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Volume 22
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Int. J. Mol. Sci., Volume 22, Issue 7 (April-1 2021) – 512 articles

Cover Story (view full-size image): Alveolar macrophages (AMs) are the most abundant cell type in the bronchioalveolar space. With every breath, the human organism is confronted with a variety of pathogens, particles, or molecules. Here, AMs sense these various invaders and, thus, represent a crucial cell type within innate pulmonary immunity. AMs have been shown to be a highly plastic cell type, able to orchestrate not only pulmonary host response but also to maintain lung tissue homeostasis. Thus, dysfunction and/or impaired development of AMs can lead to various lung diseases, ranging from increased susceptibility to pathogens, congenital lung diseases or progressive lung disorders. Thus, insights into the role of AMs have laid the foundation for new drugs and even macrophage-based immunotherapies. View this paper.
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14 pages, 1316 KiB  
Review
Granulocytes and Cells of Granulocyte Origin—The Relevant Players in Colorectal Cancer
by Izabela Siemińska, Ewa Poljańska and Jarek Baran
Int. J. Mol. Sci. 2021, 22(7), 3801; https://doi.org/10.3390/ijms22073801 - 6 Apr 2021
Cited by 13 | Viewed by 8361
Abstract
Colorectal cancer (CRC) is one of the most common malignancy and cause of cancer death worldwide, and it still remains a therapeutic challenge for western medicine. There is strong evidence that, in addition to genetic predispositions, environmental factors have also a substantial impact [...] Read more.
Colorectal cancer (CRC) is one of the most common malignancy and cause of cancer death worldwide, and it still remains a therapeutic challenge for western medicine. There is strong evidence that, in addition to genetic predispositions, environmental factors have also a substantial impact in CRC development. The risk of CRC is attributed, among others to dietary habits, alcohol consumption, whereas physical activity, food containing dietary fiber, dairy products, and calcium supplements have a protective effect. Despite progress in the available therapies, surgery remains a basic treatment option for CRC. Implementation of additional methods of treatment such as chemo- and/or targeted immunotherapy, improved survival rates, however, the results are still far from satisfactory. One of the reasons may be the lack of deeper understanding of the interactions between the tumor and different types of cells, including tumor infiltrating granulocytes. While the role of neutrophils is quite well explored in many cancers, role of eosinophils and basophils is often underestimated. As part of this review, we focused on the function of different granulocyte subsets in CRC, emphasizing the beneficial role of eosinophils and basophils, as well as dichotomic mode of neutrophils action. In addition, we addressed the current knowledge on cells of granulocyte origin, specifically granulocytic myeloid derived suppressor cells (Gr-MDSCs) and their role in development and progression of CRC. Full article
(This article belongs to the Special Issue Tumor Microenvironment: Interactions and Therapeutic Response)
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<p>Pro-(pink) and anti-tumorigenic (greenish) activities of granulocyte populations in CRC.</p> Full article ">Figure 2
<p>Granulocytes role in CRC. Eosinophils have an anti-tumor role in CRC and their infiltration into the tumor is considered as a positive prognostic marker. They are recruited to the tumor site by CCL11, CCL24, and IL-25 where they are activated by IFNγ to secrete ECP, EDN, EPO, TNF, granzyme A, and produce ROS, all with direct cytotoxicity against cancer cells. Tumoricidal effect of eosinophils depends on cell-to-cell contact and IL-18, which in the autocrine loop enhances the LFA-1—ICAM-1 mediated eosinophil adhesion to the target cells. Basophils are also beneficial in CRC and their role is related to the secretion of the granules content, including histamine and proinflammatory cytokines—e.g., TNFα, Il-6, IL-1β—augmenting inflammatory reaction, recruiting cancer-specific CD8+ T cells (CCL3 and CCL4) into the tumor and enhancing cancer cell apoptosis. Neutrophils possess both anti-tumor and pro-tumorigenic activities. Under the influence of CXCL8 (IL-8) they are recruited from peripheral blood to the tumor site and prompted for NETs formation, which support tumor spread and liver metastasis. Additionally, neutrophils are recruited into the tumor by CCL15-CCR1 pathway, where CCR1+ TANs with ARG-1 and MMP-9 activity are associated with lung metastasis. In tumors, TANs are involved in local immunosuppression through prostaglandin PGE2 and TGFβ secretion, supporting the tumor growth. Unlike N2, N1 TANs possess anti-tumorigenic functions. Their increased infiltration was shown in the early stage of CRC, where they affect the colon microbiome and hamper IL-17 dependent tumor development. Moreover, activated by type I IFNs N1 TANs inhibit angiogenesis and effectively eliminate tumor cells via antibody dependent cell-mediated cytotoxicity (ADCC) or phagocytosis. Gr-MDSCs are potent inhibitors of T cell proliferation and anti-tumor response. Their mechanisms involve ARG-1 activity, ROS production, an increased fatty acid uptake and activated fatty acid oxidation (FAO). Moreover, Gr-MDSCs produce high amounts of IL-6 for sustained inflammation in colon epithelium, thereby promoting CRC progression. Pro-tumorigenic action of Gr-MDSCs also includes secretion of exosomes containing S100A9, which additionally stimulate the tumor growth.</p> Full article ">
39 pages, 1677 KiB  
Review
Antibacterial Titanium Implants Biofunctionalized by Plasma Electrolytic Oxidation with Silver, Zinc, and Copper: A Systematic Review
by Ingmar A. J. van Hengel, Melissa W. A. M. Tierolf, Lidy E. Fratila-Apachitei, Iulian Apachitei and Amir A. Zadpoor
Int. J. Mol. Sci. 2021, 22(7), 3800; https://doi.org/10.3390/ijms22073800 - 6 Apr 2021
Cited by 44 | Viewed by 5777
Abstract
Patients receiving orthopedic implants are at risk of implant-associated infections (IAI). A growing number of antibiotic-resistant bacteria threaten to hamper the treatment of IAI. The focus has, therefore, shifted towards the development of implants with intrinsic antibacterial activity to prevent the occurrence of [...] Read more.
Patients receiving orthopedic implants are at risk of implant-associated infections (IAI). A growing number of antibiotic-resistant bacteria threaten to hamper the treatment of IAI. The focus has, therefore, shifted towards the development of implants with intrinsic antibacterial activity to prevent the occurrence of infection. The use of Ag, Cu, and Zn has gained momentum as these elements display strong antibacterial behavior and target a wide spectrum of bacteria. In order to incorporate these elements into the surface of titanium-based bone implants, plasma electrolytic oxidation (PEO) has been widely investigated as a single-step process that can biofunctionalize these (highly porous) implant surfaces. Here, we present a systematic review of the studies published between 2009 until 2020 on the biomaterial properties, antibacterial behavior, and biocompatibility of titanium implants biofunctionalized by PEO using Ag, Cu, and Zn. We observed that 100% of surfaces bearing Ag (Ag-surfaces), 93% of surfaces bearing Cu (Cu-surfaces), 73% of surfaces bearing Zn (Zn-surfaces), and 100% of surfaces combining Ag, Cu, and Zn resulted in a significant (i.e., >50%) reduction of bacterial load, while 13% of Ag-surfaces, 10% of Cu-surfaces, and none of Zn or combined Ag, Cu, and Zn surfaces reported cytotoxicity against osteoblasts, stem cells, and immune cells. A majority of the studies investigated the antibacterial activity against S. aureus. Important areas for future research include the biofunctionalization of additively manufactured porous implants and surfaces combining Ag, Cu, and Zn. Furthermore, the antibacterial activity of such implants should be determined in assays focused on prevention, rather than the treatment of IAIs. These implants should be tested using appropriate in vivo bone infection models capable of assessing whether titanium implants biofunctionalized by PEO with Ag, Cu, and Zn can contribute to protect patients against IAI. Full article
(This article belongs to the Special Issue Antimicrobial Materials with Medical Applications)
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<p>A graphical presentation of the outline of this systematic review.</p> Full article ">Figure 2
<p>An overview of the (<b>A</b>) biomaterial, (<b>B</b>) antibacterial, and (<b>C</b>) cytocompatibility specifications of the studies included in this systematic review of the literature. Combi: combination of Ag, Cu, and/or Zn, HA: hydroxyapatite, NR: not reported.</p> Full article ">Figure 3
<p>(<b>A</b>) A schematic drawing of the plasma electrolytic oxidation (PEO) setup with a cathode and an anode (implant). (<b>B</b>) During PEO processing, initially the titanium oxide layer grew outwards. After dielectric breakdown, plasma discharge occurred at the surface, resulting in a highly porous structure.</p> Full article ">Figure 4
<p>(<b>A</b>) SEM images of the typical surface morphology of titanium implants after PEO processing. (<b>B</b>) EDS analysis of the implant surface to characterize its chemical composition with spectrum of Cu (blue) and Ag (red) nanoparticles.</p> Full article ">Figure 5
<p>The relation between the antibacterial activity, cytocompatibility, and surface content for titanium surfaces biofunctionalized by PEO with Ag, Cu, or Zn. The reported antibacterial activity as a function of surface content and cytocompatibility is depicted by the blue dots. The green, red, and yellow projections enable a comparison between the parameters. Cytocompatibility is depicted as cytotoxicity (−), no effect (0), or enhanced cytocompatibility (+).</p> Full article ">
12 pages, 925 KiB  
Review
Altered Metabolic Flexibility in Inherited Metabolic Diseases of Mitochondrial Fatty Acid Metabolism
by Sara Tucci, Khaled Ibrahim Alatibi and Zeinab Wehbe
Int. J. Mol. Sci. 2021, 22(7), 3799; https://doi.org/10.3390/ijms22073799 - 6 Apr 2021
Cited by 14 | Viewed by 5337
Abstract
In general, metabolic flexibility refers to an organism’s capacity to adapt to metabolic changes due to differing energy demands. The aim of this work is to summarize and discuss recent findings regarding variables that modulate energy regulation in two different pathways of mitochondrial [...] Read more.
In general, metabolic flexibility refers to an organism’s capacity to adapt to metabolic changes due to differing energy demands. The aim of this work is to summarize and discuss recent findings regarding variables that modulate energy regulation in two different pathways of mitochondrial fatty metabolism: ?-oxidation and fatty acid biosynthesis. We focus specifically on two diseases: very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and malonyl-CoA synthetase deficiency (acyl-CoA synthetase family member 3 (ACSF3)) deficiency, which are both characterized by alterations in metabolic flexibility. On the one hand, in a mouse model of VLCAD-deficient (VLCAD?/?) mice, the white skeletal muscle undergoes metabolic and morphologic transdifferentiation towards glycolytic muscle fiber types via the up-regulation of mitochondrial fatty acid biosynthesis (mtFAS). On the other hand, in ACSF3-deficient patients, fibroblasts show impaired mitochondrial respiration, reduced lipoylation, and reduced glycolytic flux, which are compensated for by an increased ?-oxidation rate and the use of anaplerotic amino acids to address the energy needs. Here, we discuss a possible co-regulation by mtFAS and ?-oxidation in the maintenance of energy homeostasis. Full article
(This article belongs to the Special Issue Energy Metabolism in Health and Disease)
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<p>Schematic representation of the mitochondrial fatty acid β-oxidation. Long-chain fatty acids need to be transported into the mitochondria by an active transport system. Because fatty acids in the cytoplasm are activated as acyl-CoA esters, they must be bound to carnitine for transport across the mitochondrial membranes. In contrast, medium- and short-chain fatty acids do not need an active transport system and can easily undergo β-oxidation. This pathway consists of four cyclic steps. Each step shortens the acyl-CoA by two carbons. The electrons generated by each oxidative reaction are transferred to FAD or NAD+ and are forwarded to ubiquinone or the respiratory chain, respectively. (1) Acyl-CoA dehydrogenase (very long-, long-, medium-, and short-chain acyl-CoA dehydrogenase, i.e., VLCAD, long-chain acetyl-CoA dehydrogenase (LCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and SCAD, respectively).; (2) 2,3-Enoyl-CoA hydratase (LCEH and SCEH). (3) 3-Hydroxyacyl-CoA dehydrogenases (LCHAD and SCHAD). (4) β-Ketoacyl-CoA thiolase (LCKAT, MCKAT, and SCKAT). MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane.</p> Full article ">Figure 2
<p>The human mitochondrial fatty acid biosynthesis (mtFAS) pathway. The mFAS pathway is shown in blue, and the lipoic acid biosynthesis pathway is shown in green. The end product of mFAS is octanoyl-ACP, which is used as a substrate for the biosynthesis of lipoic acid. Acyl-ACP actively participates in oxidative phosphorylation (OXPHOS) assembly. CII, complex II of the electron transport chain; LA, lipoic acid; ACSF3, acyl-CoA synthetase family member 3; MCAT, malonyl CoA-acyl carrier protein transacylase; OXSM, 3-oxoacyl-ACP synthase; CBR4, carbonyl reductase 4; HSD17β, 3-ketoacyl-ACP reductase alpha subunit; HTD2, hydroxyacyl-thioester dehydratase type 2; MECR, mitochondrial 2-enoyl thioester reductase; LIAS, lipoic acid synthetase; Lipt1, lipoyltransferase 1; DLAT, dihydrolipoamide S-acetyltransferase; PDHX, pyruvate dehydrogenase protein X component; DLST, dihydrolipoyllysine residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex; DBT, dihydrolipoyl acyltransferase.</p> Full article ">
18 pages, 745 KiB  
Review
Cannabidiol and the Canonical WNT/?-Catenin Pathway in Glaucoma
by Alexandre Vallée, Yves Lecarpentier and Jean-Noël Vallée
Int. J. Mol. Sci. 2021, 22(7), 3798; https://doi.org/10.3390/ijms22073798 - 6 Apr 2021
Cited by 25 | Viewed by 5426
Abstract
Glaucoma is a progressive neurodegenerative disease which constitutes the main frequent cause of irreversible blindness. Recent findings have shown that oxidative stress, inflammation and glutamatergic pathway play key roles in the causes of glaucoma. Recent studies have shown a down regulation of the [...] Read more.
Glaucoma is a progressive neurodegenerative disease which constitutes the main frequent cause of irreversible blindness. Recent findings have shown that oxidative stress, inflammation and glutamatergic pathway play key roles in the causes of glaucoma. Recent studies have shown a down regulation of the WNT/?-catenin pathway in glaucoma, associated with overactivation of the GSK-3? signaling. WNT/?-catenin pathway is mainly associated with oxidative stress, inflammation and glutamatergic pathway. Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid derived from Cannabis sativa plant which possesses many therapeutic properties across a range of neuropsychiatric disorders. Since few years, CBD presents an increased interest as a possible drug in anxiolytic disorders. CBD administration is associated with increase of the WNT/?-catenin pathway and decrease of the GSK-3? activity. CBD has a lower affinity for CB1 but can act through other signaling in glaucoma, including the WNT/?-catenin pathway. CBD downregulates GSK3-? activity, an inhibitor of WNT/?-catenin pathway. Moreover, CBD was reported to suppress pro-inflammatory signaling and neuroinflammation, oxidative stress and glutamatergic pathway. Thus, this review focuses on the potential effects of cannabidiol, as a potential therapeutic strategy, on glaucoma and some of the presumed mechanisms by which this phytocannabinoid provides its possible benefit properties through the WNT/?-catenin pathway. Full article
(This article belongs to the Special Issue Towards an Understanding of Retinal Diseases and Novel Treatment)
14 pages, 2988 KiB  
Article
Combining Magnetic Resonance Imaging with Systemic Monocyte Evaluation for the Implementation of GBM Management
by Carolina Giordano, Giovanni Sabatino, Simona Romano, Giuseppe Maria Della Pepa, Martina Tufano, Quintino Giorgio D’Alessandris, Simone Cottonaro, Marco Gessi, Mario Balducci, Maria Fiammetta Romano, Alessandro Olivi, Simona Gaudino and Cesare Colosimo
Int. J. Mol. Sci. 2021, 22(7), 3797; https://doi.org/10.3390/ijms22073797 - 6 Apr 2021
Cited by 7 | Viewed by 3225
Abstract
Magnetic resonance imaging (MRI) is the gold standard for glioblastoma (GBM) patient evaluation. Additional non-invasive diagnostic modalities are needed. GBM is heavily infiltrated with tumor-associated macrophages (TAMs) that can be found in peripheral blood. FKBP51s supports alternative-macrophage polarization. Herein, we assessed FKBP51s expression [...] Read more.
Magnetic resonance imaging (MRI) is the gold standard for glioblastoma (GBM) patient evaluation. Additional non-invasive diagnostic modalities are needed. GBM is heavily infiltrated with tumor-associated macrophages (TAMs) that can be found in peripheral blood. FKBP51s supports alternative-macrophage polarization. Herein, we assessed FKBP51s expression in circulating monocytes from 14 GBM patients. The M2 monocyte phenotype was investigated by qPCR and flow cytometry using antibodies against PD-L1, CD163, FKBP51s, and CD14. MRI assessed morphologic features of the tumors that were aligned to flow cytometry data. PD-L1 expression on circulating monocytes correlated with MRI tumor necrosis score. A wider expansion in circulating CD163/monocytes was measured. These monocytes resulted in a dramatic decrease in patients with an MRI diagnosis of complete but not partial surgical removal of the tumor. Importantly, in patients with residual tumor, most of the peripheral monocytes that in the preoperative stage were CD163/FKBP51s? had turned into CD163/FKBP51s+. After Stupp therapy, CD163/FKBP51s+ monocytes were almost absent in a case of pseudoprogression, while two patients with stable or true disease progression showed sustained levels in such circulating monocytes. Our work provides preliminary but meaningful and novel results that deserve to be confirmed in a larger patient cohort, in support of potential usefulness in GBM monitoring of CD163/FKBP51s/CD14 immunophenotype in adjunct to MRI. Full article
(This article belongs to the Special Issue Frontiers in Neuro-Oncology)
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<p>M2 gene expression profile in peripheral blood of glioblastoma (GBM) patients (<b>a</b>) Box plots of ARG1, MSR1, MRC1, TBR II, and tumor necrosis factor-α (TNFA) transcript levels in RNA extracted by 11 patients and 9 healthy donors’ peripheral blood mononuclear cells (PBMCs). (<b>b</b>–<b>d</b>) T1 TSE post-gadolinium images, axial section (<b>b</b>), and sagittal (<b>c</b>) and coronal (<b>d</b>) plane reconstructions: Region of interest (ROI) 1 (blue) indicate the tumor volume, ROI 2 (yellow) the necrosis volume. (<b>e</b>) MSR1, MRC1, TBR II, and TNFA transcript levels according to the necrosis score (N#3 = 4, N#1 + #2 = 8). Graphs were generated using Microsoft Excel.app 16.43.</p> Full article ">Figure 2
<p>PD-L1+ peripheral monocytes in GBM patients (<b>a</b>) Box plots of counts of the whole PD-L1+ and CD163+ monocytes and their subsets co-expressing FKBP51s+ or a combination of PD-L1 and CD163 in the study population (14 patients and 14 healthy donors). Counts were performed on CD14-gated PBMCs. The graph was generated using Microsoft Excel.app 16.43. (<b>b</b>) Linear regression with Pearson correlation of M2 monocyte subsets and necrosis score. Graphs were generated using Prism GraphPad 7.0a Macintosh. NS = necrosis score.</p> Full article ">Figure 3
<p>Effect of tumor removal on M2 immunophenotype (<b>a</b>) Boxplots of pre-operative and post-operative counts of whole PD-L1+ monocytes and their subgroups. (<b>b</b>) Boxplots of pre-operative and post-operative counts of whole CD163+ monocytes or CD163/FKBP51s+ monocytes. (<b>c</b>–<b>f</b>) Boxplots of pre-operative and post-operative counts are shown according to total or subtotal tumor removal: whole PD-L1+ monocytes (<b>c</b>), PD-L1/FKBP51s+ monocytes (<b>d</b>), whole 163+ monocytes (<b>e</b>), CD163/FKBP51s monocytes (<b>f</b>). In any panel, the line graph on the right shows the post-operative changes for each patient. Boxplots were generated using Prism GraphPad 7.0a Macintosh; line graphs were generated using Microsoft Excel.app 16.43. (<b>g</b>) T1 W post-gadolinium images of a patient with total resection and (<b>f</b>) a case of subtotal resection (<b>h</b>). Blue arrow indicates the residual part of the tumor. Before-surgery images (BS) and after-surgery images (AS) are shown along with flow cytometry histograms of CD163/FKBP51s+ monocytes (highlighted in red).</p> Full article ">Figure 4
<p>MRI and CD163/FKBP51s+ monocytes on follow-up. Axial T1 images after contrast medium administration: the glioblastoma before surgery in A, the immediate post-operative image in B, post-radio-chemotherapy in C, and post-adjuvant temozolomide (TMZ) in D. On the right, correspondent flow cytometry of CD163/FKBP51s+ monocytes. (<b>a</b>) The glioblastoma before surgery in the right temporal lobe. The immediate post-operative image shows a macroscopically complete exeresis of the tumor. Images C and D show no disease progression. (<b>b</b>) The glioblastoma before surgery in the right temporal lobe. The immediate post-operative image shows a residual tumor. Images C and D show the stability of the residual tumor. (<b>c</b>) The glioblastoma before surgery in the left temporal lobe. The immediate post-operative image shows a residual tumor. Image C highlights a disease progression, which tends to be stable in subsequent D control. (<b>d</b>) The glioblastoma before surgery in the right temporal lobe. The post-operative image shows a macroscopically complete exeresis of the tumor. C and D images show a thick impregnation area along the deepest portion of the surgical cave suspected for disease progression, which tended to regress in subsequent controls (E, F).</p> Full article ">Figure 5
<p>Visual summary of the main findings of the article. The cartoon shows that FKBP51s marked CD163+ monocytes (green) or PD-L1+ monocytes (red) that responded in a different way to tumor necrosis score (<b>a</b>) or extent of surgical resection (<b>b)</b>. (<b>a</b>) PD-L1+FKBP51s+ monocytes increased with the tumor necrosis score. By contrast, CD163+ monocytes decreased with the necrosis score. This trend was reversed, albeit not significantly, in CD163+FKBP51s+ monocytes. (<b>b</b>) The PD-L1+FKBP51s+ subset had a postoperative increasing tendency, both in the cases of total and subtotal tumor removal. The CD163<sup>+</sup>FKBP51s+ monocytes decreased upon total removal, while they increased after subtotal removal.</p> Full article ">Figure 6
<p>Workflow of the studied GBM patients.</p> Full article ">
11 pages, 4555 KiB  
Article
Differential Expression of Sphingolipid Metabolizing Enzymes in Spontaneously Hypertensive Rats: A Possible Substrate for Susceptibility to Brain and Kidney Damage
by Giuseppe Pepe, Maria Cotugno, Federico Marracino, Susy Giova, Luca Capocci, Maurizio Forte, Rosita Stanzione, Franca Bianchi, Simona Marchitti, Alba Di Pardo, Sebastiano Sciarretta, Speranza Rubattu and Vittorio Maglione
Int. J. Mol. Sci. 2021, 22(7), 3796; https://doi.org/10.3390/ijms22073796 - 6 Apr 2021
Cited by 9 | Viewed by 3048
Abstract
Alterations in the metabolism of sphingolipids, a class of biologically active molecules in cell membranes with direct effect on vascular homeostasis, are increasingly recognized as important determinant in different vascular disorders. However, it is not clear whether sphingolipids are implicated in the pathogenesis [...] Read more.
