scholarly journals Photoacoustic Spectral Sensing Technique for Diagnosis of Biological Tissue Coagulation: In-Vitro Study

Diagnostics ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 133
Author(s):  
Deblina Biswas ◽  
George C. K. Chen ◽  
Hyoung Won Baac ◽  
Srivathsan Vasudevan
Keyword(s):  
Biological Tissue ◽  
In Vitro Study ◽  
Fold Increase ◽  
Dominant Frequency ◽  
Cancer Tissue ◽  
Thermal Coagulation ◽  
Tissue Heating ◽  
Coagulation Therapy ◽  
Spectral Sensing

Thermal coagulation of abnormal tissues has evolved as a therapeutic technique for different diseases including cancer. Tissue heating beyond 55 °C causes coagulation that leads to cell death. Noninvasive diagnosis of thermally coagulated tissues is pragmatic for performing efficient therapy as well as reducing damage of surrounding healthy tissues. We propose a noninvasive, elasticity-based photoacoustic spectral sensing technique for differentiating normal and coagulated tissues. Photoacoustic diagnosis is performed for quantitative differentiation of normal and coagulated excised chicken liver and muscle tissues in vitro by characterizing a dominant frequency of photoacoustic frequency spectrum. Pronounced distinction in the spectral parameter (i.e., dominant frequency) was observed due to change in tissue elastic property. We confirmed nearly two-fold increase in dominant frequencies for the coagulated muscle and liver tissues as compared to the normal ones. A density increase caused by tissue coagulation is clearly reflected in the dominant frequency composition. Experimental results were consistent over five different sample sets, delineating the potential of proposed technique to diagnose biological tissue coagulation and thus monitor thermal coagulation therapy in clinical applications.

scholarly journals Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study

2000 ◽  
Vol 350 (3) ◽  
pp. 723-734 ◽  
Author(s):  
Christine LACABARATZ-PORRET ◽  
Sophie LAUNAY ◽  
Elisabeth CORVAZIER ◽  
Raymonde BREDOUX ◽  
Béla PAPP ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Western Blotting ◽  
Time Course ◽  
In Vitro Study ◽  
Fold Increase ◽  
Distinct Pattern ◽  
Platelet Glycoprotein ◽  
Protein Patterns ◽  
Fold Induction

The endoplasmic reticulum (ER) plays a key role in Ca2+ signalling through Ca2+ release via inositol 1,4,5-trisphosphate receptors (InsP3-Rs) and Ca2+ uptake by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs). Here, we investigated the organization of platelet ER and its biogenesis during megakaryocytopoiesis. First, erythro/megakaryoblastic MEG 01, UT7, M-O7e and CHRF 288-11 cell lines, platelets and thrombopoietin-induced UT7-Mpl cells were selected for the study of SERCA2b and SERCA3 proteins by Western blotting using the antibodies IID8 and PL/IM430, respectively. As judged by platelet glycoprotein IIIa (GPIIIa) expression, an increase in SERCA3 proteins was observed while that of SERCA2b remained unchanged throughout maturation. Second, these studies were extended to the newly described alternatively spliced SERCA3a–c RNAs and InsP3-Rs using the in vitro model of PMA-induced differentiation of MEG 01 cells. Time-course and dose–response studies showed a maximal approx. 4-fold up-regulation of SERCA3 proteins using 10-8 M PMA for 3 days, which paralleled induction of GPIIIa expression. SERCA3 induction was found to occur at the level of mRNA. The modulation of the different SERCA3 species (i.e. 3a, 3b and 3c) was isoform-specific: while SERCA3a was slightly increased, an approx. 3-fold induction of SERCA3b, and a 4-fold induction of SERCA3c, was observed after 24h of PMA treatment. Isoform-specific Western blotting and/or reverse transcriptase PCR studies showed that InsP3-R types I, II and III are expressed in MEG 01 cells, as well as in platelets. Study of the expression of these InsP3-R types in PMA-induced MEG 01 cells revealed that: (i) InsP3-RI protein and mRNA showed no changes; (ii) InsP3-RII mRNA was up-regulated and peaked at hour 48 and (iii) InsP3-RIII mRNA and protein showed a transitory maximal 3- and 2.3-fold increase at hours 6 and 30, respectively. Upon PMA treatment of CHRF 288-11 cells, in which GPIIIa is not induced upon treatment, a similar pattern of regulation of InsP3-R types II and III was seen, but a distinct pattern of SERCA3 regulation was observed. These results suggest a profound reorganization of ER-protein patterns during megakaryocytopoiesis and underline the role of SERCA3 gene regulation in the control of Ca2+-dependent platelet functions.

scholarly journals Zinc and the Zinc Transporter SLC39A10/ZIP10 Are Required for Heme Synthesis in Developing Erythroid Progenitors

