scholarly journals Non-invasive and label-free 3D-visualization shows in vivo oligomerization of the staphylococcal alkaline shock protein 23 (Asp23)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Inga Petersen ◽  
Rabea Schlüter ◽  
Katharina J. Hoff ◽  
Volkmar Liebscher ◽  
Gert Bange ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
3D Visualization ◽  
Bacterial Cells ◽  
Label Free ◽  
Serial Sections ◽  
Non Invasive ◽  
Native Proteins ◽  
Fluorescence Tags

AbstractFluorescence-tags, commonly used to visualize the spatial distribution of proteins within cells, can influence the localization of the tagged proteins by affecting their stability, interaction with other proteins or the induction of oligomerization artifacts. To circumvent these obstacles, a protocol was developed to generate 50 nm thick serial sections suitable for immunogold labeling and subsequent reconstruction of the spatial distribution of immuno-labeled native proteins within individual bacterial cells. Applying this method, we show a cellular distribution of the staphylococcal alkaline shock protein 23 (Asp23), which is compatible with filament formation, a property of Asp23 that we also demonstrate in vitro.

scholarly journals Label-free imaging of macrophage phenotypes and phagocytic activity in the human dermis in vivo using two-photon excited FLIM

2021 ◽  
Author(s):  
Marius Kröger ◽  
Jörg Scheffel ◽  
Evgeny A. Shirshin ◽  
Johannes Schleusener ◽  
Martina C Meinke ◽  
...  
Keyword(s):  
Fluorescence Lifetime Imaging ◽  
Label Free ◽  
Immune Effector ◽  
Effector Cells ◽  
Two Photon ◽  
Non Invasive ◽  
Human Dermis ◽  
Main Components

Macrophages (MΦs) are important immune effector cells that promote (M1 MΦs) or inhibit (M2 MΦs) inflammation and are involved in numerous physiological and pathogenic immune responses. Their precise role and relevance, however, is not fully understood because of the lack of non-invasive quantification methods. Here, we show that two-photon excited fluorescence lifetime imaging (TPE-FLIM), a label-free non-invasive method, can visualize MΦs in human dermis in vivo. We demonstrate in vitro that human dermal MΦs exhibit specific TPE-FLIM properties that distinguish them from the main components of the extracellular matrix and other dermal cells. We visualized MΦs, their phenotypes and phagocytosis in the skin of healthy individuals in vivo using TPE-FLIM. Additionally, machine learning identified M1 and M2 MΦs with a sensitivity of 0.88±0.04 and 0.82±0.03 and a specificity of 0.89±0.03 and 0.90±0.03, respectively. In clinical research, TPE-FLIM can advance the understanding of the role of MΦs in health and disease.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

Dosimetry and Radioenhancement Comparison of Gold Nanoparticles in Kilovoltage and Megavoltage Radiotherapy using MAGAT Polymer Gel Dosimeter

2019 ◽  
Vol 9 (2Apr) ◽  
Author(s):  
S Farahani ◽  
N Riyahi Alam ◽  
S Haghgoo ◽  
M Khoobi ◽  
Gh Geraily ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
3D Visualization ◽  
Energy Levels ◽  
Polymer Gel ◽  
External Radiotherapy ◽  
Dose Enhancement ◽  
Internal Radiotherapy ◽  
Boost Radiotherapy

