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Int. J. Mol. Sci., Volume 23, Issue 3 (February-1 2022) – 914 articles

Cover Story (view full-size image): Glioblastoma is the most lethal brain tumor with the poorest overall survival. Current standard treatments are not effective. Hence, the identification of novel strategies to treat this devastating pathology is crucial. In this context, the dysregulation of alternative splicing process represents a valuable source for the identification of novel diagnostic and prognostic tools, as well as therapeutic targets, in different tumor pathologies. This study unveils that the truncated splicing variant of the somatostatin receptor subtype 5 (sst5TMD4) is overexpressed and associated with enhanced malignancy features in human glioblastoma, demonstrating the potential utility of sst5TMD4 as a potential diagnostic and prognostic biomarker. Furthermore, sst5TMD4 also represents a novel therapeutic target for glioblastoma patients. View this paper
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28 pages, 2686 KiB  
Review
Mitochondrial-Targeted Therapy for Doxorubicin-Induced Cardiotoxicity
by Bin Bin Wu, Kam Tong Leung and Ellen Ngar-Yun Poon
Int. J. Mol. Sci. 2022, 23(3), 1912; https://doi.org/10.3390/ijms23031912 - 9 Feb 2022
Cited by 70 | Viewed by 10520
Abstract
Anthracyclines, such as doxorubicin, are effective chemotherapeutic agents for the treatment of cancer, but their clinical use is associated with severe and potentially life-threatening cardiotoxicity. Despite decades of research, treatment options remain limited. The mitochondria is commonly considered to be the main target [...] Read more.
Anthracyclines, such as doxorubicin, are effective chemotherapeutic agents for the treatment of cancer, but their clinical use is associated with severe and potentially life-threatening cardiotoxicity. Despite decades of research, treatment options remain limited. The mitochondria is commonly considered to be the main target of doxorubicin and mitochondrial dysfunction is the hallmark of doxorubicin-induced cardiotoxicity. Here, we review the pathogenic mechanisms of doxorubicin-induced cardiotoxicity and present an update on cardioprotective strategies for this disorder. Specifically, we focus on strategies that can protect the mitochondria and cover different therapeutic modalities encompassing small molecules, post-transcriptional regulators, and mitochondrial transfer. We also discuss the shortcomings of existing models of doxorubicin-induced cardiotoxicity and explore advances in the use of human pluripotent stem cell derived cardiomyocytes as a platform to facilitate the identification of novel treatments against this disorder. Full article
(This article belongs to the Special Issue Metabolic Therapies for Heart Failure)
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<p>The chemical structure of anthracyclines.</p> Full article ">Figure 2
<p>Schematic diagram of the mechanisms of DOX-induced cardiotoxicity. DOX can suppress the function of the ETC, and thereby reduce ATP levels. Increased ROS production induces the opening of the mPTP and the release of pro-apoptotic proteins such as cyt C. DOX can also induce calcium overload by altering the levels and activities of Ca<sup>2+</sup> handling proteins such as TRPC, RYR2, and SERCA2A. DOX can disturb iron uptake and storage, leading to iron overload, apoptosis, and ferroptosis. DOX can complex with topoisomerase to induce DNA damage. DOX, doxorubicin; ER, endoplasmic reticulum; TRPC, transient receptor potential canonical; IRP, iron-regulatory protein; TfR, transferrin receptor; DMT1, divalent metal transporter 1; mPTP, mitochondrial permeability transition pore; Δψm, mitochondrial membrane potential; TOP2β, topoisomerase 2β; RYR2, ryanodine receptor 2; SERCA2A, sarcoplasmic/endoplasmic reticulum calcium ATPase 2; Mfrn2, mitoferrin-2; ABCB8, ABC protein B8; ETC, electron transport chain; Cyt C, cytochrome C. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 28 December 2021).</p> Full article ">Figure 3
<p>Schematic diagram of DOX-induced intracellular ROS generation. DOX was reduced to semiquinone by cellular oxidoreductases, and then parent quinone was regenerated by transferring an electron to oxygen (O<sub>2</sub>). The resulting superoxide (O<sub>2</sub><sup>−</sup>) radical initiates the formation of ROS. NO, nitric oxide; RNS, reactive nitrogen species; SOD, superoxide dismutase.</p> Full article ">Figure 4
<p>DOX-induced ferroptosis. DOX promotes iron uptake and accumulation, increases ROS production, and induces ferroptosis. IRP, iron-regulatory protein. Up arrow, increase. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 28 December 2021).</p> Full article ">Figure 5
<p>DOX can induce calcium overload by increasing calcium influx and intracellular calcium release. SR, sarcoplasmic reticulum; ER, endoplasmic reticulum. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 28 December 2021).</p> Full article ">Figure 6
<p>Research models for DOX-induced cardiotoxicity. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 28 December 2021).</p> Full article ">
16 pages, 2136 KiB  
Review
Recent Advance in Biological Responsive Nanomaterials for Biosensing and Molecular Imaging Application
by Zhenqi Jiang, Xiao Han, Chen Zhao, Shanshan Wang and Xiaoying Tang
Int. J. Mol. Sci. 2022, 23(3), 1923; https://doi.org/10.3390/ijms23031923 - 8 Feb 2022
Cited by 100 | Viewed by 2765
Abstract
In recent decades, as a subclass of biomaterials, biologically sensitive nanoparticles have attracted increased scientific interest. Many of the demands for physiologically responsive nanomaterials in applications involving the human body cannot be met by conventional technologies. Due to the field’s importance, considerable effort [...] Read more.
In recent decades, as a subclass of biomaterials, biologically sensitive nanoparticles have attracted increased scientific interest. Many of the demands for physiologically responsive nanomaterials in applications involving the human body cannot be met by conventional technologies. Due to the field’s importance, considerable effort has been expended, and biologically responsive nanomaterials have achieved remarkable success thus far. This review summarizes the recent advancements in biologically responsive nanomaterials and their applications in biosensing and molecular imaging. The nanomaterials change their structure or increase the chemical reaction ratio in response to specific bio-relevant stimuli (such as pH, redox potentials, enzyme kinds, and concentrations) in order to improve the signal for biologically responsive diagnosis. We use various case studies to illustrate the existing issues and provide a clear sense of direction in this area. Furthermore, the limitations and prospects of these nanomaterials for diagnosis are also discussed. Full article
(This article belongs to the Special Issue Nano-Materials and Methods 3.0)
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<p>Schematic illustration of Fe<sub>3</sub>O<sub>4</sub> nanozyme-strip for the detection of EBOV. Adapted from ref [<a href="#B18-ijms-23-01923" class="html-bibr">18</a>], with permission from Copyright © 2015, Elsevier B.V. All rights reserved.</p> Full article ">Figure 2
<p>Nanoscale ZIF-8/Fer for AβO sensing utilizing electrochemical and optical methods. Adapted from ref [<a href="#B21-ijms-23-01923" class="html-bibr">21</a>], with permission from Copyright © 2019, American Chemical Society.</p> Full article ">Figure 3
<p>Synthesis and characterization of Mn-SS NCPs, as well as the GSH, triggered nanoparticle decomposition, drug release, and Mn<sup>2+</sup>-enhanced MRI. Adapted from ref [<a href="#B27-ijms-23-01923" class="html-bibr">27</a>], with permission from Copyright © 2017, American Chemical Society.</p> Full article ">Figure 4
<p>ALP-triggered self-assembly of near-infrared nanoparticles for the enhanced PA imaging of tumors. Adapted from ref [<a href="#B29-ijms-23-01923" class="html-bibr">29</a>], with permission from Copyright © 2018, American Chemical Society.</p> Full article ">
16 pages, 2073 KiB  
Article
Enhanced Fear Memories and Altered Brain Glucose Metabolism (18F-FDG-PET) following Subanesthetic Intravenous Ketamine Infusion in Female Sprague–Dawley Rats
by Kennett D. Radford, Rina Y. Berman, Shalini Jaiswal, Sharon Y. Kim, Michael Zhang, Haley F. Spencer and Kwang H. Choi
Int. J. Mol. Sci. 2022, 23(3), 1922; https://doi.org/10.3390/ijms23031922 - 8 Feb 2022
Cited by 2 | Viewed by 3107
Abstract
Although women and men are equally likely to receive ketamine following traumatic injury, little is known regarding sex-related differences in the impact of ketamine on traumatic memory. We previously reported that subanesthetic doses of an intravenous (IV) ketamine infusion following fear conditioning impaired [...] Read more.
Although women and men are equally likely to receive ketamine following traumatic injury, little is known regarding sex-related differences in the impact of ketamine on traumatic memory. We previously reported that subanesthetic doses of an intravenous (IV) ketamine infusion following fear conditioning impaired fear extinction and altered regional brain glucose metabolism (BGluM) in male rats. Here, we investigated the effects of IV ketamine infusion on fear memory, stress hormone levels, and BGluM in female rats. Adult female Sprague–Dawley rats received a single IV ketamine infusion (0, 2, 10, or 20 mg/kg, over a 2-h period) following auditory fear conditioning (three pairings of tone and footshock). Levels of plasma stress hormones, corticosterone (CORT) and progesterone, were measured after the ketamine infusion. Two days after ketamine infusion, fear memory retrieval, extinction, and renewal were tested over a three-day period. The effects of IV ketamine infusion on BGluM were determined using 18F-fluoro-deoxyglucose positron emission tomography (18F-FDG-PET) and computed tomography (CT). The 2 and 10 mg/kg ketamine infusions reduced locomotor activity, while 20 mg/kg infusion produced reduction (first hour) followed by stimulation (second hour) of activity. The 10 and 20 mg/kg ketamine infusions significantly elevated plasma CORT and progesterone levels. All three doses enhanced fear memory retrieval, impaired fear extinction, and enhanced cued fear renewal in female rats. Ketamine infusion produced dose-dependent effects on BGluM in fear- and stress-sensitive brain regions of female rats. The current findings indicate that subanesthetic doses of IV ketamine produce robust effects on the hypothalamic–pituitary–adrenal (HPA) axis and brain energy utilization that may contribute to enhanced fear memory observed in female rats. Full article
(This article belongs to the Special Issue Modeling Neurological Disorders in Experimental Animals: New Insights and Emerging Roles 2.0)
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<p>Effects of subanesthetic IV ketamine infusion on locomotor activity in female rats. (<b>A</b>) Experimental design of fear conditioning/ketamine infusion and fear memory testing. (<b>B</b>) Freezing behavior during fear conditioning (fear acquisition). Freezing gradually increased to repeated tone and footshock pairing but there were no group differences at each time point. (<b>C</b>) Time course of locomotor activity during the ketamine infusion (2 h). (<b>D</b>) Total activity during the first hour of ketamine infusion. (<b>E</b>) Total activity during the second hour of ketamine infusion. * <span class="html-italic">p</span> &lt; 0.05 compared to the saline controls.</p> Full article ">Figure 2
<p>Effects of subanesthetic IV ketamine infusion on fear memory retrieval, extinction, and renewal in female rats. (<b>A</b>) Ketamine infusion increased cued fear memory retrieval with all three doses (2, 10, and 20 mg/kg, 2 h). (<b>B</b>) Ketamine infusion impaired fear extinction retrieval in female rats. Each Block consists of average freezing of two consecutive auditory tone (CS) presentations. (<b>C</b>) Higher doses of ketamine (10 and 20 mg/kg) increased contextual fear memory when animals were tested in the conditioning chamber (context A). (<b>D</b>) Higher doses of ketamine (10 and 20 mg/kg) increased cued fear memory renewal (context A) after two sessions of cued fear extinction (context B). * <span class="html-italic">p</span> &lt; 0.05 compared to saline controls.</p> Full article ">Figure 3
<p>No effects of estrous cycle on ketamine-induced fear memory enhancement. (<b>A</b>) Representative images of vaginal cell types (from top to bottom): Cornified epithelial cells (C), Leukocytes (L), and Nucleated epithelial cells (N). Scale: 30 µm. (<b>B</b>) Freezing behavior during Cue Test 1. (<b>C</b>) Freezing during Cue Test 2. (<b>D</b>) Freezing behavior during contextual fear test 3. (<b>E</b>): Freezing behavior during cued fear renewal test.</p> Full article ">Figure 4
<p>Effects of subanesthetic doses of IV ketamine infusion on plasma stress hormone (CORT and progesterone) levels in female rats. (<b>A</b>) Higher doses of ketamine (10 and 20 mg/kg) significantly elevated plasma CORT levels. (<b>B</b>) Higher doses of ketamine (10 and 20 mg/kg) also elevated plasma progesterone levels. * <span class="html-italic">p</span> &lt; 0.05 compared to saline controls.</p> Full article ">Figure 5
<p>Effects of subanesthetic doses of IV ketamine infusion on BGluM of female rats. (<b>A</b>) Representative images of <sup>18</sup>F-FDG-PET/CT of a rat brain. Three images including sagittal, planar, and coronal sections from left to right are shown in the first image. The second image shows a coronal section with a rat brain atlas registered to it. (<b>B</b>) Both 10 and 20 mg/kg ketamine increased BGluM in the cortex. (<b>C</b>) The 10 mg/kg dose reduced BGluM in the thalamus. (<b>D</b>) The 20 mg/kg dose reduced BGluM in the hypothalamus. (<b>E</b>) Both 10 and 20 mg/kg ketamine doses reduced BGluM in the midbrain. Baseline BGluM levels in these brain regions are not significantly different between the groups as shown in scan 1. * <span class="html-italic">p</span> &lt; 0.05 compared to saline controls.</p> Full article ">
14 pages, 1888 KiB  
Article
Functional Transient Receptor Potential Ankyrin 1 and Vanilloid 1 Ion Channels Are Overexpressed in Human Oral Squamous Cell Carcinoma
by Fruzsina Kiss, Viktória Kormos, Éva Szőke, Angéla Kecskés, Norbert Tóth, Anita Steib, Árpád Szállási, Bálint Scheich, Balázs Gaszner, József Kun, Gábor Fülöp, Krisztina Pohóczky and Zsuzsanna Helyes
Int. J. Mol. Sci. 2022, 23(3), 1921; https://doi.org/10.3390/ijms23031921 - 8 Feb 2022
Cited by 16 | Viewed by 3135
Abstract
Oral squamous cell carcinoma (OSCC) is a common cancer with poor prognosis. Transient Receptor Potential Ankyrin 1 (TRPA1) and Vanilloid 1 (TRPV1) receptors are non-selective cation channels expressed on primary sensory neurons and epithelial and immune cells. TRPV1 mRNA and immunopositivity, as well [...] Read more.
Oral squamous cell carcinoma (OSCC) is a common cancer with poor prognosis. Transient Receptor Potential Ankyrin 1 (TRPA1) and Vanilloid 1 (TRPV1) receptors are non-selective cation channels expressed on primary sensory neurons and epithelial and immune cells. TRPV1 mRNA and immunopositivity, as well as TRPA1-like immunoreactivity upregulation, were demonstrated in OSCC, but selectivity problems with the antibodies still raise questions and their functional relevance is unclear. Therefore, here, we investigated TRPA1 and TRPV1 expressions in OSCC and analyzed their functions. TRPA1 and TRPV1 mRNA were determined by RNAscope in situ hybridization and qPCR. Radioactive 45Ca2+ uptake and ATP-based luminescence indicating cell viability were measured in PE/CA-PJ41 cells in response to the TRPA1 agonist allyl-isothiocyanate (AITC) and TRPV1 agonist capsaicin to determine receptor function. Both TRPA1 and TRPV1 mRNA are expressed in the squamous epithelium of the human oral mucosa and in PE/CA-PJ41 cells, and their expressions are significantly upregulated in OSCC compared to healthy mucosa. TRPA1 and TRPV1 activation (100 µM AITC, 100 nM capsaicin) induced 45Ca2+-influx into PE/CA-PJ41 cells. Both AITC (10 nM–5 µM) and capsaicin (100 nM–45 µM) reduced cell viability, reaching significant decrease at 100 nM AITC and 45 µM capsaicin. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the OSCC and confirm the expression of TRPV1 channel. These channels are functionally active and might regulate cancer cell viability. Full article
(This article belongs to the Section Molecular Biology)
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<p>Representative images of <span class="html-italic">Transient Receptor Potential Ankyrin 1</span> (<span class="html-italic">TRPA1</span>) and <span class="html-italic">Vanilloid 1</span> (<span class="html-italic">TRPV1</span>) mRNA in normal human oral epithelium (<b>A</b>,<b>B</b>), in the PE/CA-PJ41 cell line (<b>C</b>,<b>D</b>) and human squamous cell carcinoma (<b>E</b>,<b>F</b>). <span class="html-italic">TRPA1</span> mRNA (red) and <span class="html-italic">TRPV1</span> mRNA (green) by RNAscope and citokeratin-14 protein (white) by immunofluorescence were depicted and counterstained with DAPI (blue) for nuclei. Scale bar: 30 µm for all images.</p> Full article ">Figure 2
<p>Relative gene expression ratios of <span class="html-italic">Transient Receptor Potential Ankyrin 1</span> (<span class="html-italic">TRPA1</span>) and <span class="html-italic">Vanilloid 1</span> (<span class="html-italic">TRPV1</span>) receptors normalized to the <span class="html-italic">Importin 8 (IPO8)</span> reference gene in the healthy control oral mucosa (n = 10), compared to oral squamous cell carcinoma (OSCC; n = 15). Columns represent the mean + SEM, *** <span class="html-italic">p</span> &lt; 0.001, Mann–Whitney U test; (<b>A</b>). Expression of <span class="html-italic">TRPA1</span> and <span class="html-italic">TRPV1</span> mRNA in the PE/CA-PJ41 cell line and <span class="html-italic">TRPA1</span> and <span class="html-italic">TRPV1</span> expressing CHO cells (positive controls) (n = 4/group; (<b>B</b>)).</p> Full article ">Figure 3
<p>Effect of allyl-isothiocianate (AITC) and capsaicin (CAPS) on <sub>45</sub>Ca<sup>2+</sup> uptake (count per minute: CPM) of CHO cells expressing the cloned Transient Receptor Potential Ankyrin 1 (TRPA1, (<b>A</b>)) and Vanilloid 1 (TRPV1, (<b>B</b>)) receptors and PE/CA-PJ41 cells. CAPS and AITC responses were antagonized by capsazepine (10 µM) and HC-030031 (10 µM), respectively. <sub>45</sub>Ca<sup>2+</sup> accumulations are presented as a percentage of agonist control. Each column represents the mean ± SEM of n = 9, ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001 (vs. control, one-way ANOVA, Dunnett’s post hoc test); #### <span class="html-italic">p</span> &lt; 0.0001 (vs. 100 nM CAPS (<b>A</b>) or 100 µM AITC (<b>B</b>), one-way ANOVA, Dunnett’s post hoc test).</p> Full article ">Figure 4
<p>Effect of AITC and capsaicin in comparison with the solvent dimethyl sulfoxide (DMSO) on the viability of PE/CA-PJ41 cells, assessed by the CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay. Data show the average results of three independent experiments ± SEM. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.005, **** <span class="html-italic">p</span> &lt; 0.0001 (vs. control, one-way ANOVA, Dunnett’s post hoc test).</p> Full article ">
19 pages, 2718 KiB  
Article
Erythrocyte Membrane Nanomechanical Rigidity Is Decreased in Obese Patients
by Jesús Sot, Aritz B. García-Arribas, Beatriz Abad, Sara Arranz, Kevin Portune, Fernando Andrade, Alicia Martín-Nieto, Olaia Velasco, Eunate Arana, Itziar Tueros, Carla Ferreri, Sonia Gaztambide, Félix M. Goñi, Luis Castaño and Alicia Alonso
Int. J. Mol. Sci. 2022, 23(3), 1920; https://doi.org/10.3390/ijms23031920 - 8 Feb 2022
Cited by 8 | Viewed by 2723
Abstract
This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in [...] Read more.
This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. Forty-nine control volunteers (16 male, 33 female, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m2, respectively) and 52 obese subjects (16 male and 36 female, BMI 38.2± 11.0 and 40.7 ± 8.7 kg/m2, respectively) were examined. The two physical techniques applied were atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces, and fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA-DPH), which provides information on lipid order at the membrane polar–nonpolar interface. These techniques, in combination with lipidomic studies, revealed a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. Lipidomic data show an increase in the cholesterol/phospholipid mole ratio and a decrease in sphingomyelin contents in obese membranes. ω-3 fatty acids (e.g., docosahexaenoic acid) appear to be less prevalent in obese patient RBCs, and this is the case for both the global fatty acid distribution and for the individual major lipids in the membrane phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Moreover, some ω-6 fatty acids (e.g., arachidonic acid) are increased in obese patient RBCs. The switch from ω-3 to ω-6 lipids in obese subjects could be a major factor explaining the higher interfacial fluidity in obese patient RBC membranes. Full article
(This article belongs to the Special Issue State-of-the-Art Biochemistry in Spain)
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<p>Representative AFM force–distance curve of an RBC. The AFM tip performs an indentation process on a supported RBC, initiated from X = 0 along the red line (trace), up to the maximum force (20 nN in this case), and coming back to the initial position long the blue line (retrace). The trace line has three distinct phases: (i) first, an elastic deformation of the cell occurs (the force required for this process depends on the cell cytoskeleton); (ii) then, after further compression, the AFM tip pierces immediately through both RBC membranes (distal and proximal); and (iii) finally the tip achieves maximum force against the support, without further X-axis displacement. Membrane rupture is achieved at a definite force, marked by the sudden appearance of small peaks, at a Y-axis value that can be statistically quantitated (performing 50–75 curves for each sample). These experiments were performed at room temperature.</p> Full article ">Figure 2
<p>AFM force spectroscopy experiments on RBC. These measurements were performed at room temperature. Obese patient RBC are significantly less resistant to AFM punch-through experiments, pointing to a decrease in stiffness (number of patients <span class="html-italic">n</span> = 20 for control, <span class="html-italic">n</span> = 22 for obese; 50–75 measurements for each patient). Average values ± S.D. (*) Significance according to Student’s t-test: <span class="html-italic">p</span> = 0.03.</p> Full article ">Figure 3
<p>TMA-DPH anisotropy measurements of RBC membranes. The black bars represent measurements at 20 °C, while gray ones represent those at 37 °C. At both temperatures, a clear decrease for anisotropy values was detected for obese patient RBC, which indicates a higher membrane fluidity (<span class="html-italic">n</span> = 49 for control, <span class="html-italic">n</span> = 52 for obese). Average values ± S.D. Significance according to Student’s t-test: (**) <span class="html-italic">p</span> &lt; 0.01; (***) <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>Lipidomic quantitation of global fatty acid presence in mature RBCs. Empty boxes refer to control (normal weight) group, while gray boxes represent obese patients. Significant differences are detected for dihomo-γ-linolenic acid (DGLA), arachidonic acid, DHA levels, SFA/MUFA, and ω-6/ω-3 ratios, pointing to a metabolic switch for obese patient RBC membranes. Significance according to Student’s t-test: (*) <span class="html-italic">p</span> &lt; 0.05; (**) <span class="html-italic">p</span> &lt; 0.01; (***) <span class="html-italic">p</span> &lt; 0.001. (<span class="html-italic">n</span> = 49 for control, <span class="html-italic">n</span> = 52 for obese).</p> Full article ">Figure 5
<p>Lipidomic quantitation of specific lipid species in RBC. Species studied were PC (<b>A</b>), PE (<b>B</b>), PS (<b>C</b>), SM (<b>D</b>), and Chol (<b>E</b>). Chol/total phospholipid mol ratio is shown in panel (<b>F</b>). Black bars refer to control RBC group, while gray bars represent the obese patient RBC group. A significant decrease in SM and an increase in Chol/phospholipid ratio were detected for obese patient RBC. Average values ± S.D. Significance according to Student’s t-test: (*) <span class="html-italic">p</span> &lt; 0.05. <span class="html-italic">n</span> = 8.</p> Full article ">Figure 6
<p>Lipidomic analysis of ω-3 and ω-6 presence in specific lipid species. Percent ω-3 and/or ω-6 in PC (<b>A</b>), PE (<b>B</b>), and PS (<b>C</b>). Black bars refer to control RBC group, while gray bars represent the obese patient RBC group. While total values for combined ω-3 + ω-6 are constant, both a decrease in ω-3 and an increase in ω-6 are detected for each lipid species in obese patient RBC. Average values ± S.D. Significance according to Student’s t-test: (*) <span class="html-italic">p</span> &lt; 0.05; (**) <span class="html-italic">p</span> &lt; 0.01; (***) <span class="html-italic">p</span> &lt; 0.001. <span class="html-italic">n</span> = 8.</p> Full article ">Figure 7
<p>PA probe measurements of blood plasma. Red/blue intensity ratio (RBIR) values for control and obese blood plasma revealed a highly significant reduction in obese patients. Experiments performed at 37 °C (<span class="html-italic">n</span> = 35 for control and <span class="html-italic">n</span> = 39 for obese). Average values ± S.D. Significance according to Student’s t-test: (***) <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
14 pages, 640 KiB  
Review
Interactions between Radiation and One-Carbon Metabolism
by Navyateja Korimerla and Daniel R. Wahl
Int. J. Mol. Sci. 2022, 23(3), 1919; https://doi.org/10.3390/ijms23031919 - 8 Feb 2022
Cited by 5 | Viewed by 4959
Abstract
Metabolic reprogramming is a hallmark of cancer. Cancer cells rewire one-carbon metabolism, a central metabolic pathway, to turn nutritional inputs into essential biomolecules required for cancer cell growth and maintenance. Radiation therapy, a common cancer therapy, also interacts and alters one-carbon metabolism. This [...] Read more.