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 1320-1320
Author(s):  
Juyoung Kim ◽  
Jaekwon Lee ◽  
Moon-Suhn Ryu
Keyword(s):  
In Vitro Study ◽  
Fold Increase ◽  
Zinc Transporter ◽  
The Body ◽  
Zinc Homeostasis ◽  
Mean Corpuscular Hemoglobin ◽  
Erythroid Progenitors ◽  
Heme Synthesis ◽  
Cellular Zinc

Abstract Objectives Zinc is an essential nutrient for diverse biological processes in the body. Cellular zinc homeostasis is established through differential expressions of the transmembrane zinc transporter proteins, ZnTs and ZIPs. The aims of the current studies were to elucidate the roles of cellular zinc in erythrocyte maturation, and to determine the zinc transporters essential to erythroid zinc homeostasis. Methods G1E-ER4 mouse cells were employed as an in vitro study model of terminal erythroid differentiation. A cell-impermeable zinc chelator, diethylenetriamine pentaacetate (DTPA), was used to limit extracellular zinc availability. For gene silencing, gene-specific siRNAs were introduced to cells via Nucleofection. Functional impacts of zinc and gene deficiency were assessed via ICP-MS-based metal quantitation, heme assays, and gene expression assays using RNA-seq, qPCR, and western analyses. Results G1E-ER4 cells featured a 1.7-fold increase in total cellular zinc contents after 48 h of differentiation. Restriction of zinc import by 50 µM of DTPA led to less red coloration and lower increases in mean corpuscular hemoglobin contents by development. The heme deficiency by DTPA was fully restored by the addition of equimolar zinc, and was not due to changes in cellular iron contents. Zinc-deficient G1E-ER4 cells differentiated with normal Alas2 and Hbb-b1 transcript responses, but less Alad and alpha-globin expressions. Among the 24 zinc transporter genes, Zip10 produced the most prominent response to zinc restriction in differentiating erythroid cells. ZIP10-deficient G1E-ER4 cells were less efficient than control cells in hemoglobin production under zinc restriction. ZIP10 deficiency alone had no effects on the molecular indices of red cell hemoglobinization. Conclusions Our studies characterize zinc as a nutrient essential to normal erythroid maturation and heme synthesis. Moreover, we have identified a compensatory role of ZIP10 for erythroid zinc homeostasis during zinc restriction. Thus, poor zinc status and ZIP10 mutations might serve as potential risk factors and thus new therapeutic targets for anemia and other erythrocyte-related disorders. Funding Sources Supported by CFANS Graduate Fellowship to JK, and the Allen Foundation, Inc. and USDA NIFA Hatch Funds to M-SR.

scholarly journals Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential

2021 ◽  
Vol 22 (16) ◽  
pp. 8764
Author(s):  
Stefano Pulcini ◽  
Lucia Merolle ◽  
Chiara Marraccini ◽  
Eleonora Quartieri ◽  
Daniele Mori ◽  
...  
Keyword(s):  
Growth Factors ◽  
Platelet Rich Plasma ◽  
In Vitro Study ◽  
Fold Increase ◽  
Positive Control ◽  
Osteogenic Potential ◽  
Standardized Protocol ◽  
Proliferation And Differentiation ◽  
Potential Methods

Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects’ treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-β and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets.

scholarly journals Cell-penetrating doxorubicin released from Elastin-like polypeptide kills doxorubicin-resistant cancer cells in vitro study

2020 ◽  
Author(s):  
Jung Su Ryu ◽  
Felix Kratz ◽  
Drazen Raucher
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
In Vitro Study ◽  
Fold Increase ◽  
Membrane Penetration ◽  
Cancer Cell Death ◽  
Cell Penetrating ◽  
Resistant Cells

Abstract Background: Elastin-like polypeptide (ELP) undergoes its characteristic of phase transitioning in response to ambient temperature. ELP therefore has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be targeted to hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer, with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of ELP, a matrix metalloproteinase (MMP) substrate, a cell penetrating peptide (CPP), and 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This construct may be initially targeted to the tumor by application of mild heat after administration. Within the hyperthermic tumor, then this construct is cleaved by MMP, releasing CPP-Dox, which can infiltrate tumor tissues and penetrate cell membranes.Methods: We produced the construct in E.coli and examined its cleavage by MMP enzymes in vitro. Flow cytometry and confocal analysis were used to verify the facilitated uptake of the digested cell-penetrating Dox by breast cancer cells and Dox-resistant cells. Cytotoxicity tests further demonstrated improvements in bioavailability of cell-penetrating Dox following the enhanced cellular uptake of the cancer cells. Comparisons with the non-cleavable ELP counterpart were paralleled.Results: This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells than the ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES/ADR), ELP-released, cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells than ELP-Dox and free Dox. Conclusion: MMP-digested CPP-Dox shows better membrane penetration and induces more cancer cell death in vitro. This CPP-complexed Dox released from ELP kills even dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

scholarly journals Bovine gamma/delta T cells are stimulated by bovine coronavirus antigens identified by a soluble T cells receptor