Background: Numerous unique characteristics of the nanosized gold, including high atomic number, low toxicity, and high biocompatibility make it one of the most appropriate nanostructures to boost radiotherapy efficacy. Many in-vivo and in-vitro investigations have indicated that gold nanoparticles (AuNPs) can significantly increase tumor injuries in low kilovoltage radiotherapy. While deep-lying tumors require much higher energy levels with greater penetration power, and investigations carried out in megavoltage energy range show contradictory results.Objective: In this study, we quantitatively assess and compare dose enhancement factors (DEFs) obtained through AuNPs under radiation of Cobalt-60 source (1.25MeV) versus Iridium-192 source (0.380 KeV) using MAGAT gel dosimeter.Material and Methods: MAGAT polymer gel in both pure and combined with 0.2 mM AuNPs was synthesized. In order to quantify the effect of energy on DEF, irradiation was carried out by Co-60 external radiotherapy and Ir-192 internal radiotherapy. Finally, readings of irradiated and non-irradiated gels were performed by MR imaging.Result: The radiation-induced R2 (1/T2) changes of the gel tubes doped with AuNPs compared to control samples, upon irradiation of beams released by Ir-192 source showed a significant dose enhancement (15.31% ±0.30) relative to the Co-60 external radiotherapy (5.85% ±0.14).Conclusion: This preliminary study suggests the feasibility of using AuNPs in radiation therapy (RT), especially in low-energy sources of brachytherapy. In addition, MAGAT polymer gel, as a powerful dosimeter, could be used for 3D visualization of radiation dose distribution of AuNPs in radiotherapy.

scholarly journals Non-invasive skin sampling of tryptophan/kynurenine ratio in vitro towards a skin cancer biomarker

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Skaidre Jankovskaja ◽  
Johan Engblom ◽  
Melinda Rezeli ◽  
György Marko-Varga ◽  
Tautgirdas Ruzgas ◽  
...  
Keyword(s):  
Skin Cancer ◽  
Relative Increase ◽  
Cancer Biomarker ◽  
Cancer Diagnostics ◽  
Sampling Time ◽  
Skin Permeability ◽  
Experimental Conditions ◽  
Non Invasive

AbstractThe tryptophan to kynurenine ratio (Trp/Kyn) has been proposed as a cancer biomarker. Non-invasive topical sampling of Trp/Kyn can therefore serve as a promising concept for skin cancer diagnostics. By performing in vitro pig skin permeability studies, we conclude that non-invasive topical sampling of Trp and Kyn is feasible. We explore the influence of different experimental conditions, which are relevant for the clinical in vivo setting, such as pH variations, sampling time, and microbial degradation of Trp and Kyn. The permeabilities of Trp and Kyn are overall similar. However, the permeated Trp/Kyn ratio is generally higher than unity due to endogenous Trp, which should be taken into account to obtain a non-biased Trp/Kyn ratio accurately reflecting systemic concentrations. Additionally, prolonged sampling time is associated with bacterial Trp and Kyn degradation and should be considered in a clinical setting. Finally, the experimental results are supported by the four permeation pathways model, predicting that the hydrophilic Trp and Kyn molecules mainly permeate through lipid defects (i.e., the porous pathway). However, the hydrophobic indole ring of Trp is suggested to result in a small but noticeable relative increase of Trp diffusion via pathways across the SC lipid lamellae, while the shunt pathway is proposed to slightly favor permeation of Kyn relative to Trp.

scholarly journals In vivo and in vitro tracking of erosion in biodegradable materials using non-invasive fluorescence imaging

2011 ◽  
Vol 10 (9) ◽  
pp. 890-890 ◽  
Author(s):  
Natalie Artzi ◽  
Nuria Oliva ◽  
Cristina Puron ◽  
Sagi Shitreet ◽  
Shay Artzi ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Biodegradable Materials ◽  
Non Invasive

scholarly journals Non-invasive, real-time reporting drug release in vitro and in vivo

2015 ◽  
Vol 51 (32) ◽  
pp. 6948-6951 ◽  
Author(s):  
Yanfeng Zhang ◽  
Qian Yin ◽  
Jonathan Yen ◽  
Joanne Li ◽  
Hanze Ying ◽  
...  
Keyword(s):  
Drug Release ◽  
Real Time ◽  
Near Infrared ◽  
Reporting System ◽  
Real Time Monitoring ◽  
Near Infrared Fluorescence ◽  
Non Invasive ◽  
Fluorescence Dye

Anin vitroandin vivodrug-reporting system is developed for real-time monitoring of drug release via the analysis of the concurrently released near-infrared fluorescence dye.

scholarly journals Menstrual flow as a non-invasive source of endometrial organoids