Metabolic reprogramming is a hallmark of cancer. Cancer cells rewire one-carbon metabolism, a central metabolic pathway, to turn nutritional inputs into essential biomolecules required for cancer cell growth and maintenance. Radiation therapy, a common cancer therapy, also interacts and alters one-carbon metabolism. This review discusses the interactions between radiation therapy, one-carbon metabolism and its component metabolic pathways. Full article
(This article belongs to the Special Issue Radiation Biology and Molecular Radiation Oncology)
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<p>Overview of radiation-induced changes in one-carbon metabolism. Enzymes, metabolites and pathways affected by radiation directly or indirectly are highlighted in the figure. Dihydrofolate (DHF), Tetrahydrofolate (THF), serine hydroxymethyltransferase (SHMT), 10-formyltetrahydrofolate (10-fTHF), 5-methyltetrahydrofolate (5-mTHF) methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS), methionine adenosyltransferase 2A (MAT2A), S-adenosylmethionine (SAM), methyltransferases(MT), S-adenosylhomocysteine (SAH), adenosylhomocysteinase (ACHY), methylthioadenosine phosphorylase (MTAP), 5′-Methylthioadenosine (MTA), 5-deoxy-5-(methylthio)ribose (MTR), betaine-homocysteine methyltransferase (BHMT), homocysteine (HCY), cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE). Enzymes, metabolites and pathways affected by radiation directly/indirectly are highlighted in red.</p> Full article ">
16 pages, 3360 KiB  
Article
Genome-Wide Identification and Expression Analysis of Heat Shock Protein 70 (HSP70) Gene Family in Pumpkin (Cucurbita moschata) Rootstock under Drought Stress Suggested the Potential Role of these Chaperones in Stress Tolerance
by Marzieh Davoudi, Jinfeng Chen and Qunfeng Lou
Int. J. Mol. Sci. 2022, 23(3), 1918; https://doi.org/10.3390/ijms23031918 - 8 Feb 2022
Cited by 25 | Viewed by 3827
Abstract
Heat shock protein 70s (HSP70s) are highly conserved proteins that are involved in stress responses. These chaperones play pivotal roles in protein folding, removing the extra amounts of oxidized proteins, preventing protein denaturation, and improving the antioxidant system activities. This conserved family has [...] Read more.
Heat shock protein 70s (HSP70s) are highly conserved proteins that are involved in stress responses. These chaperones play pivotal roles in protein folding, removing the extra amounts of oxidized proteins, preventing protein denaturation, and improving the antioxidant system activities. This conserved family has been characterized in several crops under drought stress conditions. However, there is no study on HSP70s in pumpkin (Cucurbita moschata). Therefore, we performed a comprehensive analysis of this gene family, including phylogenetic relationship, motif and gene structure analysis, gene duplication, collinearity, and promoter analysis. In this research, we found 21 HSP70s that were classified into five groups (from A to E). These genes were mostly localized in the cytoplasm, chloroplast, mitochondria, nucleus, and endoplasmic reticulum (ER). We could observe more similarity in closely linked subfamilies in terms of motifs, the number of introns/exons, and the corresponding cellular compartments. According to the collinearity analysis, gene duplication had occurred as a result of purifying selection. The results showed that the occurrence of gene duplication for all nine gene pairs was due to segmental duplication (SD). Synteny analysis revealed a closer relationship between pumpkin and cucumber than pumpkin and Arabidopsis. Promoter analysis showed the presence of various cis-regulatory elements in the up-stream region of the HSP70 genes, such as hormones and stress-responsive elements, indicating a potential role of this gene family in stress tolerance. We furtherly performed the gene expression analysis of the HSP70s in pumpkin under progressive drought stress. Pumpkin is widely used as a rootstock to improve stress tolerance, as well as fruit quality of cucumber scion. Since stress-responsive mobile molecules translocate through vascular tissue from roots to the whole plant body, we used the xylem of grafted materials to study the expression patterns of the HSP70 (potentially mobile) gene family. The results indicated that all CmoHSP70s had very low expression levels at 4 days after stress (DAS). However, the genes showed different expression patterns by progressing he drought period. For example, the expression of CmoHSP70-4 (in subgroup E) and CmoHSP70-14 (in subgroup C) sharply increased at 6 and 11 DAS, respectively. However, the expression of all genes belonging to subgroup A did not change significantly in response to drought stress. These findings indicated the diverse roles of this gene family under drought stress and provided valuable information for further investigation on the function of this gene family, especially under stressful conditions. Full article
(This article belongs to the Section Molecular Plant Sciences)
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<p>Phylogenetic analysis of HSP70 proteins of pumpkin, cucumber, and Arabidopsis. The HSP70s for pumpkin, Arabidopsis, and cucumber are shown in black, green, and red colors, respectively. The subgroups have been shown in different colors and indicated with the letters A to E.</p> Full article ">Figure 2
<p>Phylogenetic tree of CmoHSP70 proteins and their motif analysis. (<b>A</b>) Phylogenetic tree of CmoHSP70s in pumpkin. The CmoHSP70s were classified in five subgroups, from A to E, based on their similarities to Arabidopsis genes. (<b>B</b>) Ten conserved motif proteins of the CmoHSP70s, each small box indicating a motif. All 10 motif logos are shown below the figure. (<b>C</b>) Visualization of conserved domains of identified CmoHSP70s using TBtools. Each color represents a specific domain. The corresponding domain names have been shown below the figure.</p> Full article ">Figure 3
<p>Gene structure analysis of CmoHSP70s in pumpkin. The structures of intron and exon and untranslated regions (UTR) are shown in black line and yellow and green boxes, respectively. The scale is helpful for gene length estimation.</p> Full article ">Figure 4
<p>Chromosomal location of the identified <span class="html-italic">CmoHSP70s</span> in pumpkin. The genes with the same color indicate that they belong to the same subgroup based on the phylogenetic tree. The chromosome numbers have been shown below them.</p> Full article ">Figure 5
<p>Collinearity analysis of <span class="html-italic">HSP70</span> gene family in pumpkin.</p> Full article ">Figure 6
<p>Synteny analysis of <span class="html-italic">HSP70</span> family between pumpkin and two other species. The red lines show the <span class="html-italic">HSP70</span> orthologous genes between two species, and the gray lines indicate all orthologous genes. The numbers in the figure indicate the chromosome numbers.</p> Full article ">Figure 7
<p>Cis-regulatory elements related to hormones and stress in promoter region of <span class="html-italic">CmoHSP70</span> genes. (<b>A</b>) The regulatory elements of HSP70 related to hormones and (<b>B</b>) indicating the stress-related cis elements. AuxRE (auxin responsive element), ABRE (ABA responsive element), GARE (Gibberellin responsive element), MeJARE (methyl jasmonate responsive element), SARE (salicylic acid responsive element). (<b>C</b>) Sequence logo of HSE in the promoter region of CmoHSP70s.</p> Full article ">Figure 8
<p>Expression patterns of 21 identified <span class="html-italic">CmoHSP70</span> genes in response to drought stress. The samples belonged to the xylem tissues below the graft union of pumpkin rootstock and were collected at 4, 6, and 11 DAS (days after drought stress). Each value is an average of three replications, and each replicate contained three individuals. Green and low colors show low and high relative expression levels, respectively.</p> Full article ">
16 pages, 966 KiB  
Article
Transferrin Saturation/Hepcidin Ratio Discriminates TMPRSS6-Related Iron Refractory Iron Deficiency Anemia from Patients with Multi-Causal Iron Deficiency Anemia
by Hilde van der Staaij, Albertine E. Donker, Dirk L. Bakkeren, Jan M. J. I. Salemans, Lisette A. A. Mignot-Evers, Marlies Y. Bongers, Jeanne P. Dieleman, Tessel E. Galesloot, Coby M. Laarakkers, Siem M. Klaver and Dorine W. Swinkels
Int. J. Mol. Sci. 2022, 23(3), 1917; https://doi.org/10.3390/ijms23031917 - 8 Feb 2022
Cited by 6 | Viewed by 4944
Abstract
Pathogenic TMPRSS6 variants impairing matriptase-2 function result in inappropriately high hepcidin levels relative to body iron status, leading to iron refractory iron deficiency anemia (IRIDA). As diagnosing IRIDA can be challenging due to its genotypical and phenotypical heterogeneity, we assessed the transferrin saturation [...] Read more.
Pathogenic TMPRSS6 variants impairing matriptase-2 function result in inappropriately high hepcidin levels relative to body iron status, leading to iron refractory iron deficiency anemia (IRIDA). As diagnosing IRIDA can be challenging due to its genotypical and phenotypical heterogeneity, we assessed the transferrin saturation (TSAT)/hepcidin ratio to distinguish IRIDA from multi-causal iron deficiency anemia (IDA). We included 20 IRIDA patients from a registry for rare inherited iron disorders and then enrolled 39 controls with IDA due to other causes. Plasma hepcidin-25 levels were measured by standardized isotope dilution mass spectrometry. IDA controls had not received iron therapy in the last 3 months and C-reactive protein levels were <10.0 mg/L. IRIDA patients had significantly lower TSAT/hepcidin ratios compared to IDA controls, median 0.6%/nM (interquartile range, IQR, 0.4–1.1%/nM) and 16.7%/nM (IQR, 12.0–24.0%/nM), respectively. The area under the curve for the TSAT/hepcidin ratio was 1.000 with 100% sensitivity and specificity (95% confidence intervals 84–100% and 91–100%, respectively) at an optimal cut-off point of 5.6%/nM. The TSAT/hepcidin ratio shows excellent performance in discriminating IRIDA from TMPRSS6-unrelated IDA early in the diagnostic work-up of IDA provided that recent iron therapy and moderate-to-severe inflammation are absent. These observations warrant further exploration in a broader IDA population. Full article
(This article belongs to the Special Issue New Advances in Iron Metabolism, Ferritin and Hepcidin Research)
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<p>Flow chart selection process. SATURNUS, an acronym for the ‘Transferrin Saturation/Hepcidin ratio: a study on the diagnostic utility in the differentiation of Iron Refractory Iron Deficiency Anemia (IRIDA) from Iron Deficiency Anemia (IDA)’; MMC, Máxima Medical Center; Hb, hemoglobin; MCV, mean corpuscular volume; TSAT, transferrin saturation; CRP, C-reactive protein. * Severe chronic kidney disease, estimated Glomerular Filtration Rate (eGFR) &lt; 30 mL/min/1.73 m<sup>2</sup>, ** Chronic liver disease, cirrhosis, or alanine aminotransferase (ALT) &gt; 40 U/L.</p> Full article ">Figure 2
<p>(<b>a</b>–<b>c</b>) Transferrin saturation (TSAT), plasma hepcidin levels, and TSAT/hepcidin ratio in the total IRIDA group (<span class="html-italic">n</span> = 20), biallelic IRIDA patients (<span class="html-italic">n</span> = 11), monoallelic IRIDA patients (<span class="html-italic">n</span> = 9) and IDA controls (<span class="html-italic">n</span> = 39). Box and whisker plots present the quartiles (box), the medians (bold line), and the minimum and maximum (whiskers). (<b>d</b>) Receiver Operating Characteristic (ROC) curve analysis (see also <a href="#app1-ijms-23-01917" class="html-app">Supplemental Table S1</a>) comparing the TSAT/hepcidin ratio (%/nM) in IDA controls versus the total IRIDA group (red), versus the biallelic IRIDA group (purple), and versus the monoallelic IRIDA group (blue). Ns, not significant; * = <span class="html-italic">p</span> &lt; 0.05; <span class="html-italic">** = p &lt;</span> 0.001, by non-parametric Mann–Whitney U test. AUC, area under the curve.</p> Full article ">
14 pages, 2052 KiB  
Article
Plasma Metabolomics Profile of “Insulin Sensitive” Male Hypogonadism after Testosterone Replacement Therapy
by Lello Zolla and Marcello Ceci
Int. J. Mol. Sci. 2022, 23(3), 1916; https://doi.org/10.3390/ijms23031916 - 8 Feb 2022
Cited by 4 | Viewed by 2440
Abstract
Male hypogonadism is a disorder characterized by low levels of testosterone, but patients can either show normal insulin (insulin-sensitive (IS)) or over time they can become insulin-resistant (IR). Since the two groups showed different altered metabolisms, testosterone replacement therapy (TRT) could achieve different [...] Read more.
Male hypogonadism is a disorder characterized by low levels of testosterone, but patients can either show normal insulin (insulin-sensitive (IS)) or over time they can become insulin-resistant (IR). Since the two groups showed different altered metabolisms, testosterone replacement therapy (TRT) could achieve different results. In this paper, we analyzed plasma from 20 IS patients with low testosterone (<8 nmol/L) and HOMAi < 2.5. The samples, pre- and post-treatment with testosterone for 60 days, were analyzed by UHPLC and mass spectrometry. Glycolysis was significantly upregulated, suggesting an improved glucose utilization. Conversely, the pentose phosphate pathway was reduced, while the Krebs cycle was not used. Branched amino acids and carnosine metabolism were positively influenced, while β-oxidation of fatty acids (FFA) was not activated. Cholesterol, HDL, and lipid metabolism did not show any improvements at 60 days but did so later in the experimental period. Finally, both malate and glycerol shuttle were reduced. As a result, both NADH and ATP were significantly lower. Interestingly, a significant production of lactate was observed, which induced the activation of the Cori cycle between the liver and muscles, which became the main source of energy for these patients without involving alanine. Thus, the treatment must be integrated with chemicals which are not restored in order to reactivate energy production. Full article
(This article belongs to the Special Issue Insulin Sensitivity/Resistance: From Physiology to Disease)
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<p>(<b>A</b>) Metabolic Set Enrichment Analysis (MSEA) showing the most altered metabolites revealed in the plasma of hypogonadal men before and after testosterone replacement treatment (TRT). Color intensity (white-to-red) reflects increasing statistical significance, while the circle diameter covaries with pathway impact. The graph was obtained by plotting on the <span class="html-italic">y</span>-axis the –log of <span class="html-italic">p</span>-values from pathway enrichment analysis and on the x-axis the pathway impact values derived from pathway topology analysis. (<b>B</b>) Metabolic Pathway Analysis (MetPA). All the matched pathways are displayed as circles. The color and size of each circle are based on the <span class="html-italic">p</span>-value and pathway impact value, respectively. The graph was obtained by plotting on the <span class="html-italic">y</span>-axis the −log of <span class="html-italic">p</span>-values from the pathway enrichment analysis and on the <span class="html-italic">x</span>-axis the pathway impact values derived from the pathway topology analysis.</p> Full article ">Figure 2
<p>Metabolomic profile of glucose metabolism. (<b>A</b>) All glycolytic intermediates were upregulated after TRT. (<b>B</b>) Intermediates of pentose phosphate pathway were restored to that similar to control. (<b>C</b>) Decrease of glutathione disulphide as a marker of the improvement of oxidative stress. Deregulation of level of NAD and NADH. (<b>D</b>) The glycerol shuttle was not active to contribute to the oxidative phosphorylation pathway in the mitochondria. It was used for phospholipid synthesis. All data are shown as mean ± SEM of fold-change relative to the metabolite levels in controls. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 3
<p>Metabolism involved in acetyl-CoA catabolism. Intermediates of Tricarboxylic acid (TCA) cycle measured in the plasma of hypogonadal patients. We found an overall decreased level of TCA-cycle metabolites. (<b>A</b>) Level of mevalonic acid increased after therapy, yet the concentration of cholesterol did not improve. (<b>B</b>) Acetyl-carnitine and fatty acid oxidation are significantly reduced after TRT. (<b>C</b>) Levels of energy metabolites production AMP and ATP. (<b>D</b>) Intermediates of the glutaminolysis pathway. This stepwise became downregulated after TRT. All data are shown as mean ± SEM of fold-change relative to the metabolite levels in controls. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>(<b>A</b>) The downregulation of glutaminolysis led to a stop of the activation of the malate aspartate cycle, as shown by the levels of glutamate and aspartate. (<b>B</b>) Decreased levels of branched-significantly decreased. (<b>C</b>) Plasma amino acids that were significantly decreased. (<b>D</b>) Plasma amino acids that were significantly increased. (<b>E</b>) Restoration of proline and lysine involved in collagen fibers formation. All data are shown as mean ± SEM of fold-change relative to the metabolite levels in controls. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p>Schematic model summarizing change in carnosine metabolism. The carnosine production from β-alanine increased in response to testosterone therapy. All data are shown as mean ± SEM of fold-change relative to the metabolite levels in controls. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 6
<p>Overview of the energy production in hypogonadic insulin-sensivity male after TRT. Pyruvate produced by glycolysis is converted into lactate rather than alanine. Lactate can both be directly used as main energy source in highly oxidative cells such as brain, lung, heart or enter in liver or kidney and converted in glucose, that is used to produce energy (Cori Cycle).</p> Full article ">
15 pages, 5457 KiB  
Article
Screening of Differentially Expressed Genes and Localization Analysis of Female Gametophyte at the Free Nuclear Mitosis Stage in Pinus tabuliformis Carr.
by Zaixin Gong, Hailin Hu, Li Xu, Yuanyuan Zhao and Caixia Zheng
Int. J. Mol. Sci. 2022, 23(3), 1915; https://doi.org/10.3390/ijms23031915 - 8 Feb 2022
Viewed by 2122
Abstract
Female sterility is a common phenomenon in the plant world, and systematic research has not been carried out in gymnosperms. In this study, the ovules of No. 28 sterile line and No. 15 fertile line Pinus tabuliformis were used as materials, and a [...] Read more.
Female sterility is a common phenomenon in the plant world, and systematic research has not been carried out in gymnosperms. In this study, the ovules of No. 28 sterile line and No. 15 fertile line Pinus tabuliformis were used as materials, and a total of 18 cDNA libraries were sequenced by the HiSeqTM 4000 platform to analyze the differentially expressed genes (DEGs) and simple sequence repeats (SSRs) between the two lines. In addition, this study further analyzed the DEGs involved in the signal transduction of plant hormones, revealing that the signal pathways related to auxin, cytokinin, and gibberellin were blocked in the sterile ovule. Additionally, real-time fluorescent quantitative PCR verified that the expression trend of DEGs related to plant hormones was consistent with the results of high-throughput sequencing. Frozen sections and fluorescence in situ hybridization (FISH) were used to study the temporal and spatial expression patterns of PtRab in the ovules of P. tabuliformis. It was found that PtRab was significantly expressed in female gametophytes and rarely expressed in the surrounding diploid tissues. This study further explained the molecular regulation mechanism of female sterility in P. tabuliformis, preliminarily mining the key factors of ovule abortion in gymnosperms at the transcriptional level. Full article
(This article belongs to the Collection Recent Advances in Plant Molecular Science in China 2021)
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<p>Background description of tissues and workflow. (<b>A</b>) Microscopic observations of ovules; FER: female fertile line, STE: female sterile line, FNM: the process of free nuclear mitosis, FG: female gametophyte, FN: free nucleus, V: vacuole. (<b>B</b>) Schematic overview of transcriptome data generation and analyses workflow.</p> Full article ">Figure 2
<p>RNA sequencing data from ovules in <span class="html-italic">P. tabuliformis</span>. (<b>A</b>) Differentially expressed genes between FER and STE in FNM1, FNM2, and FNM3. (<b>B</b>) Venn diagram of unigene annotation in <span class="html-italic">P. tabuliformis</span> ovules.</p> Full article ">Figure 3
<p>GO and KEGG annotation of unigenes of different free nuclear mitosis stages in ovules of <span class="html-italic">P. tabuliformis</span>. (<b>A</b>) GO annotation of unigenes of the ovule of <span class="html-italic">P. tabuliformis</span>. (<b>B</b>–<b>D</b>) GO annotation of differentially expressed genes between FER and STE in the stage of FNM1, FNM2, and FNM3 of <span class="html-italic">P. tabuliformis</span>. (<b>E</b>–<b>G</b>) KEGG pathway enrichment of differentially expressed genes in the stage of FNM1, FNM2, and FNM3 of <span class="html-italic">P. tabuliformis</span>.</p> Full article ">Figure 4
<p>Analysis of expression levels of the differentially expressed genes. Expression patterns of differentially expressed genes related to the plant hormone. Data for the gene expression level were normalized to Z-score (<b>A</b>). Candidate DEGs expression levels revealed by qRT-PCR (<b>B</b>). Auxin-responsive protein IAA (<span class="html-italic">IAA</span>); auxin-responsive protein IAA26 (<span class="html-italic">IAA26</span>); auxin-responsive protein SAUR32-like (<span class="html-italic">SAUR32</span>); histidine kinase 2 (<span class="html-italic">AHK2</span>); two-component response regulator ARR2-like (<span class="html-italic">ARR-B</span>); gibberellin receptor GID1-like protein (<span class="html-italic">GID1</span>); gibberellin DELLA protein (<span class="html-italic">DELLA</span>); Rab-type small GTP-binding protein (<span class="html-italic">Rab</span>).</p> Full article ">Figure 5
<p>DEGs with opposite expression patterns during the FNM process in STE and FER; DEGs up-regulated in FER and down-regulated in STE, Venn diagram (<b>A</b>), and heat map (<b>B</b>). GO classification of DEGs with opposite expression levels (<b>C</b>). KEGG pathway enrichment of DEGs with opposite expression levels (<b>D</b>).</p> Full article ">Figure 6
<p>Fluorescence in situ hybridization of <span class="html-italic">PtRab</span> in <span class="html-italic">P. tabuliformis</span> ovules. (<b>A</b>–<b>C</b>) The spatiotemporal expression of <span class="html-italic">PtRab</span> in ovules from FNM1 to FNM3 period in fertile line. (<b>D</b>–<b>F</b>) The spatiotemporal expression of <span class="html-italic">PtRab</span> in ovules from FNM1 to FNM3 period in sterile line. FG: female gametophyte, FN: free nucleus, I: integument.</p> Full article ">Figure 7
<p>Speculative network model revealing the ovule abortion in the female sterile line of <span class="html-italic">Pinus tabuliformis</span> Carr.</p> Full article ">
22 pages, 3284 KiB  
Article
Fusarium graminearum Infection Strategy in Wheat Involves a Highly Conserved Genetic Program That Controls the Expression of a Core Effectome
by Florian Rocher, Tarek Alouane, Géraldine Philippe, Marie-Laure Martin, Philippe Label, Thierry Langin and Ludovic Bonhomme
Int. J. Mol. Sci. 2022, 23(3), 1914; https://doi.org/10.3390/ijms23031914 - 8 Feb 2022
Cited by 12 | Viewed by 4452
Abstract
Fusarium graminearum, the main causal agent of Fusarium Head Blight (FHB), is one of the most damaging pathogens in wheat. Because of the complex organization of wheat resistance to FHB, this pathosystem represents a relevant model to elucidate the molecular mechanisms underlying [...] Read more.
