Response differences of HepG2 and Primary Mouse Hepatocytes to morphological changes in electrospun PCL scaffolds

AbstractLiver disease cases are rapidly expanding across the globe and the only effective cure for end-stage disease is a transplant. Transplant procedures are costly and current supply of donor livers does not satisfy demand. Potential drug treatments and regenerative therapies that are being developed to tackle these pressing issues require effective in-vitro culture platforms. Electrospun scaffolds provide bio-mimetic structures upon which cells are cultured to regulate function in-vitro. This study aims to shed light on the effects of electrospun PCL morphology on the culture of an immortalised hepatic cell line and mouse primary hepatocytes. Each cell type was cultured on large 4–5 µm fibres and small 1–2 µm fibres with random, aligned and highly porous cryogenically spun configurations. Cell attachment, proliferation, morphology and functional protein and gene expression was analysed. Results show that fibre morphology has a measurable influence on cellular morphology and function, with the alteration of key functional markers such as CYP1A2 expression.

Download Full-text

Evidence for diagnosis of early chronic pancreatitis after three episodes of acute pancreatitis: a cross-sectional multicentre international study with experimental animal model

Scientific Reports ◽

10.1038/s41598-020-80532-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Péter J. Hegyi ◽

Alexandra Soós ◽

Emese Tóth ◽

Attila Ébert ◽

Viktória Venglovecz ◽

...

Keyword(s):

Acute Pancreatitis ◽

Chronic Pancreatitis ◽

Morphological Changes ◽

International Study ◽

Study Data ◽

Cross Sectional ◽

Clinical Biomarkers ◽

Stage Disease ◽

Significant Difference ◽

End Stage

AbstractChronic pancreatitis (CP) is an end-stage disease with no specific therapy; therefore, an early diagnosis is of crucial importance. In this study, data from 1315 and 318 patients were analysed from acute pancreatitis (AP) and CP registries, respectively. The population from the AP registry was divided into AP (n = 983), recurrent AP (RAP, n = 270) and CP (n = 62) groups. The prevalence of CP in combination with AP, RAP2, RAP3, RAP4 and RAP5 + was 0%, 1%, 16%, 50% and 47%, respectively, suggesting that three or more episodes of AP is a strong risk factor for CP. Laboratory, imaging and clinical biomarkers highlighted that patients with RAP3 + do not show a significant difference between RAPs and CP. Data from CP registries showed 98% of patients had at least one AP and the average number of episodes was four. We mimicked the human RAPs in a mouse model and found that three or more episodes of AP cause early chronic-like morphological changes in the pancreas. We concluded that three or more attacks of AP with no morphological changes to the pancreas could be considered as early CP (ECP).The new diagnostic criteria for ECP allow the majority of CP patients to be diagnosed earlier. They can be used in hospitals with no additional costs in healthcare.

Download Full-text

The effect of in vitro exposure to antisense oligonucleotides on macrophage morphology and function

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2011.3515 ◽

2011 ◽

Vol 2 (1) ◽

pp. 12 ◽

Cited By ~ 4

Author(s):

Ann Brasey ◽

Raouf Igue ◽

Loubna Djemame ◽

Serge Séguin ◽

Paolo Renzi ◽

...

Keyword(s):

Antisense Oligonucleotides ◽

Morphological Changes ◽

Lower Airway ◽

Functional Responses ◽

Phagocytic Capacity ◽

Proinflammatory Mediators ◽

Functional Consequences ◽

And Function

Antisense oligonucleotides (AON) delivered via inhalation are in drug development for respiratory diseases. In rodents and monkeys, repeated exposure to high doses of inhaled phosphorothioate (PS) AON can lead to microscopic changes in the lungs, including accumulation of alveolar macrophages in the lower airway that have a foamy appearance. The functional consequences that result from this morphological change are unclear as there is controversy whether the vacuoles/inclusion bodies reflect normal clearance of the inhaled AON or are early indicators of lung toxicity. The morphological and functional responses of macrophage to PS AON were characterized in vitro using the comparator drug amiodarone, as a known inducer of foamy macrophages. Morphological changes of increased vacuolization with the presence of lamellated structures were observed in macrophages in response to both amiodarone and AON treatment. Functional responses to the drugs clearly differed with amiodarone treatment leading to apoptosis of cells and cell death, release of proinflammatory mediators IL-1RA, MIP-1α and TNFα, decrease in IP-10, a cytokine shown to be involved in protection against pulmonary fibrosis and altered phagocytosis capacity of the cells. In contrast, AON in concentrations up to 30 μM, had no effect on cell viability or apoptosis, had minimal effects on pro-inflammatory cytokines, increased IP-10 levels and did not alter the phagocytic capacity of the cells. Exposure of macrophages to AON in vitro, led to morphological changes of increased vacuolization, but did not lead to functional consequences which were observed with another vacuolization-inducing drug, suggesting that the in vivo phenotypic changes observed following inhalation of AON may be consistent with a clearance mechanism and not an activation or impairment of macrophages.

