An in vitro Mechanical Damage Model of Isolated Myofibers in a Floating Culture Condition

Abstract Background Diabetic retinopathy (DR) has become a worldwide concern because of the rising prevalence rate of diabetes mellitus (DM). Despite much energy has been committed to DR research, it remains a difficulty for diabetic patients all over the world. Since apoptosis of retinal microvascular pericytes (RMPs) is the early characteristic of DR, this study aimed to reveal the mechanism of Shuangdan Mingmu (SDMM) capsule, a Chinese patent medicine, on oxidative stress-induced apoptosis of pericytes implicated with poly (ADP-ribose) polymerase (PARP) / glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pathway. Methods Network pharmacology approach was performed to predict biofunction of components of SDMM capsule dissolved in plasma on DR. Both PARP1 and GAPDH were found involved in the hub network of protein-protein interaction (PPI) of potential targets and were found to take part in many bioprocesses, including responding to the regulation of reactive oxygen species (ROS) metabolic process, apoptotic signaling pathway, and response to oxygen levels through enrichment analysis. Therefore, in vitro research was carried out to validate the prediction. Human RMPs cultured with media containing 0.5 mM hydrogen oxide (H2O2) for 4 h was performed as an oxidative-damage model. Different concentrations of SDMM capsule, PARP1 inhibitor, PARP1 activation, and GAPDH inhibitor were used to intervene the oxidative-damage model with N-Acetyl-L-cysteine (NAC) as a contrast. Flow cytometry was performed to determine the apoptosis rate of cells and the expression of ROS. Cell counting kit 8 (CCK8) was used to determine the activity of pericytes. Moreover, nitric oxide (NO) concentration of cells supernatant and expression of endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), B cell lymphoma 2 (BCL2), vascular endothelial growth factor (VEGF), endothelin 1 (ET1), PARP1, and GAPDH were tested through RT-qPCR, western blot (WB), or immunocytochemistry (ICC). Results Overproduction of ROS, high apoptotic rate, and attenuated activity of pericytes were observed after cells were incubated with media containing 0.5 mM H2O2. Moreover, downregulation of SOD, NO, BCL2, and GAPDH, and upregulation of VEGFA, ET1, and PARP1 were discovered after cells were exposed to 0.5 mM H2O2 in this study, which could be improved by PARP1 inhibitor and SDMM capsule in a dose-dependent way, whereas worsened by PARP1 activation and GAPDH inhibitor. Conclusions SDMM capsule may attenuate oxidative stress-induced apoptosis of pericytes through downregulating PARP expression and upregulating GAPDH expression.

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Glycine ameliorates mitochondrial dysfunction caused by ABT-199 in porcine oocytes

Journal of Animal Science ◽

10.1093/jas/skab072 ◽

2021 ◽

Author(s):

Sicong Yu ◽

Lepeng Gao ◽

Yang Song ◽

Xin Ma ◽

Shuang Liang ◽

...

Keyword(s):

Oxidative Stress ◽

Mitochondrial Dysfunction ◽

Oocyte Maturation ◽

Mitochondrial Function ◽

Protective Action ◽

Damage Model ◽

Developmental Competence ◽

Free Calcium ◽

Maturation In Vitro

Abstract Mitochondria play an important role in controlling oocyte developmental competence. Our previous studies showed that glycine can regulate mitochondrial function and improve oocyte maturation in vitro. However, the mechanisms by which glycine affects mitochondrial function during oocyte maturation in vitro have not been fully investigated. In this study, we induced a mitochondrial damage model in oocytes with the Bcl-2-specific antagonist ABT-199. We investigated whether glycine could reverse the mitochondrial dysfunction induced by ABT-199 exposure and whether it is related to calcium regulation. Our results showed that ABT-199 inhibited cumulus expansion, decreased the oocyte maturation rate and the intracellular glutathione (GSH) level, caused mitochondrial dysfunction, induced oxidative stress, which was confirmed by decreased mitochondrial membrane potential (Δ⍦m) and the expression of mitochondrial function-related genes (PGC-1α), and increased reactive oxygen species (ROS) levels and the expression of apoptosis-associated genes (Bax, caspase-3, CytC). More importantly, ABT-199-treated oocytes showed an increase in the intracellular free calcium concentration ([Ca 2+]i) and had impaired cortical type 1 inositol 1,4,5-trisphosphate receptors (IP3R1) distribution. Nevertheless, treatment with glycine significantly ameliorated mitochondrial dysfunction, oxidative stress and apoptosis, glycine also regulated [Ca 2+]i levels and IP3R1 cellular distribution, which further protects oocyte maturation in ABT-199-induced porcine oocytes. Taken together, our results indicate that glycine has a protective action against ABT-199-induced mitochondrial dysfunction in porcine oocytes.

