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26 pages, 7287 KiB

Open AccessArticle

Mitochondrial Genome Insights into Evolution and Gene Regulation in Phragmites australis

by Jipeng Cui, Qianhui Yang, Jiyue Zhang, Chuanli Ju and Suxia Cui

Int. J. Mol. Sci. 2025, 26(2), 546; https://doi.org/10.3390/ijms26020546 - 10 Jan 2025

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As a globally distributed perennial Gramineae, Phragmites australis can adapt to harsh ecological environments and has significant economic and environmental values. Here, we performed a complete assembly and annotation of the mitogenome of P. australis using genomic data from the PacBio and BGI [...] Read more.

As a globally distributed perennial Gramineae, Phragmites australis can adapt to harsh ecological environments and has significant economic and environmental values. Here, we performed a complete assembly and annotation of the mitogenome of P. australis using genomic data from the PacBio and BGI platforms. The P. australis mitogenome is a multibranched structure of 501,134 bp, divided into two circular chromosomes of 325,493 bp and 175,641 bp, respectively. A sequence-simplified succinate dehydrogenase 4 gene was identified in this mitogenome, which is often translocated to the nuclear genome in the mitogenomes of gramineous species. We also identified tissue-specific mitochondrial differentially expressed genes using RNAseq data, providing new insights into understanding energy allocation and gene regulatory strategies in the long-term adaptive evolution of P. australis mitochondria. In addition, we studied the mitogenome features of P. australis in more detail, including repetitive sequences, gene Ka/Ks analyses, codon preferences, intracellular gene transfer, RNA editing, and multispecies phylogenetic analyses. Our results provide an essential molecular resource for understanding the genetic characterisation of the mitogenome of P. australis and provide a research basis for population genetics and species evolution in Arundiaceae. Full article

(This article belongs to the Special Issue Transcriptional Regulation in Plant Development: 2nd Edition)

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15 pages, 3793 KiB

Open AccessArticle

Panduratin A Inhibits TNF Alpha-Stimulated Endothelial Cell Activation Through Suppressing the NF-κB Pathway

by Kriangkrai Kiatsoonthon, Nitchakarn Phimthong, Saranyapin Potikanond, Nitwara Wikan and Wutigri Nimlamool

Biomolecules 2025, 15(1), 34; https://doi.org/10.3390/biom15010034 - 30 Dec 2024

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Abstract

Upon exposure to inflammatory stimuli including TNF-α, endothelial cells are activated leading to the adhesion of monocytes to their surface. These events are involved in the pathophysiology of atherosclerosis. Since TNF-α activates the NF-κB pathway, which contributes to atherosclerosis, targeting this signaling pathway [...] Read more.

Upon exposure to inflammatory stimuli including TNF-α, endothelial cells are activated leading to the adhesion of monocytes to their surface. These events are involved in the pathophysiology of atherosclerosis. Since TNF-α activates the NF-κB pathway, which contributes to atherosclerosis, targeting this signaling pathway may help prevent the risk of developing the disease. The current study elucidated the inhibitory effect of panduratin A (PA) on TNF-α-induced endothelial activation and monocyte adhesion. We discovered that PA reduced the level of pro-inflammatory cytokine IL-6 and chemokine MCP-1 in the media collected from endothelial cells stimulated with TNF-α. In addition, PA inhibited the expression of ICAM-1 and VCAM-1 on the surface of TNF-α-induced endothelial cells resulting in a decrease in the number of monocytes attached to endothelial cell surface. Mechanistically, PA prevented IκB degradation and specifically suppressed NF-κB phosphorylation and nuclear translocation in endothelial cells. However, PA had no inhibitory effect on the phosphorylation of AKT, ERK1/2, p38, and JNK. Taken together, PA blocked the production of cytokine and chemokine, adhesion molecules, and monocyte adhesion in response to TNF-α stimulation, in part, through NF-κB inhibition. Our study suggests that PA may possibly be effective in blocking the pathophysiology of atherosclerosis. Full article

(This article belongs to the Special Issue The Value of Natural Compounds as Therapeutic Agents: 2nd Edition)

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15 pages, 4063 KiB

Open AccessArticle

HDAC1 and HDAC2 Are Involved in Influenza A Virus-Induced Nuclear Translocation of Ectopically Expressed STAT3-GFP

by Jessica Leong and Matloob Husain

Viruses 2025, 17(1), 33; https://doi.org/10.3390/v17010033 - 29 Dec 2024

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Abstract

Influenza A virus (IAV) remains a pandemic threat. Particularly, the evolution and increased interspecies and intercontinental transmission of avian IAV H5N1 subtype highlight the importance of continuously studying the IAV and identifying the determinants of its pathogenesis. Host innate antiviral response is the [...] Read more.

Influenza A virus (IAV) remains a pandemic threat. Particularly, the evolution and increased interspecies and intercontinental transmission of avian IAV H5N1 subtype highlight the importance of continuously studying the IAV and identifying the determinants of its pathogenesis. Host innate antiviral response is the first line of defense against IAV infection, and the transcription factor, the signal transducer and activator of transcription 3 (STAT3), has emerged as a critical component of this response. Also, histone deacetylase 1 (HDAC1) and HDAC2 have been identified as important components of IAV-induced host innate antiviral response. Upon IAV infection, STAT3 is activated and translocated to the nucleus to initiate the transcription of innate response genes. Also, the HDAC1 and HDAC2 are localized to the nucleus. In this study, we sought to investigate the role of HDAC1 and HDAC2 in IAV-induced STAT3 nuclear translocation. We employed a quantitative confocal microscopy approach and analyzed the nuclear translocation of plasmid-expressed STAT3-GFP in IAV-infected cells depleted with the expression of HDAC1 or HDAC2. We found that the depletion of both HDAC1 and HDAC2 expression inhibits the IAV-induced nuclear translocation of STAT3-GFP. These findings will help elucidate the significance of the emerging role of acetylation in IAV infection and disease severity. Full article

(This article belongs to the Special Issue Interplay between Influenza Virus and Host Factors)