Fusarium graminearum, the main causal agent of Fusarium Head Blight (FHB), is one of the most damaging pathogens in wheat. Because of the complex organization of wheat resistance to FHB, this pathosystem represents a relevant model to elucidate the molecular mechanisms underlying plant susceptibility and to identify their main drivers, the pathogen’s effectors. Although the F. graminearum catalog of effectors has been well characterized at the genome scale, in planta studies are needed to confirm their effective accumulation in host tissues and to identify their role during the infection process. Taking advantage of the genetic variability from both species, a RNAseq-based profiling of gene expression was performed during an infection time course using an aggressive F. graminearum strain facing five wheat cultivars of contrasting susceptibility as well as using three strains of contrasting aggressiveness infecting a single susceptible host. Genes coding for secreted proteins and exhibiting significant expression changes along infection progress were selected to identify the effector gene candidates. During its interaction with the five wheat cultivars, 476 effector genes were expressed by the aggressive strain, among which 91% were found in all the infected hosts. Considering three different strains infecting a single susceptible host, 761 effector genes were identified, among which 90% were systematically expressed in the three strains. We revealed a robust F. graminearum core effectome of 357 genes expressed in all the hosts and by all the strains that exhibited conserved expression patterns over time. Several wheat compartments were predicted to be targeted by these putative effectors including apoplast, nucleus, chloroplast and mitochondria. Taken together, our results shed light on a highly conserved parasite strategy. They led to the identification of reliable key fungal genes putatively involved in wheat susceptibility to F. graminearum, and provided valuable information about their putative targets. Full article
(This article belongs to the Special Issue Host-Pathogen Interaction 3.0)
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<p>In planta expression signature of <span class="html-italic">F. graminearum</span> genes coding for putative secreted proteins. Barplots represent the structure of the gene sets coding for putative secreted proteins expressed in planta for the HostV (<b>A</b>) and the PathoV (<b>B</b>) experiments. HostV barplot displays the number of genes expressed by the strain MDC_Fg1 in all the infected hosts, in some hosts (Accessory) and in only a specific host: ‘Arche’ (ARC) specific, ‘Courtot’ (COU) specific, ‘Chinese Spring’ (CS) specific, ‘Recital’ (REC) specific or ‘Renan’ (REN) specific. PathoV barplot displays the number of genes expressed by the three strains MDC_Fg1, MDC_Fg13 and MDC_FgU1, by two strains only (Accessory) and by only one strain (MDC_Fg1 specific, MDC_Fg13 specific, MDC_FgU1 specific). The Venn diagram (<b>C</b>) represents the intersection of the gene sets expressed in all the hosts (HostV) and expressed in all the strains (PathoV).</p> Full article ">Figure 2
<p>Number of secretome genes from the HostV (<b>A</b>) and the PathoV (<b>B</b>) experiments significantly impacted by the different effects tested in the differential expression (DE) analysis. For each factor of the DE analysis, the Venn diagrams indicate the number of genes displaying significant expression variations. Significance threshold: <span class="html-italic">p</span>-value corrected by Benjamini–Hochberg method &lt; 0.001.</p> Full article ">Figure 3
<p>Structure of the HostV (<b>A</b>) and PathoV (<b>B</b>) effectome gene sets. These sets gather the genes significantly regulated along infection progress and coding for putative secreted proteins. (<b>A</b>) The flower plot displays the number of genes expressed by the strain MDC_Fg1 in all the infected hosts (center circle), in some hosts (annulus) and in only a specific host (petals). (<b>B</b>) The flower plot displays the number of genes expressed by the three strains MDC_Fg1, MDC_Fg13 and MDC_FgU1 (center circle), by two strains only (annulus) and by only one strain (petals).</p> Full article ">Figure 4
<p>Discrimination of HostV (<b>A</b>) and PathoV (<b>B</b>) effectome gene sets expressed in all the hosts or by all the strains according to the experimental conditions. PLS-DA method was applied on the 433 genes expressed in all the hosts for HostV (<b>A</b>) to predict the host–infection progress combinations and on the 682 genes expressed by all the strains for PathoV (<b>B</b>) to predict the strain–infection progress combinations. The plots of the individuals extracted from the PLS-DA are represented on the two first components. For each condition, confidence ellipses are plotted to highlight discrimination strength (level set to 95%).</p> Full article ">Figure 5
<p>Expression regulation patterns of the core effectome genes along with the infection progress in HostV (<b>A</b>) and PathoV (<b>B</b>) data sets. The structure of gene and sample data sets were determined by HAC based on Ward’s minimum variance method using the z-score transformed gene expression values. Heatmap color scales represent the z-score transformed expression values of the genes from the core effectome gene set for each sample. The clustering on top of the heatmap represents the experimental conditions which are labeled according to the factors Infection Progress and Host for the HostV experiment, and Infection Progress and Strain for the PathoV experiment. The clustering on the right side of the heatmap represents the genes, which are colored according to their cluster membership.</p> Full article ">Figure 6
<p>Localization within the host of the fungal proteins encoded by the core effectome gene set according to the expression timing during the infection progress. Barplots represent the frequencies of the predicted localizations within the host of the proteins coded by <span class="html-italic">F. graminearum</span> genes expressed at the early stages of the infection (Early Expression), at intermediate stages of infection (Intermediate Expression), at latter stages of infection (Late Expression) in both HostV and PathoV experiments or expressed with different dynamics between the HostV and PathoV experiments.</p> Full article ">Figure 7
<p>Model summarizing the conserved and complex <span class="html-italic">F. graminearum</span> infection strategy on wheat spikes. As a whole, 357 effector genes were identified as the key drivers of FHB infection expressed by all the strains and in all the infected hosts; they represent the <span class="html-italic">F. graminearum</span> core effectome. These genes were expressed at very specific infection stages in a per-wave manner, including genes highly expressed at the very beginning of the interaction with the wheat tissues and others highly expressed in the later stages of the infection. The timing of gene expression was mostly conserved independently of the strain or the host. Targeted processes within the host are highly diverse with a wide array of targeted compartments and predicted functions.</p> Full article ">
12 pages, 1663 KiB  
Article
ERAP2 Inhibition Induces Cell-Surface Presentation by MOLT-4 Leukemia Cancer Cells of Many Novel and Potentially Antigenic Peptides
by Ioannis Temponeras, George Stamatakis, Martina Samiotaki, Dimitris Georgiadis, Harris Pratsinis, George Panayotou and Efstratios Stratikos
Int. J. Mol. Sci. 2022, 23(3), 1913; https://doi.org/10.3390/ijms23031913 - 8 Feb 2022
Cited by 7 | Viewed by 2692
Abstract
Recent studies have linked the activity of ER aminopeptidase 2 (ERAP2) to increased efficacy of immune-checkpoint inhibitor cancer immunotherapy, suggesting that pharmacological inhibition of ERAP2 could have important therapeutic implications. To explore the effects of ERAP2 inhibition on the immunopeptidome of cancer cells, [...] Read more.
Recent studies have linked the activity of ER aminopeptidase 2 (ERAP2) to increased efficacy of immune-checkpoint inhibitor cancer immunotherapy, suggesting that pharmacological inhibition of ERAP2 could have important therapeutic implications. To explore the effects of ERAP2 inhibition on the immunopeptidome of cancer cells, we treated MOLT-4 T lymphoblast leukemia cells with a recently developed selective ERAP2 inhibitor, isolated Major Histocompatibility class I molecules (MHCI), and sequenced bound peptides by liquid chromatography tandem mass spectrometry. Inhibitor treatment induced significant shifts on the immunopeptidome so that more than 20% of detected peptides were either novel or significantly upregulated. Most of the inhibitor-induced peptides were 9mers and had sequence motifs and predicted affinity consistent with being optimal ligands for at least one of the MHCI alleles carried by MOLT-4 cells. Such inhibitor-induced peptides could serve as triggers for novel cytotoxic responses against cancer cells and synergize with the therapeutic effect of immune-checkpoint inhibitors. Full article
(This article belongs to the Special Issue The Pathway of Antigen Processing and Presentation)
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<p>Activity of the phosphinic inhibitor DG011A. Panel (<b>A</b>), titration of DG011A inhibits ERAP2 with a 70-fold higher potency than ERAP1. Panel (<b>B</b>), DG011A shows no toxicity versus MOLT-4 cells up to 100 μM as measured by the MTT assay. Panel (<b>C</b>), representative FACS traces used for the quantitation of the presence of HLA molecules (stained by the W6/32 antibody) on the surface of MOLT-4 cells incubated with 1 μM DG011A. Panel (<b>D</b>), quantitation of the geometric mean of the signal from the FACS experiments.</p> Full article ">Figure 2
<p>Effects of the DG011A on the immunopeptidome of MOLT-4 cells. Panel (<b>A</b>), scatterplot of the signal of detected peptides isolated from the HLA molecules of MOLT-4 cells under control conditions or after incubation with DG011A. Each circle represents a unique peptide sequence. Circles along the diagonal represent peptides unchanged between the two conditions and circles in the region close to each axis represent peptides detected only in a single condition (either control or inhibitor). Panel (<b>B</b>), heatplot showing the distribution of detected peptide signals (log<sub>10</sub>) in both conditions for each of the replicates measured (three biological replicates, each measured in three technical replicates, totaling 9 measurements per condition). Panel (<b>C</b>), volcano plot, indicating the statistical significance of the observed differences between the two conditions. Each circle represents a unique peptide sequence. The middle section represents peptides detected in both conditions but at different intensities and the outermost sections peptides detected in only a single condition. Peptides that fall within the green- and cyan-colored regions have a <span class="html-italic">p</span> value of &lt;0.05 and are considered statistically significant. Panel (<b>D</b>), Venn diagram summarizing the observed numerical shifts of the immunopeptidome of MOLT-4 cells after incubation with DG011A.</p> Full article ">Figure 3
<p>Effects of the inhibitor DG011A on the length and affinity of peptides presented by MOLT-4 cells. Panel (<b>A</b>), distribution of lengths of peptide eluted from the MHC class I molecules on the surface of MOLT-4 cells that are unaffected by the presence of the inhibitor. Panel (<b>B</b>), same as in panel A but for peptides that were induced by the inhibitor. Panel (<b>C</b>), distribution of predicted affinities of each identified peptide for the HLA alleles present in MOLT-4 cells <span class="html-italic">(HLA-A*01:01, HLA-A*25:01, HLA-B*18:01, HLA-B*57:01, HLA-C*06:02, HLA-C*12:03</span>) [<a href="#B28-ijms-23-01913" class="html-bibr">28</a>]. Each circle denotes a unique peptide sequence. Peptides are grouped as in Panel (<b>A</b>).</p> Full article ">Figure 4
<p>Weblogo type plots based on Gibbs cluster analysis of 9mer sequences of identified peptides. Analysis was performed using the GibbsCluster-2.0 Server and plotted using the Seq2logo server. Amino acids are colored based on their physicochemical properties (negatively charged = red, positively charged = blue, hydrophobic/aromatic = black, hydrophilic = green). The size of the letter representation of each amino acid single letter code indicates the probability of observation at the particular position of each cluster. Positive value on the y-axis suggests a higher-than-random prevalence of the particular residue at that position. Positions that show the enhanced presence of residues that correspond to anchor residues of particular HLA alleles are indicated with arrows. Panels (<b>A</b>,<b>B</b>) indicate the two major clusters observed for peptides common in both control and inhibitor conditions and panels (<b>C</b>,<b>D</b>) indicate the two major clusters observed for peptides unique in the inhibitor-treated sample.</p> Full article ">Figure 5
<p>Synthetic strategy for DG011. (a) HMDS, 110 °C, 1 h, then 90 °C, 3 h, then EtOH, 70 °C, 30 min; (b) aq. NaOH, EtOH, rt, 24 h, then H<sub>3</sub>O+; (c) 2 × recrystallizations by AcOEt, 46%, three steps; (d) HBr/AcOH 33%, rt, 1 h; I Boc<sub>2</sub>O, Et<sub>3</sub>N, DMF, rt, 24 h, 92%, two steps; (e) H-(L)Ser(TBS)-NH<sub>2</sub>, EDC∙HCl, HOBt, DIPEA, CH<sub>2</sub>Cl<sub>2</sub>, rt, 4 h; (f) TFA/CH<sub>2</sub>Cl<sub>2</sub>/TIS/H<sub>2</sub>O 48:49:2:1, rt, 2 h, 43%, two steps.</p> Full article ">
14 pages, 2112 KiB  
Article
AKR1B10, One of the Triggers of Cytokine Storm in SARS-CoV2 Severe Acute Respiratory Syndrome
by Clovis Chabert, Anne-Laure Vitte, Domenico Iuso, Florent Chuffart, Candice Trocme, Marlyse Buisson, Pascal Poignard, Benjamin Lardinois, Régis Debois, Sophie Rousseaux, Jean-Louis Pepin, Jean-Benoit Martinot and Saadi Khochbin
Int. J. Mol. Sci. 2022, 23(3), 1911; https://doi.org/10.3390/ijms23031911 - 8 Feb 2022
Cited by 10 | Viewed by 2942
Abstract
Preventing the cytokine storm observed in COVID-19 is a crucial goal for reducing the occurrence of severe acute respiratory failure and improving outcomes. Here, we identify Aldo-Keto Reductase 1B10 (AKR1B10) as a key enzyme involved in the expression of pro-inflammatory cytokines. The analysis [...] Read more.
Preventing the cytokine storm observed in COVID-19 is a crucial goal for reducing the occurrence of severe acute respiratory failure and improving outcomes. Here, we identify Aldo-Keto Reductase 1B10 (AKR1B10) as a key enzyme involved in the expression of pro-inflammatory cytokines. The analysis of transcriptomic data from lung samples of patients who died from COVID-19 demonstrates an increased expression of the gene encoding AKR1B10. Measurements of the AKR1B10 protein in sera from hospitalised COVID-19 patients suggests a significant link between AKR1B10 levels and the severity of the disease. In macrophages and lung cells, the over-expression of AKR1B10 induces the expression of the pro-inflammatory cytokines Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and Tumor Necrosis Factor a (TNFα), supporting the biological plausibility of an AKR1B10 involvement in the COVID-19-related cytokine storm. When macrophages were stressed by lipopolysaccharides (LPS) exposure and treated by Zopolrestat, an AKR1B10 inhibitor, the LPS-induced production of IL-6, IL-1β, and TNFα is significantly reduced, reinforcing the hypothesis that the pro-inflammatory expression of cytokines is AKR1B10-dependant. Finally, we also show that AKR1B10 can be secreted and transferred via extracellular vesicles between different cell types, suggesting that this protein may also contribute to the multi-organ systemic impact of COVID-19. These experiments highlight a relationship between AKR1B10 production and severe forms of COVID-19. Our data indicate that AKR1B10 participates in the activation of cytokines production and suggest that modulation of AKR1B10 activity might be an actionable pharmacological target in COVID-19 management. Full article
(This article belongs to the Special Issue Coronavirus Disease (COVID-19): Pathophysiology)
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Figure 1

Figure 1
<p>AKR1B10 is among the most frequently overexpressed genes in post-mortem lungs from severe COVID-19 patients. (<b>A</b>) Volcano plot showing the differential gene expression in lung tissue from deceased COVID-19 patients vs. healthy donors, reduced to genes over- or under-expressed with an FDR &lt; 0.02 (from available transcriptomic data [<a href="#B28-ijms-23-01911" class="html-bibr">28</a>]).<span style="color:#5B9BD5"> ●</span> corresponds to an adjusted <span class="html-italic">p</span>-value &lt; 0.001; <span style="color:#00B050">●</span> corresponds to a fold change &gt; 5; and <span style="color:#FF3E11">●</span> corresponds to both. (<b>B</b>) GeneSet Enrichment Analysis (GSEA) plots of two genesets associated with the respective Gene Ontology terms CYTOKINE_PRODUCTION (NES: 6.18; FDR &lt; 0.001) and POSITIVE_REGULATION_OF_INFLAMMATORY_RESPONSE (NES: 7.79; FDR &lt; 0.001) illustrating two major components of the transcriptomic signature in lungs from severe COVID-19 patients.</p> Full article ">Figure 2
<p>AKR1B10 concentration in sera is tightly associated with COVID-19 severity and correlated with other biological parameters known to be related to cytokine storm. (<b>A</b>) ELISA dosages of AKR1B10 in the blood of COVID-19 patients stratified into three groups corresponding to: “Non-COVID” (patients without COVID-19; <span class="html-italic">n</span> = 16, including 6 healthy individuals, 4 COPD and 6 cancer patients); “Non-ICU” (patients hospitalised in a non-ICU respiratory ward; <span class="html-italic">n</span> = 61), and “ICU” (patients admitted in an Intensive Care Unit; <span class="html-italic">n</span> = 43); (<b>B</b>) respective balanced proportions of patients of the non-COVID, non-ICU and ICU groups in each of the four quartiles (from Q1, the lowest, to Q4 the highest) of AKR1B10 sera concentrations; (<b>C</b>) correlations between AKR1B10 concentrations and Lymphocyte counts, CRP or LDH levels in the blood of COVID-19 patients. LDH: Lactate Dehydrogenase; Lympho: Lymphocyte counts; #: difference compared to Non-COVID individuals (###: <span class="html-italic">p</span> &lt; 0.001); *: difference compared to non-ICU patients (**: <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 3
<p>AKR1B10 is a key regulator of the cytokines production in RAW264.7 and H1299 cells, whose activity may be counteracted by pharmacological inhibitors. (<b>A</b>) Expression of the cytokines IL-6, TNFα and IL-1β, measured by RT-PCR in RAW264.7 macrophage cells after 12 h of 0 µg (Lipofectamine), 1 µg, 2 µg or 3 µg of peGFP-AKR1B10<sub>GFP</sub> plasmid transfection; (<b>B</b>) expression of the cytokines IL-6, TNFα and IL-1β, measured by RT-PCR in lung cancer cells H1299 after 12 h of 0 (Lipofectamine) or 1 µg of peGFP-AKR1B10<sub>GFP</sub> plasmid transfection; (<b>C</b>) effect of an AKR1B10 inhibitor (Zopolrestat at the indicated concentrations in mM) on cytokines expression in RAW264.7 cells stressed for 6 h by LPS, at the concentration of 0.5 µg·mL<sup>−1</sup>; LPS: Lipopolysaccharides; Zopol: Zopolrestat; (<span class="html-italic">n</span> = 3–5; mean ± SEM). #: difference compared to Control and pEGFP-AKR1B10<sub>GFP</sub> [0 µg] ( ###: <span class="html-italic">p</span> &lt; 0.001); *: difference compared to pEGFP-AKR1B10<sub>GFP</sub> [3 µg] (*: <span class="html-italic">p</span> &lt; 0.05); †: difference compared to LPS [0.5 µg·mL<sup>−1</sup>] and Zopolrestat [40 mM] (†: <span class="html-italic">p</span> &lt; 0.05); §: difference compared to Control (§: <span class="html-italic">p</span> &lt; 0.05; §§: <span class="html-italic">p</span> &lt; 0.01); µ: difference compared to pEGFP-AKR1B10<sub>GFP</sub> [0 µg] (µ: <span class="html-italic">p</span> &lt; 0.05); ‡: difference compared to LPS [0.5 µg·mL<sup>−1</sup>] and Zopolrestat [0 mM] (‡: <span class="html-italic">p</span> &lt; 0.05; ‡‡: <span class="html-italic">p</span> &lt; 0.01; ‡‡‡: <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 4
<p>AKR1B10 can be transferred between different cells types via Extracellular Vesicles (EVs); (<b>A</b>) AKR1B10<sub>GFP</sub> protein level in large EVs and exosomes extracted by centrifugation (Large EV: 10,000× <span class="html-italic">g</span> × 30 min; Exosomes: 100,000× <span class="html-italic">g</span> × 70 min) in the media of H1299 cells transfected with AKR1B10<sub>GFP</sub> and sorted according to the terciles of GFP signal (respectively low, medium and high); (<b>B</b>) FACS measurements of GFP signal (FL1-H) of RAW264.7 cells exposed to extracellular vesicles extracted from H1299Ct and H1299High; (<b>C</b>) mean fluorescence intensity of the FL1-H signal measured by FACS (<span class="html-italic">n</span> = 3; mean ± SEM); EVs: Extracellular Vesicles. *: difference compared to EVs from AKR1B10<sub>GFP</sub> Ct (**: <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">
15 pages, 2886 KiB  
Article
Why Monoamine Oxidase B Preferably Metabolizes N-Methylhistamine over Histamine: Evidence from the Multiscale Simulation of the Rate-Limiting Step
by Aleksandra Maršavelski, Janez Mavri, Robert Vianello and Jernej Stare
Int. J. Mol. Sci. 2022, 23(3), 1910; https://doi.org/10.3390/ijms23031910 - 8 Feb 2022
Cited by 6 | Viewed by 2720
Abstract
Histamine levels in the human brain are controlled by rather peculiar metabolic pathways. In the first step, histamine is enzymatically methylated at its imidazole Nτ atom, and the produced N-methylhistamine undergoes an oxidative deamination catalyzed by monoamine oxidase B (MAO-B), as [...] Read more.
Histamine levels in the human brain are controlled by rather peculiar metabolic pathways. In the first step, histamine is enzymatically methylated at its imidazole Nτ atom, and the produced N-methylhistamine undergoes an oxidative deamination catalyzed by monoamine oxidase B (MAO-B), as is common with other monoaminergic neurotransmitters and neuromodulators of the central nervous system. The fact that histamine requires such a conversion prior to oxidative deamination is intriguing since MAO-B is known to be relatively promiscuous towards monoaminergic substrates; its in-vitro oxidation of N-methylhistamine is about 10 times faster than that for histamine, yet this rather subtle difference appears to be governing the decomposition pathway. This work clarifies the MAO-B selectivity toward histamine and N-methylhistamine by multiscale simulations of the rate-limiting hydride abstraction step for both compounds in the gas phase, in aqueous solution, and in the enzyme, using the established empirical valence bond methodology, assisted by gas-phase density functional theory (DFT) calculations. The computed barriers are in very good agreement with experimental kinetic data, especially for relative trends among systems, thereby reproducing the observed MAO-B selectivity. Simulations clearly demonstrate that solvation effects govern the reactivity, both in aqueous solution as well as in the enzyme although with an opposing effect on the free energy barrier. In the aqueous solution, the transition-state structure involving histamine is better solvated than its methylated analog, leading to a lower barrier for histamine oxidation. In the enzyme, the higher hydrophobicity of N-methylhistamine results in a decreased number of water molecules at the active side, leading to decreased dielectric shielding of the preorganized catalytic electrostatic environment provided by the enzyme. This renders the catalytic environment more efficient for N-methylhistamine, giving rise to a lower barrier relative to histamine. In addition, the transition state involving N-methylhistamine appears to be stabilized by the surrounding nonpolar residues to a larger extent than with unsubstituted histamine, contributing to a lower barrier with the former. Full article
(This article belongs to the Section Physical Chemistry and Chemical Physics)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Free energy profiles for the calibrated EVB gas-phase simulations for histamine (HIS/blue) and <span class="html-italic">N</span>-methylhistamine (NMH/red), each given in a batch of 10 replicas. <span class="html-italic">ε</span> is the generalized reaction coordinate defined by the difference between the potential surfaces of the reactant and product states. The tunable EVB parameters were determined by fitting such that the activation barrier and reaction free energy averaged over the replicas match the values obtained by DFT calculations (<a href="#ijms-23-01910-t001" class="html-table">Table 1</a>).</p> Full article ">Figure 2
<p>EVB free energy profiles of histamine (HIS/blue) and <span class="html-italic">N</span>-methylhistamine (NMH/red) oxidation in aqueous solution, both acquired from 10 simulation replicas. The average barrier and reaction free energy are listed in <a href="#ijms-23-01910-t002" class="html-table">Table 2</a>. <span class="html-italic">ε</span> is the generalized reaction coordinate defined by the difference between the potential surfaces of the reactant and product state. Note that the displayed profiles pertain to the reaction step and do not include the contribution associated with deprotonation of the substrate. The corresponding deprotonation corrections are 3.20 and 2.95 kcal/mol for HIS and NMH, respectively. The total free energy barriers are listed in <a href="#ijms-23-01910-t002" class="html-table">Table 2</a>.</p> Full article ">Figure 3
<p>Running average number of water molecules within 7 Å of the C4 atom of the imidazole ring (marked with purple star) of histamine (HIS/blue) and <span class="html-italic">N</span>-methylhistamine (NMH/red) computed over reaction simulation.</p> Full article ">Figure 4
<p>EVB free energy profiles for histamine (HIS/blue) and N-methylhistamine (NMH/red) oxidation in monoamine oxidase B (MAO-B), both acquired from 10 simulation replicas. The average barrier and reaction free energy are listed in <a href="#ijms-23-01910-t002" class="html-table">Table 2</a>. <span class="html-italic">ε</span> is the generalized reaction coordinate defined by the difference between the potential surfaces of the reactant and product state. Note that the displayed profiles pertain to the reaction step and do not include the contribution associated with deprotonation of the substrate. The corresponding deprotonation corrections are 3.20 and 2.95 kcal/mol for HIS and NMH, respectively. The total free energy barriers are listed in <a href="#ijms-23-01910-t002" class="html-table">Table 2</a>.</p> Full article ">Figure 5
<p>Average number (in 0.25 ns blocks) of hydrophobic residues within 5 Å of the common ring nitrogen atom (N<sup>τ</sup>) of both substrates (histamine: HIS/blue; <span class="html-italic">N</span>-methylhistamine: NMH/red) during simulation of the reaction.</p> Full article ">Figure 6
<p><span class="html-italic">N</span>-methylhistamine (NMH) in the active site of monoamine oxidase B during reaction simulation, with surrounding hydrophobic residues and Tyr326 aromatic residue indicated.</p> Full article ">Figure 7
<p>Structure of the solvated monoamine oxidase B (MAO-B) with the reacting <span class="html-italic">N</span>-methylhistamine (NMH) and flavin adenine dinucleotide (FAD) prosthetic group in a spherical simulation cell centered at the N5 atom of FAD.</p> Full article ">Scheme 1
<p>Rate limiting step of histamine (R = H) and <span class="html-italic">N</span>-methylhistamine (R = CH<sub>3</sub>) oxidation catalyzed by the MAO-B enzyme, proceeding by the hydride transfer mechanism.</p> Full article ">
16 pages, 3560 KiB  
Article
Oxidative DNA Damage and Cisplatin Neurotoxicity Is Exacerbated by Inhibition of OGG1 Glycosylase Activity and APE1 Endonuclease Activity in Sensory Neurons
by Adib Behrouzi, Hanyu Xia, Eric L. Thompson, Mark R. Kelley and Jill C. Fehrenbacher
Int. J. Mol. Sci. 2022, 23(3), 1909; https://doi.org/10.3390/ijms23031909 - 8 Feb 2022
Cited by 5 | Viewed by 3282
Abstract
Cisplatin can induce peripheral neuropathy, which is a common complication of anti-cancer treatment and negatively impacts cancer survivors during and after completion of treatment; therefore, the mechanisms by which cisplatin alters sensory neuronal function to elicit neuropathy are the subject of much investigation. [...] Read more.