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Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

Nature Communications ◽

10.1038/s41467-021-23320-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Bei Liu ◽

Shi Chen ◽

Yaxing Xu ◽

Yulin Lyu ◽

Jinlin Wang ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Culture Condition ◽

Human Fibroblast ◽

Culture System ◽

Disease Modeling ◽

Directed Differentiation

AbstractExtended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

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PDCD4–MAPK–NF-κB Positive Loop Simultaneously Promotes Microglia Activation and Neuron Apoptosis During Neuroinflammation

10.21203/rs.3.rs-348335/v1 ◽

2021 ◽

Author(s):

Quan Chen ◽

Hongjian Lu ◽

Chengwei Duan ◽

Xiangyang Zhu ◽

Yi Zhang ◽

...

Keyword(s):

Nuclear Translocation ◽

Inflammatory Diseases ◽

Damage Model ◽

Pdcd4 Expression ◽

Proapoptotic Protein ◽

Inflammatory Activation ◽

Elevated Expression ◽

The Central Nervous System ◽

Signaling Activation

Abstract Neuroinflammation and neuron injury are common features of the central nervous system (CNS) diseases. It is of great significance to identify their shared regulatory mechanisms and explore the potential therapeutic targets. Programmed cell death factor 4 (PDCD4), an apoptosis-related molecule, extensively participates in tumorigenesis and inflammatory diseases, but its expression and biological function during CNS neuroinflammation remain unclear. In the present study, utilizing the lipopolysaccharide (LPS)-induced neuroinflammation model in mice, we reported an elevated expression of PDCD4 both in injured neurons and activated microglia of the inflamed brain. A similar change in PDCD4 expression was observed in vitro in the microglial activation model. Silencing PDCD4 by shRNA significantly inhibited the phosphorylation of MAPKs (p38, ERK, and JNK), prevented the phosphorylation and nuclear translocation of NF-κB p65, and thus attenuated the LPS-induced microglial inflammatory activation. Interestingly, LPS also required the MAPK/NF-κB signaling activation to boost PDCD4 expression in microglia, indicating the presence of a positive loop. Moreover, a persistent elevation of PDCD4 expression was detected in the H2O2-induced neuronal oxidative damage model. Knocking down PDCD4 significantly inhibited the expression of proapoptotic protein BAX, suggesting the proapoptotic activity of PDCD4 in neurons. Taken together, our data indicated that PDCD4 may serve as a hub regulatory molecule that simultaneously promotes the microglial inflammatory activation and the oxidative stress-induced neuronal apoptosis within CNS. The microglial PDCD4–MAPK–NF-κB positive feedback loop may exaggerate the vicious cycle of neuroinflammation and neuronal injury and thus may become a potential therapeutic target for neuroinflammatory diseases.

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A Rational Methodology for Determination of Service Life for a Delayed Coker Drum

Volume 3: Design and Analysis ◽

10.1115/pvp2015-45028 ◽

2015 ◽

Author(s):

John J. Aumuller ◽

Jie Chen ◽

Vincent A. Carucci

Keyword(s):