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Plasmid-expressed STAT3-GFP translocates to the nucleus in response to interferon treatment. HeLa cells were transfected with plasmid pEGFP-N1-STAT3 for 48 h, and then treated with IFN-α, IFN-β, or IFN-γ (500 IU). After 1 h, cells were fixed and stained with DAPI (4′,6-diamidino-2-phenylindole), and then imaged using a confocal microscope under 40× magnification. Plot profiles of cells treated with IFN-α, IFN-β, and IFN-γ (next to respective rows) were generated using ImageJ 1.54g; red lines in Merged column indicate the profiled area; x-axes and y-axes on graphs represent distance and pixel intensity, respectively, along the lines; green and blue lines on graphs represent plot profiles of STAT3-GFP and DAPI, respectively. Full article ">Figure 2
Plasmid-expressed STAT3-GFP translocates to the nucleus in response to IAV infection. HeLa cells were transfected with plasmid pEGFP-N1-STAT3 for 48 h, and then infected with influenza virus A/Puerto Rico/8/1934(H1N1), hereafter referred to as PR8, at the multiplicity of infection (MOI) of 1.0, 3.0, or 5.0. After 6 h, cells were fixed, permeabilized, and stained with Goat anti-IAV nucleoprotein (NP) antibody followed by Donkey anti-Goat IgG conjugated with Alexa Fluor 647. Subsequently, cells were stained with DAPI and imaged using a confocal microscope under 40× magnification. Plot profiles of uninfected (UNI) and infected (INF) cells (next to respective rows) were generated using ImageJ 1.54g; red lines in Merged column indicate the profiled area; x-axes and y-axes on graphs represent distance and pixel intensity, respectively, along the lines; green and blue lines on graphs represent plot profiles of STAT3-GFP and DAPI, respectively. Full article ">Figure 3
The IAV-induced nuclear translocation of STAT3-GFP is inhibited in HDAC1-depleted cells. HeLa cells were first transfected with either control siRNA or HDAC1 siRNA for 30 h and then transfected with plasmid pEGFP-N1-STAT3 for a further 30 h. Cells were then infected with PR8 at MOI of 3.0. After 6 h, cells were fixed, permeabilized, and stained with Mouse anti-HDAC1 antibody followed by Donkey anti-Mouse IgG conjugated with Alexa Fluor 594. Subsequently, cells were stained with Goat anti-NP antibody followed by Donkey anti-Goat IgG conjugated with Alexa Fluor 647. Finally, cells were stained with DAPI and imaged using a confocal microscope under 40× magnification. UNI, uninfected; INF, infected. Full article ">Figure 4
Western blots showing the siRNA-mediated depletion of HDAC1 and HDAC2 expression in uninfected and infected cells. HeLa cells were transfected with either control (CT), HDAC1 (HD1), or HDAC2 (HD2) siRNA for 30 h. Cells were then infected with PR8 at MOI of 3.0. After 6 h, HDAC1 (62 kDa), HDAC2 (60 kDa), NP (56 kDa), and Actin (42 kDa) were detected in total cell lysates by Western blotting. UNI, uninfected; INF, infected; kDa, kilodalton. Full article ">Figure 5
The nuclear/cytoplasmic ratio of STAT3-GFP is similar in HDAC1-depleted uninfected cells and HDAC1-depleted infected cells. The pixel density of STAT3-GFP in the nucleus and the cytoplasm of Control siRNA-transfected and HDAC1 siRNA-transfected uninfected as well as infected cells was quantified using Arivis Vision 4D ver.4.1.2 software (Zeiss) integrated with Cellpose. Then, STAT3-GFP pixel density per µm2 of nucleus was divided by STAT3-GFP pixel density per µm2 of cytoplasm to calculate the STAT3-GFP nuclear/cytoplasmic (N:C) ratio. The data presented are the Mean ± SD of N:C ratio of cells imaged across three biological replicates; n = 102 (36 from each replicate). The p-value was calculated using two-way Analysis of Variance (ANOVA) employing Šídák’s multiple comparisons test. **** p = <0.0001, ns, not significant. Full article ">Figure 6
The IAV-induced nuclear translocation of STAT3-GFP is inhibited in HDAC2-depleted cells. HeLa cells were first transfected with either Control siRNA or HDAC2 siRNA for 30 h and then transfected with plasmid pEGFP-N1-STAT3 for a further 30 h. Cells were then infected with PR8 at MOI of 3.0. After 6 h, cells were fixed, permeabilized, and stained with Mouse anti-HDAC2 antibody followed by Donkey anti-Mouse IgG conjugated with Alexa Fluor 594. Subsequently, cells were stained with Goat anti-NP antibody followed by Donkey anti-Goat IgG conjugated with Alexa Fluor 647. Finally, cells were stained with DAPI and imaged using a confocal microscope under 40× magnification. UNI, uninfected; INF, infected. Full article ">Figure 7
The nuclear/cytoplasmic ratio of STAT3-GFP is similar in HDAC2-depleted uninfected cells and HDAC2-depleted infected cells. The pixel density of STAT3-GFP in the nucleus and the cytoplasm of Control siRNA-transfected and HDAC2 siRNA-transfected uninfected as well as infected cells was quantified using Arivis Vision 4D ver.4.1.2 software (Zeiss) integrated with Cellpose. Then, STAT3-GFP pixel density per µm2 of nucleus was divided by STAT3-GFP pixel density per µm2 of cytoplasm to calculate the STAT3-GFP N:C ratio. The data presented are the Mean ± SD of N:C ratio of cells imaged across three biological replicates; n = 138 (46 from each replicate). The p-value was calculated using two-way ANOVA employing Šídák’s multiple comparisons test. **** p = <0.0001; ns, not significant. Full article ">

17 pages, 4463 KiB

Open AccessArticle

Rutin Alleviates Zearalenone-Induced Endoplasmic Reticulum Stress and Mitochondrial Pathway Apoptosis in Porcine Endometrial Stromal Cells by Promoting the Expression of Nrf2

by Chuangjiang Chen, Chenlong Wang, Hui Jiang, Mengya Wang, Sajid Ur Rahman, Changjiang Chen, Hongyan Ding, Chang Zhao, Wanyue Huang and Xichun Wang

Toxins 2025, 17(1), 7; https://doi.org/10.3390/toxins17010007 - 26 Dec 2024

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Abstract

Zearalenone (ZEA) is a mycotoxin commonly found in moldy cereals and has a range of toxic effects that have seriously affected animal husbandry. Rutin, a natural flavonoid with antioxidant activities, has been studied for its potential involvement in mitigating ZEA-induced apoptosis in porcine [...] Read more.

Zearalenone (ZEA) is a mycotoxin commonly found in moldy cereals and has a range of toxic effects that have seriously affected animal husbandry. Rutin, a natural flavonoid with antioxidant activities, has been studied for its potential involvement in mitigating ZEA-induced apoptosis in porcine endometrial stromal cells (ESCs) and its potential molecular mechanism, particularly concerning the expression of Nrf2. This study investigates the molecular pathways by which rutin alleviates ZEA-induced ESC apoptosis, focusing on the role of Nrf2. Experimental data reveal that ZEA suppresses Nrf2 nuclear translocation and reduces mitochondrial membrane potential (MMP), leading to oxidative stress, endoplasmic reticulum stress (ERS), and mitochondrial pathway-driven apoptosis. Notably, rutin mitigates ZEA-induced apoptosis through Nrf2 activation. These findings highlight Nrf2 as a critical factor in rutin’s protective effects against ZEA-induced apoptosis, offering valuable insights for the clinical prevention and treatment of ZEA toxicity. Full article

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14 pages, 16811 KiB

Open AccessArticle

Exogenous Melatonin Boosts Heat Tolerance in Rosa hybrida via RhCOMT1 Modulation

by Chenyang Li, Zhiyin Ding, Zipeng Cai, Yongying Ruan, Peitao Lü and Yang Liu

Plants 2025, 14(1), 29; https://doi.org/10.3390/plants14010029 - 25 Dec 2024

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Abstract

Rosa hybrida is one the most commonly cultivated ornamental plant of economic importance and faces major challenges under heat stress. Melatonin has been widely shown to regulate plant stress response; however, the exact mechanism involved in heat stress in R. hybrida has yet [...] Read more.

Rosa hybrida is one the most commonly cultivated ornamental plant of economic importance and faces major challenges under heat stress. Melatonin has been widely shown to regulate plant stress response; however, the exact mechanism involved in heat stress in R. hybrida has yet to be determined. Here, we observed that R. hybrida in vitro plantlets supplemented with melatonin in the culture medium exhibited higher chlorophyll content, relative ion leakage, and fresh weight after 12 d of high-temperature treatment; the optimal concentration was established at 5 mg/L. Using molecular and biochemical techniques, we explored the roles of a melatonin synthase gene RhCOMT1, which expression was influenced by heat stress and melatonin. RhCOMT1 was located in the nuclear-cytoplasmic under ambient conditions, while heat stress translocated the distribution of RhCOMT1 to chloroplasts. Overexpression of RhCOMT1 in rose petal enhanced thermotolerance, and silencing of RhCOMT1 reduced thermotolerance via affect H₂O₂ content and relative ion leakage. These findings collectively emphasize the pivotal role of melatonin in enhancing thermotolerance to R. hybrida by alleviation of oxidative stress, through modulation of RhCOMT1 expression and location. Full article

(This article belongs to the Topic Plant Physiological and Ecological Responses to Environmental Stress)

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Phenotypic characterization of the buds and sterilized stems under heat stress. (A) Phenotypes of the Rosa hybrida buds under heat stress. Scale bar = 5 cm. (B,C) Flower diameters and (B) petal numbers (C) of CK, HT, MT + HT groups. (D) Phenotypes of the R. hybrida in vitro sterilized stems in different concentrations of melatonin medium under high-temperature treatment. Scale bar = 1 cm. (E–G) ChlorophyII content, (E) relative ion leakage, and (F) relative fresh weight (G) of the R. hybrida sterilized stems grow in different concentrations of melatonin medium under 12 d of HT. CK, control group; HT, 42 °C high-temperature treatment group; MT, melatonin treatment; error bars represent ± SD (n = 3). Significant differences compared to the 0 mg/L melatonin treatment group are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance, Student’s t-test). Full article ">Figure 2
Expression pattern and bioinformatics analysis of RhCOMT1. (A) The expression levels of RhCOMT1 under high-temperature treatment. (B) The expression levels of RhCOMT1 under melatonin treatment of different concentrations. (C) Phylogenetic relationships between RhCOMT1 and other COMT proteins. The data are presented as the means ± SDs (n = 9). Significant differences compared to the 0 h high-temperature treatment and 0 mg/L melatonin treatment group are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance, Student’s t-test). Full article ">Figure 3
Subcellular localization assay of RhCOMT1 under RT and HT. The location of RhCOMT1 is based on visualization of GFP in tobacco leaves transformed with a fusion construct (pSuper1300:GFP-RhCOMT1) or empty vector (pSuper1300:GFP). Microscopic images were taken under bright field and fluorescence. The overlapped images are shown on the right. RT, room temperature; HT, high-temperature treatment; GFP, green fluorescent protein. Full article ">Figure 4
Thermotolerance assay using RhCOMT1-overexpressing petal discs. (A) The phenotypes of rose petal discs under room temperature (RT, 25 °C) and post high-temperature treatment (HT, 42 °C, 36 h) with DAB staining. The experiment was repeated three times, with 27 rose petal discs per experiment. Scale bar = 1 cm. (B) Detection of RhCOMT1 expression in RhCOMT1-overexpression petal discs. (C,D) Determination of H2O2 content and (C) relative ion leakage (D) of petal discs post HT (42 °C, 36 h). The data are presented as the means ± SD for three replicates (** p < 0.01, *** p < 0.001, Student’s t-test, the pSuper1300:RhCOMT1 compared with the pSuper1300 control post HT, respectively). RT, room temperature; HT, high-temperature treatment; DAB, 3,3′diaminobenzidine. Full article ">Figure 5
Thermotolerance assay using RhCOMT1-silenced petal discs. (A) The phenotypes of rose petal discs under room temperature (RT, 25 °C) and high-temperature treatment (HT, 42 °C, 36 h) with DAB staining. The experiment was repeated three times, with 27 rose petal discs per experiment. Scale bar = 1 cm. (B) Detection of RhCOMT1 expression in TRV-VIGS petal discs. (C,D) Determination of H2O2 content and (C) relative ion leakage (D) of petal discs post HT (42 °C, 36 h). The data are presented as the means ± SDs for three replicates (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test, the pTRV2-RhCOMT1 compared with the pTRV2-control post HT, respectively). RT, room temperature; HT, high-temperature treatment; DAB, 3,3′diaminobenzidine. Full article ">Figure 6
Model for RhCOMT responding to heat stress of R. hybrida. RhCOMT1 can be significantly induced by heat stress. The over-expression of RhCOMT1 has been shown to enhance heat stress tolerance by increasing the capability to scavenge ROS. Furthermore, RhCOMT1 has been observed to localise in chloroplasts at high temperatures, which has the potential to improve chloroplast chlorophyll stability and photosynthetic efficiency. Full article ">

