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APL Bioengineering Jun 2024Activation of fibroblasts is pivotal for wound healing; however, persistent activation leads to maladaptive processes and is a hallmark of fibrosis, where disease...
Activation of fibroblasts is pivotal for wound healing; however, persistent activation leads to maladaptive processes and is a hallmark of fibrosis, where disease mechanisms are only partially understood. Human model systems complement animal models for both hypothesis testing and drug evaluation to improve the identification of therapeutics relevant to human disease. Despite advances, a challenge remains in understanding the dynamics of human fibroblast responses to complex microenvironment stimuli, motivating the need for more advanced tools to investigate fibrotic mechanisms. This work established approaches for assessing the temporal dynamics of these responses using genetically encoded fluorescent reporters of alpha smooth muscle actin expression, an indicator of fibroblast activation. Specifically, we created a toolset of human lung fibroblast reporter cell lines from different origins (male, female; healthy, idiopathic pulmonary fibrosis) and used three different versions of the reporter with the fluorescent protein modified to exhibit different temporal stabilities, providing temporal resolution of protein expression processes over a range of timescales. Using this toolset, we demonstrated that reporters provide insight into population shifts in response to both mechanical and biochemical cues that are not detectable by traditional end point assessments with differential responses based on cell origin. Furthermore, individual cells can also be tracked over time, with opportunities for comparison to complementary end point measurements. The establishment of this reporter toolset enables dynamic cell investigations that can be translated into more complex synthetic culture environments for elucidating disease mechanisms and evaluating therapeutics for lung fibrosis and other complex biological processes more broadly.
PubMed: 38938687
DOI: 10.1063/5.0166152 -
Turk Patoloji Dergisi Jun 2024Pancreatic stellate cells (PSC) have been defined to be the key players in pancreatic fibrogenesis and carcinogenesis. They undergo myofibroblast-like differentiation,...
OBJECTIVE
Pancreatic stellate cells (PSC) have been defined to be the key players in pancreatic fibrogenesis and carcinogenesis. They undergo myofibroblast-like differentiation, express α-smooth muscle actin (α-SMA), and play a crucial role in injury and inflammation sites. This study aims to evaluate the relationship between α-SMA expression and histopathological parameters of pancreatic ductal adenocarcinoma (PDAC), and investigate their association with prognosis.
MATERIAL AND METHODS
Eighty-one consecutive pancreatectomies diagnosed as usual pancreatic ductal adenocarcinoma were included. The stromal density was scored as loose, moderate, or dense, and α-SMA expression was evaluated immunohistochemically.
RESULTS AND CONCLUSION
Mean survival was 19.6 months. Male gender, larger tumor diameter ( > 3.7 cm), and older age ( > 64 years) were identified as independent poor prognostic factors. Perineural invasion significantly effected survival. A statistically significant correlation was found between high α-SMA expression and the presence of angioinvasion (p=0.01). Stromal α-SMA expression in PDAC may help determine the risk of angioinvasion.
PubMed: 38938104
DOI: 10.5146/tjpath.2024.13521 -
Journal of Digestive Diseases Jun 2024We aimed to disclose the molecular mechanism of snail1 in liver fibrosis.
OBJECTIVE
We aimed to disclose the molecular mechanism of snail1 in liver fibrosis.
METHODS
Carbon tetrachloride (CCl) was used to induce a liver fibrosis model in mice whereby serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were evaluated, and liver pathological alternations were assessed. Rat hepatic stellate cells (HSC-T6) were irritated with transforming growth factor (TGF)-β1, followed by assessment of cell viability and migration. The levels of snail1, ALKBH5, and lysine specific demethylase 4C (KDM4C) were quantified by immunohistochemistry, western blot, or reverse transcription-quantitative polymerase chain reaction, in addition to α-smooth muscle actin (SMA), anti-collagen type I α1 (COL1A1), vimentin, and E-cadherin. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and RNA stability were evaluated to determine the relationship between ALKBH5 and snail1. Changes in KDM4C-bound ALKBH5 promoter and enrichment of histone H3 lysine 9 trimethylation (H3K9me3) at the ALKBH5 promoter were determined using chromatin immunoprecipitation.
