-inflammatory status (IL-6 and NF-kB) was determined by RT-PCR and immunofluorescence. The data demonstrated the effectiveness of mangiferin loaded in nanosystems to protect cells from damage. Finally, to gain insight into the keratinocytes’ uptake of ethosome and transethosome, transmission electron microscopy analyses were conducted, showing that both nanosystems were able to pass intact within the cells.

Download Full-text

Facile synthesis of Au/ZnO/Ag nanoparticles using Glechoma hederacea L. extract, and their activity against leukemia

Biomedical Microdevices ◽

10.1007/s10544-021-00557-0 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Renata Dobrucka ◽

Aleksandra Romaniuk-Drapała ◽

Mariusz Kaczmarek

Keyword(s):

Electron Microscopy ◽

Ag Nanoparticles ◽

Mononuclear Cells ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Mtt Test ◽

Transmission Electron ◽

Almost All ◽

Glechoma Hederacea

AbstractMetal combinations have been attracting the attention of scientists for some time. They usually exhibit new characteristics that are different from the ones possessed by their components. In this work, Au/ZnO/Ag nanoparticles were synthesized biologically using Glechoma hederacea L. extract. The synthesized Au/ZnO/Ag nanoparticles were characterized by UV-Vis, Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), and Atomic Force Microscopy (AFM). The microscopic methods confirmed the presence of spherical nanoparticles of 50–70 nm. The influence of biologically synthesized Au/ZnO/Ag nanoparticles on the vitality of human cells was evaluated in vitro with the use of established human Acute T Cell Leukemia cell line, Jurkat (ATCC® TIB-152™), as well as mononuclear cells isolated from peripheral blood (PBMC) of voluntary donors. Cell survival and the half-maximal inhibitory concentration index (IC50) were analyzed by the MTT test. The studies showed that the total loss of cell viability occurred at the Au/ZnO/Ag nanoparticle concentration range of 10 µmol–50 µmol. The use of Au/ZnO/Ag nanoparticles at the concentration of 100 µmol eliminated almost all living cells from the culture in 24h. The above observation confirms the result obtained during the MTT test.

Download Full-text

Cemp1-p3 Peptide Promotes the Transformation of Octacalcium Phosphate into Hydroxyapatite Crystals

Crystals ◽

10.3390/cryst10121131 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1131

Author(s):

Maricela Santana ◽

Gonzalo Montoya ◽

Raúl Herrera ◽

Lía Hoz ◽

Enrique Romo ◽

...

Keyword(s):

Electron Microscopy ◽

Octacalcium Phosphate ◽

Sequence Motifs ◽

Simple Method ◽

Transmission Electron ◽

Precursor Phase ◽

Mineralized Tissues ◽

Potent Growth

Dental cementum contains unique molecules that regulate the mineralization process in vitro and in vivo, such as cementum protein 1 (CEMP1). This protein possesses amino acid sequence motifs like the human recombinant CEMP1 with biological activity. This novel cementum protein 1-derived peptide (CEMP1-p3, from the CEMP1’s N-terminal domain: (QPLPKGCAAVKAEVGIPAPH), consists of 20 amino acids. Hydroxyapatite (HA) crystals could be obtained through the combination of the amorphous precursor phase and macromolecules such as proteins and peptides. We used a simple method to synthesize peptide/hydroxyapatite nanocomposites using OCP and CEMP1-p3. The characterization of the crystals through scanning electron microscopy (SEM), powder X-ray diffraction (XRD), high--resolution transmission electron microscopy (HRTEM), and Raman spectroscopy revealed that CEMP1-p3 transformed OCP into hydroxyapatite (HA) under constant ionic strength and in a buffered solution. CEMP1-p3 binds and highly adsorbs to OCP and is a potent growth stimulator of OCP crystals. CEMP1-p3 fosters the transformation of OCP into HA crystals with crystalline planes (300) and (004) that correspond to the cell of hexagonal HA. Octacalcium phosphate crystals treated with CEMP1-p3 grown in simulated physiological buffer acquired hexagonal arrangement corresponding to HA. These findings provide new insights into the potential application of CEMP1-p3 on possible biomimetic approaches to generate materials for the repair and regeneration of mineralized tissues, or restorative materials in the orthopedic field.