14 pages, 2758 KiB

Open AccessReview

The Expression Regulation and Cancer-Promoting Roles of RACGAP1

by Jiacheng Lin, Yuhao Zhu, Zhaoping Lin, Jindong Yu, Xiaobing Lin, Weiyuan Lai, Beibei Tong, Liyan Xu, Enmin Li and Lin Long

Biomolecules 2025, 15(1), 3; https://doi.org/10.3390/biom15010003 - 24 Dec 2024

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Abstract

RACGAP1 is a Rho-GTPase-activating protein originally discovered in male germ cells to inactivate Rac, RhoA and Cdc42 from the GTP-bound form to the GDP-bound form. GAP has traditionally been known as a tumor suppressor. However, studies increasingly suggest that overexpressed RACGAP1 activates Rac [...] Read more.

RACGAP1 is a Rho-GTPase-activating protein originally discovered in male germ cells to inactivate Rac, RhoA and Cdc42 from the GTP-bound form to the GDP-bound form. GAP has traditionally been known as a tumor suppressor. However, studies increasingly suggest that overexpressed RACGAP1 activates Rac and RhoA in multiple cancers to mediate downstream oncogene overexpression by assisting in the nuclear translocation of signaling molecules and to promote cytokinesis by regulating the cytoskeleton or serving as a component of the central spindle. Contradictorily, it was also reported that RACGAP1 in gastric cancer could inactivate Rac and RhoA. In addition, studies have revealed that RACGAP1 can be a biomarker for prognosis, and its role in reducing doxorubicin sensitivity poses difficulties for treatment, while the current drug targets mainly focus on its downstream molecule. This article mainly reviews the expression regulation of RACGAP1 and its cancer-promoting functions through oncogene expression mediation and Rho-GTPase activation. Full article

(This article belongs to the Section Molecular Biology)

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RACGAP1 promotes cancer through the Hippo pathway and JAK/STAT pathway. In the Hippo pathway, the phosphorylated form of MST1/2 activates LAST1/2 via phosphorylation. YAP can be deactivated by the activated form of LAST when phosphorylated. YAP in the non-phosphorylated state can enter the nucleus, bind to the promoter region of TPR with TEAD and promote its transcription. TPR, RACGAP1 and AURKB form complexes that promote spindle formation. Moreover, RACGAP1 inhibits the Hippo pathway by promoting the inactivation of LAST1/2 through F-actin. RACGAP1 forms a ternary complex together with STAT3 and Rac1 to phosphorylate STAT3, and accomplishes the nuclear transport of STAT3 as a nuclear chaperone. RACGAP1 binds JAK2 to enable the phosphorylation of STAT3 by JAK2. Rac1 and p-STAT3 activate the nuclear localization signal (NLS) in RACGAP1 so that it can promote the nuclear translocation and the transcriptional activity of p-STAT3. RACGAP1 can inactivate Rac1, but after binding with APPL2, it cannot inactivate Rac1. Full article ">Figure 2
RACGAP1 can activate Rho-GTPases. (a) RACGAP1 inhibits Rac1, Cdc42 and RhoA. (b) Phosphorylated RACGAP1 can activate Rac1. (c) RACGAP1 can activate RhoA in hepatocellular carcinoma. (d) RACGAP1 can form a complex with Rac1 and STAT3, and with ECT and MKLP1, the latter complex of which can activate RhoA. (e) In NIH3T3 cells, Rac1 can inhibit RhoA. (f) In Swiss 3T3 fibroblasts, RhoA can activate Rac1. Full article ">Figure 3
RACGAP1 binding ECT2 to promotes actomyosin ring formation. Plk phosphorylates RACGAP1 at 2 sites so that RACGAP1 binds to ECT2 with the help of MKLP1, and the complex activates RhoA, Myosin II and ERK. PP2A-B′ can bind to RACGAP1 to break the interaction between RACGAP1 and ECT2. Full article ">

11 pages, 1974 KiB

Open AccessArticle

Nuclear Factor-κB Signaling Regulates the Nociceptin Receptor but Not Nociceptin Itself

by Lan Zhang, Ulrike M. Stamer, Robin Moolan-Vadackumchery and Frank Stüber

Cells 2024, 13(24), 2111; https://doi.org/10.3390/cells13242111 - 20 Dec 2024

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Abstract

The nociceptin receptor (NOP) and nociceptin are involved in the pathways of pain and inflammation. The potent role of nuclear factor-κB (NFκB) in the modulation of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β on the nociceptin system in human THP-1 cells under inflammatory [...] Read more.

The nociceptin receptor (NOP) and nociceptin are involved in the pathways of pain and inflammation. The potent role of nuclear factor-κB (NFκB) in the modulation of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β on the nociceptin system in human THP-1 cells under inflammatory conditions were investigated. Cells were stimulated without/with phorbol-myristate-acetate (PMA), TNF-α, IL-1β, or PMA combined with individual cytokines. To examine NFκB’s contribution to the regulation of the nociceptin system, PMA-stimulated cells were treated with NFκB inhibitor BAY 11-7082, JSH-23, or anacardic acid before culturing with TNF-α or IL-1β. NOP and prepronociceptin (ppNOC) mRNA were quantified by RT-qPCR; cell membrane NOP and intracellular nociceptin protein levels were measured by flow cytometry. Phosphorylation and localization of NFκB/p65 were determined using ImageStream. PMA + TNF-α decreased NOP mRNA compared to stimulation with PMA alone, while PMA + IL-1β did not. BAY 11-7082 and JSH-23 reversed the repression of NOP by PMA + TNF-α. TNF-α and IL-1β attenuated PMA’s upregulating effects on ppNOC. None of the inhibitors preserved the upregulation of ppNOC in PMA + TNF-α and PMA + IL-1β cultures. TNF-α strongly mediated the nuclear translocation of NFκB/p65 in PMA-treated cells, while IL-1β did not. Proinflammatory cytokines suppressed NOP and ppNOC mRNA in PMA-induced human THP-1 cells. NFκB signaling seems to be an important regulator controlling the transcription of NOP. These findings suggest that the nociceptin system may play an anti-inflammatory role during immune responses. Full article