Cisplatin can induce peripheral neuropathy, which is a common complication of anti-cancer treatment and negatively impacts cancer survivors during and after completion of treatment; therefore, the mechanisms by which cisplatin alters sensory neuronal function to elicit neuropathy are the subject of much investigation. Our previous work suggests that the DNA repair activity of APE1/Ref-1, the rate-limiting enzyme of the base excision repair (BER) pathway, is critical for neuroprotection against cisplatin. A specific role for 8-oxoguanine DNA glycosylase-1 (OGG1), the glycosylase that removes the most common oxidative DNA lesion, and putative coordination of OGG1 with APE1/Ref-1 in sensory neurons, has not been investigated. We investigated whether inhibiting OGG1 glycosylase activity with the small molecule inhibitor, TH5487, and/or APE1/Ref-1 endonuclease activity with APE Repair Inhibitor III would alter the neurotoxic effects of cisplatin in sensory neuronal cultures. Sensory neuron function was assessed by calcitonin gene-related peptide (CGRP) release, as a marker of sensitivity and by neurite outgrowth. Cisplatin altered neuropeptide release in an inverse U-shaped fashion, with low concentrations enhancing and higher concentrations diminishing CGRP release. Pretreatment with BER inhibitors exacerbated the functional effects of cisplatin and enhanced 8oxo-dG and adduct lesions in the presence of cisplatin. Our studies demonstrate that inhibition of OGG1 and APE1 endonuclease activity enhances oxidative DNA damage and exacerbates neurotoxicity, thus limiting oxidative DNA damage in sensory neurons that might alleviate cisplatin-induced neuropathy. Full article
(This article belongs to the Special Issue Genome Stability and Neurological Disease)
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Figure 1
<p>CGRP release from neuronal cultures is altered following exposure to cisplatin. (<b>A</b>). Columns represent the mean ± SEM of CGRP release stimulated by a 10 min exposure to 30 nM capsaicin following a 24 h exposure to increasing concentrations of cisplatin (30 μM). * <span class="html-italic">p</span> &lt; 0.0001 comparing stimulated release in CIS to no treatment control, two-way ANOVA with Tukey’s posttest. (<b>B</b>). Each column represents the mean ± SEM of the total CGRP content following exposure to cisplatin treatments. * <span class="html-italic">p</span> &lt; 0.05 comparing content in CIS (100 μM) to no treatment control, one-way ANOVA with Dunnett’s posttest.</p> Full article ">Figure 2
<p>CGRP release from neuronal cultures is altered following exposure to cisplatin and the changes in stimulated CGRP release following exposure to base excision repair inhibitors. (<b>A</b>). Columns represent the mean ± SEM of CGRP release stimulated by a 10 min exposure to 30 nM capsaicin following a 24 h exposure to cisplatin (30 μM). * <span class="html-italic">p</span> &lt; 0.0001 comparing stimulated release in CIS to VEH controls; † <span class="html-italic">p</span> &lt; 0.001 comparing CIS effects in the presence and absence of base excision repair inhibitors, two-way ANOVA with Tukey’s posttest. (<b>B</b>). Each column represents the mean ± SEM of the total CGRP content following the indicated treatments.</p> Full article ">Figure 3
<p>Effects of cisplatin and base excision repair inhibitors on 8-oxodG levels in cells adjacent to sensory neurons. (<b>A</b>), Representative immunostaining for 8-oxodG (green) in sensory neuron cultures. The original magnification was ×20. Scale bar represents 200 µm. (<b>B</b>), Quantitative analysis of the integrated density of 8-oxodG staining within PGP9.5+ regions of the image field, acquired as relative fluorescence units using Cytation5 software. * <span class="html-italic">p</span> &lt; 0.0001 comparing TH and ARI3 to VEH; † <span class="html-italic">p</span> &lt; 0.0001 comparing TH/CIS and ARI3/CIS to inhibitors alone, two-way ANOVA with Tukey’s posttest.</p> Full article ">Figure 4
<p>Effects of cisplatin and base excision repair inhibitors on cisplatin adduct levels in cells adjacent to sensory neurons. (<b>A</b>) Representative immunostaining for cisplatin adducts (green) in sensory neuron cultures. The original magnification was ×20. Scale bar represents 200 µm. (<b>B</b>) Quantitative analysis of the integrated density of adduct staining within PGP9.5+ regions of the image field, acquired as relative fluorescence units using Cytation5 software. † <span class="html-italic">p</span> &lt; 0.0001 comparing TH/CIS and ARI3/CIS to TH/VEH and ARI3/VEH, respectively, * <span class="html-italic">p</span> &lt; 0.0001 comparing VEH/CIS to ARI3/CIS; two-way ANOVA with Tukey’s posttest.</p> Full article ">Figure 5
<p>Effects of cisplatin and base excision repair inhibitors on neurite outgrowth. (<b>A</b>) Representative immunostaining for PGP9.5 (green) in sensory neuron cultures. The original magnification was ×20. Scale bar represents 200 µm. (<b>B</b>) Quantitative analysis of the axonal area of PGP9.5 staining, acquired using Cytation5 software. * <span class="html-italic">p</span> &lt; 0.01 comparing the indicated groups to the Vehicle-treated control, two-way ANOVA with Tukey’s posttest.</p> Full article ">Figure 6
<p>A summary of the proposed roles of oxidative DNA damage, OGG1 and APE1 in sensory neurons. Figure created with Biorender.com, accessed on 29 December 2021.</p> Full article ">
17 pages, 3640 KiB  
Article
A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells
by Adam L. Valkovic, Martina Kocan, Brad Hoare, Sarah Marshall, Daniel J. Scott and Ross A. D. Bathgate
Int. J. Mol. Sci. 2022, 23(3), 1908; https://doi.org/10.3390/ijms23031908 - 8 Feb 2022
Cited by 4 | Viewed by 3187
Abstract
Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence [...] Read more.
Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types. Full article
(This article belongs to the Special Issue The Kinase Inhibitors in Human Diseases)
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Figure 1
<p>Design and expression of cyclic GMP sensor using YFP-PDE5-Rluc8 (CYGYEL), a bioluminescence resonance energy transfer (BRET)-based biosensor for cyclic guanosine monophosphate (cGMP) activity. (<b>A</b>) The isolated cGMP-binding domain from human phosphodiesterase (PDE) 5 is sandwiched between Venus and Rluc8. (<b>B</b>) HEK293T cells were transduced with CYGYEL lentivirus (HEK-CYGYEL cells) and sorted by fluorescence-activated cell sorting. A “high-expressing” population was imaged using a fluorescence microscope (20× magnification). (<b>C</b>) A spectral scan was generated after stimulating lysates of HEK-CYGYEL cells with cGMP (100 µM). Spectral scan data are the mean and standard error of the mean (SEM) of data from three independent assays.</p> Full article ">Figure 2
<p>Characterisation of the CYGYEL BRET-based biosensor for cGMP activity in live cells. Intracellular cGMP was detected in real time after stimulation of HEK-CYGYEL cells with the nitric oxide (NO) donor DEA. Cells were stimulated with diethylamine NONOate (DEA) (D; 10 or 100 μM) for 90 min (min) either (<b>A</b>) without any PDE inhibitors, (<b>B</b>) with the cGMP-specific PDE5 inhibitor vardenafil (V; 100 nM) pre-added (approximately 10 min), (<b>C</b>) with vardenafil added after 40.5 min, or (<b>D</b>) with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (I; 500 μM) pre-added (approximately 10 min). Time of ligand or vehicle addition is represented by arrows. Data are mean and SEM of three independent experiments.</p> Full article ">Figure 3
<p>Blocking and reversing the cGMP sensor signal in HEK-CYGYEL cells. Intracellular cGMP was detected in real time after stimulation of HEK293T cells stably expressing CYGYEL with the NO donor DEA (D; 10 or 100 μM) in the absence or presence of the sGC inhibitor ODQ (O; 100 μM). (<b>A</b>) Cells were stimulated with DEA without ODQ. (<b>B</b>) Cells were stimulated with DEA after preincubation (approximately 10 min) with ODQ. (<b>C</b>) Cells were stimulated with DEA for 20.5 min before addition of ODQ. Time of ligand or vehicle addition is represented by arrows. Data are mean and SEM of three independent experiments.</p> Full article ">Figure 4
<p>Activation of the CYGYEL cGMP sensor by cGMP and cAMP. Lysates from HEK293T cells stably expressing CYGYEL were stimulated with a range of concentrations of (<b>A</b>) cGMP or (<b>B</b>) cAMP for 60 min. (<b>C</b>) Concentration–response curves were generated by taking the area under the curve (AUC) from the time-course data. Live HEK293T cells stably expressing (<b>D</b>) the cAMP sensor using YFP-Epac-Rluc (CAMYEL) BRET-based cAMP biosensor or (<b>E</b>) CYGYEL were stimulated with a range of concentrations of forskolin for 60 min. (<b>F</b>) Concentration–response curves were generated by taking the area under the curve from the time-course data, normalised as a percentage of maximum forskolin response. Time of vehicle or ligand addition is represented by an arrow. Data are the mean and SEM of three or four independent experiments.</p> Full article ">Figure 5
<p>DEA-induced cGMP activity in human umbilical vein smooth muscle cells (HUVSMCs) and human umbilical vein endothelial cells (HUVECs). HUVSMCs and HUVECs stably expressing CYGYEL were stimulated with vehicle or the nitric oxide donor DEA for 60 min. Time courses were generated for (<b>A</b>) HUVSMCs and (<b>B</b>) HUVECs. (<b>C</b>) Concentration–responses curves were generated by taking the area under the curve (AUC) from the time-course data. Time of vehicle or DEA addition is represented by an arrow. Data are the mean and SEM for two (panel B) or four independent experiments.</p> Full article ">Figure 6
<p>CNP-induced cGMP activity in HUVSMCs and HUVECs. HUVSMCs and HUVECs stably expressing CYGYEL were stimulated with vehicle or C-type natriuretic peptide (CNP) for 60 min. Time courses were generated for (<b>A</b>) HUVSMCs and (<b>B</b>) HUVECs. (<b>C</b>) Concentration–response curves were generated by taking the AUC from the time-course data. Time of vehicle or CNP addition is represented by an arrow. Data are the mean and SEM for three independent experiments.</p> Full article ">
24 pages, 10245 KiB  
Article
Blood Bacteria-Free DNA in Septic Mice Enhances LPS-Induced Inflammation in Mice through Macrophage Response
by Warerat Kaewduangduen, Peerapat Visitchanakun, Wilasinee Saisorn, Ariya Phawadee, Charintorn Manonitnantawat, Chirapas Chutimaskul, Paweena Susantitaphong, Patcharee Ritprajak, Naraporn Somboonna, Thanya Cheibchalard, Dhammika Leshan Wannigama, Patipark Kueanjinda and Asada Leelahavanichkul
Int. J. Mol. Sci. 2022, 23(3), 1907; https://doi.org/10.3390/ijms23031907 - 8 Feb 2022
Cited by 17 | Viewed by 4236
Abstract
Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the [...] Read more.
Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the sources, influences, and impacts of bacteria-free DNA in sepsis. First, bacteria-free DNA and bacteriome in blood were demonstrated in cecal ligation and puncture (CLP) sepsis mice. Second, administration of bacterial lysate (a source of bacterial DNA) in dextran sulfate solution (DSS)-induced mucositis mice elevated blood bacteria-free DNA without bacteremia supported gut translocation of free DNA. The absence of blood bacteria-free DNA in DSS mice without bacterial lysate implies an impact of the abundance of bacterial DNA in intestinal contents on the translocation of free DNA. Third, higher serum cytokines in mice after injection of combined bacterial DNA with lipopolysaccharide (LPS), when compared to LPS injection alone, supported an influence of blood bacteria-free DNA on systemic inflammation. The synergistic effects of free DNA and LPS on macrophage pro-inflammatory responses, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10), pro-inflammatory genes (NFκB, iNOS, and IL-1β), and profound energy alteration (enhanced glycolysis with reduced mitochondrial functions), which was neutralized by TLR-9 inhibition (chloroquine), were demonstrated. In conclusion, the presence of bacteria-free DNA in sepsis mice is partly due to gut translocation of bacteria-free DNA into the systemic circulation, which would enhance sepsis severity. Inhibition of the responses against bacterial DNA by TLR-9 inhibition could attenuate LPS-DNA synergy in macrophages and might help improve sepsis hyper-inflammation in some situations. Full article
(This article belongs to the Special Issue Human and Animal Monocytes and Macrophages in Homeostasis and Disease 2.0)
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Figure 1
<p>Bacteremia in cecal ligation and puncture (CLP) sepsis, the impact of both intestinal infection and gut-barrier defect. Characteristics of mice after sham or CLP, as indicated by survival analysis (<b>A</b>), bacterial burdens in the blood (<b>B</b>), serum endotoxin (<b>C</b>), serum bacteria-free DNA (<b>D</b>), gut-barrier defect (FITC-dextran assay) (<b>E</b>), identified bacteria based on bacterial colony characteristics (mass-spectrometry analysis) (<b>F</b>), serum cytokines (TNF-α, IL-6, and IL-10) (<b>G</b>), organ injury (serum creatinine and alanine transaminase) (<b>H</b>), and peripheral blood leukocyte (total, neutrophils, and lymphocytes) (<b>I</b>) are demonstrated (<span class="html-italic">n</span> = 15/group for A and <span class="html-italic">n</span> = 9–10/group for <b>B</b>–<b>I</b>). *, <span class="html-italic">p</span> &lt; 0.05 vs. sham; survival analysis and the difference between two groups were determined by Log-rank test and Student’s T test, respectively.</p> Full article ">Figure 2
<p>Pathogenic proteobacteria were mainly presented in blood bacteriome of mice, both sham and cecal ligation and puncture (CLP), while Firmicutes were mainly demonstrated in fecal microbiome analysis of both groups. Characteristics of bacteriome analysis from the blood and feces of mice after sham or CLP, as indicated by bacterial abundance in phylum and species with the average abundance (<b>A</b>–<b>D</b>) and the graph presentation of some groups of bacteria (<b>E</b>) are demonstrated (<span class="html-italic">n</span> = 3–5 for sham and <span class="html-italic">n</span> = 6 for CLP). *, <span class="html-italic">p</span> &lt; 0.05 vs. sham; the difference between groups was determined by Log-rank test and Student’s T test, respectively.</p> Full article ">Figure 3
<p>The translocation of bacteria-free DNA from the gut into the blood circulation in a non-surgical intestinal tight-junction injury model using dextran sulfate solution (DSS)-induced mucositis. Characteristics of mice with DSS or regular drinking water (Water) with oral gavage by bacterial lysate or phosphate buffer solution (PBS) started on days 5–7 of the experiments, as indicated by serum bacteria-free DNA (<b>A</b>), gut-barrier defect (FITC-dextran) (<b>B</b>), serum lipopolysaccharide (LPS) (<b>C</b>), serum pro-inflammatory cytokines (TNF-α and IL-6) (<b>D</b>,<b>E</b>) and colon injury score with representative histological pictures on hematoxylin and eosin (H&amp;E) staining (<b>F</b>,<b>G</b>) are demonstrated (<span class="html-italic">n</span> = 6–8/group). The colon pathology of control PBS gavage with regular drinking water (PBS–water) is not demonstrated due to the similarity with control bacterial lysate gavage with water (lysate–water). Arrows indicate inflammatory cell infiltration in DSS-induced intestinal injury. *, <span class="html-italic">p</span> &lt; 0.05 between the indicated groups; the difference between groups was determined by one-way ANOVA with Tukey’s analysis.</p> Full article ">Figure 4
<p>Additive effect of bacteria-free DNA on LPS-induced pro-inflammation in mice and the influence of bacteria-free DNA in blood circulation. Characteristics of mice after injection with bacteria-free DNA (DNA) or lipopolysaccharide (LPS) alone or in combination (LPS + DNA), as indicated by serum bacteria-free DNA (<b>A</b>), gut-barrier defect (FITC-dextran) (<b>B</b>), serum alanine transaminase (<b>C</b>), serum cytokines (TNF-α, IL-6, and IL-10) (<b>D</b>–<b>F</b>), cytokines from liver tissue (TNF-α, IL-6, and IL-10) (<b>G</b>–<b>I</b>) are demonstrated (<span class="html-italic">n</span> = 7–9/time-point). #, <span class="html-italic">p</span> &lt; 0.05 vs. DNA; ϕ, <span class="html-italic">p</span> &lt; 0.05 vs. baseline; *, <span class="html-italic">p</span> &lt; 0.05 between the indicated groups; one-way ANOVA with Tukey’s analysis and repeated measures ANOVA were used for the analysis among groups and between different time-points within groups, respectively.</p> Full article ">Figure 5
<p>Additive effect of bacteria-free DNA on LPS-induced pro-inflammation M1 macrophage polarization and the impact of bacterial DNA in macrophage responses. Characteristics of macrophages after activation by media control, lipopolysaccharide (LPS), bacteria-free DNA (DNA), and LPS with the DNA (LPS + DNA), as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10) (<b>A</b>–<b>C</b>) and the expression of genes for inflammatory signals (<span class="html-italic">NFκB</span> and <span class="html-italic">TLR-4</span>) (<b>D</b>,<b>E</b>), M1 macrophage polarization (<span class="html-italic">iNOS</span> and <span class="html-italic">IL-1β</span>) (<b>F</b>,<b>G</b>), and M2 macrophage polarization (<span class="html-italic">FIZZ-1</span>, <span class="html-italic">Arg-1,</span> and <span class="html-italic">TGF-β</span>) (<b>H</b>–<b>J</b>) are demonstrated (independent triplicated experiments were performed). *, <span class="html-italic">p</span> &lt; 0.05 vs. all of other groups; #, <span class="html-italic">p</span> &lt; 0.05 vs. macrophages with media control group; the difference between groups was determined by one-way ANOVA with Tukey’s analysis.</p> Full article ">Figure 6
<p>Additive effect of bacteria-free DNA on LPS-mediated cell energy alteration in macrophages and the impact of bacteria-free DNA in macrophage responses. Characteristics of macrophages after activation by media control, lipopolysaccharide (LPS), bacteria-free DNA (DNA), and LPS with the DNA (LPS + DNA), as indicated by mitochondrial abundance using mitochondrial DNA (<span class="html-italic">mtDNA</span>) and MitoTracker fluorescent staining (<b>A</b>,<b>B</b>), oxygen consumption rate (OCR) of mitochondrial stress test with the graph presentation of respiratory capacity (maximal respiration) and respiratory reserve (<b>C</b>–<b>E</b>), and extracellular acidification rate (ECAR) for a glycolysis stress test with the graph presentation of glycolysis capacity (maximal glycolysis) and glycolysis reserve (<b>F</b>–<b>H</b>) are demonstrated (independent triplicated experiments were performed). *, <span class="html-italic">p</span> &lt; 0.05 vs. all of the other groups; ϕ, <span class="html-italic">p</span> &lt; 0.05 between the indicated groups; the difference between groups was determined by one-way ANOVA with Tukey’s analysis.</p> Full article ">Figure 7
<p>TLR-9 inhibitors attenuated macrophage inflammatory responses against bacteria-free DNA and the impact of TLR-9 on bacteria-free DNA-induced inflammation. Characteristics of macrophages after activation by media control, lipopolysaccharide (LPS), bacteria-free DNA (DNA), and LPS with the DNA (LPS + DNA) with incubation by media (Control) or endosomal acidification inhibitors, chloroquine (CQ) or monensin, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10) (<b>A</b>–<b>C</b>) and expression of inflammatory genes (<span class="html-italic">NFκB</span> and <span class="html-italic">TLR-4</span>) (<b>D</b>,<b>E</b>) are demonstrated (independent triplicated experiments were performed). *, <span class="html-italic">p</span> &lt; 0.05 vs. all of other groups; #, <span class="html-italic">p</span> &lt; 0.05 vs. macrophages with DNA in the control group; ϕ, <span class="html-italic">p</span> &lt; 0.05 vs. macrophages with media control within each group of the experiments; the difference between groups was determined by one-way ANOVA with Tukey’s analysis.</p> Full article ">Figure 8
<p>TLR-9 inhibitors attenuated energy modification in macrophage with bacteria-free DNA stimulation and the impact of TLR-9 on bacteria-free DNA-induced cell-energy alteration. Characteristics of macrophages after activation by media control or lipopolysaccharide with bacteria-free DNA (LPS + DNA) alone or with chloroquine (LPS + DNA + CQ) as indicated by extracellular acidification rate (ECAR) for a glycolysis stress test with the graph presentation of glycolysis capacity (maximal glycolysis) and glycolysis reserve (<b>A</b>–<b>C</b>), oxygen consumption rate (OCR) of mitochondrial stress test with the graph presentation of respiratory capacity (maximal respiration) and respiratory reserve (<b>D</b>–<b>F</b>), and mitochondrial abundance using mitochondrial DNA (<span class="html-italic">mtDNA</span>) and MitoTracker fluorescent staining (<b>G</b>,<b>H</b>) are demonstrated (independent triplicated experiments were performed). *, <span class="html-italic">p</span> &lt; 0.05 vs. all of other groups; #, <span class="html-italic">p</span> &lt; 0.05 vs. macrophages with media control group; the difference between groups was determined by one-way ANOVA with Tukey’s analysis.</p> Full article ">Figure 9
<p>Conclusion of the experiments on mouse models and macrophages with the key massages (at the bottom part of the figure). Accordingly, (i) CLP (bacteria-free DNA possibly originated from the death of bacteria in blood and gut-barrier defect); (ii) DSS mucositis with oral gavage by bacterial lysate (bacteria-free DNA in blood was transferred from the gut); (iii) LPS with and without bacteria-free DNA injection (the importance of bacteria-free DNA in blood on the induction of LPS hyper-inflammatory responses); and (iv) the synergy between LPS and bacteria-free DNA on macrophage responses and the attenuation by TLR-9 blockage. Figure created using Biorender (<a href="https://biorender.com/" target="_blank">https://biorender.com/</a>, accessed on 29 January 2022).</p> Full article ">Figure 10
<p>The working hypothesis demonstrates a simultaneous stimulation of surface TLR-4 and endosomal TLR-9 by LPS and bacteria-free DNA, respectively, which induces inflammatory responses through MyD88, and non-MyD88 (TRIF), which induces TRAF-6 [<a href="#B92-ijms-23-01907" class="html-bibr">92</a>,<a href="#B93-ijms-23-01907" class="html-bibr">93</a>]. The vigorous inflammatory stimulation, especially cytokine production, requires high cell energy, mostly by the glycolysis pathway [<a href="#B81-ijms-23-01907" class="html-bibr">81</a>], which might cause mitochondrial damage with higher inflammatory responses [<a href="#B75-ijms-23-01907" class="html-bibr">75</a>,<a href="#B94-ijms-23-01907" class="html-bibr">94</a>]. MyD88, myeloid differentiation primary response 88; TRIF, TIR-domain-containing adapter-inducing interferon-β; TRAF6, TNF receptor associated factor 6; IRF-3, interferon regulatory factor 3. Figure created using Biorender (<a href="https://biorender.com/" target="_blank">https://biorender.com/</a>, accessed on 29 January 2022).</p> Full article ">
17 pages, 5438 KiB  
Article
Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity
by Joanna Pastwińska, Kaja Karaś, Anna Sałkowska, Iwona Karwaciak, Katarzyna Chałaśkiewicz, Błażej A. Wojtczak, Rafał A. Bachorz and Marcin Ratajewski
Int. J. Mol. Sci. 2022, 23(3), 1906; https://doi.org/10.3390/ijms23031906 - 8 Feb 2022
Cited by 6 | Viewed by 2773
Abstract
RORγT is a protein product of the RORC gene belonging to the nuclear receptor subfamily of retinoic-acid-receptor-related orphan receptors (RORs). RORγT is preferentially expressed in Th17 lymphocytes and drives their differentiation from naive CD4+ cells and is involved in the regulation of the [...] Read more.