RESULTS
In fibrosis mice, snail1 was upregulated while ALKBH5 and KDM4C were downregulated. KDM4C overexpression reduced serum ALT and AST levels, liver injury, and α-SMA, COL1A1 and VIMENTIN expressions but increased E-cadherin expression. However, the aforementioned trends were reversed by concurrent overexpression of snail1. In HSC-T6 cells exposed to TGF-β1, ALKBH5 overexpression weakened cell viability and migration, downregulated α-SMA, COL1A1 and VIMENTIN, upregulated E-CADHERIN, and decreased m6A modification of snail1 and its mRNA stability. KDM4C increased ALKBH5 expression by lowering H3K9me3 level, but inhibited HSC-T6 cell activation by regulating the ALKBH5/snail1 axis.
CONCLUSION
KDM4C decreases H3K9me3 methylation to upregulate ALKBH5 and subsequently inhibits snail1, ultimately impeding liver fibrosis.
PubMed: 38938016
DOI: 10.1111/1751-2980.13291 -
Nature Communications Jun 2024Flowering plants rely on the polarized growth of pollen tubes to deliver sperm cells (SCs) to the embryo sac for double fertilization. In pollen, the vegetative nucleus...
Flowering plants rely on the polarized growth of pollen tubes to deliver sperm cells (SCs) to the embryo sac for double fertilization. In pollen, the vegetative nucleus (VN) and two SCs form the male germ unit (MGU). However, the mechanism underlying directional transportation of MGU is not well understood. In this study, we provide the first full picture of the dynamic interplay among microtubules, actin filaments, and MGU during pollen germination and tube growth. Depolymerization of microtubules and inhibition of kinesin activity result in an increased velocity and magnified amplitude of VN's forward and backward movement. Pharmacological washout experiments further suggest that microtubules participate in coordinating the directional movement of MGU. In contrast, suppression of the actomyosin system leads to a reduced velocity of VN mobility but without a moving pattern change. Moreover, detailed observation shows that the direction and velocity of VN's movement are in close correlations with those of the actomyosin-driven cytoplasmic streaming surrounding VN. Therefore, we propose that while actomyosin-based cytoplasmic streaming influences on the oscillational movement of MGU, microtubules and kinesins avoid MGU drifting with the cytoplasmic streaming and act as the major regulator for fine-tuning the proper positioning and directional migration of MGU in pollen.
Topics: Microtubules; Actin Cytoskeleton; Kinesins; Pollen; Actomyosin; Pollen Tube; Cell Nucleus; Arabidopsis; Cytoplasmic Streaming; Germination
PubMed: 38937444
DOI: 10.1038/s41467-024-49323-9 -
In Vivo (Athens, Greece) 2024Cold physical plasma (CPP) has emerged as an effective therapy in oncology by inducing cytotoxic effects in various cancer cells, including chondrosarcoma (CS), Ewing's...
BACKGROUND/AIM
Cold physical plasma (CPP) has emerged as an effective therapy in oncology by inducing cytotoxic effects in various cancer cells, including chondrosarcoma (CS), Ewing's sarcoma (ES), and osteosarcoma (OS). The current study investigated the impact of CPP on cell motility in CS (CAL-78), ES (A673), and OS (U2-OS) cell lines, focusing on the actin cytoskeleton.
MATERIALS AND METHODS
The CASY Cell Counter and Analyzer was used to study cell proliferation and determine the optimal concentrations of fetal calf serum to maintain viability without stimulation of cell proliferation. CellTiter-BlueCell viability assay was used to determine the effects of CPP on the viability of bone sarcoma cells. The Radius assay was used to determine cell migration. Staining for Deoxyribonuclease I, G-actin, and F-actin was used to assay for the effects on the cytoskeleton.
RESULTS
Reductions in cell viability and motility were observed across all cell lines following CPP treatment. CPP induced changes in the actin cytoskeleton, leading to decreased cell motility.
CONCLUSION
CPP effectively reduces the motility of bone sarcoma cells by altering the actin cytoskeleton. These findings underscore CPP's potential as a therapeutic tool for bone sarcomas and highlight the need for further research in this area.