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Effects of PMA on NOP (A) and nociceptin (B) mRNA and protein levels in THP-1 cells. Cells were treated with PMA 5 ng/mL or without PMA (controls) for 24 h. Median with interquartile range and 10–90 percentiles; mean “+”; mRNA and protein data are representative of six and twelve independent experiments, respectively. Wilcoxon tests * p < 0.05; ** p < 0.005; *** p < 0.001, compared to the respective controls. Full article ">Figure 2
Dose-dependent effects of cytokines and NFκB inhibitors. THP-1 cells were cultured without/with PMA 5 ng/mL and without/with various concentrations of individual cytokines for 24 h. mRNA expression of NOP (A) and ppNOC (B) is presented as mRNA ratio related to the respective PMA samples. (C) Dose-dependent inhibitory effects of different NFκB inhibitors on IL1B mRNA levels induced by LPS. Cells were pre-treated without/with various concentrations of BAY 11-7082 (BAY), JSH-23 (JSH), or anarcadic acid (AA) for 1 h prior to culturing without/with LPS 100 ng/mL for 6 h. IL1B mRNA levels are presented as mRNA ratio related to an untreated group. Data are from two independent experiments and measures are expressed in mean with range. One-sample t test, * p < 0.05; ** p < 0.005, compared to the PMA group (NOP and ppNOC) and compared to the LPS group (IL1B). Full article ">Figure 3
Effects of TNF-α and IL-1β on NOP and ppNOC mRNA expression. THP-1 cells were cultured without/with PMA 5 ng/mL in the presence or absence of TNF-α 10 ng/mL or IL-1β 10 ng/mL for 24 h. Quantitative PCR analysis of NOP (A) and ppNOC mRNA expression (B). Fold change values in mRNA levels are normalized against the respective PMA groups. Data are from six independent experiments and are presented as median with interquartile range and 10–90 percentiles; mean “+”. Wilcoxon test with post hoc test. ** p < 0.005. Full article ">Figure 4
Effects of NFκB inhibitors on NOP and ppNOC mRNA expression. THP-1 cells were stimulated without/with PMA 5 ng/mL for 24 h, followed by treatment without/with one of the NFκB inhibitors (BAY 11-7082 (BAY, 100 nM), JSH-23 (JSH, 100 nM), and anacardic acid (AA, 100 nM)) for 1 h before exposure to TNF-α 10 ng/mL or IL-1β 10 ng/mL for 6 h (BAY and JSH) and for 12 h (AA). NOP (A–C) and ppNOC mRNA levels (D–F) are fold-change related to the corresponding PMA groups (controls). Median with interquartile range and 10–90 percentiles, experiment using BAY or AA, n = 4; experiment using JSH, n = 7. Mann–Whitney U test with post hoc test. * p < 0.05; ** p < 0.005. Full article ">Figure 5
NFκB/p65 phosphorylation and nuclear translocation. THP-1 cells were treated without/with PMA 5 ng/mL for 24 h, followed by incubation with TNF-α 10 ng/mL or IL-1β 10 ng/mL or without additional cytokines for 1 h. (A) Composite images of representative cells (60×) in real time showing level of NFκB/p65 protein and its localization. From left to right in each panel, brightfield images of each cell, followed by the nuclear (red) and NFκB/p65 (green) images, and the merged image of the nucleus with NFκB/p65. The scale bar is 7 μm. (B) NFκB/p65 translocation into the nucleus, measured using similarity scores (SS). Histogram displaying varying SS in the cells of each group: untreated (white), TNF-α (yellow), IL-1β (green), PMA (purple), PMA + TNF-α (red) and PMA+ IL-1β (blue). (C) Median SS (MSS) for NFκB/p65 of the individual groups. Data are from three independent experiments and measures are expressed in median with interquartile range. Full article ">

16 pages, 3701 KiB

Open AccessArticle

Essential Role of Cortactin in Myogenic Differentiation: Regulating Actin Dynamics and Myocardin-Related Transcription Factor A-Serum Response Factor (MRTFA-SRF) Signaling

by Quoc Kiet Ly, Mai Thi Nguyen, Thanh Huu Phan Ngo and Wan Lee

Int. J. Mol. Sci. 2024, 25(24), 13564; https://doi.org/10.3390/ijms252413564 - 18 Dec 2024

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Abstract

Cortactin (CTTN) is an actin-binding protein regulating actin polymerization and stabilization, which are vital processes for maintaining skeletal muscle homeostasis. Despite the established function of CTTN in actin cytoskeletal dynamics, its role in the myogenic differentiation of progenitor cells remains largely unexplored. In [...] Read more.

Cortactin (CTTN) is an actin-binding protein regulating actin polymerization and stabilization, which are vital processes for maintaining skeletal muscle homeostasis. Despite the established function of CTTN in actin cytoskeletal dynamics, its role in the myogenic differentiation of progenitor cells remains largely unexplored. In this study, we investigated the role of CTTN in the myogenic differentiation of C2C12 myoblasts by analyzing its effects on actin cytoskeletal remodeling, myocardin-related transcription factor A (MRTFA) nuclear translocation, serum response factor (SRF) activation, expression of myogenic transcription factors, and myotube formation. CTTN expression declined during myogenic differentiation, paralleling the reduction in MyoD, suggesting a potential role in the early stages of myogenesis. We also found that CTTN knockdown in C2C12 myoblasts reduced filamentous actin, enhanced globular actin levels, and inhibited the nuclear translocation of MRTFA, resulting in suppressed SRF activity. This led to the subsequent downregulation of myogenic regulatory factors, such as MyoD and MyoG. Furthermore, CTTN knockdown reduced the nuclear localization of YAP1, a mechanosensitive transcription factor, further supporting its regulatory roles in cell cycle and proliferation. Consequently, CTTN depletion impeded proliferation, differentiation, and myotube formation in C2C12 myoblasts, highlighting its dual role in the coordination of cell cycle regulation and myogenic differentiation of progenitor cells during myogenesis. This study identifies CTTN as an essential regulator of myogenic differentiation via affecting the actin remodeling-MRTFA-SRF signaling axis and cell proliferation. Full article

(This article belongs to the Special Issue Exploring the Cytoskeleton and Its Regulators: From Structure to Function)

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Figure 1
Modulation of CTTN expression during myoblast differentiation. (A) Immunoblotting was conducted to assess CTTN expression levels in C2C12 myoblasts and various tissues from C57BL/6 mice, with α-tubulin as a loading control. (B) C2C12 myoblasts were harvested on specified differentiation days, and the protein expression levels of MyoD, MyoG, MyHC, and CTTN were analyzed by immunoblotting, with β-actin as a loading control. (C) Protein expression levels were normalized to β-actin, and relative expression ratios were calculated, setting day 0 as one for MyoD and CTTN, day 1 for MyoG, and day 2 for MyHC. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001). Full article ">Figure 2
CTTN knockdown led to a reduction in F-actin levels and an increase in G-actin levels. C2C12 myoblasts were transfected with 200 nM of either control scRNA or siCTTN (siCTTN-1 or siCTTN-2). (A) CTTN expression was assessed by immunoblotting 24 h after transfection. CTTN expression levels were normalized to β-actin, and relative expression ratios were calculated with the control scRNA set to one. (B) After 24 h post-transfection, cells were stained with FITC-phalloidin (green) for F-actin and Hoechst 33,342 (blue) for nuclei. Scale bar: 25 μm. Phalloidin intensities were quantified using ImageJ software, version 1.5.4. (C) F- and G-actin levels were quantified by flow cytometry after staining with FITC-phalloidin for F-actin and DNase I for G-actin, respectively. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (** p < 0.01, *** p < 0.001). Full article ">Figure 3
CTTN depletion impaired the nuclear localization of MRTFA and YAP1. C2C12 myoblasts were transfected with either control scRNA or siCTTN and analyzed 24 h post-transfection. (A) Cytoplasmic and nuclear fractions were subjected to immunoblot analysis for MRTFA, SRF, YAP1, pYAP1 (phosphorylated YAP1), and CTTN expression. For MRTFA, different exposure times were used to account for its varied distribution between cytoplasmic and nuclear compartments. α-Tubulin and lamin B2 served as cytoplasmic and nuclear markers, respectively. β-Actin was used as a loading control. (B,C) The protein expression levels were normalized to β-actin, and relative expression ratios were calculated with the control scRNA set to one. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (** p < 0.01, *** p < 0.001). Full article ">Figure 4
CTTN knockdown suppressed SRF transcriptional activity. (A) Diagram of the luciferase reporter construct featuring the truncated SMYD1 promoter region, including the CArG box for SRF binding. (B) C2C12 myoblasts were transfected with either the pGL3 vector (Vector) or pGL3 containing the SMYD1 promoter (SMYD1) along with control scRNA or siCTTN. Relative luciferase activity was measured 24 h post-transfection. (C) C2C12 myoblasts were transfected with either control scRNA or siCTTN, and mRNA levels of SRF, Vinculin, and SMYD1 were assessed by RT-qPCR, normalized to GAPDH expression 24 h post-transfection. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (** p < 0.01, *** p < 0.001); ns indicates non-significance. Full article ">Figure 5
CTTN depletion impeded cell proliferation and cell cycle progression. C2C12 myoblasts were transfected with either control scRNA or siCTTN and analyzed 24 h post-transfection. (A) Cell proliferation was evaluated by EdU incorporation (green) to label replicating cells, with Hoechst 33,342 (blue) as a nuclear counterstain. Scale bar: 50 µm. (B) The percentage of EdU-positive cells was quantified using ImageJ software. (C) Viable cell numbers were measured using a cell viability assay kit. (D) mRNA levels of proliferation markers (PCNA, cyclin B1, and cyclin D1) were assessed by RT-qPCR and normalized to GAPDH expression. (E,F) Cell cycle analysis was performed using flow cytometry with scatter plots. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001). Full article ">Figure 6
CTTN knockdown suppressed the expression of myogenic regulatory factors. (A) C2C12 myoblasts were transfected with either control scRNA or siCTTN, allowed to differentiate, and then harvested on specified differentiation days. Protein expression levels of MyoD, MyoG, MyHC, and CTTN were analyzed by immunoblotting. (B) Protein expression levels for scRNA (open column) and siCTTN (blue column) were normalized to β-actin and presented as relative ratios, with scRNA expression levels on day 0 (for CTTN and MyoD) or day 3 (for MyoG and MyHC) set to one. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001); ns indicates non-significance. Full article ">Figure 7
CTTN depletion impaired myogenic differentiation. C2C12 myoblasts were transfected with either control scRNA or siCTTN and then allowed to differentiate for 5 days. (A) Representative immunocytochemistry stained with MyHC antibody (green) and Hoechst 33,342 (blue). Scale bar: 100 μm. (B) MyHC-positive areas, differentiation indices, fusion indices, and myotube widths were determined as described in <a href="#sec4-ijms-25-13564" class="html-sec">Section 4</a>. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (*** p < 0.001). Full article ">Figure 8
Schematic illustration of the actin-MRTFA-SRF and YAP1 signaling pathway regulated by CTTN. Full article ">