Alterations in the metabolism of sphingolipids, a class of biologically active molecules in cell membranes with direct effect on vascular homeostasis, are increasingly recognized as important determinant in different vascular disorders. However, it is not clear whether sphingolipids are implicated in the pathogenesis of hypertension-related cerebrovascular and renal damage. In this study, we evaluated the existence of possible abnormalities related to the sphingolipid metabolism in the brain and kidneys of two well validated spontaneously hypertensive rat strains, the stroke-prone (SHRSP) and the stroke-resistant (SHRSR) models, as compared to the normotensive Wistar Kyoto (WKY) rat strain. Our results showed a global alteration in the metabolism of sphingolipids in both cerebral and renal tissues of both hypertensive strains as compared to the normotensive rat. However, few defects, such as reduced expression of enzymes involved in the metabolism/catabolism of sphingosine-1-phosphate and in the de novo biosynthetic pathways, were exclusively detected in the SHRSP. Although further studies are necessary to fully understand the significance of these findings, they suggest that defects in specific lipid molecules and/or their related metabolic pathways may likely contribute to the pathogenesis of hypertensive target organ damage and may eventually serve as future therapeutic targets to reduce the vascular consequences of hypertension. Full article
(This article belongs to the Special Issue Sphingolipid Metabolism and Signaling in Diseases)
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<p>Simplified schematic representation of sphingolipid metabolism. Ceramide (Cer) represents the hub in the synthesis of sphingolipids. It is generated through the de novo biosynthetic route by the action of multiple enzymes such as Serine palmitoyltransferase (SPT) 3-keto-dihydrosphingosine reductase (KDSR), ceramide synthase (CERS) and ceramide desaturase (DES). Ceramide may also derive from sphingosine, which, in turn, produces sphingosine-1-phosphate (S1P) through phosphorylation by SPHK1/2. S1P can be irreversibly catabolized into hexadecenal + phospho-ethanolamine by S1P-Lyase (SGPL1).</p> Full article ">Figure 2
<p>Expression of S1P metabolizing enzymes is defective in brain tissues from SHRSP. Representative cropped immunoblottings and densitometric analysis of SPHK1 (sphingosine kinase 1) (<b>A</b>,<b>B</b>), SPHK2 (sphingosine kinase 2) (<b>C</b>,<b>D</b>) and SGPL1 (S1P-Lyase) (<b>E</b>,<b>F</b>) in brain and kidney tissues from WKY, SHRSP and SHRSR. In each immunoblotting, all samples were run on the same gel. Non-adjacent samples are separated by a black line. Data are represented as mean ± SD, <span class="html-italic">n</span> = 6/7 for each group of rats. * <span class="html-italic">p</span><math display="inline"><semantics> <mrow> <mo> </mo> <mo>&lt;</mo> </mrow> </semantics></math> 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span><math display="inline"><semantics> <mrow> <mo> </mo> <mo>&lt;</mo> </mrow> </semantics></math> 0.001 (one-way ANOVA, Tukey post-test). The raw date for generating the figure are reported in the Supplementary Material <a href="#app1-ijms-22-03796" class="html-app">Table S1</a>.</p> Full article ">Figure 3
<p>Expression of S1P receptors is aberrant in both brain and kidney tissues from SHRSP and SHRSR. Representative immunoblottings and densitometric analysis of S1PR1 (S1P receptor 1) (<b>A</b>,<b>B</b>), S1PR2 (S1P receptor 2) (<b>C</b>,<b>D</b>) and S1PR3 (S1P receptor 3) (<b>E</b>,<b>F</b>) in brain and kidney tissues from WKY, SHRSP and SHRSR. In each immunoblotting, all samples were run on the same gel. Non-adjacent samples are separated by a black line. Data are represented as mean ± SD, <span class="html-italic">n</span> = 6/8 for each group of rats. * <span class="html-italic">p</span><math display="inline"><semantics> <mrow> <mo> </mo> <mo>&lt;</mo> <mo> </mo> </mrow> </semantics></math>0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span><math display="inline"><semantics> <mrow> <mo> </mo> <mo>&lt;</mo> <mo> </mo> </mrow> </semantics></math>0.001 (one-way ANOVA, Tukey post-test). The raw date used for generating the figure are reported in the <a href="#app1-ijms-22-03796" class="html-app">Supplementary Materials</a> <a href="#app1-ijms-22-03796" class="html-app">Table S2</a>.</p> Full article ">Figure 4
<p>Gene expression of ceramide synthetizing enzymes is defective in brain and kidney tissues from SHRSP and SHRSR. qPCR analysis of CerS1 (ceramide synthase 1) (<b>A</b>) and CerS2 (ceramide synthase 2) (<b>B</b>) in brain tissue from WKY, SHRSP and SHRSR. qPCR analysis of CerS2 (<b>C</b>) and CerS6 (ceramide synthase 6) (<b>D</b>) in kidney tissue from WKY, SHRSP and SHRSR. Data are represented as mean ± SD, <span class="html-italic">n</span> = 5/6 for each group of rats. * <span class="html-italic">p</span> <math display="inline"><semantics> <mrow> <mo>&lt;</mo> <mo> </mo> </mrow> </semantics></math>0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span><math display="inline"><semantics> <mrow> <mo> </mo> <mo>&lt;</mo> </mrow> </semantics></math> 0.001; **** <span class="html-italic">p</span> <math display="inline"><semantics> <mrow> <mo>&lt;</mo> <mo> </mo> </mrow> </semantics></math>0.0001 (one-way ANOVA, Tukey post-test). The raw date used for generating the figure are reported in the Supplementary Material <a href="#app1-ijms-22-03796" class="html-app">Table S3</a>.</p> Full article ">Figure 5
<p>Expression of rate-limiting enzyme of de novo sphingolipid biosynthesis is defective in brain and kidney tissues from SHRSP and SHRSR. Representative immunoblottings and densitometric analysis of SPTLC1 (serine palmitoyltransferase long chain base subunit 1) (<b>A</b>,<b>B</b>) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2) (<b>C</b>,<b>D</b>) in brain and kidney tissues from WKY, SHRSP and SHRSR. In each immunoblotting, all samples were run on the same gel. Non-adjacent samples are separated by a black line. Data are represented as mean ± SD, <span class="html-italic">n</span> = 6/7 for each group of rats. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01 (one-way ANOVA, Tukey post-test). The raw date used for generating the figure are reported in the <a href="#app1-ijms-22-03796" class="html-app">Supplementary Materials</a> <a href="#app1-ijms-22-03796" class="html-app">Table S4</a>.</p> Full article ">Figure 6
<p>Expression of Kdsr mRNA is reduced in kidney tissues from SHRSP and SHRSR. qPCR analysis of Kdsr (3-keto-dihydrosphingosine reductase) (<b>A</b>,<b>B</b>) and Des (ceramide desaturase) (<b>C</b>,<b>D</b>) in brain and kidney tissues from WKY, SHRSP and SHRSR. Data are represented as mean ± SD, <span class="html-italic">n</span> = 5/6 for each group of rats. * <span class="html-italic">p</span> &lt; 0.05 (one-way ANOVA, Tukey post-test). The raw date used for generating the figure are reported in the <a href="#app1-ijms-22-03796" class="html-app">Supplementary Materials</a> <a href="#app1-ijms-22-03796" class="html-app">Table S5</a>.</p> Full article ">
16 pages, 3118 KiB  
Article
Towards the Enhancement of Essential Oil Components’ Antimicrobial Activity Using New Zein Protein-Gated Mesoporous Silica Microdevices
by Elisa Poyatos-Racionero, Gemma Guarí-Borràs, María Ruiz-Rico, Ángela Morellá-Aucejo, Elena Aznar, José Manuel Barat, Ramón Martínez-Máñez, María Dolores Marcos and Andrea Bernardos
Int. J. Mol. Sci. 2021, 22(7), 3795; https://doi.org/10.3390/ijms22073795 - 6 Apr 2021
Cited by 21 | Viewed by 2998
Abstract
The development of new food preservatives is essential to prevent foodborne outbreaks or food spoilage due to microbial growth, enzymatic activity or oxidation. Furthermore, new compounds that substitute the commonly used synthetic food preservatives are needed to stifle the rising problem of microbial [...] Read more.
The development of new food preservatives is essential to prevent foodborne outbreaks or food spoilage due to microbial growth, enzymatic activity or oxidation. Furthermore, new compounds that substitute the commonly used synthetic food preservatives are needed to stifle the rising problem of microbial resistance. In this scenario, we report herein, as far as we know, for the first time the use of the zein protein as a gating moiety and its application for the controlled release of essential oil components (EOCs). The design of microdevices consist of mesoporous silica particles loaded with essential oils components (thymol, carvacrol and cinnamaldehyde) and functionalized with the zein (prolamin) protein found in corn as a molecular gate. The zein protein grafted on the synthesized microdevices is degraded by the proteolytic action of bacterial enzymatic secretions with the consequent release of the loaded essential oil components efficiently inhibiting bacterial growth. The results allow us to conclude that the new microdevice presented here loaded with the essential oil component cinnamaldehyde improved the antimicrobial properties of the free compound by decreasing volatility and increasing local concentration. Full article
(This article belongs to the Special Issue Ordered Mesoporous Materials)
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Figure 1
<p>(<b>a</b>) Normalized X-ray patterns of all the synthesized solids. From bottom to top, the reported solids are as follows: “as made” material (a.m.), calcined support M41 (<b><span class="html-italic">i</span></b>) and final materials, M41–RhB–Z (<span class="html-italic">ii</span>), M41–Thy–Z (<span class="html-italic">iii</span>), M41–Car–Z (<span class="html-italic">iv</span>), M41–Cin–Z (<span class="html-italic">v</span>). (<b>b</b>) TEM images of M41 solids (<span class="html-italic">i</span>), M41–RhB–Z (<span class="html-italic">ii</span>), M41–Thy–Z (<span class="html-italic">iii</span>), M41–Car–Z (<span class="html-italic">iv</span>), M41–Cin–Z (<span class="html-italic">v</span>). Insets correspond to a 2× magnification of the framed areas.</p> Full article ">Figure 2
<p>(<b>a</b>) Nitrogen adsorption (○)–desorption (●) isotherms of the calcined M41 solid and the final M41–RhB–Z solid at standard temperature and pressure conditions (STP); (<b>b</b>) pore distribution graph for M41 and (<b>c</b>) pore distribution graph for M41–RhB–Z.</p> Full article ">Figure 3
<p>ζ potential values of the solids in the different synthesis steps.</p> Full article ">Figure 4
<p>ChemDraw 3D MM2 energy-minimized model showing the 3D structure of thymol, carvacrol and cinnamaldehyde.</p> Full article ">Figure 5
<p>Cargo release profiles of RhB from M41–RhB–Z in H<sub>2</sub>O at pH 8 (○) and when proteolytic enzymes (pronase) are present (●). Release assays were performed in triplicate, and error bars correspond to the standard deviation.</p> Full article ">Figure 6
<p>Cargo release profiles of EOCs from M41–Thy–Z (<b>left</b>); M41–Car–Z (<b>center</b>) and M41–Cin–Z (<b>right</b>) in H<sub>2</sub>O at pH 8. (○) in the absence and (●) in the presence of proteolytic enzymes (pronase).</p> Full article ">Figure 7
<p><span class="html-italic">E. coli</span> counts after incubation with M41–EOC–Z systems (M41-Thy-Z (<b>a</b>), M41–Car–Z (<b>b</b>) and M41–Cin–Z (<b>c</b>)) and free EOCs (thymol (<b>d</b>), carvacrol (<b>e</b>) and cinnamaldehyde (<b>f</b>)) according to particle and EOC concentration. Red dots mark the coincidental concentrations between the encapsulated EOC and the free molecule. Antimicrobial assays were performed in triplicate, and error bars correspond to the standard deviation.</p> Full article ">Figure 8
<p>Fluorescence images of untreated <span class="html-italic">E. coli</span> (<b>a</b>) and the cells treated with M41–Z (<b>b</b>) as the negative control, free Cin (<b>c</b>) and M41–Cin–Z (<b>d</b>) after 5 h of incubation. The study was performed using a two-color fluorescent LIVE/DEAD<sup>®</sup> <span class="html-italic">Bac</span>Light™ assay used to visualize viable (green) and dead (red) bacteria. Red-stained bacteria are not visible when dead cells do not remain intact after treatment.</p> Full article ">Scheme 1
<p>Representation of M41–EOC–Z synthesis and cargo release. Cargo loading (EOCs) is followed by APTES-functionalization, and finally zein is attached to the amino groups. The cargo is actively released due to the hydrolyzing action of protease enzymes.</p> Full article ">
19 pages, 6008 KiB  
Article
Excitatory Effects of Calcitonin Gene-Related Peptide (CGRP) on Superficial Sp5C Neurons in Mouse Medullary Slices
by Fang Zheng, Barbara E. Nixdorf-Bergweiler, Johannes van Brederode, Christian Alzheimer and Karl Messlinger
Int. J. Mol. Sci. 2021, 22(7), 3794; https://doi.org/10.3390/ijms22073794 - 6 Apr 2021
Cited by 8 | Viewed by 3478
Abstract
The neuromodulator calcitonin gene-related peptide (CGRP) is known to facilitate nociceptive transmission in the superficial laminae of the spinal trigeminal nucleus caudalis (Sp5C). The central effects of CGRP in the Sp5C are very likely to contribute to the activation of central nociceptive pathways [...] Read more.
The neuromodulator calcitonin gene-related peptide (CGRP) is known to facilitate nociceptive transmission in the superficial laminae of the spinal trigeminal nucleus caudalis (Sp5C). The central effects of CGRP in the Sp5C are very likely to contribute to the activation of central nociceptive pathways leading to attacks of severe headaches like migraine. To examine the potential impacts of CGRP on laminae I/II neurons at cellular and synaptic levels, we performed whole-cell patch-clamp recordings in juvenile mouse brainstem slices. First, we tested the effect of CGRP on cell excitability, focusing on neurons with tonically firing action potentials upon depolarizing current injection. CGRP (100 nM) enhanced tonic discharges together with membrane depolarization, an excitatory effect that was significantly reduced when the fast synaptic transmissions were pharmacologically blocked. However, CGRP at 500 nM was capable of exciting the functionally isolated cells, in a nifedipine-sensitive manner, indicating its direct effect on membrane intrinsic properties. In voltage-clamped cells, 100 nM CGRP effectively increased the frequency of excitatory synaptic inputs, suggesting its preferential presynaptic effect. Both CGRP-induced changes in cell excitability and synaptic drives were prevented by the CGRP receptor inhibitor BIBN 4096BS. Our data provide evidence that CGRP increases neuronal activity in Sp5C superficial laminae by dose-dependently promoting excitatory synaptic drive and directly enhancing cell intrinsic properties. We propose that the combination of such pre- and postsynaptic actions of CGRP might underlie its facilitation in nociceptive transmission in situations like migraine with elevated CGRP levels. Full article
(This article belongs to the Special Issue Neuropeptides, Receptors, and Behavior)
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<p>Firing patterns of Sp5C laminae I/II cells. (<b>A</b>) Bright field micrographs illustrate a typical freshly prepared transverse slice (350 µm thickness; left), and examples of biocytin-filled neurons in lamina II (middle) and in lamina I (right, with soma location indicated by an asterisk on the left, and axon pointed by arrow). AP, area postrema; cc, central canal; IO, inferior olivary complex; NA, nucleus ambiguous; sptV, spinal tract of the trigeminal nerve; XII, hypoglossal nucleus. (<b>B</b>) Five classes of Sp5C laminae I/II cells are distinguished by their properties of action potential discharge to depolarizing currents. Cells were recorded under whole-cell current-clamp mode, with membrane potential initially held at −70 mV (by current injection). Pattern of spiking was determined by their responses (top three panels) to the serial of current injection (bottom panels). The initial burst-like spiking (arrow) was enlarged on the right. (<b>C</b>) Distribution of spiking patterns of Sp5C cells recorded in the absence and in the presence of inhibitory and excitatory synaptic blockers. Kynurenic acid (KA, 2 mM) was used to block fast glutamatergic synaptic transmission, and picrotoxin (PTX, 100 µM) plus strychnine (10 µM) were used to block fast GABAergic and glycinergic synaptic transmissions, respectively.</p> Full article ">Figure 2
<p>Calcitonin gene-related peptide (CGRP) excites tonic-firing cells in Sp5C laminae I/II. Depolarizing current (35–100 pA) was adjusted individually to evoke 4–10 APs per pulse (500 ms) in each cell before CGRP application, with membrane potential set at −70 mV. (<b>A</b>) Original traces taken before, during and after CGRP (100 nM for 5 min) illustrate the reversible enhancement of AP firing in a lamina I cell. (<b>B</b>) Representative traces from another cell show how the CGRP receptor antagonist BIBN 4096BS (10 µM) prevents the excitatory effect of CGRP. (<b>C</b>,<b>D</b>) Histograms summarize CGRP-induced changes in AP discharges (<b>C</b>) and time to first AP (<b>D</b>) in the absence and in the presence of BIBN 4096BS. Numbers in columns indicate sample size. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 3
<p>CGRP enhances Sp5C cell intrinsic excitability. Action potentials of Sp5C laminae I/II cells were evoked in the presence of kynurenic acid (KA), picrotoxin (PTX) and strychnine to block fast synaptic transmissions. (<b>A</b>,<b>B</b>) Overlapped traces from two cells illustrate the concentration-dependent effect of CGRP application on the evoked APs. Dashed line indicates the initial potential of −70 mV. (<b>C</b>) Superimposed traces are the 1st APs of the cell in B, with enlarged time scale to illustrate the CGRP-induced reduction in AP threshold and afterhyperpolarization (AHP). (<b>D</b>–<b>H</b>) Histograms characterize the impacts of CGRP on AP discharges (<b>D</b>) and time to evoke 1st AP (<b>E</b>), AP threshold (<b>F</b>), maximal rising slope of AP (indicative of available sodium channels; (<b>G</b>) and AHP (<b>H</b>). (<b>I</b>) Summary of CGRP-induced membrane depolarization depicted further by a voltage trace above the histogram. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 4
<p>Involvement of L-type calcium channels in the excitatory effect of CGRP. All recordings were performed in the cocktail of KA, PTX and strychnine to block fast synaptic transmissions. (<b>A</b>) Voltage traces were collected from a Sp5C cell in laminae II before and during CGRP (500 nM) application, in the presence of the L-type calcium channel blocker nifedipine (10 µM). (<b>B</b>,<b>C</b>) Histograms summarize that nifedipine blocks the responses of Sp5C cells to high concentration of CGRP (500 nM), manifested in AP discharges and time to evoke 1st AP (<b>B</b>), voltage threshold for AP and afterhyperpolarization (<b>C</b>).</p> Full article ">Figure 5
<p>Calcitonin gene-related peptide (CGRP) facilitates excitatory synaptic drive onto Sp5C laminae I/II cells. Spontaneously occurring excitatory postsynaptic currents (spEPSCs) were monitored under whole-cell voltage-clamp mode at −70 mV, in the presence of antagonists for GABA<sub>A</sub>Rs and GlyRs. (<b>A</b>) Raw traces of spEPSCs from a lamina I cell were collected before CGRP application (control), 5 min in CGRP (100 nM) and 10 min after wash. Superimposed traces on the right represent the averaged events from the cell before (black trace) and during drug application (red trace). (<b>B</b>) Histograms summarize the effects of acutely applied CGRP on the frequency and the averaged peak amplitude of spEPSCs. Inset in up-right corner with the normalized changes in spEPSC frequency further reinforces the dose-dependent effect of CGRP. (<b>C</b>) Typical responses of spEPSCs to CGRP (100 nM) in the presence of a potent non-peptide antagonist BIBN 4096BS (10 µM, 20 min). Kynurenic acid (KA, 2 mM) was applied at the end of this experiment to verify the glutamatergic origin of spEPSCs. (<b>D</b>) Summary of the dampening effects of CGRP receptor antagonists on spEPSCs in Sp5C I/II cells. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 6
<p>Immunohistochemically processed sections showing the outer laminae of the mouse dorsolateral Sp5C. Immunofluorescence of CGRP receptor components receptor activity modifying protein 1 (RAMP1) (<b>A</b>,<b>D</b>,<b>E</b>) and calcitonin-like receptor (CLR) (<b>B</b>,<b>G</b>–<b>I</b>) in the spinal trigeminal tract (sptV) and lamina I/II of the mouse Sp5C ((<b>A</b>–<b>C</b>,<b>G</b>) dorsolateral, (<b>D</b>,<b>I</b>) ventrolateral, (<b>E</b>,<b>H</b>) lateral); (<b>C</b>,<b>F</b>) are control stainings without first antibody but incubated with the second antibody Cy3; blue is nucleus staining (DAPI). The dotted lines in (<b>D</b>,<b>G</b>) show the approximate border between sptV and lamina (<b>I</b>). Arrows point to cell bodies that are closely approached by RAMP1/CLR immuno-positive nerve fibres, the arrowhead in G shows CLR immuno-positive fibres accompanying a penetrating medullary blood vessel, and the arrowhead in the control staining F points to an unspecific immunofluorescence in the wall of a blood vessel; magnification bars 200 µm in (<b>A</b>–<b>C</b>) and 50 µm in (<b>D</b>–<b>I</b>). No CGRP receptor component immunofluorescence of cell bodies is visible.</p> Full article ">
23 pages, 6908 KiB  
Article
Predicting the Structure and Dynamics of Membrane Protein GerAB from Bacillus subtilis
by Sophie Blinker, Jocelyne Vreede, Peter Setlow and Stanley Brul
Int. J. Mol. Sci. 2021, 22(7), 3793; https://doi.org/10.3390/ijms22073793 - 6 Apr 2021
Cited by 7 | Viewed by 3820
Abstract
Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore’s inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine [...] Read more.
Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore’s inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine activation of GerA requires all three subunits, but which binds L-alanine is unknown. To date, how GRs trigger germination is unknown, in particular due to lack of detailed structural information about B subunits. Using homology modelling with molecular dynamics (MD) simulations, we present structural predictions for the integral membrane protein GerAB. These predictions indicate that GerAB is an ?-helical transmembrane protein containing a water channel. The MD simulations with free L-alanine show that alanine binds transiently to specific sites on GerAB. These results provide a starting point for unraveling the mechanism of L-alanine mediated signaling by GerAB, which may facilitate early events in spore germination. Full article
(This article belongs to the Section Biochemistry)
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<p>Alignment of GerAB models with templates. GerAB was aligned with 3GI8 ApcT K158A (pdb code 3GI8, [<a href="#B25-ijms-22-03793" class="html-bibr">25</a>]) and GkApcT (pdb code 6F34 [<a href="#B26-ijms-22-03793" class="html-bibr">26</a>]) [<a href="#B33-ijms-22-03793" class="html-bibr">33</a>,<a href="#B35-ijms-22-03793" class="html-bibr">35</a>]. For 3GI8-SWISS as well as 6F34-SWISS, 303 amino acids were aligned. For 3GI8-RaptorX 365 amino acids were aligned. Identical amino acids are displayed in purple font, transmembrane (TM) domains as established in this research are highlighted in color, corresponding to <a href="#ijms-22-03793-f002" class="html-fig">Figure 2</a> and TM domains as currently defined by TMHMM/Phobius are underlined [<a href="#B19-ijms-22-03793" class="html-bibr">19</a>,<a href="#B20-ijms-22-03793" class="html-bibr">20</a>,<a href="#B21-ijms-22-03793" class="html-bibr">21</a>,<a href="#B22-ijms-22-03793" class="html-bibr">22</a>]. Asterisks indicate that the residues could not be aligned and gray letters on the C-terminus indicate the domains that are not present in the models.</p> Full article ">Figure 2
<p>Schematic overview of GerAB topology. (<b>A</b>) This topology prediction is based on the structures of the three GerAB models examined in this study. The helices are shown as cylinders and the possible L-alanine binding pocket is depicted as a purple star between TM1 and 6. The figure is adapted from [<a href="#B24-ijms-22-03793" class="html-bibr">24</a>]. (<b>B</b>) illustrates the organisation of the protein from the inside of the IM. Colors of the helices depicted as circles correspond to A.</p> Full article ">Figure 3
<p>Structure of GerAB models. (<b>A</b>) shows the helicity and structure of the 3GI8-RaptorX model [<a href="#B25-ijms-22-03793" class="html-bibr">25</a>], with the colors indicating the helical regions as defined in <a href="#ijms-22-03793-f002" class="html-fig">Figure 2</a>. (<b>B</b>) depicts the helicity and structure of the 3GI8-SWISS model [<a href="#B25-ijms-22-03793" class="html-bibr">25</a>]. (<b>C</b>) shows the helicity and structure of 6F34-SWISS model [<a href="#B26-ijms-22-03793" class="html-bibr">26</a>]. The helicity is defined as the distance between the backbone oxygen atom of residue n and the backbone nitrogen atom of residue n+4. The blue line indicates a distance of 0.35 nm, the distance threshold for a hydrogen bond interaction and the red blocks indicate TM regions as defined by TMHMM/Phobius, see Uniprot entry UniProtKB-P07869 (GERAB_BACSU) [<a href="#B36-ijms-22-03793" class="html-bibr">36</a>]. Helicity per model is the average of the last 50 ns of 5 runs.</p> Full article ">Figure 4
<p>Interaction of GerAB residues with potassium, membrane tails and water. (<b>A</b>–<b>C</b>) shows the proximity plots for K<sup>+</sup>, membrane tails and water to residues of GerAB. A P value of 1 indicates that water or membrane tails are within 0.6 nm of the residue for the duration of the simulation, while a value of 0 indicates that water or lipid tails are never within a distance of 0.6 nm of the residue. The proximity is calculated over 5 runs for each model. (<b>D</b>) shows a snapshot of the complex with the membrane in white, water in blue and GerAB helices in color, corresponding to <a href="#ijms-22-03793-f002" class="html-fig">Figure 2</a>.</p> Full article ">Figure 5
<p>Number of binding events and average residence time for water to GerAB residues. In (<b>A</b>) the average number of binding events (Nb) are shown for residues in 3GI8-RaptorX, 3GI8-SWISS and 6F34-SWISS. (<b>B</b>) shows the average residence time of water to residues in 3GI8-RaptorX, 3GI8-SWISS and 6F34-SWISS.</p> Full article ">Figure 6
<p>Interactions of GerAB with L-alanine. (<b>A</b>–<b>C</b>) shows the average proximity of L-alanine to each residue in the three GerAB models. Regions with higher proximity can be identified and are colored purple in the structures (<b>D</b>–<b>F</b>) Graphs (<b>G</b>–<b>I</b>) show the number of binding events of L-alanine to residues in the GerAB models. (<b>J</b>–<b>L</b>) show the average residence time of L-alanine to residues in the GerAB models.</p> Full article ">Figure A1
<p>RMSD graphs of 3GI8-Raptorx and 3GI8-SWISS, 6F34-SWISS. This figure shows the RMSD graphs for each run of the three models. RMSD is displayed in nm and time is shown in ns. Most flexibility is observed in the first 50 ns of the simulations.</p> Full article ">Figure A2
<p>In this figure the templates 3GI8 [<a href="#B25-ijms-22-03793" class="html-bibr">25</a>] (<b>A</b>,<b>B</b>) and 6F34 [<a href="#B26-ijms-22-03793" class="html-bibr">26</a>] (<b>C</b>) are compared with the created GerAB models. The structures are colored according to the topology defined in <a href="#ijms-22-03793-f002" class="html-fig">Figure 2</a>. The colors in the overlay of A are green for the template 3GI8 and cream for the 3GI8-RaptorX model. The colors in the overlay of B are purple for the template 3GI8 and cream for the 3GI8-SWISS model. In C the template 6F34 is depicted in blue and the 6F34-SWISS model again in cream.</p> Full article ">Figure A3
<p>Width of GerAB channel as function of the time. This graphs shows for each run of the three models the radius of the channel in the <span class="html-italic">z</span>-axis (in nm) over time in ns. The <span class="html-italic">z</span>-axis is perpendicular to the membrane. The size of the pore is shown on a scale from 0 to 1 nm in color ranging from green to light blue, as indicated by the color bar. Data obtained using HOLE [<a href="#B37-ijms-22-03793" class="html-bibr">37</a>].</p> Full article ">Figure A4
<p>Average residence time and number of binding events for potassium (K<sup>+</sup>) to GerAB residues. (<b>A</b>) shows the number of binding events for the three models. (<b>B</b>) depicts the average residence time of (K<sup>+</sup>) against the residue index for the three models.</p> Full article ">
31 pages, 8262 KiB  
Article
Altered microRNA Transcriptome in Cultured Human Liver Cells upon Infection with Ebola Virus
by Idrissa Diallo, Jeffrey Ho, Benoit Laffont, Jonathan Laugier, Abderrahim Benmoussa, Marine Lambert, Zeinab Husseini, Geoff Soule, Robert Kozak, Gary P. Kobinger and Patrick Provost
Int. J. Mol. Sci. 2021, 22(7), 3792; https://doi.org/10.3390/ijms22073792 - 6 Apr 2021
Cited by 14 | Viewed by 4558
Abstract
Ebola virus (EBOV) is a virulent pathogen, notorious for inducing life-threatening hemorrhagic fever, that has been responsible for several outbreaks in Africa and remains a public health threat. Yet, its pathogenesis is still not completely understood. Although there have been numerous studies on [...] Read more.
Ebola virus (EBOV) is a virulent pathogen, notorious for inducing life-threatening hemorrhagic fever, that has been responsible for several outbreaks in Africa and remains a public health threat. Yet, its pathogenesis is still not completely understood. Although there have been numerous studies on host transcriptional response to EBOV, with an emphasis on the clinical features, the impact of EBOV infection on post-transcriptional regulatory elements, such as microRNAs (miRNAs), remains largely unexplored. MiRNAs are involved in inflammation and immunity and are believed to be important modulators of the host response to viral infection. Here, we have used small RNA sequencing (sRNA-Seq), qPCR and functional analyses to obtain the first comparative miRNA transcriptome (miRNome) of a human liver cell line (Huh7) infected with one of the following three EBOV strains: Mayinga (responsible for the first Zaire outbreak in 1976), Makona (responsible for the West Africa outbreak in 2013–2016) and the epizootic Reston (presumably innocuous to humans). Our results highlight specific miRNA-based immunity pathways and substantial differences between the strains beyond their clinical manifestation and pathogenicity. These analyses shed new light into the molecular signature of liver cells upon EBOV infection and reveal new insights into miRNA-based virus attack and host defense strategy. Full article
(This article belongs to the Special Issue Host-Pathogen Interaction 2.0)
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Figure 1
<p>Relative viral replication of EBOV variants. The relative viral replication of Mayinga (blue), Makona (gold), and Reston (gray) post-infection (24 h, 96 h) were approximated by RT-qPCR using the log fold increase of GP mRNA in Huh7-infected cells, versus uninfected cells (black). Data were normalized with a reference gene (ACTB), reported to control uninfected cells and expressed with a relative quantitation method (ddCT). Data presented were calculated from three biological replicate (<span class="html-italic">n = 3</span>) measurements. The two-way analysis of variance (ANOVA) and Sidak multiple comparisons were used for statistical analysis. Statistically significant differences (uninfected vs. strains-infected cells) are indicated by stars (*), * <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">p</span> &lt; 0.0001.</p> Full article ">Figure 2
<p>Modulation of cytokines (IL-6, IL-8, IL-1b, TNF) in Huh7 cells infected or not with Ebola virus. The relative mRNA levels of four cytokines (IL-6, IL-8, IL-1b, TNF) were measured for three EBOV variants at 24 h (black) and 96 h (gray) post-infection based on the linear fold increase in Huh7-infected cells using RT-qPCR. Data are normalized with a reference gene (ACTB), reported to control (uninfected), and expressed with a relative quantitation method (ddCT). Data presented were calculated from three biological replicate measurements (<span class="html-italic">n</span> = 3). The two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons were used for statistical analysis. Statistically significant differences (uninfected vs. strains-infected cells) are indicated by stars (*), ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001; non significant differences are indicated by the abbreviation “ns”.</p> Full article ">Figure 3
<p>Modulation of IgSF Cell Adhesion Molecules (ICAM1, VCAM1) in Huh7 cells infected or not with Ebola virus. The relative levels of cell adhesion molecules ICAM-1 and VCAM-1 in EBOV-infected Huh7 cells were measured by RT-qPCR at 24 h (black) and 96 h (gray) post-infection using the linear fold increase. Data were normalized with a reference gene (ACTB), reported to control (uninfected), and expressed with a relative quantitation method (ddCT). Data presented were calculated from three biological replicate measurements (<span class="html-italic">n</span> = 3). The two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons were used for statistical analysis. Statistically significant differences (uninfected vs. variants-infected) are indicated by stars (*), *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001; non significant differences are indicated by the abbreviation “ns”.</p> Full article ">Figure 4
<p>Modulation of coagulation factors (F2, F3, F8, F10) in Huh7 cells infected or not with Ebola virus. The relative levels of coagulation factor 2, factor 3, factor 8 and factor 10 mRNAs in EBOV-infected Huh7 cells were measured by RT-qPCR at 24 h (black) and 96 h (gray) post-infection using the linear fold increase. Data were normalized with a reference gene (ACTB), reported to control (uninfected), and expressed with a relative quantitation method (ddCT). Data presented were calculated from three biological replicate measurements (<span class="html-italic">n</span> = 3). The two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons were used for statistical analysis. Statistically significant differences (uninfected vs. variants-infected) are indicated by stars (*), * <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">p</span> &lt; 0.0001; non significant differences are indicated by the abbreviation “ns”.</p> Full article ">Figure 5
<p>Modulation of apoptosis players (Bax, Bcl-2, Casp3) in Huh7 cells infected or not with Ebola virus. The relative expression of Bax, Bcl-2, and Casp3 mRNAs in EBOV-infected Huh7 cells were measured by RT-qPCR at 24 h (black) and 96 h (gray) post-infection using the linear fold increase. Data were normalized with a reference gene (ACTB), reported to control (uninfected), and expressed with a relative quantitation method (ddCT). Data presented were calculated from three biological replicate measurements (<span class="html-italic">n</span> = 3). The two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons were used for statistical analysis. Statistically significant differences (uninfected vs. variants-infected) are indicated by stars (*), * <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001; non significant differences are indicated by the abbreviation “ns”.</p> Full article ">Figure 6
<p>Scatter plot showing Huh7 miRNome modulated by Ebola variants over time. X-axis shows the controls (uninfected) at the indicated time, Y-axis is the experimental condition using the EBOV variant Mayinga (<b>A</b>), Makona (<b>B</b>) or Reston (<b>C</b>). Only a few miRNAs were modulated by the variants. PC: Pearson correlation. UP (red dots): Upregulated miRNAs, DOWN (green dots): Downregulated miRNAs. N.D.E (grey dots): Not differentially expressed.</p> Full article ">Figure 7
<p>Selective modulation of the DE miRNA expression (%) induced by the EBOV variant over time. Based on the total number of miRNAs expressed in each condition, we estimated the percentage of miRNAs that were upregulated, downregulated or no longer regulated. This graph shows the percentage (%) of upregulated and downregulated miRNAs at 24 h and 96 h, highlighting a reduction in the number of downregulated miRNAs induced by the EBOV Reston variant over time.</p> Full article ">Figure 8
<p>Venn diagrams showing unique and common DE (upregulated and downregulated) miRNAs detected (by RNA-Seq) in Huh7 cells infected with 3 ebolavirus strains (Mayinga, Makona, Reston).</p> Full article ">Figure 9
<p>Modulation of the most abundant isoform miRNAs in Huh7 cells infected or not with Ebola virus. (<b>A</b>) Heatmap of the 20 most abundant Huh7 miRNAs expression profiling tracked during EBOV infections with the Mayinga, Makona or Reston variant. TPM, transcripts per million. (<b>B</b>) Variation over time of the 3 most abundant miRNAs in Huh7 cells in the presence of the EBOV Mayinga, Makona or Reston variants. The figure was generated from the data of (<b>A</b>): <b>[</b> . TPM, transcripts per million.</p> Full article ">Figure 10
<p>Model of EBOV cell entry and of the host miRNAs targeting the EBOV RNA genome. The steps leading to the cytosolic release of the viral genome into Huh7 cells are presented. The 3 most abundant uninfected Huh7 cell miRNAs and the 6 most EBOV-upregulated miRNAs (3 miRNAs for 24 h and 3 miRNAs for 96 h for each variant) over time are predicted to target various genes of the viral genome (VP35, VP40, GP, L) in the ZEBOV (Mayinga/Makona, green) variant and the Reston (RESTV, red) variant. The predictions were performed with RNA22. Only significant (≤0.05) hits were retained. * = miRNA expressed in the early phase, (the others being expressed in the late phase).</p> Full article ">Figure 11
<p>Alignment of the top upregulated host miRNAs on the EBOV RNA genome for both ZEBOV and RESTV Scheme 7. Cells before and after infection with (<b>A</b>) ZEBOV (Mayinga, Makona) and (<b>B</b>) RESTV (Reston) were aligned with their respective genome using RNA22. Alignments with the lowest free energy (FE) in kcal/mol and the highest probability (<span class="html-italic">p</span> ≤ 0.05) were reported along with the leftmost position (LP) in nt of the alignment on the EBOV RNA genome.</p> Full article ">Figure 11 Cont.
<p>Alignment of the top upregulated host miRNAs on the EBOV RNA genome for both ZEBOV and RESTV Scheme 7. Cells before and after infection with (<b>A</b>) ZEBOV (Mayinga, Makona) and (<b>B</b>) RESTV (Reston) were aligned with their respective genome using RNA22. Alignments with the lowest free energy (FE) in kcal/mol and the highest probability (<span class="html-italic">p</span> ≤ 0.05) were reported along with the leftmost position (LP) in nt of the alignment on the EBOV RNA genome.</p> Full article ">
17 pages, 19307 KiB  
Review
Ssu72 Dual-Specific Protein Phosphatase: From Gene to Diseases
by Soeun Hwang, Min-Hee Kim and Chang-Woo Lee
Int. J. Mol. Sci. 2021, 22(7), 3791; https://doi.org/10.3390/ijms22073791 - 6 Apr 2021
Cited by 7 | Viewed by 5783
Abstract
More than 70% of eukaryotic proteins are regulated by phosphorylation. However, the mechanism of dephosphorylation that counteracts phosphorylation is less studied. Phosphatases are classified into 104 distinct groups based on substrate-specific features and the sequence homologies in their catalytic domains. Among them, dual-specificity [...] Read more.