Service Life ◽

Low Cycle Fatigue ◽

Damage Model ◽

Mechanical Damage ◽

Damage Mechanism ◽

Modern Trend ◽

Cold Spot ◽

Chromium Alloy ◽

Cycle Fatigue ◽

Service Environment

Delayed unit coker drums operate in a severe service environment that precludes long term reliability due to excessive shell bulging and cracking of shell joint and shell to skirt welds. Thermal fatigue is recognized as the leading damage mechanism and past work has provided an idealized description of the thermo-mechanical mechanism via local hot and cold spot formation to quantify a lower bound life estimate for shell weld failure. The present work extends this idealized thermo-mechanical damage model by evaluating actual field data to determine a potential upper bound life estimate. This assessment also provides insight into practical techniques for equipment operators to identify design and operational opportunities to extend the service life of coke drums for their specific service environments. A modern trend of specifying higher chromium and molybdenum alloy content for drum shell material in order to improve low cycle fatigue strength is seen to be problematic; rather, the use of lower alloy materials that are generally described as fatigue tough materials are better suited for the high strain-low cycle fatigue service environment of coke drums. Materials such as SA 204 C (C – ½ Mo) and SA 302 B (C – Mn – ½ Mo) or SA 302 C (C – Mn – ½ Mo – ½ Ni) are shown to be better candidates for construction in lieu of low chromium alloy steel materials such as SA 387 grades P11 (1¼ Cr – ½ Mo), P12 (1 Cr – ½ Mo), P22 (2¼ Cr – 1 Mo) and P21 (3 Cr – 1 Mo).

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Resveratrol-loaded peptide-hydrogels inhibit scar formation in wound healing through suppressing inflammation

Regenerative Biomaterials ◽

10.1093/rb/rbz041 ◽

2019 ◽

Cited By ~ 2

Author(s):

Chen-Chen Zhao ◽

Lian Zhu ◽

Zheng Wu ◽

Rui Yang ◽

Na Xu ◽

...

Keyword(s):

Wound Healing ◽

Damage Model ◽

Skin Wound ◽

Scar Formation ◽

Wound Dressings ◽

Skin Damage ◽

Skin Wound Healing ◽

Accelerate Wound Healing ◽

Peptide Hydrogels

Abstract Scar formation seriously affects the repair of damaged skin especially in adults and the excessive inflammation has been considered as the reason. The self-assembled peptide-hydrogels are ideal biomaterials for skin wound healing due to their similar nanostructure to natural extracellular matrix, hydration environment and serving as drug delivery systems. In our study, resveratrol, a polyphenol compound with anti-inflammatory effect, is loaded into peptide-hydrogel (Fmoc-FFGGRGD) to form a wound dressing (Pep/RES). Resveratrol is slowly released from the hydrogel in situ, and the release amount is controlled by the loading amount. The in vitro cell experiments demonstrate that the Pep/RES has no cytotoxicity and can inhibit the production of pro-inflammatory cytokines of macrophages. The Pep/RES hydrogels are used as wound dressings in rat skin damage model. The results suggest that the Pep/RES dressing can accelerate wound healing rate, exhibit well-organized collagen deposition, reduce inflammation and eventually prevent scar formation. The Pep/RES hydrogels supply a potential product to develop new skin wound dressings for the therapy of skin damage.

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Synergy of NUP98-HOXA10 Fusion Gene and NrasG12D Mutation Preserves the Stemness of Hematopoietic Stem Cells on Culture Condition

Cells ◽

10.3390/cells8090951 ◽

2019 ◽

Vol 8 (9) ◽

pp. 951 ◽

Cited By ~ 2

Author(s):

Yong Dong ◽

Chengxiang Xia ◽

Qitong Weng ◽

Tongjie Wang ◽

Fangxiao Hu ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Pluripotent Stem Cells ◽

Culture Condition ◽

Fusion Gene ◽

Nras Mutation ◽

Hematopoietic Stem ◽

Bona Fide

Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.

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Coupled Thermal-Mechanical Progressive Damage Model with Strain and Heating Rate Effects for Lightning Strike Damage Assessment

Applied Composite Materials ◽

10.1007/s10443-019-09789-z ◽

2019 ◽

Vol 26 (5-6) ◽

pp. 1437-1459 ◽

Cited By ~ 3

Author(s):

S. L. J. Millen ◽

A. Murphy ◽

G. Catalanotti ◽

G. Abdelal

Keyword(s):