30 pages, 10385 KiB

Open AccessArticle

Second-Generation Antipsychotics Induce Metabolic Disruption in Adipose Tissue-Derived Mesenchymal Stem Cells Through an aPKC-Dependent Pathway

by Marco Varalda, Jacopo Venetucci, Herald Nikaj, Chaitanya Reddy Kankara, Giulia Garro, Nazanin Keivan, Valentina Bettio, Paolo Marzullo, Annamaria Antona, Guido Valente, Sergio Gentilli and Daniela Capello

Cells 2024, 13(24), 2084; https://doi.org/10.3390/cells13242084 - 17 Dec 2024

Viewed by 573

Abstract

Metabolic syndrome (MetS) is a cluster of metabolic abnormalities, including visceral obesity, dyslipidemia, and insulin resistance. In this regard, visceral white adipose tissue (vWAT) plays a critical role, influencing energy metabolism, immunomodulation, and oxidative stress. Adipose-derived stem cells (ADSCs) are key players in [...] Read more.

Metabolic syndrome (MetS) is a cluster of metabolic abnormalities, including visceral obesity, dyslipidemia, and insulin resistance. In this regard, visceral white adipose tissue (vWAT) plays a critical role, influencing energy metabolism, immunomodulation, and oxidative stress. Adipose-derived stem cells (ADSCs) are key players in these processes within vWAT. While second-generation antipsychotics (SGAs) have significantly improved treatments for mental health disorders, their chronic use is associated with an increased risk of MetS. In this study, we explored the impact of SGAs on ADSCs to better understand their role in MetS and identify potential therapeutic targets. Our findings reveal that olanzapine disrupts lipid droplet formation during adipogenic differentiation, impairing insulin receptor endocytosis, turnover, and signaling. SGAs also alter the endolysosomal compartment, leading to acidic vesicle accumulation and increased lysosomal biogenesis through TFEB activation. PKCζ is crucial for the SGA-induced nuclear translocation of TFEB and acidic vesicle formation. Notably, inhibiting PKCζ restored insulin receptor tyrosine phosphorylation, normalized receptor turnover, and improved downstream signaling following olanzapine treatment. This activation of PKCζ by olanzapine is driven by increased phosphatidic acid synthesis via phospholipase D (PLD), following G protein-coupled receptor (GPCR) signaling activation. Overall, olanzapine and clozapine disrupt endolysosomal homeostasis and insulin signaling in a PKCζ-dependent manner. These findings highlight SGAs as valuable tools for uncovering cellular dysfunction in vWAT during MetS and may guide the development of new therapeutic strategies to mitigate the metabolic side effects of these drugs. Full article

(This article belongs to the Special Issue Adipose Tissue, Obesity, and Metabolic Diseases)

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13 pages, 2263 KiB

Open AccessArticle

10(E)-Pentadecenoic Acid Inhibits Melanogenesis Partly Through Suppressing the Intracellular MITF/Tyrosinase Axis

by Min-Kyeong Lee, Kyoung Mi Moon, Su-Yeon Park, Jaeseong Seo, Ah-Reum Kim and Bonggi Lee

Antioxidants 2024, 13(12), 1547; https://doi.org/10.3390/antiox13121547 - 17 Dec 2024

Viewed by 437

Abstract

Melanogenesis, the biological process responsible for melanin synthesis, plays a crucial role in determining skin and hair color, photoprotection, and serving as a biomarker in various diseases. While various factors regulate melanogenesis, the role of fatty acids in this process remains underexplored. This [...] Read more.

Melanogenesis, the biological process responsible for melanin synthesis, plays a crucial role in determining skin and hair color, photoprotection, and serving as a biomarker in various diseases. While various factors regulate melanogenesis, the role of fatty acids in this process remains underexplored. This study investigated the anti-melanogenic properties of 10(E)-pentadecenoic acid (10E-PDA) through both in silico and in vitro analyses. SwissSimilarity was utilized to predict the functional properties of 10E-PDA by comparing it with structurally similar lipids known to exhibit anti-melanogenic effects. Subsequent in vitro experiments demonstrated that 10E-PDA significantly reduced melanin production and intracellular tyrosinase activity in α-MSH (melanocyte-stimulating hormone)-stimulated B16F10 melanoma cells without exhibiting significant cytotoxicity at concentrations up to 15 μM. Further mechanistic studies revealed that 10E-PDA inhibited the nuclear translocation of microphthalmia-associated transcription factor (MITF), consistent with the decrease observed in p-MITF protein levels. It also decreased the mRNA levels of tyrosinase-related proteins (TRP-1, TRP-2) and tyrosinase, while reducing the protein levels of TRP-1 and tyrosinase, but not TRP-2. These findings suggest that 10E-PDA exerts its anti-melanogenic effects by modulating the MITF/tyrosinase axis, presenting potential therapeutic implications for skin pigmentation disorders. Full article

(This article belongs to the Special Issue Antioxidants for Skin Health)

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13 pages, 1326 KiB

Open AccessArticle

Sex and Age Differences in Glucocorticoid Signaling After an Aversive Experience in Mice

by Yun Li, Bin Zhang, Youhua Yang, Ping Su, James Nicholas Samsom, Albert H. C. Wong and Fang Liu

Cells 2024, 13(24), 2041; https://doi.org/10.3390/cells13242041 - 10 Dec 2024

Viewed by 788

Abstract

Background: glucocorticoids may play an important role in the formation of fear memory, which is relevant to the neurobiology of post-traumatic stress disorder (PTSD). In our previous study, we showed the glucocorticoid receptor (GR) forms a protein complex with FKBP51, which prevents translocation [...] Read more.

Background: glucocorticoids may play an important role in the formation of fear memory, which is relevant to the neurobiology of post-traumatic stress disorder (PTSD). In our previous study, we showed the glucocorticoid receptor (GR) forms a protein complex with FKBP51, which prevents translocation of GR into the nucleus to affect gene expression; this complex is elevated in PTSD patients and by fear-conditioned learning in mice, and disrupting this complex blocks the storage and retrieval of fear-conditioned memories. The timing of release of glucocorticoid relative to the formation of a traumatic memory could be important in this process, and remains poorly understood. Methods and Results: we mapped serum corticosterone over time after fear conditioning in cardiac blood samples from male and female mice, as well as adult and aged mice using ELISA. We show a significant alteration in serum corticosterone after conditioning; notably, levels spike after 30 min but drop lower than unconditioned controls after 24 h. We further investigate the effect of glucocorticoid on GR phosphorylation and localization in HEK 293T cells by Western blot. Hydrocortisone treatment promotes phosphorylation and nuclear translocation of GR. Conclusions: these data contribute to our understanding of the processes linking stress responses to molecular signals and fear memory, which is relevant to understanding the shared mechanisms related to PTSD. Full article