More than 70% of eukaryotic proteins are regulated by phosphorylation. However, the mechanism of dephosphorylation that counteracts phosphorylation is less studied. Phosphatases are classified into 104 distinct groups based on substrate-specific features and the sequence homologies in their catalytic domains. Among them, dual-specificity phosphatases (DUSPs) that dephosphorylate both phosphoserine/threonine and phosphotyrosine are important for cellular homeostasis. Ssu72 is a newly studied phosphatase with dual specificity that can dephosphorylate both phosphoserine/threonine and phosphotyrosine. It is important for cell-growth signaling, metabolism, and immune activation. Ssu72 was initially identified as a phosphatase for the Ser5 and Ser7 residues of the C-terminal domain of RNA polymerase II. It prefers the cis configuration of the serine–proline motif within its substrate and regulates Pin1, different from other phosphatases. It has recently been reported that Ssu72 can regulate sister chromatid cohesion and the separation of duplicated chromosomes during the cell cycle. Furthermore, Ssu72 appears to be involved in the regulation of T cell receptor signaling, telomere regulation, and even hepatocyte homeostasis in response to a variety of stress and damage signals. In this review, we aim to summarize various functions of the Ssu72 phosphatase, their implications in diseases, and potential therapeutic indications. Full article
(This article belongs to the Special Issue Proteins in Human Diseases: Molecular Insights into Drug Targets and Novel Therapeutic Modalities)
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<p>Summary of the functional role of Ssu72 phosphatase in transcription cycle. (<b>A</b>) During the initiation of transcription, Ssu72 can genetically and functionally interact with RNAPII subunits, TFIIB, and multiple kinases/phosphatases associated with carboxyl-terminal domain (CTD) modification, contributing to preinitiation complex (PIC) formation and transcription start site selection by RNA polymerase II (RNAPII). Furthermore, Ssu72 can regulate initiation–elongation and elongation–termination transitions by dephosphorylating both Ser5P and Ser7P. For example, Ssu72 along with Ser2P phosphatase Fcp1 can restore the hypophosphorylated form of CTD during elongation and induce Ser5P–Pro6 CTD conformation by interacting with Ess1, a proline isomerase, during transcription termination. (<b>B</b>) In the gene loop, Ssu72 can recycle RNAPII by negatively regulating the phosphorylation of Ser5P at the termination region. TFIIB directly binds to RNAPII associated with the terminator activated by Ssu72. In this way, Ssu72 can stabilize the promoter–terminator gene loop with TFIIB. In addition, Ssu72 affects promoter directionality in relation to gene loops. The ncRNA made by RNAPII often starts with bidirectional promoters that synthesize mRNA and ncRNA in opposite directions.</p> Full article ">Figure 2
<p>Schematic representation of Ssu72 phosphatase that regulates sister chromatid segregation during cell cycle. During the G1 phase, cohesin is regulated for the loading and unloading actions of NIPBL–MAU2 and PDS5–WAPL, respectively. In the subsequent step, Esco1/2 acetylates K105 and K106 of SMC3. Sororin reinforces the loading action via the recruitment of Pds5. In the late S phase, Ssu72 phosphatase can counteract the SA2 hyperphosphorylation associated with Cdk1 and Plk1, thus maintaining sister chromatid cohesion. However, the S19 residue with Ssu72 phosphatase activity is dephosphorylated through Aurora B-mediated phosphorylation in late G2 phase and prophase. Additionally, SA2 phosphorylation mediated by Plk1 can lead to the dissociation of cohesin from sister chromatids. Sororin is removed from cohesin by Aurora B. Finally, unresolved cohesins can retain their ring structure until the cleavage of the RAD21 subunit by separase. They can be reused in interphase after HDAC8 deacetylates the acetyl group of SMC3.</p> Full article ">Figure 3
<p>A physiopathological mechanism of Ssu72 regulating hepatic chromosome polyploidization and liver function during postnatal liver development. During postnatal development, Ssu72 plays an essential role in regulating hepatic chromosome polyploidization in a cell cycle-dependent manner. Depletion of Ssu72, which binds to and dephosphorylates Rb, contributes to the activation of E2F and then induces an aberrant DNA replication cycle by overriding the quiescence stage. Persistent elevation of the endoreplication process by Ssu72 depletion can facilitate the genesis of mononucleated polyploid hepatocytes, leading to extensive development of liver diseases such as NAFLD, fibrosis, and steatohepatitis. In addition, accumulation of liver damage such as from infection with HBV or HCV can affect the pathogenesis of HCC.</p> Full article ">Figure 4
<p>Ssu72 regulates autoimmune disease by controlling the homeostatic balance of T cell subsets. (<b>A</b>) Ssu72 regulates inflammatory responses by binding directly to STAT3. In the presence of Ssu72, Treg cell differentiation is increased while Th17 cell differentiation is decreased. mRNA levels of genes encoding proinflammatory cytokines, TBK1, and IKBKE are also downregulated. (<b>B</b>) Ssu72 is activated by various T cell receptor signaling molecules, including T cell receptor (TCR) and IL-2R. Activated Ssu72 can form a complex with PLCγ1, which is required for the development and function of Tregs. NFAT and ERK/JNK, downstream signaling molecules of PLCγ1, are then activated, promoting Foxp3<sup>+</sup>Treg induction. Ssu72 deficiency can prevent CD4 + T cell differentiation into Tregs at the peripheral region by inducing IL-2 and IFNγ, thus promoting CD4 + T cell activation.</p> Full article ">
14 pages, 4537 KiB  
Article
Argon Plasma Exposure Augments Costimulatory Ligands and Cytokine Release in Human Monocyte-Derived Dendritic Cells
by Sander Bekeschus, Dorothee Meyer, Kevin Arlt, Thomas von Woedtke, Lea Miebach, Eric Freund and Ramona Clemen
Int. J. Mol. Sci. 2021, 22(7), 3790; https://doi.org/10.3390/ijms22073790 - 6 Apr 2021
Cited by 16 | Viewed by 3074
Abstract
Cold physical plasma is a partially ionized gas expelling many reactive oxygen and nitrogen species (ROS/RNS). Several plasma devices have been licensed for medical use in dermatology, and recent experimental studies suggest their putative role in cancer treatment. In cancer therapies with an [...] Read more.
Cold physical plasma is a partially ionized gas expelling many reactive oxygen and nitrogen species (ROS/RNS). Several plasma devices have been licensed for medical use in dermatology, and recent experimental studies suggest their putative role in cancer treatment. In cancer therapies with an immunological dimension, successful antigen presentation and inflammation modulation is a key hallmark to elicit antitumor immunity. Dendritic cells (DCs) are critical for this task. However, the inflammatory consequences of DCs following plasma exposure are unknown. To this end, human monocyte-derived DCs (moDCs) were expanded from isolated human primary monocytes; exposed to plasma; and their metabolic activity, surface marker expression, and cytokine profiles were analyzed. As controls, hydrogen peroxide, hypochlorous acid, and peroxynitrite were used. Among all types of ROS/RNS-mediated treatments, plasma exposure exerted the most notable increase of activation markers at 24 h such as CD25, CD40, and CD83 known to be crucial for T cell costimulation. Moreover, the treatments increased interleukin (IL)-1?, IL-6, and IL-23. Altogether, this study suggests plasma treatment augmenting costimulatory ligand and cytokine expression in human moDCs, which might exert beneficial effects in the tumor microenvironment. Full article
(This article belongs to the Special Issue Plasma Biology)
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<p>Study protocol and toxicity comparison. (<b>a</b>) Image of argon plasma treatment of cells in 24-well plates; (<b>b</b>) scheme of study protocol; (<b>c</b>,<b>d</b>) overlay 4′,6-diamidino-2-phenylindole (DAPI) histograms of monocyte-derived DCs (moDCs) and lymphocytes according to the indicated treatments (<b>c</b>) and quantification of the percentage of viable moDCs and lymphocytes (<b>d</b>) treated together in a single well. Data are mean and standard error of three experiments, and statistical analysis was performed using t-test with <span class="html-italic">p</span> &lt; 0.001 (***) differing significantly or non-significantly (ns).</p> Full article ">Figure 2
<p>Argon plasma treatment and reactive oxygen and nitrogen species (ROS/RNS) have dose-dependent toxicity profiles. (<b>a</b>) Kinetic metabolic activity of moDCs treated as indicated over 6 h; (<b>b</b>–<b>f</b>) metabolic activity in response to several argon plasma treatments times or concentrations of ROS/RNS or LPS at 24 h; (<b>g</b>–<b>k</b>) viability in response to several argon plasma treatment times or concentrations of ROS/RNS or lipopolysaccharide (LPS) at 24 h; and (<b>l</b>) heatmap for comparison between reduction in metabolic activity and viability. Data are mean and standard error of 3–6 different donors.</p> Full article ">Figure 3
<p>Argon plasma treatment and ROS/RNS modulate the surface marker expression profiles. (<b>a</b>) overlay histograms of several cell surface markers of unstained, stained, stained, and LPS-pulsed human moDCs; (<b>b</b>) quantification and fold-change differences in human moDCs treated as indicated and analyzed 24 h later; (<b>c</b>) data summary using principal-component analysis. Data are mean and standard error of 3–6 different donors. Statistical analysis was done using one-way analysis of variances with <span class="html-italic">p</span> &lt; 0.05 (*), <span class="html-italic">p</span> &lt; 0.01 (**), and <span class="html-italic">p</span> &lt; 0.001 (***).</p> Full article ">Figure 4
<p>Argon plasma treatment and ROS/RNS modulate the cytokine release profiles. Cells were treated as indicated, supernatants were collected 24 h later, and absolute cytokine concentrations of 10 analytes (<b>a</b>–<b>j</b>) were assessed. All data were also related to each other using principal component analysis (<b>k</b>). Data are violin plots and median (red lines) of 3–6 different donors. Statistical analysis was done using one-way analysis of variances with <span class="html-italic">p</span> &lt; 0.05 (*). Dashed lines show values of LPS-positive controls of moDCs included for the analytes.</p> Full article ">Figure A1
<p>Standard curves from the multiplex cytokine analysis for each analyte across a range of concentrations shown together with the corresponding average mean fluorescence intensities (Avg(MFI)). All curve fittings had an <span class="html-italic">R</span><sup>2</sup> of greater 0.99, providing great accuracy for the quantification of absolute cytokine levels in moDC culture supernatants.</p> Full article ">
12 pages, 297 KiB  
Review
Chronic Low Grade Inflammation in Pathogenesis of PCOS
by Ewa Rudnicka, Katarzyna Suchta, Monika Grymowicz, Anna Calik-Ksepka, Katarzyna Smolarczyk, Anna M. Duszewska, Roman Smolarczyk and Blazej Meczekalski
Int. J. Mol. Sci. 2021, 22(7), 3789; https://doi.org/10.3390/ijms22073789 - 6 Apr 2021
Cited by 320 | Viewed by 28445
Abstract
Polycystic ovary syndrome (PCOS) is a one of the most common endocrine disorders, with a prevalence rate of 5–10% in reproductive aged women. It’s characterized by (1) chronic anovulation, (2) biochemical and/or clinical hyperandrogenism, and (3) polycystic ovarian morphology. PCOS has significant clinical [...] Read more.
Polycystic ovary syndrome (PCOS) is a one of the most common endocrine disorders, with a prevalence rate of 5–10% in reproductive aged women. It’s characterized by (1) chronic anovulation, (2) biochemical and/or clinical hyperandrogenism, and (3) polycystic ovarian morphology. PCOS has significant clinical implications and can lead to health problems related to the accumulation of adipose tissue, such as obesity, insulin resistance, metabolic syndrome, and type 2 diabetes. There is also evidence that PCOS patients are at higher risk of cardiovascular diseases, atherosclerosis, and high blood pressure. Several studies have reported the association between polycystic ovary syndrome (PCOS) and low-grade chronic inflammation. According to known data, inflammatory markers or their gene markers are higher in PCOS patients. Correlations have been found between increased levels of C-reactive protein (CRP), interleukin 18 (IL-18), tumor necrosis factor (TNF-?), interleukin 6 (IL-6), white blood cell count (WBC), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1? (MIP-1?) in the PCOS women compared with age- and BMI-matched controls. Women with PCOS present also elevated levels of AGEs and increased RAGE (receptor for advanced glycation end products) expression. This chronic inflammatory state is aggravating by obesity and hyperinsulinemia. There are studies describing mutual impact of hyperinsulinemia and obesity, hyperandrogenism, and inflammatory state. Endothelial cell dysfunction may be also triggered by inflammatory cytokines. Many factors involved in oxidative stress, inflammation, and thrombosis were proposed as cardiovascular risk markers showing the endothelial cell damage in PCOS. Those markers include asymmetric dimethylarginine (ADMA), C-reactive protein (CRP), homocysteine, plasminogen activator inhibitor-I (PAI-I), PAI-I activity, vascular endothelial growth factor (VEGF) etc. It was also proposed that the uterine hyperinflammatory state in polycystic ovary syndrome may be responsible for significant pregnancy complications ranging from miscarriage to placental insufficiency. In this review, we discuss the most importance evidence concerning the role of the process of chronic inflammation in pathogenesis of PCOS. Full article
(This article belongs to the Special Issue Polycystic Ovary Syndrome: From Molecular Mechanisms to Therapies)
20 pages, 402 KiB  
Review
Recent Perspectives on Sex Differences in Compulsion-Like and Binge Alcohol Drinking
by Anna K. Radke, Elizabeth A. Sneddon, Raizel M. Frasier and Frederic W. Hopf
Int. J. Mol. Sci. 2021, 22(7), 3788; https://doi.org/10.3390/ijms22073788 - 6 Apr 2021
Cited by 62 | Viewed by 6516
Abstract
Alcohol use disorder remains a substantial social, health, and economic problem and problem drinking levels in women have been increasing in recent years. Understanding whether and how the underlying mechanisms that drive drinking vary by sex is critical and could provide novel, more [...] Read more.
Alcohol use disorder remains a substantial social, health, and economic problem and problem drinking levels in women have been increasing in recent years. Understanding whether and how the underlying mechanisms that drive drinking vary by sex is critical and could provide novel, more targeted therapeutic treatments. Here, we examine recent results from our laboratories and others which we believe provide useful insights into similarities and differences in alcohol drinking patterns across the sexes. Findings for binge intake and aversion-resistant, compulsion-like alcohol drinking are considered, since both are likely significant contributors to alcohol problems in humans. We also describe studies regarding mechanisms that may underlie sex differences in maladaptive alcohol drinking, with some focus on the importance of nucleus accumbens (NAcb) core and shell regions, several receptor types (dopamine, orexin, AMPA-type glutamate), and possible contributions of sex hormones. Finally, we discuss how stressors such as early life stress and anxiety-like states may interact with sex differences to contribute to alcohol drinking. Together, these findings underscore the importance and critical relevance of studying female and male mechanisms for alcohol and co-morbid conditions to gain a true and clinically useful understanding of addiction and neuropsychiatric mechanisms and treatment. Full article
(This article belongs to the Special Issue Models of Drug Addiction: Translating Molecular Targets into Molecular Medicine)
15 pages, 2731 KiB  
Article
Phosphorylation of GAPVD1 Is Regulated by the PER Complex and Linked to GAPVD1 Degradation
by Hussam Ibrahim, Philipp Reus, Anna Katharina Mundorf, Anna-Lena Grothoff, Valerie Rudenko, Christina Buschhaus, Anja Stefanski, Niklas Berleth, Björn Stork, Kai Stühler, Faiza Kalfalah and Hans Reinke
Int. J. Mol. Sci. 2021, 22(7), 3787; https://doi.org/10.3390/ijms22073787 - 6 Apr 2021
Cited by 2 | Viewed by 3753
Abstract
Repressor protein period (PER) complexes play a central role in the molecular oscillator mechanism of the mammalian circadian clock. While the main role of nuclear PER complexes is transcriptional repression, much less is known about the functions of cytoplasmic PER complexes. We found [...] Read more.
Repressor protein period (PER) complexes play a central role in the molecular oscillator mechanism of the mammalian circadian clock. While the main role of nuclear PER complexes is transcriptional repression, much less is known about the functions of cytoplasmic PER complexes. We found with a biochemical screen for PER2-interacting proteins that the small GTPase regulator GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), which has been identified previously as a component of cytoplasmic PER complexes in mice, is also a bona fide component of human PER complexes. We show that in situ GAPVD1 is closely associated with casein kinase 1 delta (CSNK1D), a kinase that regulates PER2 levels through a phosphoswitch mechanism, and that CSNK1D regulates the phosphorylation of GAPVD1. Moreover, phosphorylation determines the kinetics of GAPVD1 degradation and is controlled by PER2 and a C-terminal autoinhibitory domain in CSNK1D, indicating that the regulation of GAPVD1 phosphorylation is a novel function of cytoplasmic PER complexes and might be part of the oscillator mechanism or an output function of the circadian clock. Full article
(This article belongs to the Section Molecular Biology)
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<p>Affinity liquid chromatography–mass spectrometry (LC-MS) and gene ontology (GO) enrichment analyses of Repressor protein period 2 (PER2)-interacting proteins in human HT1080 cells. (<b>A</b>) Volcano plot of all proteins identified by affinity LC-MS. Enrichment is plotted as fold change (FC) against -log10(<span class="html-italic">p</span>-value) (<span class="html-italic">t</span>-test, n = 3). Crosslines delimit proteins with FC &gt; 5 and <span class="html-italic">p</span> &lt; 0.0005 highlighted in red; (<b>B</b>) Heat map of significantly enriched PER2-interacting proteins from control (GFP#1–3) and bait (PER2-GFP#1-3) purification replicates (FC &gt; 0, <span class="html-italic">p</span> &lt; 0.05). Colors indicate the distance of a signal from the sample mean in standard deviations (Z-score). Boxes highlight protein complexes. The heat map was created using heatmapper software [<a href="#B18-ijms-22-03787" class="html-bibr">18</a>]; (<b>C</b>,<b>D</b>) GO analysis of PER2-interacting proteins purged for tubulins, proteasomal, and ribosomal proteins, which are highly abundant in cells and often create an unspecific background in LC-MS experiments. The revised protein list was analyzed with DAVID [<a href="#B19-ijms-22-03787" class="html-bibr">19</a>] for the enrichment of GO terms in the categories biological process (BP) (<b>C</b>) and cellular component (CC) (<b>D</b>). The lists are sorted by increasing <span class="html-italic">p</span>-value shown as red bars (range: BP 1.2 × 10<sup>−8</sup> − 0.004; CC 1.3 × 10<sup>−10</sup> − 0.004). The number of genes in each group (gene count) is shown as blue bars (range: BP 3 − 12; CC 3 − 58).</p> Full article ">Figure 2
<p>In situ detection of GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)-PER2 and GAPVD1-casein kinase 1 delta (CSNK1D) protein complexes by proximity ligation assay (PLA). (<b>A</b>) Immunofluorescence microscopy overlay images of PLA with primary antibodies against GAPVD1 and PER2 or (<b>B</b>) GAPVD1 and CSNK1D in wild type HT1080 cells. Red: PLA signals; cyan: DAPI. Lower panels: Quantification of PLA, including controls with only one primary antibody each. Data points represent single cells on at least five different microscopy slides in three independent experiments; **** <span class="html-italic">p</span> &lt; 5 × 10<sup>−5</sup> (<span class="html-italic">t</span>-test).</p> Full article ">Figure 3
<p>Downregulation of the PER complex components or inhibition of CSNK1D/E kinase activity modulates GAPVD1 phosphorylation levels in HeLa cells (<b>A</b>) Western blot analysis of GAPVD1 after treatment with siRNAs against CSNK1D (C1D), CSNK1D/E (C1D/E), PER2 (P2), PER1/2 (P1/2), or non-target siRNA (nt) in HeLa cells; (<b>B</b>) Western blot analysis of GAPVD1 after treatment of HeLa cells with increasing concentrations of PF670462 for 18 h. An antibody against Calnexin (CANX) was used as a control for equal loading. Lower panels: Quantification of GAPVD1 protein levels shown as the ratio between the faster (lower) and slower (higher) migrating GAPVD1 protein band normalized to cells treated with nt siRNA or mock-treated cells. Circles show single data points; error bars show standard deviations. * <span class="html-italic">p</span> &lt; 0.05 (<span class="html-italic">t</span>-test).</p> Full article ">Figure 4
<p>CSNK1D-dependent GAPVD1 phosphorylation is regulated by PER2 (<b>A</b>) Western blot analysis of GAPVD1 in HT1080 wild type cells, HT1080 cells overexpressing CSNK1D-GFP (C), PER2-GFP (P) or PER2-GFP and CSNK1D-RFP (CP) were analyzed with an antibody against human GAPVD1; (<b>B</b>) Clonal CP cells (CP#1–3) were analyzed with antibodies against GAPVD1, RFP, and GFP; (<b>C</b>) HT1080 wild type, and CP cells were analyzed untreated, mock-treated (phosphatase buffer, no calf intestinal phosphatase (CIP)) or CIP-treated (phosphatase buffer and CIP) and analyzed with an antibody against GAPVD1. HT1080 cells overexpressing GAPVD1-GFP were included as size marker; (<b>D</b>) CP cells were treated with increasing amounts (20 nM, 200 nM, 2 µM, 20 µM) of PF670462 for 18 h. Cells were harvested directly after treatment or allowed to recover for 24 h after withdrawal of the drug and analyzed with an antibody against GAPVD1. Antibodies against U2 small nuclear RNA auxiliary factor 2 (U2AF2) and Calnexin (CANX) were used as a control for equal loading.</p> Full article ">Figure 5
<p>Phosphorylation of GAPVD1 is inhibited by the C-terminal tail of CSNK1D and requires direct interaction of CSNK1D and PER2. (<b>A</b>) Western blot analysis of GAPVD1 in HT1080 wild type cells, CP cells, and different clonal cell lines expressing CSNK1D(Δ317-342)-RFP (C(ΔC)#1-2) or CSNK1D-RFP (C#1–3); (<b>B</b>) HT1080 wild type cells and cells overexpressing CSNK1D-RFP or CSNK1D(Δ317–342)-RFP together with PER2-GFP (CP and C(ΔC)P), and cells overexpressing CSNK1D-GFP alone (<b>C</b>) were analyzed with antibodies against GAPVD1, CSNK1D, and PER2; an antibody against Calnexin (CANX) was used as a control for equal loading; dashed lines indicate lane splicing within one and the same Western blot membrane; (<b>C</b>) Western blot analysis of GAPVD1 in HT1080 wild type cells and cells expressing CSNK1D-RFP together with PER2-GFP (CP) or PER2(Δ489–618)-GFP (CP(Δ)); (<b>D</b>) CSNK1D was immunoprecipitated from CP or CP(Δ) cells with or without pre-treatment with PF-670462 for 18 h. Cell lysates (Input) and immunoprecipitated proteins were analyzed with antibodies against CSNK1D and PER2; (<b>E</b>) CSNK1D or GAPVD1 were immunoprecipitated from synchronized U2OS cells at the indicated circadian times (CT) after dexamethasone treatment, and immunoprecipitated proteins were analyzed with antibodies against CSNK1D, PER2, and GAPVD1. Similar levels of the non-rhythmic CSNK1D protein indicate equal IP efficiencies in all samples.</p> Full article ">Figure 6
<p>Phosphorylation regulates the speed of GAPVD1 degradation. (<b>A</b>) The ratio of phosphorylated to unphosphorylated peptides in each sample is shown for all peptides that were identified in HT1080 wild type cells (black), CP cells (grey), or wild type cells pre-treated with 20 µM PF670462 for 18 h (white). Circles show single data points; error bars show standard deviations. Samples in which neither a phosphorylated nor an unphosphorylated peptide was detected for a specific phosphorylation site (P-site) were omitted from the dataset. The locations of P-sites relative to the RasGAP and VPS9 domains and a proline rich region (PRR) in GAPVD1 are shown underneath [<a href="#B26-ijms-22-03787" class="html-bibr">26</a>]; (<b>B</b>) Degradation kinetics of GAPVD1 after treatment with 50 µM cycloheximide in HT1080 wild type cells (black), CP cells (grey) or wild type cells pre-treated with 20 µM PF670462 for 18h (white). GAPVD1 protein levels were normalized to Calnexin, and t = 0 values were set to 1. Lines connect replicate means (n = 4); error bars show standard errors of mean. * <span class="html-italic">p</span> &lt; 0.05 (<span class="html-italic">t</span>-test).</p> Full article ">
15 pages, 4523 KiB  
Article
Hemi- and Homozygous Loss-of-Function Mutations in DSG2 (Desmoglein-2) Cause Recessive Arrhythmogenic Cardiomyopathy with an Early Onset
by Andreas Brodehl, Alexey Meshkov, Roman Myasnikov, Anna Kiseleva, Olga Kulikova, Bärbel Klauke, Evgeniia Sotnikova, Caroline Stanasiuk, Mikhail Divashuk, Greta Marie Pohl, Maria Kudryavtseva, Karin Klingel, Brenda Gerull, Anastasia Zharikova, Jan Gummert, Sergey Koretskiy, Stephan Schubert, Elena Mershina, Anna Gärtner, Polina Pilus, Kai Thorsten Laser, Valentin Sinitsyn, Sergey Boytsov, Oxana Drapkina and Hendrik Miltingadd Show full author list remove Hide full author list
Int. J. Mol. Sci. 2021, 22(7), 3786; https://doi.org/10.3390/ijms22073786 - 6 Apr 2021
Cited by 23 | Viewed by 4817
Abstract
About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown [...] Read more.