RORγT is a protein product of the RORC gene belonging to the nuclear receptor subfamily of retinoic-acid-receptor-related orphan receptors (RORs). RORγT is preferentially expressed in Th17 lymphocytes and drives their differentiation from naive CD4+ cells and is involved in the regulation of the expression of numerous Th17-specific cytokines, such as IL-17. Because Th17 cells are implicated in the pathology of autoimmune diseases (e.g., psoriasis, inflammatory bowel disease, multiple sclerosis), RORγT, whose activity is regulated by ligands, has been recognized as a drug target in potential therapies against these diseases. The identification of such ligands is time-consuming and usually requires the screening of chemical libraries. Herein, using a Tanimoto similarity search, we found corosolic acid and other pentacyclic tritepenes in the library we previously screened as compounds highly similar to the RORγT inverse agonist ursolic acid. Furthermore, using gene reporter assays and Th17 lymphocytes, we distinguished compounds that exert stronger biological effects (ursolic, corosolic, and oleanolic acid) from those that are ineffective (asiatic and maslinic acids), providing evidence that such combinatorial methodology (in silico and experimental) might help wet screenings to achieve more accurate results, eliminating false negatives. Full article
(This article belongs to the Special Issue Drug Design and Virtual Screening)
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Figure 1
<p>Identification of corosolic and asatic acids as compounds similar to ursolic acid in a virtual screening of the L1600 Kinase Inhibitor Library (TargetMol).</p> Full article ">Figure 2
<p>Tanimoto similarity values of the ursolic acid analogs.</p> Full article ">Figure 3
<p>Molecular docking analysis of the ursolic, corosolic, oleanolic, asiatic, and maslinic acids binding to the LBD of the RORγ receptor. (<b>A</b>) Histograms of the binding energies of all considered acids with the 3l0j receptor. (<b>B</b>) Boxplots of the binding energies of all considered acids with the 3l0j receptor.</p> Full article ">Figure 4
<p>Molecular docking analysis of the best (green) and the worst (yellow) stereoizomers for ursolic (<b>A</b>), corosolic (<b>B</b>), and oleanolic (<b>C</b>) acids binding to the LBD of the RORγ receptor. Hydrogen bonds are represented as dark-blue dotted lines.</p> Full article ">Figure 5
<p>Effect of ursolic acid analogs on RORγ-dependent transcription in the HEK293 cell line. HEK293 cells were cotransfected with the pGL4.35[luc2P/9XGAL4UAS/Hygro], GAL4-DBD RORγ, and pCMVSEAP vectors. Twenty-four hours later, the cells were treated with increasing concentrations of ursolic (<b>A</b>), corosolic (<b>B</b>), oleanolic (<b>C</b>), asiatic (<b>D</b>), and maslinic acids (<b>E</b>) for another 48 h. After that time, the cells were lysed, and luciferase activity was measured. Luciferase results are standardized for the transfection efficiency control, which was SEAP. Mean ± SD, <span class="html-italic">n</span> = 3. EC50 values were calculated using AAT Bioquest (Sunnyvale, CA, USA) EC50 calculator, (<a href="https://www.aatbio.com/tools/ec50-calculator/" target="_blank">https://www.aatbio.com/tools/ec50-calculator/</a>, accessed on 6 December 2021).</p> Full article ">Figure 6
<p>(<b>A</b>–<b>E</b>) Effect of ursolic acid analogs on CD4+ lymphocyte viability. CD4+ cells were isolated from buffy coats of healthy donors and subjected to Th17 polarization in the presence of increasing concentrations of ursolic, corosolic, oleanolic, asiatic, and maslinic acids for 5 days. Then, cell viability was determined using the CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay. Mean ± SD, <span class="html-italic">n</span> = 3, compared with control cells.</p> Full article ">Figure 7
<p>Effect of ursolic acid analogs on the expression of selected genes in human Th17 cells. Human naive CD4+ cells were treated with increasing concentrations of ursolic (<b>A</b>), corosolic (<b>B</b>), and oleanolic (<b>C</b>) acids and cultured under Th17-polarizing conditions for 5 days. Then, cells were collected for RNA extraction. The expression of the <span class="html-italic">IL17A</span>, <span class="html-italic">IL17F</span>, <span class="html-italic">IL21</span>, and <span class="html-italic">IL22</span> genes was determined by real-time RT–PCR. The results were normalized to the housekeeping genes <span class="html-italic">HPRT1</span>, <span class="html-italic">HMBS</span>, and <span class="html-italic">RPL13A</span>. An asterisk indicates a statistically significant difference at <span class="html-italic">p</span> &lt; 0.05 compared with control cells. The data are presented as statistical dot plots with the median value (bars) from seven independent cultures (<span class="html-italic">n</span> = 7).</p> Full article ">Figure 8
<p>The analysis of IL-17 production in supernatants of Th17 cells cultured in the presence of increasing concentrations of ursolic (<b>A</b>), corosolic (<b>B</b>), and oleanolic acids (<b>C</b>) for 5 days was determined using the Quantikine Human IL-17 Immunoassay kit (R&amp;D Systems). An asterisk indicates a statistically significant difference at <span class="html-italic">p</span> &lt; 0.05 compared with control cells. The data are presented as statistical dot plots with the median value (bars) from seven independent cultures (<span class="html-italic">n</span> = 7).</p> Full article ">Figure 9
<p>Chromatin immunoprecipitation results showing that ursolic, corosolic, and oleanolic acids decrease the levels of RORγT protein occupancy on the <span class="html-italic">IL17A</span> (<b>A</b>) and <span class="html-italic">IL17F</span> (<b>B</b>) gene promoters. Mean ± SD, <span class="html-italic">n</span> = 3, an asterisk indicates a statistically significant difference at <span class="html-italic">p</span> &lt; 0.05 compared with control.</p> Full article ">
18 pages, 3762 KiB  
Article
The Expression of TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Can Be Controlled by the Antioxidant Orchestrator NRF2 in Human Carcinoma Cells
by Helga Simon-Molas, Cristina Sánchez-de-Diego, Àurea Navarro-Sabaté, Esther Castaño, Francesc Ventura, Ramon Bartrons and Anna Manzano
Int. J. Mol. Sci. 2022, 23(3), 1905; https://doi.org/10.3390/ijms23031905 - 8 Feb 2022
Cited by 6 | Viewed by 2584
Abstract
Hyperactivation of the KEAP1-NRF2 axis is a common molecular trait in carcinomas from different origin. The transcriptional program induced by NRF2 involves antioxidant and metabolic genes that render cancer cells more capable of dealing with oxidative stress. The TP53-Induced Glycolysis and Apoptosis Regulator [...] Read more.
Hyperactivation of the KEAP1-NRF2 axis is a common molecular trait in carcinomas from different origin. The transcriptional program induced by NRF2 involves antioxidant and metabolic genes that render cancer cells more capable of dealing with oxidative stress. The TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) is an important regulator of glycolysis and the pentose phosphate pathway that was described as a p53 response gene, yet TIGAR expression is detected in p53-null tumors. In this study we investigated the role of NRF2 in the regulation of TIGAR in human carcinoma cell lines. Exposure of carcinoma cells to electrophilic molecules or overexpression of NRF2 significantly increased expression of TIGAR, in parallel to the known NRF2 target genes NQO1 and G6PD. The same was observed in TP53KO cells, indicating that NRF2-mediated regulation of TIGAR is p53-independent. Accordingly, downregulation of NRF2 decreased the expression of TIGAR in carcinoma cell lines from different origin. As NRF2 is essential in the bone, we used mouse primary osteoblasts to corroborate our findings. The antioxidant response elements for NRF2 binding to the promoter of human and mouse TIGAR were described. This study provides the first evidence that NRF2 controls the expression of TIGAR at the transcriptional level. Full article
(This article belongs to the Special Issue The Nrf2 Pathway: Regulation, Functions, and Potential Applications 2.0)
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<p>The expression of <span class="html-italic">TIGAR</span> is modulated by NRF2 activators in a transcription-dependent manner and independently of p53. (<b>A</b>) HeLa cells were treated with 5 or 20 µM (<b>A</b>) sulforaphane (SFN) (<span class="html-italic">n</span> = 4) or (<b>B</b>) dimethyl fumarate (DMF) (<span class="html-italic">n</span> = 5) for 24 h and analyzed by RT-qPCR. (<b>C</b>,<b>D</b>) HeLa cells were pretreated with 5 µg/mL actinomycin-D (Act-D) and then subsequently exposed to (<b>C</b>) 20 µM SFN (<span class="html-italic">n</span> = 4) or (<b>D</b>) 20 µM DMF (<span class="html-italic">n</span> = 5) for 24 h and analyzed by RT-qPCR. (<b>E</b>,<b>F</b>). (<b>E</b>) HCT116 40.16 (<span class="html-italic">n</span> = 3) and (<b>F</b>) HCT116 379.2 (<span class="html-italic">n</span> = 3) cell lines were treated with 20 µM sulforaphane (SFN) for 24 h and subsequently analyzed by RT-qPCR. Data are presented as the mean fold change relative to untreated cells (CT) ± SEM and differences were analyzed with (<b>A</b>,<b>B</b>) one-way ANOVA using the Holm–Sidak method for multiple comparisons, (<b>C</b>,<b>D</b>) two-way ANOVA using the Holm–Sidak method for multiple comparisons or (<b>E</b>,<b>F</b>) independent <span class="html-italic">t</span>-tests (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 2
<p>Specific modulation of NRF2 expression affects TIGAR levels. (<b>A</b>,<b>B</b>) HeLa cells were transfected with 1 µg of a pcDNA3 plasmid coding for human <span class="html-italic">NFE2L2</span> or the corresponding empty vector (pcDNA3) and subsequently analyzed after 24 h by (<b>A</b>) RT-qPCR (<span class="html-italic">n</span> = 14) or (<b>B</b>) Western blot (NRF2 and TIGAR, <span class="html-italic">n</span> = 6, G6PD <span class="html-italic">n</span> = 3). (<b>C</b>) Representative Western blot images of the conditions specified in B. (<b>D</b>,<b>E</b>) HeLa cells were transfected with 100 nM <span class="html-italic">NFE2L2</span>-targeting siRNA or the corresponding scrambled siRNA (Scr.) and analyzed after 72 h by (<b>D</b>) RT-qPCR (<span class="html-italic">n</span> = 5) or (<b>E</b>) Western blot (<span class="html-italic">n</span> = 5). (<b>F</b>) Representative Western blot images of the conditions specified in E. (<b>G</b>) H460 cells were transfected with 100 nM <span class="html-italic">NFE2L2</span>-targeting siRNA or the corresponding scrambled siRNA and analyzed after 72 h by RT-qPCR (<span class="html-italic">n</span> = 4). (<b>H</b>) A549 cells were transfected with 100 nM <span class="html-italic">NFE2L2</span>-targeting siRNA or the corresponding scrambled siRNA and analyzed after 72 h by Western blot (NRF2 <span class="html-italic">n</span> = 4, G6PD <span class="html-italic">n</span> = 3, TIGAR <span class="html-italic">n</span> = 8). Data are represented as the mean ± SEM relative to the corresponding control (pcDNA3 or Scr.) and differences were analyzed with independent <span class="html-italic">t</span>-tests (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 3
<p>In-silico study of conserved AREs in the promoter of the human <span class="html-italic">TIGAR</span> gene. The ECR Browser (available at <a href="http://ecrbrowser.dcode.org" target="_blank">http://ecrbrowser.dcode.org</a>, accessed on 10 January 2022) [<a href="#B31-ijms-23-01905" class="html-bibr">31</a>] was used to analyze the sequence from 4.428.749 to 4.430.851 bp of human chromosome 12 (−1630, +472 from the transcription start site (TSS)). The sequence was compared to the genome of Pan troglodytes (panTro3) and rhesus macaque (rheMac2). Horizontal red lines above the genomic sequences indicate evolutionary conserved regions. The ECR Browser image has been modified to highlight the most relevant elements in the present study. Antioxidant response elements (AREs) corresponding to TGACnnAGC motifs are represented by asterisks. The SP1, CREB1 and p53 binding sites are also indicated.</p> Full article ">Figure 4
<p>NRF2 enhances the transcription of human <span class="html-italic">TIGAR</span> through an ARE located on its promoter. (<b>A</b>) Schematic representation of the pGL3 promoter luciferase reporter constructs containing the antioxidant response elements (AREs) in human <span class="html-italic">TIGAR</span>: ARE1, ARE2 or both (ARE1 + ARE2). (<b>B</b>,<b>C</b>) Luciferase activity of the pGL3 luciferase reporter constructs normalized to β-galactosidase activity in (<b>B</b>) HeLa cells co-transfected with an <span class="html-italic">NFE2L2</span> overexpressing plasmid (<span class="html-italic">n</span> = 3) and (<b>C</b>) HeLa cells treated with 20 µM sulforaphane (SFN) (<span class="html-italic">n</span> = 6). Data are represented as the mean fold change relative to the corresponding control (empty pGL3 promoter vector (pGL3-Prom). (<b>D</b>,<b>E</b>) RT-qPCR analysis of <span class="html-italic">TIGAR</span> ARE1 (<b>D</b>) or <span class="html-italic">NQO1</span> ARE (<b>E</b>) in ChIP-enriched chromatin fractions from HeLa cells transfected with 100 nM <span class="html-italic">NFE2L2</span>-targeting siRNA or Scr. siRNA and analyzed after 72h. Specific antibody against NRF2 or nonspecific IgGs were used and chromatin enrichment was normalized to input fractions (<span class="html-italic">n</span> = 3). Data are represented as the mean fold change relative to the Scr. condition immunoprecipitated with anti-NRF2 ± SEM. Differences were analyzed with (<b>B</b>,<b>C</b>) one-way ANOVA or (<b>D</b>,<b>E</b>) two-way ANOVA using Tukey’s method for multiple comparisons (** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 5
<p>NRF2 regulates the expression of <span class="html-italic">Tigar</span> in mouse primary osteoblasts. (<b>A</b>,<b>B</b>) Primary mouse osteoblasts were treated with (<b>A</b>) 20 µM dimethylfumarate (DMF) or (<b>B</b>) 20 µM sulforaphane (SFN) for 48 h and the expression of <span class="html-italic">Nrf2</span>, <span class="html-italic">Txnrd1</span>, <span class="html-italic">Nqo1</span>, <span class="html-italic">Gclc</span> and <span class="html-italic">Tigar</span> was determined by RT-qPCR (<span class="html-italic">n</span> = 5). (<b>C</b>) Schematic representation of mouse <span class="html-italic">Tigar</span> promoter with the identified antioxidant response element (ARE). (<b>D</b>) Primary mouse osteoblasts were treated with 5 µM DMF for 48 h and ChIP was performed. ChIP-enriched chromatin of <span class="html-italic">Tigar</span> ARE from anti-NRF2 antibody or nonspecific IgGs fractions was analyzed by RT-qPCR and normalized to input fractions (<span class="html-italic">n</span> = 5). Data are represented as the mean ± SEM and differences were analyzed with (<b>A</b>,<b>B</b>) independent <span class="html-italic">t</span>-tests or (<b>D</b>) two-way ANOVA using Tukey’s method for multiple comparisons (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 6
<p>Graphical abstract of the main findings of this work. Transcriptional regulation of <span class="html-italic">TIGAR</span> by NRF2 in human and mouse cells. Upon exposure to the electrophilic molecules SFN and DMF, or after NRF2 overexpression, NRF2 is liberated from KEAP1 and translocates to the nucleus. NRF2 forms heterodimers with sMafs proteins in antioxidant response elements (AREs) located at the promoter of <span class="html-italic">TIGAR</span> and other antioxidant genes such as <span class="html-italic">NQO1</span> and <span class="html-italic">G6PD</span>, enhancing their transcription.</p> Full article ">
24 pages, 14577 KiB  
Article
High, in Contrast to Low Levels of Acute Stress Induce Depressive-like Behavior by Involving Astrocytic, in Addition to Microglial P2X7 Receptors in the Rodent Hippocampus
by Ya-Fei Zhao, Wen-Jing Ren, Ying Zhang, Jin-Rong He, Hai-Yan Yin, Yang Liao, Patrizia Rubini, Jan M. Deussing, Alexei Verkhratsky, Zeng-Qiang Yuan, Peter Illes and Yong Tang
Int. J. Mol. Sci. 2022, 23(3), 1904; https://doi.org/10.3390/ijms23031904 - 8 Feb 2022
Cited by 15 | Viewed by 3642
Abstract
Extracellular adenosine 5′-triphosphate (ATP) in the brain is suggested to be an etiological factor of major depressive disorder (MDD). It has been assumed that stress-released ATP stimulates P2X7 receptors (Rs) at the microglia, thereby causing neuroinflammation; however, other central nervous system (CNS) cell [...] Read more.
Extracellular adenosine 5′-triphosphate (ATP) in the brain is suggested to be an etiological factor of major depressive disorder (MDD). It has been assumed that stress-released ATP stimulates P2X7 receptors (Rs) at the microglia, thereby causing neuroinflammation; however, other central nervous system (CNS) cell types such as astrocytes also possess P2X7Rs. In order to elucidate the possible involvement of the MDD-relevant hippocampal astrocytes in the development of a depressive-like state, we used various behavioral tests (tail suspension test [TST], forced swim test [FST], restraint stress, inescapable foot shock, unpredictable chronic mild stress [UCMS]), as well as fluorescence immunohistochemistry, and patch-clamp electrophysiology in wild-type (WT) and genetically manipulated rodents. The TST and FST resulted in learned helplessness manifested as a prolongation of the immobility time, while inescapable foot shock caused lower sucrose consumption as a sign of anhedonia. We confirmed the participation of P2X7Rs in the development of the depressive-like behaviors in all forms of acute (TST, FST, foot shock) and chronic stress (UCMS) in the rodent models used. Further, pharmacological agonists and antagonists acted in a different manner in rats and mice due to their diverse potencies at the respective receptor orthologs. In hippocampal slices of mice and rats, only foot shock increased the current responses to locally applied dibenzoyl-ATP (Bz-ATP) in CA1 astrocytes; in contrast, TST and restraint depressed these responses. Following stressful stimuli, immunohistochemistry demonstrated an increased co-localization of P2X7Rs with a microglial marker, but no change in co-localization with an astroglial marker. Pharmacological damage to the microglia and astroglia has proven the significance of the microglia for mediating all types of depression-like behavioral reactions, while the astroglia participated only in reactions induced by strong stressors, such as foot shock. Because, in addition to acute stressors, their chronic counterparts induce a depressive-like state in rodents via P2X7R activation, we suggest that our data may have relevance for the etiology of MDD in humans. Full article
(This article belongs to the Special Issue Purinergic Signalling in Physiology and Pathophysiology)
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Figure 1
<p>Effects of P2X7R agonists and antagonists in tests inducing learned helplessness in mice and rats. (<b>A</b>–<b>H</b>) Experiments in mice. (<b>A</b>) dose-dependent prolongation of the TST immobility time of mice by i.c.v. applied Bz-ATP, in comparison with i.c.v.-applied PBS. (<b>B</b>) no difference in the TST immobility time between the WT and P2X7R KO mice during repeated measurements on 3 successive days. No difference in the TST immobility time between mice injected i.c.v. with A-438079 (1 µM; <b>C</b>) or i.p. with JNJ-47965567 (30 mg/kg; <b>D</b>) in comparison with their PBS-injected counterparts. Prolongation of the TST immobility time by Bz-ATP (3 µM; i.c.v.; <b>E</b>) and its blockade by co-applied A-438079 (1 µM; i.c.v.) or JNJ-47965567 (30 mg/kg; i.p.; <b>F</b>). (<b>G</b>) the TST immobility time after CUMS was applied for 30 days. When, thereafter, the TST was measured for 3 successive days, on the third day there was a significant difference between PBS- and JNJ-47965567 (30 mg/kg; i.p.)-treated mice. (<b>H</b>) no difference in the FST immobility time between the WT and P2X7R KO mice during repeated measurements on 3 successive days. * <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the PBS-injected controls (<b>A</b>,<b>E</b>) or the PBS-injected controls on the first day of application (<b>G</b>,<b>H</b>). (<b>E</b>, <span class="html-italic">t</span> = 4.238, <span class="html-italic">p</span> &lt; 0.001; Student’s <span class="html-italic">t</span>-test). (<b>A</b>, F = 4.199, 30 µM, <span class="html-italic">p</span> = 0.004; 300 µM, <span class="html-italic">p</span> = 0.003; <b>G</b>, F = 3.827, 3rd day PBS, <span class="html-italic">p</span> = 0.039; one-way ANOVA followed by the Holm–Sidak test). (<b>B</b>, F<sub>treatment</sub> = 0.261, F<sub>genotype × treatment</sub> = 1.114, <span class="html-italic">p</span> = 0.571; <b>H</b>, F<sub>treatment</sub> = 0.328, F<sub>genotype × treatment</sub> = 0.568, <span class="html-italic">p</span> = 0.300; two-way ANOVA). <sup>§</sup> <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the PBS-injected controls on the same day of application (<b>G</b>, F = 10.578, 3rd day, <span class="html-italic">p</span> = 0.008; one-way ANOVA, followed by the Holm–Sidak test). (<b>I</b>–<b>K</b>) experiments in rats. Decrease in the FST immobility time by A-438079 (1 µM; i.c.v.; <b>I</b>), or JNJ-47965567 (30 mg/kg; i.p.; <b>J</b>) in comparison with their PBS-injected counterparts. (<b>K</b>) Bz-ATP (0.03–30 µM; i.c.v.) fails to prolong the FST immobility time in the PBS-injected controls (one way ANOVA). <sup>§</sup> <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the PBS-injected controls on the same day of application (<b>I</b>, F = 10.571, 2nd day, <span class="html-italic">p</span> = 0.001, 3rd day, <span class="html-italic">p</span> &lt; 0.001; <b>J</b>, F = 14,975, 2nd day, <span class="html-italic">p</span> &lt; 0.001, 3rd day, <span class="html-italic">p</span> &lt; 0.001; one-way ANOVA followed by the Holm–Sidak test). The number of experiments is indicated throughout above each symbol (<b>A</b>) or within each column (<b>B</b>–<b>K</b>).</p> Full article ">Figure 2
<p>Effects of the TST and foot shock on the open-field behavior of mice; changes in the TST immobility time and sucrose consumption after foot shock and its antagonism by the blockade or genetic deletion of the P2X7Rs. (<b>A</b>) running traces of mice in the open-field apparatus under control conditions, immediately after TST, and 1 h after TST. The observation time was 10 min. (<b>B</b>) total running distance, time spent in the center, and time spent in the border of the open-field apparatus without TST (control), as well as immediately (0 h) and 1 h after TST. (<b>C</b>) total running distance, time spent in the center, and time spent in the border of the open-field apparatus without TST, as well as immediately (0 h), 1 h, and 4 h after foot shock. * <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the three open-field parameters measured without the application of TST or foot shock (<b>B</b>, Total distance, F = 3.526, 0 h, <span class="html-italic">p</span> = 0.046; <b>C</b>, Total distance, F = 19.412, 0 h, <span class="html-italic">p</span> &lt; 0.001, 1 h, <span class="html-italic">p</span> &lt; 0.001) one-way ANOVA, followed by the Holm–Sidak test. (<b>D</b>) the TST immobility time measured without foot shock, or alternatively 1 or 4 h after foot shock. (<b>E</b>) the TST immobility time measured without foot shock or after the co-application of JNJ-47965567 (30 mg/kg; i.p.) and foot shock. * <span class="html-italic">p</span> &lt; 0.05; statistically significant differences from the TST immobility time alone (<b>D</b>, FS + TST, F = 29.415, 1 h, <span class="html-italic">p</span> &lt; 0.001, 4 h, <span class="html-italic">p</span> &lt; 001; one-way ANOVA, followed by the Holm–Sidak test). Effect of i.c.v. Bz-ATP (10 µM) on the foot shock-induced modulation of sucrose preference when compared with the effect of the solvent PBS alone (<b>F</b>). Change in sucrose preference after foot shock (<b>G</b>) and the inhibition of this effect by JNJ-47965567 (30 mg/kg; i.p.) (<b>H</b>). In control experiments the solvent of JNJ-47965567 was applied before the foot shock (FS). The change in sucrose preference after foot shock (<b>I</b>) and no effect on the P2X7R KO animals (<b>J</b>). * <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from sucrose consumption after FS + PBS (<b>F</b>, FS + Bz-ATP, <span class="html-italic">t</span> = 3.102, <span class="html-italic">p</span> = 0.008) or untreated controls (<b>G</b>, <span class="html-italic">t</span> = 5.404, <span class="html-italic">p</span> &lt; 0.001; <b>I</b>, <span class="html-italic">t</span> = 2.531, <span class="html-italic">p</span> = 0.024); Student’s <span class="html-italic">t</span>-test. The number of experiments is indicated in the first column of each set.</p> Full article ">Figure 3