Download Full-text

Upregulated Expression of Fibronectin Receptors Underlines the Adhesive Capability of Thymocytes to Thymic Epithelial Cells During the Early Stages of Differentiation: Lessons From Sublethally Irradiated Mice

Blood ◽

10.1182/blood.v93.3.974.403k19_974_990 ◽

1999 ◽

Vol 93 (3) ◽

pp. 974-990

Author(s):

Sergio R. Dalmau ◽

Claudia S. Freitas ◽

Wilson Savino

Keyword(s):

Epithelial Cells ◽

Cell Attachment ◽

Whole Body ◽

Thymic Epithelial Cells ◽

Strongly Correlated ◽

Enhanced Expression ◽

Single Positive ◽

Young Stage ◽

And Function

A 250-cGy whole-body γ-radiation dose was used to induce thymus regression in mice, and to study the expression and function of extracellular matrix (ECM) receptors in distinct thymocyte subsets emerging during repopulation of the organ. The onset of regeneration was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable increase in the absolute counts of CD3−CD25hiCD44+ and CD3−CD25in/hiCD44−cells occurred. Enhanced expression of L-selectin, 4, and 5 integrin chains (L-selhi 4hi5hi) was also exhibited by these cells. This pattern of expression was maintained until the CD4+CD8+ (DP) young stage was achieved. Afterward, there was a general downregulation of these ECM receptors in DP as well as in CD4+ or CD8+ single positive (SP) thymocytes (L-selin 4in5in). In some recently generated SP cells, 4 expression was downregulated before the 5 chain, and L-selectin was upregulated in half of more mature cells. The expression of the 6 integrin chain was downregulated only in maturing CD4+cells. Importantly, the increased expression of L-selectin and 4 and 5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC segment of fibronectin is targeted by the 4 integrin during thymocyte/TEC adhesion; (2) that the RGD motif of the 120-kD fragment of fibronectin, a target for 5 integrin, has a secondary role in this adhesion; and (3) that the YIGSR cell attachment motif of the β1 chain of laminin/merosin recognized by a nonintegrin receptor is not used for thymocyte adherence. In conclusion, our results show that an upregulated set of receptors endows CD25+ precursors and cells up to the young DP stage with a high capability of interacting with thymic ECM components.

Download Full-text

Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

Journal of Cell Science ◽

10.1242/jcs.109.6.1479 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1479-1495 ◽

Cited By ~ 4

Author(s):

L.A. Temesvari ◽

J.M. Rodriguez-Paris ◽

J.M. Bush ◽

L. Zhang ◽

J.A. Cardelli

Keyword(s):

Morphological Changes ◽

Specific Inhibitor ◽

Fluid Phase ◽

Contractile Vacuole ◽

Dependent Manner ◽

Concanamycin A ◽

Latex Beads ◽

And Function

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

Download Full-text

Subpopulations of soluble, misfolded proteins commonly bypass chaperones: How it happens at the molecular level

10.1101/2021.08.18.456736 ◽

2021 ◽

Author(s):

Ritaban Halder ◽

Daniel A. Nissley ◽

Ian Sitarik ◽

Edward P. O’Brien

Keyword(s):