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Figure 1
Successful establishment of fear-conditioned mouse model. (A) A schematic illustration schedule for fear conditioning. The conditioned stimulus (CS) was a white light illuminated for 30 s, the unconditioned stimulus (US) was a 1 s 0.5 mA foot shock. Conditioned animals received 5 CS-US pairings, control animals received the CS alone. (B) Time spent freezing during the 3 min CS presentation on day 5 in adult male (8-week-old) mice. Conditioned animals showed significantly more freezing behavior compared to controls. **** p < 0.0001, n = 16, permutation test. Full article ">Figure 2
Fear conditioning affects the time course of corticosterone levels in mice. (A) Corticosterone protein-expression response curves over 24 h in unconditioned (Control) and fear-conditioned animals (Conditioned). Comparison of corticosterone levels over time in conditioned (dashed) and unconditioned (solid) adult (8-week-old) male (B), adult female (C), aged (64-week-old) male (D), and aged female (E) mice. Data are shown as mean ± SEM, 4-way ANOVA (n = 4 *, female aged control n = 3), -corrected post hoc marginal means. Significance indicators: # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 relative to 0 h; $ p < 0.05, $$$ p < 0.001, $$$$ p < 0.0001 relative to 0.5 h; %% p < 0.01 relative to 1 h; ^ p < 0.05, ^^ p < 0.01 relative to 2 h, &&& p < 0.001 relative to 4 h, † p < 0.05, †† p < 0.005, †††† p < 0.0001 relative to 8 h; ‡ p < 0.05 relative to 12 h. * Control relative to Conditioned; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">Figure 3
Female mice have increased corticosterone over time, relative to males, after fear conditioning. Comparison of corticosterone levels over time in male (solid) and female (dashed) adult (8-week-old) unconditioned control mice (A), adult conditioned (B), aged (64-week-old) control (C), and aged conditioned (D) mice. Data are shown as mean ± SEM, 4-way ANOVA (n = 4 *, female aged control n = 3), fdr-corrected post hoc marginal means, * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 4
Age reduces corticosterone levels and reactivity to fear conditioning. Comparison of corticosterone levels over time in adult (8-week-old) (solid) and aged (64-week-old) (dashed) male unconditioned control mice (A), male conditioned (B), female control (C), and female conditioned (D) mice. Data are shown as mean ± SEM, 4-way ANOVA (n = 4 *, female aged control n = 3), fdr-corrected post hoc marginal means, * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 5
Hydrocortisone treatment stimulates Ser211 phosphorylation of glucocorticoid receptor (GR) and promotes nuclear translocation of GR in HEK 293T cells. (A) Representative Western blots (top) and densitometric analysis (bottom) of phosphorylated GR-S211 (pGRSer211) in HEK 293T whole-cell lysates stimulated by different concentrations of hydrocortisone. The level of pGRSer211 is expressed as a ratio relative to levels of unphosphorylated GR normalized to the vehicle (DMSO) alone condition. (B) Representative Western blots (top) and densitometric analysis (bottom) of the levels of pGRSer211 in HEK 293T cells stimulated by hydrocortisone (100 nM) over time. pGRSer211/GR ratios were normalized to time 0. (C) Representative Western blots (top) and densitometric analysis (bottom) of the changes in cytoplasmic GR protein expression in HEK 293T cells stimulated by different concentrations of hydrocortisone. GR levels expressed relative to α–tubulin and normalized to vehicle (DMSO). (D) Representative Western blots (top) and densitometric analysis (bottom) of the changes in nuclear GR protein expression in HEK 293T cells stimulated by different concentrations of hydrocortisone. GR levels expressed relative to histone H3 and normalized to vehicle (DMSO). Data are shown as mean ± SEM, one-way ANOVA (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">Figure 5 Cont.
Hydrocortisone treatment stimulates Ser211 phosphorylation of glucocorticoid receptor (GR) and promotes nuclear translocation of GR in HEK 293T cells. (A) Representative Western blots (top) and densitometric analysis (bottom) of phosphorylated GR-S211 (pGRSer211) in HEK 293T whole-cell lysates stimulated by different concentrations of hydrocortisone. The level of pGRSer211 is expressed as a ratio relative to levels of unphosphorylated GR normalized to the vehicle (DMSO) alone condition. (B) Representative Western blots (top) and densitometric analysis (bottom) of the levels of pGRSer211 in HEK 293T cells stimulated by hydrocortisone (100 nM) over time. pGRSer211/GR ratios were normalized to time 0. (C) Representative Western blots (top) and densitometric analysis (bottom) of the changes in cytoplasmic GR protein expression in HEK 293T cells stimulated by different concentrations of hydrocortisone. GR levels expressed relative to α–tubulin and normalized to vehicle (DMSO). (D) Representative Western blots (top) and densitometric analysis (bottom) of the changes in nuclear GR protein expression in HEK 293T cells stimulated by different concentrations of hydrocortisone. GR levels expressed relative to histone H3 and normalized to vehicle (DMSO). Data are shown as mean ± SEM, one-way ANOVA (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">

18 pages, 2463 KiB

Open AccessArticle

Interaction Between the Matrix Protein and the Polymerase Complex of Respiratory Syncytial Virus

by Elliot B. Atchison, Sarah N. Croft, Cynthia Mathew, Daniel W. Brookes, Matthew Coates, Kazuhiro Ito and Reena Ghildyal

Viruses 2024, 16(12), 1881; https://doi.org/10.3390/v16121881 - 4 Dec 2024

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Abstract

The global burden of respiratory syncytial virus (RSV) and severe associated disease is prodigious. RSV-specific vaccines have been launched recently but there is no antiviral medicine commercially available. RSV polymerase (L) protein is one of the promising antiviral targets, along with fusion and [...] Read more.

The global burden of respiratory syncytial virus (RSV) and severe associated disease is prodigious. RSV-specific vaccines have been launched recently but there is no antiviral medicine commercially available. RSV polymerase (L) protein is one of the promising antiviral targets, along with fusion and nucleocapsid proteins. During medicinal chemistry campaigns, two potent L-protein inhibitors (PC786 and PC751) were identified. Both compounds inhibited the RSV A/B-induced cytopathic effect in HEp-2 cells equally, but PC786 was more potent than PC751 in bronchial epithelial cells. Repeated treatment with escalating concentrations on RSV A2-infected HEp-2 cells revealed both inhibitors led to a Y1631H mutation in the L protein, but only PC786 induced a mutation in the M protein (V153A). By L protein fragment and M protein binding analysis, we showed that the M protein interacts with the 1392–1735 amino acid region of the L protein, where PC786 potentially binds. In addition, PC786 treatment or PC786-induced mutant RSV was found to increase M-protein nuclear localisation later in infection, concomitant with delayed fusion protein localisation at the budding viral filaments. As M protein is known to play a key role in virus assembly and budding late in infection, our data suggests that disrupting the interaction between the M and L proteins could provide a novel target for antiviral development. Full article

(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)

