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Int. J. Mol. Sci., Volume 24, Issue 13 (July-1 2023) – 820 articles

Cover Story (view full-size image): Hepatocellular carcinoma (HCC) is often diagnosed at an unresectable advanced stage. Advanced HCC is treated with systemic chemotherapy as well as transarterial chemoembolization and hepatic transarterial infusion chemotherapy with cisplatin, which have long been the standard of care for patients with unresectable HCC, but have been limited to the treatment of intrahepatic disease. Recently, systemic chemotherapy with molecularly targeted agents and immune checkpoint inhibitors has been reported to be effective in treating unresectable HCC, and the treatment of unresectable HCC has undergone a major paradigm shift. This review summarizes the action and resistance mechanisms of cisplatin and describes the treatment of major hepatobiliary cancers with cisplatin. View this paper
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16 pages, 5545 KiB  
Article
Mosaic Genome of a British Cider Yeast
by Beatrice Bernardi, Florian Michling, Jürgen Fröhlich and Jürgen Wendland
Int. J. Mol. Sci. 2023, 24(13), 11232; https://doi.org/10.3390/ijms241311232 - 7 Jul 2023
Viewed by 1797
Abstract
Hybrid formation and introgressions had a profound impact on fermentative yeasts domesticated for beer, wine and cider fermentations. Here we provide a comparative genomic analysis of a British cider yeast isolate (E1) and characterize its fermentation properties. E1 has a Saccharomyces uvarum genome [...] Read more.
Hybrid formation and introgressions had a profound impact on fermentative yeasts domesticated for beer, wine and cider fermentations. Here we provide a comparative genomic analysis of a British cider yeast isolate (E1) and characterize its fermentation properties. E1 has a Saccharomyces uvarum genome into which ~102 kb of S. eubayanus DNA were introgressed that replaced the endogenous homologous 55 genes of chromosome XIV between YNL182C and YNL239W. Sequence analyses indicated that the DNA donor was either a lager yeast or a yet unidentified S. eubayanus ancestor. Interestingly, a second introgression event added ~66 kb of DNA from Torulaspora microellipsoides to the left telomere of SuCHRX. This region bears high similarity with the previously described region C introgression in the wine yeast EC1118. Within this region FOT1 and FOT2 encode two oligopeptide transporters that promote improved nitrogen uptake from grape must in E1, as was reported for EC1118. Comparative laboratory scale grape must fermentations between the E1 and EC1118 indicated beneficial traits of faster consumption of total sugars and higher glycerol production but low acetic acid and reduced ethanol content. Importantly, the cider yeast strain produced high levels of fruity ester, including phenylethyl and isoamyl acetate. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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Figure 1

Figure 1
<p>Non-reciprocal translocation and introgressions into the E1 genome. The four non-reciprocal translocations are shown as ribbons connecting segments found on one chromosome in CBS 7001 and translocated to a new position in E1, e.g., an approx. 38 kb fragment found on <span class="html-italic">CHRIII</span> in CBS 7001 was translocated to chromosome XI in E1. Five introgressions (1 to 5) are indicated as arrows originating from non-<span class="html-italic">Saccharomyces uvarum</span> source chromosomes and pointing to insertion sites in E1, e.g., for translocation 1: an 8.7 kb DNA fragment from <span class="html-italic">S. eubayanus</span> chromosome II was introgressed into E1 chromosome II. Fold read depth track is based on counting binned short reads aligned to the draft assembly. (Pairwise) Similarity track is based on Tamura and Nei two-parameter metric without γ correction [<a href="#B45-ijms-24-11232" class="html-bibr">45</a>] in consecutive 1 kb windows containing at least 900 aligned positions; only values &gt;98% are shown (similarity was &lt;98% in 147 of 11,419 windows). Cyan lines are median fold read depth and median similarity on the corresponding tracks. LCB track shows Locally Collinear Blocks between E1 draft genome and reference genome contigs. Reference genome contigs are in clockwise orientation and E1 contigs are oriented counterclockwise. %). Contig extent in the E1 draft genome is shown (grey subdivisions on green pseudochromosomes). <span class="html-italic">Su</span>—<span class="html-italic">Saccharomyces uvarum</span>; <span class="html-italic">Se</span>—<span class="html-italic">S. eubayanus</span>; <span class="html-italic">Sk</span>—<span class="html-italic">S. kudriavzevii</span>; <span class="html-italic">Tm—Torulaspora microellipsoides</span>; region C—region C from <span class="html-italic">T. microellipsoides</span>; and LCB—Locally Collinear Block (from Whole Genome Alignment).</p> Full article ">Figure 2
<p><span class="html-italic">Saccharomyces uvarum</span> E1 genome structure and the positions of five introgressed, non-<span class="html-italic">Saccharomyces uvarum</span> DNA fragments. The E1 contigs were aligned to the 16 chromosomes of CBS 7001. E1 is a diploid yeast strain, and all introgressions were also present in both of the respective chromosomes. The positions of introgressions are marked by a triangle. <span class="html-italic">Su</span>—<span class="html-italic">Saccharomyces uvarum</span>; <span class="html-italic">Se</span>—<span class="html-italic">S. eubayanus</span>; <span class="html-italic">Sk</span>—<span class="html-italic">S. kudriavzevii</span>; and region C represents the region C from <span class="html-italic">Torulaspora microellipsoides</span>.</p> Full article ">Figure 3
<p>Distribution of similarity of <span class="html-italic">Saccharomyces uvarum</span> E1 DNA with the respective reference genomes. Similarity was calculated for consecutive 1 kb windows containing at least 900 aligned positions (Tamura and Nei two-parameter similarity without γ correction [<a href="#B46-ijms-24-11232" class="html-bibr">46</a>]). Lower extent of vertical axis (similarity) was restricted to only display values &gt; 95% (similarity &lt; 95% was observed only in a total of 23 windows). The number of bases derived from each species is listed at the bottom.</p> Full article ">Figure 4
<p>Large introgression of <span class="html-italic">S. eubayanus CHRXIV</span> DNA into E1. DNA sequence identity of the 102 kb <span class="html-italic">S. eubayanus</span> introgression into E1 to Saaz group lager yeast <span class="html-italic">S. eubayanus</span> subgenomes (<b>A</b>) and <span class="html-italic">S. uvarum</span> CBS 7001 (<b>B</b>) and sequence similarity of the 102 kb <span class="html-italic">S. eubayanus</span> insert to natural <span class="html-italic">S. eubayanus</span> strains or industrial hybrids (<b>C</b>). Pairwise similarity was calculated for 500 nt consecutive windows. Cumulative mismatch plots illustrate the origin of the introgressed segment; i.e., few mismatches accumulated along the aligned E1 DNA sequence when compared to a DNA sequence conspecific to the introgression donor. Black line—sequence similarity E1/CBS 1513 or E1/CBS 7001 (panels A and B, respectively); orange line—sequence similarity E1/other lager yeasts; green line (panel B)—E1/CBS 7001.</p> Full article ">Figure 5
<p>E1 region C variant. The <span class="html-italic">T. microellipsoides</span> (<span class="html-italic">Tm</span>) introgression into E1 contains an additional 1785 nt <span class="html-italic">Tm</span>-DNA missing in the wine strain EC1118, from which region C was first described. <span class="html-italic">Su</span>—<span class="html-italic">Saccharomyces uvarum</span>; <span class="html-italic">Sc</span>—<span class="html-italic">S. cerevisiae.</span> Genes and their transcriptional orientation of the 5′-part of region C are indicated by arrows.</p> Full article ">Figure 6
<p>Comparative fermentations between the cider yeast strain and EC1118. Müller-Thurgau must was fermented at either 10 °C (<b>A</b>) or 18 °C (<b>B</b>) by the cider yeast E1 (red lines) or the wine yeast EC1118 (blue). CO<sub>2</sub> release was measured by monitoring daily mass loss of the fermentation vessels. HPLC analysis of ethanol, glycerol and acetic acid are shown.</p> Full article ">Figure 7
<p>Volatile aroma compounds (VOCs) production. At the end of fermentation, compounds produced by E1 and EC1118 were analyzed by GS/MS. Fermentations were performed in triplicate (A,B,C) at 10 °C and 18 °C. Concentrations of VOCs produced are normalized to logarithmic scale and converted into a color-coded heat map indicating consistency of fermentations and allowing for better comparison between E1 and EC1118.</p> Full article ">Figure 8
<p>Growth and N assimilation by <span class="html-italic">fot1/fot2</span> deletion strains. (<b>A</b>) Growth of the <span class="html-italic">fot1/fot2</span> deletion strains (G166–G168) on beer plates was compared to the E1 wild type strain. Images were obtained after two and three days of growth at 25 °C. (<b>B</b>) Amino nitrogen content at the end of fermentation with the <span class="html-italic">fot1/fot2</span> deletion strains was compared to E1. Error bars indicate standard deviation.</p> Full article ">
12 pages, 2514 KiB  
Article
Effect of Trace Metal Ions on the Conformational Stability of the Visual Photoreceptor Rhodopsin
by Feifei Wang, Pol Fernandez-Gonzalez, Eva Ramon, Patricia Gomez-Gutierrez, Margarita Morillo and Pere Garriga
Int. J. Mol. Sci. 2023, 24(13), 11231; https://doi.org/10.3390/ijms241311231 - 7 Jul 2023
Cited by 1 | Viewed by 1520
Abstract
Trace metals are essential elements that play key roles in a number of biochemical processes governing human visual physiology in health and disease. Several trace metals, such as zinc, have been shown to play important roles in the visual phototransduction process. In spite [...] Read more.
Trace metals are essential elements that play key roles in a number of biochemical processes governing human visual physiology in health and disease. Several trace metals, such as zinc, have been shown to play important roles in the visual phototransduction process. In spite of this, there has been little research conducted on the direct effect of trace metal elements on the visual photoreceptor rhodopsin. In the current study, we have determined the effect of several metal ions, such as iron, copper, chromium, manganese, and nickel, on the conformational stability of rhodopsin. To this aim, we analyzed, by means of UV-visible and fluorescence spectroscopic methods, the effects of these trace elements on the thermal stability of dark rhodopsin, the stability of its active Metarhodopsin II conformation, and its chromophore regeneration. Our results show that copper prevented rhodopsin regeneration and slowed down the retinal release process after illumination. In turn, Fe3+, but not Fe2+, increased the thermal stability of the dark inactive conformation of rhodopsin, whereas copper ions markedly decreased it. These findings stress the important role of trace metals in retinal physiology at the photoreceptor level and may be useful for the development of novel therapeutic strategies to treat retinal disease. Full article
(This article belongs to the Special Issue Molecular Basis of Sensory Transduction in Health and Disease 2.0)
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Figure 1

Figure 1
<p>UV-vis absorption spectrum of immunopurified Rho from bovine ROS, in 2 mM sodium phosphate (NaPi), pH 6.0, and 0.05% n-dodecyl-β-<span class="html-small-caps">d</span>-maltoside (DM). The spectrum shows the characteristic bands at 280 nm (opsin) and 500 nm (11CR bound to opsin).</p> Full article ">Figure 2
<p>The UV-vis absorption spectra of Rho were obtained following pre-treatment with various metals under different experimental conditions. Spectra of samples were recorded in the dark state (dark, solid line), after metal addition (dark, dashed red line), upon photobleaching for 30 s (light, dashed line) and after acidification with 2N H<sub>2</sub>SO<sub>4</sub> (acid, dotted line). Samples were, respectively, treated with Fe<sup>3+</sup>, Cu<sup>2+</sup>, and Fe<sup>2+</sup> at a final concentration of 50 μM. All the above experiments were conducted at 20 °C.</p> Full article ">Figure 3
<p>Effects of selected trace metals on the chromophore thermal stability of Rho in vitro. Under dark conditions, each trace metal was added separately to the sample at a final concentration of 50 μM. The absorbance of the sample was recorded in the wavelength range of 250 nm to 650 nm at 48 °C, with measurements taken every 2 min for a total duration of 100 min. The absorbance at 500 nm was plotted, and the t<sub>1/2</sub> of the process was calculated based on the fitted curves. Mean and standard error of mean values were derived from independent repeated experiments (n = 3, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 4
<p>Effect of trace elements on the chromophore regeneration of Rho. The impact of trace elements on Rho regeneration was determined. Different metals and 11CR were added and the dark spectra were measured. Samples were subsequently photobleached, and spectra were recorded every 2 min at 20 °C min. The absorbance at 500 nm was plotted, and the mean and standard error of mean were calculated based on independent repeated experiments. (<b>a</b>) The t<sub>1/2</sub> of the process was calculated from the fitted curves. (<b>b</b>) Regeneration percentage of Rho. The ratio of the amount of regenerated Rho to the original Rho was calculated. Mean and standard error of mean values were derived from independent repeated experiments (n = 3).</p> Full article ">Figure 5
<p>Meta II stability of Rho treated with or without metals. Fluorescence curves were recorded on a PTI Quanta Master 4 spectrofluorometer with a sample 0.5 µM Rho in the absence and the presence of metals at 20 °C. The fluorescence signal of Trp265 gradually increased over time, as a result of retinal leaving its binding pocket, until it reached a plateau.</p> Full article ">Figure 6
<p>Effects of trace elements on Meta II t<sub>1/2</sub>. Upon excitation with light at 295 nm, Rho undergoes conformational changes, causing the release of bound retinal from the binding pocket and the fluorescence emission of a previously shielded Trp265. The fluorescence signal increase was recorded using a spectrofluorometer, and the t<sub>1/2</sub> of the Meta II decay process was determined. Mean and standard error of mean values were derived from independent repeated experiments (n = 3, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 7
<p>Fe<sup>3+</sup> and Fe<sup>2+</sup> binding sites on the surface of the crystal structure of bovine Rho (PDB ID: 1U19) determined by GRID22 program as implemented in MOE software (version MOE2020.09). Left models correspond to a full protein view, whereas the right models correspond to the magnified retinal binding pocket domain and are depicted in an inverted manner with regard to the left images for better visualization of the 11-<span class="html-italic">cis</span>-retinal chromophore. The secondary structure is represented in blue-colored ribbon, whereas retinal is shown in green using CPK (<b>left</b>) or stick representation (<b>right</b>). Surfaces in orange and purple are the calculated interaction potential surfaces for Fe<sup>3+</sup> and Fe<sup>2+</sup>, respectively, using an iso-contour level of −12.5 kcal/mol.</p> Full article ">
19 pages, 7389 KiB  
Article
EPs® 7630 Stimulates Tissue Repair Mechanisms and Modifies Tight Junction Protein Expression in Human Airway Epithelial Cells
by Lei Fang, Liang Zhou, Žarko Kulić, Martin D. Lehner, Michael Tamm and Michael Roth
Int. J. Mol. Sci. 2023, 24(13), 11230; https://doi.org/10.3390/ijms241311230 - 7 Jul 2023
Cited by 4 | Viewed by 2380
Abstract
Airway epithelium repair after infection consists of wound repair, re-synthesis of the extracellular matrix (ECM), and tight junction proteins. In humans, EPs® 7630 obtained from Pelargonium sidoides roots reduces the severity and duration of acute respiratory tract infections. The effect of EPs [...] Read more.
Airway epithelium repair after infection consists of wound repair, re-synthesis of the extracellular matrix (ECM), and tight junction proteins. In humans, EPs® 7630 obtained from Pelargonium sidoides roots reduces the severity and duration of acute respiratory tract infections. The effect of EPs® 7630 on tissue repair of rhinovirus-16 (RV-16) infected and control human airway epithelial cells was assessed for: (i) epithelial cell proliferation by manual cell counts, (ii) epithelial wound repair by “scratch assay”, (iii) ECM composition by Western-blotting and cell-based ELISA, and (iv) epithelial tight junction proteins by Western-blotting. EPs® 7630 stimulated cell proliferation through cAMP, CREB, and p38 MAPK. EPs® 7630 significantly improved wound repair. Pro-inflammatory collagen type-I expression was reduced by EPs® 7630, while fibronectin was increased. Virus-binding tight junction proteins desmoglein2, desmocollin2, ZO-1, claudin1, and claudin4 were downregulated by EPs® 7630. The RV16-induced shift of the ECM towards the pro-inflammatory type was prevented by EPs® 7630. Most of the effects of EPs® 7630 on tissue repair and regeneration were sensitive to inhibition of cAMP-induced signaling. The data suggest that EPs® 7630-dependent modification of epithelial cell metabolism and function might underlie the faster recovery time from viral infections, as reported by others in clinical studies. Full article
(This article belongs to the Special Issue Bioactive Compounds from Natural Products for the Prevention and Treatment of Chronic Diseases 2.0)
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Figure 1

Figure 1
<p>EPs<sup>®</sup> 7630 stimulates epithelial cell proliferation and intracellular signaling. (<b>A</b>) EPs<sup>®</sup> 7630 increased epithelial cell counts (48 h) and compensated cell loss of RV16-infected cells. * indicates significant difference compared to control 48 h. The effect of the different treatments was determined by direct cell count (upper panel) and by MTT assay (lower panel). (<b>B</b>) Role of signaling mediators on EPs<sup>®</sup> 7630 induced cell proliferation. The effect of the different treatments was determined by direct cell count (upper panel) and by MTT assay (lower panel). (<b>C</b>) EPs<sup>®</sup> 7630 stimulated the formation of intracellular cAMP. (<b>D</b>) Kinetic of EPs<sup>®</sup> 7630 dependent shift of Akt to phosphorylated (p-)Akt. (<b>E</b>) Kinetic of EPs<sup>®</sup> 7630 dependent shift of cyclic AMP response element binding protein (CREB) to p-CREB. (<b>F</b>) Kinetic of EPs<sup>®</sup> 7630 dependent shift of protein-38 (p38) to p-p38 MAPK. (<b>G</b>) Kinetic of EPs<sup>®</sup> 7630 dependent shift of mammalian target of rapamycin (mTOR) to p-mTOR. (<b>H</b>) Representative Western-blots used to generate the above shown bar charts of signaling proteins. Bars show mean ± S.E.M. of experiments performed in four different epithelial cell strains.</p> Full article ">Figure 1 Cont.
<p>EPs<sup>®</sup> 7630 stimulates epithelial cell proliferation and intracellular signaling. (<b>A</b>) EPs<sup>®</sup> 7630 increased epithelial cell counts (48 h) and compensated cell loss of RV16-infected cells. * indicates significant difference compared to control 48 h. The effect of the different treatments was determined by direct cell count (upper panel) and by MTT assay (lower panel). (<b>B</b>) Role of signaling mediators on EPs<sup>®</sup> 7630 induced cell proliferation. The effect of the different treatments was determined by direct cell count (upper panel) and by MTT assay (lower panel). (<b>C</b>) EPs<sup>®</sup> 7630 stimulated the formation of intracellular cAMP. (<b>D</b>) Kinetic of EPs<sup>®</sup> 7630 dependent shift of Akt to phosphorylated (p-)Akt. (<b>E</b>) Kinetic of EPs<sup>®</sup> 7630 dependent shift of cyclic AMP response element binding protein (CREB) to p-CREB. (<b>F</b>) Kinetic of EPs<sup>®</sup> 7630 dependent shift of protein-38 (p38) to p-p38 MAPK. (<b>G</b>) Kinetic of EPs<sup>®</sup> 7630 dependent shift of mammalian target of rapamycin (mTOR) to p-mTOR. (<b>H</b>) Representative Western-blots used to generate the above shown bar charts of signaling proteins. Bars show mean ± S.E.M. of experiments performed in four different epithelial cell strains.</p> Full article ">Figure 2
<p>Effect of EPs<sup>®</sup> 7630 on the expression of fibronectin and collagen type-I. (<b>A</b>) Cell-based ELISA analysis of fibronectin deposition induced by EPs<sup>®</sup> 7630. (<b>B</b>) Role of cell signaling on EPs<sup>®</sup> 7630-induced fibronectin deposition. (<b>C</b>) Inhibitory effect of EPs<sup>®</sup> 7630 on collagen type-I deposition. (<b>D</b>) Collagen type-I expression influenced by different cell signaling inhibitors. Bars show mean ± S.E.M. of n = 4 cell lines.</p> Full article ">Figure 3
<p>EPs<sup>®</sup> 7630 modifies the expression of tight junction proteins and cell differentiation. Representative Western-blots and the image analysis of four Western-blots are presented as bar charts of the mean ± S.E.M. for: (<b>A</b>) claudin1, (<b>B</b>) claudin4, (<b>C</b>) occludin, (<b>D</b>) desmocolin2, (<b>E</b>) desmoglein2, and (<b>F</b>) ZO-1. The loading control, α-tubulin, was the same for all six tight junction proteins. Statistics were calculated using Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 4
<p>EPs<sup>®</sup> 7630 modifies the expression of tight junction proteins and cell differentiation. Representative Western-blots and subsequent image analysis of four independent experiments are shown for (<b>A</b>) E-cadherin, (<b>B</b>) gelsolin, (<b>C</b>) ICAM-1, and (<b>D</b>) β-defensin-1. The loading control, α-tubulin, was the same for all five tight junction proteins. Bar charts represent mean ± S.E.M., and <span class="html-italic">p</span>-values were calculated using Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 5
<p>Effect of EPs<sup>®</sup> 7630 on wound repair in human primary epithelial cells. (<b>A</b>) Representative phase-contrast photographs and measurement of wound area (area in between the two blue lines) over 6 h. Similar results were obtained in 2 additional cell lines. (<b>B</b>) Wound repair kinetic of naïve cells over 24 h. (<b>C</b>) Wound repair kinetic of RV16-infected cells over 24 h. Data points represent the mean ± S.E.M. of three cell lines; each cell line and time point were measured in duplicate experiments. <span class="html-italic">p</span>-values indicate significant differences comparing untreated to EPs<sup>®</sup> 7630 pre-treated cells.</p> Full article ">Figure 6
<p>Summary of EPs<sup>®</sup> 7630 induced cAMP-dependent regulation of epithelial barrier function and reduced expression of virus-adhering tight junction proteins. “P” in blue circle indicates phosphorylation of the corresponding signaling protein. Red arrow indicates upregulation and blue arrow indicates downregulation.</p> Full article ">Figure 7
<p>(<b>A</b>) LC-UV-HRMS fingerprint of EPs<sup>®</sup> 7630 detected at different UV wavelengths and in negative and positive ion modes as annotated. The substance assignments a-w are given in <a href="#ijms-24-11230-t001" class="html-table">Table 1</a>. The prodelphinidins are visible at 254 nm and 280 nm as a bulge between ca. 12 and 38 min. (<b>B</b>) HPTLC fingerprint of EPs<sup>®</sup> 7630 native and with different staining reagents as annotated, detected at visible light and 366 nm, respectively. Umckalin is the uppermost spot at an R<sub>f</sub> of ca. 0.95. The dark spot at the start (R<sub>f</sub> = 0) represents the prodelphinidins.</p> Full article ">Figure 8
<p><sup>1</sup>H- and <sup>13</sup>C-NMR fingerprints of EPs<sup>®</sup> 7630 in two different solvents as annotated. The prodelphinidins have broad signals due to the oligomeric nature and the manifold structures of the substance class. In contrast, the benzopyranons and a carbohydrate portion have sharp signals, as characteristic for small molecules. In DMSO-d<sub>6</sub>, the OH groups of the prodelphinidins are visible as broad signals in the <sup>1</sup>H spectrum between 7 and 9 ppm. These signals are absent in the <sup>1</sup>H spectrum measured in D<sub>2</sub>O/MeOD-d<sub>4</sub> because the prodelphinidin OH protons exchange with the deuterons of the solvent.</p> Full article ">
20 pages, 973 KiB  
Review
Stem/Progenitor Cells and Related Therapy in Bronchopulmonary Dysplasia
by Manuela Marega, Natalia El-Merhie, Mira Y. Gökyildirim, Valerie Orth, Saverio Bellusci and Cho-Ming Chao
Int. J. Mol. Sci. 2023, 24(13), 11229; https://doi.org/10.3390/ijms241311229 - 7 Jul 2023
Cited by 6 | Viewed by 2824
Abstract
Bronchopulmonary dysplasia (BPD) is a chronic lung disease commonly seen in preterm infants, and is triggered by infection, mechanical ventilation, and oxygen toxicity. Among other problems, lifelong limitations in lung function and impaired psychomotor development may result. Despite major advances in understanding the [...] Read more.