Tertiary Structure ◽

Molecular Level ◽

Meta Analysis ◽

Hydrophobic Surface ◽

Misfolded Proteins ◽

Similar Degree ◽

Functional Protein ◽

Folded Proteins ◽

And Function

ABSTRACTSubpopulations of soluble, misfolded proteins can bypass chaperones within cells. The scope of this phenomenon and the lifetimes of these states have not been experimentally quantified, and how such misfolding happens at the molecular level is poorly understood. We address the first issue through a meta-analysis of the experimental literature. We find that in all quantitative protein refolding-function studies, there is always a subpopulation of soluble but misfolded and less-functional protein that does not fold in the presence of one or more chaperones. This subpopulation ranges from 8% to 50% of the soluble protein molecules in solution. Fitting the experimental time traces to a kinetic model, we find these chaperone-bypassing misfolded states take months or longer to fold and function in the presence of different chaperones. We next addressed how, at the molecular level, some misfolded proteins can evade chaperones by simulating six different proteins interacting with E. coli’s GroEL and HtpG chaperones when those proteins are in folded, unfolded, or long-lived, soluble, misfolded states. We observe that both chaperones strongly bind the unfolded state and weakly bind the folded and misfolded states to a similar degree. Thus, these chaperones cannot distinguish between the folded and long-lived misfolded states of these proteins. A structural analysis reveals the misfolded states are highly similar to the native state – having a similar size, amount of exposed hydrophobic surface area, and level of tertiary structure formation. These results demonstrate that in vitro it is common for appreciable subpopulations of proteins to remain misfolded, soluble, and evade the refolding action of chaperones for very long times. Further, these results suggest that this happens because these misfolded subpopulations are near-native and therefore interact with chaperones to a similar extent as properly folded proteins. More broadly, these results indicate a mechanism in which long-time scale changes in protein structure and function can persist in cells because some protein’s non-native states can bypass components of the proteostasis machinery.TEASERNear-native, misfolded protein conformations explain why some soluble proteins fail to refold in the presence of chaperones.

Download Full-text

Human endothelial cell morphology and autacoid expression

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.268.4.h1613 ◽

1995 ◽

Vol 268 (4) ◽

pp. H1613-H1620

Author(s):

C. J. de Groot ◽

V. A. Chao ◽

J. M. Roberts ◽

R. N. Taylor

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Morphological Changes ◽

Umbilical Vein ◽

Three Dimensional ◽

Endothelin 1 ◽

And Function ◽

Cellular Fibronectin ◽

Cells Cultured

Human umbilical vein endothelial (HUVE) cells plated on plastic or gelatin-coated dishes grow as a “cobblestone” monolayer. By contrast, endothelial cells cultured on a complex matrix (e.g., Matrigel) form three-dimensional, capillary-like structures. In the current study, we verified the capillary phenotype of the latter structures and asked whether the morphological changes induced by extracellular matrix also affect human endothelial gene expression and function in vitro. Concentrations of cellular fibronectin, prostacyclin, and endothelin-1 were measured in the conditioned media by enzyme-linked immunosorbent and radioimmunoassays. Steady-state concentrations of HUVE mRNA were estimated by reverse transcription-polymerase chain reaction and quantified by Northern analyses to assess fibronectin and endothelin-1 gene expression. We found that the subjacent extracellular matrix affects the morphology, proliferation, and differentiation of HUVE cells in vitro. Cells cultured on gelatin were more mitotically active, expressed significantly less cellular fibronectin, made similar amounts of prostacyclin, and secreted significantly more endothelin-1 compared with the same cells grown on a Matrigel substrate.

Download Full-text

Scaffolds from Surgically Removed Kidneys as a Potential Source of Organ Transplantation

BioMed Research International ◽

10.1155/2015/325029 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Marek Karczewski ◽

Tomasz Malkiewicz

Keyword(s):

Cell Attachment ◽

Kidney Tissue ◽

Point Of View ◽

Common Disease ◽

Kidney Graft ◽

Binding Domains ◽

Ecm Architecture ◽

Stage Renal Disease ◽

End Stage

End stage renal disease (ESRD) is a common disease, which relates to nearly 600 million people in the total population. What is more, it seems to be a crucial problem from the epidemiological point of view. These facts lead to a further necessity of renal replacement therapy development connected with rising expenditures for the health care system. The aim of kidney tissue engineering is to develop and innovate methods of obtaining renal extracellular matrix (ECM) scaffolds derived from kidney decellularization. Recently, progress has been made towards developing a functional kidney graftin vitroon demand. In fact, decellularized tissues constitute ideal natural scaffolds, due to the preservation of native ECM architecture, as well as of cell-ECM binding domains critical in promoting cell attachment, migration, and proliferation. One of the potential sources of the natural scaffolds is the kidney, which cannot be transplanted immediately after excision.

Download Full-text

Multiple prebiotic metals mediate translation

10.1101/256958 ◽

2018 ◽

Cited By ~ 1

Author(s):

Marcus S. Bray ◽

Timothy K. Lenz ◽

Jay William Haynes ◽

Jessica C. Bowman ◽

Anton S. Petrov ◽

...

Keyword(s):

Messenger Rna ◽

Tertiary Structure ◽

Cell Function ◽

Living Organism ◽

Translation System ◽

Ancestral State ◽

Functional Protein ◽

Functional Roles ◽

And Function

ABSTRACTToday, Mg2+is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2’HydroxylAcylation analyzed byPrimerExtension) was used to compare the effect of Mg2+, Fe2+, and Mn2+on the tertiary structure of rRNA. Then, we usedin vitrotranslation reactions to test whether Fe2+or Mn2+could mediate protein production, and quantified ribosomal metal content. We found that: (i) Mg2+, Fe2+, and Mn2+had strikingly similar effects on rRNA folding; (ii) Fe2+and Mn2+can replace Mg2+as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+and Mn2+were abundant, these findings suggest that Fe2+and Mn2+played a role in early ribosomal evolution.SIGNIFICANCERibosomes are found in every living organism where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ~4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O2, high Fe2+, high Mn2+). Our results expand the roles of Fe2+and Mn2+in ancient and extant biochemistry as cofactors for ribosomal structure and function.