Topics: Humans; Cell Movement; Plasma Gases; Cell Line, Tumor; Bone Neoplasms; Cell Survival; Cell Proliferation; Cytoskeleton; Actin Cytoskeleton; Osteosarcoma; Actins; Sarcoma
PubMed: 38936915
DOI: 10.21873/invivo.13607 -
Cellular Signalling Jun 2024Tumor-associated macrophages (TAMs) secrete cytokines, chemokines, and growth factors in the tumor microenvironment (TME) to support cancer progression. Higher TAM...
Tumor-associated macrophages (TAMs) secrete cytokines, chemokines, and growth factors in the tumor microenvironment (TME) to support cancer progression. Higher TAM infiltration in the breast TME is associated with a poor prognosis. Previous studies have demonstrated the role of macrophages in stimulating long-range intercellular bridges referred to as tunneling nanotubes (TNTs) in cancer cells. Intercellular communication between cancer cells via TNTs promotes cancer growth, invasion, metastasis, and therapy resistance. Given the important role of TNTs and macrophages in cancer, the role of macrophage-induced TNTs in chemotherapy drug doxorubicin resistance is not known. Furthermore, the mechanism of macrophage-mediated TNT formation is elusive. In this study, it is shown that the macrophage-conditioned medium (MΦCM) partially mimicked inflammatory TME, induced an EMT phenotype, and increased migration in MCF-7 breast cancer cells. Additionally, secreted proteins in MΦCM induced TNT formation in MCF-7 cells, which led to increased resistance to doxorubicin. Transcriptomic analysis of MΦCM-treated MCF-7 cells showed enrichment of the NF-κB and focal adhesion pathways, as well as upregulation of genes involved in EMT, extracellular remodeling, and actin cytoskeleton reorganization. Interestingly, inhibitors of PKC, Src, NF-κB, and p38 decreased macrophage-induced TNT formation in MCF-7 cells. These results reveal the novel role of PKC and Src in inducing TNT formation in cancer cells and suggest that inhibition of PKC and Src activity may likely contribute to reduced macrophage-breast cancer cell interaction and the potential therapeutic strategy of cancer.
PubMed: 38936787
DOI: 10.1016/j.cellsig.2024.111274 -
European Journal of Pharmacology Jun 2024Given estrogen's recognized regulatory influence on diverse metabolic and immune functions, this study sought to explore its potential impact on fibrosis and elucidate...
AIM
Given estrogen's recognized regulatory influence on diverse metabolic and immune functions, this study sought to explore its potential impact on fibrosis and elucidate the underlying metabolic regulations.
METHODS
Female mice underwent ovary removal surgery, followed by carbon tetrachloride (CCl) administration to induce liver injury. Biochemical index analysis and histopathological examination were then conducted. The expression levels of alpha-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), and collagen type 1 alpha 1 chain (COL1A1) were assessed using western blotting to further elucidate the extent of liver injury. Finally, metabolite extraction and metabolomic analysis were performed to evaluate metabolic changes.
RESULTS
Ovary removal exacerbated CCl-induced liver damage, while estrogen supplementation provided protection against hepatic changes resulting from OVX. Furthermore, estrogen mitigated liver injury induced by CCl treatment in vivo. Estrogen supplementation significantly restored liver damage induced by OVX and CCl. Comparative analysis revealed significant alterations in pathways including aminoacyl-tRNA biosynthesis, glycine, serine, and threonine metabolism, lysine degradation, and taurine and hypotaurine metabolism in estrogen treatment.
CONCLUSION
Estrogen supplementation alleviates liver injury induced by OVX and CCl, highlighting its protective effects against fibrosis and associated metabolic alterations.
PubMed: 38936452
DOI: 10.1016/j.ejphar.2024.176774 -
Annals of Hematology Jun 2024There are significant differences in the activated partial thromboplastin time (APTT) critical values reported in different studies, most of which does not make...
INTRODUCTION
There are significant differences in the activated partial thromboplastin time (APTT) critical values reported in different studies, most of which does not make recommendations for any specific clear detection systems. The International Council for Standardization in Hematology (ICSH) recommends that APTT critical values be established based on the reagent type, coagulation factor sensitivity and heparin response. The objective of this study was to establish APTT critical values by using different reagents and based on single coagulation factor deficiencies.