About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown and a genetic contribution might be underestimated. Here, we used a next-generation sequencing (NGS) approach and in addition single nucleotide polymor-phism (SNP) arrays for the genetic analysis of two independent index patients without familial medical history. Of note, this genetic strategy revealed a homozygous splice site mutation (DSG2–c.378+1G>T) in the first patient and a nonsense mutation (DSG2–p.L772X) in combination with a large deletion in DSG2 in the second one. In conclusion, a recessive inheritance pattern is likely for both cases, which might contribute to the hidden medical history in both families. This is the first report about these novel loss-of-function mutations in DSG2 that have not been previously identi-fied. Therefore, we suggest performing deep genetic analyses using NGS in combination with SNP arrays also for ACM index patients without obvious familial medical history. In the future, this finding might has relevance for the genetic counseling of similar cases. Full article
(This article belongs to the Special Issue Genetic Basis and Molecular Mechanisms of Heart Rhythm Disorders)
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<p>Pedigrees of the described families. (<b>A</b>) Family A has a South Asian origin. The male index patient (II-1) received his diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) at the age of 12. (<b>B</b>) Family B has a Russian origin. Circles represent females, squares males. Black-filled symbols indicate a cardiac phenotype and white symbols indicate healthy family members. +/− indicates heterozygous, +/+ homozygous, and +/0 hemizygous status. Index patients are marked with an arrow. Obligate carriers are shown with a black dot in the pedigree symbol.</p> Full article ">Figure 2
<p>Clinical data of II-I, Family A. (<b>A–B</b>) Cardiac magnetic resonance imaging (MRI) revealed a dilated right ventricle with reduced global function and segmental bulging. Hematoxylin and eosin (<b>C</b>) and trichrome staining (<b>D</b>) of a right-ventricular biopsy revealed subendocardial fibrosis and degeneration of cardiomyocytes. Scale bars represent 50 µm. (<b>E–F</b>) Right axis deviation, at the age of 12 years still incomplete, later complete right bundle branch block, inverted T-waves in the right precordial leads and non-sustained ventricular tachycardia were present in the 12-lead electrocardiogram (ECG).</p> Full article ">Figure 3
<p>Speckle tracking echocardiography (STE) demonstrated dyssynchrony of the septal and right ventricular free-wall segments (see also <a href="#app1-ijms-22-03786" class="html-app">Video S1, Supplementary Data</a>).</p> Full article ">Figure 4
<p>Clinical data of Family B. (<b>A–I</b>) Cardiac magnetic resonance imaging (MRI) of index patient (II-2, Family B). Asterisk indicates thinning and aneurysmal protrusion of right ventricular wall (<b>A–C</b>). Arrows indicate ischemic changes in left ventricle (<b>D,F</b>). Dotted arrows indicate thrombosis in the right ventricle (<b>B,E</b>). Ellipse shows fibrosis of the right ventricle (<b>F</b>). Triangle demonstrates LV non-compaction (<b>G–I</b>). Rhombus demonstrates RV non-compaction (<b>G,I</b>). (<b>J</b>) Epsilon waves were present in the 12-lead ECG. (<b>K</b>) Tachycardia was present in the Holter monitoring-ECG.</p> Full article ">Figure 5
<p>Cardiac magnetic resonance imaging (MRI) of the I-1 (<b>A</b>), I-2 (<b>B,D–F</b>), and II -1 (<b>C</b>) of Family B. Asterisk indicates non-compaction layer.</p> Full article ">Figure 6
<p>Genetic analysis of II-1 (Family A). (<b>A</b>) Integrated genome view of exon 4 in <span class="html-italic">DSG2</span> of patient II-1. (<b>B</b>) Electropherogram of exon 4 in <span class="html-italic">DSG2</span> (II-1). The mutation DSG2–c.378+1G&gt;T is affecting the donor splice site and is found in a homozygous status. (<b>C</b>) Karyoview of II-1. LOH = loss of heterozygosity. (<b>D</b>) Detailed ideogram of human chromosome 18 revealing a 15.6 Mb loss of heterozygosity (LOH).</p> Full article ">Figure 7
<p>(<b>A–I</b>) Genetic analysis of Family B. (<b>A</b>) Integrated genome view of exon 14 in <span class="html-italic">DSG2</span> of patient II-2 (Family B). (<b>B–F</b>) Partial electropherograms of <span class="html-italic">DSG2</span> exon 14 revealed for I-1 two heterozygous variants p.L772X and p.R773K (<b>B</b>), for I‑2 a wild-type sequence (<b>C</b>), for II-1 the heterozygous variant p.R773K (<b>D</b>), for II-2 hemizygous p.L772X (<b>E</b>), and for II-3 hemizygous p.R773K (<b>F</b>). (<b>G</b>) Karyoview of II-2 (Family B). LOH = loss of heterozygosity. (<b>H–I</b>) Detailed ideogram of human chromosome 18 revealing an additional deletion on the second chromosome affecting nearly the complete <span class="html-italic">DSG2</span> gene.</p> Full article ">
18 pages, 2129 KiB  
Review
In Vitro Activation Early Follicles: From the Basic Science to the Clinical Perspectives
by Kim Cat Tuyen Vo and Kazuhiro Kawamura
Int. J. Mol. Sci. 2021, 22(7), 3785; https://doi.org/10.3390/ijms22073785 - 6 Apr 2021
Cited by 36 | Viewed by 8604
Abstract
Development of early follicles, especially the activation of primordial follicles, is strictly modulated by a network of signaling pathways. Recent advance in ovarian physiology has been allowed the development of several therapies to improve reproductive outcomes by manipulating early folliculogenesis. Among these, in [...] Read more.
Development of early follicles, especially the activation of primordial follicles, is strictly modulated by a network of signaling pathways. Recent advance in ovarian physiology has been allowed the development of several therapies to improve reproductive outcomes by manipulating early folliculogenesis. Among these, in vitro activation (IVA) has been recently developed to extend the possibility of achieving genetically related offspring for patients with premature ovarian insufficiency and ovarian dysfunction. This method was established based on basic science studies of the intraovarian signaling pathways: the phosphoinositide 3-kinase (PI3K)/Akt and the Hippo signaling pathways. These two pathways were found to play crucial roles in folliculogenesis from the primordial follicle to the early antral follicle. Following the results of rodent experiments, IVA was implemented in clinical practice. There have been multiple recorded live births and ongoing pregnancies. Further investigations are essential to confirm the efficacy and safety of IVA before used widely in clinics. This review aimed to summarize the published literature on IVA and provide future perspectives for its improvement. Full article
(This article belongs to the Special Issue Molecular Basis of Fertility Preservation and Restoration 3.0)
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<p>The schematic folliculogenesis from primordial to ovulatory stages.</p> Full article ">Figure 2
<p>The phosphoinositide 3-kinase (PI3K)/Akt/ forkhead box O3 (FOXO3) pathway in oocytes regulates PF activation. Upon growth factors binding to tyrosine kinase receptors (Kit ligand, insulin-like growth factor (IGF), epidermal growth factor (EGF), etc.), the autophosphorylation of intracellular regions of these receptors takes place. Activated receptors then stimulate PI3K activity, leading to increases in phosphatidylinositol-3,4,5-triphosphate (PIP3) levels and phosphatidylinositol-dependent kinase 1 (PDK1) as well as Akt stimulation. Activated Akt then migrates to the cell nucleus and suppresses FOXO3 actions to promote primordial follicle growth [<a href="#B8-ijms-22-03785" class="html-bibr">8</a>,<a href="#B10-ijms-22-03785" class="html-bibr">10</a>]. Akt also promotes the phosphorylation of tuberous sclerosis 2 (TCS2), which enhances mammalian target of rapamycin complex (mTORC1) activation. S6 kinase (S6K), ribosomal protein S6 (rpS6) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) are the downstream substrates of mTORC1 which promote the translation of related mRNAs following by cell growth and proliferation.</p> Full article ">Figure 3
<p>Tissue fragmentation and mechanical manipulation disrupt ovarian Hippo signaling pathway and promote follicle growth. (<b>Left</b>) Without tissue fragmentation and mechanical manipulation, Hippo signaling is activated. Kinase complexes consisting of the mammalian sterile-20 like serine/threonine kinase 1/2 (MST 1/2) and Salvador (SAV) are phosphorylated. Subsequently, large tumor suppressor 1/2 (LATS1/2) phosphorylates Yes-associated protein (YAP)/ transcriptional co-activator PDZ-binding motif (TAZ), leading to the degradation of YAP/TAZ or retention of YAP/TAZ in the cytoplasm. Consequently, transcriptional enhanced associate domains (TEADs) cannot promote the expression of target genes. (<b>Right</b>) Ovarian fragmentation leads to actin polymerization, resulting in nuclear translocation of YAP. Nuclear YAP interacted with TEADs to increase the expression CCN growth factors and baculoviral inhibitors of apoptosis repeat containing (BIRC) apoptosis inhibitors, resulting in follicle growth.</p> Full article ">Figure 4
<p>Comparison of the clinical approach between conventional IVA and drug-free IVA. In conventional IVA, one or both ovaries from POI patients were removed under laparoscopic surgery and cut into strips before vitrification. After thawing, strips were fragmented into 1–2 mm cubes, before incubation with Akt stimulators. Two days later, cultured cubes were autografted under second laparoscopic surgery beneath the serosa of Fallopian tubes. In drug-free IVA, the ovarian cortical tissues were removed and fragmented into 1–2 mm cubes followed by grafting back without culture in remaining ovaries and beneath the serosa of Fallopian tubes within the same operation.</p> Full article ">Figure 5
<p>The schematic hypothesis for correlation of polycystic ovary syndrome (PCOS) and Hippo signaling pathway. (1) The mechanical damage and/or degrading enzyme approaches or (2) local administration of the Hippo downstream CCN growth factors and/or actin polymerization drugs for disruption of Hippo signaling aim to ameliorate the dysregulation of Hippo signaling pathway in PCOS patients for resumption of follicle growth.</p> Full article ">
13 pages, 2259 KiB  
Article
Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells
by Véronique Noé and Carlos J. Ciudad
Int. J. Mol. Sci. 2021, 22(7), 3784; https://doi.org/10.3390/ijms22073784 - 6 Apr 2021
Cited by 5 | Viewed by 2444
Abstract
Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells [...] Read more.
Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases. Full article
(This article belongs to the Special Issue Oligonucleotide, Therapy, and Applications 2.0)
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Graphical abstract
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<p>Organization of the pD22 <span class="html-italic">DHFR</span> minigene stably transfected in NB6 cells. A 0.8-kb PstI–BstEII genomic DNA fragment containing exon 2 and flanks of the Chinese hamster <span class="html-italic">DHFR</span> gene was cloned into the PstI site in intron 1 of pDCH1P to obtain the pD22 construct as described in Chen and Chasin [<a href="#B25-ijms-22-03784" class="html-bibr">25</a>].</p> Full article ">Figure 2
<p>General approach for the design of the different editing PPRHs to edit the extra sequence (in light blue) including the additional exon 2 present in the <span class="html-italic">DHFR</span> minigene pD22. The complete structure for each editing PPRH contained a PPRH core and a sequence tail in its 5’ end homologous to 20 nt upstream (in purple) and/or 20 nt downstream (in blue) of the PstI restriction site present in the original pDCHIP minigene.</p> Full article ">Figure 3
<p>(<b>A</b>) PCR products from genomic DNA using a pair of primers in DHFR exon 1 and exon 3, respectively, obtained from either NB6 cells or random edited colonies, which were used in the sequencing reactions. (<b>B</b>) Highlighted in blue, the <span class="html-italic">DHFR</span> DNA sequence from exon 1 to exon 3 in the edited colonies compared to the original pD22 minigene, in which the extra sequence including the additional copy of exon 2 and its flanking regions is highlighted in grey. The sequences corresponding to the primers used to amplify the genomic DNA are underlined. Exons are indicated in uppercase whereas the intronic sequence is indicated in lowercase.</p> Full article ">Figure 4
<p>DHFR mRNA analysis in the edited clones. (<b>A</b>) Lanes 1 to 5, PCR products representative of random edited colonies from LDSHpPrI1UDPstI; lanes 6 to 11, PCR products representative of random edited colonies from LDSHpE3I1UDPstI. The species originating from NB6, UA21 and K1 cells (wildtype), respectively, correspond to the three last lanes. (<b>B</b>) Lanes 1 to 5, PCR products representative of random edited colonies from LDSHpE6I1UDPstI; lanes 6 to 13, PCR products representative of random edited colonies obtained with the combination of LDSHpPrI1UDPstI and LDSHpE3I1UDPstI. The species originating from NB6, UA21 and K1 cells (wildtype), respectively, are shown in the last three lanes.</p> Full article ">Figure 5
<p>Intron retention in DHFR mRNA from edited clones. (<b>A</b>) Map of the locations of hybridization of the primers used in the analysis. (<b>B</b>) and (<b>C</b>) Lanes 1 and 2, PCR products representative of random edited colonies from LDSHpPrI1UDPstI; lanes 3 and 4, PCR products representative of random edited colonies from LDSHpE3I1UDPstI; lanes 6 and 7, PCR products representative of random edited colonies from LDSHpE6I1UDPstI; and lanes 8 to 13, PCR products representative of random edited colonies originating from the combination of LDSHpPrI1UDPstI and LDSHpE3I1UDPstI; lane NB6, species originating from NB6 cells.</p> Full article ">Figure 6
<p>Levels of the DHFR protein. (<b>A</b>) Representative blot of the DHFR protein in total extracts from edited colonies obtained upon transfection with either LDHpPrI1UDPstI, LDHpE3I1UDPstI or LDHpE6I1UDPstI, NB6 or UA21 cells. (<b>B</b>) Representative blot of the DHFR protein in total extracts from edited colonies obtained upon transfection with the combination of LDHpPrI1UDPstI and LDHpE3I1UDPstI, NB6 or UA21 cells. Tubulin signal was used in all samples to normalize the results.</p> Full article ">Figure 7
<p>DHFR activity in control NB6 cells and random edited colonies obtained from transfections with the different editing PPRHs. Results are represented as the means ± SD values of three independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">
14 pages, 3062 KiB  
Article
Novel and Potent Small Molecules against Melanoma Harboring BRAF Class I/II/III Mutants for Overcoming Drug Resistance
by Namkyoung Kim, Injae Shin, Jiwon Lee, Eunhye Jeon, Younghoon Kim, Seongshick Ryu, Eunhye Ju, Wonjeong Cho and Taebo Sim
Int. J. Mol. Sci. 2021, 22(7), 3783; https://doi.org/10.3390/ijms22073783 - 6 Apr 2021
Cited by 10 | Viewed by 4140
Abstract
Melanoma accounts for the majority of skin cancer deaths. About 50% of all melanomas are associated with BRAF mutations. BRAF mutations are classified into three classes with regard to dependency on RAF dimerization and RAS signaling. The most frequently occurring class I BRAF [...] Read more.