2017 ◽  
Vol 73 (7) ◽  
pp. 412-417
Author(s):  
Felix N. Toka
Keyword(s):  
T Cells ◽  
Mhc Class Ii ◽  
Γδ T Cells ◽  
In Vitro Study ◽  
Fold Increase ◽  
In Vitro Assays ◽  
Gamma Delta ◽  
Bovine Coronavirus ◽  
Gm Csf

Gamma/delta (γδ) T cells in cattle account for an abundant T cell population. However, little is known regarding the function of γδ T cells as immune cells compared to αβ T cells. Not many pathogen-related antigens have been defined and known to stimulate γδ T cells. To address this information gap, we constructed a soluble receptor for bovine γδ T cells (sγδTCR) that was later used to identify two proteins (156 kDa and 102 kDa) or protein fragments expressed by bovine coronavirus (BCov). The molecular weight of the larger protein suggests it could be the spike glycoprotein of BCov. Subsequently, we used the identified viral proteins to study the reactivity of bovine γδ T cells. In vitro assays showed that purified preparations of the two proteins stimulated WC1+ γδ T cells isolated from cattle. A 4-fold increase in IFN production and a significantly higher expression of MHC class II was observed. However, these viral ligands could not stimulate γδ T cells to synthesize IL-8 or GM-CSF, known to be produced by γδ T cells when stimulated with bacterial antigens. Although the γδ T cells assessed here appeared activated by way of IFN and MHC II expression, surface markers such as CD2, CD25, CD44, CD62L and CD335 were not expressed at significant levels. Further, the activation elicited by viral ligands was not sufficient to induce cytotoxic capability in γδ T cells in vitro as measured by a flow cytometry-based cytotoxicity assay. This in vitro study shows that WC1+ γδ T cells can directly recognize viral antigen

Temperature sensing of adipose tissue heating with the luminescent upconversion nanoparticles as nanothermometer: in vitro study

2017 ◽  
Author(s):  
I. Yu. Yanina ◽  
E. K. Volkova ◽  
A. M. Zaharevich ◽  
J. G. Konyukhova ◽  
V. I. Kochubey ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
In Vitro Study ◽  
Temperature Sensing ◽  
Vitro Study ◽  
Upconversion Nanoparticles ◽  
Tissue Heating

scholarly journals Cell-Penetrating Doxorubicin Released from Elastin-Like Polypeptide Kills Doxorubicin-Resistant Cancer Cells in In Vitro Study

2021 ◽  
Vol 22 (3) ◽  
pp. 1126
Author(s):  
Jung Su Ryu ◽  
Felix Kratz ◽  
Drazen Raucher
Keyword(s):  
Cancer Cells ◽  
In Vitro Study ◽  
Fold Increase ◽  
Membrane Penetration ◽  
Cancer Cell Death ◽  
Cell Penetrating ◽  
Free Doxorubicin ◽  
Novel Strategy ◽  
Resistant Cells

Elastin-like polypeptides (ELPs) undergo a characteristic phase transition in response to ambient temperature. Therefore, it has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be used to target hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of an ELP, a matrix metalloproteinase (MMP) substrate, a cell-penetrating peptide (CPP), and a 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells compared to ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES-SA/Dx5), ELP-released cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells compared to ELP-Dox and free Dox. MMP-digested CPP-Dox showed better membrane penetration and induced more cancer cell death in vitro. This CPP-complexed Dox released from the ELP killed even Dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

scholarly journals Role of atrial tissue remodeling on rotor dynamics: an in vitro study

2015 ◽  
Vol 309 (11) ◽  
pp. H1964-H1973 ◽  
Author(s):  
Andreu M. Climent ◽  
María S Guillem ◽  
Lucia Fuentes ◽  
Peter Lee ◽  
Christian Bollensdorff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cultures ◽  
Cardiac Tissue ◽  
Early Stage ◽  
Tissue Remodeling ◽  
In Vitro Study ◽  
Rotor Dynamics ◽  
Dominant Frequency ◽  
Atrial Tissue