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Tereza Cindrova-Davies ◽  
Xiaohui Zhao ◽  
Kay Elder ◽  
Carolyn J. P. Jones ◽  
Ashley Moffett ◽  
...  
Keyword(s):  
Early Pregnancy ◽  
Recurrent Miscarriage ◽  
Milk Proteins ◽  
Receptor Expression ◽  
In Vitro Fertilisation ◽  
Non Invasive ◽  
Menstrual Flow ◽  
Pregnancy Hormones

AbstractAssessment of the endometrium often necessitates a biopsy, which currently involves an invasive, transcervical procedure. Here, we present an alternative technique based on deriving organoids from menstrual flow. We demonstrate that organoids can be derived from gland fragments recovered from menstrual flow. To confirm they faithfully reflect the in vivo state we compared organoids derived from paired scratch biopsies and ensuing menstrual flow from patients undergoing in vitro fertilisation (IVF). We demonstrate that the two sets of organoids share the same transcriptome signature, derivation efficiency and proliferation rate. Furthermore, they respond similarly to sex steroids and early-pregnancy hormones, with changes in morphology, receptor expression, and production of ‘uterine milk’ proteins that mimic those during the late-secretory phase and early pregnancy. This technique has wide-ranging impact for non-invasive investigation and personalised approaches to treatment of common gynaecological conditions, such as endometriosis, and reproductive disorders, including failed implantation after IVF and recurrent miscarriage.

scholarly journals Distinct roles of tumor associated mutations in collective cell migration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rachel M. Lee ◽  
Michele I. Vitolo ◽  
Wolfgang Losert ◽  
Stuart S. Martin
Keyword(s):  
Collective Behavior ◽  
Metastatic Potential ◽  
Breast Epithelial Cell ◽  
Collective Cell Migration ◽  
Label Free ◽  
Collective Migration ◽  
Contrast Imaging ◽  
Pten Deletion

AbstractRecent evidence suggests that groups of cells are more likely to form clinically dangerous metastatic tumors, emphasizing the importance of understanding mechanisms underlying collective behavior. The emergent collective behavior of migrating cell sheets in vitro has been shown to be disrupted in tumorigenic cells but the connection between this behavior and in vivo tumorigenicity remains unclear. We use particle image velocimetry to measure a multidimensional migration phenotype for genetically defined human breast epithelial cell lines that range in their in vivo behavior from non-tumorigenic to aggressively metastatic. By using cells with controlled mutations, we show that PTEN deletion enhances collective migration, while Ras activation suppresses it, even when combined with PTEN deletion. These opposing effects on collective migration of two mutations that are frequently found in patient tumors could be exploited in the development of novel treatments for metastatic disease. Our methods are based on label-free phase contrast imaging, and thus could easily be applied to patient tumor cells. The short time scales of our approach do not require potentially selective growth, and thus in combination with label-free imaging would allow multidimensional collective migration phenotypes to be utilized in clinical assessments of metastatic potential.

scholarly journals OMRT-11. The effect of microenvironment on glioblastoma stem cells therapeutic resistance

2021 ◽  
Vol 3 (Supplement_2) ◽  
pp. ii9-ii9
Author(s):  
Tamara Lah Turnsek ◽  
Barbara Breznik ◽  
Bernarda Majc ◽  
Metka Novak ◽  
Andrej Porčnik ◽  
...  
Keyword(s):  
Stem Cells ◽  
Epithelial To Mesenchymal Transition ◽  
Combined Treatment ◽  
Cell Plasticity ◽  
Glioblastoma Stem Cells ◽  
Mesenchymal Transition ◽  
Non Invasive ◽  
Differential Gene