METHODS
The APTT values were determined in commercial endogenous coagulation factor-deficient plasma at concentrations of 1 IU/dL, 2 IU/dL, 5 IU/dL, 10 IU/dL, 20 IU/dL, and 30 IU/dL by using four assay systems. The retrospective collection of data from patients who lacked factor VIII (FVIII), FIX, or FXI alone was performed. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic accuracy of APTT for identifying patients with an endogenous coagulation factor activity < 5 IU/dL.
RESULTS
The APTT values in the plasma samples with the same concentrations of endogenous coagulation factors were significantly different among the four assay systems (P < 0.001). The suggested critical values of APTT were 40.0 s for Sysmex CS5100 (Actin FSL), 58.0 s for Sysmex CS5100 (Actin), 51.8 s for STA-R Evolution (STA-PTTA), and 64.8 s for ACL TOP 700 (HemosIL SynthasIL). On the basis of the ROC curve, the optimal threshold values for APTT (STA-PTTA) were 55.8 s in patients with a simple deficiency of FVIII (sensitivity = 100%, specificity = 85.7%, area under the ROC curve (AUC) = 0.982), 54.3 s in patients with a simple deficiency of FIX (sensitivity = 100%, specificity = 92.9%, AUC = 0.986), and 71.7 s in patients with a simple deficiency of FXI (sensitivity = 100%, specificity = 94.1%, AUC = 0.992), which were closer (difference of 0.6-2.5 s) to the cutoff points for commercial plasma at equal factor levels.
CONCLUSIONS
APTT critical values need to be established for different reagents based on the presence of a single coagulation factor deficiency.
PubMed: 38935318
DOI: 10.1007/s00277-024-05718-8 -
Naunyn-Schmiedeberg's Archives of... Jun 2024The dreaded nosocomial pathogen Clostridioides difficile causes diarrhea and severe inflammation of the colon, especially after the use of certain antibiotics. The...
The dreaded nosocomial pathogen Clostridioides difficile causes diarrhea and severe inflammation of the colon, especially after the use of certain antibiotics. The bacterium releases two deleterious toxins, TcdA and TcdB, into the gut, which are mainly responsible for the symptoms of C. difficile-associated diseases (CDADs). Both toxins are capable of entering independently into various host cells, e.g., intestinal epithelial cells, where they mono-O-glucosylate and inactivate Rho and/or Ras GTPases, important molecular switches for various cellular functions. We have shown recently that the cellular uptake of the Clostridioides difficile toxins TcdA and TcdB (TcdA/B) is inhibited by the licensed class III antiarrhythmic drug amiodarone (Schumacher et al. in Gut Microbes 15(2):2256695, 2023). Mechanistically, amiodarone delays the cellular uptake of both toxins into target cells most likely by lowering membrane cholesterol levels and by interfering with membrane insertion and/or pore formation of TcdA/B. However, serious side effects, such as thyroid dysfunction and severe pulmonary fibrosis, limit the clinical use of amiodarone in patients with C. difficile infection (CDI). For that reason, we aimed to test whether dronedarone, an amiodarone derivative with a more favorable side effect profile, is also capable of inhibiting TcdA/B. To this end, we tested in vitro with various methods the impact of dronedarone on the intoxication of Vero and CaCo-2 cells with TcdA/B. Importantly, preincubation of both cell lines with dronedarone for 1 h at concentrations in the low micromolar range rendered the cells less sensitive toward TcdA/B-induced Rac1 glucosylation, collapse of the actin cytoskeleton, cell rounding, and cytopathic effects, respectively. Our study points toward the possibility of repurposing the already approved drug dronedarone as the preferable safer-to-use alternative to amiodarone for inhibiting TcdA/B in the (supportive) therapy of CDADs.
PubMed: 38935126
DOI: 10.1007/s00210-024-03248-8 -
The Journal of Cell Biology Oct 2024Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes...
Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.
Topics: Animals; Actin-Related Protein 2-3 Complex; Actins; Cell Membrane; Centrosome; Drosophila melanogaster; Drosophila Proteins; Dyneins; Exocytosis; Microtubules
PubMed: 38935075
DOI: 10.1083/jcb.202403115