Bronchopulmonary dysplasia (BPD) is a chronic lung disease commonly seen in preterm infants, and is triggered by infection, mechanical ventilation, and oxygen toxicity. Among other problems, lifelong limitations in lung function and impaired psychomotor development may result. Despite major advances in understanding the disease pathologies, successful interventions are still limited to only a few drug therapies with a restricted therapeutic benefit, and which sometimes have significant side effects. As a more promising therapeutic option, mesenchymal stem cells (MSCs) have been in focus for several years due to their anti-inflammatory effects and their secretion of growth and development promoting factors. Preclinical studies provide evidence in that MSCs have the potential to contribute to the repair of lung injuries. This review provides an overview of MSCs, and other stem/progenitor cells present in the lung, their identifying characteristics, and their differentiation potential, including cytokine/growth factor involvement. Furthermore, animal studies and clinical trials using stem cells or their secretome are reviewed. To bring MSC-based therapeutic options further to clinical use, standardized protocols are needed, and upcoming side effects must be critically evaluated. To fill these gaps of knowledge, the MSCs’ behavior and the effects of their secretome have to be examined in more (pre-) clinical studies, from which only few have been designed to date. Full article
(This article belongs to the Special Issue Pediatric Lung Diseases: Novel Molecular Insights and Therapeutic Advances)
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Figure 1

Figure 1
<p>Schematic overview of the different cell types within the respiratory epithelium. Trachea to bronchi: the epithelial surfaces of the trachea and proximal airways are lined by a pseudostratified columnar epithelium consisting of basal, brush, ciliated, goblet, and club cells, along with the less frequent pulmonary neuroendocrine cells (PNECs) and ionocytes. Bronchioles to alveoli: small airways are lined by a simple columnar or cuboidal epithelium that consists of ciliated, club, and a few goblet cells. Alveoli: the alveoli are lined by either squamous alveolar type 1 (AT1) or cuboidal alveolar type 2 (AT2) cell.</p> Full article ">Figure 2
<p>Schematic representation of the regenerative potential of the lung stem/progenitor cells (<b>A</b>) MSCs support the stem cell niche under physiological conditions and upon injury; basal cells differentiate in the secretory and ciliated cells. Variant cells can contribute to the secretory cell pool, and the secretory cells can come from the BASCs and secretory cells themselves. BASCs can differentiate in ciliated cells as well. AT2 cells can differentiate from the BASCs, and they represent the primary source of AT1 cells, and additionally they can maintain themselves; MSCs contribute to suppressing inflammation, inhibiting immune cells, such as monocytes, neutrophils, and lymphocytes (decreased level of pro-inflammatory molecules, TNF-α, IL-6 e.g.,) (<b>B</b>) MSCs give rise to the entire mesenchymal compartment, including lipofibroblasts (LIFs) and myofibroblasts (MYFs), with the activation of the lipogenic or myogenic program that can contribute to the progression or the resolution of injuries.</p> Full article ">
13 pages, 3066 KiB  
Article
Elevation of Arginase-II in Podocytes Contributes to Age-Associated Albuminuria in Male Mice
by Guillaume Ajalbert, Andrea Brenna, Xiu-Fen Ming, Zhihong Yang and Duilio M. Potenza
Int. J. Mol. Sci. 2023, 24(13), 11228; https://doi.org/10.3390/ijms241311228 - 7 Jul 2023
Cited by 1 | Viewed by 1491
Abstract
One of the manifestations of renal aging is podocyte dysfunction and loss, which are associated with proteinuria and glomerulosclerosis. Studies show a male bias in glomerular dysfunction and chronic kidney diseases, and the underlying mechanisms remain obscure. Recent studies demonstrate the role of [...] Read more.
One of the manifestations of renal aging is podocyte dysfunction and loss, which are associated with proteinuria and glomerulosclerosis. Studies show a male bias in glomerular dysfunction and chronic kidney diseases, and the underlying mechanisms remain obscure. Recent studies demonstrate the role of an age-associated increase in arginase-II (Arg-II) in proximal tubules of both male and female mice. However, it is unclear whether Arg-II is also involved in aging glomeruli. The current study investigates the role of the sex-specific elevation of Arg-II in podocytes in age-associated increased albuminuria. Young (3–4 months) and old (20–22 months) male and female mice of wt and arginase-II knockout (arg-ii−/−) were used. Albuminuria was employed as a readout of glomerular function. Cellular localization and expression of Arg-II in glomeruli were analyzed using an immunofluorescence confocal microscope. A more pronounced age-associated increase in albuminuria was found in male than in female mice. An age-associated induction of Arg-II in glomeruli and podocytes (as demonstrated by co-localization of Arg-II with the podocyte marker synaptopodin) was also observed in males but not in females. Ablation of the arg-ii gene in mice significantly reduces age-associated albuminuria in males. Also, age-associated decreases in podocyte density and glomerulus hypertrophy are significantly prevented in male arg-ii−/− but not in female mice. However, age-associated glomerulosclerosis is not affected by arg-ii ablation in both sexes. These results demonstrate a role of Arg-II in sex-specific podocyte injury in aging. They may explain the sex-specific differences in the development of renal disease in humans during aging. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Kidney Injury 2.0)
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<p>Arg-II knockout decreases albuminuria in aged male mice. Urinary creatinine (<b>A</b>,<b>D</b>) and albumin (<b>B</b>,<b>E</b>) were measured in young and old <span class="html-italic">wt</span> and <span class="html-italic">arg-ii<sup>−/−</sup></span> mice of both sexes. Glomerular function (<b>C</b>,<b>F</b>) monitored via albuminuria is reported as the ratio of urinary albumin/creatinine (uACR). <span class="html-italic">n</span> = 11–13. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> ≤ 0.001, **** <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">Figure 2
<p>Serum creatinine is not affected by aging and sex. Serum creatinine levels were measured to evaluate glomerular dysfunction. <span class="html-italic">n</span> = 5.</p> Full article ">Figure 3
<p>Podocyte number and density. (<b>A</b>,<b>D</b>) Representative confocal images of kidney glomeruli of all the groups showing WT1 (white) and synaptopodin (SNPT, red) co-staining. DAPI was used to stain the nuclei (blue). (<b>B</b>,<b>E</b>) Quantification of the podocyte density in glomeruli of male (<b>B</b>) and female mice (<b>E</b>). (<b>C</b>,<b>F</b>) Podocyte density was calculated as the average of all analyzed glomeruli per kidney. Scale bar: 30 µm. <span class="html-italic">n</span> = 10. * <span class="html-italic">p</span> ≤ 0.05, **** <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">Figure 4
<p>Glomerulosclerosis score. (<b>A</b>) Representative image for each score category obtained with PAS staining. PAS-positive tissue is stained magenta; nuclei are counterstained with hematoxylin (dark blue). Scale bar: 20 µm. (<b>B</b>,<b>C</b>) Quantification of glomerulosclerosis scores for male (<b>B</b>) and female mice (<b>C</b>). The total glomerulosclerosis score was calculated from the average score of 100 glomeruli per kidney. <span class="html-italic">n</span> = 9. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> ≤ 0.001, **** <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">Figure 5
<p><span class="html-italic">WT</span> old male glomeruli show Arg-II expression, while female glomeruli do not. (<b>A</b>) Co-immunostaining of glomeruli for Arg-II (green) and SNPT (red). Nuclei were stained with DAPI (blue). A merged image is shown. Scale bar: 20 µm. (<b>B</b>) Quantification of Arg-II-positive glomeruli. (<b>C</b>) The enlarged image of the indicated insert in (<b>A</b>) reveals the co-localization of Arg-II with the podocyte marker SNPT in the glomeruli of aged male mice. Arrows indicate co-localization. Scale bar: 10 µm. **** <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">
33 pages, 4978 KiB  
Review
AB Toxins as High-Affinity Ligands for Cell Targeting in Cancer Therapy
by Ana Márquez-López and Mónica L. Fanarraga
Int. J. Mol. Sci. 2023, 24(13), 11227; https://doi.org/10.3390/ijms241311227 - 7 Jul 2023
Cited by 2 | Viewed by 2930
Abstract
Conventional targeted therapies for the treatment of cancer have limitations, including the development of acquired resistance. However, novel alternatives have emerged in the form of targeted therapies based on AB toxins. These biotoxins are a diverse group of highly poisonous molecules that show [...] Read more.
Conventional targeted therapies for the treatment of cancer have limitations, including the development of acquired resistance. However, novel alternatives have emerged in the form of targeted therapies based on AB toxins. These biotoxins are a diverse group of highly poisonous molecules that show a nanomolar affinity for their target cell receptors, making them an invaluable source of ligands for biomedical applications. Bacterial AB toxins, in particular, are modular proteins that can be genetically engineered to develop high-affinity therapeutic compounds. These toxins consist of two distinct domains: a catalytically active domain and an innocuous domain that acts as a ligand, directing the catalytic domain to the target cells. Interestingly, many tumor cells show receptors on the surface that are recognized by AB toxins, making these high-affinity proteins promising tools for developing new methods for targeting anticancer therapies. Here we describe the structure and mechanisms of action of Diphtheria (Dtx), Anthrax (Atx), Shiga (Stx), and Cholera (Ctx) toxins, and review the potential uses of AB toxins in cancer therapy. We also discuss the main advances in this field, some successful results, and, finally, the possible development of innovative and precise applications in oncology based on engineered recombinant AB toxins. Full article
(This article belongs to the Special Issue Bacterial Toxins and Cancer)
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<p>Schematic representation of the cellular effects of highly potent biotoxins. (<b>a</b>) Ricin toxin (Rtx), a protein produced by the seeds of the castor bean plant, has inhibitory effects on protein synthesis. When Rtx is ingested or inhaled, it leads to tissue damage in various organs. (<b>b</b>) Staphylococcal enterotoxins (SEs) are pyrogenic proteins that cause gastroenteritis and emesis. They induce the release of inflammatory molecules by immune system cells. (<b>c</b>) Examples of neurotoxins. Batrachotoxin, a steroid alkaloid found in the skin of the Columbine poison frog, exhibits selectivity in binding to sodium channels. This binding action causes the channels to remain open, leading to heightened permeability to sodium ions. Consequently, it induces cardiotoxicity and neurotoxicity. Among the bacterial toxins, Botulinum neurotoxins (BoNTs) and Tetanus neurotoxin (TeNT) are noteworthy examples. Produced by Clostridium species, these toxins hinder the presynaptic release of the neurotransmitter acetylcholine, thereby resulting in reversible paralysis. BoNTs primarily impede acetylcholine release in peripheral cholinergic terminals, while TeNT targets inhibitory interneurons within the spinal cord. In the graphical representation, inhibition is denoted by a red cross, while activation is indicated by a green tick. Rtx, SEs, BoNTs, and TeNT are represented as cartoon structures using Biorender software. Toxin protein structures Data Bank IDs are 2AAI for Ricin, 1SXT for SEs, 1S0F for BoNTs, and 5N0C for TeNT.</p> Full article ">Figure 2
<p>Scheme of the active forms of the different types of AB toxins. (<b>a)</b> AB toxins have two moieties: A and B. The A moiety is the active domain with enzymatic activity, while the B domain has the binding receptor property. Depending on the type of AB toxins, they can also have a linker between A and B that usually consists of a peptide and/or a disulfide bond. AB toxins active form results from a proteolytic cleavage between A and B moieties or within the A or B moiety. These toxins can be classified according to their A:B stoichiometry. (<b>b</b>) Single-chain AB toxins are produced as single polypeptide chains. They have a 1:1 A B stoichiometry, and both subunits remain linked by an interchain disulfide bond in the active form. (<b>c</b>) When the B domain is in an oligomeric state, the A and B moieties are produced as separate proteins, which assemble later on. In AB<sub>2</sub> and AB<sub>5</sub> toxins, A and B are assembled after the synthesis to form the holotoxin. In the active form, proteolytic cleavage occurs within the A domain, giving rise to the A1 and A2 domains that remain linked by a disulfide bond. (<b>d</b>) In A + B toxins, A and B are assembled into the de-active form after the B moiety suffers proteolytic cleavage. In this case, the holotoxin is usually in AB<sub>7</sub> form.</p> Full article ">Figure 3
<p>AB toxins classification, according to their structure and A translocation into the cytosol. (<b>a</b>) Toxins of group 1 (Dtx, BoNTs, TeNT) are produced as single-polypeptide precursors. These proteins are proteolytically cleaved to generate a di-chain linked by an inter-chain disulfide bond. In the endosome membrane, the B subunit forms a pore to translocate the A subunit into the cytosol. (<b>b</b>) Toxins of group 2 (Atx, C2 toxin) are produced as separated A and B proteins. The B moiety is activated by proteolytic cleavage by host furin proteases and assembles into heptamer-shaped ring structures that can bind the A moiety. B heptamers form a pore in the membrane of the endosome to translocate the A subunit into the cytosol. (<b>c</b>) Toxins of group 3 (Stx, Ctx), as in group 2, are synthesized as independent A and B proteins. However, proteolytic cleavage occurs in the A moiety, resulting in two fragments that remain linked by a disulfide bond. In the ER, A1 is translocated to the cytosol with the assistance of ER machinery (Sec proteins). This step requires the reduction of the disulfide bond. (<b>d</b>) Finally, toxins of group 4 (Rtx, Exotoxin A) are similar to toxins of group 1, but translocation of the A subunit occurs in the ER instead of the endosomes. Scissors indicate the proteolytic cleavage spot on the polypeptide toxins.</p> Full article ">Figure 4
<p>Molecular structure of Dtx and proHB-EGF, binding sites in the B subunit, and mechanism of action. (<b>a</b>) Crystallized structure of the proHB-EGF (purple) extracellular domain corresponding to residues 107–147. (<b>b</b>) Crystallized structure of Dtx. It consists of three different domains: R in pink, T in green, and C in light blue. The R and T domains are the B moiety of the toxin (binding and translocation domains), and the C domain corresponds to the A moiety. (<b>c</b>) Crystallized structure of proHB-EGF<sub>107–147</sub>–DtxR binding. All protein structures were depicted using PyMOL software, employing a cartoon representation. The Protein Data Bank IDs are the following: 1XDT for the extracellular HB-EGF domain, 1F0L for Dtx, and 1XDT for Dtx-HB-EGF binding. (<b>d</b>) Receptor-mediated endocytosis (RME) is the mechanism through which Dtx reaches the cytosol. It binds to the pro-HB-EGF receptor on the surface of host cells and enters the endolysosomal pathway via clathrin-coated pits. Once inside the endosome, the complex encounters an acidic environment, and the T-domain is inserted in the membrane of the endosome, forming a channel, and allowing the catalytic subunit DtxA to reach the cytosol. DtxA catalyzes NAD<sup>+</sup>–dependent ADP-ribosylation of EF-2, inhibiting protein synthesis. Dtx is represented as cartoon structures using Biorender software.</p> Full article ">Figure 5
<p>Molecular structure of Atx and TEM8 and binding sites in the B subunit. (<b>a</b>) Crystallized structure of the tripartite Atx: PA in pink and brown, EF in light purple, and LF in blue. PA is the B subunit of the toxin, and LF and EF are the catalytic subunits. (<b>b</b>) Crystallized structure of the TEM8 vWA extracellular domain corresponding to residues 36–220. This domain consists of six α-helices (α1–α6) surrounding a central hydrophobic core formed by six stranded β-sheets (β1–β6). From left to right, side, top, and bottom views of the vWA domain. Mg<sup>2+</sup> ion is represented as a pink sphere bound in the MIDAS motif. (<b>c</b>) Crystallized structure of TEM<sub>836–220</sub>-PA Binding. TEM8 binds to Atx through hydrophobic interactions with residues of domains II and IV of PA. Residues 153–158 located in the β3–β4 loop interact with Leu<sub>340</sub> and Ala<sub>341</sub> of PA domain IV. Residues 113 and 115 located in the α2–α3 loop, interact with a hydrophobic cleft comprised of Leu<sub>687</sub>, Ile<sub>689</sub>, Ile<sub>646</sub>, Phe<sub>678</sub>, and Ile<sub>656</sub> of PA domain IV. Residues 87 and 88 located in the β2–β3 loop interact with Asp<sub>657</sub>, Arg<sub>658</sub>, Asp<sub>714</sub>, and Thr<sub>715</sub> of PA domain IV. Finally, residues 65 and 57 located in helix α1 interact with Tyr<sub>688</sub> in PA domain II. After binding, a 20 kDa peptide (PA<sub>20</sub>, brown) is cut from PA<sub>83</sub> by a furin protease of the host cell. PA<sub>63</sub> forms a ring-shaped heptamer where EF and LF can bind. All protein structures are shown as cartoon representations using PyMOL software. The Protein Data Bank IDs are the following: 3N2N for the TEM8 extracellular domain, 1ACC for PA, 1J7N for LF, 1XFY for EF, and 1T6B for TEM8-PA binding.</p> Full article ">Figure 6
<p>Atx entry mechanisms and cellular effects. Receptor-mediated endocytosis (RME) is also the mechanism through which Atx reaches the cytosol. It binds to TEM8/CMG2 on the surface of host cells and enters the endolysosomal pathway via clathrin-coated pits. Once inside the endosome, the complex encounters an acidic environment, and a channel is formed in the membrane of the endosome, allowing the catalytic subunits LF and EF to reach the cytosol. LF cleaves MKKs, and EF acts as a calmodulin-dependent adenylyl cyclase to increase cAMP concentrations.</p> Full article ">Figure 7
<p>Molecular structure of Stx and Ctx and their receptors, and binding sites in the B subunit. Stx and Ctx bind glycan receptors overexpressed in some solid tumors. (<b>a</b>) Crystallized structure of the Stx. It consists of a 32 kDa A subunit (light purple and brown) and five identical 7.7 kDa B subunits (green, yellow, orange, blue, and pink) that form a pentamer. In green, the two cysteine residues form the disulfide bond that links StxA1 (light purple) and the alpha-helix StxA2 (brown). (<b>b</b>) Crystallized structure of the StxB-Gb3 binding. Each monomer of the B pentamer can bind up to three Gb3 molecules in the terminal galactose disaccharide moiety of the receptor. (<b>c</b>) Schematic representation of the Gb3 receptor structure. Gb3 is composed of a ceramide, which consists of sphingosine (light yellow) and a fatty acid molecule (light red), a molecule of D-glucose (purple), and two molecules of D-galactose (turquoise). (<b>d</b>) Crystallized structure of the Ctx. It consists of a 28 kDa A subunit (light purple and brown) and five identical 11 kDa B subunits (green, yellow, orange, blue, and pink) that form a pentamer. In green, the two cysteine residues form the disulfide bond that links CtxA1 (light purple) and the alpha-helix CtxA2 (brown). (<b>e</b>) Crystallized structure of the CtxB-GM1 binding. Each monomer of the B pentamer can only bind one GM1 molecule. (<b>f</b>) Schematic representation of the GM1 receptor. GM1, like Gb3, is composed of the ceramide group; however, the sugar moiety is different. It consists of one molecule of D-glucose, two molecules of D-galactose, one of <span class="html-italic">N</span>-acetylneuraminic acid (NANA, green), and one molecule of <span class="html-italic">N</span>-acetyl-D-galactosamine (GalNac, yellow). All protein structures were depicted using PyMOL software, employing a cartoon representation. The Protein Data Bank IDs are the following: 1R4Q for Stx, 1BOS for StxB-Gb3 binding, 1XTC for Ctx, and 5ELD for CtxB-GM1 binding.</p> Full article ">Figure 8
<p>Stx and Ctx entry mechanisms and cellular effects. The mechanism by which Stx and Ctx reach the cytosol is also receptor-mediated endocytosis. They bind glycans on the surface of cells but follow a different entry mechanism than Dtx or Atx. Once they are bound to Gb3 and GM1 receptors on the surface of the host cell, they can enter via clathrin-coated pits or by invaginations of the plasma membrane. Although they are delivered to early endosomes, they undergo retrograde vesicular transport via the trans-Golgi network (TGN) and the Golgi stack to reach the ER lumen, from which the catalytic subunits StxA1 and CtxA1 are released into the cytosol. Once in the cytosol, StxA1 inhibits protein synthesis, while CtxA1 increases the amount of cAMP, leading to the dehydration of cells.</p> Full article ">Figure 9
<p>Principal biomedical applications of AB toxins. All toxins described in this review are used for cancer therapy and diagnosis, taking advantage of the B subunit binding to overexpressed receptors or as immunotoxins. However, there are more biomedical applications based on the B subunit, such as blood–brain barrier (BBB) drug delivery agents or the development of vaccines. Applications are colored in red for Diphtheria toxin, in blue for Anthrax toxin, in yellow for Cholera toxin and in green for Shiga toxin.</p> Full article ">
27 pages, 1733 KiB  
Review
Eukaryotic Cell Membranes: Structure, Composition, Research Methods and Computational Modelling
by Anatoly Zhukov and Valery Popov
Int. J. Mol. Sci. 2023, 24(13), 11226; https://doi.org/10.3390/ijms241311226 - 7 Jul 2023
Cited by 14 | Viewed by 7210
Abstract
This paper deals with the problems encountered in the study of eukaryotic cell membranes. A discussion on the structure and composition of membranes, lateral heterogeneity of membranes, lipid raft formation, and involvement of actin and cytoskeleton networks in the maintenance of membrane structure [...] Read more.
This paper deals with the problems encountered in the study of eukaryotic cell membranes. A discussion on the structure and composition of membranes, lateral heterogeneity of membranes, lipid raft formation, and involvement of actin and cytoskeleton networks in the maintenance of membrane structure is included. Modern methods for the study of membranes and their constituent domains are discussed. Various simplified models of biomembranes and lipid rafts are presented. Computer modelling is considered as one of the most important methods. This is stated that from the study of the plasma membrane structure, it is desirable to proceed to the diverse membranes of all organelles of the cell. The qualitative composition and molar content of individual classes of polar lipids, free sterols and proteins in each of these membranes must be considered. A program to create an open access electronic database including results obtained from the membrane modelling of individual cell organelles and the key sites of the membranes, as well as models of individual molecules composing the membranes, has been proposed. Full article
(This article belongs to the Section Biochemistry)
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<p>Types of lipid rafts in membranes. Lipid rafts include (<b>a</b>) the Lo zone in one monolayer of the membrane; (<b>b</b>) the Lo zone in one monolayer and GPI-anchored protein bound to membrane phosphatidylinositol; (<b>c</b>) the Lo zone in both monolayers opposite each other; (<b>d</b>) the Lo zone in one monolayer around the integral protein; (<b>e</b>) the Lo zone in one monolayer around the integral protein and GPI-anchored protein bound to membrane phosphatidylinositol; (<b>f</b>) the Lo zone around the transmembrane protein in both monolayers opposite each other. Lo and Ld—liquid-ordered and liquid-disordered lipid phases in membranes, respectively. The interfacial zone buffers a significant difference in ordering between Lo and Ld zones.</p> Full article ">Figure 2
<p>A possible scheme of the “encyclopaedia of biomembranes”. The database should contain information about models of all biological membranes, including the outer and inner membranes of organelles (nucleus, chloroplast, mitochondria), as well as their individual sites with special properties (contact platforms). For each membrane, models of typical membrane sections, microdomains, nanodomains, molecules of biomembrane components, as well as models of interaction of molecules at the scales from 1 μm to 0.5 nm should be presented. PM: plasma membrane; ER: endoplasmic reticulum.</p> Full article ">
17 pages, 1542 KiB  
Article
Transforming Psoriasis Care: Probiotics and Prebiotics as Novel Therapeutic Approaches
by Mihaela Cristina Buhaș, Rareș Candrea, Laura Ioana Gavrilaș, Doina Miere, Alexandru Tătaru, Andreea Boca and Adrian Cătinean
Int. J. Mol. Sci. 2023, 24(13), 11225; https://doi.org/10.3390/ijms241311225 - 7 Jul 2023
Cited by 25 | Viewed by 8763
Abstract
Psoriasis is a chronic inflammatory skin disease with autoimmune pathological characteristics. Recent research has found a link between psoriasis, inflammation, and gut microbiota dysbiosis, and that probiotics and prebiotics provide benefits to patients. This 12-week open-label, single-center clinical trial evaluated the efficacy of [...] Read more.