The objective of this article is to present an in vitro model of atrial cardiac tissue that could serve to study the mechanisms of remodeling related to atrial fibrillation (AF). We analyze the modification on gene expression and modifications on rotor dynamics following tissue remodeling. Atrial murine cells (HL-1 myocytes) were maintained in culture after the spontaneous initiation of AF and analyzed at two time points: 3.1 ± 1.3 and 9.7 ± 0.5 days after AF initiation. The degree of electrophysiological remodeling (i.e., relative gene expression of key ion channels) and structural inhomogeneity was compared between early and late cell culture times both in nonfibrillating and fibrillating cell cultures. In addition, the electrophysiological characteristics of in vitro fibrillation [e.g., density of phase singularities (PS/cm2), dominant frequency, and rotor meandering] analyzed by means of optical mapping were compared with the degree of electrophysiological remodeling. Fibrillating cell cultures showed a differential ion channel gene expression associated with atrial tissue remodeling (i.e., decreased SCN5A, CACN1C, KCND3, and GJA1 and increased KCNJ2) not present in nonfibrillating cell cultures. Also, fibrillatory complexity was increased in late- vs. early stage cultures (1.12 ± 0.14 vs. 0.43 ± 0.19 PS/cm2, P < 0.01), which was associated with changes in the electrical reentrant patterns (i.e., decrease in rotor tip meandering and increase in wavefront curvature). HL-1 cells can reproduce AF features such as electrophysiological remodeling and an increased complexity of the electrophysiological behavior associated with the fibrillation time that resembles those occurring in patients with chronic AF.

The Role of Ca2+I in Procoagulant Surface Expression and Apoptosis in Human Platelets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3569-3569
Author(s):  
Adam M. Gwozdz ◽  
Hong Wang ◽  
K.W. Annie Bang ◽  
Marian A. Packham ◽  
John Freedman ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Fold Increase ◽  
Surface Expression ◽  
Human Platelets ◽  
Apoptotic Pathway ◽  
Outer Leaflet ◽  
Activated Platelets

Abstract Asymmetry of phospholipids across the plasma membrane bilayer is a feature of all eukaryotic cells. When platelets are stimulated with certain agonsists, phospholipids are randomized by the action of a Ca2+-dependent scramblase enzyme, resulting in exposure of the anionic aminophospholipid phosphatidylserine (PS) on the outer leaflet that provides a procoagulant surface, catalyzing thrombin formation. We have previously demonstrated that the procoagulant surface of activated platelets persists in vitro for at least 4 hrs (Blood100:63b, 2002). Such persistence may propagate thrombosis in vivo when activated procoagulant platelets re-enter the circulation after fibrinolysis. There is currently little information concerning the mechanisms by which the procoagulant surface persists on activated platelets. In this in vitro study, the Ca2+-chelator BAPTA (0.1 μmol/109 platelets) was used to investigate the role of intracellular Ca2+ (Ca2+i) in procoagulant surface expression and persistence; PS expression was determined flow cytometrically by the binding of annexin A5-FITC. Unexpectedly, chelation of Ca2+i resulted in a 2–2.5x-fold increase in PS expression on the surface of platelets 5 min after activation with thrombin or thrombin+collagen (T+C), and this persisted for up to 4 hrs (last time point tested). Since PS expression is a hallmark of apoptosis in nucleated cells, we also examined another platelet apoptosis marker, the collapse of the mitochondrial inner membrane potential (ΔΨm), by flow cytometry using the potential-sensitive dye TMRM; PS expression was measured concurrently. This allowed us to distinguish between activated platelets expressing PS with an intact ΔΨm and apoptotic platelets expressing PS with a dissipated ΔΨm. 70–85% of the thrombin- or T+C-activated platelets expressing PS had an intact ΔΨm, which persisted for up to 4 hrs after activation. Thus, PS expression can occur independently of ΔΨm loss. However, chelation of Ca2+i with BAPTA resulted in 60–70% of the thrombin- or T+C-activated platelets persistently expressing PS to also have a collapsed ΔΨm, indicating that apoptotic pathways similar to those found in nucleated cells may modulate PS expression in platelets and may depend on Ca2+i concentrations. Caspases and calpain are centrally involved in apoptotic signaling and execution in nucleated cells. Caspases-9 and -3 have been identified in human platelets and may be responsible for downstream activation of calpain. We examined the effects of Ca2+i chelation in thrombin- and T+C- activated platelets on the activation of procaspases and calpain by Western blotting. In keeping with our observations of increased PS expression with concurrent ΔΨm loss in activated platelets with Ca2+i chelation, we observed cleavage of both procaspase-9, procaspase-3 and calpain, which did not occur in activated platelets without Ca2+i chelation. Taken together, our results indicate that Ca2+i levels in activated platelets may serve as a decisional checkpoint for the apoptotic pathway in human platelets, where procaspase-9 and procaspase-3 along with downstream calpain may function in a Ca2+-sensitive manner to protect platelets against PS exposure and ΔΨm collapse.

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