Download Full-text

Biocompatibility Characteristics of Titanium Coated with Multi Walled Carbon Nanotubes—Hydroxyapatite Nanocomposites

Materials ◽

10.3390/ma12020224 ◽

2019 ◽

Vol 12 (2) ◽

pp. 224 ◽

Cited By ~ 5

Author(s):

Jung-Eun Park ◽

Yong-Seok Jang ◽

Tae-Sung Bae ◽

Min-Ho Lee

Keyword(s):

Electron Microscopy ◽

Carbon Nanotubes ◽

Sol Gel ◽

Pure Titanium ◽

Cell Proliferation And Differentiation ◽

Transmission Electron ◽

Proliferation And Differentiation ◽

Multi Walled Carbon Nanotubes ◽

Walled Carbon Nanotubes

Multi walled carbon nanotubes-hydroxyapatite (MWCNTs-HA) with various contents of MWCNTs was synthesized using the sol-gel method. MWCNTs-HA composites were characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). HA particles were generated on the surface of MWCNT. Produced MWCNTs-HA nanocomposites were coated on pure titanium (PT). Characteristic of the titanium coated MWCNTs-HA was evaluated by field-emission scanning electron microscopy (FE-SEM) and XRD. The results show that the titanium surface was covered with MWCNTs-HA nanoparticles and MWCNTs help form the crystalized hydroxyapatite. Furthermore, the MWCNTs-HA coated titanium was investigated for in vitro cellular responses. Cell proliferation and differentiation were improved on the surface of MWCNT-HA coated titanium.

Download Full-text

Abstract 531: Tie-2/Cre--Mediated Conditional Deletion of Lipid Phosphate Phosphatase-3 Reveals Its Role in Endothelial Cell Apoptosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a531 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Ishita Chatterjee ◽

Kishore K Wary

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Genome Wide Association Study ◽

Vascular Endothelial Cell ◽

Protein Levels ◽

Conditional Deletion ◽

Transmission Electron ◽

Increased Risk

Rationale: A recent genome-wide association study (GWAS) has linked a frequently occurring variation in the LPP3 (also known as PPAP2b) loci to increased risk of coronary heart disease (CAD). However, the in vivo function of LPP3 in vascular endothelial cell is incompletely understood. Goal: To address the endothelial cell (EC) specific function of Lpp3 in mice. Results: Tie-2/Cre mediated Lpp3 deletion did not affect normal vasculogenesis in early embryonic development, in contrast, in late embryonic stages it led to impaired angiogenesis associated with hemorrhage, edema and late embryonic lethal phenotype. Immunohistochemical staining followed by microscopic analyses of mutant embryos revealed reduced fibronectin and VE-cadherin expression throughout different vascular bed, and increased apoptosis in CD31+ vascular structures. Transmission electron microscopy (TEM) showed the presence of apoptotic endothelial cells and disruption of adherens junctions in mutant embryos. LPP3-knockdown in vitro showed an increase in p53 and p21 protein levels, with concomitant decrease in cell proliferation. LPP3-knockdown also decreased transendothelial electrical resistance (TER), interestingly re-expression of ß-catenin cDNA into LPP3-depleted endothelial cells partially restored the effect of loss of LPP3. Conclusion: These results suggest the ability of LPP3 to regulate survival and apoptotic activities of endothelial cells during patho/physiological angiogenesis.

Download Full-text

Human Islet Amyloid Polypeptide Fibril Binding to Catalase: A Transmission Electron Microscopy and Microplate Study

The Scientific World JOURNAL ◽

10.1100/tsw.2010.73 ◽

2010 ◽

Vol 10 ◽

pp. 879-893 ◽

Cited By ~ 10

Author(s):

Nathaniel G. N. Milton ◽

J. Robin Harris

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Islet Amyloid Polypeptide ◽

Amyloid Β ◽

Human Islet ◽

Islet Amyloid ◽

Human Islet Amyloid Polypeptide ◽

Transmission Electron

The diabetes-associated human islet amyloid polypeptide (IAPP) is a 37-amino-acid peptide that forms fibrilsin vitroandin vivo. Human IAPP fibrils are toxic in a similar manner to Alzheimer's amyloid-β (Aβ) and prion protein (PrP) fibrils. Previous studies have shown that catalase binds to Aβ fibrils and appears to recognize a region containing the Gly-Ala-Ile-Ile sequence that is similar to the Gly-Ala-Ile-Leu sequence found in human IAPP residues 24-27. This study presents a transmission electron microscopy (TEM)—based analysis of fibril formation and the binding of human erythrocyte catalase to IAPP fibrils. The results show that human IAPP 1-37, 8-37, and 20-29 peptides form fibrils with diverse and polymorphic structures. All three forms of IAPP bound catalase, and complexes of IAPP 1-37 or 8-37 with catalase were identified by immunoassay. The binding of biotinylated IAPP to catalase was high affinity with a KDof 0.77nM, and could be inhibited by either human or rat IAPP 1-37 and 8-37 forms. Fibrils formed by the PrP 118-135 peptide with a Gly-Ala-Val-Val sequence also bound catalase. These results suggest that catalase recognizes a Gly-Ala-Ile-Leu—like sequence in amyloid fibril-forming peptides. For IAPP 1-37 and 8-37, the catalase binding was primarily directed towards fibrillar rather than ribbon-like structures, suggesting differences in the accessibility of the human IAPP 24-27 Gly-Ala-Ile-Leu region. This suggests that catalase may be able to discriminate between different structural forms of IAPP fibrils. The ability of catalase to bind IAPP, Aβ, and PrP fibrils demonstrates the presence of similar accessible structural motifs that may be targets for antiamyloid therapeutic development.

Download Full-text