Psoriasis is a chronic inflammatory skin disease with autoimmune pathological characteristics. Recent research has found a link between psoriasis, inflammation, and gut microbiota dysbiosis, and that probiotics and prebiotics provide benefits to patients. This 12-week open-label, single-center clinical trial evaluated the efficacy of probiotics (Bacillus indicus (HU36), Bacillus subtilis (HU58), Bacillus coagulans (SC208), Bacillus licheniformis (SL307), and Bacillus clausii (SC109)) and precision prebiotics (fructooligosaccharides, xylooligosaccharides, and galactooligosaccharides) in patients with psoriasis receiving topical therapy, with an emphasis on potential metabolic, immunological, and gut microbiota changes. In total, 63 patients were evaluated, with the first 42 enrolled patients assigned to the intervention group and the next 21 assigned to the control group (2:1 ratio; non-randomized). There were between-group differences in several patient characteristics at baseline, including age, psoriasis severity (the incidence of severe psoriasis was greater in the intervention group than in the control group), the presence of nail psoriasis, and psoriatic arthritis, though it is not clear whether or how these differences may have affected the study findings. Patients with psoriasis receiving anti-psoriatic local therapy and probiotic and prebiotic supplementation performed better in measures of disease activity, including Psoriasis Area and Severity Index, Dermatology Life Quality Index, inflammatory markers, and skin thickness compared with those not receiving supplementation. Furthermore, in the 15/42 patients in the intervention group who received gut microbiota analysis, the gut microbiota changed favorably following 12 weeks of probiotic and prebiotic supplementation, with a shift towards an anti-inflammatory profile. Full article
(This article belongs to the Special Issue Psoriatic Arthritis and Skin Diseases: Pathogenesis and Therapies)
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<p>Abundances that showed a significant difference (<span class="html-italic">p</span> &lt; 0.05) from baseline to Week 12 of probiotic and prebiotic administration ((<b>A</b>) phylum level, (<b>B</b>) species level, (<b>C</b>) bacteria level).</p> Full article ">Figure 2
<p>Design and flow of the study.</p> Full article ">
14 pages, 2114 KiB  
Review
The Zebrafish Embryo as a Model Organism for Testing mRNA-Based Therapeutics
by Tjessa Bondue, Sante Princiero Berlingerio, Lambertus van den Heuvel and Elena Levtchenko
Int. J. Mol. Sci. 2023, 24(13), 11224; https://doi.org/10.3390/ijms241311224 - 7 Jul 2023
Cited by 7 | Viewed by 5221
Abstract
mRNA-based therapeutics have revolutionized the world of molecular therapy and have proven their potential in the vaccination campaigns for SARS-CoV2 and clinical trials for hereditary disorders. Preclinical studies have mainly focused on in vitro and rodent studies. However, research in rodents is costly [...] Read more.
mRNA-based therapeutics have revolutionized the world of molecular therapy and have proven their potential in the vaccination campaigns for SARS-CoV2 and clinical trials for hereditary disorders. Preclinical studies have mainly focused on in vitro and rodent studies. However, research in rodents is costly and labour intensive, and requires ethical approval for all interventions. Zebrafish embryonic disease models are not always classified as laboratory animals and have been shown to be extremely valuable for high-throughput drug testing. Zebrafish larvae are characterized by their small size, optical transparency and high number of embryos, and are therefore also suited for the study of mRNA-based therapeutics. First, the one-cell stage injection of naked mRNA can be used to assess the effectivity of gene addition in vivo. Second, the intravascular injection in older larvae can be used to assess tissue targeting efficiency of (packaged) mRNA. In this review, we describe how zebrafish can be used as a steppingstone prior to testing mRNA in rodent models. We define the procedures that can be employed for both the one-cell stage and later-stage injections, as well as the appropriate procedures for post-injection follow-up. Full article
(This article belongs to the Special Issue Zebrafish as a Model for Biomedical Studies)
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<p>Evaluation of mRNA-based therapies in the zebrafish model. Synthetic mRNA can be produced with desired modifications by in vitro mRNA transcription (IVT mRNA) from a cDNA template with the sequence of interest. Modified IVT mRNA is equipped with a 5′ cap and 3′ poly-A tail and can be injected in the one-cell stage as naked mRNA or packaged in a delivery vehicle. mRNA can also be injected into older zebrafish embryos. After injection, mRNA can be detected and protein expression confirmed. Phenotypic screens can be carried out post-injection to assess effectiveness. hpf = hours post-fertilization. This figure was created with BioRender.com.</p> Full article ">Figure 2
<p>The structure of a functional mRNA molecule. A functional mRNA model is capped on the 5′ side to protect from exonuclease attack. The coding sequence is flanked by the 5′ and 3′ untranslated regions (UTRs), which influence translation efficiency and stability. Finally, a long stretch of adenine residues (poly-A tail) at the 3′-end provides further stabilization and is a main determinant of mRNA half-life.</p> Full article ">Figure 3
<p>Early development of the zebrafish embryo. After fertilization, the blastomere undergoes cleavage and gastrulation. Between 10.33 hpf and 24 hpf, the somites are formed (segmentation) and, at the 13-somite stage (24 h), the pronephros begins to form. The circulatory system can be seen by the presence of a heartbeat and organogenesis continues with the formation of the neuromeres, otoliths, liver anlage and intestine anlage. At 48 h, pectoral fins are formed and the embryo is touch reactive. Hatching occurs between 48 hpf and 72 hpf, and the early organogenesis is finalized. The free-swimming embryo has a protruding mouth with a functional pronephros and intestinal tract. Intravenous delivery can be performed from 24 h–48 h onwards. This figure was created with BioRender.com.</p> Full article ">Figure 4
<p>Duct of Cuvier injection results in circulation of the injected solution. The 72 h-old embryos were injected in the duct of Cuvier with a fluorescence-labelled molecule (rhodamine B isothiocyanate–dextran) and evaluated with fluorescence microscopy. Successful injection can be validated within a few minutes as the presence of labelled compound in the circulatory system (arrow). This figure was created with BioRender.com.</p> Full article ">Figure 5
<p>The mRNA’s coding for a fluorescent protein can be used to follow up protein expression during development. Synthetic mRNA that codes for a GFP was injected at the one-cell stage and fluorescence followed up during the first 24 h post-injection. The GFP protein can be seen in the blastomeres within 3 h post-injection and in the embryo body at 24 h post-injection. As no translation happens in the yolk (arrow), there is no protein observed there. As expected with one-cell stage injection, the protein expression is ubiquitous. Scalebar = 100 µm.</p> Full article ">
23 pages, 4275 KiB  
Review
Numerous Trigger-like Interactions of Kinases/Protein Phosphatases in Human Skeletal Muscles Can Underlie Transient Processes in Activation of Signaling Pathways during Exercise
by Alexander Yu. Vertyshev, Ilya R. Akberdin and Fedor A. Kolpakov
Int. J. Mol. Sci. 2023, 24(13), 11223; https://doi.org/10.3390/ijms241311223 - 7 Jul 2023
Viewed by 1851
Abstract
Optimizing physical training regimens to increase muscle aerobic capacity requires an understanding of the internal processes that occur during exercise that initiate subsequent adaptation. During exercise, muscle cells undergo a series of metabolic events that trigger downstream signaling pathways and induce the expression [...] Read more.
Optimizing physical training regimens to increase muscle aerobic capacity requires an understanding of the internal processes that occur during exercise that initiate subsequent adaptation. During exercise, muscle cells undergo a series of metabolic events that trigger downstream signaling pathways and induce the expression of many genes in working muscle fibers. There are a number of studies that show the dependence of changes in the activity of AMP-activated protein kinase (AMPK), one of the mediators of cellular signaling pathways, on the duration and intensity of single exercises. The activity of various AMPK isoforms can change in different directions, increasing for some isoforms and decreasing for others, depending on the intensity and duration of the load. This review summarizes research data on changes in the activity of AMPK, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and other components of the signaling pathways in skeletal muscles during exercise. Based on these data, we hypothesize that the observed changes in AMPK activity may be largely related to metabolic and signaling transients rather than exercise intensity per se. Probably, the main events associated with these transients occur at the beginning of the exercise in a time window of about 1–10 min. We hypothesize that these transients may be partly due to putative trigger-like kinase/protein phosphatase interactions regulated by feedback loops. In addition, numerous dynamically changing factors, such as [Ca2+], metabolite concentration, and reactive oxygen and nitrogen species (RONS), can shift the switching thresholds and change the states of these triggers, thereby affecting the activity of kinases (in particular, AMPK and CaMKII) and phosphatases. The review considers the putative molecular mechanisms underlying trigger-like interactions. The proposed hypothesis allows for a reinterpretation of the experimental data available in the literature as well as the generation of ideas to optimize future training regimens. Full article
(This article belongs to the Special Issue The Physiology of Striated Muscle Tissue 2.0)
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<p>A simplified scheme of Ca<sup>2+</sup>- and AMPK-dependent signaling. Exercise causes numerous changes at the metabolic level. An increase in [AMP] and [Ca<sup>2+</sup>] concentrations, indicated by an arrow (↑), activates coupled signaling pathways that form a complex network with numerous feedback loops and protein–protein interactions. This leads to changes in gene expression.</p> Full article ">Figure 2
<p>AMPK subunit-associated activity in response to exercise: trends based on data from [<a href="#B16-ijms-24-11223" class="html-bibr">16</a>]. A decrease in α1 AMPK activity after high-intensity sprinting exercise. Significant: (*).</p> Full article ">Figure 3
<p>The progressive increase of AMPK activity (trends). Based on data from [<a href="#B12-ijms-24-11223" class="html-bibr">12</a>,<a href="#B15-ijms-24-11223" class="html-bibr">15</a>,<a href="#B20-ijms-24-11223" class="html-bibr">20</a>,<a href="#B23-ijms-24-11223" class="html-bibr">23</a>,<a href="#B25-ijms-24-11223" class="html-bibr">25</a>,<a href="#B26-ijms-24-11223" class="html-bibr">26</a>]. Axes X and Y indicate the time of the corresponding exercise and the fold increase of the AMPK activity compared to the pre-exercise state, respectively.</p> Full article ">Figure 4
<p>A decrease in the activity of various isoforms of AMPK at exercise onset (cycling exercise for 90 min at 67% VO<sub>2peak</sub>), trends based on data from [<a href="#B12-ijms-24-11223" class="html-bibr">12</a>]. Significant: (*), different from the preceding time point: (+).</p> Full article ">Figure 5
<p>Effect of exercise duration on skeletal muscle CaMKII autonomous activity and phosphorylation, based on data from [<a href="#B27-ijms-24-11223" class="html-bibr">27</a>]. Significant: (*).</p> Full article ">Figure 6
<p>The nonlinear increase of AMPK activity during moderate-intensity exercise. (<b>A</b>) The increase of α2 AMPK activity, based on data from [<a href="#B24-ijms-24-11223" class="html-bibr">24</a>]. (<b>B</b>,<b>C</b>) Simulation results for the percentage of all α2 phosphorylated proteins (dashed) and of the phosphorylated γ3 heterotrimers (solid) in type I fibers and type II fibers, respectively, as an indirect measure of AMPK activity.</p> Full article ">Figure 7
<p>The activity of AMPK (α2β2γ1) that was phosphorylated by CaMKKβ (also known as CaMKK2) with subsequent incubation with recombinant protein phosphatase 1γ (PP1γ) or recombinant protein phosphatase 2Cα (PP2Cα), based on data from [<a href="#B38-ijms-24-11223" class="html-bibr">38</a>,<a href="#B39-ijms-24-11223" class="html-bibr">39</a>].</p> Full article ">Figure 8
<p>An approximate distribution of protein phosphatase’s regulatory factors in three time domains. For distribution, the dynamics of the response after stimulation, shown in the figures in the original papers, were used. To model transient processes during exercise, factors from the “fast” group were mainly considered as candidates.</p> Full article ">Figure 9
<p>Coupled kinase and phosphatase switches can produce the tristability required for long-term potentiation (LTP) and long-term depression (LTD) of synapses, based on data from [<a href="#B45-ijms-24-11223" class="html-bibr">45</a>]. Dependence of the sign of synaptic modification on Ca<sup>2+</sup> level during stimulation.</p> Full article ">Figure 10
<p>Proposed trigger interactions of kinases/protein phosphatases in the human skeletal muscle. Green lines indicate activation processes, while red ones designate inhibition. Objects with a blue border already exist in a recently developed model [<a href="#B3-ijms-24-11223" class="html-bibr">3</a>], while objects with a pink border are still not presented in the modular model. Objects with yellow shading designate kinases and phosphatases, while blue ones mean metabolites and hormones. The figure shows the main factors that change during exercise and rest periods between exercise bouts that can affect thresholds for switching potential triggers. Increase of concentrations is indicated by an arrow (↑), and decrease is indicated by an arrow (↓). Abbreviations: PKA, protein kinase A; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinases.</p> Full article ">Figure 11
<p>The effect of exercise modality on phosphorylation of CaMKII, AMPK, and p38 mitogen-activated protein kinase (p38-MAPK) proteins immediately after (+0 h) isocaloric exercises. Based on data from [<a href="#B92-ijms-24-11223" class="html-bibr">92</a>]. The phosphorylation of CaMKII, AMPK, and p38-MAPK markedly increases after intermittent exercise only. Significant: (*).</p> Full article ">Figure 12
<p>Proposed trigger interactions of kinases/protein phosphatases in human skeletal muscle in the context of the substrate specificity of protein kinases and protein phosphatases. Isoforms of protein phosphatase 2A are represented by the type of its regulatory subunit. Green lines indicate activation processes, while red ones designate inhibition. Other designations are the same as in <a href="#ijms-24-11223-f010" class="html-fig">Figure 10</a>. Solid lines represent confirmed interactions using published data [<a href="#B48-ijms-24-11223" class="html-bibr">48</a>,<a href="#B49-ijms-24-11223" class="html-bibr">49</a>,<a href="#B51-ijms-24-11223" class="html-bibr">51</a>,<a href="#B62-ijms-24-11223" class="html-bibr">62</a>,<a href="#B89-ijms-24-11223" class="html-bibr">89</a>,<a href="#B90-ijms-24-11223" class="html-bibr">90</a>,<a href="#B118-ijms-24-11223" class="html-bibr">118</a>,<a href="#B119-ijms-24-11223" class="html-bibr">119</a>,<a href="#B120-ijms-24-11223" class="html-bibr">120</a>,<a href="#B121-ijms-24-11223" class="html-bibr">121</a>,<a href="#B122-ijms-24-11223" class="html-bibr">122</a>,<a href="#B123-ijms-24-11223" class="html-bibr">123</a>,<a href="#B124-ijms-24-11223" class="html-bibr">124</a>,<a href="#B125-ijms-24-11223" class="html-bibr">125</a>]. Dashed lines represent possible interactions. Increase of concentrations is indicated by an arrow (↑). Long green arrows indicate stimulation of activity. Question marks (?) indicate missing required data.</p> Full article ">
14 pages, 7402 KiB  
Article
Bacterial Outer Membrane Vesicles Loaded with Perhexiline Suppress Tumor Development by Regulating Tumor-Associated Macrophages Repolarization in a Synergistic Way
by Shoujin Jiang, Wei Fu, Sijia Wang, Guanshu Zhu, Jufang Wang and Yi Ma
Int. J. Mol. Sci. 2023, 24(13), 11222; https://doi.org/10.3390/ijms241311222 - 7 Jul 2023
Cited by 8 | Viewed by 1927
Abstract
Tumor-associated macrophages (TAMs) promote tumor development and metastasis and are categorized into M1-like macrophages, suppressing tumor cells, and M2-like macrophages. M2-like macrophages, occupying a major role in TAMs, can be repolarized into anti-tumoral phenotypes. In this study, outer membrane vesicles (OMVs) secreted by [...] Read more.
Tumor-associated macrophages (TAMs) promote tumor development and metastasis and are categorized into M1-like macrophages, suppressing tumor cells, and M2-like macrophages. M2-like macrophages, occupying a major role in TAMs, can be repolarized into anti-tumoral phenotypes. In this study, outer membrane vesicles (OMVs) secreted by Escherichia coli Nissle 1917 carry perhexiline (OMV@Perhx) to explore the influence of OMVs and perhexiline on TAM repolarization. OMV@Perhx was internalized by macrophages and regulated the phenotype of TAMs from M2-like to M1-like efficiently to increase the level of tumor suppressor accordingly. Re-polarized macrophages promoted apoptosis and inhibited the mobility of tumor, cells including invasion and migration. The results indicate that OMVs improve the efficacy of perhexiline and also represent a promising natural immunomodulator. Combining OMVs with perhexiline treatments shows powerfully synergistic anti-tumor effects through co-culturing with re-polarized macrophages. This work is promising to exploit the extensive applications of OMVs and chemical drugs, therefore developing a meaningful drug carrier and immunomodulator as well as expanding the purposes of traditional chemical drugs. Full article
(This article belongs to the Section Molecular Nanoscience)
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<p>Characterization of OMVs. (<b>a</b>) Transmission electron micrograph image of <span class="html-italic">E</span>. <span class="html-italic">coli</span> Nissle 1917-derived OMVs. Scale bar, 200 nm. (<b>b</b>) Size distribution of OMVs was measured by dynamic light scattering analysis (n = 6). (<b>c</b>) Cell toxicity of OMVs was examined by Cell-Counting-Kit assay (n = 4). (<b>d</b>) The concentration changes of total protein were monitored by the BCA kit at different times (n = 6). Data were presented as the mean ± SD (one-way ANOVA comparison tests, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 2
<p>Curcumin-loaded OMVs and the internalization of OMV@Curcumin characterized by a confocal laser scanning microscope. (<b>a</b>) OMVs loading curcumin as imaged by CLSM; purple arrows, blue arrows, and red arrows represent Dil-labeled OMVs, curcumin, and OMV@Cur, respectively. Scale bar, 10 μm. (<b>b</b>) OMV@Cur internalized by macrophages. Red: Dil-stained macrophages; yellow: curcumin-labeled OMVs. Scale bar, 20 μm.</p> Full article ">Figure 3
<p>The level of polarization or repolarization was analyzed by qRT-PCR and ROS detection. (<b>a</b>,<b>b</b>) Transcription level of Arg-1 and CD206 was examined by qRT-PCR (n = 6). (<b>c</b>,<b>d</b>) IL-6 and TNF-α, biomarkers of M1-like polarization, were analyzed by qRT-PCR (n = 6). (<b>e</b>) Detection of relative ROS level in macrophages (n = 6). Control: PBS, M2: IL-4 (100 ng/mL), M2 + Perhx: IL-4 + perhexiline (2.5 μM), M2 + OMV:IL-4 + OMV (2 μg/mL), M2 + OMV@Perhx: IL-4 + OMV@Perhx (2 μg@2.5 μM). Data are presented as the mean ± SD (one-way ANOVA comparison tests, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 4
<p>The polarization and repolarization were analyzed by flow cytometry. (<b>a</b>) iNOS-Cy7 and Arg-1-PE were biomarkers for M1 and M2, respectively; the level of biomarkers was quantified by flow cytometry. (<b>b</b>,<b>c</b>) Expression of biomarkers using histogram. Data are presented as the mean ± SD (one-way ANOVA comparison tests, ** <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, n = 6).</p> Full article ">Figure 5
<p>The level of tumor suppressors secreted by diverse phenotypes of macrophages. (<b>a</b>) The relative IFN-γ concentration in cell-cultured supernatant analyzed by an ELISA kit (n = 6). (<b>b</b>) The relative CXCL10 concentration in cell-cultured supernatant analyzed by an ELISA kit (n = 6). (<b>c</b>) The level of NO in cell-cultured supernatant analyzed by Griess NO detection kit (n = 6). Control: PBS, M2: IL-4 (100 ng/mL); M2 + Perhx: IL-4 + perhexiline (2.5 μM); M2 + OMV: IL-4 + OMV (2 μg/mL); M2 + OMV@Perhx: IL-4 + OMV@Perhx (2ug@2.5 μM). Data are presented as the mean ± SD (one-way ANOVA comparison tests, ** <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>The apoptosis level of CT26 cells treated with different macrophage-cultured supernatant. M0: macrophages + PBS (24 h); M2: macrophages + IL-4 (24 h); Perhx: macrophages + IL-4 (24 h) + Perhx (24 h); OMV: macrophages + IL-4 (24 h) + OMV (24 h); OMV@Perhx: macrophages + IL-4 (24 h) + OMV@Perhx (24 h). Data are presented as the mean ± SD (one-way ANOVA comparison tests, * <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, n = 6).</p> Full article ">Figure 7
<p>The influence of repolarized macrophages on the mobility of CT26 tumor cells as verified by transwell assay. (<b>a</b>) Transwell migration assay. Macrophages stimulated with different treatments, including PBS, IL-4, IL-4 + OMVs, IL-4 + perhexiline, and IL-4 + OMV@Perhx, show distinct effects on the migration ability of CT26 tumor cells (n = 6). (<b>b</b>) Capacity of CT26 tumor cells to cross through the Matrigel as examined by transwell invasion assay upon different treatments (PBS, IL-4, IL-4 + OMVs, IL-4 + perhexiline, IL-4 + OMV@Perhx, n = 6). Data are presented as the mean ± SD (one-way ANOVA comparison tests, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001). Scale bar = 50 µm.</p> Full article ">
15 pages, 3453 KiB  
Article
Transcription Factor Nrf2 Modulates Lipopolysaccharide-Induced Injury in Bovine Endometrial Epithelial Cells
by Pengjie Song, Chen Liu, Mingkun Sun, Jianguo Liu, Pengfei Lin, Huatao Chen, Dong Zhou, Keqiong Tang, Aihua Wang and Yaping Jin
Int. J. Mol. Sci. 2023, 24(13), 11221; https://doi.org/10.3390/ijms241311221 - 7 Jul 2023
Cited by 3 | Viewed by 2081
Abstract
Endometritis in high-yield dairy cows adversely affects lactation length, milk quality, and the economics of dairy products. Endoplasmic reticulum stress (ERS) in bovine endometrial epithelial cells (BEECs) occurs as a consequence of diverse post-natal stressors, and plays a key role in a variety [...] Read more.
Endometritis in high-yield dairy cows adversely affects lactation length, milk quality, and the economics of dairy products. Endoplasmic reticulum stress (ERS) in bovine endometrial epithelial cells (BEECs) occurs as a consequence of diverse post-natal stressors, and plays a key role in a variety of inflammatory diseases. Nuclear-factor-erythroid-2-related factor 2 (Nrf2) is an important protective regulatory factor in numerous inflammatory responses. However, the mechanism by which Nrf2 modulates inflammation by participating in ERS remains unclear. The objective of the present study was to explore the role of Nrf2 in lipopolysaccharide (LPS)-induced injury to BEECs and to decipher the underlying molecular mechanisms of this injury. The expression of Nrf2- and ERS-related genes increased significantly in bovine uteri with endometritis. Isolated BEECs were treated with LPS to stimulate the inflammatory response. The expression of Nrf2 was significantly higher in cells exposed to LPS, which also induced ERS in BEECs. Activation of Nrf2 led to enhanced expression of the genes for the inflammation markers TNF-α, p65, IL-6, and IL-8 in BEECs. Moreover, stimulation of Nrf2 was accompanied by activation of ERS. In contrast, Nrf2 knockdown reduced the expression of TNF-α, p65, IL-6, and IL-8. Additionally, Nrf2 knockdown decreased expression of ERS-related genes for the GRP78, PERK, eIF2α, ATF4, and CHOP proteins. Collectively, our findings demonstrate that Nrf2 and ERS are activated during inflammation in BEECs. Furthermore, Nrf2 promotes the inflammatory response by activating the PERK pathway in ERS and inducing apoptosis in BEECs. Full article
(This article belongs to the Section Molecular Immunology)
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<p>ERS and increased Nrf2 expression occurred in bovine uterine tissues with endometritis. (<b>A</b>) Histopathological characterization of samples from the uteri of healthy (Health) and endometriotic (Endom) dairy cows (Bar = 50 μm). (<b>B</b>–<b>D</b>) The expression of inflammation-related factors <span class="html-italic">IL-6</span>, <span class="html-italic">IL-8</span>, and <span class="html-italic">TNF-α</span> in uterine tissue was assessed by RT-qPCR. (<b>E</b>) The mRNA expression of <span class="html-italic">Nrf2</span> was detected in uteri with and without endometritis by RT-qPCR. (<b>F</b>) Immunohistochemical analysis was used to visualize Nrf2 expression in dairy cow uteri (Bar = 100 μm). (<b>G</b>–<b>I</b>) Western blotting was used to analyze GRP78 and eIF2α levels in dairy cow uteri. The experiments were repeated in triplicate and expression data were normalized to those of <span class="html-italic">β-actin</span>. The unpaired Student’s <span class="html-italic">t</span>-test was used to compare the results between the healthy and endometritis groups. Statistical significance was set at <span class="html-italic">p</span> &lt; 0.05 (* <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>LPS-induced inflammatory response and ERS response in BEECs. (<b>A</b>–<b>C</b>) Western blot and RT-qPCR analysis of Nrf2 expression in BEECs treated with LPS. (<b>D</b>–<b>J</b>) Western blot analysis of the expression of ERS-related proteins (GRP78, eIF2α, PERK, ATF4, and ATF6) in BEECs with and without LPS treatment. (<b>K</b>–<b>N</b>) The mRNA levels of <span class="html-italic">GRP78</span>, <span class="html-italic">eIF2α</span>, <span class="html-italic">ATF4</span>, and <span class="html-italic">ATF6</span> in BEECs with and without LPS treatment were assessed by RT-qPCR. (<b>O</b>–<b>Q</b>) The mRNA levels of inflammatory cytokines <span class="html-italic">IL-8</span> and <span class="html-italic">IL-10</span> in BEECs with and without LPS treatment were assessed by RT-qPCR. (<b>R</b>,<b>S</b>) Immunofluorescence of Nrf2 and DAPI-staining of BEECs with and without LPS treatment (Bar = 20 μm). LPS was used at 10 μg/mL in all cases. One-way ANOVA and Tukey’s post hoc test were used for statistical analysis. Data are the mean ± SEM (<span class="html-italic">n</span> = 3). ns no significance, * <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 3
<p>Knockdown of Nrf2 attenuated the LPS-induced inflammatory response and ERS in BEECs. (<b>A</b>,<b>B</b>) BEECs were transfected with Nrf2 siRNA (siNrf2) or negative control siRNA (siNC) for 24 h and Nrf2 protein expression was detected using Western blot analysis. (<b>C</b>) BEECs were transfected with siNrf2 and the mRNA expression of the gene for Nrf2 was assessed by RT−qPCR. (<b>D</b>,<b>E</b>) Detection of cell cycle using annexin V−FITC and PI staining assay and flow cytometry. (<b>F</b>–<b>H</b>) BEECs were transfected with siNrf2 and the mRNA expression of inflammatory factors IL−6, IL−8, and TNF−α was assessed by RT−qPCR. (<b>I</b>–<b>M</b>) BEECs were transfected with siNrf2 or siNC for 24 h and ERS-related protein expression (GRP78, PERK, ATF4, and eIF2α) was detected using Western blot analysis. (<b>N</b>) Immunofluorescence analysis of p65 in control and Nrf2 knockdown cells (Bar = 10 μm). Data are presented as mean ± SEM (<span class="html-italic">n</span> = 3). One−way ANOVA analysis was used. * <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 4
<p>Activation of Nrf2 aggravates the LPS-induced inflammatory response and ERS in BEECs. (<b>A</b>–<b>C</b>) BEECs were pretreated with the indicated concentrations of TBHQ for 2 h and mRNA and protein levels for Nrf2 were detected by RT−qPCR and Western blotting, respectively. (<b>D</b>) Cell viability of BEECs after treatment with different concentrations of TBHQ was assessed by CCK−8 analysis. (<b>E</b>–<b>I</b>) Western blotting analysis of p65− and ERS−related protein expression in BEECs with or without TBHQ and LPS treatment. (<b>J</b>–<b>L</b>) RT-qPCR analysis of inflammatory factors in BEECs treated with or without LPS and TBHQ treatment. (<b>M</b>,<b>N</b>) Immunofluorescence analysis of p65 in BEECs in control and Nrf2−activated cells with or without LPS treatment (Bar = 10 μm). One-way ANOVA analysis was used. Data are the mean ± SEM (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 5
<p>Effects of LPS on Ca<sup>2+</sup> mobilization and ER function in BEECs. (<b>A</b>,<b>D</b>) BEECs in control and Nrf2−knockdown cells with or without LPS and TBHQ treatment were assessed for Ca<sup>2+</sup> efflux from the ER into the cytoplasm using a Ca<sup>2+</sup> detection kit (Bar = 5 μm). (<b>B</b>) Cell apoptosis was analyzed by an apoptosis detection kit with Mito-Tracker Red CMXRos and Annexin V-FITC (Bar = 20 μm). (<b>C</b>,<b>F</b>,<b>G</b>) Apoptosis-related protein expression levels were assessed in Nrf2−activated BEECs treated with or without LPS. (<b>B</b>,<b>E</b>,<b>H</b>,<b>I</b>) Western blot was used to analyze apoptosis−related protein expression in Nrf2-knockdown BEECs treated with or without LPS. One−way ANOVA analysis was used. Data are the mean ± SEM (<span class="html-italic">n</span> = 3). ns: no significance, * <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 ">
16 pages, 10457 KiB  
Article
Preparation of Xanthene-TEMPO Dyads: Synthesis and Study of the Radical Enhanced Intersystem Crossing
by Wenhui Zhu, Yanran Wu, Yiyan Zhang, Andrey A. Sukhanov, Yuqi Chu, Xue Zhang, Jianzhang Zhao and Violeta K. Voronkova
Int. J. Mol. Sci. 2023, 24(13), 11220; https://doi.org/10.3390/ijms241311220 - 7 Jul 2023
Viewed by 1677
Abstract
We prepared a rhodamine-TEMPO chromophore-radical dyad (RB-TEMPO) to study the radical enhanced intersystem crossing (REISC). The visible light-harvesting chromophore rhodamine is connected with the TEMPO (a nitroxide radical) via a C–N bond. The UV-vis absorption spectrum indicates negligible electron interaction between the two [...] Read more.