<p>Bz-ATP-induced current responses in the hippocampal CA1 astrocytes of rats and mice, and their modification after the development of learned helplessness. Concentration-dependent increase of the current amplitudes (<b>A</b>) and current durations (<b>A</b>, <b>left inset</b>) caused by Bz-ATP, in preparations taken from 25-day old rats; representative tracings recorded at a holding potential of −80 mV. Bz-ATP (30–3000 µM) was applied every 3 min for 10 s. (<b>A</b>, <b>left inset</b>) superimposed current responses are shown at a longer time scale than those displayed in <b>A</b>. (<b>A</b>, <b>right inset</b>) injection of gradually increasing current pulses evoked only electrotonic changes in membrane potential and characterized astrocytes as belonging to the passive group. (<b>B</b>) concentration-dependent increase of the current amplitudes caused by Bz-ATP (100–3000 µM) in preparations taken from 25-day old mice. (<b>C</b>) plot of the Bz-ATP concentration against the current amplitude in astrocytes from rats, WT mice and P2X7R KO mice. Mean ± SEM of the indicated number of concentration-response relationships are shown. There was no response to Bz-ATP when hippocampal slices were prepared from the P2X7R KO mice. * <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the effect of Bz-ATP (3000 µM) in WT mice (<b>C</b>, t = 4.033, <span class="html-italic">p</span> = 0.001; Student’s <span class="html-italic">t</span>-test). <sup>§</sup> <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the effect of Bz-ATP (3000 µM) in the P2X7R KO mice (<b>C</b>, T = 144.00, <span class="html-italic">p</span> &lt; 0.001; Mann–Whitney rank sum test). Current responses to NMDA (100 µM) and Bz-ATP (300, 1000 µM) of astrocytes prepared from 12-day (<b>D</b>) or 25-day old (<b>E</b>) rats; representative tracings. (<b>F</b>) mean ± SEM of the indicated number of experiments. * <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the effect of NMDA (100 µM), in 12–14-day old astrocytes (<b>B</b>, F = 5.725, Bz-ATP 300 µM, <span class="html-italic">p</span> = 0.019, Bz-ATP 1000 µM, <span class="html-italic">p</span> = 0.014; one-way ANOVA, followed by the Holm–Sidak test), and from the effect of NMDA (100 µM) in 23–25-day old astrocytes (<b>B</b>, Bz-ATP 300 µM, <span class="html-italic">p</span> &lt; 0.001, Bz-ATP 1000 µM, <span class="html-italic">p</span> = 0.002; one-way ANOVA, followed by the Holm–Sidak test, respectively). <sup>§</sup> <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the effect of the corresponding NMDA (100 µM) and Bz-ATP (1000 µM) effects in the 12–14-day old group of astrocytes. (<b>B</b>, NMDA-NMDA, T = 165.00, <span class="html-italic">p</span> &lt; 0.001, BzATP-BzATP, T = 165.00, <span class="html-italic">p</span> &lt; 0.001; Mann–Whitney rank sum test). (<b>G</b>–<b>I</b>) current responses to AMPA (100 µM), Bz-ATP (300 µM) and muscimol (100 µM) in hippocampal CA1 astrocytes prepared from 20–25-day old rats. Representative tracings from brain slices taken from untreated (<b>G</b>, <b>left panel</b>), TST-treated (<b>H</b>, <b>left panel</b>) and foot shock-treated (<b>I</b>, <b>left panel</b>) rats. In the respective right panels, the mean ± SEM of identical experiments are shown for AMPA, Bz-ATP and muscimol. * <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from Bz-ATP (<b>H</b>, <b>right panel</b>, F = 12.285, TST, <span class="html-italic">p</span> = 0.029, foot shock, <span class="html-italic">p</span> = 0.018) and muscimol (<b>I</b>, <b>right panel</b>, F = 10.170, TST, <span class="html-italic">p</span> = 0.046, foot shock, <span class="html-italic">p</span> &lt; 0.001) currents in control CA1 astrocytes (one-way ANOVA, followed by the Holm–Sidak test).</p> Full article ">Figure 4
<p>Effects of learned helplessness on Bz-ATP-induced current amplitudes in hippocampal CA1 astrocytes of rats and mice. (<b>A</b>) current responses of astrocytes to Bz-ATP (300, 1000 µM), prepared from rats which underwent no stress (control), or restraint stress, immediately or 1 day before preparing their hippocampal slices for recordings. Representative tracings. (<b>B</b>) mean ± SEM of current amplitudes in brain slices from rats unstressed (control) or stressed by TST. Current measurements were made immediately after TST (0 d), 1-day after TST (1 d), immediately after the last TST in a series of 4, applied on each consecutive day, and 1 day after a series of such stimulations. In this and all further experiments, two responses to Bz-ATP (300 µM) were averaged for further calculations. (<b>C</b>) mean ± SEM of current amplitudes in brain slices from rats unstressed or stressed by restraint. Measurements were immediately after restraint, 1-day after restraint, immediately after the last restraint in a series of 4, applied on each consecutive day, and 1 day after such a series of stimulations. (<b>D</b>) current responses of astrocytes to Bz-ATP (300, 1000 µM), prepared from rats which underwent no stress, or inescapable foot shock, immediately or 1, 3 and 7 days before preparing their hippocampal slices for recording. Representative tracings. (<b>E</b>) mean ± SEM of current amplitudes in brain slices from rats unstressed or stressed by foot shock. Current measurements were made immediately after foot shock with 1 mA or 2 mA current strength, as well as 1, 3 and 7 days after foot shock with 2 mA current strength. (<b>F</b>) mean ± SEM of current amplitudes in brain slices from mice unstressed or stressed by foot shock. Current measurements were made immediately after foot shock with 1 mA current strength, as well as 1, 3 and 7 days after foot shock. * <span class="html-italic">p</span> &lt; 0.05; statistically significant differences from the effect of Bz-ATP (300 µM) in control preparations (<b>B</b>, F = 3.571, TST (0 h), <span class="html-italic">p</span> = 0.027; <b>C</b>, F = 4.852, restraint (0 h), <span class="html-italic">p</span> = 0.008; <b>E</b>, F = 4.473, foot shock (1 d), <span class="html-italic">p</span> = 0.001; <b>F</b>, F = 3.044, foot shock 1 mA (0 h), <span class="html-italic">p</span> = 0.025, foot shock (1 day), <span class="html-italic">p</span> = 0.033). <sup>+</sup> <span class="html-italic">p</span> &lt; 0.05; statistically significant differences from the effect of Bz-ATP (1000 µM) in control preparations. (<b>B</b>, F = 5.073, TST (0 h), <span class="html-italic">p</span> = 0.005; <b>C</b>, F = 4.070, restraint (0 h), <span class="html-italic">p</span> = 0.033, 4× restraint (0 h), <span class="html-italic">p</span> = 0.030; <b>E</b>, F = 4.505, foot shock (1 d), <span class="html-italic">p</span> = 0.002; <b>F</b>, F = 4.318, foot shock (1 mA), <span class="html-italic">p</span> = 0.006, foot shock (1 d), <span class="html-italic">p</span> = 0.006). One-way ANOVA followed by the Holm–Sidak test in each case. The number of experiments is indicated throughout in each pair of columns (<b>B</b>,<b>C</b>,<b>E</b>,<b>F</b>).</p> Full article ">Figure 5
<p>Effects of learned helplessness on NMDA and Bz-ATP-induced currents in hippocampal CA1 pyramidal neurons. (<b>A</b>) action potential firing caused by gradually increasing current injection into neurons discriminated thereby from astrocytes. Representative tracings. (<b>B</b>) the holding potential of the neurons was set to −70 mV and then NMDA (100 µM), and Bz-ATP (300, 1000 µM) was applied for 10 s every 3 min. Representative tracing. (<b>C</b>) there was no change in the NMDA- or Bz-ATP-induced current amplitudes after the TST, when compared with the unstressed (control) preparations. (<b>D</b>) there was no change in the NMDA- or Bz-ATP-induced current amplitudes after foot shock when compared with the unstressed preparations. * <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the respective current response to NMDA under control conditions (<b>C</b>, F = 23.398, Bz-ATP 300, <span class="html-italic">p</span> &lt; 0.001, Bz-ATP 1000, <span class="html-italic">p</span> &lt; 0.001; <b>D</b>, F = 10.332, Bz-ATP 300, <span class="html-italic">p</span> &lt; 0.001, Bz-ATP 1000, <span class="html-italic">p</span> = 0.002). <sup>+</sup> <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the respective current response to NMDA after the TST or foot shock (<b>C</b>, F = 23.398, Bz-ATP 300, <span class="html-italic">p</span> &lt; 0.001, Bz-ATP 1000, <span class="html-italic">p</span> &lt; 0.001, <b>D</b>, F = 10.332, Bz-ATP 300, <span class="html-italic">p</span> &lt; 0.001, Bz-ATP 1000, <span class="html-italic">p</span> = 0.002). There was no statistically significant difference between any of the agonist effects between that measured under control conditions or after the TST or foot shock, respectively. One-way ANOVA, followed by the Holm-Sidak test. The number of experiments is indicated in the first set of columns.</p> Full article ">Figure 6
<p>Effects of the TST and foot shock on P2X7R-immunoreactivities (IRs), with microglial (Iba1), astroglial (GFAP), and (pro)oligodendroglial (Olig2, NG2) markers as well as their co-localization with 4’,6-diamidino-2-phenylindol (DAPI) in the hippocampal CA1 region of 3-week old rats. (<b>A</b>) fluorescence microscopic pictures of immunopositive cells and DAPI-labelled nuclei. The astrocytic marker glial fibrillary acidic protein (GFAP; green fluorescence)-IR co-stains with the red fluorescent P2X7R-IR, and the blue fluorescent DAPI. Triple-labelling of all three IRs is displayed in the right row of the picture as shown. One representative snapshot obtained from the hippocampi of 8 animals. Scale bars, 100 µm. (<b>B</b>–<b>F</b>) The stained sections were examined under 400× magnification of the microscope over the whole image; the IF was evaluated with ImageJ software and, after subtracting the background, it was expressed as a % of the whole area. (<b>B</b>,<b>C</b>) the Iba1/P2X7-IR was increased by the TST and foot shock both with and without minocycline pretreatment of mice (see <a href="#sec4-ijms-23-01904" class="html-sec">Section 4</a>). However, minocycline largely decreased the number and density of the microglial cells. (<b>E</b>,<b>F</b>) oligodendrocytes and NG2 glial cells responded to the TST and foot shock in a similar manner as microglia did. (<b>D</b>) neither the GFAP- nor the GFAP/P2X7R-IR changed after TST or foot shock stimulation in comparison with the unstressed controls. * <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the first column in a triad of columns in each panel (<b>B</b>, F = 2.635, <span class="html-italic">p</span> = 0.090; <b>C</b>, F = 9.681, TST, <span class="html-italic">p</span> = 0.002, foot shock, <span class="html-italic">p</span> = 0.005; <b>D</b>, F = 0.751, <span class="html-italic">p</span> = 0.484; <b>E</b>, F = 20.661, TST, <span class="html-italic">p</span> &lt; 0.001, foot shock, <span class="html-italic">p</span> &lt; 0.001; <b>F</b>, F = 4.258, TST, <span class="html-italic">p</span> = 0.093, foot shock, <span class="html-italic">p</span> = 0.032). <sup>+</sup> <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the second column in a triad of columns in each panel (<b>B</b>, F = 6.206, TST, <span class="html-italic">p</span> = 0.016, foot shock, <span class="html-italic">p</span> = 0.010; <b>C</b>, F = 7.581, TST, <span class="html-italic">p</span> = 0.009, foot shock, <span class="html-italic">p</span> = 0.006; <b>D</b>, F = 3.265, <span class="html-italic">p</span> = 0.058; <b>E</b>, F = 41.398, TST, <span class="html-italic">p</span> &lt; 0.001, foot shock, <span class="html-italic">p</span> &lt; 0.001). One-way ANOVA, followed by the Holm–Sidak test in each case. The number of experiments is indicated in the first set of columns.</p> Full article ">Figure 7
<p>Effects of Bz-ATP in behavioral tests after the pretreatment of mice with drugs inhibiting microglial activation or interfering with astrocytic metabolism or oligodendrocytic proliferation. The immobility time in the tail suspension test and the sucrose preference after inescapable foot shock was measured in the left and right panels of <b>A</b>–<b>C</b>, respectively. Bz-ATP (10 µM) was applied unilaterally to the lateral ventricle of the brain. (<b>A</b>) blockade of microglial activation by minocycline (see <a href="#sec4-ijms-23-01904" class="html-sec">Section 4</a>) abolishes the prolongation of the TST immobility time (left panel) and the foot shock-induced decrease in sucrose preference (right panel), normally observed in response to Bz-ATP. (<b>B</b>) selective damage to astrocytes by L-α-aminoadipate does not change the prolongation of the TST immobility time (left panel) but abolishes the foot shock-induced decrease in sucrose preference (right panel), normally observed in response to Bz-ATP. (<b>C</b>) preferential blockade of oligodendrocytic proliferation by cytosine-β-arabinoside (Ara C), has no effect either on the prolongation of the TST immobility time (left panel), or the foot shock-induced decrease in sucrose preference (right panel), normally observed in response to Bz-ATP. * <span class="html-italic">p</span> &lt; 0.05; statistically significant difference from the preceding column (<b>A</b>, <b>left panel</b>, Mino, t = 4.258, <span class="html-italic">p</span> &lt; 0.001, BzATP, t = 1.065, <span class="html-italic">p</span> = 0.305; <b>A</b>, <b>right panel</b>, Mino, t = 3.913, <span class="html-italic">p</span> = 0.002, BzATP, t = 0.253, <span class="html-italic">p</span> = 0.804; <b>B</b>, <b>left panel</b>, α-Amino, t = 4.153, <span class="html-italic">p</span> &lt; 0.001, BzATP, t = 2.109, <span class="html-italic">p</span> = 0.046; <b>B</b>, <b>right panel</b>, α-Amino, t = 1.265, <span class="html-italic">p</span> = 0.227, BzATP, t = 0.782, <span class="html-italic">p</span> = 0.447; <b>C</b>, <b>left panel</b>, Ara, t = 2.196, <span class="html-italic">p</span> = 0.045, BzATP, t = 2.707; <span class="html-italic">p</span> = 0.0170; <b>C</b>, <b>right panel</b>, Ara, t = 4.636, <span class="html-italic">p</span> &lt; 0.001, BzATP, t = 2.366, <span class="html-italic">p</span> = 0.033); Student’s <span class="html-italic">t</span>-test. The number of experiments is indicated for each pair of columns.</p> Full article ">
14 pages, 16887 KiB  
Article
The Biosynthesis and Transport of Ophiobolins in Aspergillus ustus 094102
by Jingjing Yan, Jiamin Pang, Jianjia Liang, Wulin Yu, Xuequn Liao, Ayikaimaier Aobulikasimu, Xinrui Yi, Yapeng Yin, Zixin Deng and Kui Hong
Int. J. Mol. Sci. 2022, 23(3), 1903; https://doi.org/10.3390/ijms23031903 - 8 Feb 2022
Cited by 6 | Viewed by 2604
Abstract
Ophiobolins are a group of sesterterpenoids with a 5-8-5 tricyclic skeleton. They exhibit a significant cytotoxicity and present potential medicinal prospects. However, the biosynthesis and transport mechanisms of these valuable compounds have not been fully resolved. Herein, based on a transcriptome analysis, gene [...] Read more.
Ophiobolins are a group of sesterterpenoids with a 5-8-5 tricyclic skeleton. They exhibit a significant cytotoxicity and present potential medicinal prospects. However, the biosynthesis and transport mechanisms of these valuable compounds have not been fully resolved. Herein, based on a transcriptome analysis, gene inactivation, heterologous expression and feeding experiments, we fully explain the biosynthesis pathway of ophiobolin K in Aspergillus ustus 094102, especially proved to be an unclustered oxidase OblCAu that catalyzes dehydrogenation at the site of C16 and C17 of both ophiobolin F and ophiobolin C. We also find that the intermediate ophiobolin C and final product ophiobolin K could be transported into a space between the cell wall and membrane by OblDAu to avoid the inhibiting of cell growth, which is proved by a fluorescence observation of the subcellular localization and cytotoxicity tests. This study completely resolves the biosynthesis mechanism of ophiobolins in strain A. ustus 094102. At the same time, it is revealed that the burden of strain growth caused by the excessive accumulation and toxicity of secondary metabolites is closely related to compartmentalized biosynthesis. Full article
(This article belongs to the Section Molecular Pharmacology)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Chemical structure of a part of ophiobolins. Ophiobolin F (<b>1</b>); ophiobolin K (<b>2</b>); ophiobolin C (<b>3</b>); 6-pei-ophiobolin C (<b>4</b>); 6-epi-ophiobolin N (<b>5</b>); 6-epi-ophiobolin K (<b>6</b>); 6-epi-ophiobolin G (<b>7</b>); 21,21-O-dihydro-6-epi-ophiobolin G (<b>8</b>); 21-deoxyophiobolin K (<b>9</b>).</p> Full article ">Figure 2
<p>Functional confirmation of genes in ophiobolin biosynthesis gene cluster of <span class="html-italic">A</span><span class="html-italic">spergillus ustus</span> 094102 (<span class="html-italic">obl<sub>Au</sub></span>). (<b>a</b>) Ophiobolin biosynthetic gene cluster in <span class="html-italic">A. ustus</span> 094102. (<b>b</b>) HPLC profiles of standards and culture extracts from wild-type (WT) strain and mutants at the wavelength of 234 nm. (<b>c</b>) HPLC analysis of WT strain, Δ<span class="html-italic">oblB,</span> Ao-pTAex3 and Ao-<span class="html-italic">oblA</span> at the wavelength of 200 nm. (<b>d</b>) LC–ESI-HRMS analysis of culture extracts from <span class="html-italic">Aspergillus oryzae</span> transformants. Peaks of m/z 369 (red) and m/z 387 (green) were extracted.</p> Full article ">Figure 3
<p>Confirmation of <span class="html-italic">oblC<sub>Au</sub></span> gene function. (<b>a</b>) HPLC profiles of standards and culture extracts from wild-type strain and Δ<span class="html-italic">oblC</span> mutant at the wavelength of 234 nm. (<b>b</b>) LC–ESI-HRMS analysis of culture extracts from <span class="html-italic">A. oryzae</span> transformants. Peaks of m/z 367(orange), m/z 369 (red), m/z 385 (gray) and m/z 387 (green) were extracted.</p> Full article ">Figure 4
<p>Functional characterization of OblC<sub>Au</sub>. (<b>a</b>) HPLC detection of crude extracts of <span class="html-italic">A. oryzae</span> expressing <span class="html-italic">oblA<sub>Au</sub></span>-<span class="html-italic">D<sub>Au</sub></span>. (<b>b</b>) GC-MS analysis of extracts from Ao-<span class="html-italic">oblA<sub>Au</sub></span>-<span class="html-italic">oblC<sub>Au</sub></span>. (<b>c</b>) MS spectra of m/z 358 was extracted. (<b>d</b>) MS spectra of m/z 356 was extracted.</p> Full article ">Figure 5
<p>Fluorescence observation of subcellular localization of OblA<sub>Au</sub>, OblB<sub>Au</sub>, OblC<sub>Au</sub> and OblD<sub>Au</sub>. <b>a/b/c/d–i</b>: multiple nuclei localization by DAPI; <b>a/b/c/d–ii</b>: eGFP-OblA<sub>Au</sub>, eGFP-OblB<sub>Au</sub>, eGFP-OblC<sub>Au</sub> and eGFP-OblD<sub>Au</sub> localization, respectively; <b>a/c/d–iii</b> and <b>b–v</b>: bright fields. <b>a-iv</b>: merge of <b>a–i</b>, <b>a–ii</b> and <b>a–iii</b>; <b>b–iii</b>: localization of Mito-Tracker Red CMXRos; <b>b–iv</b>: merge of <b>b–i</b>, <b>b–ii</b>, <b>b–iii</b> and <b>b–iv</b>; <b>c–iv</b>: merge of <b>c–i</b>, <b>c–ii</b> and <b>c–iii</b>; <b>d–iv</b>: merge of <b>d–i</b>, <b>d–ii</b> and <b>d–iii</b>.</p> Full article ">Scheme 1
<p>Proposed ophiobolin biosynthetic pathway. NET: non-enzymatic transformation.</p> Full article ">
21 pages, 1652 KiB  
Review
Calcium Signalling in Breast Cancer Associated Bone Pain
by Andrea Bortolin, Estrela Neto and Meriem Lamghari
Int. J. Mol. Sci. 2022, 23(3), 1902; https://doi.org/10.3390/ijms23031902 - 8 Feb 2022
Cited by 5 | Viewed by 4390
Abstract
Calcium (Ca2+) is involved as a signalling mediator in a broad variety of physiological processes. Some of the fastest responses in human body like neuronal action potential firing, to the slowest gene transcriptional regulation processes are controlled by pathways involving calcium [...] Read more.
Calcium (Ca2+) is involved as a signalling mediator in a broad variety of physiological processes. Some of the fastest responses in human body like neuronal action potential firing, to the slowest gene transcriptional regulation processes are controlled by pathways involving calcium signalling. Under pathological conditions these mechanisms are also involved in tumoral cells reprogramming, resulting in the altered expression of genes associated with cell proliferation, metastatisation and homing to the secondary metastatic site. On the other hand, calcium exerts a central function in nociception, from cues sensing in distal neurons, to signal modulation and interpretation in the central nervous system leading, in pathological conditions, to hyperalgesia, allodynia and pain chronicization. It is well known the relationship between cancer and pain when tumoral metastatic cells settle in the bones, especially in late breast cancer stage, where they alter the bone micro-environment leading to bone lesions and resulting in pain refractory to the conventional analgesic therapies. The purpose of this review is to address the Ca2+ signalling mechanisms involved in cancer cell metastatisation as well as the function of the same signalling tools in pain regulation and transmission. Finally, the possible interactions between these two cells types cohabiting the same Ca2+ rich environment will be further explored attempting to highlight new possible therapeutical targets. Full article
(This article belongs to the Special Issue Cell Signalling in Cancer: Organelles and Beyond)
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Figure 1

Figure 1
<p>Schematic overview of the main Ca<sup>2+</sup> signalling mechanisms. Calcium release from the intracellular calcium stores is mainly mediated by inositol-1,4,5-triphosphate receptors (IP<sub>3</sub>Rs) and ryanodine receptors (RYRs), sensitive to signals present both inside and outside the endoplasmic/sarcoplasmic reticulum (ER/SR). Inositol-1,4,5-triphosphate (IP<sub>3</sub>) is produced by different isoforms of phospholipase C (PLC) and binding to IP<sub>3</sub>Rs can trigger calcium release. The CD38 ADP ribosyl cyclase has both synthase (S) and hydrolase (H) functions producing cyclic ADP ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP) and has been proposed to act as a cellular metabolism sensor since ATP and NADH can inhibit the hydrolase enzymatic function. The exact way cADPr and NAADP influence calcium release is still unclear but they seem to indirectly act on both RYRs and IP<sub>3</sub>Rs. cADPr might enhance sarco(endo)plasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA) pump activity, increasing the level of Ca<sup>2+</sup> in the ER/SR, sensitising the RYRs. NAADP triggers calcium release from a channel located on lysosomes-related organelles, locally increasing the intracellular calcium concentration which can in turn result in direct stimulation of RYRs and IP<sub>3</sub>Rs or in an indirect stimulation through an increase of Ca<sup>2+</sup> concentration in ER/SR similarly to what happens with cADPr. PI, phosphatidylinositol-4,5-bisphosphate. Adapted from [<a href="#B5-ijms-23-01902" class="html-bibr">5</a>].</p> Full article ">Figure 2
<p>Calcium sources and sinks. Calcium can enter neurons from the extracellular space through many membrane channels as NMDARs and AMPARs, P2XRs, ASICs, TRP channels and VDCCs while PMCA pumps and NCX Ca<sup>2+</sup> exchanger extrude calcium to the extracellular environment to maintain the basal membrane potential and regulating the intracellular calcium concentration. The activation of menbrane Gq-coupled GPCRs and TKRs can trigger calcium release from the ER via IP<sub>3</sub>Rs and ryanodine receptors (RYRs) where again SERCA and NCX are responsible for calcium re-uptake. Mitochondrial NCXL and MCU contribute to cytoplasmic calcium signalling by releasing and removing it respectively, while all calcium binding proteins (CaBP) act as calcium buffers amongst all stimuli, dynamically releasing and removing calcium ions depending on the concentration. Through nuclear pores cytosolic calcium can enter the nuclear space where it can regulate the activity of transcription factors acting therefore on genes expression. Adapted from [<a href="#B14-ijms-23-01902" class="html-bibr">14</a>].</p> Full article ">Figure 3
<p>Vicious cycle of osteolytic metastases. Tumoural cells secrete soluble factors as interleukines (IL), tumour-necrosis factor alpha (TNF-<math display="inline"><semantics> <mi>α</mi> </semantics></math>) and parathyroid-hormone related peptide (PTHrP), promoting osteoblasts mediated receptor activator of nuclear factor kappa-B ligand (RANKL) secretion. RANKL stimulates pre-osteoclasts differentiation into fully mature osteoclasts increasing therefore the bone matrix degradation and the release of the soluble factors trapped in it, like transforming-growth factor beta (TGF-<math display="inline"><semantics> <mi>β</mi> </semantics></math>), bone morphogenic protein (BMP), fibroblasts growth factor (FGF), insuline-like growth factor (IGF) and platelet-derived growth factor (PDGF). Finally, these growth factors stimulate tumoural growth resulting therefore in a feed forward vicious cycle.</p> Full article ">Figure 4
<p>Mechanisms of extracellular acidification: Tumoural cells secrete H<sup>+</sup> ions and import HCO<sub>3</sub><sup>−</sup> to counter the intracellular acidification resulting from the increased glycolysis (Warburg Effect). This actively regulated process involves many plasma membrane proteins like HCO<sub>3</sub><sup>−</sup> transporters, co-transporters and exchangers, proton pupms and exchangers, and monocarboxylate transporters (MCT). At the same time osteoclasts actively secrete protons to degrade the minaralized bone matrix acidifying the bone extracellular environment. Bicarbonate transporters (BTs), sodium-bicarbonate co-transporters (NBCs). anions exchangers (AEs), sodium-hydrogen exchangers (NHEs), vacuolar proton-translocating ATPase (V-ATPase), carbonic anhydrase 2 (CA2), glucose transporter 1 (GLUT1). Adapted from [<a href="#B134-ijms-23-01902" class="html-bibr">134</a>].</p> Full article ">
24 pages, 6074 KiB  
Article
Inhibition of the Monocarboxylate Transporter 1 (MCT1) Promotes 3T3-L1 Adipocyte Proliferation and Enhances Insulin Sensitivity
by Tracey Bailey, Ainhoa Nieto and Patricia McDonald
Int. J. Mol. Sci. 2022, 23(3), 1901; https://doi.org/10.3390/ijms23031901 - 8 Feb 2022
Cited by 4 | Viewed by 3419
Abstract
Enlarged, hypertrophic adipocytes are less responsive to insulin and are a hallmark feature of obesity, contributing to many of the negative metabolic consequences of excess adipose tissue. Although the mechanisms remain unclear, the adipocyte size appears to be inversely correlated with insulin sensitivity [...] Read more.