Heating Rate ◽

Thermal Loading ◽

Damage Model ◽

Applied Pressure ◽

Mechanical Damage ◽

Failure Criteria ◽

Lightning Strike ◽

Progressive Damage ◽

Rate Effects ◽

Progressive Damage Model

AbstractThis paper proposes a progressive damage model incorporating strain and heating rate effects for the prediction of composite specimen damage resulting from simulated lightning strike test conditions. A mature and robust customised failure model has been developed. The method used a scaling factor approach and non-linear degradation models from published works to modify the material moduli, strength and stiffness properties to reflect the effects of combined strain and thermal loading. Hashin/Puck failure criteria was used prior to progressive damage modelling of the material. Each component of the method was benchmarked against appropriate literature. A three stage modelling framework was demonstrated where an initial plasma model predicts specimen surface loads (electrical, thermal, pressure); a coupled thermal-electric model predicts specimen temperature resulting from the electrical load; and a third, dynamic, coupled temperature-displacement, explicit model predicts the material state due to the thermal load, the resulting thermal-expansion and the lightning plasma applied pressure loading. Unprotected specimen damage results were presented for two SAE lightning test Waveforms (B & A); with the results illustrating how thermal and mechanical damage behaviour varied with waveform duration and peak current.

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Gradient enhanced thermo-mechanical damage model for concrete at high temperatures including transient thermal creep

International Journal for Numerical and Analytical Methods in Geomechanics ◽

10.1002/nag.376 ◽

2004 ◽

Vol 28 (78) ◽

pp. 715-735 ◽

Cited By ~ 27

Author(s):

Chris J. Pearce ◽

Claus V. Nielsen ◽

N. Bićanić

Keyword(s):

High Temperatures ◽

Damage Model ◽

Mechanical Damage ◽

Thermal Creep ◽

Transient Thermal

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Evaluation of Role for flt3L in Human Early B and T/NK Lymphopoiesis in a Novel Stromal Cell Culture System

Blood ◽

10.1182/blood.v118.21.2385.2385 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2385-2385

Author(s):

Yoshiki Nakamori ◽

Kohshi Ohishi ◽

Bing Liu ◽

Masahiro Masuya ◽

Hirofumi Hamada ◽

...

Keyword(s):

Gene Expression ◽

Stromal Cells ◽

Culture Condition ◽

Culture System ◽

Nk Cell ◽

Cell Development ◽

Cellular Interaction ◽

Differentiation Potential ◽

Vitro Assay

Abstract Abstract 2385 The regulatory mechanism of human early lymphopoiesis remains less defined. A major limitation of conventional in vitro assay is that B and T lymphopoiesis cannot be assessed simultaneously. We exploded a novel culture assay and found that the hTERT-transduced telomerized human stromal cells support the generation of CD19+CD34lo/-CD10+cyCD79a+CD20+/−VpreB− pro B- and CD7+CD34+CD45RA+CD56−cyCD3− early T-cell precursors from human hematopoietic progenitors without cytokines, which was enhanced by flt3L (2010, ASH). We further characterized the generated lymphoid precursors, and verified that lineage-specific transcription factors for B (Pax-5 and EBF) and T/NK-cell precursors (GATA-3, HEB, Id2) were expressed by the CD19+ and CD7+CD56− cells, respectively. The CD7+CD56− cells showed the differentiation potential to T- and NK-lineage cells, by replating with OP9-Delta1 in the presence of flt3L+IL-7, and with the telomerized-stromal cells in the presence of flt3L+IL-7+IL-15, respectively. In serum-free culture condition, B-cell differentiation was minimally supported by the stromal cells, while low number of CD7+ cells was observed. Nonetheless, a significant number of CD7+, CD19-cyCD79a+, and CD19+ cells developed in the presence of flt3L, suggesting an important role for flt3L in the generation of early T/NK- and, particularly, B-lineage cell precursors on the stromal cells. The cellular interaction between Notch and Notch ligand Delta-1 or -4 in the presence of appropriate cytokines is considered to be crucial in T-lineage cell development in mice. We therefore examined whether Notch pathway is involved in the T/NK-cell development under this culture condition. While the gene expression of Delta-1 or -4 was detected in the telomerized stromal cells, the protein expression of these ligands was not detected with Western-blotting analysis. After co-culture of hematopoietic progenitor cells with the telomerized stromal cells, the gene expression of Notch target gene, HES1, was not increased. These data suggest that Notch signaling is not involved in the generation of early T/NK cell precursors on the stromal cells. This novel in vitro culture system suggests that the development of early B- and T/NK- cell precursors from hematopoietic precursors take places on the stromal cells in a Notch-independent manner and that flt3L plays a principal role in the stimulation of the early B and T/NK lymphopoiesis. Disclosures: No relevant conflicts of interest to declare.

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