We prepared a rhodamine-TEMPO chromophore-radical dyad (RB-TEMPO) to study the radical enhanced intersystem crossing (REISC). The visible light-harvesting chromophore rhodamine is connected with the TEMPO (a nitroxide radical) via a C–N bond. The UV-vis absorption spectrum indicates negligible electron interaction between the two units at the ground state. Interestingly, the fluorescence of the rhodamine moiety is strongly quenched in RB-TEMPO, and the fluorescence lifetime of the rhodamine moiety is shortened to 0.29 ns, from the lifetime of 3.17 ns. We attribute this quenching effect to the intramolecular electron spin–spin interaction between the nitroxide radical and the photoexcited rhodamine chromophore. Nanosecond transient absorption spectra confirm the REISC in RB-TEMPO, indicated by the detection of the rhodamine chromophore triplet excited state; the lifetime was determined as 128 ns, which is shorter than the native rhodamine triplet state lifetime (0.58 μs). The zero-field splitting (ZFS) parameters of the triplet state of the chromophore were determined with the pulsed laser excited time-resolved electron paramagnetic resonance (TREPR) spectra. RB-TEMPO was used as a photoinitiator for the photopolymerization of pentaerythritol triacrylate (PETA). These studies are useful for the design of heavy atom-free triplet photosensitizers, the study of the ISC, and the electron spin dynamics of the radical-chromophore systems upon photoexcitation. Full article
(This article belongs to the Special Issue Recent Advances in Free Radicals, Radical Ions and Radical Pairs)
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<p>(<b>a</b>) UV–vis absorption spectra of the compounds in ethanol (EtOH); (<b>b</b>) UV-Vis absorption spectra change with the addition of trifluoroacetic acid added to RB-TEMPO solution in EtOH; (<b>c</b>) Kinetics of the lactam/opened amide transformation of the Rho moiety for these compounds, monitored at 555 nm upon addition of TFA (96 mM) in EtOH. <span class="html-italic">c</span> = 1.0 × 10<sup>−5</sup> M, 20 °C.</p> Full article ">Figure 2
<p>(<b>a</b>) Fluorescence emission spectra of RB and RB-TEMPO in EtOH with TFA (48 mM) added. Optically matched solutions were used in each panel (each of the solutions gives the same absorbance at the excitation wavelength: <span class="html-italic">λ</span><sub>ex</sub> = 300 nm, <span class="html-italic">A</span><sub>300nm</sub> = 0.101); (<b>b</b>) the corresponding fluorescence decay traces of RB and RB-TEMPO with TFA (48 mM) at 570 nm in EtOH. The black and blue lines are the measured curves, and the red lines are the fitted curves. <span class="html-italic">λ</span><sub>ex</sub> = 510 nm. <span class="html-italic">c</span> = 1.0 × 10<sup>−5</sup> M, 20 °C.</p> Full article ">Figure 3
<p>(<b>a</b>) Fluorescence emission spectra change with incremental amount of TEMPO added to RB solution in EtOH. Optically matched solutions were used in each panel (each of the solutions gives the same absorbance at the excitation wavelength: <span class="html-italic">λ</span><sub>ex</sub> = 300 nm, <span class="html-italic">A</span><sub>300nm</sub> = 0.101) and 20 °C; (<b>b</b>) the corresponding fluorescence decay traces of RB and RB with 50 eq. of TEMPO at 570 nm in EtOH. The blue line is the measured curve, and the red line is the fitted curve. <span class="html-italic">λ</span><sub>ex</sub> = 510 nm. <span class="html-italic">c</span> = 1.0 × 10<sup>−5</sup> M, 20 °C.</p> Full article ">Figure 4
<p>(<b>a</b>) Nanosecond transient absorption spectra of RB−TEMPO upon pulsed laser excitation, the direction of the arrow indicates the increase of the delay time. (<span class="html-italic">λ</span><sub>ex</sub> = 550 nm) and (<b>b</b>) the corresponding decay trace of RB-TEMPO monitored at 545 nm, (The red line is obtained by fitting the black decay curve), <span class="html-italic">c</span> = 1.0 × 10<sup>–5</sup> M, in de-aerated methanol, 20 °C.</p> Full article ">Figure 5
<p>(<b>a</b>) Nanosecond time-resolved transient absorption spectra of Eosin Y upon pulsed laser excitation, the direction of the arrow indicates the increase of the delay time. (<span class="html-italic">λ</span><sub>ex</sub> = 520 nm). (<b>b</b>) Decay trace of Eosin Y at 525 nm, <span class="html-italic">c</span> = 1.0 × 10<sup>–5</sup> M, in de-aerated methanol. (<b>c</b>) Triplet state lifetime after TEMPO quenching of Eosin Y at 525 nm (TEMPO radical quenched triplet lifetime), the red lines are obtained by exponential fitting the black decay curve, <span class="html-italic">c</span> [EY] = 1.0 × 10<sup>–5</sup> M, <span class="html-italic">c</span> [TEMPO] = 5.0 × 10<sup>–4</sup> M in de-aerated methanol, 20 °C.</p> Full article ">Figure 6
<p>Experimental and simulation TR EPR spectra of (<b>a</b>) RB and (<b>b</b>) Eosin Y. Simulation parameters are presented in the table below. Excited with nanosecond pulsed laser at 525 nm with energy 1 mJ. <span class="html-italic">c</span> = 1.0 × 10<sup>−4</sup> M in ethanol, in frozen solution at 80 K.</p> Full article ">Figure 7
<p>The photopolymerization of the monomer PETA under N<sub>2</sub>, upon photoirradiation using a 35 W Xenon lamp (unfiltered white light intensity: 80 mW/cm<sup>2</sup>, light dose: 4.8 × 10<sup>3</sup> mJ/cm<sup>2</sup>) with different photoinitiators and co-initiators. The first two rows are RB-TEMPO photopolymerization experiments for the solution of (<b>a</b>)/(<b>e</b>) PETA alone, (<b>b</b>)/(<b>f</b>) PETA/RB-TEMPO, (<b>c</b>)/(<b>g</b>) PETA/NB, (<b>d</b>)/(<b>h</b>) PETA/RB-TEMPO/NB. The first row is before the photoirradiation and the second row is after the photoirradiation. The third and fourth rows are the photopolymerization experiments with the <b>RB</b> as the photoinitiator under similar conditions. (<b>i</b>)/(<b>m</b>) PETA alone, (<b>j</b>)/(<b>n</b>) PETA/RB, (<b>k</b>)/(<b>o</b>) PETA/NB, (<b>l</b>)/(<b>p</b>) PETA/RB/NB. The third row are the samples before the photoirradiation and the fourth row is after the photoirradiation. Photosensitizer: 6 wt%. The photopolymerization activity is shown in the gelation of liquid blends upon photoirradiation.</p> Full article ">Scheme 1
<p>Synthesis of the Compounds. Key: (a) Rhodamine B, phosphorus oxychloride, dry DCM, 45 °C, reflux 5 h under N<sub>2</sub>, directly used in the next reaction. (b) 4-Amino-TEMPO, dry triethylamine, dry acetonitrile, 80 °C, reflux 21 h under N<sub>2</sub>, directly used in the next reaction. Yield: 10%. (c) trifluoroacetic acid.</p> Full article ">Scheme 2
<p>Photochemical mechanisms of a generation of radicals for the RB/NB system. Note for RB-TEMPO, the ion exchange reaction may exist as well; therefore intramolecular electron transfer may occur, which leads to the generation of the hexyl radical and photopolymerization results as well. (“*” represents the excited state of the photosensitizer, “1” represents the singlet state, “3” represents the triplet state.)</p> Full article ">
15 pages, 3076 KiB  
Communication
Neurodegenerative Disease Associated Pathways in the Brains of Triple Transgenic Alzheimer’s Model Mice Are Reversed Following Two Weeks of Peripheral Administration of Fasudil
by Richard Killick, Christina Elliott, Elena Ribe, Martin Broadstock, Clive Ballard, Dag Aarsland and Gareth Williams
Int. J. Mol. Sci. 2023, 24(13), 11219; https://doi.org/10.3390/ijms241311219 - 7 Jul 2023
Cited by 6 | Viewed by 2385
Abstract
The pan Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor fasudil acts as a vasodilator and has been used as a medication for post-cerebral stroke for the past 29 years in Japan and China. More recently, based on the involvement of ROCK inhibition in synaptic [...] Read more.
The pan Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor fasudil acts as a vasodilator and has been used as a medication for post-cerebral stroke for the past 29 years in Japan and China. More recently, based on the involvement of ROCK inhibition in synaptic function, neuronal survival, and processes associated with neuroinflammation, it has been suggested that the drug may be repurposed for neurodegenerative diseases. Indeed, fasudil has demonstrated preclinical efficacy in many neurodegenerative disease models. To facilitate an understanding of the wider biological processes at play due to ROCK inhibition in the context of neurodegeneration, we performed a global gene expression analysis on the brains of Alzheimer’s disease model mice treated with fasudil via peripheral IP injection. We then performed a comparative analysis of the fasudil-driven transcriptional profile with profiles generated from a meta-analysis of multiple neurodegenerative diseases. Our results show that fasudil tends to drive gene expression in a reverse sense to that seen in brains with post-mortem neurodegenerative disease. The results are most striking in terms of pathway enrichment analysis, where pathways perturbed in Alzheimer’s and Parkinson’s diseases are overwhelmingly driven in the opposite direction by fasudil treatment. Thus, our results bolster the repurposing potential of fasudil by demonstrating an anti-neurodegenerative phenotype in a disease context and highlight the potential of in vivo transcriptional profiling of drug activity. Full article
(This article belongs to the Special Issue Neurodegenerative Diseases: Molecular Mechanisms and Therapies)
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<p>Self-organising maps of the transcriptional data reveal the global effects of fasudil treatment. In the top left, the SOM weights are regressed against treatment status, and the corresponding Z scores are shown, coloured to represent the scores. It is clear that there are islands of positive and negative correlation corresponding to genes that are up- and down-regulated, respectively. On the top right, the weights are correlated with the mouse sex and show a significant sex effect that is, however, muted relative to that of fasudil. The expression data with the variance explained by sex subtracted out leaves a residual expression profile for which the SOM is shown below. As expected, sex no longer shows any significant correlation, bottom right, with the weights, and interestingly, the fasudil correlations are stronger, bottom left.</p> Full article ">Figure 2
<p>Fasudil-driven gene expression changes tend to be the reverse of those in multiple neurodegenerative conditions. The significant gene expression Z scores (<math display="inline"><semantics> <mrow> <mrow> <mo>|</mo> <mi>Z</mi> <mo>|</mo> </mrow> <mo>&gt;</mo> <mn>2</mn> </mrow> </semantics></math>) are shown for AD, PD, and HD together with those for the fasudil treatment; non-significant values are left blank, and entries are coloured according to the scores. The most down-regulated genes in AD are shown on the left, and the most up-regulated on the right. As expected, there is a high degree of consistency across the other neurodegenerative conditions, PD and HD. Fasudil shows an up-regulation of all but four genes of those most down-regulated in AD. The reversal is less significant for those genes up-regulated in AD, with only 14 out of 25 driven down with fasudil.</p> Full article ">Figure 3
<p>Fasudil regulates pathways in an opposite sense to that seen in multiple neurodegenerative conditions. The significant pathway enrichment Z scores (<math display="inline"><semantics> <mrow> <mrow> <mo>|</mo> <mi>Z</mi> <mo>|</mo> </mrow> <mo>&gt;</mo> <mn>2</mn> </mrow> </semantics></math>) are shown for AD, PD, and HD together with those for the fasudil treatment; non-significant enrichment scores are left blank with entries coloured according to the scores. The most down-regulated pathways in AD are shown on the left, and the most up-regulated on the right. As expected, there is a high degree of consistency across the other neurodegenerative conditions, PD and HD. Fasudil shows an up-regulation of all the pathways most down-regulated in AD and a down-regulation of 76% of the most up-regulated pathways in AD.</p> Full article ">Figure 4
<p>Pathway and gene-based analyses of the CMAP drug-driven transcription profiles in cancer cell lines in relation to target profiles from AD, PD, and HD. The drugs and target profiles are shown as points on a plane with distances between drugs given by <math display="inline"><semantics> <mrow> <mfrac> <mn>1</mn> <mn>2</mn> </mfrac> <mrow> <mo>(</mo> <mrow> <mn>1</mn> <mo>−</mo> <mi>c</mi> </mrow> <mo>)</mo> </mrow> </mrow> </semantics></math>, where the correlation is <math display="inline"><semantics> <mrow> <mi>c</mi> <mo>=</mo> <mfrac> <mrow> <mi>U</mi> <mi>U</mi> <mo>+</mo> <mi>D</mi> <mi>D</mi> <mo>−</mo> <mi>U</mi> <mi>D</mi> <mo>−</mo> <mi>D</mi> <mi>U</mi> </mrow> <mrow> <mi>U</mi> <mi>U</mi> <mo>+</mo> <mi>D</mi> <mi>D</mi> <mo>+</mo> <mi>U</mi> <mi>D</mi> <mo>+</mo> <mi>D</mi> <mi>U</mi> </mrow> </mfrac> </mrow> </semantics></math>, with <math display="inline"><semantics> <mi>U</mi> </semantics></math> and <math display="inline"><semantics> <mi>D</mi> </semantics></math> the number of shared pathways/genes regulated upwards and downwards, respectively. The distance to the target profile is <math display="inline"><semantics> <mrow> <mfrac> <mn>1</mn> <mn>2</mn> </mfrac> <mrow> <mo>(</mo> <mrow> <mn>1</mn> <mo>+</mo> <mi>c</mi> </mrow> <mo>)</mo> </mrow> </mrow> </semantics></math>, such that proximity now indicates anti-correlation. Points are mapped to the plane with the target at the centre, in black, and distances to the target are preserved. Angular separations of the compounds are optimised through steepest descent iteration. The compounds are scattered in a more diffuse pattern for the pathway-based analysis. Interestingly, fasudil, shown in violet, emerges as a repurposing candidate only in the pathway-based analysis. In particular, the contingency table for AD is <math display="inline"><semantics> <mrow> <mrow> <mo>[</mo> <mrow> <mtable> <mtr> <mtd> <mrow> <mn>12</mn> </mrow> </mtd> <mtd> <mrow> <mn>20</mn> </mrow> </mtd> </mtr> <mtr> <mtd> <mrow> <mn>41</mn> </mrow> </mtd> <mtd> <mrow> <mn>11</mn> </mrow> </mtd> </mtr> </mtable> </mrow> <mo>]</mo> </mrow> </mrow> </semantics></math> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.00017</mn> </mrow> </semantics></math>, that for PD is <math display="inline"><semantics> <mrow> <mrow> <mo>[</mo> <mrow> <mtable> <mtr> <mtd> <mrow> <mn>17</mn> </mrow> </mtd> <mtd> <mrow> <mn>13</mn> </mrow> </mtd> </mtr> <mtr> <mtd> <mrow> <mn>24</mn> </mrow> </mtd> <mtd> <mn>6</mn> </mtd> </mtr> </mtable> </mrow> <mo>]</mo> </mrow> </mrow> </semantics></math> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.047</mn> </mrow> </semantics></math>, and that for HD is <math display="inline"><semantics> <mrow> <mrow> <mo>[</mo> <mrow> <mtable> <mtr> <mtd> <mrow> <mn>20</mn> </mrow> </mtd> <mtd> <mrow> <mn>12</mn> </mrow> </mtd> </mtr> <mtr> <mtd> <mrow> <mn>36</mn> </mrow> </mtd> <mtd> <mn>6</mn> </mtd> </mtr> </mtable> </mrow> <mo>]</mo> </mrow> </mrow> </semantics></math> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.016</mn> </mrow> </semantics></math>.</p> Full article ">
16 pages, 3076 KiB  
Review
Reviewing PTBP1 Domain Modularity in the Pre-Genomic Era: A Foundation to Guide the Next Generation of Exploring PTBP1 Structure–Function Relationships
by Christine Carico and William J. Placzek
Int. J. Mol. Sci. 2023, 24(13), 11218; https://doi.org/10.3390/ijms241311218 - 7 Jul 2023
Viewed by 1783
Abstract
Polypyrimidine tract binding protein 1 (PTBP1) is one of the most well-described RNA binding proteins, known initially for its role as a splicing repressor before later studies revealed its numerous roles in RNA maturation, stability, and translation. While PTBP1’s various biological roles have [...] Read more.
Polypyrimidine tract binding protein 1 (PTBP1) is one of the most well-described RNA binding proteins, known initially for its role as a splicing repressor before later studies revealed its numerous roles in RNA maturation, stability, and translation. While PTBP1’s various biological roles have been well-described, it remains unclear how its four RNA recognition motif (RRM) domains coordinate these functions. The early PTBP1 literature saw extensive effort placed in detailing structures of each of PTBP1’s RRMs, as well as their individual RNA sequence and structure preferences. However, limitations in high-throughput and high-resolution genomic approaches (i.e., next-generation sequencing had not yet been developed) precluded the functional translation of these findings into a mechanistic understanding of each RRM’s contribution to overall PTBP1 function. With the emergence of new technologies, it is now feasible to begin elucidating the individual contributions of each RRM to PTBP1 biological functions. Here, we review all the known literature describing the apo and RNA bound structures of each of PTBP1’s RRMs, as well as the emerging literature describing the dependence of specific RNA processing events on individual RRM domains. Our goal is to provide a framework of the structure–function context upon which to facilitate the interpretation of future studies interrogating the dynamics of PTBP1 function. Full article
(This article belongs to the Section Biochemistry)
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<p>Topologic organization and key features of RRM1. (<b>a</b>) Apo structure of RRM1 (PDB: 1SJQ [<a href="#B30-ijms-24-11218" class="html-bibr">30</a>]) with residues comprising the RNP1 sequence on β3 shaded green (K94, N95, Q96, A97, F98, I99, E100, M101) and residues of RNP2 on β1 shaded in teal (I61, H62, I63, R64, K65, L66). C terminal residue (L136; blue) is stabilized by hydrophobic contacts with several residues across the β-sheet (V60, L89, F98, N87, E100; red). (<b>b</b>) Key residues that interact with the minimal RNA binding register YCN. Residues that make stacking interactions (H62, R64) are colored sand yellow. Residues that make hydrogen bonds (Q129, F130, S131, N132) are colored purple. Note that N132 also makes a stacking interaction with the C<sub>3</sub> nucleotide, but is colored based on hydrogen bond in this figure. Residues that engage in hydrophobic interactions (L89, L91, F98, H133, L136) are colored red.</p> Full article ">Figure 2
<p>Topologic organization and key features of RRM2. (<b>a</b>) Apo structure of RRM2 (PDB: 1SJR [<a href="#B30-ijms-24-11218" class="html-bibr">30</a>]) with residues comprising the RNP1 sequence on β3 shaded green (Q221, F222, Q223, A224, L225, L226, Q227, Y228) and residues of RNP2 on β1 shaded teal (I186, I187, V188, E189, N190, L191). C-terminal residues (R263, V265; blue) are stabilized by hydrophobic contacts with several residues across the β-sheet (V183, I214, L225, K212, Q227, S272, D274; red). Residues Y267, Y268 and N269 for a pseudo-β6 strand (pink). (<b>b</b>) Key residues that interact with the minimal RNA binding register CU(N)N. Residues that make stacking interactions (R185, K259) are colored in sand yellow. Residues that make hydrogen bonds (S258) are colored purple. Note that the main chain of K259 also forms an H-bond, but is colored based on stacking interaction in this figure. Residues that engage in hydrophobic interactions (I214, F216, L225, L260, L263) are colored red. Residues with undefined contacts with RNA (K66, Y267, K271) are colored black.</p> Full article ">Figure 3
<p>Topologic organization and key features of apo RRM3-4. Apo structure of the RRM3-4 didomain (PDB: 2EVZ [<a href="#B33-ijms-24-11218" class="html-bibr">33</a>]) (<b>a</b>), with individual views of RRM4 (<b>b</b>) and RRM3 (<b>c</b>). Residues comprising the RNP1 sequences on β3 shaded green (RRM3: K400, E401, N402, A403, L404, V405, Q406, M407; RRM4: R517, K518, M19, A520, L521, I522, Q523, M524) and residues of RNP2 on β1 shaded teal (RRM3: L365, L366, V367, S368, N369, L370; RRM4: L482, H483, L484, S485, N486, L487). The C-terminal region of RRM4 α2 (L534, I535, H538, N539; hot pink) interacts with L461 (hot pink) of the interdomain linker, as well as with the N-terminal region of RRM3 α1 (Q378, F381, I382, V386; hot pink), positioning these helices perpendicularly to one another. The N-terminal region of RRM4 α2 (V527 (salmon), E528 (blue), V531 (salmon)) interacts with the α2-β4 loop of RRM3 (H423 (salmon), K424 (blue), L425 (salmon), H426 (salmon)). Most contacts between the interdomain linker (F464, F472, I475, P477, and P478; not individually identified in this figure) are between α1 (V386, Y387, D389; pink) and α2 of RRM3 (Q412, L415, H419; purple), along with H423 (salmon) of the following loop, with an additional contact with RRM4 (F552 of β4 of RRM4; purple). Finally, this interaction is stabilized by an ion pair (K424 on RRM3 and E528 on RRM4; blue). Note that colors correspond to residues that interact with one another, and residues with multiple contacts (V386, H423) are reported twice in the appropriate colors.</p> Full article ">Figure 4
<p>RNA interactions of RRM3-4. (<b>a</b>) Structure of RRM3-4 didomain (PDB: 2EVZ [<a href="#B33-ijms-24-11218" class="html-bibr">33</a>]) with key residues that interact with the minimal binding register of RRM4 (YCN) and RRM3 (YCUUN), highlighted in colors that reflect the nature of their chemical interaction with RNA nucleotides. Residues that make stacking interactions are colored sand yellow, residues that engage in hydrophobic interactions are colored red, and residues that make hydrogen bonds are colored purple (see descriptions below). (<b>b</b>) Key residues of RRM4 that interact with the minimal RNA binding register (YCN). Residue H483 makes a stacking interaction. Residues N448, S553, K554, I557 make hydrogen bonds. Residues K511, F513, K515, L521 engage in hydrophobic interactions. (<b>c</b>) Key residues of RRM3 that interact with the minimal RNA binding register (YCUNN). Residue F397 makes a stacking interaction. Residues T433, S435, K436, H437, V440 make hydrogen bonds. Residues K394, L396, L404 engage in hydrophobic interactions. Residues L366—RNP2 position 2, L396, R431, L452, P443, and R444 have undefined contacts with RNA and are colored black.</p> Full article ">
21 pages, 1127 KiB  
Review
Investigating the Crime Scene—Molecular Signatures in Inflammatory Bowel Disease
by Vibeke Andersen, Tue B. Bennike, Corinna Bang, John D. Rioux, Isabelle Hébert-Milette, Toshiro Sato, Axel K. Hansen and Ole H. Nielsen
Int. J. Mol. Sci. 2023, 24(13), 11217; https://doi.org/10.3390/ijms241311217 - 7 Jul 2023
Cited by 3 | Viewed by 2614
Abstract
Inflammatory bowel diseases (IBD) are without cure and troublesome to manage because of the considerable diversity between patients and the lack of reliable biomarkers. Several studies have demonstrated that diet, gut microbiota, genetics and other patient factors are essential for disease occurrence and [...] Read more.