Enlarged, hypertrophic adipocytes are less responsive to insulin and are a hallmark feature of obesity, contributing to many of the negative metabolic consequences of excess adipose tissue. Although the mechanisms remain unclear, the adipocyte size appears to be inversely correlated with insulin sensitivity and glucose tolerance, wherein smaller adipocytes are insulin-sensitive and larger adipocytes develop insulin resistance and exhibit an impaired glucose uptake. Thus, pharmacological strategies aimed at regulating adipocyte hypertrophy (increase in adipocyte size) in favor of promoting hyperplasia (increase in adipocyte number) have the potential to improve adipocyte insulin sensitivity and provide therapeutic benefits in the context of metabolic disorders. As white adipose tissue can metabolize large amounts of glucose to lactate, using transcriptomics and in vitro characterization we explore the functional consequences of inhibiting monocarboxylate transporter 1 (MCT1) activity in fully differentiated adipocytes. Our studies show that the pharmacological inhibition of MCT1, a key regulator of the cellular metabolism and proliferation, promotes the re-entry of mature adipocytes into the cell cycle. Furthermore, we demonstrate that inhibitor-treated adipocytes exhibit an enhanced insulin-stimulated glucose uptake as compared with untreated adipocytes, and that this outcome is dependent on the cyclin-dependent kinase 1 (CDK1) activity. In summary, we identify a mechanism though which MCT1 inhibition improves the insulin sensitivity of mature adipocytes by inducing cell cycle re-entry. These results provide the foundation for future studies investigating the role MCT1 plays in adipocyte hyperplasia, and its therapeutic potential as a drug target for obesity and metabolic disease. Full article
(This article belongs to the Section Biochemistry)
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Graphical abstract

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<p>Influence of monocarboxylate transporter 1(MCT1) inhibition on preadipocyte differentiation. (<b>A</b>) cDNA generated from 3T3-L1 wildtype (WT), 3T3-L1-shMCT1, and 3T3-L1-shScramble was subjected to RT-qPCR with MCT1 primers (**** <span class="html-italic">p</span> &lt; 0.0001). (<b>B</b>) Cell lysates were subjected to Western blot analysis using anti-MCT1 antibodies (*** <span class="html-italic">p</span> &lt; 0.001). (<b>C</b>) Quantification of MCT1 protein expression. (<b>D</b>) Oil Red O staining of 3T3-L1 (WT) cells treated with or without 1 µM AZD3965, 3T3-L1-shMCT1, and 3T3-L1-shScramble during differentiation. Scale bars depict 100 µm. (<b>E</b>) Quantification of Oil Red O staining. (<b>F</b>) Immunoblotting of hormone sensitive lipase (HSL), peroxisome proliferator-activated receptor γ (PPARγ), and fatty acid-binding protein 4 (FABP4) in 3T3-L1 (WT) and 3T3-L1-shMCT1 cells undergoing differentiation in the absence or presence of 1 µM AZD3965 at indicated time points. (<b>G</b>) Quantification of HSL, PPARγ, and FABP4 expression on day 10. (** <span class="html-italic">p</span> &lt; 0.01). Values presented represent mean ± SEM for 3 biological replicates.</p> Full article ">Figure 2
<p>MCT1 inhibition in fully differentiated adipocytes. (<b>A</b>) Oil Red O staining of differentiated 3T3-L1 (WT) cells treated with or without 1 µM AZD3965 for 24 h, 48 h, or 72 h. (<b>B</b>) Quantification of Oil Red O staining (**** <span class="html-italic">p</span> &lt; 0.0001) (*** <span class="html-italic">p</span> &lt; 0.001) (** <span class="html-italic">p</span> &lt; 0.01). (<b>C</b>) Cell viability of differentiated 3T3-L1 (WT) cells treated with or without 1 µM AZD3965 for 24 h, 48 h, or 72 h. (<b>D</b>) Relative intracellular concentrations of NADP+ and NADPH following treatment with or without 1 µM AZD3965 for 24 h, 48 h, or 72 h (* <span class="html-italic">p</span> &lt; 0.05). (<b>E</b>) Intracellular glycerol was quantified following treatment with or without 1 µM AZD3965 for 24 h, 48 h, or 72 h. (<b>F</b>) mRNA levels of HSL, PPARγ, and FABP4 in 3T3-L1 cells treated with or without 1 µM AZD3965 for 24 h (** <span class="html-italic">p</span> &lt; 0.01). (<b>G</b>) Quantifications of immunoblotting experiments (immunoblots showing relative protein expression levels of HSL, PPARγ, and FABP4 in differentiated 3T3-L1 (WT) cells treated with 1 µM AZD3965 for 0 to 72 h (immunoblots can be found in <a href="#app1-ijms-23-01901" class="html-app">Supplementary Figure S1A</a>. (<b>H</b>) Differentiated adipocytes treated with or without 1 µM AZD3965 for 72 h. Fresh media was conditioned for 24 h prior to collection and analysis with an adipokine antibody array (antibody array blots can be found in <a href="#app1-ijms-23-01901" class="html-app">Supplementary Figure S1B</a>). Quantification of the resulting chemiluminescent signals are presented in (H). Values presented represent mean ± SEM for 3 biological replicates.</p> Full article ">Figure 3
<p>Phenotypic and transcriptomic analyses of adipocytes following MCT1 inhibition. (<b>A</b>) Intracellular lactate levels of differentiated adipocytes treated with 1 µM AZD3965 for 15 min to 24 h. (**** <span class="html-italic">p</span> &lt; 0.0001) (*** <span class="html-italic">p</span> &lt; 0.001) (** <span class="html-italic">p</span> &lt; 0.01) (* <span class="html-italic">p</span> &lt; 0.05) (<b>B</b>) Intracellular pyruvate levels of differentiated adipocytes treated with 1 µM AZD3965 for 15 min to 24 hr. (<b>C</b>) Following the same treatment paradigm, intracellular NAD<sup>+</sup> and NADH were quantified and are presented as a ratio. (<b>D</b>) Significantly altered molecular functions predicted by IPA plotted against significance. (<b>E</b>) Heat map of transcript level changes in DEGs involved in cell cycle. Values presented represent mean ± SEM for 3 biological replicates.</p> Full article ">Figure 4
<p>Analysis of adipocyte proliferation. (<b>A</b>) RT-qPCR analysis of mRNA levels of indicated genes performed on cDNA of adipocytes differentiated with or without1 µM AZD3965 for 24 h (**** <span class="html-italic">p</span> &lt; 0.001), (*** <span class="html-italic">p</span> &lt; 0.001). (<b>B</b>) p-CDK1y15, total CDK1, and α-tubulin expression in cell lysates collected from differentiated 3T3-L1 treated with or without 1 µM AZD3965 for 24 h, 48 h, or 72 h. (<b>C</b>) quantification of relative p-Tyr15-CDK1 expression normalized to total CDK1 and α-tubulin (** <span class="html-italic">p</span> &lt; 0.01), (* <span class="html-italic">p</span> &lt; 0.05). (<b>D</b>) Proliferation of differentiated 3T3-L1 cells treated with indicated concentrations of AZD3965 for 24 h, 48 h, or 72 h. (<b>E</b>) Assessment of proliferation in preadipocytes, differentiated 3T3-L1, and 3T3-L1-shMCT1 cells treated with the indicated concentrations of AZD3965 for 72 h. Quantification of (<b>H</b>) where (<b>F</b>) average pixel area of Ki67 (Cy5) per nuclei was calculated and presented in microns and (<b>G</b>) fluorescence intensity of Ki67 (Cy5) per nuclei was calculated. Values presented represent mean ± SEM for 3 biological replicates. (<b>H</b>) Differentiated adipocytes treated with or without 1 µM AZD3965 for 24 h were fixed and stained, and nucleus, Ki67, and lipid droplets visualized using confocal microscopy. DAPI nucleus/DNA staining (blue); BODIPY lipid droplet staining (green); Ki67 staining (red). Scale bars depict 10 µm.</p> Full article ">Figure 5
<p>Insulin responsiveness. Glucose uptake in differentiated 3T3-L1 (WT) cells. (<b>A</b>) Glucose uptake in 3T3-L1 cells treated with 1 µM AZD3965 for 0 h (solid bars) or 24 h, 48 h, and 72 h (striped bars) were treated with (red bars) or without (blue bars) insulin for 30 min (**** <span class="html-italic">p</span> &lt; 0.001), (*** <span class="html-italic">p</span> &lt; 0.001), (** <span class="html-italic">p</span> &lt; 0.01). (<b>B</b>) Experiment performed in (<b>A</b>) repeated in the presence of 10 µM RO-3306 (checked bars). (<b>C</b>) Experiment performed as in (<b>A</b>) with 3T3-L1 shMCT1 cells. (<b>D</b>) Experiment performed as in (<b>A</b>) with 3T3-L1-shScramble cells (** <span class="html-italic">p</span> &lt; 0.01), (* <span class="html-italic">p</span> &lt; 0.05). (<b>E</b>) Differentiated adipocytes were treated with or without (basal) 1 µM AZD3965 for 24 h, 48 h, or 72 h, as indicated. Adipocytes were then treated with or with insulin for 72 h. Following the treatment time course, total triglyceride content was assessed and is presented as a fold change normalized to basal (without AZD3965 and no insulin). Values presented represent mean ± SEM for 3 biological replicates.</p> Full article ">
20 pages, 1989 KiB  
Article
Insights into the Steps of Breast Cancer–Brain Metastases Development: Tumor Cell Interactions with the Blood–Brain Barrier
by Fabienne Hamester, Christine Stürken, Ceren Saygi, Minyue Qi, Karen Legler, Christian Gorzelanny, José R. Robador, Barbara Schmalfeldt, Elena Laakmann, Volkmar Müller, Isabell Witzel and Leticia Oliveira-Ferrer
Int. J. Mol. Sci. 2022, 23(3), 1900; https://doi.org/10.3390/ijms23031900 - 8 Feb 2022
Cited by 8 | Viewed by 3500
Abstract
Brain metastases (BM) represent a growing problem for breast cancer (BC) patients. Recent studies have demonstrated a strong impact of the BC molecular subtype on the incidence of BM development. This study explores the interaction between BC cells of different molecular subtypes and [...] Read more.
Brain metastases (BM) represent a growing problem for breast cancer (BC) patients. Recent studies have demonstrated a strong impact of the BC molecular subtype on the incidence of BM development. This study explores the interaction between BC cells of different molecular subtypes and the blood–brain barrier (BBB). We compared the ability of BC cells of different molecular subtypes to overcome several steps (adhesion to the brain endothelium, disruption of the BBB, and invasion through the endothelial layer) during cerebral metastases formation, in vitro as well as in vivo. Further, the impact of these cells on the BBB was deciphered at the molecular level by transcriptome analysis of the triple-negative (TNBC) cells themselves as well as of hBMECs after cocultivation with BC cell secretomes. Compared to luminal BC cells, TNBC cells have a greater ability to influence the BBB in vitro and consequently develop BM in vivo. The brain-seeking subline and parental TNBC cells behaved similarly in terms of adhesion, whereas the first showed a stronger impact on the brain endothelium integrity and increased invasive ability. The comparative transcriptome revealed potential brain-metastatic-specific key regulators involved in the aforementioned processes, e.g., the angiogenesis-related factors TNXIP and CXCL1. In addition, the transcriptomes of the two TNBC cell lines strongly differed in certain angiogenesis-associated factors and in several genes related to cell migration and invasion. Based on the present study, we hypothesize that the tumor cell’s ability to disrupt the BBB via angiogenesis activation, together with increased cellular motility, is required for BC cells to overcome the BBB and develop brain metastases. Full article
(This article belongs to the Section Molecular Oncology)
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<p>Properties of BC cell lines during brain metastatic cascade. (<b>A</b>) BC cell (MCF-7, MDA-MB-231, and MDA-MB-231-BR) adhesion to activated (+TNF-α, 10 ng/mL for 4 h) and not activated (-TNF-α, untreated) hBMECs was analyzed under static conditions. Relative amount (to MCF-7 untreated situation = 1) of adhesive cells is shown (representative experiment; <span class="html-italic">n</span> = 5); (<b>B</b>) immunofluorescence staining of ZO-1 (green) and nuclei (DAPI, blue) in hBMECs, magnification 60×; (<b>C</b>) the effect of different BC cells on BBB integrity are shown as normalized resistance values at 4 kHz (values were set at 1 before treatment (=0 min)) measured with the ECIS system over 20 min (representative experiment; <span class="html-italic">n</span> = 3); <b>(D</b>) bar graphs displaying relative resistance values under the influence of different tumor cells (<span class="html-italic">n</span> = 3); (<b>E</b>) invasion potential of MCF-7, MDA-MB-231, and MDA-MB-231-BR through hBMECs measured in a transwell assay (<span class="html-italic">n</span> = 3). Values are means ± s.d. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.005.</p> Full article ">Figure 2
<p>BM development in an intracardiac mouse model depending on different BC cell lines. (<b>A</b>) Kaplan–Meier plot of mouse survival, intracardiac injected with 1 × 10<sup>6</sup> cells of each BC cell line (MCF-7: <span class="html-italic">n</span> = 5, MDA-MB-231: <span class="html-italic">n</span> = 2, MDA-MB-231-BR: <span class="html-italic">n</span> = 12); (<b>B</b>) representative BLI pictures of whole mice of each group 21 days after injection; (<b>C</b>) ex vivo BLI signal quantification from brains of all three test groups (MCF-7, MDA-MB-231, and MDA-MB-231-BR); (<b>D</b>) representative pictures of BLI-measured brains at the final time point of each group; (<b>E</b>) sagittal sections of mouse brains immunohistochemically stained on luciferase. From each test group, one representative picture of the whole brain and detailed metastases staining are shown. Corresponding scales are indicated above the pictures. Values are means ± s.d. ** <span class="html-italic">p</span> &lt; 0.005.</p> Full article ">Figure 3
<p>Transcriptome analysis of hBMECs after treatment with BC secretomes. (<b>A</b>) Schematic representation of the experimental design: hBMECs were treated with CM of BC cell lines MCF-7, MDA-MB-231, and their corresponding brain metastatic subline (MDA-MB-231-BR), and RNA sequencing was subsequently performed; (<b>B</b>) Venn diagrams showing RNA sequencing results (<span class="html-italic">n</span> = 4). A total of 58.611 genes were analyzed from each data set. The number of up- and downregulated and overlapping genes compared between different groups of treatment (hBMEC<sup>Ctrl</sup>, hBMEC<sup>MCF-7</sup>, hBMEC<sup>MDA-MB-231</sup>, and hBMEC<sup>MDA-MB-231-BR</sup>) are displayed; (<b>C</b>) reactome pathway analysis represents pathways enriched due to different treatments; (<b>D</b>) heatmap displaying log2FC values of the strongest differential gene expression by the influence of TNBC secretomes relative to control (untreated).</p> Full article ">Figure 4
<p>Brain metastatic-specific effects of BC cells on the brain endothelium. (<b>A</b>) Heatmap displaying log2FC values corresponding to all genes included in signaling by NTRK1, interleukin-7 signaling, interleukin-6 family signaling, and gap junction assembly pathways for hBMEC<sup>MDA-MB-231-BR</sup> as well as hBMEC<sup>MDA-MB-231</sup>, both vs. hBMEC<sup>Ctrl</sup>; (<b>B</b>) list of endothelial genes significantly deregulated by MDA-MB-231-BR in comparison to the parental cell line MDA-MB-231; (<b>C</b>) relative brain endothelial expression of <span class="html-italic">CXCL1</span> and <span class="html-italic">TXNIP</span> after 4 h of treatment with CM of MDA-MB-231 (hBMEC<sup>MDA-MB-231</sup>) and CM of MDA-MB-231-BR (hBMEC<sup>MDA-MB-231-BR</sup>); (<b>D</b>) gene concept network displaying significantly differentially expressed genes of MDA-MB-231-BR cells compared with MDA-MB-231 in enriched GO-terms cell adhesion and locomotion. Genes of interest are marked up. Values are normalized to corresponding <span class="html-italic">GAPDH</span> expression (<span class="html-italic">n</span> = 3). Values are means ± s.d. ** <span class="html-italic">p</span> &lt; 0.005.</p> Full article ">
17 pages, 59158 KiB  
Article
Proglumide Reverses Nonalcoholic Steatohepatitis by Interaction with the Farnesoid X Receptor and Altering the Microbiome
by Martha D. Gay, Hong Cao, Narayan Shivapurkar, Sivanesan Dakshanamurthy, Bhaskar Kallakury, Robin D. Tucker, John Kwagyan and Jill P. Smith
Int. J. Mol. Sci. 2022, 23(3), 1899; https://doi.org/10.3390/ijms23031899 - 8 Feb 2022
Cited by 5 | Viewed by 2820
Abstract
Nonalcoholic steatohepatitis (NASH) is associated with obesity, metabolic syndrome, and dysbiosis of the gut microbiome. Cholecystokinin (CCK) is released by saturated fats and plays an important role in bile acid secretion. CCK receptors are expressed on cholangiocytes, and CCK-B receptor expression increases in [...] Read more.
Nonalcoholic steatohepatitis (NASH) is associated with obesity, metabolic syndrome, and dysbiosis of the gut microbiome. Cholecystokinin (CCK) is released by saturated fats and plays an important role in bile acid secretion. CCK receptors are expressed on cholangiocytes, and CCK-B receptor expression increases in the livers of mice with NASH. The farnesoid X receptor (FXR) is involved in bile acid transport and is a target for novel therapeutics for NASH. The aim of this study was to examine the role of proglumide, a CCK receptor inhibitor, in a murine model of NASH and its interaction at FXR. Mice were fed a choline deficient ethionine (CDE) diet to induce NASH. Some CDE-fed mice received proglumide-treated drinking water. Blood was collected and liver tissues were examined histologically. Proglumide’s interaction at FXR was evaluated by computer modeling, a luciferase reporter assay, and tissue FXR expression. Stool microbiome was analyzed by RNA-Sequencing. CDE-fed mice developed NASH and the effect was prevented by proglumide. Computer modeling demonstrated specific binding of proglumide to FXR. Proglumide binding in the reporter assay was consistent with a partial agonist at the FXR with a mean binding affinity of 215 nM. FXR expression was significantly decreased in livers of CDE-fed mice compared to control livers, and proglumide restored FXR expression to normal levels. Proglumide therapy altered the microbiome signature by increasing beneficial and decreasing harmful bacteria. These data highlight the potential novel mechanisms by which proglumide therapy may improve NASH through interaction with the FXR and consequent alteration of the gut microbiome. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Fatty Liver and Metabolic/Malignant Events)
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<p>Effects of CDE diet and proglumide on food intake and body weight. (<b>A</b>) Weight of food in grams consumed by each group weekly. (<b>B</b>) Mean ± SEM of food intake during the study. Significantly different from control and CDE/Prog groups ** <span class="html-italic">p</span> = 0.001. (<b>C</b>) Final body weights (mean ± SEM) for each group at the termination of the study. Significantly different from control and CDE/Prog groups ** <span class="html-italic">p</span> &lt; 0.01. (<b>A</b>,<b>B</b>) modified from reference [<a href="#B21-ijms-23-01899" class="html-bibr">21</a>] with permission.</p> Full article ">Figure 2
<p>Representative H&amp;E-stained liver tissues from mouse groups. (<b>A</b>) H&amp;E section from the liver of a control mouse shows normal hepatic histology, scale 500 μm. (<b>B</b>) H&amp;E image from a mouse in the CDE/Reg group reveals steatosis, inflammation, and fibrosis, scale 500 μm. Hepatocellular carcinoma developed in this mouse after 18 weeks on the CDE diet (arrow). (<b>C</b>) H&amp;E image from the liver of a mouse fed the CDE diet for 18 weeks but also treated with proglumide (CDE/Prog) shows some steatosis but less inflammation and fibrosis compared to the liver of the CDE/Reg mouse, scale 500 μm. (<b>D</b>) Histologic section from the liver of a CDE/Reg mouse shows typical features of NASH in this perivenular area of the liver including macro- and micro-steatosis, inflammation, fibrosis and balloon degeneration, scale 200 μm. (<b>E</b>) Image from the liver of a CDE/Prog-fed mouse shows less inflammation, steatosis, and fibrosis in the liver, scale 200 μm.</p> Full article ">Figure 3
<p>Effects of CDE diet and proglumide on genes involved in proliferation and fibrosis of the liver. Mean ± SEM of mRNA expression for (<b>A</b>) <span class="html-italic">CK19</span>, (<b>B</b>) <span class="html-italic">collagen-1α</span>, (<b>C</b>) <span class="html-italic">collagen-4,</span> and (<b>D</b>) <span class="html-italic">TGBβR2</span> were all significantly increased in the livers of CDE-fed mice by qRT-PCR (blue columns). Proglumide decreased the mRNA expression of these genes while mice were on the CDE diet (red column). Significantly different from livers of control mice ** <span class="html-italic">p</span> = 0.015 and *** <span class="html-italic">p</span> &lt; 0.0005.</p> Full article ">Figure 4
<p>Expression of liver chemokines and cytokines in CDE-fed mouse livers compared to controls. (<b>A</b>) mRNA expression of several cytokines and chemokines are significantly increased in livers of CDE-fed mice with NASH compared to control mice with normal liver histology. (<b>B</b>) Chemokine and cytokine mRNA expression in livers of CDE-fed mice are reversed with proglumide therapy. (The figure was reproduced with the permission from Rightslink<sup>®</sup>, published in Cancer Prevention research [<a href="#B27-ijms-23-01899" class="html-bibr">27</a>].)</p> Full article ">Figure 5
<p>Computer modeling of proglumide and the FXR. (<b>A</b>) Magnified view of structural model proglumide complex with FXR. The FXR amino acids interacting with proglumide are shown as stick model. Hydrogen bonds are shown in broken lines (red). Proglumide carbon atoms are colored green. (<b>B</b>) Image of a bile acid superimposed with the structural model of proglumide complex with FXR. The FXR amino acids interacting with proglumide are shown as stick model. Proglumide carbon atoms are colored green. Bile acid carbon atoms are colored in magenta.</p> Full article ">Figure 6
<p>Proglumide’s interaction with FXR. (<b>A</b>) Results of proglumide interaction at the FXR as evaluated by the FXR reporter assay shows the expected sigmoidal curve of the FXR agonist, GW4064, with an EC<sub>50</sub>~312.7 nM. Proglumide reacts with the FXR receptor with a characteristic agonist curve similar to GW4064 and an EC<sub>50</sub>~214.9 nM. (<b>B</b>) Results of the reporter assay showing characteristic plot with the agonist (GW1464) compared to that of the FXR antagonist DY268. (<b>C-a</b>) FXR protein expression by Western blot is shown for NASH livers of mice on CDE/Reg diet (a) <span class="html-italic">N</span> = 10) and from livers of mice on CDE/Prog diet ((<b>C-b</b>), <span class="html-italic">N</span> = 10)). Protein expression is normalized with β-actin. (<b>D</b>) Densitometry analysis of the Western blot above for FXR protein expression is analyzed and plotted as a ratio normalized to β-actin. FXR expression is significantly increased in the mice on the CDE diet treated with proglumide compared to mice on the CDE diet with untreated water (* <span class="html-italic">p</span> = 0.03). (<b>E</b>) FXR mRNA expression as measured by qRT-PCR shows a decrease in the FXR expression in the CDE/Reg-fed mouse livers compared to FXR expression in the normal mouse liver. Restoration of the mRNA levels to control levels is shown in the livers of mice treated with proglumide (* <span class="html-italic">p</span> = 0.042).</p> Full article ">Figure 7
<p>Microbiome analysis by RNAseq in control mice compared to that of CDE/Reg and CDE/Prog mice. (<b>A</b>) The number of reads for the top 24 genera represented in stacked columns with each color representing a different genus and the height of the color represents the number of reads for that genus. The individual results for each mouse are shown for the control mice (<span class="html-italic">N</span> = 10 (<b>A-a</b>)), the CDE/Reg mice (<span class="html-italic">N</span> = 7, (<b>A-b</b>)), and the CDE/Prog mice (<span class="html-italic">N</span> = 7, (<b>A-c</b>)). (<b>B</b>) The mean number of reads in stacked columns for each group is shown with an increase in pathogenic bacteria (i.e., Bacteroides) in the CDE/Reg mice compared to controls and this level is decreased in mice in the CDE/Prog group. (<b>C</b>) A list of the represented genus by name with color coding is shown and correlates with (<b>A</b>,<b>B</b>). (<b>D</b>) The number of individual reads and mean ± SEM of three different bacteria (Alistipes, Akkermansia, and Dorea) that were specifically altered in the microbiome of mice with NASH on the CDE/Reg diet. In mice on the CDE/Prog diet, the beneficial bacteria Alistipes (** <span class="html-italic">p</span> = 0.01, *** <span class="html-italic">p</span> = 0.0005) and Akkermansia (** <span class="html-italic">p</span> = 0.025) increase in the number of reads while the pro-inflammatory bacteria Dorea decreases with proglumide therapy (** <span class="html-italic">p</span> = 0.008).</p> Full article ">
31 pages, 1369 KiB  
Review
Blood–Brain Barrier Transporters: Opportunities for Therapeutic Development in Ischemic Stroke
by Kelsy L. Nilles, Erica I. Williams, Robert D. Betterton, Thomas P. Davis and Patrick T. Ronaldson
Int. J. Mol. Sci. 2022, 23(3), 1898; https://doi.org/10.3390/ijms23031898 - 8 Feb 2022
Cited by 28 | Viewed by 4720
Abstract
Globally, stroke is a leading cause of death and long-term disability. Over the past decades, several efforts have attempted to discover new drugs or repurpose existing therapeutics to promote post-stroke neurological recovery. Preclinical stroke studies have reported successes in identifying novel neuroprotective agents; [...] Read more.