Melanoma accounts for the majority of skin cancer deaths. About 50% of all melanomas are associated with BRAF mutations. BRAF mutations are classified into three classes with regard to dependency on RAF dimerization and RAS signaling. The most frequently occurring class I BRAF V600 mutations are sensitive to vemurafenib whereas class II and class III mutants, non-V600 BRAF mutants are resistant to vemurafenib. Herein we report six pyrimido[4,5-d]pyrimidin-2-one derivatives possessing highly potent anti-proliferative activities on melanoma cells harboring BRAF class I/II/III mutants. Novel and most potent derivative, SIJ1777, possesses not only two-digit nanomolar potency but also 2 to 14-fold enhanced anti-proliferative activities compared with reference compound, GNF-7 against melanoma cells (SK-MEL-2, SK-MEL-28, A375, WM3670, WM3629). Moreover, SIJ1777 substantially inhibits the activation of MEK, ERK, and AKT and remarkably induces apoptosis and significantly blocks migration, invasion, and anchorage-independent growth of melanoma cells harboring BRAF class I/II/II mutations while both vemurafenib and PLX8394 have little to no effects on melanoma cells expressing BRAF class II/III mutations. Taken together, our six GNF-7 derivatives exhibit highly potent activities against melanoma cells harboring class I/II/III BRAF mutations compared with vemurafenib as well as PLX8394. Full article
(This article belongs to the Special Issue Precision Oncology in Melanoma Progression)
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<p>Docking model prediction of SIJ1777 on BRAF V600E mutant. H-bond interactions are indicated with dashed lines.</p> Full article ">Figure 2
<p>Chemical structures of GNF-7 and its derivatives.</p> Full article ">Figure 3
<p>The effect of SIJ1777 on AKT and MAPK signaling pathways in melanoma cell lines harboring various BRAF mutation status (<b>A</b>) SK-MEL-2 (wt) (<b>B</b>) SK-MEL-28 (class I) (<b>C</b>) C8161 (class II) (<b>D</b>) WM3670, WM3629 (class III). Cells were treated with 0.01, 0.1, 1 μM of SIJ1777, and 1 μM of vemurafenib, PLX8394, GNF-7, and SIJ1227 for 2 h. Cell lysates were subjected to western blot analysis to estimate the phospho- or total- form of AKT, MEK, ERK levels, and GAPDH was used as the internal loading controls.</p> Full article ">Figure 4
<p>The effect of SIJ1777 on apoptosis induction. (<b>A</b>) Western blot for pro-apoptotic marker level (cleaved PARP) in melanoma cell lines. GAPDH was used as the internal loading control. (<b>B</b>) Quantification graphs of western blot results by ImageJ (<span class="html-italic">n</span> = 3). (<b>C</b>) Apoptotic cell (annexin V-positive) population was measured by flow cytometry analysis against melanoma cell lines (<span class="html-italic">n</span> = 3). Cells were treated with indicated substances for 24 h. Statistical significances were determined using a one-way ANOVA analysis (* <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">p</span> &lt; 0.0001).</p> Full article ">Figure 5
<p>The effect of SIJ1777 on migration and invasion of melanoma cells harboring BRAF wt or class I/II/II mutations. (<b>A</b>) Scratch assay results for assessing migration capability. After scratching each cell monolayer, indicated compounds at 0.01 μM concentration were incubated for 12 h. Migration ratio was analyzed using migrated area using ImageJ (<span class="html-italic">n</span> = 3). (<b>B</b>) Boyden chamber assay for assessing invasion capability using cell invasion kit (QCM ECMatrix Cell Invasion Assay, <span class="html-italic">n</span> = 3). Statistical significances were determined using a one-way ANOVA analysis (* <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">p</span> &lt; 0.0001).</p> Full article ">Figure 6
<p>Clonogenic assay analysis of SIJ1777 in C8161. (<b>A</b>,<b>B</b>) 2D clonogenic assay (colony formation assay) results of the compounds on C8161 melanoma cell. After incubation with test compounds for 14 days, colonies were photographed without magnification. (<b>C</b>,<b>D</b>) 3D clonogenic assay (soft agar assay) results of test compounds on C8161 melanoma cell. Cells embedded within 0.35% low melting agar and incubated with the indicated compounds for 14 days and observed without magnification. (<b>B</b>,<b>D</b>) Number of colonies were determined automatically by ImageJ (<span class="html-italic">n</span> = 3, respectively). (<b>E</b>) Western blot analysis of SIJ1777 in C8161. Cells were treated with 0.01, 0.1 μM of SIJ1777, and 0.1 μM of vemurafenib, PLX8394, GNF-7, and SIJ1227 for 24 h. Cell lysates were subjected to western blot analysis to estimate the phospho- or total- form of AKT, MEK, ERK levels, and GAPDH was used as the internal loading controls (left panel). Quantification result (<span class="html-italic">n</span> = 3) of western blot result by ImageJ (right panel). Statistical significances were determined using a one-way ANOVA analysis (* <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">p</span> &lt; 0.0001).</p> Full article ">
20 pages, 6382 KiB  
Article
The Fibronectin Expression Determines the Distinct Progressions of Malignant Gliomas via Transforming Growth Factor-Beta Pathway
by Chih-Wei Chen, Cheng-Han Yang, Yuan-Ho Lin, Ya-Chin Hou, Tain-Junn Cheng, Sheng-Tsung Chang, Yu-Hua Huang, Shang-Ting Chung, Chung-Ching Chio, Yan-Shen Shan, Hung-Chi Cheng and Wen-Tsan Chang
Int. J. Mol. Sci. 2021, 22(7), 3782; https://doi.org/10.3390/ijms22073782 - 6 Apr 2021
Cited by 5 | Viewed by 3107
Abstract
Due to the increasing incidence of malignant gliomas, particularly glioblastoma multiforme (GBM), a simple and reliable GBM diagnosis is needed to screen early the death-threaten patients. This study aimed to identify a protein that can be used to discriminate GBM from low-grade astrocytoma [...] Read more.
Due to the increasing incidence of malignant gliomas, particularly glioblastoma multiforme (GBM), a simple and reliable GBM diagnosis is needed to screen early the death-threaten patients. This study aimed to identify a protein that can be used to discriminate GBM from low-grade astrocytoma and elucidate further that it has a functional role during malignant glioma progressions. To identify proteins that display low or no expression in low-grade astrocytoma but elevated levels in GBM, glycoprotein fibronectin (FN) was particularly examined according to the mining of the Human Protein Atlas. Web-based open megadata minings revealed that FN was mainly mutated in the cBio Cancer Genomic Portal but dominantly overexpressed in the ONCOMINE (a cancer microarray database and integrated data-mining platform) in distinct tumor types. Furthermore, numerous different cancer patients with high FN indeed exhibited a poor prognosis in the PrognoScan mining, indicating that FN involves in tumor malignancy. To investigate further the significance of FN expression in glioma progression, tumor specimens from five malignant gliomas with recurrences that received at least two surgeries were enrolled and examined. The immunohistochemical staining showed that FN expression indeed determined the distinct progressions of malignant gliomas. Furthermore, the expression of vimentin (VIM), a mesenchymal protein that is strongly expressed in malignant cancers, was similar to the FN pattern. Moreover, the level of epithelial–mesenchymal transition (EMT) inducer transforming growth factor-beta (TGF-?) was almost recapitulated with the FN expression. Together, this study identifies a protein FN that can be used to diagnose GBM from low-grade astrocytoma; moreover, its expression functionally determines the malignant glioma progressions via TGF-?-induced EMT pathway. Full article
(This article belongs to the Special Issue Biochemistry, Molecular Biology and Druggability of Proteins)
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<p>Fibronectin (FN) displays low-to-medium levels in distinct tissues and organs but exhibits either no or high expression in gliomas. (<b>A</b>) Protein and (<b>B</b>) RNA levels of FN in different tissues and organs obtained from the Human Protein Atlas database (<a href="https://www.proteinatlas.org/" target="_blank">https://www.proteinatlas.org/</a>, accessed on 1 November 2017) mining. (<b>C</b>) Statistical pattern of FN expression in distinct cancer types by immunohistochemical (IHC) staining acquired from the Human Protein Atlas database mining. (<b>D</b>) Expression of FN in 12 glioma specimens by IHC staining provided by the Human Protein Atlas database mining. Red * indicates the statistical expression pattern of FN in glioma tumors.</p> Full article ">Figure 2
<p>FN is mainly mutated in the genomic DNA but dominantly overexpressed in the gene expression in many different cancer types. (<b>A</b>) The type and (<b>B</b>) distribution of FN genomic DNA alteration in numerous distinct tumor types analyzed from the cBio Cancer Genomic Portal database (<a href="http://www.cbioportal.org/" target="_blank">http://www.cbioportal.org/</a>, accessed on 1 November 2017) mining. (<b>C</b>) The statistical FN expression levels compared between normal and cancerous tissues analyzed from the ONCOMINE database (<a href="https://www.oncomine.org/resource/" target="_blank">https://www.oncomine.org/resource/</a>, accessed on 1 November 2017) mining in different cancer types. Red * indicates the statistical expression pattern of FN in glioma tumors.</p> Full article ">Figure 3
<p>A high level of FN expression is frequently correlated with poor prognosis in numerous distinct tumor types. Survival analysis of FN expression level in different cancer types, including brain (<b>A</b>), breast (<b>B</b>), colorectal (<b>C</b>), lung (<b>D</b>), multiple myeloma (<b>E</b>), ovarian (<b>F</b>), prostate (<b>G</b>), and soft tissue (<b>H</b>) cancers, by the PrognoScan database (<a href="http://www.abren.net/PrognoScan/" target="_blank">http://www.abren.net/PrognoScan/</a>, accessed on 1 November 2017) mining. The red dotted line indicates high level of FN expression and the blue dotted line indicates low level of FN expression. The difference between the two groups of low and high expression levels was recognized as statistically significant when <span class="html-italic">p</span>-value was &lt;0.05.</p> Full article ">Figure 4
<p>FN, vimentin (VIM), and transforming growth factor-beta (TGF-β) expressions are increased during the progression of low-grade astrocytoma into glioblastoma multiforme (GBM). Serial images including first and follow-up examinations of a patient with GBM are shown by using either brain computerized tomography (CT) on 25 October 2010 (<b>A</b>) and 8 December 2010 (<b>C</b>) or head MRI on 25 October 2010 (<b>B</b>), 5 May 2011 (<b>D</b>), 28 December 2012 (<b>E</b>) and 17 May 2013 (<b>F</b>). The IHC staining images indicate the expression level of FN (<b>G</b> and <b>H</b>), VIM (<b>I</b> and <b>J</b>), and TGF-β (<b>K</b> and <b>L</b>) proteins in the origin of low-grade astrocytoma (<b>G</b>, <b>I</b>, and <b>K</b>) and local recurrence of GBM (<b>H</b>, <b>J</b>, and <b>L</b>) tumor specimens.</p> Full article ">Figure 5
<p>Enhanced FN, VIM, and TGF-β expressions are associated with the local recurrence of GBM. Serial images including first and follow up examinations of a patient with GBM are shown by using either brain CT on 10 July 2009 (<b>A</b>) and 7 August 2009 (<b>C</b>) or head MRI on 10 July 2009 (<b>B</b>), 30 November 2009 (<b>D</b>), 16 April 2010 (<b>E</b>), and 26 November 2010 (<b>F</b>). The IHC staining images indicate the expression level of FN (<b>G</b> and <b>H</b>), VIM (<b>I</b> and <b>J</b>), and TGF-β (<b>K</b> and <b>L</b>) proteins in origin (<b>G</b>, <b>I</b>, and <b>K</b>) and local recurrence (<b>H</b>, <b>J</b>, and <b>L</b>) of GBM tumor specimens.</p> Full article ">Figure 6
<p>Expression of FN, VIM, and TGF-β is elevated during local recurrence and then remote brain metastasis of GBM. Serial images including first and follow up examinations of a patient with GBM are shown by using either brain CT on 1 August 2010 (<b>A</b>) or head MRI on 1 August 2010 (<b>B</b>), 11 April 2011 (<b>C</b>), 19 July 2012 (<b>D</b>), 30 January 2013 (<b>E</b>) and 3 June 2013 (<b>F</b>). The IHC staining images indicate the expression level of FN (<b>G</b>–<b>I</b>), VIM (<b>J</b>–<b>L</b>), and TGF-β (<b>M</b>–<b>O</b>) proteins in origin (<b>G</b>, <b>J</b>, and <b>M</b>), local recurrence (<b>H</b>, <b>K</b>, and <b>N</b>) and then remote brain metastasis (<b>I</b>, <b>L</b>, and <b>O</b>) of GBM tumor specimens.</p> Full article ">Figure 7
<p>Increased FN, VIM, and TGF-β expressions are associated with spinal metastasis of GBM. Serial images including first and follow-up examinations of a patient with GBM are shown by using either brain CT on 11 May 2013 (<b>A</b>) and 17 August 2014 (<b>D</b>) or head MRI on 11 May 2013 (<b>B</b>), 26 July 2013 (<b>C</b>) and 17 August 2014 (<b>E</b> and <b>F</b>). The IHC staining images indicate the expression level of glial fibrillary acidic protein (GFAP) (<b>G</b> and <b>H</b>), Ki-67 (<b>I</b> and <b>J</b>), FN (<b>K</b> and <b>L</b>), VIM (<b>M</b> and <b>N</b>), and TGF-β (<b>O</b> and <b>P</b>) proteins in origin (<b>G</b>, <b>I</b>, <b>K</b>, <b>M</b>, and <b>O</b>) and spine metastasis (<b>H</b>, <b>J</b>, <b>L</b>, <b>N</b>, and <b>P</b>) of GBM tumor specimens.</p> Full article ">Figure 8
<p>FN, VIM, and TGF-β expressions are increased in local recurrence but decreased during the progression of GBM into low-grade astrocytoma. Serial images including first and follow up examinations of a patient with GBM are shown by using either brain CT on 17 December 2009 (<b>A</b>) and 31 May 2016 (<b>G</b>) or head MRI on 17 December 2009 (<b>B</b>), 26 January 2010 (<b>C</b>), 4 June 2010 (<b>D</b>), 29 October 2014 (<b>E</b>), 30 October 2015 (<b>F</b>), and 13 September 2016 (<b>H</b>). The IHC staining images indicate the expression level of FN (<b>I</b>–<b>K</b>), VIM (<b>L</b>–<b>N</b>), and TGF-β (<b>O</b>–<b>Q</b>) proteins in origin (<b>I</b>, <b>L</b>, and <b>O</b>) and local recurrence (<b>J</b>, <b>M</b>, and <b>P</b>) of GBM, and progression into low-grade astrocytoma (<b>K</b>, <b>N</b>, and <b>Q</b>) tumor specimens.</p> Full article ">
24 pages, 406 KiB  
Review
Polymorphisms of Dopamine Receptor Genes and Parkinson’s Disease: Clinical Relevance and Future Perspectives
by Luca Magistrelli, Marco Ferrari, Alessia Furgiuele, Anna Vera Milner, Elena Contaldi, Cristoforo Comi, Marco Cosentino and Franca Marino
Int. J. Mol. Sci. 2021, 22(7), 3781; https://doi.org/10.3390/ijms22073781 - 6 Apr 2021
Cited by 29 | Viewed by 5578
Abstract
Parkinson’s disease (PD) is a neurodegenerative disease caused by loss of dopaminergic neurons in the midbrain. PD is clinically characterized by a variety of motor and nonmotor symptoms, and treatment relies on dopaminergic replacement. Beyond a common pathological hallmark, PD patients may present [...] Read more.
Parkinson’s disease (PD) is a neurodegenerative disease caused by loss of dopaminergic neurons in the midbrain. PD is clinically characterized by a variety of motor and nonmotor symptoms, and treatment relies on dopaminergic replacement. Beyond a common pathological hallmark, PD patients may present differences in both clinical progression and response to drug therapy that are partly affected by genetic factors. Despite extensive knowledge on genetic variability of dopaminergic receptors (DR), few studies have addressed their relevance as possible influencers of clinical heterogeneity in PD patients. In this review, we summarized available evidence regarding the role of genetic polymorphisms in DR as possible determinants of PD development, progression and treatment response. Moreover, we examined the role of DR in the modulation of peripheral immunity, in light of the emerging role of the peripheral immune system in PD pathophysiology. A better understanding of all these aspects represents an important step towards the development of precise and personalized disease-modifying therapies for PD. Full article
(This article belongs to the Special Issue Genomics of Brain Disorders 3.0)
27 pages, 5472 KiB  
Article
Genome and Pangenome Analysis of Lactobacillus hilgardii FLUB—A New Strain Isolated from Mead
by Klaudia Gustaw, Piotr Koper, Magdalena Polak-Berecka, Kamila Rachwał, Katarzyna Skrzypczak and Adam Waśko
Int. J. Mol. Sci. 2021, 22(7), 3780; https://doi.org/10.3390/ijms22073780 - 6 Apr 2021
Cited by 5 | Viewed by 4684
Abstract
The production of mead holds great value for the Polish liquor industry, which is why the bacterium that spoils mead has become an object of concern and scientific interest. This article describes, for the first time, Lactobacillus hilgardii FLUB newly isolated from mead, [...] Read more.
The production of mead holds great value for the Polish liquor industry, which is why the bacterium that spoils mead has become an object of concern and scientific interest. This article describes, for the first time, Lactobacillus hilgardii FLUB newly isolated from mead, as a mead spoilage bacteria. Whole genome sequencing of L. hilgardii FLUB revealed a 3 Mbp chromosome and five plasmids, which is the largest reported genome of this species. An extensive phylogenetic analysis and digital DNA-DNA hybridization confirmed the membership of the strain in the L. hilgardii species. The genome of L. hilgardii FLUB encodes 3043 genes, 2871 of which are protein coding sequences, 79 code for RNA, and 93 are pseudogenes. L. hilgardii FLUB possesses three clustered regularly interspaced short palindromic repeats (CRISPR), eight genomic islands (44,155 bp to 6345 bp), and three (two intact and one incomplete) prophage regions. For the first time, the characteristics of the genome of this species were described and a pangenomic analysis was performed. The concept of the pangenome was used not only to establish the genetic repertoire of this species, but primarily to highlight the unique characteristics of L. hilgardii FLUB. The core of the genome of L. hilgardii is centered around genes related to the storage and processing of genetic information, as well as to carbohydrate and amino acid metabolism. Strains with such a genetic constitution can effectively adapt to environmental changes. L. hilgardii FLUB is distinguished by an extensive cluster of metabolic genes, arsenic detoxification genes, and unique surface layer proteins. Variants of MRS broth with ethanol (10–20%), glucose (2–25%), and fructose (2–24%) were prepared to test the strain’s growth preferences using Bioscreen C and the PYTHON script. L. hilgardii FLUB was found to be more resistant than a reference strain to high concentrations of alcohol (18%) and sugars (25%). It exhibited greater preference for fructose than glucose, which suggests it has a fructophilic nature. Comparative genomic analysis supported by experimental research imitating the conditions of alcoholic beverages confirmed the niche specialization of L. hilgardii FLUB to the mead environment. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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<p>Tree inferred with FastME 2.1.6.1 from GBDP distances calculated from 16S rDNA gene sequences. The branch lengths are scaled in terms of the GBDP distance formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values &gt;60% from 100 replications, with an average branch support of 83.1%. The tree was rooted at the midpoint.</p> Full article ">Figure 2
<p>Tree inferred with FastME 2.1.6.1 from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values &gt;60% from 100 replications, with an average branch support of 16.6%. The tree was rooted at the midpoint.</p> Full article ">Figure 3
<p>Phylogenetic tree based on PGFams, visualized in iTOL.</p> Full article ">Figure 4
<p>Circular map of the <span class="html-italic">L. hilgardii</span> FLUB chromosome and plasmids. The outer circle shows the scale in megabases (Mb). The representation, from outer to inner circle, is as follows: forward and reverse strand CDSs (the color gradient represents the percentage of GC; the green stripes represent RNAs genes), GC content, and GC skew. The genome map was visualized using the CGView circular genome visualization tool.</p> Full article ">Figure 5
<p>Distribution of clusters of orthologous groups of proteins (COGs) among accessory, core and singleton gene groups.</p> Full article ">Figure 6
<p>A snapshot of a Krona hierarchical data circle graph showing COG distribution in the complete sequence of <span class="html-italic">L. hilgardii</span> FLUB. A multi-layered interactive version with zoom and four-step depth adjustment is available via link or in the <a href="#app1-ijms-22-03780" class="html-app">Supplementary Materials</a>, as well as graphs for the individual replicons or pangenome.</p> Full article ">Figure 7
<p>Growth kinetics of <span class="html-italic">L. hilgrardii</span> FLUB (green, F) and <span class="html-italic">L. hilgardii</span> DSMZ (blue, D) visualized as a three-dimensional scatter plot using plotly (Plotly Technologies INC., Montréal , QC, Canada): (<b>A</b>) growth kinetics on MRS medium (without dextrose) enriched with fructose in the concentration range of 2–25%, (<b>B</b>) growth on MRS supplemented with glucose (2–25%). An interactive version and parameters calculated according to Hoeflinger et al. are available in the <a href="#app1-ijms-22-03780" class="html-app">Supplementary Materials</a> [<a href="#B65-ijms-22-03780" class="html-bibr">65</a>].</p> Full article ">Figure 8
<p>Growth curves of <span class="html-italic">L. hilgrardii</span> FLUB (<b>A</b>) and <span class="html-italic">L. hilgardii</span> DSMZ (<b>B</b>) on a medium supplemented with 12–20% ethanol, and control (MRS).</p> Full article ">
20 pages, 3039 KiB  
Review
Endogenous Opioid Peptides and Alternatively Spliced Mu Opioid Receptor Seven Transmembrane Carboxyl-Terminal Variants
by Anna Abrimian, Tamar Kraft and Ying-Xian Pan
Int. J. Mol. Sci. 2021, 22(7), 3779; https://doi.org/10.3390/ijms22073779 - 6 Apr 2021
Cited by 16 | Viewed by 7588
Abstract
There exist three main types of endogenous opioid peptides, enkephalins, dynorphins and ?-endorphin, all of which are derived from their precursors. These endogenous opioid peptides act through opioid receptors, including mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opioid receptor (KOR), [...] Read more.