Inflammatory bowel diseases (IBD) are without cure and troublesome to manage because of the considerable diversity between patients and the lack of reliable biomarkers. Several studies have demonstrated that diet, gut microbiota, genetics and other patient factors are essential for disease occurrence and progression. Understanding the link between these factors is crucial for identifying molecular signatures that identify biomarkers to advance the management of IBD. Recent technological breakthroughs and data integration have fuelled the intensity of this research. This research demonstrates that the effect of diet depends on patient factors and gut microbial activity. It also identifies a range of potential biomarkers for IBD management, including mucosa-derived cytokines, gasdermins and neutrophil extracellular traps, all of which need further evaluation before clinical translation. This review provides an update on cutting-edge research in IBD that aims to improve disease management and patient quality of life. Full article
(This article belongs to the Special Issue Inflammatory Bowel Disease: From Pre-clinial Models into Translation)
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<p>Graphical abstract demonstrating how diet, gut microbes and patient factors affect individuals at risk of IBD or diagnosed with IBD. Understanding the link between these factors is crucial to identify molecular signatures, create diagnostic, prognostic, predictive and disease-monitoring biomarkers, and develop new drugs to manage IBD. Created with Biorender.com (accessed on 2 May 2023).</p> Full article ">Figure 2
<p>(<b>A</b>) Mucusa, (<b>B</b>) Epithelium, (<b>C</b>) Epithelial cell. Diet, gut microbes and patient factors interact at the mucosal surface. Schematic diagram of the intestinal mucosa constituting the intestinal barrier and immune system [<a href="#B1-ijms-24-11217" class="html-bibr">1</a>]. From the luminal side, it consists of the mucus and epithelial lining overlying the connective tissue. 1. (healthy) and 2. (UC). The outermost layer from the lumen side is the mucus. In the healthy gut, commensal microorganisms interact with the outer mucus layer and do not reach the inner mucus layer or epithelial cells. In IBD, the number of GCs is reduced, and this barrier is compromised, giving rise to the condition commonly described as a “leaky gut”. Certain microbial molecules activate the Toll-like receptors (TLR), and certain metabolites such as short-chain fatty acids (SCFA) activate the G protein-coupled receptors (GPR) on the intestinal epithelial cells. Enteroendocrine cells (EEC) monitor the gut microbiota and regulate inflammatory processes [<a href="#B27-ijms-24-11217" class="html-bibr">27</a>]. These processes stimulate the innate immune system resulting in gut inflammation by the pro-inflammatory IL-17 stimulating the IL-17 receptor A (IL-17RA), and neutrophilic granulocytes (NG) accumulate in the intestinal mucosa. EEC, enteroendocrine cells; GC, goblet cells; IL-17, interleukin-17; IL-17-RA, IL-17 receptor A; NF-ĸβ, nuclear-factor kappa beta; NG, neutrophilic granulocytes; GPR, G protein-coupled receptors; SCFA, short-chain fatty acids; TLR, Toll-like receptors; UC, ulcerative colitis. Created with Biorender.com (accessed on 2 May 2023).</p> Full article ">
15 pages, 2608 KiB  
Article
In Vitro Evaluation of the Antiamoebic Activity of Kaempferol against Trophozoites of Entamoeba histolytica and in the Interactions of Amoebae with Hamster Neutrophils
by David Levaro-Loquio, Jesús Serrano-Luna, Maritza Velásquez-Torres, Germán Higuera-Martínez, Ivonne Maciel Arciniega-Martínez, Aldo Arturo Reséndiz-Albor, Nadia Mabel Pérez-Vielma and Judith Pacheco-Yépez
Int. J. Mol. Sci. 2023, 24(13), 11216; https://doi.org/10.3390/ijms241311216 - 7 Jul 2023
Cited by 2 | Viewed by 1635
Abstract
Entamoeba histolytica (E. histolytica) is a parasite in humans that provokes amoebiasis. The most employed drug is metronidazole (MTZ); however, some studies have reported that this drug induces genotoxic effects. Therefore, it is necessary to explore new compounds without toxicity that [...] Read more.
Entamoeba histolytica (E. histolytica) is a parasite in humans that provokes amoebiasis. The most employed drug is metronidazole (MTZ); however, some studies have reported that this drug induces genotoxic effects. Therefore, it is necessary to explore new compounds without toxicity that can eliminate E. histolytica. Flavonoids are polyphenolic compounds that have demonstrated inhibition of growth and dysregulation of amoebic proteins. Despite the knowledge acquired to date, action mechanisms are not completely understood. The present work evaluates the effect of kaempferol against E. histolytica trophozoites and in the interactions with neutrophils from hamster, which is a susceptibility model. Our study demonstrated a significant reduction in the amoebic viability of trophozoites incubated with kaempferol at 150 μM for 90 min. The gene expression analysis showed a significant downregulation of Pr (peroxiredoxin), Rr (rubrerythrin), and TrxR (thioredoxin reductase). In interactions with amoebae and neutrophils for short times, we observed a reduction in ROS (reactive oxygen species), NO (nitric oxide), and MPO (myeloperoxidase) neutrophil activities. In conclusion, we confirmed that kaempferol is an effective drug against E. histolytica through the decrease in E. histolytica antioxidant enzyme expression and a regulator of several neutrophil mechanisms, such as MPO activity and the regulation of ROS and NO. Full article
(This article belongs to the Special Issue Neutrophil in Cell Biology and Diseases 2.0)
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<p>Trophozoite viability of <span class="html-italic">E. histolytica</span> at different times and concentrations of kaempferol or MTZ. Percentage of viability of trophozoites of <span class="html-italic">E. histolytica</span> in the presence of different concentrations of kaempferol or MTZ (90, 100, 110, 120, 130, 140, and 150 μM) for 90 min was determined with WST-1 assay. DMSO (Dimethylsulfoxide) was used as a vehicle. Data represent the mean ± SD (triplicate). <span class="html-italic">p</span>-values were determined with ANOVA (**** <span class="html-italic">p</span> &lt; 0.0001; ** <span class="html-italic">p</span> &lt; 0.01), compared with the control group (Ctrl), while those compared with the MTZ <span class="html-italic">p</span>-values were determined with ANOVA (**** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 2
<p>Percentage of VERO cells’ viability with kaempferol at 60, 90, 180, and 360 min was determined using WST-1 assay. Data represent the mean ± SD (triplicate). <span class="html-italic">p</span>-values were determined with ANOVA.</p> Full article ">Figure 3
<p>Cell lysate of <span class="html-italic">E. histolytica</span> incubated with kaempferol or MTZ at 150 μM for 90 min at 37 °C and electrophoresed on a 10% SDS-polyacrylamide gel. (A) Total cell lysate, (B) total cell lysate incubated with MTZ, (C) total cell lysate incubated with kaempferol for 90 min. MW = molecular weight.</p> Full article ">Figure 4
<p>Differential expression of the antioxidant enzyme genes in trophozoites of <span class="html-italic">E. histolytica</span>. Gene expression of antioxidant enzymes (Rr, Prx, TrxR, and Trx) in trophozoites in the presence of kaempferol at 150 μM for 90 min was determined using qRT-PCR. There is a statistically significant difference between Rr, Prx, and TrxR genes in kaempferol compared with those in the control and the MTZ groups. Statistical analyses were determined with Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01). Data represent the mean ± SD (triplicate). <span class="html-italic">p</span>-values were determined using Student’s <span class="html-italic">t</span>-test and using Bonferroni’s test.</p> Full article ">Figure 5
<p>Determination of MPO activity in the interactions of <span class="html-italic">E. histolytica</span> and neutrophil of hamsters. MPO activity in neutrophils in the presence of trophozoites of <span class="html-italic">E. histolytica</span> incubated with kaempferol or MTZ at 150 μM at different times was determined. DMSO was used as a vehicle, PMA (phorbol 12-myristate 13-acetate) was used as a positive control, and MPO inhibitor 100 mM (ABAH) was used as MPO activity control. Data represent the mean ± SD of the three independent experiments. <span class="html-italic">p</span>-values were determined using two-way ANOVA (**** <span class="html-italic">p</span> &lt; 0.0001; * <span class="html-italic">p</span> &lt; 0.05), comparing N+T with the N+T+kaempferol group, while compared with the MTZ group, <span class="html-italic">p</span>-values were determined using ANOVA (**** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 6
<p>Determination of ROS and NO produced in the interaction between neutrophils and trophozoites of <span class="html-italic">E. histolytica</span> in the presence of kaempferol. (<b>a</b>) Quantification of total ROS using dichlorodihydrofluorescein diacetate (DCFH-DA) assay kit in the interaction of neutrophils in presence of trophozoites incubated with kaempferol at 150 μM for 90 min. DMSO and LPS were used as controls. <span class="html-italic">p</span>-values were determined using two-way ANOVA (**** <span class="html-italic">p</span> &lt; 0.0001) to compare the N+T group with the N+T+kaempferol group, while compared with the MTZ group, <span class="html-italic">p</span>-values were determined using two-way ANOVA (**** <span class="html-italic">p</span> &lt; 0.0001). (<b>b</b>) Detection of NO using the Griess method in the interaction of neutrophils in the presence of <span class="html-italic">E. histolytica</span> incubated with kaempferol at 150 μM for 90 min. Data represent the mean ± SD of the three independent experiments. <span class="html-italic">p</span>-values were determined using two-way ANOVA (**** <span class="html-italic">p</span> &lt; 0.0001), comparing the N+T group with the N+T+kaempferol group, while compared with the MTZ group, <span class="html-italic">p</span>-values were determined using ANOVA (**** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">
16 pages, 3087 KiB  
Article
Cathepsin B Is Not an Intrinsic Factor Related to Asparaginase Resistance of the Acute Lymphoblastic Leukemia REH Cell Line
by Iris Munhoz Costa, Brian Effer, Tales Alexandre Costa-Silva, Chen Chen, Michael F. Ciccone, Adalberto Pessoa, Camila O. dos Santos and Gisele Monteiro
Int. J. Mol. Sci. 2023, 24(13), 11215; https://doi.org/10.3390/ijms241311215 - 7 Jul 2023
Cited by 1 | Viewed by 1649
Abstract
L-Asparaginase (ASNase) is a biopharmaceutical used as an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Yet, some cases of ALL are naturally resistant to ASNase treatment, which results in poor prognosis. The REH ALL cell line, used as a model [...] Read more.
L-Asparaginase (ASNase) is a biopharmaceutical used as an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Yet, some cases of ALL are naturally resistant to ASNase treatment, which results in poor prognosis. The REH ALL cell line, used as a model for studying the most common subtype of ALL, is considered resistant to treatment with ASNase. Cathepsin B (CTSB) is one of the proteases involved in the regulation of in vivo ASNase serum half-life and it has also been associated with the progression and resistance to treatment of several solid tumors. Previous works have shown that, in vitro, ASNase is degraded when incubated with REH cell lysate, which is prevented by a specific CTSB inhibitor, suggesting a function of this protease in the ASNase resistance of REH cells. In this work, we utilized a combination of CRISPR/Cas9 gene targeting and enzymatic measurements to investigate the relevance of CTSB on ASNase treatment resistance in the ALL model cell line. We found that deletion of CTSB in REH ALL cells did not confer ASNase treatment sensitivity, thus suggesting that intrinsic expression of CTSB is not a mechanism that drives the resistant nature of these ALL cells to enzymes used as the first-line treatment against leukemia. Full article
(This article belongs to the Special Issue State-of-the-Art Molecular Oncology in Brazil 2.0)
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Figure 1

Figure 1
<p>Monitoring of REHcas9 cell infection and CTSB edition. (<b>A</b>) Representative image of REHcas9 infection using lentivirus particles sgRNA-Rosa26. The same procedure was carried out for all lentiviruses produced (sgRNA-1–5 for <span class="html-italic">CTSB</span> gene and sgRNA-RPA3). The infection was confirmed by GFP expression in inverted fluorescence microscope. The figure shows the uninfected cells and infected cells with sgRNA-Rosa26 lentivirus expressing GFP under bright light and green channel excitation at 488 nm. Scale bar, 100 µm. (<b>B</b>) Schematic representation of gene and qPCR-based assay to estimate gene editing efficiency using CRISPR/Cas9 editing tool. Five different primers pairs were designed to flank the target region for the knockout of each sgRNA to validate the editing of the <span class="html-italic">CTSB</span> gene by qPCR. (<b>C</b>) Confirmation of <span class="html-italic">CTSB</span> gene editing by qPCR. Derivate melting curves from qPCR of REHcas9 cells infected with different sgRNAs. The genomic DNA of each cell was used for <span class="html-italic">CTSB</span> editing validation. Uninfected REHcas9 cells and REHcas9 cells infected with sgRNA-Rosa26 (REHcas9 + sgRNA-Rosa26) were used as a negative control (no editing in <span class="html-italic">CTSB</span> gene). The specific primer for the cyclin-dependent kinase 7 (<span class="html-italic">CDKL7</span>) gene was used as an internal control.</p> Full article ">Figure 2
<p>Evaluation of CTSB protein amount and activity in REHcas9 + sgRNAs. (<b>A</b>) Western blotting assay image using REHcas9 + sgRNAs. Uninfected REHcas9 and REHcas9 + sgRNAs cells were lysed, 150 µg of total protein was used in each sample, and the film was exposed for 2.5 and 5 min. α-Tubulin protein (60 kDa) was used as a total protein load control. (<b>B</b>) Cathepsin B activity assay of uninfected (in the presence or absence of CTSB inhibitor) and infected cells with sgRNAs (1–5) and Rosa26 using the Cathepsin B Activity Fluorometric Assay Kit (BioVision, Waltham, MA, USA). (<b>C</b>) CTSB activity assay using Sigma-Aldrich reagents (see <a href="#sec4-ijms-24-11215" class="html-sec">Section 4</a>) for analysis of lysates of REHcas9 cells and cells infected with sgRNAs-1, 2, and 4 and Rosa26. Results represent the mean ± standard deviation of experiments performed in triplicate. One-way ANOVA statistical analysis followed by Tukey’s post hoc test showed a significant difference (<span class="html-italic">p</span> &lt; 0.05) between the CTSB activities of sgRNA-infected cells (1–5) when compared to the uninfected control REHcas9 cells (*) or when compared to cells treated with CTSB inhibitor (#).</p> Full article ">Figure 3
<p>Analysis of <span class="html-italic">CTSB</span> gene editing in confirmed REHcas9 cells with <span class="html-italic">CTSB</span> knockout by sequencing. (<b>A</b>) Analysis of gene editing by sequencing of REHcas9 + sgRNA-1 and REHcas9 + sgRNA-4. The plasmids obtained from TOPO + PCR constructs were sequenced using the TOPO M13 vector primer and aligned with the <span class="html-italic">CTSB</span> gene sequence (NG_009217.2) using Geneious<sup>®</sup> 11.1.5 software. (<b>B</b>) Human CTSB protein sequence (Uniprot code: P07858). The signal peptide sequence is identified in red; the propeptide sequence is identified in blue; the mature protein sequence is identified in black; the C-terminal propeptide sequence is identified in green. The arrows represent the position at the beginning of the changes in the amino-acid sequence caused by the editing of sgRNA-1 (orange arrow) and sgRNA-4 (purple arrow).</p> Full article ">Figure 4
<p>Monitoring of REHcas9-sgRNA cell proliferation by flow cytometry. (<b>A</b>) Cells infected with different sgRNAs were monitored by flow cytometer MACSQuant<sup>®</sup> (Miltenyi Biotec) for 27 days. (<b>B</b>) After 27 days, cells were sorted by fluorescence-activated cell sorting (FACS) and monitored for additional 15 days.</p> Full article ">Figure 5
<p>Purification and activity assay of ASNases. (<b>A</b>) Evaluation of purity of ErA_WT, ErA_DM, and EcA_WT proteoforms by 12% SDS-PAGE gel electrophoresis. ErA_WT and ErA_DM proteins have ~37 kDa and the EcA_WT protein has ~35 kDa. MW indicates the molecular weight Dual Color Standards <sup>TM</sup> (Bio-Rad). (<b>B</b>) Specific activity of ErA_WT, ErA_DM, and EcA_ WT proteoforms measured by Nessler’s reagent. Graph of reaction speed in μmol/min as a function of the amount of protein in milligram. The slope of the line equation represents the specific activity for each enzyme given in U/mg. One unit (U) is equal to 1 µmol of ammonium produced per minute at 37 °C. The points represent the mean ± standard error (n = 3).</p> Full article ">Figure 6
<p>In vitro cytotoxicity assay of CTSB KO cells in the presence of ASNase. REHcas9 (uninfected) and REHcas + sgRNA-1 cells were treated with different concentrations of EcA_WT, ErA_WT, and ErA_DM enzymes for 72 h. Cells without treatment and with the addition of the buffer in which the enzymes were diluted are named “control”. The assay was performed in analytical triplicate, and the data represent the mean ± standard deviation (n = 3).</p> Full article ">
22 pages, 5106 KiB  
Article
Dopamine D3 Receptor Modulates Akt/mTOR and ERK1/2 Pathways Differently during the Reinstatement of Cocaine-Seeking Behavior Induced by Psychological versus Physiological Stress
by Aurelio Franco-García, Rocío Guerrero-Bautista, Juana María Hidalgo, Victoria Gómez-Murcia, María Victoria Milanés and Cristina Núñez
Int. J. Mol. Sci. 2023, 24(13), 11214; https://doi.org/10.3390/ijms241311214 - 7 Jul 2023
Viewed by 1559
Abstract
Stress triggers relapses in cocaine use that engage the activity of memory-related nuclei, such as the basolateral amygdala (BLA) and dentate gyrus (DG). Preclinical research suggests that D3 receptor (D3R) antagonists may be a promising means to attenuate cocaine reward and relapse. As [...] Read more.
Stress triggers relapses in cocaine use that engage the activity of memory-related nuclei, such as the basolateral amygdala (BLA) and dentate gyrus (DG). Preclinical research suggests that D3 receptor (D3R) antagonists may be a promising means to attenuate cocaine reward and relapse. As D3R regulates the activity of the Akt/mTOR and MEK/ERK1/2 pathways, we assessed the effects of SB-277011-A, a D3R antagonist, on the activity of these kinases during the reinstatement of cocaine-induced conditioned place preference (CPP) induced by psychological (restraint) and physiological (tail pinch) stress. Both stimuli reactivated an extinguished cocaine-CPP, but only restrained animals decreased their locomotor activity during reinstatement. Cocaine-seeking behavior reactivation was correlated with decreased p-Akt, p-mTOR, and p-ERK1/2 activation in both nuclei of restrained animals. While a D3R blockade prevented stress-induced CPP reinstatement and plasma corticosterone enhancement, SB-277011-A distinctly modulated Akt, mTOR, and ERK1/2 activation depending on the stressor and the dose used. Our data support the involvement of corticosterone in the SB-277011-A effects in restrained animals. Additionally, the ratios p-mTOR/mTOR and/or p-ERK1/2 /ERK1/2 in the BLA during stress-induced relapse seem to be related to the locomotor activity of animals receiving 48 mg/kg of the antagonist. Hence, our study indicates the D3R antagonist’s efficacy to prevent stress-induced relapses in drug use through distinct modulation of Akt/mTOR and MEK/ERK1/2 pathways in memory-processing nuclei. Full article
(This article belongs to the Special Issue Role of Dopamine in Health and Disease: Biological Aspect 2.0)
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Figure 1
<p>The antagonism of D3R blocked the reactivation of cocaine-induced CPP induced by both restraint and tail pinch. (<b>A</b>) Schematic of the experimental timeline and behavioral procedures. (<b>B</b>) Mean preference time spent in cocaine-paired chamber during pre-conditioning, post-conditioning, Extinction and Reinstatement of control mice (vehicle, no stressor; Cnt), animals that experienced restraint but did not receive the D3R antagonist (vehicle, restraint; Cnt R), and animals that experienced restraint and received the D3R antagonist at two different doses, 24 or 48 mg/kg, restraint; (SB24 R or SB48 R). Repeated measures one-way ANOVA showed differences among tests within Cnt group (F (2.182, 17.46) = 12.86; P = 0.0003), Cnt R group (F (1.461, 13.15) = 23.59; P = 0.0001), SB48 R (F (1.435, 12.91) = 7.776; P = 0.0098) but not SB24 R group (F (1.432, 10.02) = 3.418; P = 0.0845). * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001 vs. pre-conditioning; <sup>+</sup> P &lt; 0.05, <sup>++</sup> P &lt; 0.01, <sup>+++</sup> P &lt; 0.001 vs. Post-conditioning; <sup>##</sup> P &lt; 0.01 vs. Extinction (Tukey’s test). (<b>C</b>) Reinstatement score expressed as time in cocaine-paired chamber during Reinstatement test minus the same in Extinction test in Cnt, Cnt R, SB24 R and SB48 R animals. One-way ANOVA revealed significant differences among group means (F (3, 32) = 4.913; P = 0.0064). * P &lt; 0.05 vs. Cnt; <sup>++</sup> P &lt; 0.01 vs. Cnt R (Tukey’s test). (<b>D</b>) Mean preference time spent in cocaine-paired chamber during pre-conditioning, post-conditioning, Extinction, and Reinstatement of control mice (vehicle, no stressor; Cnt), animals that experienced tail pinch but did not receive the D3R antagonist (vehicle, tail pinch; Cnt TP), and animals that experienced tail pinch and received the D3R antagonist at two different doses SB-277011-B, 24 or 48 mg/kg, restraint; SB24 TP or SB48 TP). Repeated measures one-way ANOVA showed differences among tests within Cnt group (F (2.440, 17.08) = 14.24; P = 0.0001), Cnt TP group (F (1.917, 15.34) = 30.18; P &lt; 0.0001), SB48 TP group (F (1.644, 11.51) = 6.606; P = 0.0154) but not SB24 TP group (F (1.314, 14.45) = 3.561; P = 0.0706). * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001 vs. pre-conditioning; <sup>+</sup> P &lt; 0.05, <sup>++</sup> P &lt; 0.01 vs. post-conditioning; <sup>###</sup> P &lt; 0.001 vs. Extinction (Tukey’s test). (<b>E</b>) Reinstatement score expressed as time in cocaine-paired chamber during Reinstatement test minus the same time in Extinction test in Cnt, Cnt TP, SB24 TP and SB48 TP animals. One-way ANOVA revealed significant differences among group means (F (3, 30) = 5.505; P = 0.0039). * P &lt; 0.05 vs. Cnt; <sup>+</sup> P &lt; 0.05, <sup>++</sup> P &lt; 0.01 vs. Cnt TP (Tukey’s test). (<b>F</b>–<b>H</b>) Locomotor activity during Extinction and Reinstatement tests for Cnt, Cnt R, SB24 R and SB48 R animals. Graphics show the number of entries to saline-paired (paired Student’s <span class="html-italic">t</span> test: Cnt (t<sub>8</sub> = 0.5483, P = 0.5985), Cnt R (t<sub>9</sub> = 2.930, P = 0.0167), SB24 R (t<sub>7</sub> = 10.74, P &lt; 0.0001) and SB48 R (t<sub>8</sub> = 8.321, P &lt; 0.0001)) (<b>F</b>) and cocaine-paired (paired Student’s <span class="html-italic">t</span> test: Cnt (t<sub>8</sub> = 0.4024, P = 0.6979), Cnt R (t<sub>8</sub> = 3.608, P = 0.0069), SB24 R (t<sub>7</sub> = 11.87, P &lt; 0.0001) and SB48 R (t<sub>8</sub> = 8.168, P &lt; 0.0001)) (<b>G</b>) compartments and total entries (paired Student’s <span class="html-italic">t</span> test: Cnt (t<sub>8</sub> = 0.02346, P = 0.9819), Cnt R (t<sub>9</sub> = 2.878, P = 0.0182), SB24 R (t<sub>7</sub> = 14.27, P &lt; 0.0001) and SB48 R (t<sub>8</sub> = 8.602, P &lt; 0.0001)) (<b>H</b>). (<b>F</b>–<b>H</b>) * P &lt; 0.05, *** P &lt; 0.001 vs. Extinction test (Tukey’s test). (<b>I</b>) Total entries score in Cnt, Cnt R, SB24 R and SB48 R animals, expressed as the difference in total entries during Reinstatement test minus total entries during Extinction test. One-way ANOVA: (F (3, 32) = 8.240; P = 0.0003). (<b>J</b>–<b>L</b>) Locomotor activity during Extinction and Reinstatement tests for Cnt, Cnt TP, SB24 TP and SB48 TP animals. Graphics show the number of entries to saline (sal)-paired (paired Student’s <span class="html-italic">t</span> test: Cnt (t<sub>7</sub> = 1.619, P = 0.1495), Cnt TP (t<sub>8</sub> = 0.5979, P = 0.5664), SB24 TP (t<sub>11</sub> = 7.794, P &lt; 0.0001) and SB48 TP (t<sub>7</sub> = 14.24, P &lt; 0.001) (<b>J</b>), cocaine (coc)-paired (paired Student’s <span class="html-italic">t</span> test: Cnt (t<sub>7</sub> = 1.015, P = 0.3438), Cnt TP (t<sub>8</sub> = 0.5979, P = 0.5377), SB24 TP (t<sub>11</sub> = 6.390, P &lt; 0.0001) and SB48 TP (t7 = 6.312, P = 0.0004) (<b>K</b>) compartments and total entries (paired Student’s <span class="html-italic">t</span> test: Cnt (t<sub>7</sub> = 1.405, P = 0.2028), Cnt TP (t<sub>8</sub> = 0.6641, P = 0.5253), SB24 TP (t<sub>11</sub> = 7.386, P &lt; 0.0001) and SB48 TP (t<sub>7</sub> = 10.19, P &lt; 0.0001) (<b>L</b>). (<b>J</b>–<b>L</b>) *** P &lt; 0.001 vs. Extinction test. (<b>M</b>) Total entries score in Cnt, Cnt TP, SB24 TP and SB48 TP animals, expressed as the difference in total entries during Reinstatement test minus total entries during Extinction test. One-way ANOVA: (F (3, 30) = 2.297; P = 0.0977). Data are shown as mean ± S.E.M (<span class="html-italic">n</span> = 7–12 per group).</p> Full article ">Figure 2