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Changes in viral load in vehicle, PC786- and PC751-treated ALI-cultured bronchial epithelium post RSV (0.002 MOI) apical inoculation. PC786 or PC751 was added to the apical surface, once daily from Day 0 (1 h after virus inoculation) to Day 6. The assay was conducted in triplicate, and the graph indicates geometric mean with 95% CI. Full article ">Figure 2
SNP genotype analysis of the L-gene of RSV A2 treated with PC786 (A), PC751 (B) or wild-type RSV A2 (passage 6), and also of the M-gene of RSV A2 treated with PC786 (C). The axes show the relative fluorescence of the probes specific to either the WT or mutant sequence of the L-gene or M-gene. Full article ">Figure 3
PC786 treatment results in increased nuclear retention of GFP-M. Cells transfected to express GFP-Mwt were treated with 100 nM PC751 or PC786, DMSO (vehicle), or left untreated (NT) for 12 h before imaging live at 24 h post transfection. Fn/c for M protein was calculated using the formula Fn/c = (Fn − Fb)/(Fc − Fb), where Fn = nuclear fluorescence, Fc = cytoplasmic fluorescence, Fb = background autofluorescence. Unpaired Student’s t test was used to determine statistical differences indicated on the graphs. Statistical significance was set at p < 0.05. **—p < 0.001, ***—p < 0.0001, ****—p < 0.00001, ns—non significant. Full article ">Figure 4
A2-786 has abnormal assembly and budding compared to wild-type A2. HEp2 cells were infected with A2 wild-type or its mutants, A2-751 (mutation in L only), A2-786 (mutation in L and M) at MOI = 1. Cells were fixed at different times, permeabilised and probed with anti-RSV antibody (recognises F, N, P, M proteins) and either anti-F protein or anti-M protein antibodies. Cells were incubated with Alexa488 (green) or Alexa547 (red) secondary antibodies, stained with Hoechst (blue nuclear stain) and mounted on slides. Slides were viewed on Nikon Ti confocal system and digital images collected as described in text. Single images representative of at least 15 images from two different experiments are shown. Colocaliation is denoted by yellow colour in the images labelled ‘merge’. (A,B) Cells fixed at 18 h post infection. (C,D) Cells fixed at 30 h (A2-751) or 36 h (A2-786) post-infection. (E) A2-786 infected cells fixed at 42 h post-infection. (F) The Fn/c for M at 18 h and 30 h post-infection was calculated (as in <a href="#viruses-16-01881-f003" class="html-fig">Figure 3</a>) for digital images such as shown in (A–D). Unpaired Student’s t-test was used to determine statistical differences indicated on the graph. Statistical significance was set at p < 0.05. **—p < 0.001, ns—non significant. Full article ">Figure 5
M protein binds to fragment 6 (1518 aa to 1863 aa) of L protein. (A) Schematic showing the position of the L-protein fragments and the mutation in fragment 6. The cDNA is shown, with L protein below. Red lines indicate the length of each fragment. Arrows point to mutation. (B) Each of the L-gene fragments shown in A was cloned into pET30a vector for expression in bacteria. Each clone was expressed in E. coli and lysate separated into soluble (SF) and insoluble (IF) fractions using B-PER reagent (Pierce). Equivalent volumes of each fraction were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Molecular weights in kDa are shown on the left and fragment identity on the top of the gel. (C) Protein–protein overlay binding assay. Proteins separated on SDS-PAGE as in B were transferred to a nitrocellulose membrane. The membrane was blocked and probed with purified wild-type M protein. Bound M protein was detected with mouse antibody to M protein followed by secondary antibody and development using ECL. Luminescent bands were detected on a LiCor Odyssey Fc. Molecular weights in kDa are shown on the left and fragment identity above the blot. Full article ">Figure 6
Interaction between fragment 6 of L and M protein is modulated by Y1631 and V153 in L and M proteins, respectively. (A) L-gene fragments 5, 6, and 7, shown in <a href="#viruses-16-01881-f005" class="html-fig">Figure 5</a>, were cloned into pmCherry-C1, with a N-terminal mCherry (red) fusion tag. Each plasmid was co-transfected into COS-7 cells with wild-type M (Mwt) cloned in frame with an N-terminal EGFP (green) tag. Images were acquired 24 h later by live cell confocal microscopy on a Nikon Ti system using NIS Elements software. Single cells for each co-transfection are shown, representative of at least 20 cells and three independent experiments. Colocalization is indicated by yellow colour in the images labelled ‘merge’. (B) Site-directed mutagenesis was used to mutate fragment 6 (Y1631H) and Mwt (V153A). Mutants were cloned into pmCherry-C1 and pEGFP-DestC, respectively. Plasmids were co-transfected into COS-7 cells to express the requisite L fragment with Mwt or M (V153A), followed by live cell microscopy as for A. (C) Images such as those in A, B were analysed for co-localisation of red and green pixels using the Coloc2 plugin within Fiji. Mean ± SEM of Pearson’s correlation coefficient is depicted. L6m—mutated fragment 6 (Y1631H). Mm—mutated M (V153A). Statistical significance was set at p < 0.05. **—p < 0.001, ns—non significant. Full article ">

23 pages, 7444 KiB

Open AccessArticle

Monocyte/Macrophage-Specific Loss of ARNTL Suppresses Chronic Kidney Disease-Associated Cardiac Impairment

by Yuya Yoshida, Naoki Nishikawa, Kohei Fukuoka, Akito Tsuruta, Kaita Otsuki, Taiki Fukuda, Yuma Terada, Tomohito Tanihara, Taisei Kumamoto, Ryotaro Tsukamoto, Takumi Nishi, Kosuke Oyama, Kengo Hamamura, Kouta Mayanagi, Satoru Koyanagi, Shigehiro Ohdo and Naoya Matsunaga

Int. J. Mol. Sci. 2024, 25(23), 13009; https://doi.org/10.3390/ijms252313009 - 3 Dec 2024

Viewed by 710

Abstract

Defects in Aryl hydrocarbon receptor nuclear translocator-like 1 (ARNTL), a central component of the circadian clock mechanism, may promote or inhibit the induction of inflammation by monocytes/macrophages, with varying effects on different diseases. However, ARNTL’s role in monocytes/macrophages under chronic kidney disease (CKD), [...] Read more.

Defects in Aryl hydrocarbon receptor nuclear translocator-like 1 (ARNTL), a central component of the circadian clock mechanism, may promote or inhibit the induction of inflammation by monocytes/macrophages, with varying effects on different diseases. However, ARNTL’s role in monocytes/macrophages under chronic kidney disease (CKD), which presents with systemic inflammation, is unclear. Here, we report that the expression of Arntl in monocytes promoted CKD-induced cardiac damage. The expression of G-protein-coupled receptor 68 (GPR68), which exacerbates CKD-induced cardiac disease, was regulated by ARNTL. Under CKD conditions, GPR68 expression was elevated via ARNTL, particularly in the presence of PU.1, a transcription factor specific to monocytes and macrophages. In CKD mouse models lacking monocyte-specific ARNTL, GPR68 expression in monocytes was reduced, leading to decreased cardiac damage and fibrosis despite no improvement in renal excretory capacity or renal fibrosis and increased angiotensin II production. The loss of ARNTL did not affect the expression of marker molecules, indicating the origin or differentiation of cardiac macrophages, but affected GPR68 expression only in cardiac macrophages derived from mature monocytes, highlighting the significance of the interplay between GPR68 and ARNTL in monocytes/macrophages and its influence on cardiac pathology. Understanding this complex relationship between circadian clock mechanisms and disease could help uncover novel therapeutic strategies. Full article

(This article belongs to the Topic Molecular and Cellular Mechanisms of Heart Disease)

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24 pages, 2915 KiB

Open AccessArticle

Detection of Hepatitis C Virus Infection from Patient Sera in Cell Culture Using Semi-Automated Image Analysis

by Noemi Schäfer, Paul Rothhaar, Christian Heuss, Christoph Neumann-Haefelin, Robert Thimme, Julia Dietz, Christoph Sarrazin, Paul Schnitzler, Uta Merle, Sofía Pérez-del-Pulgar, Vibor Laketa and Volker Lohmann

Viruses 2024, 16(12), 1871; https://doi.org/10.3390/v16121871 - 30 Nov 2024

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Abstract

The study of hepatitis C virus (HCV) replication in cell culture is mainly based on cloned viral isolates requiring adaptation for efficient replication in Huh7 hepatoma cells. The analysis of wild-type (WT) isolates was enabled by the expression of SEC14L2 and by inhibitors [...] Read more.

The study of hepatitis C virus (HCV) replication in cell culture is mainly based on cloned viral isolates requiring adaptation for efficient replication in Huh7 hepatoma cells. The analysis of wild-type (WT) isolates was enabled by the expression of SEC14L2 and by inhibitors targeting deleterious host factors. Here, we aimed to optimize cell culture models to allow infection with HCV from patient sera. We used Huh7-Lunet cells ectopically expressing SEC14L2, CD81, and a GFP reporter with nuclear translocation upon cleavage by the HCV protease to study HCV replication, combined with a drug-based regimen for stimulation of non-modified wild-type isolates. RT-qPCR-based quantification of HCV infections using patient sera suffered from a high background in the daclatasvir-treated controls. We therefore established an automated image analysis pipeline based on imaging of whole wells and iterative training of a machine learning tool, using nuclear GFP localization as a readout for HCV infection. Upon visual validation of hits assigned by the automated image analysis, the method revealed no background in daclatasvir-treated samples. Thereby, infection events were found for 15 of 34 high titer HCV genotype (gt) 1b sera, revealing a significant correlation between serum titer and successful infection. We further show that transfection of viral RNA extracted from sera can be used in this model as well, albeit with so far limited efficiency. Overall, we generated a robust serum infection assay for gt1b isolates using semi-automated image analysis, which was superior to conventional RT-qPCR-based quantification of viral genomes. Full article

(This article belongs to the Special Issue Hepatitis C Virus 2024)