There exist three main types of endogenous opioid peptides, enkephalins, dynorphins and ?-endorphin, all of which are derived from their precursors. These endogenous opioid peptides act through opioid receptors, including mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opioid receptor (KOR), and play important roles not only in analgesia, but also many other biological processes such as reward, stress response, feeding and emotion. The MOR gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms. One type of these splice variants, the full-length 7 transmembrane (TM) Carboxyl (C)-terminal variants, has the same receptor structures but contains different intracellular C-terminal tails. The pharmacological functions of several endogenous opioid peptides through the mouse, rat and human OPRM1 7TM C-terminal variants have been considerably investigated together with various mu opioid ligands. The current review focuses on the studies of these endogenous opioid peptides and summarizes the results from early pharmacological studies, including receptor binding affinity and G protein activation, and recent studies of ?-arrestin2 recruitment and biased signaling, aiming to provide new insights into the mechanisms and functions of endogenous opioid peptides, which are mediated through the OPRM1 7TM C-terminal splice variants. Full article
(This article belongs to the Special Issue Opioid Receptors and Endorphinergic Systems 2.0)
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<p>Schematic of the major endogenous peptides processed from human proenkephalin (PENK), prodynorphin (PDYN) and proopiomelanocortin (POMC). BAM: bovine adrenal medulla peptide; MSH: melanocyte stimulating hormone; ACTH: adrenocorticotropic hormone; CLIP: corticotropin-like intermediate lobe peptide; LPH: lipotropin.</p> Full article ">Figure 2
<p>Predicted amino acid sequences from 7TM <span class="html-italic">C</span>-terminal variants (modified from [<a href="#B17-ijms-22-03779" class="html-bibr">17</a>]. The top panel is an animation that shows structures of MORs and adjacent proteins on membrane. TM domains are indicated by cylinders. Splice junctions are shown by arrows. Calcium (Ca++) and potassium (K+) channels are indicated by opened canals across membrane. Gα, Gβ and Gγ: G proteins; PLCβ: phospholipase Cβ; PLA2: phospholipase A2; The bottom panel listed predicted amino acid sequences encoded by downstream exons of exon 3 in mouse (mMOR), rat (rMOR) and human (hMOR) splice variants. Italic red S, T and Y are predicted phosphorylation sites. Underlined sequences are predicted phosphorylation codes, PxPxxE/D or PxxPxxE/D, for β-arrestin binding based on crystal G protein coupled receptors (GPCR) structures [<a href="#B72-ijms-22-03779" class="html-bibr">72</a>].</p> Full article ">Figure 3
<p>Correlation of the EC<sub>50</sub> values with % maximum stimulation (% Max) in [<sup>35</sup>S]GTPγS binding and with the K<sub>i</sub> values in receptor binding among mouse Oprm1 7TM <span class="html-italic">C</span>-terminal variants. A). Correlations of the K<sub>i</sub> values in receptor binding from <a href="#ijms-22-03779-t002" class="html-table">Table 2</a> with the EC<sub>50</sub> values in [<sup>35</sup>S]GTPγS binding from <a href="#ijms-22-03779-t003" class="html-table">Table 3</a>. Correlation coefficients (<span class="html-italic">r<sup>2</sup></span>) were calculated for each drug by linear regression (Prism 8, GraphPad). There was no significant correlation between binding site affinity (K<sub>i</sub>) and potency (EC<sub>50</sub>) in the [<sup>35</sup>S]GTPγS binding. DAMGO, <span class="html-italic">r<sup>2</sup></span> = 0.03; Morphine, <span class="html-italic">r<sup>2</sup></span> = 0.01; β-endorphin, <span class="html-italic">r<sup>2</sup></span> = 0.24; Dynorphin A, <span class="html-italic">r<sup>2</sup></span> = 0.16; Endomorphin-1, <span class="html-italic">r<sup>2</sup></span> = 0.01; Endomorphin-2, <span class="html-italic">r<sup>2</sup></span> = 0.44. B). Correlation of the EC<sub>50</sub> values and % maximum stimulation (% Max) in the [<sup>35</sup>S]GTPγS binding. No significant correlation between the EC<sub>50</sub> and % Max was observed. Morphine, <span class="html-italic">r<sup>2</sup></span> = 0.00; β-endorphin, <span class="html-italic">r<sup>2</sup></span> = 0.05; Dynorphin A, <span class="html-italic">r<sup>2</sup></span> = 0.16; Endomorphin-1, <span class="html-italic">r<sup>2</sup></span> = 0.04; Endomorphin-2, <span class="html-italic">r<sup>2</sup></span> = 0.07.</p> Full article ">Figure 4
<p>Heatmap of biased factors (adopted from [<a href="#B81-ijms-22-03779" class="html-bibr">81</a>]). Biased factors were calculated using the Black and Leff Operational Model by using different normalization methods, as described in [<a href="#B81-ijms-22-03779" class="html-bibr">81</a>]. (<b>A</b>). Normalized with respect to DAMGO at MOR−1 for a comparison between drugs and variants. (<b>B</b>). Normalized with respect to each drug at mMOR−1 for a comparison across variants. The negative (blue) values indicate β-arrestin2 bias whereas the positive bias (red) values indicate G protein bias.</p> Full article ">
15 pages, 2243 KiB  
Article
Study on Demethoxycurcumin as a Promising Approach to Reverse Methicillin-Resistance of Staphylococcus aureus
by Qian-Qian Li, Ok-Hwa Kang and Dong-Yeul Kwon
Int. J. Mol. Sci. 2021, 22(7), 3778; https://doi.org/10.3390/ijms22073778 - 6 Apr 2021
Cited by 8 | Viewed by 2650
Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) has always been a threatening pathogen. Research on phytochemical components that can replace antibiotics with limited efficacy may be an innovative method to solve intractable MRSA infections. The present study was devoted to investigate the antibacterial activity of the [...] Read more.
Methicillin-resistant Staphylococcus aureus (MRSA) has always been a threatening pathogen. Research on phytochemical components that can replace antibiotics with limited efficacy may be an innovative method to solve intractable MRSA infections. The present study was devoted to investigate the antibacterial activity of the natural compound demethoxycurcumin (DMC) against MRSA and explore its possible mechanism for eliminating MRSA. The minimum inhibitory concentrations (MICs) of DMC against MRSA strains was determined by the broth microdilution method, and the results showed that the MIC of DMC was 62.5 ?g/mL. The synergistic effects of DMC and antibiotics were investigated by the checkerboard method and the time–kill assay. The ATP synthase inhibitors were employed to block the metabolic ability of bacteria to explore their synergistic effect on the antibacterial ability of DMC. In addition, western blot analysis and qRT-PCR were performed to detect the proteins and genes related to drug resistance and S. aureus exotoxins. As results, DMC hindered the translation of penicillin-binding protein 2a (PBP2a) and staphylococcal enterotoxin and reduced the transcription of related genes. This study provides experimental evidences that DMC has the potential to be a candidate substance for the treatment of MRSA infections. Full article
(This article belongs to the Special Issue Antimicrobial Resistance-New Insights)
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<p>The chemical structure of demethoxycurcumin (DMC).</p> Full article ">Figure 2
<p>Time–kill curves of DMC and gentamicin (GEN) alone and in combinations against MRSA ((<b>a</b>) MRSA ATCC 33591; (<b>b</b>) MRSA DPS-2).</p> Full article ">Figure 3
<p>The effect of the ATP synthase inhibitors, DCCD (25 μg/mL) and NaN<sub>3</sub> (125 μg/mL), on the susceptibility of MRSA strain ATCC 33591 to DMC treatment (15.6 μg/mL). The data are means ± SD of triplicate determinations. Different letters in each bars indicate significant statistical differences between treatments (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 4
<p>The effect of DMC and GEN on the expression of penicillin-binding protein 2a (PBP2a) in MRSA strain ATCC 33591, as analyzed by western blot. Loading differences were normalized by anti-GAPDH antibody. Lane 1, control (untreated); lane 2, DMC 7.8 μg/mL (1/8MIC); lane 3, DMC 12.5 μg/mL (1/4 MIC); lane 4, DMC 25 μg/mL (1/2 MIC); lane 5, GEN 3.9 μg/mL. The data are means ± SD of triplicate determinations. Different letters in each bars indicate significant statistical differences between treatments (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 5
<p>The effect of DMC and GEN on the mRNA expression of <span class="html-italic">mec</span> operon (<b>a</b>) and <span class="html-italic">bla</span> operon (<b>b</b>), as analyzed by qRT-PCR. ATCC 33591 were treated with serial dilution of DMC (7.8 μg/mL and 31.25 μg/mL) and GEN (3.9 μg/mL). The data are means ± SD of triplicate determinations. Different letters in each bars indicate significant statistical differences between treatments (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>The effect of DMC on the protein (<b>a</b>) and mRNA (<b>b</b>) expression of staphylococcal enterotoxin A (SEA), as analyzed by western blot and qRT-PCR. ATCC 33591 were treated with serial dilution of DMC (7.8 μg/mL, 15.6 μg/mL and 31.25 μg/mL) and GEN (3.9 μg/mL). The data are means ± SD of triplicate determinations. Different letters in each bars indicate significant statistical differences between treatments (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
15 pages, 5356 KiB  
Article
Characterization of the mbsA Gene Encoding a Putative APSES Transcription Factor in Aspergillus fumigatus
by Yong-Ho Choi, Sang-Cheol Jun, Min-Woo Lee, Jae-Hyuk Yu and Kwang-Soo Shin
Int. J. Mol. Sci. 2021, 22(7), 3777; https://doi.org/10.3390/ijms22073777 - 6 Apr 2021
Cited by 10 | Viewed by 2753
Abstract
The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, five genes predicted to [...] Read more.
The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, five genes predicted to encode APSES TFs are present. Here, we report the characterization of one of these genes, called mbsA (Afu7g05620). The deletion (?) of mbsA resulted in significantly decreased hyphal growth and asexual sporulation (conidiation), and lowered mRNA levels of the key conidiation genes abaA, brlA, and wetA. Moreover, ?mbsA resulted in reduced spore germination rates, elevated sensitivity toward Nikkomycin Z, and significantly lowered transcripts levels of genes associated with chitin synthesis. The mbsA deletion also resulted in significantly reduced levels of proteins and transcripts of genes associated with the SakA MAP kinase pathway. Importantly, the cell wall hydrophobicity and architecture of the ?mbsA asexual spores (conidia) were altered, notably lacking the rodlet layer on the surface of the ?mbsA conidium. Comparative transcriptomic analyses revealed that the ?mbsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes, which was corroborated by elevated levels of GT production in the mutant. While the ?mbsA mutant produced higher amount of GT, ?mbsA strains showed reduced virulence in the murine model, likely due to the defective spore integrity. In summary, the putative APSES TF MbsA plays a multiple role in governing growth, development, spore wall architecture, GT production, and virulence, which may be associated with the attenuated SakA signaling pathway. Full article
(This article belongs to the Section Molecular Microbiology)
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<p>Summary of MbsA. (<b>A</b>) Schematic presentation of the domain structure of the MbsA-like proteins using SMART (<a href="http://smart.embl-heidelberg.de" target="_blank">http://smart.embl-heidelberg.de</a>, accessed on 10 February 2021). (<b>B</b>) A phylogenetic tree of the MbsA-like proteins in various <span class="html-italic">Aspergillus</span> and <span class="html-italic">Saccharomyces cerevisiae</span> was constructed based on the matrix of pair-wise distances between the KilA-N domain sequences. (<b>C</b>) Levels of <span class="html-italic">mbsA</span> mRNA during the lifecycle of <span class="html-italic">A</span>. <span class="html-italic">fumigatus</span> wild type (WT, AF293). The vegetative stage (V0) and time (hours) of incubation in post asexual developmental induction is shown.</p> Full article ">Figure 2
<p>MbsA is required for proper growth, development, and spore germination. (<b>A</b>) Colony photographs and radial growth rate of wild type (WT), Δ<span class="html-italic">mbsA</span>, and complemented (C′) strains point-inoculated on solid glucose minimal medium with 0.1% yeast extract (MMY) and grown for 3 days. (<b>B</b>) Conidia numbers produced by each strain per plate. (<b>C</b>) mRNA levels of the key asexual developmental regulators in the Δ<span class="html-italic">mbsA</span> strain relative to WT at 3 days determined by RT-qPCR. Fungal cultures were done in solid MMY and mRNA levels were normalized using the <span class="html-italic">ef1α</span> gene. (<b>D</b>) Germination rates of <span class="html-italic">A</span>. <span class="html-italic">fumigatus</span> strains when inoculated in liquid CM at 37 °C. The number of conidia showing germ-tube protrusion was recorded at 2 h intervals and which is represented as a percentage of the total number of conidia in each microscope field. Data are presented as the mean ± standard deviation from three independent experiments. ANOVA test: ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 3
<p>MbsA is necessary for the proper regulation of chitin synthesis. (<b>A</b>) Radial growth of WT, Δ<span class="html-italic">mbsA</span>, and C′ strains in the presence of Nikkomycin Z (NZ, 50 µg/mL) following incubation at 37 °C for 72 h. (<b>B</b>) Chitin synthesis–related genes in WT, Δ<span class="html-italic">mbsA</span>, and C′ strains analyzed by RT-qPCR. The <span class="html-italic">ef1α</span> gene as the endogenous control. Statistical differences between strains were evaluated with ANOVA test: ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 4
<p>MbsA positively affects the SakA MAPK pathway. (<b>A</b>) Conidia of the indicated strains were grown for 14 h in MMY medium and treated with 200 µg/mL of Calcofluor White (CFW) (+) or not (−). Aliquots of cell were harvested after 20 min and used to prepare total protein extracts. Protein extracts were analyzed by immunoblotting with anti-phospho-p38 and anti-phoshpo-p42/44 antibodies. (<b>B</b>) mRNA levels of SakA MAP kinase pathway-related genes in WT, Δ<span class="html-italic">mbsA</span>, and C′ strains analyzed by RT-qPCR at the same culture condition. The <span class="html-italic">ef1α</span> gene as the endogenous control. Statistical differences between strains were evaluated with ANOVA test: ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p>MbsA is needed for proper conidia hydrophobicity. (<b>A</b>) Hydrophobicity test for each strain. Strains were cultured on MMY agar plates for 4 days at 37 °C and 10 μL of a detergent solution (0.2% SDS, 50 mM EDTA) were dropped onto the surface of a colony. The droplets were observed to penetrate into the colonies. (<b>B</b>) Percentage hydrophobicity of three strains by MATS test. (<b>C</b>) SDS-PAGE analysis of the hydrophobin, RodA of relevant strains. RodA was extracted from the dried conidia (10<sup>9</sup>) with hydrofluoric acid (HF). RodA*: degraded form of RodA due to HF treatment. (<b>D</b>) RT-qPCR analysis of hydrophobin genes in WT, Δ<span class="html-italic">mbsA</span>, and C′ strains. The <span class="html-italic">ef1α</span> gene as the endogenous control. Statistical differences between strains were evaluated with ANOVA test: ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 6
<p>MbsA is needed for proper cell wall architecture and rodlet layer. (<b>A</b>) Transmission electron micrographs of conidia. The conidia were harvested from the cells cultured on MMY medium for 5 days, and they were fabricated as ultra-thin specimens for transmission electron microscopy. (<b>B</b>) Atomic force microscopy (AFM) image of conidial surface of three strains. Upper: amplitude image; Lower: phase image. Rodlets showed only in WT and C′ strains (arrows). Bar indicates 100 nm.</p> Full article ">Figure 7
<p>MbsA down-regulates gliotoxin (GT) production. (<b>A</b>) Heat map of those genes encoding toxin-related proteins. Most of gliotoxin biosynthetic genes were up-regulated by the loss of <span class="html-italic">mbsA</span>. (<b>B</b>) Determination of GT production in WT, Δ<span class="html-italic">mbsA</span>, and C′ strains. The culture supernatant of each strain was extracted with chloroform and subjected to TLC. (<b>C</b>) RT-qPCR analysis of GT-related genes in WT, Δ<span class="html-italic">mbsA</span>, and C′ strains. The <span class="html-italic">ef1α</span> gene as the endogenous control. Statistical differences between WT and mutant strains were evaluated with ANOVA test: ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 8
<p>MbsA’s role in virulence. (<b>A</b>) Schematic presentation of <span class="html-italic">A</span>. <span class="html-italic">fumigatus</span> infection in immunocompromised mouse model. Six-week-old female ICR mice were immunocompromised by treatment of cyclophosphamide (Cy, 250 mg/kg at day −3 and −1 and 125 mg/kg at day +1) and cortisone acetate (CA, 250 mg/kg at day −1 and 125 mg/kg at day +3). On day 0 mice were intranasally infected. (<b>B</b>) Survival curve of mice infected with WT, Δ<span class="html-italic">mbsA</span>, and C′ strains (<span class="html-italic">n</span> = 10/group). (<b>C</b>) Lung sections after each strains infection were stained with Hematoxylin and Eosin (H&amp;E) or Periodic-acid Schiff (PAS). Arrows indicate fungal mycelium. Bars indicate 200 µm. (<b>D</b>) Fungal burden in the lungs of mice infected with WT and Δ<span class="html-italic">mbsA</span>, and C′ strains. Data are represented as mean ± standard deviation from three independent experiments. ANOVA test: ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 9
<p>A genetic model depicting the role of MbsA in <span class="html-italic">A</span>. <span class="html-italic">fumigatus</span>. MbsA positively regulates asexual sporulation, <span class="html-italic">rodA</span> expression, the SakA MAP kinase pathway, and virulence, but negatively controls expression of gliotoxin-related genes and GT production.</p> Full article ">
16 pages, 1029 KiB  
Review
Application of the Antibody-Inducing Activity of Glycosphingolipids to Human Diseases
by Tetsuya Okuda
Int. J. Mol. Sci. 2021, 22(7), 3776; https://doi.org/10.3390/ijms22073776 - 6 Apr 2021
Cited by 9 | Viewed by 3565
Abstract
Glycosphingolipids (GSLs) are composed of a mono-, di-, or oligosaccharide and a ceramide and function as constituents of cell membranes. Various molecular species of GSLs have been identified in mammalian cells due to differences in the structures of oligosaccharides. The oligosaccharide structure can [...] Read more.