<p>(<b>A</b>) Analysis of corticosterone levels in plasma of control mice (vehicle, no stressor; Cnt), animals that experienced tail pinch but did not receive the D3R antagonist (vehicle, tail pinch; Cnt TP), and animals that experienced tail pinch and received the D3R antagonist at two different doses (SB-277011-B, 24 or 48 mg/kg, tail pinch; SB24 TP or SB48 TP). One-way ANOVA: F (3, 32) = 42.59); P &lt; 0.001. ** P &lt; 0.01, *** P &lt; 0.001 vs. Cnt, <sup>+++</sup> P &lt; 0.001 vs. Cnt TP (Tukey’s test). Cohen’s <span class="html-italic">d</span> values: Cnt TP vs. Cnt [<span class="html-italic">d</span>: 2.38; effect size r: 0.77]; SB24 TP vs. Cnt TP [<span class="html-italic">d</span>: −3.21; effect size r: −0.85]; SB48 TP vs. Cnt TP [<span class="html-italic">d</span>: −4.30; effect size r: −0.91]; SB48 TP vs. Cnt [<span class="html-italic">d</span>: −2.80; effect size r: −0.81]. (<b>B</b>) Analysis of corticosterone levels in plasma of control mice (vehicle, no stressor; Cnt), animals that experienced restraint but did not receive the D3R antagonist (vehicle, restraint; Cnt R), and animals that experienced restraint and received the D3R antagonist at two different doses SB-277011-B,24 or 48 mg/kg, restraint; SB24 R or SB48 R). One-way ANOVA: F (3, 31) = 27.29; P &lt; 0.001. *** P &lt; 0.001 vs. Cnt, <sup>+++</sup> P &lt; 0.001 vs. Cnt R (Tukey’s test). Cohen’s <span class="html-italic">d</span> values: Cnt R vs. Cnt [<span class="html-italic">d</span>: 2.04; effect size r: 0.71]; SB24 TP vs. Cnt TP [<span class="html-italic">d</span>: −2.88; effect size r: −0.82]; SB48 TP vs. Cnt TP [<span class="html-italic">d</span>: −3.58; effect size r: −0.87]. (<b>C</b>) Comparison of corticosterone levels in plasma between Cnt TP and Cnt R groups. Unpaired Student’s <span class="html-italic">t</span> test: t<sub>17</sub> = 2.809, P = 0.0121. * P &lt; 0.05 vs. Cnt TP. Cohen’s <span class="html-italic">d</span> values: Cnt R vs. Cnt TP [<span class="html-italic">d</span>: 1.31; effect size r: 0.55]. (<b>D</b>) Correlation analysis between plasmatic corticosterone and reinstatement score in tail pinched animals, measured as the time spent in cocaine-paired chamber during reinstatement test minus the same during extinction test. (<b>E</b>) Correlation analysis between plasmatic corticosterone and reinstatement score in restrained animals. (<b>F</b>) Correlation analysis between plasmatic corticosterone and total entries in tail pinched animals, measured as the total entries during reinstatement test minus those during extinction test. (<b>G</b>) Correlation analysis between plasmatic corticosterone and total entries in restrained animals. (<b>H</b>) Correlation analysis between reinstatement score and total entries in tail pinched animals. (<b>I</b>) Correlation analysis between reinstatement score and total entries in restrained animals. Correlation analyses were revealed through Pearson’s test. Data are shown as mean ± S.E.M (<span class="html-italic">n</span> = 7–12 per group).</p> Full article ">Figure 3
<p>Basolateral amygdala analysis in restraint and tail-pinch-stress paradigms. (<b>A</b>–<b>C</b>) Semiquantitative analysis and representative immunoblot of p-Akt/β-actin, Akt/β-actin, and p-Akt/Akt levels in restraint paradigm. One-way ANOVA: F (3, 21) = 5.454; P = 0.0062 (<b>A</b>); F (3, 21) = 1.774; P = 0.1829 (<b>B</b>); F (3, 21) = 6.916; P = 0.002 (<b>C</b>). (<b>D</b>–<b>F</b>) Semiquantitative analysis and representative immunoblot of p-mTOR/β-actin, mTOR/β-actin, and p-mTOR/mTOR levels in restraint paradigm. One-way ANOVA: F (3, 21) = 4.588 (<b>D</b>); F (3, 22) = 0.9630, P = 0.4278 (<b>E</b>); F (3, 22) = 11.79; P &lt; 0.0001 (<b>F</b>). (<b>G</b>–<b>I</b>) Semiquantitative analysis and representative immunoblot of p-ERK<sub>1/2</sub>/β-actin, ERK<sub>1/2</sub>/β-actin, and p-ERK<sub>1/2</sub>/ERK<sub>1/2</sub> levels in restraint paradigm. One-way ANOVA: F (3, 19) = 2.94; P = 0.0494 (<b>G</b>); F (3, 21) = 0.5587; P = 0.6481 (<b>H</b>), F (3, 22) = 2290; P = 0.1064 (<b>I</b>). (<b>A</b>–<b>I</b>) * P &lt; 0.05, ** P &lt; 0.01 vs. Cnt; <sup>+</sup> P &lt; 0.05, <sup>++</sup> P &lt; 0.01 vs. Cnt R; <sup>#</sup> P &lt; 0.05, <sup>##</sup> P &lt; 0.05 vs. SB24 R (Tukey’s test). (<b>J</b>–<b>L</b>) Semiquantitative analysis and representative immunoblot of p-Akt/β-actin, Akt/β-actin and p-Akt/Akt levels in tail-pinch paradigm. One-way ANOVA: F (3, 23) = 0.7468; P = 0.5353 (<b>J</b>); F (3, 23) = 0.3078; P = 0.8195 (<b>K</b>); F (3, 24) = 0.9801; P = 0.4186 (<b>L</b>). (<b>M</b>–<b>O</b>) Semiquantitative analysis and representative immunoblot of p-mTOR/β-actin, mTOR/β-actin, and p-mTOR/mTOR levels in tail-pinch paradigm. One-way ANOVA: F (3, 24) = 0.4483; P = 0.7208 (<b>M</b>); F (3, 24) = 0.6504; P = 0.5904 (<b>N</b>); F (3, 24) = 1.067; P = 0.3817 (<b>O</b>). (<b>P</b>–<b>R</b>) Semiquantitative analysis and representative immunoblot of p-ERK<sub>1/2</sub>/β-actin, ERK<sub>1/2</sub>/β-actin and p-ERK<sub>1/2</sub>/ERK<sub>1/2</sub> levels in tail-pinch paradigm. One-way ANOVA: F (3, 22) = 0.8227; P = 0.4953 (<b>P</b>); F (3, 23) = 2.744; P = 0.0663 (<b>Q</b>); F (3, 23) = 6.016; P = 0.0035 (<b>R</b>). (<b>J</b>–<b>R</b>) <sup>+</sup> P &lt; 0.05 vs. Cnt TP (Tukey’s test). Data are shown as mean ± S.E.M (<span class="html-italic">n</span> = 4–8 per group).</p> Full article ">Figure 4
<p>Dentate gyrus analysis in restraint and tail-pinch-stress paradigms (<b>A</b>–<b>C</b>) Semiquantitative analysis and representative immunoblot of p-Akt/β-actin, Akt/β-actin, and p-Akt/Akt levels in restraint paradigm. One-way ANOVA: F (3, 24) = 3838; P = 0.0224 (<b>A</b>); F (3, 24) = 4.951; P = 0.0081 (<b>B</b>); F (3, 24) = 2.086; P = 0.1287 (<b>C</b>). (<b>D</b>–<b>F</b>) Semiquantitative analysis and representative immunoblot of p-mTOR/β-actin, mTOR/β-actin, and p-mTOR/mTOR levels in restraint paradigm. One-way ANOVA: F (3, 24) = 3.663; P = 0.0264 (<b>D</b>); F (3, 24) = 0.5961; P = 0.6237 (<b>E</b>); F (3, 23) = 0.9933; P = 0.4135 (<b>F</b>). (<b>G</b>–<b>I</b>) Semiquantitative analysis and representative immunoblot of p-ERK<sub>1/2</sub>/β-actin, ERK<sub>1/2</sub>/β-actin and p-ERK<sub>1/2</sub>/ERK<sub>1/2</sub> levels in restraint paradigm. One-way ANOVA: F (3, 23) = 4.390; P = 0.0139 (<b>G</b>); F (3, 24) = 0.7024; P = 0.5599 (<b>H</b>); F (3, 23) = 5.051; P = 0.0078 (<b>I</b>). (<b>A</b>–<b>I</b>) * P &lt; 0.05 vs. Cnt; <sup>+</sup> P &lt; 0.05, <sup>++</sup> P &lt; 0.01 vs. Cnt R (Tukey’s test). (<b>J</b>–<b>L</b>) Semiquantitative analysis and representative immunoblot of p-Akt/β-actin, Akt/β-actin, and p-Akt/Akt levels in tail-pinch paradigm. One-way ANOVA: F (3, 23) = 9.915; P = 0.0002 (<b>J</b>); F (3, 23) = 1.442; P = 0.2563 (<b>K</b>); F (3, 24) = 4.678; P = 0.0104 (<b>L</b>). (<b>M</b>–<b>O</b>) Semiquantitative analysis and representative immunoblot of p-mTOR/β-actin, mTOR/β-actin, and p-mTOR/mTOR levels in tail-pinch paradigm. One-way ANOVA: F (3, 24) = 1.513; P = 0.2366 (<b>M</b>); F (3, 24) = 1.806; P = 0.1730 (<b>N</b>); F (3, 22) = 0.2821; P = 0.8377 (<b>O</b>). (<b>P</b>–<b>R</b>) Semiquantitative analysis and representative immunoblot of p-ERK<sub>1/2</sub>/β-actin, ERK<sub>1/2</sub>/β-actin, and p-ERK<sub>1/2</sub>/ERK<sub>1/2</sub> levels in tail-pinch paradigm. One-way ANOVA: F (3, 24) = 2.817; P = 0.0606 (<b>P</b>); F (3, 24) = 0.3584; P = 0.7835 (<b>Q</b>); F (3, 24) = 2.188; P = 0.1157 (<b>R</b>). (<b>J</b>–<b>R</b>) <sup>+</sup> P &lt; 0.05, <sup>++</sup> P &lt; 0.01, <sup>+++</sup> P &lt; 0.001 vs. Cnt TP (Tukey’s test). Data are shown as mean ± S.E.M (<span class="html-italic">n</span> = 4–8 animals per group).</p> Full article ">Figure 5
<p>(<b>A</b>) Correlation analysis of corticosterone in plasma of animals that experienced restraint and received the D3R antagonist at 48 mg/kg (SB48 R); p-Akt/Akt and p-mTOR/mTOR levels in basolateral amygdala. (<b>B</b>) Correlation analysis of corticosterone in plasma of animals that experienced restraint and received the D3R antagonist at 24 mg/kg (SB24 R) and p-mTOR/mTOR levels in dentate gyrus. (<b>C</b>) Correlation analysis of corticosterone in plasma and p-Akt/Akt levels in dentate gyrus of SB48 R mice. (<b>D</b>) Correlation analysis of total entries, expressed as the total entries during reinstatement test minus those during extinction test, and p-mTOR/mTOR in basolateral amygdala of SB48 R animals. (<b>E</b>) Correlation analysis of total entries and p-ERK<sub>1/2</sub>/ERK<sub>1/2</sub> levels in basolateral amygdala of animals that experienced tail pinch and received the SB-277011-A at 48 mg/kg (SB48 TP). Correlation analyses were performed through Pearson’s test (<span class="html-italic">n</span> = 4–8 animals per group).</p> Full article ">
22 pages, 7910 KiB  
Article
Identification of Regulatory Molecular “Hot Spots” for LH/PLOD Collagen Glycosyltransferase Activity
by Daiana Mattoteia, Antonella Chiapparino, Marco Fumagalli, Matteo De Marco, Francesca De Giorgi, Lisa Negro, Alberta Pinnola, Silvia Faravelli, Tony Roscioli, Luigi Scietti and Federico Forneris
Int. J. Mol. Sci. 2023, 24(13), 11213; https://doi.org/10.3390/ijms241311213 - 7 Jul 2023
Cited by 2 | Viewed by 2521
Abstract
Hydroxylysine glycosylations are post-translational modifications (PTMs) essential for the maturation and homeostasis of fibrillar and non-fibrillar collagen molecules. The multifunctional collagen lysyl hydroxylase 3 (LH3/PLOD3) and the collagen galactosyltransferase GLT25D1 are the human enzymes that have been identified as being responsible for the [...] Read more.
Hydroxylysine glycosylations are post-translational modifications (PTMs) essential for the maturation and homeostasis of fibrillar and non-fibrillar collagen molecules. The multifunctional collagen lysyl hydroxylase 3 (LH3/PLOD3) and the collagen galactosyltransferase GLT25D1 are the human enzymes that have been identified as being responsible for the glycosylation of collagen lysines, although a precise description of the contribution of each enzyme to these essential PTMs has not yet been provided in the literature. LH3/PLOD3 is thought to be capable of performing two chemically distinct collagen glycosyltransferase reactions using the same catalytic site: an inverting beta-1,O-galactosylation of hydroxylysines (Gal-T) and a retaining alpha-1,2-glucosylation of galactosyl hydroxylysines (Glc-T). In this work, we have combined indirect luminescence-based assays with direct mass spectrometry-based assays and molecular structure studies to demonstrate that LH3/PLOD3 only has Glc-T activity and that GLT25D1 only has Gal-T activity. Structure-guided mutagenesis confirmed that the Glc-T activity is defined by key residues in the first-shell environment of the glycosyltransferase catalytic site as well as by long-range contributions from residues within the same glycosyltransferase (GT) domain. By solving the molecular structures and characterizing the interactions and solving the molecular structures of human LH3/PLOD3 in complex with different UDP-sugar analogs, we show how these studies could provide insights for LH3/PLOD3 glycosyltransferase inhibitor development. Collectively, our data provide new tools for the direct investigation of collagen hydroxylysine PTMs and a comprehensive overview of the complex network of shapes, charges, and interactions that enable LH3/PLOD3 glycosyltransferase activities, expanding the molecular framework and facilitating an improved understanding and manipulation of glycosyltransferase functions in biomedical applications. Full article
(This article belongs to the Special Issue Recent Advances in Collagen Proteins)
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<p>A direct assay to probe Lys-to-Glc-Gal-Hyl conversion. The reaction schematic monitored by the assay is depicted in (<b>A</b>). The MS spectra show the results obtained by incubating a synthetic GIKGIKGIKGIK peptide (MW 1254 Da) with enzymes and cofactors, as shown in the figure legends. All peaks identified are doubly charged, resulting in nominal masses corresponding to half of the expected MW. (<b>B</b>) Using LH3/PLOD3 and LH activity cofactors (i.e., 2-OG and Fe<sup>2+</sup>), MS peaks corresponding to a singly hydroyxylated Lys on the peptide (i.e., 635 Da) appear. (<b>C</b>) The addition of Gal-T activity cofactors (i.e., UDP-Gal and Mn<sup>2+</sup>) to the same mixture as in (<b>B</b>) does not yield additional MS peaks. (<b>D</b>) When the mixture in (<b>C</b>) is incubated with GLT25D1, the MS peaks corresponding to Gal-Hyl are found (i.e., 716 Da). (<b>E</b>) When the same mixture as in (<b>D</b>) also contains UDP-Glc, the peaks corresponding to Glc-Gal-Hyl appear (i.e., 797 Da).</p> Full article ">Figure 2
<p>Structural and functional features of the LH3/PLOD3 glycosyltransferase (GT) domain. (<b>A</b>) Cartoon representation of the LH3/PLOD3 GT domain (PDB ID: 6FXR) showing the key residues shaping the catalytic site as sticks. The PolyAsp motif (brown) and the glycoloop (cyan) involved in the binding of UDP-sugar donor substrates are shown. The residues implicated in the catalytic activity and investigated in this works are colored, while the residues depicted in gray have already been shown to be essential in Mn<sup>2+</sup> (purple sphere) and UDP (black sticks) coordination. (<b>B</b>) Summary of the evaluation of the Glc-T activity of LH3/PLOD3 mutants compared to the wild-type using MS direct assays. (<b>C</b>) Evaluation of the Glc-T activity of LH3/PLOD3 mutants compared to wild-type using luminescence-based indirect assays. Each graph shows the enzymatic activity detected in the absence (i.e., “uncoupled”, light blue) or presence of gelatin, which was used as the acceptor substrate. The plotted data are baseline-corrected, where the baseline was the background control. In both (<b>B</b>,<b>C</b>) panels, the error bars represent standard deviations from the averages of independent experiments (N &gt; 3).</p> Full article ">Figure 3
<p>Structural characterization of LH3/PLOD3 mutants. (<b>A</b>) Crystal structure of the LH3/PLOD3 p.(Val80Lys) mutant in complex with UDP-glucose and Mn<sup>2+</sup>. Electron density is visible for the mutated lysine and the UDP portion of the donor substrate (green mesh, 2<span class="html-italic">F<sub>o</sub></span>−<span class="html-italic">F<sub>c</sub></span> omit electron density map, contoured at 1.3 σ). Catalytic residues shaping the enzyme cavity are shown as sticks; Mn<sup>2+</sup> is shown as a purple sphere. Consistent with what was observed in the crystal structure of wild-type LH3/PLOD3, the glucose moiety of the donor substrate is not visible in the experimental electron density. (<b>B</b>) Crystal structure of the LH3/PLOD3 p.(Asp190Ser) mutant in complex with UDP-glucose and Mn<sup>2+</sup>. Electron density is visible for the mutated Serine and for the entire donor substrate, including the sugar moiety (green mesh, 2<span class="html-italic">F<sub>o</sub></span>−<span class="html-italic">F<sub>c</sub></span> omit electron density map, contoured at 1.3 σ). Colors and representations as in (<b>A</b>). (<b>C</b>) Superposition of wild-type, p.(Val80Lys), and p.(Asp190Ser) LH3/PLOD3 available crystal structures in substrate-free (cyan for wild-type, yellow for p.(Val80Lys), respectively) and with UDP-glucose bound (marine for wild-type, orange for p.(Val80Lys), magenta for p.(Asp190Ser), respectively) states. Notably, the conformations adopted by the side chain of Trp145 upon ligand binding are consistent in the wild-type and in the mutant enzyme. As the glycoloop is flexible in substrate-free structures, the side chains of Val/Lys80 are only visible in the in UDP-sugar-bound structures.</p> Full article ">Figure 4
<p>Binding mode of the glucose moiety of the UDP-Glc donor substrate observed in the crystal structure of LH3/PLOD3 p.(Asp190Ser). (<b>A</b>) Highlight of the amino acid network surrounding the Glc moiety of the donor substratein the crystal structure. UDP-Glc is shown as thick yellow sticks, whereas amino acids found at less than 5 Å distance from the Glc moiety are shown as thin blue/green sticks. (<b>B</b>) Overview of the interaction network surrounding the UDP-Glc donor substrate in the co-crystal structure with LH3/PLOD3 p.(Asp190Ser). Colors are as in (<b>A</b>). Figure made with LIGPLOT+ [<a href="#B36-ijms-24-11213" class="html-bibr">36</a>]. (<b>C</b>) The conformation adopted by the Glc moiety of the UDP-Glc substrate in the glycosyltransferase catalytic site of LH3/PLOD3 p.(Asp190Ser) leaves an empty cavity that is geometrically and sizably compatible with the Gal moiety of the acceptor substrate. Shown is a surface rendering of the GT domain of LH3/PLOD3 p.(Asp190Ser) colored by electrostatic potential, with highlights of the UDP-Glc donor substrate shown as sticks.</p> Full article ">Figure 5
<p>Ser178 in LH1/PLOD1, corresponding to Asp190 in LH3/PLOD3, is a key residue for Glc-T activity for both enzyme isoforms. (<b>A</b>) Direct MS-based assays comparing the signal associated with Glc-Gal-Hyl using wild-type and Ser178Asp LH1/PLOD1 variants. (<b>B</b>) Evaluation of the Glc-T activity of LH1 wild-type and Ser178Asp using luminescence-based indirect assays. The analysis of coupled and uncoupled enzymatic activities is as in <a href="#ijms-24-11213-f002" class="html-fig">Figure 2</a>C. In both panels, the error bars represent standard deviations from the averages of independent experiments (N &gt; 3).</p> Full article ">Figure 6
<p>Characterization of UDP-sugar analogs. (<b>A</b>) Thermal stability of LH3 wild-type (solid green) using differential scanning fluorimetry (DSF) in the presence of various Mn<sup>2+</sup> and several UDP-sugars. A prominent stabilization effect is achieved in the presence of the biological donor substrates UDP-galactose (solid blue), UDP-Glucose (solid purple), and free UDP (solid black). A milder stabilization effect is also obtained with UDP-xylose (red dash) and UDP-glucuronic acid (green dash). (<b>B</b>) Luminescence-based competition assays evaluating the binding of increasing concentrations of UDP-GlcA or UDP-Xyl to wild-type LH3/PLOD3 in the presence of either UDP-Gal (<b>left</b>) or UDP-Glc (<b>right</b>) and acceptor substrates (i.e., gelatin). (<b>C</b>) Crystal structure of LH3 wild-type in complex with Mn<sup>2+</sup> and UDP-glucuronic acid shows clear electron density for UDP (2<span class="html-italic">F<sub>o</sub></span>−<span class="html-italic">F<sub>c</sub></span> omit electron density maps, green mesh, contour level 1.2 σ). The glucuronic acid (shown in yellow) can be modelled even if with the partial electron density. (<b>D</b>) Crystal structure of the LH3 Val80Lys mutant in complex with Mn<sup>2+</sup> and UDP-glucuronic acid. While the UDP backbone can be modelled in the electron density (black sticks) (2<span class="html-italic">F<sub>o</sub></span>−<span class="html-italic">F<sub>c</sub></span> omit electron density maps, green mesh, contour level 1.2 σ), in this case, no electron density is present for the glucuronic acid (shown in yellow). In addition, the portion of the glycoloop containing the mutated lysine is flexible from residue 79 to 83 (shown as cyan spheres). (<b>E</b>) Crystal structure of LH3 wild-type in complex with Mn<sup>2+</sup> and UDP-xylose. Similar to UDP-GlcA, UDP shows clear electron density (2<span class="html-italic">F<sub>o</sub></span>−<span class="html-italic">F<sub>c</sub></span> omit electron density maps, green mesh, contour level 1.2 σ), whereas partial density is shown for the xylose moiety (shown in pink).</p> Full article ">
17 pages, 1710 KiB  
Review
The Role of Autophagy and Apoptosis in Affected Skin and Lungs in Patients with Systemic Sclerosis
by Vesna Spasovski, Marina Andjelkovic, Marina Parezanovic, Jovana Komazec, Milena Ugrin, Kristel Klaassen and Maja Stojiljkovic
Int. J. Mol. Sci. 2023, 24(13), 11212; https://doi.org/10.3390/ijms241311212 - 7 Jul 2023
Cited by 3 | Viewed by 1976
Abstract
Systemic sclerosis (SSc) is a complex autoimmune inflammatory disorder with multiple organ involvement. Skin changes present the hallmark of SSc and coincide with poor prognosis. Interstitial lung diseases (ILD) are the most widely reported complications in SSc patients and the primary cause of [...] Read more.