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Figure 1
Establishment of a cell culture model to assess HCV WT replication: (A,B,D,F) Subgenomic reporter replicons based on Con1 WT and GLT1 WT were electroporated into Huh7-Lunet C/M/S cells and treated with either DMSO or the indicated concentrations of (A) H479 combined with PI4KA-F1 (PCi), or with PCi and the indicated concentrations of DCV (B)/Legalon SIL (D) or Alisporivir (F) at 4 h after electroporation. Cells were harvested 72 h later and firefly luciferase activity from cell lysates (RLU) was quantified as a correlate of RNA replication efficiency and normalized to 4 h. A replication-deficient Con1 variant (ΔGDD) served as negative control and is indicated with a dashed grey line. Values are shown as means from two (A,B) or three (D,F) independent experiments with two technical replicates each. (C,E,G) Huh7-Lunet C/M/S cells were seeded into 96-well plates and treated with the indicated concentrations of DCV (C)/Legalon SIL (E) or Alisporivir (G). After 72 h, cell viability in cell lysates was measured via Cell-titer-Glow and normalized to the DMSO control. Values are shown as means from at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired two-tailed Student’s t-test). Full article ">Figure 2
Comparison of luciferase activity and RT-qPCR-based readouts in persistent replicon cells and HCV infected cells: (A) Cell lines harboring the indicated replicons were treated with 250 µM SIL, 1 µM Alisporivir, 1 nM DCV, or were left untreated, and harvested at the indicated time points. Upper panels: Firefly luciferase activity from cell lysates (RLU) was quantified as a correlate of RNA replication efficiency and normalized to 4 h. Values are shown as means from two independent experiments with two technical replicates each. Lower panels: After RNA extraction, HCV RNA copies were quantified using RT-qPCR. HCV RNA copy numbers (CN) were normalized on GAPDH and are shown as CN per well. Values are shown as means from two independent experiments with three technical replicates each. (B) Huh7-Lunet C/M/S cells were infected with either GLT1 patient serum or JC1cc infectious particles (MOI of 10 at the first repetition, MOI of 1 at the second and third repetition). After 72 h, samples were harvested, and HCV RNA replication was measured via RT-qPCR. HCV RNA copy numbers (CN) were normalized on GAPDH and are shown as CN per well. Values are shown as means of four (GLT1: untr. + DCV), three (JC1cc: untr. + DCV), or two (GLT1 and JC1cc: SIL and Alisporivir treatment) independent experiments with three technical replicates each. p-values from unpaired two-tailed Student’s t-test are indicated for all experiments with 3 or more biological replicates. Full article ">Figure 3
Workflow for serum infection and establishment of semi-automated image analysis: (A) Workflow for serum infection. (B) Principle of automated image analysis by machine learning. Infected cells are identified by nuclear GFP due to co-localization with DAPI. Positive cells were marked in red by ilastik, facilitating visual validation. (C) Huh7-Lunet C/M/S cells were infected with JC1cc (MOI = 1) alone or additionally treated with DCV, fixed, and stained with DAPI 72 h post-infection. A group of infected cells is indicated by a white box and additionally displayed with a higher magnification. Percentage of positive cells (right panel) was obtained by automated image analysis. *** p < 0.001 (unpaired two-tailed Student’s t-test). (B,C) Scale bar indicates a length of 100 µm. (D) Optimization of the automated image analysis. False-positive background in images of either uninfected or DCV-treated cells was reduced by iterative training of the machine learning tool, including adjustment of cell count per well and refocusing strategy. (E) Workflow of automated image analysis (AIA) followed by visual validation. Full article ">Figure 4
Comparison of HCV infection using high-titer sera by RT-qPCR or microscopic analysis: (A–C) Huh7-Lunet C/M/S cells treated with PCi and infected with patient sera in presence or absence of DCV; 72 h after infection, cells were fixed, stained with DAPI. (A) Total RNA was extracted 72 h after infection and HCV RNA was quantified. (B) 72 h after infection, quantification by automated image analysis was performed. (C) Truly infected cells detected after visual validation of hits generated by the automated image analysis. Note that no bona fide infected cells were detected in the DCV-treated samples. All values are means and SD of three or two (serum 44) independent biological replicates. Note that RT-qPCR data from the GLT1 serum were taken from <a href="#viruses-16-01871-f002" class="html-fig">Figure 2</a>B to allow a direct comparison (n = 4). n.d.: not detectable. * p < 0.05, ** p < 0.01 (unpaired two-tailed Student’s t-test). (D) Results of the semi-automated image-based analysis of all data shown in panel (C) and in Figure 6C were analyzed regarding the impact of RNA titer and post-transplant status on infection outcome. The anonymized abbreviation of each serum is given next to the respective dot (see <a href="#viruses-16-01871-t001" class="html-table">Table 1</a> and <a href="#viruses-16-01871-t002" class="html-table">Table 2</a>). Samples were judged positive if at least one infected cell was found via the automated image analysis combined with visual validation. * p < 0.05 (unpaired two-tailed Student’s t-test). Full article ">Figure 5
Establishment of an HCV WT RNA transfection protocol. In vitro transcribed RNA of GLT1 WT (gt1b), TN WT (gt1a), and DBN WT (gt3a) was transfected into Huh7-Lunet C/M/S cells with different amounts of input RNA, ranging from 107 to 1010 RNA copies per transfection, either by electroporation (A,C) or by lipid-based transfection (B,D). RT-qPCR (A,B) and the automated image analysis served as readouts (C,D). Samples were measured in technical triplicates. In the image-based analysis (C,D), the samples transfected with 108 RNA copies of TN WT and DBN WT were set as false-positive backgrounds, since no infected cells could be detected upon visual validation, depicted as a dashed grey line. Numbers of cells visually validated represent only DCV- samples. (A,C) Electroporation: Samples were harvested after 72 h. Values are taken from two (TN WT, DBN WT) or three (GLT1 WT) independent experiments. (B,D) Lipid-based transfection: Cells were split 24 h post-transfection, at this time point DCV was added. Samples were harvested after 2 (to stick to the total incubation time of 72 h) or 3 (to ensure DCV treatment for 72 h) days. Since there were no relevant differences in the results with varying incubation times observed, the data of the two experiments are shown in the same graph. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (unpaired two-tailed Student’s t-test). Full article ">Figure 6
Results of RNA transfection in comparison to serum infection. Ten patient sera were tested using RNA transfection and serum infection in parallel. All samples were harvested after 72 h, treatment with DCV served as negative control. Samples were analyzed via RT-qPCR (A), AIA (B), and with AIA followed by visual validation (C). Values are shown as means of three biological replicates and were measured in technical triplicates in RT-qPCR. Data of the serum infection assay of sera 2130, 2213, AU, and GLT1 were taken from <a href="#viruses-16-01871-f004" class="html-fig">Figure 4</a> for better comparison. n.d.: not detectable. ** p < 0.01 (unpaired two-tailed Student’s t-test). Full article ">

30 pages, 16811 KiB

Open AccessFeature PaperArticle

Unveiling Smyd-2’s Role in Cytoplasmic Nrf-2 Sequestration and Ferroptosis Induction in Hippocampal Neurons After Cerebral Ischemia/Reperfusion

by Daohang Liu and Yizhun Zhu

Cells 2024, 13(23), 1969; https://doi.org/10.3390/cells13231969 - 28 Nov 2024

Viewed by 784

Abstract

SET and MYND Domain-Containing 2 (Smyd-2), a specific protein lysine methyltransferase (PKMT), influences both histones and non-histones. Its role in cerebral ischemia/reperfusion (CIR), particularly in ferroptosis—a regulated form of cell death driven by lipid peroxidation—remains poorly understood. This study identifies the expression of [...] Read more.

SET and MYND Domain-Containing 2 (Smyd-2), a specific protein lysine methyltransferase (PKMT), influences both histones and non-histones. Its role in cerebral ischemia/reperfusion (CIR), particularly in ferroptosis—a regulated form of cell death driven by lipid peroxidation—remains poorly understood. This study identifies the expression of Smyd-2 in the brain and investigates its relationship with neuronal programmed cell death (PCD). We specifically investigated how Smyd-2 regulates ferroptosis in CIR through its interaction with the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway. Smyd-2 knockout protects HT-22 cells from Erastin-induced ferroptosis but not TNF-α + Smac-mimetic-induced apoptosis/necroptosis. This neuroprotective effect of Smyd-2 knockout in HT-22 cells after Oxygen–Glucose Deprivation/Reperfusion (OGD/R) was reversed by Erastin. Smyd-2 knockout in HT-22 cells shows neuroprotection primarily via the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway, despite the concurrent upregulation of Smyd-2 and Nrf-2 observed in both the middle cerebral artery occlusion (MCAO) and OGD/R models. Interestingly, vivo experiments demonstrated that Smyd-2 knockout significantly reduced ferroptosis and lipid peroxidation in hippocampal neurons following CIR. Moreover, the Nrf-2 inhibitor ML-385 abolished the neuroprotective effects of Smyd-2 knockout, confirming the pivotal role of Nrf-2 in ferroptosis regulation. Cycloheximide (CHX) fails to reduce Nrf-2 expression in Smyd-2 knockout HT-22 cells. Smyd-2 knockout suppresses Nrf-2 lysine methylation, thereby promoting the Nrf-2/Keap-1 pathway without affecting the PKC-δ/Nrf-2 pathway. Conversely, Smyd-2 overexpression disrupts Nrf-2 nuclear translocation, exacerbating ferroptosis and oxidative stress, highlighting its dual regulatory role. This study underscores Smyd-2’s potential for ischemic stroke treatment by disrupting the Smyd-2/Nrf-2-driven antioxidant capacity, leading to hippocampal neuronal ferroptosis. By clarifying the intricate interplay between ferroptosis and oxidative stress via the Nrf-2/Keap-1 pathway, our findings provide new insights into the molecular mechanisms of CIR and identify Smyd-2 as a promising therapeutic target. Full article

(This article belongs to the Special Issue Oxidative Stress and Antioxidant Strategies: Relationships and Cellular Pathways for Human Health)

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