Globally, stroke is a leading cause of death and long-term disability. Over the past decades, several efforts have attempted to discover new drugs or repurpose existing therapeutics to promote post-stroke neurological recovery. Preclinical stroke studies have reported successes in identifying novel neuroprotective agents; however, none of these compounds have advanced beyond a phase III clinical trial. One reason for these failures is the lack of consideration of blood–brain barrier (BBB) transport mechanisms that can enable these drugs to achieve efficacious concentrations in ischemic brain tissue. Despite the knowledge that drugs with neuroprotective properties (i.e., statins, memantine, metformin) are substrates for endogenous BBB transporters, preclinical stroke research has not extensively studied the role of transporters in central nervous system (CNS) drug delivery. Here, we review current knowledge on specific BBB uptake transporters (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents); organic cation transporters (OCTs in humans; Octs in rodents) that can be targeted for improved neuroprotective drug delivery. Additionally, we provide state-of-the-art perspectives on how transporter pharmacology can be integrated into preclinical stroke research. Specifically, we discuss the utility of in vivo stroke models to transporter studies and considerations (i.e., species selection, co-morbid conditions) that will optimize the translational success of stroke pharmacotherapeutic experiments. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Cerebrovascular Diseases)
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<p>Anatomy of the neurovascular unit.</p> Full article ">Figure 2
<p>Localization of ATP-Binding Cassette (ABC) transporters at the Blood-Brain Barrier (BBB). ABC efflux transporters that are known to play a critical role in central nervous system (CNS) drug disposition are shown. All of these transporters function as primary active transporters and utilize ATP as an energy source to move drug molecules against their concentration gradient. Current knowledge in the field implies that P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) function in synergy to restrict blood-to-brain transport of therapeutics [<a href="#B6-ijms-23-01898" class="html-bibr">6</a>,<a href="#B69-ijms-23-01898" class="html-bibr">69</a>].</p> Full article ">Figure 3
<p>Localization of drug-transporting organic anion transporting polypeptides (OATPs/Oatps) at the Blood-Brain Barrier (BBB). The rodent Oatp isoform Oatp1a4 and its human orthologue OATP1A2 are expressed at the luminal and abluminal plasma membrane of brain microvascular endothelial cells [<a href="#B71-ijms-23-01898" class="html-bibr">71</a>,<a href="#B76-ijms-23-01898" class="html-bibr">76</a>,<a href="#B77-ijms-23-01898" class="html-bibr">77</a>,<a href="#B78-ijms-23-01898" class="html-bibr">78</a>]. The driving force for these transporters is the transmembrane concentration gradient. Therefore, they will primarily facilitate blood-to-brain uptake of transport substrates when a therapeutic is administered via the systemic circulation.</p> Full article ">Figure 4
<p>Proposed localization of organic cation transporters (OCTs/Octs) and multidrug and toxin extruders (MATEs/Mates) at the Blood-Brain Barrier (BBB). Due to their polarized nature, OCT/Oct isoforms are believed to be localized to the luminal plasma membrane in brain microvascular endothelial cells while MATE/Mate transporters are localized to the abluminal plasma membrane. These SLC transporters function as secondary active transporters that are coupled to a proton gradient to drive substrate transport in the blood-to-brain direction.</p> Full article ">
16 pages, 5315 KiB  
Article
Overexpression of MdZAT5, an C2H2-Type Zinc Finger Protein, Regulates Anthocyanin Accumulation and Salt Stress Response in Apple Calli and Arabidopsis
by Da-Ru Wang, Kuo Yang, Xun Wang, Xiao-Lu Lin, Lin Rui, Hao-Feng Liu, Dan-Dan Liu and Chun-Xiang You
Int. J. Mol. Sci. 2022, 23(3), 1897; https://doi.org/10.3390/ijms23031897 - 8 Feb 2022
Cited by 30 | Viewed by 3909
Abstract
Zinc finger proteins are widely involved and play an important role in plant growth and abiotic stress. In this research, MdZAT5, a gene encoding C2H2-type zinc finger protein, was cloned and investigated. The MdZAT5 was highly expressed in flower tissues by qRT-PCR [...] Read more.
Zinc finger proteins are widely involved and play an important role in plant growth and abiotic stress. In this research, MdZAT5, a gene encoding C2H2-type zinc finger protein, was cloned and investigated. The MdZAT5 was highly expressed in flower tissues by qRT-PCR analyses and GUS staining. Promoter analysis showed that MdZAT5 contained multiple response elements, and the expression levels of MdZAT5 were induced by various abiotic stress treatments. Overexpression of MdZAT5 in apple calli positively regulated anthocyanin accumulation by activating the expressions of anthocyanin biosynthesis-related genes. Overexpression of MdZAT5 in Arabidopsis also enhanced the accumulation of anthocyanin. In addition, MdZAT5 increased the sensitivity to salt stress in apple calli. Ectopic expression of MdZAT5 in Arabidopsis reduced the expression of salt-stress-related genes (AtNHX1 and AtABI1) and improved the sensitivity to salt stress. In conclusion, these results suggest that MdZAT5 plays a positive regulatory role in anthocyanin accumulation and negatively regulates salt resistance. Full article
(This article belongs to the Section Molecular Biology)
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Figure 1
<p>Basic information about the <span class="html-italic">MdZAT5</span> sequence. (<b>A</b>) Conserved sequence of MdZAT5 protein. The blue rectangle indicates the zinc finger domain. The numbers represent the length of amino acids. (<b>B</b>,<b>C</b>) predicted the secondary and tertiary protein structures of MdZAT5, respectively. The numbers denote the length of amino acids.</p> Full article ">Figure 2
<p>Phylogenetic tree, amino acid sequence alignment, and conserved motifs analysis. (<b>A</b>) The phylogenetic tree of ZAT5 proteins from 18 different plants. (<b>B</b>) Comparison of amino acid sequences of ZAT5 proteins from 10 different plants. The red triangle represents MdZAT5. The red box represents a conserved domain. They all have two conserved zinc finger domains and an EAR motif.</p> Full article ">Figure 3
<p>Tissue expression analysis of <span class="html-italic">MdZAT5</span>. (<b>A</b>) The relative expression level of <span class="html-italic">MdZAT5</span> in different tissues (roots, stems, leaves, flowers, and fruits) by qRT-PCR. (<b>B</b>) Tissue expression analysis of <span class="html-italic">MdZAT5</span> via GUS staining in transgenic <span class="html-italic">Arabidopsis</span>. Different lowercase letters represent a significant difference (<span class="html-italic">p</span> &lt; 0.05). Data are the mean ± SD of three independent replicates.</p> Full article ">Figure 4
<p>The expression pattern of <span class="html-italic">MdZAT5</span> under different treatment conditions. The relative expression of <span class="html-italic">MdZAT5</span> in 150 mM NaCl (<b>A</b>), 10% PEG6000 (<b>B</b>), 4 °C (<b>C</b>), and 100 μM ABA (<b>D</b>), respectively. Different lowercase letters represent a significant difference (<span class="html-italic">p</span> &lt; 0.05). Data are the mean ± SD of three independent replicates.</p> Full article ">Figure 5
<p>GUS staining in <span class="html-italic">ProMdZAT5::GUS</span> transgenic calli and <span class="html-italic">Arabidopsis</span>. The <span class="html-italic">ProMdZAT5::GUS</span> transgenic <span class="html-italic">Arabidopsis</span> (<b>A</b>) and calli (<b>B</b>), treated with 24 °C, 150 mM NaCl, 6% PEG, 4 °C, 100 μM ABA, and high light. (<b>C</b>) The GUS activity of <span class="html-italic">MdZAT5</span> of (<b>B</b>). Different lowercase letters represent a significant difference (<span class="html-italic">p</span> &lt; 0.05). Data are the mean ± SD of three independent replicates.</p> Full article ">Figure 6
<p>Overexpression of <span class="html-italic">MdZAT5</span> in apple calli and <span class="html-italic">Arabidopsis</span> promoted anthocyanin accumulation. The phenotypes (<b>A</b>) and anthocyanin content (<b>B</b>) of WT and <span class="html-italic">MdZAT5-OVX</span>. Expression analysis of <span class="html-italic">MdZAT5</span> (<b>D</b>) and genes involved in anthocyanin biosynthesis-related genes (<span class="html-italic">MdANR</span>, <span class="html-italic">MdCHI</span>, <span class="html-italic">MdCHS</span>, <span class="html-italic">MdDFR</span>, <span class="html-italic">MdF3H,</span> and <span class="html-italic">MdUFGT</span>) (<b>C</b>) in WT and <span class="html-italic">MdZAT5-OVX</span>. The phenotypes (<b>E</b>) and anthocyanin content (<b>G</b>) of Col-0 and <span class="html-italic">MdZAT5-OE</span>. (<b>F</b>) Expression analysis of <span class="html-italic">MdZAT5</span> in Col-0 and <span class="html-italic">MdZAT5-OE</span>. Different lowercase letters represent a significant difference (<span class="html-italic">p</span> &lt; 0.05). Data are the mean ± SD of three independent replicates.</p> Full article ">Figure 7
<p><span class="html-italic">MdZAT5</span> enhanced the sensitivity to salt in apple calli. (<b>A</b>) The phenotypes of WT and <span class="html-italic">MdZAT5-OVX</span> with 100 mM NaCl. Fresh weight (<b>B</b>), MDA content (<b>C</b>), relative electronic conductivity (<b>D</b>) of WT, and <span class="html-italic">MdZAT5-OVX</span>. Different lowercase letters represent a significant difference (<span class="html-italic">p</span> &lt; 0.05). Data are the mean ± SD of three independent replicates.</p> Full article ">Figure 8
<p><span class="html-italic">MdZAT5</span> enhanced the sensitivity to salt in transgenic <span class="html-italic">Arabidopsis</span>. (<b>A</b>) The phenotypes of <span class="html-italic">Arabidopsis</span> seedlings treated with MS medium, 150 mM NaCl treatment. Lateral root numbers (<b>B</b>) and primary root length (<b>C</b>) in Col-0 and <span class="html-italic">MdZAT5-OE</span>. (<b>D</b>) Phenotypes of <span class="html-italic">Arabidopsis</span> treated with 150 mM NaCl after 14 days and MDA content (<b>E</b>). The expression level of <span class="html-italic">AtNHX1</span> (<b>F</b>) and <span class="html-italic">AtABI1</span> (<b>G</b>) in Col-0 and <span class="html-italic">MdZAT5-OE</span>. Different lowercase letters represent a significant difference (<span class="html-italic">p</span> &lt; 0.05). Data are the mean ± SD of three independent replicates.</p> Full article ">Figure 9
<p>Ectopic expression of <span class="html-italic">MdZAT5</span> enhances ROS buildup under salt treatment. DAB staining for H<sub>2</sub>O<sub>2</sub> (<b>A</b>) and NBT staining for O<sub>2</sub><sup>−</sup> (<b>B</b>) in Col-0 and <span class="html-italic">MdZAT5-OE Arabidopsis</span> leaves after 14 days of salt treatment. H<sub>2</sub>O<sub>2</sub> content (<b>C</b>) and O<sub>2</sub><sup>−</sup> generation rates (<b>D</b>) in Col-0 and <span class="html-italic">MdZAT5-OE Arabidopsis</span> after 14 days of salt treatment. Different lowercase letters represent a significant difference (<span class="html-italic">p</span> &lt; 0.05). Data are the mean ± SD of three independent replicates.</p> Full article ">
14 pages, 14581 KiB  
Article
Exposure to b-LED Light While Exerting Antimicrobial Activity on Gram-Negative and -Positive Bacteria Promotes Transient EMT-like Changes and Growth Arrest in Keratinocytes
by Michela Terri, Nicoletta Mancianti, Flavia Trionfetti, Bruno Casciaro, Valeria de Turris, Giammarco Raponi, Giulio Bontempi, Claudia Montaldo, Alessandro Domenici, Paolo Menè, Maria Luisa Mangoni and Raffaele Strippoli
Int. J. Mol. Sci. 2022, 23(3), 1896; https://doi.org/10.3390/ijms23031896 - 8 Feb 2022
Cited by 2 | Viewed by 2460
Abstract
While blue LED (b-LED) light is increasingly being studied for its cytotoxic activity towards bacteria in therapy of skin-related infections, its effects on eukaryotic cells plasticity are less well characterized. Moreover, since different protocols are often used, comparing the effect of b-LED towards [...] Read more.
While blue LED (b-LED) light is increasingly being studied for its cytotoxic activity towards bacteria in therapy of skin-related infections, its effects on eukaryotic cells plasticity are less well characterized. Moreover, since different protocols are often used, comparing the effect of b-LED towards both microorganisms and epithelial surfaces may be difficult. The aim of this study was to analyze, in the same experimental setting, both the bactericidal activity and the effects on human keratinocytes. Exposure to b-LED induced an intense cytocidal activity against Gram-positive (i.e, Staphylococcus aureus) and Gram-negative (i.e., Pseudomonas aeruginosa) bacteria associated with catheter-related infections. Treatment with b-LED of a human keratinocyte cell line induced a transient cell cycle arrest. At the molecular level, exposure to b-LED induced a transient downregulation of Cyclin D1 and an upregulation of p21, but not signs of apoptosis. Interestingly, a transient induction of phosphor-histone γ-H2Ax, which is associated with genotoxic damages, was observed. At the same time, keratinocytes underwent a transient epithelial to mesenchymal transition (EMT)-like phenotype, characterized by E-cadherin downregulation and SNAIL/SLUG induction. As a functional readout of EMT induction, a scratch assay was performed. Surprisingly, b-LED treatment provoked a delay in the scratch closure. In conclusion, we demonstrated that b-LED microbicidal activity is associated with complex responses in keratinocytes that certainly deserve further analysis. Full article
(This article belongs to the Special Issue Advances in Radiation Toxicity)
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Figure 1
<p>Number of viable cells of <span class="html-italic">S. aureus</span> ATCC 25923, <span class="html-italic">P. aeruginosa</span> ATCC 27853 and the clinical isolate <span class="html-italic">P. aeruginosa</span> 19595, after 4 h of treatment with b-LED (blue bars). Controls were untreated samples at time 0 (black bars) and 4 h (grey bars). The data reported are the mean ± standard deviation (SD) of three independent experiments. The level of statistical significance between samples was determined by the multiple <span class="html-italic">t</span> test (GraphPad Prism v.8.0.1, GraphPad Software, La Jolla, CA, USA), and indicated as follows: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001 and ns, not significant.</p> Full article ">Figure 2
<p>Exposure to b-LED causes a transient block in cell proliferation. (<b>A</b>,<b>B</b>) Cell viability test of HaCaT cells exposed to b-LED. HaCaT cells were pretreated with calcein AM (2 µM) and exposed to b-LED for 4 h. Cells were then analyzed 4, 24 and 48 h after beginning of exposure to b-LED. Cells were fixed and stained with DAPI. Images were acquired by fluorescence microscopy. (<b>A</b>) Calcein AM/DAPI-positive cells; (<b>B</b>) calcein AM-positive cells. Bars represent the mean ± SEM from two independent experiments. Twelve fields containing at least 30 nuclei per field were analyzed. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01. (<b>C</b>) Western blot showing the expression of cleaved caspase 3 in HaCaT cells exposed to b-LED for 4 h. Cells were analyzed 4, 24 and 48 h after beginning of exposure to b-LED. Western blot analysis was performed on total lysates. Tubulin was detected as a loading control. Data are representative of three independent experiments. (<b>D</b>) Cell proliferation assay of HaCaT cells exposed to b-LED. HaCaT cells were left untreated or were exposed to b-LED for 4 h and were then evaluated for cell proliferation assays 4, 24 and 48 h after beginning of exposure to b-LED using a SpectraMax 13 microplate reader. The experiment was performed in triplicate and was repeated twice. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; ns, not significant. (<b>E</b>–<b>H</b>) HaCaT cells exposed to b-LED for 4 h and then were analyzed 4 and 24 h after beginning of exposure. (<b>E</b>) Quantitative RT-PCR expression analysis of p21 in. (<b>F</b>) Western blot expression of p21 in HaCaT cells. (<b>G</b>) quantitative RT-PCR expression analysis of Cyclin D1 in HaCaT cells. (<b>H</b>) Western blot expression of Cyclin D1 in HaCaT cells. Quantitative RT-PCR was performed on total RNA. L34 mRNA levels were used for normalization. Results are expressed in terms of fold change; bars represent the mean ± SEM of duplicate determinations in four independent experiments. * <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.001 and ns, not significant. Western blot analysis was performed on total lysates. Tubulin was detected as a loading control. Data are representative of three independent experiments.</p> Full article ">Figure 3
<p>Exposure to b-LED causes a transient induction of γ -H2AX histone in HaCaT cells. (<b>A</b>) WB showing the expression of γ-H2AX histone (phospho S139) in HaCaT cells exposed to b-LED for 4 h and then analyzed at the indicated times. Etoposide (10 µM) was used as positive control. Western blot analysis was performed on total lysates. Tubulin was detected as a loading control. (<b>B</b>) Densitometric quantification of the experiment shown above. Results are expressed in terms of fold change. (<b>C</b>) Confocal immunofluorescence showing the expression of γ-H2AX histone (phospho S139) in cells stimulated as in (<b>A</b>). (<b>D</b>) quantification of the experiment shown in (<b>C</b>). At least 120 nuclei were quantified from 2 independent experiments. Bars represent the mean ± SEM of duplicate determinations in four independent experiments. * <span class="html-italic">p</span> &lt; 0.05, **** <span class="html-italic">p</span> &lt; 0.0001 and ns, not significant.</p> Full article ">Figure 4
<p>Exposure to b-LED causes a transient EMT phenotype with delay in wound closure. (<b>A</b>) Quantitative RT-PCR expression analysis of SNAIL, SLUG and E-cadherin in HaCaT cells exposed to b-LED for 4 h and then were analyzed 4 and 24 h after beginning of exposure. Quantitative RT-PCR was performed on total RNA. L34 mRNA levels were used for normalization. Results are expressed in term of fold change bars represent the mean ± SEM of duplicate determinations in at least five independent experiments. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01. (<b>B</b>) Western blot showing the expression of SLUG and E-cadherin in HaCaT cells exposed to b-LED for 4 h. Cells were analyzed 4, 24 and 48 h after the beginning of exposure to b-LED. Western blot analysis was performed on total lysates. Tubulin was detected as a loading control. Data are representative of three independent experiments. (<b>C</b>) Effect of exposure to b-LED on wound closure. HaCaT cells were left to reach 100% confluency in Ibidi μ-Dish plates. MCs were exposed to b-LED for 4 h. Then, 24 or 48 h after the beginning of exposure the insert was removed and after 18 h cells were fixed and stained with phalloidin (green) or Hoechst33342 (blue) to stain nuclei. Representative experiment is shown of three performed. (<b>D</b>) Quantification of the experiment shown in (<b>C</b>). Bars represent the mean ± SEM from three independent experiments. Two corresponding fields at time 0 and 24 h per experiments were measured. *** <span class="html-italic">p</span> &lt; 0.001, ns, not significant.</p> Full article ">
14 pages, 3159 KiB  
Article
Synergistic Effect of Co-Delivering Ciprofloxacin and Tetracycline Hydrochloride for Promoted Wound Healing by Utilizing Coaxial PCL/Gelatin Nanofiber Membrane
by Mengxia Lin, Yuan Liu, Junwei Gao, Donghui Wang, Dan Xia, Chunyong Liang, Ning Li and Ruodan Xu
Int. J. Mol. Sci. 2022, 23(3), 1895; https://doi.org/10.3390/ijms23031895 - 8 Feb 2022
Cited by 39 | Viewed by 3834
Abstract
Combining multiple drugs or biologically active substances for wound healing could not only resist the formation of multidrug resistant pathogens, but also achieve better therapeutic effects. Herein, the hydrophobic fluoroquinolone antibiotic ciprofloxacin (CIP) and the hydrophilic broad-spectrum antibiotic tetracycline hydrochloride (TH) were introduced [...] Read more.
Combining multiple drugs or biologically active substances for wound healing could not only resist the formation of multidrug resistant pathogens, but also achieve better therapeutic effects. Herein, the hydrophobic fluoroquinolone antibiotic ciprofloxacin (CIP) and the hydrophilic broad-spectrum antibiotic tetracycline hydrochloride (TH) were introduced into the coaxial polycaprolactone/gelatin (PCL/GEL) nanofiber mat with CIP loaded into the PCL (core layer) and TH loaded into the GEL (shell layer), developing antibacterial wound dressing with the co-delivering of the two antibiotics (PCL-CIP/GEL-TH). The nanostructure, physical properties, drug release, antibacterial property, and in vitro cytotoxicity were investigated accordingly. The results revealed that the CIP shows a long-lasting release of five days, reaching the releasing rate of 80.71%, while the cumulative drug release of TH reached 83.51% with a rapid release behavior of 12 h. The in vitro antibacterial activity demonstrated that the coaxial nanofiber mesh possesses strong antibacterial activity against E. coli and S. aureus. In addition, the coaxial mats showed superior biocompatibility toward human skin fibroblast cells (hSFCs). This study indicates that the developed PCL-CIP/GEL-TH nanofiber membranes hold enormous potential as wound dressing materials. Full article
(This article belongs to the Special Issue Biocompatible Materials for the Application to Medical Diagnoses and Therapeutics)
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Figure 1

Figure 1
<p>The preparation of coaxial nanofibers with co-delivering of CIP and TH antibiotics.</p> Full article ">Figure 2
<p>(<b>a</b>–<b>d</b>) SEM images of different coaxial nanofiber membranes; (<b>e</b>–<b>h</b>) TEM images of different core–shell nanofibers; (<b>i</b>–<b>l</b>) diameter distribution of different nanofibers; (<b>m</b>) EDS mapping of the PCL-CIP/GEL-TH nanofibers.</p> Full article ">Figure 3
<p>Physico-chemical characterization of different nanofibers. (<b>a</b>) FTIR spectra; (<b>b</b>) stress–strain curves; (<b>c</b>) water contact angles; (<b>d</b>) swelling behaviors. (<span class="html-italic">n</span> = 3); *—<span class="html-italic">p</span> &lt; 0.05; **—<span class="html-italic">p</span> &lt; 0.01; ***—<span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>In vitro drug release of CIP and TH at different periods. (<b>a</b>) 24 h; (<b>b</b>) 120 h.</p> Full article ">Figure 5
<p>Antibacterial activity of coaxial nanofibers against <span class="html-italic">E. coli</span> and <span class="html-italic">S. aureus</span>. (<b>a</b>) Inhibition zone assay; (<b>b</b>,<b>c</b>) bar diagrams of antibacterial efficacy of different membranes (<span class="html-italic">n</span> = 3); (<b>d</b>) SEM images of the morphologies of <span class="html-italic">E. coli</span> and <span class="html-italic">S. aureus</span> on different nanofibers; (<b>e</b>,<b>f</b>) the long-lasting antibacterial effects evaluation of different nanofibers by CCK-8 assay (<span class="html-italic">n</span> = 3). *—<span class="html-italic">p</span> &lt; 0.05; **—<span class="html-italic">p</span> &lt; 0.01; ***—<span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p>(<b>a</b>–<b>d</b>) The fluorescent images and (<b>e</b>) the viability of hSFCs cultured for 24 h on different samples.</p> Full article ">
21 pages, 1826 KiB  
Review
The Potential Effect of Lidocaine, Ropivacaine, Levobupivacaine and Morphine on Breast Cancer Pre-Clinical Models: A Systematic Review
by Ana Catarina Matos, Inês Alexandra Marques, Ana Salomé Pires, Ana Valentim, Ana Margarida Abrantes and Maria Filomena Botelho
Int. J. Mol. Sci. 2022, 23(3), 1894; https://doi.org/10.3390/ijms23031894 - 8 Feb 2022
Cited by 8 | Viewed by 4313
Abstract
Breast cancer (BC) is one of the most common types of cancer and the second leading cause of death in women. Local anaesthetics (LAs) and opioids have been shown to influence cancer progression and metastasis formation in several pre-clinical studies. However, their effects [...] Read more.
Breast cancer (BC) is one of the most common types of cancer and the second leading cause of death in women. Local anaesthetics (LAs) and opioids have been shown to influence cancer progression and metastasis formation in several pre-clinical studies. However, their effects do not seem to promote consensus. A systematic review was conducted using the databases Medline (via PubMed), Scopus, and Web of Science (2010 to December 2021). Search terms included “lidocaine”, “ropivacaine”, “levobupivacaine”, “morphine”, “methadone”, “breast cancer”, “breast carcinoma” and “breast neoplasms” in diverse combinations. The search yielded a total of 784 abstracts for initial review, 23 of which met the inclusion criteria. Here we summarise recent studies on the effect of analgesics and LAs on BC cell lines and animal models and in combination with other treatment regimens. The results suggest that local anaesthetics have anti-tumorigenic properties, hence their clinical application holds therapeutic potential. Regarding morphine, the findings are conflicting, but this opioid appears to be a tumour-promoting agent. Methadone-related results are scarce. Additional research is clearly required to further study the mechanisms underlying the controversial effects of each analgesic or LA to establish the implications upon the outcome and prognosis of BC patients’ treatment. Full article
(This article belongs to the Special Issue Challenges of Radiation Biology for Cancer Management)
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Figure 1
<p>PRISMA flow diagram of the study research methodology in the literature. Adapted from PRISMA Group [<a href="#B27-ijms-23-01894" class="html-bibr">27</a>]. * Clinical studies, reviews, meta-analyses, editorials, opinion pieces, and articles not providing the effect of pain medicine on therapeutic response to BC, articles written in languages other than English, written before 2010 or unavailable as complete articles were excluded.</p> Full article ">Figure 2
<p>Possible mechanisms underlying lidocaine’s anti-cancer effects. Ca<sup>2+</sup>—Calcium; CXCR4—C-X-C Chemokine Receptor type 4; DNA—Deoxyribonucleic acid; FGF9—Fibroblast Growth Factor 9; MMP-2—Matrix Metallopeptidase 2; MMP-9—Matrix Metallopeptidase 9; PARP—Poly (ADP-ribose) Polymerase; TRPM7—Transient receptor potential cation channel subfamily M member 7; TRPV6—Transient receptor potential cation channel subfamily V member 6.</p> Full article ">Figure 3
<p>Possible mechanisms underlying ropivacaine’s anti-cancer effects. ATP—Adenosine triphosphate; DNA—Deoxyribonucleic acid; Gsk3β—Glycogen synthase kinase 3 beta; miR-27b3p—microRNA-27b3p; PI3K-Akt—Phosphatidylinositol 3-kinase—Protein kinase B; RhoA—Ras homolog family member A; YAP—Yes-associated protein.</p> Full article ">Figure 4
<p>Possible mechanisms underlying levobupivacaine’s anti-cancer effects. DNA—Deoxyribonucleic acid; Gsk3β—Glycogen synthase kinase 3 beta; mTOR—Mammalian target of rapamycin; PI3K-Akt—Phosphatidylinositol 3-kinase—Protein kinase B; RhoA—Ras homolog family member A.</p> Full article ">Figure 5
<p>Possible mechanisms underlying morphine’s effects on breast cancer cells. EMT—Epithelial-mesenchymal transition; MOR—μ-opioid receptors; NET-1—Neuroepithelial Cell Transforming 1; PI3K-Akt—Phosphatidylinositol 3-kinase—Protein kinase B; RhoA—Ras homolog family member A; SP—Substance P; TSP-1—Thrombospondin-1.</p> Full article ">
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