Systemic sclerosis (SSc) is a complex autoimmune inflammatory disorder with multiple organ involvement. Skin changes present the hallmark of SSc and coincide with poor prognosis. Interstitial lung diseases (ILD) are the most widely reported complications in SSc patients and the primary cause of death. It has been proposed that the processes of autophagy and apoptosis could play a significant role in the pathogenesis and clinical course of different autoimmune diseases, and accordingly in SSc. In this manuscript, we review the current knowledge of autophagy and apoptosis processes in the skin and lungs of patients with SSc. Profiling of markers involved in these processes in skin cells can be useful to recognize the stage of fibrosis and can be used in the clinical stratification of patients. Furthermore, the knowledge of the molecular mechanisms underlying these processes enables the repurposing of already known drugs and the development of new biological therapeutics that aim to reverse fibrosis by promoting apoptosis and regulate autophagy in personalized treatment approach. In SSc-ILD patients, the molecular signature of the lung tissues of each patient could be a distinctive criterion in order to establish the correct lung pattern, which directly impacts the course and prognosis of the disease. In this case, resolving the role of tissue-specific markers, which could be detected in the circulation using sensitive molecular methods, would be an important step toward development of non-invasive diagnostic procedures that enable early and precise diagnosis and preventing the high mortality of this rare disease. Full article
(This article belongs to the Special Issue Pathogenesis, Immunity and Therapy of Systemic Sclerosis: Molecular Aspects)
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<p>Schematic representation of apoptosis regulation in SSc. In the skin of SSc patients, both intrinsic and extrinsic apoptotic pathways are deregulated. In response to biochemical stimulation, both canonical and non-canonical TGF-β pathways are activated, and XIAP is shown to be a link between them. XIAP also inhibits caspase-3 and 7. As a result of constitutively activated autocrine TGFβ signaling, PP2A level is downregulated in SSc fibroblasts, leading to increased AKT and ERK1/2 phosphorylation. Extrinsic apoptotic pathway is deregulated through cIAP and XIAP proteins. Their higher expression was shown to abrogate caspase-3 and 7 in late-stage fibroblast populations from SSc patients. Matrix stiffness through the activity of fibroblast integrins transmits the mechanical force from the matrix to the actin cytoskeleton through focal adhesion-associated protein FAK. FAK activation and its constitutive phosphorylation of downstream molecules drives profibrotic gene expression. In addition, target genes regulated through this mechanism also include proapoptotic BCL-2 family members Bcl-2, BIM, and PUMA, which activate to prime the cell for apoptosis. In response to this, extensive activation of antiapoptotic Bcl-XL protein takes place, in order to ensure cell survival.</p> Full article ">Figure 2
<p>Factors that influence fibroblast-to-myofibroblast transition.</p> Full article ">Figure 3
<p>The proposed processes of apoptosis, autophagy, and cellular stress in SSc-ILD patients. Schematic representation of apoptosis, autophagy, and oxidative stress processes in SSc-ILD, where apoptosis is marked in green, autophagy in blue, and oxidative stress in orange. Downregulation of HIPK2 kinase in NSIP and p53-Mdm2 conjugate in UIP/IPF patients indicate the differences between apoptotic pathways involved in the pathogenesis of NSIP and UIP/IPF, respectively. PRDX1 is solely upregulated in NSIP-specific areas in comparison with IPF, suggesting more severe oxidative stress in IPF patients.</p> Full article ">
17 pages, 3762 KiB  
Article
Transcriptome Analysis of Diurnal and Nocturnal-Warmed Plants, the Molecular Mechanism Underlying Cold Deacclimation Response in Deschampsia antarctica
by Dariel López, Giovanni Larama, Patricia L. Sáez and León A. Bravo
Int. J. Mol. Sci. 2023, 24(13), 11211; https://doi.org/10.3390/ijms241311211 - 7 Jul 2023
Cited by 2 | Viewed by 1671
Abstract
Warming in the Antarctic Peninsula is one of the fastest on earth, and is predicted to become more asymmetric in the near future. Warming has already favored the growth and reproduction of Antarctic plant species, leading to a decrease in their freezing tolerance [...] Read more.
Warming in the Antarctic Peninsula is one of the fastest on earth, and is predicted to become more asymmetric in the near future. Warming has already favored the growth and reproduction of Antarctic plant species, leading to a decrease in their freezing tolerance (deacclimation). Evidence regarding the effects of diurnal and nocturnal warming on freezing tolerance-related gene expression in D. antarctica is negligible. We hypothesized that freezing tolerance-related gene (such as CBF-regulon) expression is reduced mainly by nocturnal warming rather than diurnal temperature changes in D. antarctica. The present work aimed to determine the effects of diurnal and nocturnal warming on cold deacclimation and its associated gene expression in D. antarctica, under laboratory conditions. Fully cold-acclimated plants (8 °C/0 °C), with 16h/8h thermoperiod and photoperiod duration, were assigned to four treatments for 14 days: one control (8 °C/0 °C) and three with different warming conditions (diurnal (14 °C/0 °C), nocturnal (8 °C/6 °C), and diurnal-nocturnal (14 °C/6 °C). RNA-seq was performed and differential gene expression was analyzed. Nocturnal warming significantly down-regulated the CBF transcription factors expression and associated cold stress response genes and up-regulated photosynthetic and growth promotion genes. Consequently, nocturnal warming has a greater effect than diurnal warming on the cold deacclimation process in D. antarctica. The eco-physiological implications are discussed. Full article
(This article belongs to the Special Issue Abiotic Stresses in Plants: From Molecules to Environment)
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<p>Number of differentially expressed genes (DEGs). The amounts of differentially expressed genes in warming treatments, up-regulated (light yellow) and down-regulated (orange) in relation to the control condition of cold-acclimated plants, 8 °C/0 °C (CA). Warming treatments received the following thermoperiods: 8 °C/6 °C, nocturnal warming (NW+); 14 °C/0 °C, diurnal warming (DW+); and 14 °C/6 °C, diurnal-nocturnal warming (DNW+). The plants were cultivated in chambers under controlled conditions at a photoperiod and thermoperiod of 18 h/6 h duration.</p> Full article ">Figure 2
<p>Venn diagram of the number of differentially expressed genes (DEGs). The number and percentage of down-regulated genes (<b>A</b>) and up-regulated genes (<b>B</b>) in warming treatments in relation to the control condition of cold-acclimated plants, 8 °C/0 °C (CA). Warming treatments received the following thermoperiods: 8 °C/6 °C, nocturnal warming (NW+); 14 °C/0 °C, diurnal warming (DW+); and 14 °C/6 °C, diurnal-nocturnal warming (DNW+). The plants were cultivated in chambers under controlled conditions at a photoperiod and thermoperiod of 18 h/6 h duration.</p> Full article ">Figure 3
<p>Clusters obtained from principal component analysis and self-organizing maps (PCA-SOM). The nodes group transcripts up-regulated (<b>A</b>) and down-regulated (<b>B</b>) by nocturnal warming, and the enriched gene ontology categories with their corresponding <span class="html-italic">p</span>-values are shown. The boxes represent the interquartile range (middle 50% of the data), its midline indicates the median value, and the whiskers indicate the ranges of the upper and lower 25%, excluding outliers. Corresponding to treatments with the following thermoperiods (day/night): 8 °C/0 °C, plants acclimated to cold (AF); 8 °C/6 °C, night heating (CN); 14 °C/0 °C, daytime warming (CD); and 14 °C/6 °C, daytime-night warming (CDN). The plants were cultivated in chambers under controlled conditions at a photoperiod and thermoperiod of 18 h/6 h duration.</p> Full article ">Figure 4
<p>Heat-map of co-expressed genes’ response to nocturnal warming. The genes’ relative abundance is presented by the z-score of the log<sub>10</sub> FPKM, after 2 h of temperature drop during the nocturnal period, of plants with a cold acclimation of 8 °C/0 °C (CA); nocturnal warming of 8 °C/6 °C (NW+); diurnal warming of 14 °C/0 °C (DW+); and diurnal-nocturnal warming of 14 °C/6 °C (DNW+). The plants were cultivated in chambers under controlled conditions at a photoperiod and thermoperiod of 18 h/6 h duration.</p> Full article ">Figure 5
<p>Relative expression of genes related to freezing tolerance. The variation on gene relative expression was evaluated by qRT-PCR. Values represent the fold change mean ± standard error (n = 3), in relation to cold-acclimated (CA) plants 8 °C/0 °C, at the beginning of the nocturnal period (0 h). Transcripts representing variation included: (<b>A</b>) ‘Dehydration-responsive element-binding protein 1A’ (DRE1A/CBF3), (<b>B</b>) ‘Dehydration-responsive element-binding protein 1D’ (DRE1D/CBF4), (<b>C</b>) ‘Dehydrin 1’ (DHN1), (<b>D</b>) ‘E3 ubiquitin-protein ligase RGLG5’ (RGLG5), (<b>E</b>) ‘UDP-glycosyltransferase 73B3’ (U73B3), (<b>F</b>) ‘Vacuolar cation/proton exchanger 1’ (CAX1), (<b>G</b>) ‘Ornithine aminotransferase’ (OAT), (<b>H</b>) ‘Sucrose synthase 4’ (SUS4), and (<b>I</b>) ‘Sucrose phosphate synthase 4’ (SPS4F). Warming treatments correspond to: nocturnal warming, 8 °C/6 °C (NW+); diurnal warming, 14 °C/0 °C (DW+); and diurnal-nocturnal warming, 14 °C/6 °C (DNW+), with 18 h/6 h photoperiod and thermoperiods duration. Significant differences among factors are shown as different lower cases (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>Relative expression of up-regulated genes by nocturnal warming. The variation in relative gene expression was evaluated by qRT-PCR. Values represent the fold change mean ± standard error (n = 3), in relation to cold-acclimated (CA) plants 8 °C/0 °C, at the beginning of the nocturnal period (0 h). Transcript variation shown includes: (<b>A</b>) ‘IAA-amino acid hydrolase ILR1-like 2’ (ILL2), (<b>B</b>) ‘1-deoxy-D-xylulose-5-phosphate synthase 1’ (DXS1), (<b>C</b>) ‘Triose phosphate/phosphate translocator’ (TPT), (<b>D</b>) ‘Oxygen-evolving enhancer protein 2’ (PSBP2), (<b>E</b>) ‘Fructose-bisphosphate aldolase 5’ (FAB5), and (<b>F</b>) ‘Ribulose bisphosphate carboxylase small subunit 1’ (RBCS1). Warming treatments correspond to: nocturnal warming, 8 °C/6 °C (NW+); diurnal warming, 14 °C/0 °C (DW+); and diurnal-nocturnal warming, 14 °C/6 °C (DNW+), with 18 h/6 h photoperiod and thermoperiods duration. Significant differences among factors are shown as different lower cases (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
26 pages, 5380 KiB  
Article
LncRNA 220: A Novel Long Non-Coding RNA Regulates Autophagy and Apoptosis in Kupffer Cells via the miR-5101/PI3K/AKT/mTOR Axis in LPS-Induced Endotoxemic Liver Injury in Mice
by Ying Yang, Tian Tian, Shan Li, Nanhong Li, Haihua Luo and Yong Jiang
Int. J. Mol. Sci. 2023, 24(13), 11210; https://doi.org/10.3390/ijms241311210 - 7 Jul 2023
Cited by 1 | Viewed by 1918
Abstract
Sepsis is a severe medical condition distinguished by immune systematic dysfunction and multiple organic injury, or even failure, resulting from an acute systemic inflammatory response. Acute liver injury (ALI) could be considered as a notable inflammatory outcome of sepsis. Studies have demonstrated the [...] Read more.
Sepsis is a severe medical condition distinguished by immune systematic dysfunction and multiple organic injury, or even failure, resulting from an acute systemic inflammatory response. Acute liver injury (ALI) could be considered as a notable inflammatory outcome of sepsis. Studies have demonstrated the essential roles played by long non-coding RNAs (lncRNAs) in mediating the processes of various diseases, including their ability to engage in interactions with microRNAs (miRNAs) as complexes of competing endogenous RNA (ceRNA) to modulate signaling pathways. In this study, a newly discovered lncRNA, named 220, was identified to function in regulating autophagy and apoptosis in Kupffer cells treated with lipopolysaccharide (LPS). This was achieved through sponging miR-5101 as a ceRNA complex, as identified via high-throughput sequencing. The expression of 220 was found to be significantly different in the hepatic tissues of endotoxemic mice that were treated with LPS for 8 h, ultimately modulating the ALI process. Our studies have collectively demonstrated that 220 is a novel regulator that acts on LPS-induced autophagy and apoptosis in Kupffer cells, thereby mediating the ALI process induced by LPS. Furthermore, the validation of our findings using clinical databases suggests that 220 could potentially serve as a molecular target of clinical, diagnostic, and therapeutic significance in septic liver injury. Full article
(This article belongs to the Special Issue Immuno-Metabolism of Sepsis)
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<p>Identification and annotation of lncRNA 220. (<b>A</b>) H&amp;E staining for the livers of mice treated with LPS. (<b>B</b>) Volcano plots generated to illustrate the differentially expressive lncRNAs in the livers of mice treated with LPS at different time points (0 h, 2 h, 8 h, and 24 h). (<b>C</b>) Heat map of differentially expressive lncRNAs in the livers of mice treated with LPS for 8 h (the lncRNA exhibiting the highest degree of differential expression is depicted within the red-dashed enclosure). (<b>D</b>) Expressive trend plot of lncRNA TCONS_00127483. (<b>E</b>) Chromosomal location of lncRNA TCONS_00127483 in the mouse genome. (<b>F</b>) Expressions of 220 in the livers of mice treated with LPS at different time points (0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h). (<b>G</b>) Expressions of 220 in the livers of WT and TLR4<sup>−/−</sup> mice treated with LPS for 8 h. (<b>H</b>) Expressions of 220 in the livers of WT and MyD88<sup>−/−</sup>, TRIF<sup>−/−</sup> mice treated with LPS for 4 h (*, <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; 0.0001; ns, not significant).</p> Full article ">Figure 2
<p>Identification and annotation of lncRNA 220. (<b>A</b>) FISH assay utilized to examine the distribution of 220 at the tissue level. (<b>B</b>) Expressions of 220 in Kupffer cells and AML-12 cells treated with LPS at different time points (0 h, 2 h, 4 h, 8 h, 12 h, and 24 h). (<b>C</b>) Nucleocytoplasmic distribution of 220 in Kupffer cells at the normal condition. (<b>D</b>) Variable trend of cytoplasmic expressions of 220 in Kupffer cells treated with LPS at different time points (0 h, 2 h, 8 h, 12 h, and 24 h). (<b>E</b>) Variable trend of nuclear expressions of 220 in Kupffer cells treated with LPS at different time points (0 h, 2 h, 8 h, 12 h, and 24 h). (<b>F</b>,<b>G</b>) FISH assay utilized to examine the distribution of 220 at the cellular level (*, <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; ns, not significant).</p> Full article ">Figure 3
<p>Prediction of the ceRNA complex constructed by lncRNA 220 and its targeted miRNAs. (<b>A</b>) Prediction of the targeted miRNAs of 220. (<b>B</b>) Venn plot of the overlapping downstream mRNA targets of miR-5101, miR-669b-5p, and miR-7234-5p. (<b>C</b>) Interactive plot of downstream targeted mRNAs of 5101. (<b>D</b>) Interactive plot of 10 hub targeted mRNAs of 5101 screened out by Cytoscape (the hue of the icon symbolizes the significance of genes, with a deeper red hue indicating a greater degree of importance). (<b>E</b>) GO/KEGG enrichment analysis for the hub targeted mRNAs (the manifestation of the hub targeted mRNAs’ participation in the PI3K pathway is exhibited within the red-dashed enclosure). (<b>F</b>) Sankey plot of ceRNA complex formed by 220 and 5101 (the color used in this plot is primarily employed for the purpose of designating each variable and its interrelation). (<b>G</b>) Prediction of the regulatory pathway involving the interaction of 220 and 5101.</p> Full article ">Figure 3 Cont.
<p>Prediction of the ceRNA complex constructed by lncRNA 220 and its targeted miRNAs. (<b>A</b>) Prediction of the targeted miRNAs of 220. (<b>B</b>) Venn plot of the overlapping downstream mRNA targets of miR-5101, miR-669b-5p, and miR-7234-5p. (<b>C</b>) Interactive plot of downstream targeted mRNAs of 5101. (<b>D</b>) Interactive plot of 10 hub targeted mRNAs of 5101 screened out by Cytoscape (the hue of the icon symbolizes the significance of genes, with a deeper red hue indicating a greater degree of importance). (<b>E</b>) GO/KEGG enrichment analysis for the hub targeted mRNAs (the manifestation of the hub targeted mRNAs’ participation in the PI3K pathway is exhibited within the red-dashed enclosure). (<b>F</b>) Sankey plot of ceRNA complex formed by 220 and 5101 (the color used in this plot is primarily employed for the purpose of designating each variable and its interrelation). (<b>G</b>) Prediction of the regulatory pathway involving the interaction of 220 and 5101.</p> Full article ">Figure 4
<p>Decoy of miRNA 5101 by lncRNA 220 as a ceRNA complex. (<b>A</b>) Expressions of 5101 in the livers of mice treated with LPS for 8 h. (<b>B</b>) RNA pull-down assay conducted to confirm the interaction between 220 and 5101. (<b>C</b>) Dual-luciferase reporter assay conducted to confirm the interaction between 220 and 5101 (the complementary interaction sequence between 220 WT and 5101, as well as the mutation sequence of the corresponding 220 MUT are exhibited in the red-fonts box). (<b>D</b>) Interfering efficiency of 220. (<b>E</b>) Expressions of 5101 after knockdown of 220. (<b>F</b>) Overexpression efficiency of 5101. (<b>G</b>) Interfering efficiency of 5101. (<b>H</b>) Expressions of 220 after overexpression 5101. (<b>I</b>) Expressions of 220 after knockdown of 5101. (<b>J</b>) Assessment for the mRNA levels of inflammatory cytokines and Pik3ca after overexpression and knockdown of 5101 (*, <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; ns, not significant).</p> Full article ">Figure 5
<p>Up-regulation of miRNA 5101 on LPS-induced autophagy in Kupffer cells. (<b>A</b>,<b>B</b>) WB results pertaining to the expressive levels of pertinent proteins within the PI3K/AKT/mTOR signaling pathway and cell autophagy process after overexpression of 5101. (<b>C</b>,<b>D</b>) WB results pertaining to the expressive levels of pertinent proteins within the PI3K/AKT/mTOR signaling pathway and cell autophagy process after knockdown of 5101. (<b>E</b>,<b>F</b>) Immunofluorescence co-localization assay conducted to detect the autophagic flux after overexpression and knockdown of 5101 (*, <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; ns, not significant).</p> Full article ">Figure 6
<p>Up-regulation of miRNA 5101 on LPS-induced apoptosis in Kupffer cells. (<b>A</b>,<b>B</b>) WB results pertaining to the expressive levels of pertinent proteins of cell apoptosis process after overexpression of 5101. (<b>C</b>,<b>D</b>) WB results pertaining to the expressive levels of pertinent proteins of cell apoptosis process after knockdown of 5101. (<b>E</b>,<b>F</b>) TUNEL assay after overexpression and knockdown of 5101. (<b>G</b>,<b>H</b>) Flow cytometry after overexpression and knockdown of 5101 (*, <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; ns, not significant).</p> Full article ">Figure 7
<p>The clinical significance of lncRNA 220. (<b>A</b>) Homologous sequences of 220 on chromosome 21 in human. (<b>B</b>) Validation of the expressions of 220 in human cells. (<b>C</b>) Prediction of the downstream targeted miRNAs of 220 in human. (<b>D</b>) Correlation between PIK3R1 (highlighted within the red-dashed box) screened out by Cytoscape and miR-5100 (the hue of the icon symbolizes the significance of genes, with a deeper red hue indicating a greater degree of importance). (<b>E</b>) Correlation between PTEN (highlighted within the red-dashed box) screened out by Cytoscape and miR-3616-3p. (<b>F</b>) Expressions of PIK3R1 on the fifth day compared to the first day after admission in normal guys, survivors, and non-survivors with sepsis, respectively. (<b>G</b>) Expressions of PTEN on the fifth day compared to the first day after admission in normal guys, survivors, and non-survivors with sepsis, respectively. (<b>H</b>) Immune cell infiltrative analysis in peripheral blood conducted on non-survivors with sepsis after admission, comparing the first day to the fifth day. (<b>I</b>) Correlative lollipop plot between the mRNA expression of PIK3R1 and the infiltrative proportions of various immune cells. (<b>J</b>) ROC diagnostic curve of PIK3R1 in non-survivors with sepsis. (<b>K</b>) ROC diagnostic curve of PTEN in non-survivors with sepsis (*, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ns, not significant).</p> Full article ">Figure 8
<p>Regulatory mechanism of lncRNA 220. Upon LPS treatment, the TLR4-MyD88 dependent pathway functions as the primary downstream regulatory pathway. The TLR4 receptors situated on the Kupffer cell membranes transmit signals to the nucleus via phosphorylated MyD88, thereby promoting the transcription of 220. A minor proportion of 220 is transported through the nuclear pores to the cytoplasm, where it complexes with 5101 to form the ceRNA complex. During inflammatory conditions, the complex is responsible for regulating the downstream PI3K/AKT/mTOR signaling pathway by modulating the levels of phosphorylated PIK3R1, AKT, and mTOR, which in turn contributes to the autophagy and apoptosis processes in Kupffer cells.</p> Full article ">
17 pages, 3342 KiB  
Article
Albuminuria-Related Genetic Biomarkers: Replication and Predictive Evaluation in Individuals with and without Diabetes from the UK Biobank
by Marisa Cañadas-Garre, Andrew T. Kunzmann, Kerry Anderson, Eoin P. Brennan, Ross Doyle, Christopher C. Patterson, Catherine Godson, Alexander P. Maxwell and Amy Jayne McKnight
Int. J. Mol. Sci. 2023, 24(13), 11209; https://doi.org/10.3390/ijms241311209 - 7 Jul 2023
Cited by 2 | Viewed by 2433
Abstract
Increased albuminuria indicates underlying glomerular pathology and is associated with worse renal disease outcomes, especially in diabetic kidney disease. Many single nucleotide polymorphisms (SNPs), associated with albuminuria, could be potentially useful to construct polygenic risk scores (PRSs) for kidney disease. We investigated the [...] Read more.
Increased albuminuria indicates underlying glomerular pathology and is associated with worse renal disease outcomes, especially in diabetic kidney disease. Many single nucleotide polymorphisms (SNPs), associated with albuminuria, could be potentially useful to construct polygenic risk scores (PRSs) for kidney disease. We investigated the diagnostic accuracy of SNPs, previously associated with albuminuria-related traits, on albuminuria and renal injury in the UK Biobank population, with a particular interest in diabetes. Multivariable logistic regression was used to evaluate the influence of 91 SNPs on urine albumin-to-creatinine ratio (UACR)-related traits and kidney damage (any pathology indicating renal injury), stratifying by diabetes. Weighted PRSs for microalbuminuria and UACR from previous studies were used to calculate the area under the receiver operating characteristic curve (AUROC). CUBN-rs1801239 and DDR1-rs116772905 were associated with all the UACR-derived phenotypes, in both the overall and non-diabetic cohorts, but not with kidney damage. Several SNPs demonstrated different effects in individuals with diabetes compared to those without. SNPs did not improve the AUROC over currently used clinical variables. Many SNPs are associated with UACR or renal injury, suggesting a role in kidney dysfunction, dependent on the presence of diabetes in some cases. However, individual SNPs or PRSs did not improve the diagnostic accuracy for albuminuria or renal injury compared to standard clinical variables. Full article
(This article belongs to the Section Molecular Informatics)
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<p>Diagram showing the genes with the most significant associations (<span class="html-italic">p</span> &lt; 0.001) across phenotypes and cohorts.</p> Full article ">Figure 2
<p>Summary of the SNPs found to be associated with the different phenotypes in each cohort. DM: diabetic cohort. KDIGO: Kidney Disease: Improving Global Outcomes. nonDM: non-diabetic cohort. SNP: single nucleotide polymorphism. UACR: urine albumin/creatinine ratio. SNP information is shown as chromosome|gene|rs identifier|counted allele|alternate allele. * Number of models including the SNP. ** Number of phenotypes associated with the SNP. Green color means the counted allele increases the beta coefficient and red is used for alleles decreasing the beta coefficient.</p> Full article ">Figure 3
<p>Beta coefficients and standard errors for the five single nucleotide polymorphisms with more associations across phenotypes. SNP information is shown as chromosome|gene|rs identifier|counted allele|alternate allele. The absence of dots and values means no association of the SNP with that phenotype/cohort. SNP: single nucleotide polymorphism. UACR: urine albumin-to-creatinine ratio.</p> Full article ">
10 pages, 11095 KiB  
Communication
Conjugated Linoleic Acid-Mediated Connexin-43 Remodeling and Sudden Arrhythmic Death in Myocardial Infarction
by Natia Qipshidze Kelm, Jane C. Solinger, Kellianne M. Piell and Marsha P. Cole
Int. J. Mol. Sci. 2023, 24(13), 11208; https://doi.org/10.3390/ijms241311208 - 7 Jul 2023
Cited by 2 | Viewed by 1467
Abstract
Connexin 43 (Cx43) is expressed in the left and right ventricles and is primarily responsible for conducting physiological responses in microvasculature. Studies have demonstrated that NADPH oxidase (NOX) enzymes are essential in cardiac redox biology and are responsible for the generation of reactive [...] Read more.