Download Full-text

Drosophila PS integrins recognize vertebrate vitronectin and function as cell-substratum adhesion receptors in vitro

Development ◽

10.1242/dev.113.3.1007 ◽

1991 ◽

Vol 113 (3) ◽

pp. 1007-1016 ◽

Cited By ~ 6

Author(s):

S. Hirano ◽

K. Ui ◽

T. Miyake ◽

T. Uemura ◽

M. Takeichi

Keyword(s):

Cell Attachment ◽

Collagen Type I ◽

Matrix Proteins ◽

Type I ◽

Rgd Peptides ◽

Drosophila Cell ◽

Type Iv ◽

And Function

Using the Drosophila cell line MLDmBG-1, a monoclonal antibody aBG-1 that can inhibit not only cell clumping but also cell spreading was generated. This antibody immunoprecipitates a complex of molecules consisting of a major 120 × 10(3) Mr and other components. To characterize the 120 × 10(3) Mr component, we purified it, generated antibodies to it, and cloned its cDNA. Sequencing of this cDNA suggests that the 120 × 10(3) Mr molecule is identical to PS beta, a beta chain of Drosophila integrins. The other components immunoprecipitated included two alpha chains of Drosophila integrins, PS1 alpha and PS2 alpha, as revealed using specific antibodies to these molecules. These suggest that aBG-1 recognizes the PS beta associated with PS1 alpha or PS2 alpha. However, immunostaining of embryos and larvae with aBG-1 showed that the staining pattern is similar to that for PS2 alpha but not for PS beta, suggesting that the antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. We then attempted to characterize the ligands for these integrin complexes, using culture dishes coated with various vertebrate matrix proteins. These cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. This spreading was partially inhibited by aBG-1, but not by other control antibodies or RGD peptides. The cell attachment to these substrata was not affected by the antibody. The cells also can attach to dishes coated with laminin but without spreading, and this attachment was not inhibited by aBG-1. Furthermore, they do not attach to dishes coated with collagen type I, type IV, and fibrinogen. These results indicate that Drosophila PS integrins can recognize vertebrate vitronectin, and also fibronectin with a weaker affinity, at sites other than RGD sequences, and thus can function in cell-substratum adhesion.

Download Full-text

PrPSc with Seeding Activity Extensively Overlaps with Proteinase-Resistant PrPSc Rather than Infectious PrPSc

Pathogens ◽

10.3390/pathogens9030241 ◽

2020 ◽

Vol 9 (3) ◽

pp. 241 ◽

Cited By ~ 1

Author(s):

Yoshifumi Iwamaru ◽

Yuichi Matsuura ◽

Kohtaro Miyazawa

Keyword(s):

Spongiform Encephalopathy ◽

Prion Infection ◽

Infectious Titer ◽

Stage Disease ◽

Amplification Assay ◽

End Stage ◽

Dose Levels ◽

The Relationship ◽

Conformational Conversion

The disease-associated prion protein (PrPSc) has the ability to seed the conformational conversion of normal prion proteins into the amyloid fibril form. This prion seeding activity can be measured using an in vitro amplification assay termed real-time quaking-induced conversion (RT-QuIC). There is a strong correlation between RT-QuIC positivity and prion infection; however, the relationship between seeding activity and infectivity remains elusive. In this study, we used endpoint dilution RT-QuIC on the brain homogenates from wild-type mice with mouse-adopted bovine spongiform encephalopathy (mBSE) at defined intervals during the incubation period and evaluated the temporal relationship among prion seeding dose, levels of proteinase-resistant PrPSc (PrPres), and infectious titer. We found that the infectious titer reached a plateau by 100 days postinfection, whereas seeding dose and PrPres levels were continuously elevated. Our calculation showed that the doubling time (dt) for seeding dose from 40 to 100 days postinoculation was closer to the dt for PrPres levels than to the dt for prion titer. Although an uncoupling of seeding doses and PrPres levels was observed at end-stage disease in this model, our findings suggest that there is substantial but not complete overlap between PrPSc with seeding activity and PrPres rather than infectious PrPSc.

Download Full-text