Abstract Epithelial-to-mesenchymal transition (EMT) is an essential molecular and cellular process in physiologic processes and invasion of various types of carcinoma and glioblastoma (GBM) cells. EMT is activated and regulated by specific endogenous triggers in complex network of intercellular interactions and signaling pathways. The hallmark of cancer-linked EMT are intermediate states that show notable cell plasticity, characteristic of cancer stem cells (CSCs), including glioblastoma stem cells – GSCs. GSCs resistance to irradiation (IR) and temozolomide (TMZ) chemotherapy is responsible for early relapses, even at distant brain sites. As GSCs are mostly homing to their “niches” as slowly-dividing GSC-subtype, mimicking a proneural-like non- invasive phenotype PN-genotype, we assume that this, by undergoing an EMT-like transition, GSCs are-reprogrammed to an invasive mesenchymal (MES) GBs/GSCs phenotype in a processes, called PMT (1). However, it is not known, if and by which environmental cues within the niche, this transition of GSCs is induced in vivo. In this work, we are presenting the transriptome data obtained when we exposed GSC spheroids to irradiation alone, TMZ alone and to the combined treatment in vitro and compared their differential genetic fingerprints related to EMT/PMT transition to the GSCs PMT transition, when embedded in their natural microenvironment in the GBM organoid model. The differential gene expression upon GSCs therapeutic perturbation (when alone and vs in the tumoroid microenvironment) will reveal the effects of the major candidate genes, associated with micronevironmendt stromal cells and matrix are contributing their observed EMT/PMT transition of GSCs in vivo. •1. Majc, B., Sever, T., Zarić, M, Breznik, B., Turk, B, Lah Turnšek, T. Epithelial- to-mesenchymal transition as the driver of changing carcinoma and glioblastoma microenvironment. DOI: 10.1016/j.bbamcr.2020.118782

scholarly journals Targeting Cancer Cell Tight Junctions Enhances PLGA-Based Photothermal Sensitizers’ Performance In Vitro and In Vivo

Pharmaceutics ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 43
Author(s):  
Victoria O. Shipunova ◽  
Vera L. Kovalenko ◽  
Polina A. Kotelnikova ◽  
Anna S. Sogomonyan ◽  
Olga N. Shilova ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
3D Culture ◽  
Intercellular Junctions ◽  
Cell Junctions ◽  
Multicellular Spheroids ◽  
Mesenchymal Transition ◽  
Non Invasive ◽  
Order Of Magnitude

The development of non-invasive photothermal therapy (PTT) methods utilizing nanoparticles as sensitizers is one of the most promising directions in modern oncology. Nanoparticles loaded with photothermal dyes are capable of delivering a sufficient amount of a therapeutic substance and releasing it with the desired kinetics in vivo. However, the effectiveness of oncotherapy methods, including PTT, is often limited due to poor penetration of sensitizers into the tumor, especially into solid tumors of epithelial origin characterized by tight cellular junctions. In this work, we synthesized 200 nm nanoparticles from the biocompatible copolymer of lactic and glycolic acid, PLGA, loaded with magnesium phthalocyanine, PLGA/Pht-Mg. The PLGA/Pht-Mg particles under the irradiation with NIR light (808 nm), heat the surrounding solution by 40 °C. The effectiveness of using such particles for cancer cells elimination was demonstrated in 2D culture in vitro and in our original 3D model with multicellular spheroids possessing tight cell contacts. It was shown that the mean inhibitory concentration of such nanoparticles upon light irradiation for 15 min worsens by more than an order of magnitude: IC50 increases from 3 µg/mL for 2D culture vs. 117 µg/mL for 3D culture. However, when using the JO-4 intercellular junction opener protein, which causes a short epithelial–mesenchymal transition and transiently opens intercellular junctions in epithelial cells, the efficiency of nanoparticles in 3D culture was comparable or even outperforming that for 2D (IC50 = 1.9 µg/mL with JO-4). Synergy in the co-administration of PTT nanosensitizers and JO-4 protein was found to retain in vivo using orthotopic tumors of BALB/c mice: we demonstrated that the efficiency in the delivery of such nanoparticles to the tumor is 2.5 times increased when PLGA/Pht-Mg nanoparticles are administered together with JO-4. Thus the targeting the tumor cell junctions can significantly increase the performance of PTT nanosensitizers.

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