Glycosphingolipids (GSLs) are composed of a mono-, di-, or oligosaccharide and a ceramide and function as constituents of cell membranes. Various molecular species of GSLs have been identified in mammalian cells due to differences in the structures of oligosaccharides. The oligosaccharide structure can vary depending on cell lineage, differentiation stage, and pathology; this property can be used as a cell identification marker. Furthermore, GSLs are involved in various aspects of the immune response, such as cytokine production, immune signaling, migration of immune cells, and antibody production. GSLs containing certain structures exhibit strong immunogenicity in immunized animals and promote the production of anti-GSL antibodies. By exploiting this property, it is possible to generate antibodies that recognize the fine oligosaccharide structure of specific GSLs or glycoproteins. In our study using artificially synthesized GSLs (artGSLs), we found that several structural features are correlated with the antibody-inducing activity of GSLs. Based on these findings, we designed artGSLs that efficiently induce the production of antibodies accompanied by class switching and developed several antibodies that recognize not only certain glycan structures of GSLs but also those of glycoproteins. This review comprehensively introduces the immune activities of GSLs and their application as pharmaceuticals. Full article
(This article belongs to the Special Issue Sphingolipid Metabolism and Signaling in Diseases)
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<p>Chemical structure and mammalian glycosphingolipid (GSL) biosynthetic pathway. (<b>A</b>) Chemical structure of a typical mammalian GSL, globotriaosylceramide (Gb3Cer), shown as an example. (<b>B</b>) Arrows and blue font indicate biosynthetic pathway and catalytic enzymes, respectively. Abbreviations: GlcCer, Glcβ1,1Cer; GalCer, Galβ1,1Cer; LacCer, Galβ1,4Glcβ1,1Cer; GM3, Siaα2,3Galβ1,4Glcβ1,1Cer; UGCG, UDP-glucose ceramide glucosyltransferase; UGT8, UDP glycosyltransferase 8; GAL3ST1, galactose-3-<span class="html-italic">O</span>-sulfotransferase 1; B4GALT5/6, β-1,4-galactosyltransferase 5/6; B3GNT5, UDP-GlcNAc: βGal β-1,3-<span class="html-italic">N</span>-acetylglucosaminyltransferase 5; A4GALT, α-1,4-galactosyltransferase; B4GALNT1, β-1,4-<span class="html-italic">N</span>-acetyl-galactosaminyltransferase 1; ST3GAL5, ST3 β-galactoside α-2,3-sialyltransferase 5; ST8SIA1, ST8 α-<span class="html-italic">N</span>-acetyl-neuraminide α-2,8-sialyltransferase 1.</p> Full article ">Figure 2
<p>Antibody-inducing activity of mammalian GSLs. (<b>A</b>) Chemical structures of GSLs used for immunization experiments. GM3 containing C18:0 stearic acid (GM3-C18) was prepared by chemical synthesis to be a uniform structure. Bovine milk-derived GM3 (GM3-BM) and human erythrocyte-derived Gb4Cer (Gb4Cer-HE) predominantly contain very-long-chain fatty acids with 22–24 carbons. (<b>B</b>) Reactivity of mice serum IgMs against immunizing GSL. Mice were immunized with each GSL, and serum samples were prepared 7 days after immunization, as described previously [<a href="#B15-ijms-22-03776" class="html-bibr">15</a>]. Reactivity of serum antibodies against immunizing GSLs was analyzed by ELISA (A450). Abbreviations; BG, serum from untreated mice; D7, serum prepared from mice immunized with each GSL 7 days after immunization. Diamonds indicate individual mouse serum samples (n = 6–11). Solid lines indicate average reactivity of serum samples. Values include previously reported data [<a href="#B10-ijms-22-03776" class="html-bibr">10</a>].</p> Full article ">Figure 3
<p>Antibody-inducing activity of artificially synthesized GSLs (artGSLs). (<b>A</b>) Chemical structures of artGSLs used for immunization experiments. The ceramide mimetics C12S and C12L are composed of a saturated C12-sphingosine mimetic and stearic acid (C18:0) or lignoceric acid (C24:0), respectively. These ceramide mimetics are bound to the oligosaccharide via a β-linkage. Abbreviations: 6SLN, 6′-sialyl LacNAc/Neu5Acα2,6Galβ1,4GlcNAc; sLe<sup>X</sup>, sialyl Lewis<sup>X</sup>/Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAc; Le<sup>X</sup>, Lewis<sup>X</sup>/Galβ1,4(Fucα1,3)GlcNAc; CF4, core-fucosylated tetrasaccharide/Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAc. (<b>B</b>) Reactivity of mice serum IgMs against immunizing GSL. Mice were immunized with each artGSL, and serum samples were prepared 7 days after immunization, as described previously [<a href="#B15-ijms-22-03776" class="html-bibr">15</a>]. Reactivity of serum antibodies against immunizing GSLs was analyzed by ELISA (A450). Abbreviations; BG, serum from untreated mice; D7, serum prepared from mice immunized with each GSL 7 days after immunization. Diamonds indicate individual mouse serum samples (n = 8–13). Solid lines indicate average reactivity of serum samples. Values include previously reported data [<a href="#B13-ijms-22-03776" class="html-bibr">13</a>,<a href="#B15-ijms-22-03776" class="html-bibr">15</a>].</p> Full article ">Figure 4
<p>IgG-inducing activity of CF4-C12L and its derivatives. (<b>A</b>) Chemical structures of CF4-C12L derivatives used for immunization experiments. The ceramide mimetic C182L is composed of C18-phytosphingosine and lignoceric acid. The ceramide mimetic is bound to the oligosaccharide via a β-linkage. Abbreviations: CF4, core-fucosylated tetrasaccharide/Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAc; CF3, core-fucosylated trisaccharide/GlcNAcβ1,4(Fucα1,6)GlcNAc. (<b>B</b>) Reactivity of mice serum IgGs against immunizing GSL. Mice were immunized with each artGSL, and serum samples were prepared 7 days after immunization, as described previously [<a href="#B15-ijms-22-03776" class="html-bibr">15</a>]. Reactivity of serum antibodies against immunizing GSLs was analyzed by ELISA (A450). Abbreviations; BG, serum from untreated mice; D7, serum prepared from mice immunized with each GSL 7 days after immunization. Diamonds indicate individual mouse serum samples (n = 6–10). Solid lines indicate average reactivity of serum samples. Values include previously reported data [<a href="#B15-ijms-22-03776" class="html-bibr">15</a>].</p> Full article ">
21 pages, 1510 KiB  
Review
Cancer-Associated Adipocytes in Breast Cancer: Causes and Consequences
by Ilona Rybinska, Nunzia Mangano, Elda Tagliabue and Tiziana Triulzi
Int. J. Mol. Sci. 2021, 22(7), 3775; https://doi.org/10.3390/ijms22073775 - 6 Apr 2021
Cited by 64 | Viewed by 6996
Abstract
Breast cancer progression is highly dependent on the heterotypic interaction between tumor cells and stromal cells of the tumor microenvironment. Cancer-associated adipocytes (CAAs) are emerging as breast cancer cell partners favoring proliferation, invasion, and metastasis. This article discussed the intersection between extracellular signals [...] Read more.
Breast cancer progression is highly dependent on the heterotypic interaction between tumor cells and stromal cells of the tumor microenvironment. Cancer-associated adipocytes (CAAs) are emerging as breast cancer cell partners favoring proliferation, invasion, and metastasis. This article discussed the intersection between extracellular signals and the transcriptional cascade that regulates adipocyte differentiation in order to appreciate the molecular pathways that have been described to drive adipocyte dedifferentiation. Moreover, recent studies on the mechanisms through which CAAs affect the progression of breast cancer were reviewed, including adipokine regulation, metabolic reprogramming, extracellular matrix remodeling, and immune cell modulation. An in-depth understanding of the complex vicious cycle between CAAs and breast cancer cells is crucial for designing novel strategies for new therapeutic interventions. Full article
(This article belongs to the Special Issue Unraveling the Involvement of the Adipose Tissue in Breast Cancer Progression)
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<p>Transcriptional regulators of adipogenesis. Multipotent mesenchymal stem cells (MSC) upon adipogenic stimuli give rise to preadipocytes that, after clonal expansion and subsequent differentiation, become mature adipocytes. Molecules relevant in this process are shown with their approximate induction and duration reflected by lines. The preadipocyte factor 1 (Pref-1) is expressed in preadipocytes and participates in the maintenance of this state. It is an inhibitor of adipogenesis and its expression must be reduced during adipocyte differentiation. CCAAT-enhancer-binding protein C/EBPβ is involved in adipogenesis at an early phase and, together with C/EBPδ, regulates the transcription of the peroxisome proliferator-activated receptor γ (PPARγ). PPARγ with C/EBPα cooperatively induces adipocyte differentiation, regulating the expression of adipocyte-specific genes.</p> Full article ">Figure 2
<p>Extracellular regulators of adipogenesis. Signals from activators and repressors of adipogenesis are integrated in the nucleus by transcription factors that directly or indirectly activate (red arrows) or inhibit (blue lines) the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα). The induction of PPARγ by p38 is related to the BMP2 pathway, and the red arrow from ERK is related to insulin and FGF pathways. AKT, protein kinase B; BMP, bone morphogenetic protein; CREB, cAMP response element-binding protein; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; FGFR, fibroblast growth-factor receptor; FOXO, forkhead protein; FZD, frizzled family receptors; GATA, GATA binding protein; GC, glucocorticoids; GCR, GC receptor; Gli, GLI family zinc finger; IL, interleukin; IR, insulin receptor; JAK, Janus kinase; JNK, Jun <span class="html-italic">n</span>-terminal kinase; MEK, mitogen-activated MAPK/ERK kinase; mTOR, mammalian target of rapamycin; N1ICD, Notch1 intracellular domain; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; Notch1; Notch homolog 1, translocation-associated; p38, protein 38 MAPK; PI3K, phosphatidylinositol-3 kinase; PTC, patched; SHN2, schnurri-2; SHH, sonic hedgehog; SMAD, SMAD family member; STAT, signal transducer and activator of transcription; TCF7, T-cell factor 7; TNF, tumor necrosis factor; TNFR, TNF receptor; TGFβ, transforming-growth factor β; WNT, wingless-related integration site.</p> Full article ">Figure 3
<p>Signaling regulation of adipocyte dedifferentiation. Pathways described to be involved in adipocyte dedifferentiation in cancer and in other pathologies like fibrosis and liposarcoma. ?, indicates that the molecule(s) contained in exosomes and involved in inducing the adipocyte pro-inflammatory phenotype are unknown and have only pro-inflammatory effects (<sup>#</sup>); *, IL-6 was described to induce lipolysis and, thus, to reduce triglyceride (TG) content, while adrenomedullin (ADM) was described to induce the phosphorylation of hormone-sensitive lipase (HSL), a step necessary for lipolysis induction that reduces the TG content; ^, miRs were demonstrated to induce PPARγ reduction. Blue and red arrows indicate reduction and increase, respectively. miR, microRNA; FA, fatty acid; pHSL, phosphorylated hormone sensitive lipase; TGF, transforming growth factor; N1ICD, Notch1 intracellular domain; WNT, wingless-related integration site.</p> Full article ">Figure 4
<p>Pro-tumoral factors released by cancer-associated adipocytes (CAAs). CAAs release free or exosome-associated molecules that directly or indirectly, through the modulation of the immune cells, induce the acquisition of aggressive features in breast cancer (BC) cells. The blue and red arrows indicate the increased and decreased release compared to mature adipocytes. In the central panel, blue lines indicate that cells are blocked in their function; red arrows indicate that cells are recruited/activated. BHB, β-hydroxybutyrate; DC, dendritic cells; FFAs, free fatty acids; IL, interleukin; HGF, hepatocyte growth factor; IGF, insulin growth factor; IGFBP2, IGF binding protein 2; M, macrophages; MDSC, myeloid derived suppressor cells; NK, natural killer cells; PAI-1, plasminogen activator inhibitor 1; PLOD2, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2; TGFβ, transforming growth factor β; TAN, tumor associated neutrophils; TNF, tumor necrosis factor; T Reg, regulatory T cells; VEGF, vascular endothelial growth factor.</p> Full article ">
14 pages, 2630 KiB  
Article
Reduced Claudin-12 Expression Predicts Poor Prognosis in Cervical Cancer
by Abidur Rahman, Makoto Kobayashi, Kotaro Sugimoto, Yuta Endo, Manabu Kojima, Shigenori Furukawa, Takafumi Watanabe, Shu Soeda, Yuko Hashimoto, Keiya Fujimori and Hideki Chiba
Int. J. Mol. Sci. 2021, 22(7), 3774; https://doi.org/10.3390/ijms22073774 - 6 Apr 2021
Cited by 10 | Viewed by 3754
Abstract
Background: Within the claudin (CLDN) family, CLDN12 mRNA expression is altered in various types of cancer, but its clinicopathological relevance has yet to be established due to the absence of specific antibodies (Abs) with broad applications. Methods: We generated a monoclonal Ab (mAb) [...] Read more.
Background: Within the claudin (CLDN) family, CLDN12 mRNA expression is altered in various types of cancer, but its clinicopathological relevance has yet to be established due to the absence of specific antibodies (Abs) with broad applications. Methods: We generated a monoclonal Ab (mAb) against human/mouse CLDN12 and verified its specificity. By performing immunohistochemical staining and semiquantification, we evaluated the relationship between CLDN12 expression and clinicopathological parameters in tissues from 138 cases of cervical cancer. Results: Western blot and immunohistochemical analyses revealed that the established mAb selectively recognized the CLDN12 protein. Twenty six of the 138 cases (18.8%) showed low CLDN12 expression, and the disease-specific survival (DSS) and recurrence-free survival rates were significantly decreased compared with those in the high CLDN12 expression group. We also demonstrated, via univariable and multivariable analyses, that the low CLDN12 expression represents a significant prognostic factor for the DSS of cervical cancer patients (HR 3.412, p = 0.002 and HR 2.615, p = 0.029, respectively). Conclusions: It can be concluded that a reduced CLDN12 expression predicts a poor outcome for cervical cancer. The novel anti-CLDN12 mAb could be a valuable tool to evaluate the biological relevance of the CLDN12 expression in diverse cancer types and other diseases. Full article
(This article belongs to the Section Biochemistry)
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<p>Generation of rat monoclonal antibodies (mAbs) against human/mouse claudin-12 (CLDN12). (<b>A</b>) Topology of CLDN12 (<b>left</b>) and amino acid sequences of the C-terminal cytoplasmic domains of human and mouse CLDN12 (<b>right</b>). The C-terminal region that correspond to an antigenic polypeptide is indicated in red. (<b>B</b>,<b>C</b>) HEK293T cells were transfected with the CLDN12 or empty expression vector, and cell blocks were subjected to immunohistochemical and Western blot analyses using the indicated anti-CLDN12 mAb clones. (<b>D</b>) Amino acid sequences of the antigenic peptide of the C-terminal cytoplasmic domain of human CLDN12 and the corresponding regions of the closely related CLDNs. Conserved amino acids are shown in red. (<b>E</b>) HEK293T cells were transfected with individual CLDN expression vector, and subjected to Western blot analysis using the indicated anti-CLDN12 Abs. (<b>F</b>) Normal human liver tissues were immunohistochemically stained with the indicated anti-CLDN12 Abs. Arrows and arrowheads reveal cytoplasmic and membranous signals, respectively. Scale bars, 100 μm.</p> Full article ">Figure 2
<p>Expression of CLDN12 protein in normal and premalignant epithelial tissues of the uterine cervix. (<b>A</b>) Normal human cervical tissues and (<b>B</b>) squamous intraepithelial lesion (SIL) tissues were immunohistochemically stained with the anti-CLDN12 mAb. HE, hematoxylin-eosin; LSIL, low-grade SIL; HSIL, high-grade SIL. Scale bars, 100 μm.</p> Full article ">Figure 3
<p>CLDN12 protein expression in cervical cancer tissues. (<b>A</b>) Squamous cell carcinoma (SCC) and (<b>B</b>) adenocarcinoma (ADCA) tissues were immunohistochemically stained with the anti-CLDN12 mAb. HE, hematoxylin-eosin. Scale bars, 100 μm.</p> Full article ">Figure 4
<p>Low CLDN12 expression is associated with poor outcome in cervical cancer patients. (<b>A</b>) The disease-specific and (<b>B</b>) recurrence-free survival for low and high expression of CLDN12 protein in cervical cancer subjects are indicated.</p> Full article ">
18 pages, 2547 KiB  
Review
Corticosteroids for COVID-19 Therapy: Potential Implications on Tuberculosis
by Radha Gopalaswamy and Selvakumar Subbian
Int. J. Mol. Sci. 2021, 22(7), 3773; https://doi.org/10.3390/ijms22073773 - 6 Apr 2021
Cited by 57 | Viewed by 12157
Abstract
On 11 March 2020, the World Health Organization announced the Corona Virus Disease-2019 (COVID-19) as a global pandemic, which originated in China. At the host level, COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), affects the respiratory system, with the clinical [...] Read more.
On 11 March 2020, the World Health Organization announced the Corona Virus Disease-2019 (COVID-19) as a global pandemic, which originated in China. At the host level, COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), affects the respiratory system, with the clinical symptoms ranging from mild to severe or critical illness that often requires hospitalization and oxygen support. There is no specific therapy for COVID-19, as is the case for any common viral disease except drugs to reduce the viral load and alleviate the inflammatory symptoms. Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb), also primarily affects the lungs and has clinical signs similar to pulmonary SARS-CoV-2 infection. Active TB is a leading killer among infectious diseases and adds to the burden of the COVID-19 pandemic worldwide. In immunocompetent individuals, primary Mtb infection can also lead to a non-progressive, asymptomatic latency. However, latent Mtb infection (LTBI) can reactivate symptomatic TB disease upon host immune-suppressing conditions. Importantly, the diagnosis and treatment of TB are hampered and admixed with COVID-19 control measures. The US-Center for Disease Control (US-CDC) recommends using antiviral drugs, Remdesivir or corticosteroid (CST), such as dexamethasone either alone or in-combination with specific recommendations for COVID-19 patients requiring hospitalization or oxygen support. However, CSTs can cause immunosuppression, besides their anti-inflammatory properties. The altered host immunity during COVID-19, combined with CST therapy, poses a significant risk for new secondary infections and/or reactivation of existing quiescent infections, such as LTBI. This review highlights CST therapy recommendations for COVID-19, various types and mechanisms of action of CSTs, the deadly combination of two respiratory infectious diseases COVID-19 and TB. It also discusses the importance of screening for LTBI to prevent TB reactivation during corticosteroid therapy for COVID-19. Full article
(This article belongs to the Collection Feature Papers in Molecular Pharmacology)
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<p>Canonical glucocorticoid receptor signaling underpinning the mechanism of action of corticosteroids. Corticosteroid (CSTs) exert their downstream effects on host cells by activating glucocorticoid receptor (GR), which modulate the transcription of several target genes. The binding of CST with GR leads to conformational changes in GR; the GR-complex translocate to the nucleus, where GR binds to GR-response elements, including positive (blue arrows) and negative (red lines) transcriptional regulators, receptors and enzymes. The downstream effects, including mitochondrial functions, metabolism, stress response, and anti-inflammatory effects of CST, are mediated through differential regulation of various effector molecules (dotted lines with arrow).</p> Full article ">Figure 2
<p>Summary of pathological events associated with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and <span class="html-italic">Mycobacterium tuberculosis</span> (Mtb) co-infection and the effect of corticosteroid therapy. (<b>A</b>). SARS-CoV-2 enters through the pulmonary route, reaches the alveoli, and interacts with host immune cells, including alveolar macrophages (AM) and pneumocytes (P). Infection of AM produces cytokines/chemokines that recruit and activate other immune cells such as T cells (T), monocytes (M), neutrophils (N) from the blood to the infection site. Activated T cells also produce inflammatory cytokines, leading to a “cytokine storm”. In COVID-19 cases with mild and moderate symptoms, the cytokine storm-mediated inflammation is either controlled and the patient recovers; or the infection progresses to active disease with severe symptoms. The lungs of COVID-19 cases with severe symptoms showed elevated immune cell recruitment and activation, leading to pneumonia. As the disease progresses, the accumulation of fluid in the lungs results in inflammatory pulmonary edema, leading to acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. Corticosteroid (CST) treatment blocks inflammation caused by the cytokine storm and prevents the formation of pneumonia, edema, ARDS, and fibrosis in severe COVID-19 cases. (<b>B</b>). Infection of lungs by <span class="html-italic">Mycobacterium tuberculosis</span> (Mtb) causes either symptomatic cavitary progressive tuberculosis (active TB) in individuals with dampened protective immunity or non-progressive LTBI (blue lines) in those with strong immune control. LTBI cases can reactivate to symptomatic TB upon immune-suppressing host conditions. The SARS-CoV-2 infection would happen either in the context of active TB or LTBI (no symptoms). In the SARS-CoV-2 and Mtb co-infected cases (green lines), the host protective immunity is further dampened, leading to exacerbated disease among active TB cases. Besides, SARS-CoV-2 infection of LTBI cases may result in reactivation to active TB (green lines). Corticosteroid (CST) therapy can potentially cause immune-suppression (red line), which augments the dampened protective immunity (purple line), accelerating disease pathology in active TB cases (green line) and reactivates LTBI into symptomatic TB (purple line). Mtb-mediated disease pathologies can be further worsened by SARS-CoV-2 infection; thus, both TB and SARS-CoV-2 promote the disease caused by each of these pathogens.</p> Full article ">
33 pages, 497 KiB  
Review
Manmade Electromagnetic Fields and Oxidative Stress—Biological Effects and Consequences for Health
by David Schuermann and Meike Mevissen
Int. J. Mol. Sci. 2021, 22(7), 3772; https://doi.org/10.3390/ijms22073772 - 6 Apr 2021
Cited by 109 | Viewed by 33840
Abstract
Concomitant with the ever-expanding use of electrical appliances and mobile communication systems, public and occupational exposure to electromagnetic fields (EMF) in the extremely-low-frequency and radiofrequency range has become a widely debated environmental risk factor for health. Radiofrequency (RF) EMF and extremely-low-frequency (ELF) MF [...] Read more.
Concomitant with the ever-expanding use of electrical appliances and mobile communication systems, public and occupational exposure to electromagnetic fields (EMF) in the extremely-low-frequency and radiofrequency range has become a widely debated environmental risk factor for health. Radiofrequency (RF) EMF and extremely-low-frequency (ELF) MF have been classified as possibly carcinogenic to humans (Group 2B) by the International Agency for Research on Cancer (IARC). The production of reactive oxygen species (ROS), potentially leading to cellular or systemic oxidative stress, was frequently found to be influenced by EMF exposure in animals and cells. In this review, we summarize key experimental findings on oxidative stress related to EMF exposure from animal and cell studies of the last decade. The observations are discussed in the context of molecular mechanisms and functionalities relevant to health such as neurological function, genome stability, immune response, and reproduction. Most animal and many cell studies showed increased oxidative stress caused by RF-EMF and ELF-MF. In order to estimate the risk for human health by manmade exposure, experimental studies in humans and epidemiological studies need to be considered as well. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Genotoxicity)
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