Connexin 43 (Cx43) is expressed in the left and right ventricles and is primarily responsible for conducting physiological responses in microvasculature. Studies have demonstrated that NADPH oxidase (NOX) enzymes are essential in cardiac redox biology and are responsible for the generation of reactive oxygen species (ROS). NOX2 is linked to left ventricular remodeling following myocardial infarction (MI). It was hypothesized that conjugated linoleic acid (cLA) treatment increases NOX-2 levels in heart tissue and disrupts connexins between the myocytes in the ventricle. Data herein demonstrate that cLA treatment significantly decreases survival in a murine model of MI. The observance of cLA-induced ventricular tachyarrhythmia’s (VT) led to the subsequent investigation of the underlying mechanism in this MI model. Mice were treated with cLA for 12 h, 24 h, 48 h, or 72 h to determine possible time-dependent changes in NOX and Cx43 signaling pathways in isolated left ventricles (LV) extracted from cardiac tissue. The results suggest that ROS generation, through the stimulation of NOX2 in the LV, triggers a decrease in Cx43 levels, causing dysfunction of the gap junctions following treatment with cLA. This cascade of events may initiate VT and subsequent death during MI. Taken together, individuals at risk of MI should use caution regarding cLA consumption. Full article
(This article belongs to the Section Molecular Biology)
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<p>cLA treatment induces ventricular fibrillation following MI. ECG data from control and cLA-treated mice (<b>A</b>) before MI, (<b>B</b>) 5 min after MI in control mice, and (<b>C</b>) 5 min after MI in cLA-treated mice demonstrate changes in ST segment elevation following LAD. MI surgery in untreated mice (n = 15); MI surgery in cLA-treated mice (n = 35).</p> Full article ">Figure 2
<p>cLA treatment does not affect cardiac dysfunction after 72 h of treatment. Treatment with cLA does not affect cardiac function, FS in (<b>A</b>) control and (<b>B</b>) cLA-treated mice were 53 ± 3 and 51 ± 4, respectively. MI surgery in untreated mice (n = 15); MI surgery in cLA-treated mice (n = 35).</p> Full article ">Figure 3
<p>cLA treatment alters Cx-43 level and increases NOX2 level in heart tissue. cLA decreases levels of Cx-43expression in time point treated mice (<b>A</b>). Conversely, it increases levels of NOX-expression (<b>B</b>). MI surgery in untreated mice (n = 15); MI surgery in cLA-treated mice (n = 35). * <span class="html-italic">p</span> &lt; 0.05 vs. control, # <span class="html-italic">p</span> &lt; 0.05 vs. cLA 12 h.</p> Full article ">Figure 4
<p>cLA exacerbates connexin-43 disruption in mice after 72 h of treatment. Cx-43 is stained in red, in control (<b>A</b>) Cx-43 is represents linear staining, which is disrupted in cLA-treated mice after 72 h of treatment (<b>A</b>). Quantitated protein expression reveals that cLA lowers CX-43 expression in mice treated with cLA after 72 h of treatment (<b>B</b>). MI surgery in untreated mice (n = 15); MI surgery in cLA-treated mice (n = 35).</p> Full article ">Figure 5
<p>cLA alters survival in mice during myocardial infarction (MI). cLA decreases survival during MI. * <span class="html-italic">p</span> &lt; 0.05 vs. control, # <span class="html-italic">p</span> &lt; 0.05 vs. MI. MI surgery in untreated mice results in a cumulative survival of 80%, however mice treated with cLA had a cumulative survival of 20% during MI surgery. Numbers on graph represent the number of animals analyzed in each group.</p> Full article ">Figure 6
<p>cLA treatment increases ROS generation after 72 h of treatment. Immunohistochemistry images demonstrate MitoTracker staining, which measures ROS generation, which was increased in treated mice with cLA after 72 h of treatment (<b>A</b>). Quantitated expression of MitoTracker (<b>B</b>).</p> Full article ">
13 pages, 2348 KiB  
Article
Atomic Layer Deposition of Alumina-Coated Thin-Film Cathodes for Lithium Microbatteries
by Aaron O’Donoghue, Micheál Shine, Ian M. Povey and James F. Rohan
Int. J. Mol. Sci. 2023, 24(13), 11207; https://doi.org/10.3390/ijms241311207 - 7 Jul 2023
Cited by 1 | Viewed by 1739
Abstract
This work shows the electrochemical performance of sputter-deposited, binder-free lithium cobalt oxide thin films with an alumina coating deposited via atomic layer deposition for use in lithium-metal-based microbatteries. The Al2O3 coating can improve the charge–discharge kinetics and suppress the phase [...] Read more.
This work shows the electrochemical performance of sputter-deposited, binder-free lithium cobalt oxide thin films with an alumina coating deposited via atomic layer deposition for use in lithium-metal-based microbatteries. The Al2O3 coating can improve the charge–discharge kinetics and suppress the phase transition that occurs at higher potential limits where the crystalline structure of the lithium cobalt oxide is damaged due to the formation of Co4+, causing irreversible capacity loss. The electrochemical performance of the thin film is analysed by imposing 4.2, 4.4 and 4.5 V upper potential limits, which deliver improved performances for 3 nm of Al2O3, while also highlighting evidence of Al doping. Al2O3-coated lithium cobalt oxide of 3 nm is cycled at 147 µA cm−2 (~2.7 C) to an upper potential limit of 4.4 V with an initial capacity of 132 mAh g−1 (65.7 µAh cm−2 µm−1) and a capacity retention of 87% and 70% at cycle 100 and 400, respectively. This shows the high-rate capability and cycling benefits of a 3 nm Al2O3 coating. Full article
(This article belongs to the Special Issue Material Design and Mechanisms of Lithium-Ion Batteries)
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<p>CV profiles of bare and 3 nm Al<sub>2</sub>O<sub>3</sub>-coated LCO at (<b>a</b>) 0.05 mV s<sup>−1</sup> (cycle 10); (<b>b</b>) 0.2 mV s<sup>−1</sup> (cycle 40); and (<b>c</b>) 0.5 mV s<sup>−1</sup> (cycle 70).</p> Full article ">Figure 2
<p>Discharge profiles of bare, 1, 2 and 3 nm coated Al<sub>2</sub>O<sub>3</sub> at LCO at 63 μA/cm<sup>2</sup>.</p> Full article ">Figure 3
<p>CV profiles at 0.2 mV s<sup>−1</sup> for (<b>a</b>) 1 nm Al<sub>2</sub>O<sub>3</sub>-coated LCO, (<b>b</b>) 2 nm Al<sub>2</sub>O<sub>3</sub>-coated LCO, (<b>c</b>) 3 nm Al<sub>2</sub>O<sub>3</sub>-coated LCO and (<b>d</b>) overlay of cycle 20 for 1–3 nm Al<sub>2</sub>O<sub>3</sub>-coating thickness.</p> Full article ">Figure 4
<p>CV profiles of 3 nm Al<sub>2</sub>O<sub>3</sub>-coated LCO at 0.2 mV s<sup>−1</sup> (<b>a</b>) 1-day air exposure (cycle 1 to 30), (<b>b</b>) 1-day vs. 3-week air exposure (cycle 1), (<b>c</b>) 1-day vs. 3-week air exposure (cycle 10) and (<b>d</b>) 1-day vs. 3-week air exposure (cycle 20).</p> Full article ">Figure 5
<p>(<b>a</b>) Discharge profile of 3 nm Al<sub>2</sub>O<sub>3</sub>-coated LCO and (<b>b</b>) the measured capacity and coulombic efficiency.</p> Full article ">
20 pages, 1265 KiB  
Review
A Cross-Talk about Radioresistance in Lung Cancer—How to Improve Radiosensitivity According to Chinese Medicine and Medicaments That Commonly Occur in Pharmacies
by Paulina Nowak, Iwona Bil-Lula and Mariola Śliwińska-Mossoń
Int. J. Mol. Sci. 2023, 24(13), 11206; https://doi.org/10.3390/ijms241311206 - 7 Jul 2023
Cited by 6 | Viewed by 2430
Abstract
Lung cancer is one of the most common cancers in the population and is characterized by non-specific symptoms that delay the diagnosis and reduce the effectiveness of oncological treatment. Due to the difficult placement of the tumor, one of the main methods of [...] Read more.
Lung cancer is one of the most common cancers in the population and is characterized by non-specific symptoms that delay the diagnosis and reduce the effectiveness of oncological treatment. Due to the difficult placement of the tumor, one of the main methods of lung cancer treatment is radiotherapy, which damages the DNA of cancer cells, inducing their apoptosis. However, resistance to ionizing radiation may develop during radiotherapy cycles, leading to an increase in the number of DNA points of control that protect cells from apoptosis. Cancer stem cells are essential for radioresistance, and due to their ability to undergo epithelial–mesenchymal transition, they modify the phenotype, bypassing the genotoxic effect of radiotherapy. It is therefore necessary to search for new methods that could improve the cytotoxic effect of cells through new mechanisms of action. Chinese medicine, with several thousand years of tradition, offers a wide range of possibilities in the search for compounds that could be used in conventional medicine. This review introduces the potential candidates that may present a radiosensitizing effect on lung cancer cells, breaking their radioresistance. Additionally, it includes candidates taken from conventional medicine—drugs commonly available in pharmacies, which may also be significant candidates. Full article
(This article belongs to the Special Issue Radiation Damage in Biomolecules and Cells 3.0)
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<p>The main mechanisms of radiotherapy response improvement due to active compounds. The figure explains the main mechanisms of TCM and conventional compounds, divided into two sections: cell death and protective properties. All substances reviewed possess pro-apoptotic functions. Metformin, rosiglitazone, lovastatin, and simvastatin act radioprotectively. Tashinone may induce autophagy, whereas oridonine suppresses cancer stemness.</p> Full article ">
15 pages, 2198 KiB  
Review
SLO3: A Conserved Regulator of Sperm Membrane Potential
by Maximilian D. Lyon, Juan J. Ferreira, Ping Li, Shweta Bhagwat, Alice Butler, Kelsey Anderson, Maria Polo and Celia M. Santi
Int. J. Mol. Sci. 2023, 24(13), 11205; https://doi.org/10.3390/ijms241311205 - 7 Jul 2023
Cited by 5 | Viewed by 3254
Abstract
Sperm cells must undergo a complex maturation process after ejaculation to be able to fertilize an egg. One component of this maturation is hyperpolarization of the membrane potential to a more negative value. The ion channel responsible for this hyperpolarization, SLO3, was first [...] Read more.
Sperm cells must undergo a complex maturation process after ejaculation to be able to fertilize an egg. One component of this maturation is hyperpolarization of the membrane potential to a more negative value. The ion channel responsible for this hyperpolarization, SLO3, was first cloned in 1998, and since then much progress has been made to determine how the channel is regulated and how its function intertwines with various signaling pathways involved in sperm maturation. Although Slo3 was originally thought to be present only in the sperm of mammals, recent evidence suggests that a primordial form of the gene is more widely expressed in some fish species. Slo3, like many reproductive genes, is rapidly evolving with low conservation between closely related species and different regulatory and pharmacological profiles. Despite these differences, SLO3 appears to have a conserved role in regulating sperm membrane potential and driving large changes in response to stimuli. The effect of this hyperpolarization of the membrane potential may vary among mammalian species just as the regulation of the channel does. Recent discoveries have elucidated the role of SLO3 in these processes in human sperm and provided tools to target the channel to affect human fertility. Full article
(This article belongs to the Special Issue Recent Advances in the Physiology of Ion Channels in Sperm Cells)
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<p>Amino acid sequence homology of mouse (mSLO3), human (hSLO3), and bovine (bSLO3) SLO3. Conserved regions are highlighted in blue. Dark highlighting indicates conservation between three species, light highlighting indicates conservation between two species. Sequence alignment performed using Jalview Version 2 [<a href="#B47-ijms-24-11205" class="html-bibr">47</a>,<a href="#B48-ijms-24-11205" class="html-bibr">48</a>,<a href="#B49-ijms-24-11205" class="html-bibr">49</a>,<a href="#B50-ijms-24-11205" class="html-bibr">50</a>].</p> Full article ">Figure 2
<p>Human SLO3 gating ring structure determined by X-ray crystallography. (<b>a</b>) Cartoon of domain topology of two opposing SLO3 α-subunits. (<b>b</b>) Crystal structure of the gating ring of a hSLO3 tetramer with RCK1 and RCK2 domains colored in blue and red, respectively. (<b>c</b>) A single subunit of the hSLO3 channel and (<b>d</b>) highlight of RCK1. (<b>e</b>) A closeup of the hSLO3 assembly interface and (<b>f</b>) the corresponding region of SLO1 bound to Ca<sup>2+</sup>. The RCK1 N-terminal residue that connects to the transmembrane pore is shown as a green sphere. Ca<sup>2+</sup> ion is shown as a yellow sphere. Reprinted/adapted with permission from [<a href="#B69-ijms-24-11205" class="html-bibr">69</a>].</p> Full article ">Figure 3
<p>Models of mouse and human SLO3 activity. (<b>a</b>) Mouse: The exposure to a more alkaline pH and high [HCO<sub>3</sub><sup>−</sup>] concentrations in the female tract contribute to an increase in pH<sub>i</sub>, potentially through the activation of the sNHE. This rise in pH<sub>i</sub> leads to the activation of SLO3 channels, resulting in membrane hyperpolarization. This hyperpolarization enhances calcium influx through CatSper channels, possibly through two distinct mechanisms: Firstly, by increasing the inward driving force of calcium. Secondly, it may further activate sNHE to elevate intracellular pH even more. (<b>b</b>) Human: In human sperm, exposure to an elevated external pH could potentially activate the Hv1 channel, resulting in an increase in pH<sub>i</sub> and contributing to the activation of SLO3 and CatSper channels. However, it is important to note that in humans, SLO3 channels are primarily activated by calcium, while CatSper channels are activated by progesterone. On the other hand, activation of SLO3 leads to membrane hyperpolarization, which has been proposed to remove [Ca<sup>2+</sup>]<sub>i</sub> oscillations that inhibit CatSper activation. This raises the question of whether SLO3 is activated upstream or downstream of CatSper channels.</p> Full article ">
16 pages, 5206 KiB  
Article
Exploring the Pathophysiologic Cascade Leading to Osteoclastogenic Activation in Gaucher Disease Monocytes Generated via CRISPR/Cas9 Technology
by Maximiliano Emanuel Ormazabal, Eleonora Pavan, Emilio Vaena, Dania Ferino, Jessica Biasizzo, Juan Marcos Mucci, Fabrizio Serra, Adriana Cifù, Maurizio Scarpa, Paula Adriana Rozenfeld and Andrea Elena Dardis
Int. J. Mol. Sci. 2023, 24(13), 11204; https://doi.org/10.3390/ijms241311204 - 7 Jul 2023
Cited by 1 | Viewed by 2022
Abstract
Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid β-glucosidase gene (GBA1), leading to a deficiency in the β-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features [...] Read more.
Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid β-glucosidase gene (GBA1), leading to a deficiency in the β-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features in GD patients. Although both defective bone formation and increased bone resorption due to osteoblast and osteoclast dysfunction contribute to GD bone pathology, the molecular bases are not fully understood, and bone disease is not completely resolved with currently available specific therapies. For this reason, using editing technology, our group has developed a reliable, isogenic, and easy-to-handle cellular model of GD monocytes (GBAKO-THP1) to facilitate GD pathophysiology studies and high-throughput drug screenings. In this work, we further characterized the model showing an increase in proinflammatory cytokines (Interleukin-1β and Tumor Necrosis Factor-α) release and activation of osteoclastogenesis. Furthermore, our data suggest that GD monocytes would display an increased osteoclastogenic potential, independent of their interaction with the GD microenvironment or other GD cells. Both proinflammatory cytokine production and osteoclastogenesis were restored at least, in part, by treating cells with the recombinant human GCase, a substrate synthase inhibitor, a pharmacological chaperone, and an anti-inflammatory compound. Besides confirming that this model would be suitable to perform high-throughput screening of therapeutic molecules that act via different mechanisms and on different phenotypic features, our data provided insights into the pathogenic cascade, leading to osteoclastogenesis exacerbation and its contribution to bone pathology in GD. Full article
(This article belongs to the Collection Feature Papers in “Molecular Biology”)
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<p>Osteoclastogenic differentiation assay in THP-1 wt and GBAKO-THP1 monocytes. (<b>A</b>) Schematic representation of the osteoclast differentiation assay. After inducing macrophage differentiation by incubating cells for 48 h with PMA, cells were cultured in the presence of M-CSF and RANKL to induce osteoclastogenesis. After 7 days, TRAP staining was used to identify the osteoclast-like cells. (<b>B</b>) TRAP staining (brown) and hematoxylin (purple) staining for osteoclasts and nuclei identification, respectively. Arrows indicate osteoclasts (i.e., TRAP+ cells with 3 or more nuclei); scale bar: 100 µm. (<b>C</b>) Quantification of generated osteoclasts expressed as the percentage of the total number of cells that tested TRAP-positive. Data are shown as mean ± SD of three independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 <span class="html-italic">t</span>-test. Abbreviations: PMA: phorbol-12 myristate-13 acetate; M-CSF: recombinant human macrophage colony-stimulating factor; RANKL: recombinant human sRANK ligand; TRAP: tartrate-resistant acid phosphatase.</p> Full article ">Figure 2
<p>IL-1β and TNF-α levels in the culture supernatant of THP-1 wt and GBAKO-THP1 monocytes. The levels of IL-1β and TNF-α released to the culture media of THP-1 wt and GBAKO-THP1 cells were quantified using a Simple Plex assay (ELLA). Data were normalized via the number of cultured cells and expressed as means ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.1 ** <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">t</span>-test.</p> Full article ">Figure 3
<p>Effect of IL-1 β antagonist on the osteoclastogenic potential of GBAKO-THP1 monocytes. GBAKO-THP1 monocytes were differentiated to osteoclasts in the presence or absence of the IL-1 β receptor antagonist Anakinra. The osteoclasts generated were identified as TRAP-positive cells and quantified. The results were expressed as the percentage of the total number of cells that tested TRAP-positive. Data are shown as mean ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.1 ** <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">t</span>-test.</p> Full article ">Figure 4
<p>Effect of treatments on GCase activity in GBAKO-THP1 monocytes. GBAKO-THP1 cells were treated with 1.6 µM of rhGCase or 100 µM of ABX. After 48 or 96 h, the GCase activity was assessed. Only rhGCase treatment resulted in increased GCase activity, while ABX did not show any effect. Data are expressed as the percentage of the GCase activity detected in wild-type cells and are shown as mean ± SD of three independent experiments. **** <span class="html-italic">p</span> &lt; 0.0001 one-way ANOVA.</p> Full article ">Figure 5
<p>Effect of different treatments on intracellular GlcSph levels of GBAKO-THP1 monocytes. GBAKO-THP1 cells were treated with rhGCase (1.6 µM), D, L-threo-PDMP (as SRT, 20 µM), ABX (100 µM), and PPS (5 μg/mL) for 48 and 96 h. Intracellular GlcSph was measured using LC-MS/MS. rhGCase, SRT, and ABX treatments significantly reduced intracellular GlcSph levels, but PPS had no effect. Data were normalized by the intracellular protein amount and shown as mean ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.1 ** <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 one-way ANOVA.</p> Full article ">Figure 6
<p>Effect of Ambroxol on extracellular GlcSph levels of GBAKO-THP1 monocytes. Cells were treated with 100 µM of ABX for 48 and 96 h, and the GlcSph levels in the culture media were measured using LC-MS/MS. Ambroxol treatment increased GlSph release to the culture media. GlcSph levels were normalized by the number of cultured cells, and data are expressed as mean ± SD of three independent experiments. ** <span class="html-italic">p</span> &lt; 0.01 **** <span class="html-italic">p</span> &lt; 0.0001 one-way ANOVA.</p> Full article ">Figure 7
<p>Effect of the different treatments on GBAKO-THP1 monocyte osteoclastogenic potential. GBAKO-THP1 cells were treated using rhGCase (1.6 µM), D, L-threo-PDMP (as SRT, 20 µM), ABX (100 µM), and PPS (5 μg/mL) throughout the osteoclastogenic differentiation process. TRAP assay was used to identify the osteoclast-like cells obtained after 7 days. (<b>A</b>) Representative TRAP staining (brown) and hematoxylin (purple) staining for osteoclasts and nuclei identification, respectively. Arrows indicate osteoclasts (i.e., TRAP+ cells with 3 or more nuclei); scale bar: 100 µm. (<b>B</b>) All treatments induced a reduction in the number of osteoclast-like cells differentiated from the GBAKO-THP1 cells. The results are expressed as the percentage of total cells that resulted in TRAP-positive at the end of the differentiation protocol. Data are shown as mean ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.1 *** <span class="html-italic">p</span> &lt; 0.001 **** <span class="html-italic">p</span> &lt; 0.0001 one-way ANOVA.</p> Full article ">Figure 7 Cont.
<p>Effect of the different treatments on GBAKO-THP1 monocyte osteoclastogenic potential. GBAKO-THP1 cells were treated using rhGCase (1.6 µM), D, L-threo-PDMP (as SRT, 20 µM), ABX (100 µM), and PPS (5 μg/mL) throughout the osteoclastogenic differentiation process. TRAP assay was used to identify the osteoclast-like cells obtained after 7 days. (<b>A</b>) Representative TRAP staining (brown) and hematoxylin (purple) staining for osteoclasts and nuclei identification, respectively. Arrows indicate osteoclasts (i.e., TRAP+ cells with 3 or more nuclei); scale bar: 100 µm. (<b>B</b>) All treatments induced a reduction in the number of osteoclast-like cells differentiated from the GBAKO-THP1 cells. The results are expressed as the percentage of total cells that resulted in TRAP-positive at the end of the differentiation protocol. Data are shown as mean ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.1 *** <span class="html-italic">p</span> &lt; 0.001 **** <span class="html-italic">p</span> &lt; 0.0001 one-way ANOVA.</p> Full article ">Figure 8
<p>Effect of the different treatments on IL-1β and TNF-α release in GBAKO-THP1 monocytes. GBAKO-THP1 cells were treated with rhGCase (1.6 µM), D, L-threo-PDMP (as SRT, 20 µM), ABX (100 µM), and PPS (5 μg/mL) for 48 h. IL-1β (<b>A</b>) and TNF-α (<b>B</b>) were assessed in the supernatant of cultured cells using Simple Plex assay (ELLA). Results were normalized via the number of cultured cells. Treated GBAKO-THP1 showed decreased levels of one or both cytokines in comparison with untreated GBAKO-THP1. Data are shown as mean ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.1 ** <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 <span class="html-italic">t</span>-test.</p> Full article ">Figure 9
<p>Schematic pathophysiology in the new monocyte GD model.</p> Full article ">
14 pages, 2549 KiB  
Article
The Combined Use of Copper Sulfate and Trichlorfon Exerts Stronger Toxicity on the Liver of Zebrafish
by Jianlu Zhang, Mingzhen Zhu, Qijun Wang and Hui Yang
Int. J. Mol. Sci. 2023, 24(13), 11203; https://doi.org/10.3390/ijms241311203 - 7 Jul 2023
Cited by 6 | Viewed by 1850
Abstract
In aquaculture, copper sulphate and trichlorfon are commonly used as disinfectants and insecticide, sometimes in combination. However, improper use can result in biotoxicity and increased ecological risks. The liver plays a crucial role in detoxification, lipid metabolism, nutrient storage, and immune function in [...] Read more.
In aquaculture, copper sulphate and trichlorfon are commonly used as disinfectants and insecticide, sometimes in combination. However, improper use can result in biotoxicity and increased ecological risks. The liver plays a crucial role in detoxification, lipid metabolism, nutrient storage, and immune function in fish. Selecting the liver as the main target organ for research helps to gain an in-depth understanding of various aspects of fish physiology, health, and adaptability. In the present study, zebrafish were exposed to Cu (0.5 mg/L) and Tri (0.5 mg/L) alone and in combination for 21 days. The results demonstrate that both Cu and Tri caused hepatocyte structure damage in zebrafish after 21 days of exposure, with the combination showing an even greater toxicity. Additionally, the antioxidant and immune enzyme activities in zebrafish liver were significantly induced on both day 7 and day 21. A transcriptome analysis revealed that Cu and Tri, alone and in combination, impacted various physiological activities differently, including metabolism, growth, and immunity. Overall, Cu and Tri, either individually or in combination, can induce tissue damage by generating oxidative stress in the body, and the longer the exposure duration, the stronger the toxic effects. Moreover, the combined exposure to Cu and Tri exhibits enhanced toxicity. This study provides a theoretical foundation for the combined use of heavy metal disinfectants and other drugs. Full article
(This article belongs to the Special Issue Molecular Toxicity of Drugs in Human and Animal Organs)
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<p>Effect of copper sulfate and trichlorfon on the liver of zebrafish on the 21st day. (<b>A</b>–<b>D</b>) Liver tissue paraffin sections; (<b>A</b>) Con; (<b>B</b>) Cu; (<b>C</b>) Tri; (<b>D</b>) Cu + Tri. (<b>E</b>–<b>H</b>) Liver tissue transparent electron microscopy; (<b>E</b>) Con; (<b>F</b>) Cu; (<b>G</b>) Tri; (<b>H</b>,<b>I</b>) Cu + Tri. (<b>J</b>) Relative number of hepatocytes per unit area of the hepatic slice. Bars indicate mean ± SD (n = 3). * indicates <span class="html-italic">p</span> &lt; 0.05. The arrow indicates the gap between hepatocytes. * indicates autophagolysosome; triangle indicates intracellular vacuoles; N: nucleus; M: mitochondria; ER: endoplasmic reticulum.</p> Full article ">Figure 2
<p>Effect of copper sulfate and trichlorfon on antioxidant and immune enzyme activity. (<b>A</b>) Catalase (CAT); (<b>B</b>) superoxide dismutase (SOD); (<b>C</b>) glutathione peroxidase (GPX); (<b>D</b>) alkaline phosphatase (AKP). Con is the control group. Cu is the copper sulfate exposure group. Tri is the trichlorfon exposure group. Cu + Tri is the combination exposure group. Bars indicate mean ± SD (n = 3). * indicates <span class="html-italic">p</span> &lt; 0.05. The term “statistical significance” indicates that there is a significant difference when comparing each group of data to the control group.</p> Full article ">Figure 3
<p>(<b>A</b>) Transcriptome sequencing analysis of number of DEGs in three treatment groups compared to the control. (<b>B</b>–<b>D</b>) Venn analysis for DEGs.</p> Full article ">Figure 4
<p>RT-qPCR verification of nine DEGs. <span class="html-italic">ef1α</span> and <span class="html-italic">actβ</span> were used as the reference genes; bars indicate mean ± SE (n = 3).</p> Full article ">Figure 5
<p>Heatmap analysis (columns represent samples, and rows represent genes. The red region indicates up-regulation of genes, and the blue region indicates down-regulation of genes). (<b>A</b>) Comparison between control and copper sulfate + trichlorfon group. (<b>B</b>) Comparison between control and copper sulfate group. (<b>C</b>) Comparison between control and trichlorfon group.</p> Full article ">Figure 6
<p>Actual Cu<sup>2+</sup> and trichlorfon concentration in water samples. (<b>A</b>) represents the concentration of copper ions. (<b>B</b>) represents the concentration of trichlorfon. The first measurement refers to the initial determination of copper sulfate and trichlorfon concentrations immediately after adding them to the water. The second measurement refers to the determination of concentrations prior to changing the water for the experiment.</p> Full article ">
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