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Volume 22
Issue 6
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Int. J. Mol. Sci., Volume 22, Issue 6 (March-2 2021) – 505 articles

Cover Story (view full-size image): Enteric fever is a major global healthcare issue caused largely by Salmonella enterica serovars Typhi and Paratyphi A. The objective of this study was to develop a novel, bivalent oral vaccine capable of protecting against both serovars. Our approach centred on genetically engineering the attenuated S. Typhi ZH9 strain, to introduce two S. Paratyphi A immunogenic elements: flagellin H:a and lipopolysaccharide (LPS) O:2. The resulting new strain, ZH9PA, incorporated these two genetic changes and exhibited comparable growth kinetics to the parental ZH9 strain. A formulation containing both ZH9 and ZH9PA strains together constitutes a new bivalent vaccine candidate that targets both S. Typhi and S. Paratyphi A antigens to address a major global healthcare gap for enteric fever prophylaxis. View this paper
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20 pages, 3909 KiB  
Review
Transcriptional Regulation of Postnatal Cardiomyocyte Maturation and Regeneration
by Stephanie L. Padula, Nivedhitha Velayutham and Katherine E. Yutzey
Int. J. Mol. Sci. 2021, 22(6), 3288; https://doi.org/10.3390/ijms22063288 - 23 Mar 2021
Cited by 23 | Viewed by 5487
Abstract
During the postnatal period, mammalian cardiomyocytes undergo numerous maturational changes associated with increased cardiac function and output, including hypertrophic growth, cell cycle exit, sarcomeric protein isoform switching, and mitochondrial maturation. These changes come at the expense of loss of regenerative capacity of the [...] Read more.
During the postnatal period, mammalian cardiomyocytes undergo numerous maturational changes associated with increased cardiac function and output, including hypertrophic growth, cell cycle exit, sarcomeric protein isoform switching, and mitochondrial maturation. These changes come at the expense of loss of regenerative capacity of the heart, contributing to heart failure after cardiac injury in adults. While most studies focus on the transcriptional regulation of embryonic or adult cardiomyocytes, the transcriptional changes that occur during the postnatal period are relatively unknown. In this review, we focus on the transcriptional regulators responsible for these aspects of cardiomyocyte maturation during the postnatal period in mammals. By specifically highlighting this transitional period, we draw attention to critical processes in cardiomyocyte maturation with potential therapeutic implications in cardiovascular disease. Full article
(This article belongs to the Special Issue Cardiac Repair and Regeneration: New Molecular Mechanisms and Therapeutics)
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Figure 1

Figure 1
<p>Transcriptional control of nucleation, cell cycling, and hypertrophic growth in embryonic, neonatal, and juvenile/adult rodent cardiomyocytes. (<b>A</b>) Embryonic/fetal cardiomyocytes are primarily mononucleated and proliferate to drive cardiac growth prenatally due to high levels (red) of Tead1, E2f1, Foxm1, Myc. After birth, karyokinesis in the absence of cytokinesis increases as cardiomyocytes mature and become multinucleated. This is concomitant with the downregulation (green) of cell cycle promoting factors such as Tead1, E2f1, Foxm1, Myc; together with the upregulation (red) of cell cycle inhibitory factors such as Foxo1, Meis1, p57, p21. (<b>B</b>) Hypertrophic growth increases after birth with the induction of multinucleation. This is associated with increased levels of T3 thyroid hormone and Meis1 in adult cardiomyocytes. Refer to text for citations.</p> Full article ">Figure 2
<p>Transcriptional control of mitochondrial maturation and sarcomeric protein isoform expression in embryonic, neonatal, and juvenile/adult rodent cardiomyocytes. (<b>A</b>) As cardiomyocytes mature, mitochondria number and size increase, as does the number of cristae. These maturational changes are associated with downregulation (green) of embryonic transcription factors including Hif-1α and Hand1, and upregulation (red) of neonatal/adult transcription factors including PPARs, ERRs, and PGC1α. (<b>B</b>) Embryonic cardiomyocytes express immature Tnni1, Myh7, and Myl7, which are replaced by Tnni3, Myh6 and Myl2 during the postnatal period. Sarcomere number also increases during postnatal maturation and hypertrophic growth. This isoform switching during maturation is associated with downregulation of transcription factors, such as Mef2c and Mef2d, and upregulation of Mef2a and Mef2b. Refer to text for citations.</p> Full article ">
15 pages, 2195 KiB  
Article
Engineering a Novel Bivalent Oral Vaccine against Enteric Fever
by Annelise Soulier, Claudia Prevosto, Mary Chol, Livija Deban and Rocky M. Cranenburgh
Int. J. Mol. Sci. 2021, 22(6), 3287; https://doi.org/10.3390/ijms22063287 - 23 Mar 2021
Cited by 4 | Viewed by 3844
Abstract
Enteric fever is a major global healthcare issue caused largely by Salmonella enterica serovars Typhi and Paratyphi A. The objective of this study was to develop a novel, bivalent oral vaccine capable of protecting against both serovars. Our approach centred on genetically engineering [...] Read more.
Enteric fever is a major global healthcare issue caused largely by Salmonella enterica serovars Typhi and Paratyphi A. The objective of this study was to develop a novel, bivalent oral vaccine capable of protecting against both serovars. Our approach centred on genetically engineering the attenuated S. Typhi ZH9 strain, which has an excellent safety record in clinical trials, to introduce two S. Paratyphi A immunogenic elements: flagellin H:a and lipopolysaccharide (LPS) O:2. We first replaced the native S. Typhi fliC gene encoding flagellin with the highly homologous fliC gene from S. Paratyphi A using Xer-cise technology. Next, we replaced the S. Typhi rfbE gene encoding tyvelose epimerase with a spacer sequence to enable the sustained expression of O:2 LPS and prevent its conversion to O:9 through tyvelose epimerase activity. The resulting new strain, ZH9PA, incorporated these two genetic changes and exhibited comparable growth kinetics to the parental ZH9 strain. A formulation containing both ZH9 and ZH9PA strains together constitutes a new bivalent vaccine candidate that targets both S. Typhi and S. Paratyphi A antigens to address a major global healthcare gap for enteric fever prophylaxis. This vaccine is now being tested in a Phase I clinical trial (NCT04349553). Full article
(This article belongs to the Special Issue Antimicrobial Resistance, Molecular Mechanisms and Fight Strategies)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Replacing the <span class="html-italic">S.</span> Typhi (H:d) flagellin with <span class="html-italic">S.</span> Paratyphi A (H:a) flagellin. (<b>a</b>) The genetic engineering process to generate <span class="html-italic">S.</span> Typhi ZH9 expressing <span class="html-italic">S.</span> Paratyphi A flagellin (ZH9PF). Adapted with permission from Bloor and Cranenburgh, 2006 [<a href="#B23-ijms-22-03287" class="html-bibr">23</a>]. (<b>b</b>) Fluorescence microscopy with <span class="html-italic">S.</span> Typhi ZH9 and the derivative strain, ZH9PF, probed with H:d antiserum (anti-<span class="html-italic">S.</span> Typhi) or H:a antiserum (anti-<span class="html-italic">S.</span> Paratyphi A) plus Dylight 488 secondary antibodies; the left column images are phase contrast images, and the right column images are immuno-fluorescence images. Images were taken at 100× magnification. Scale bars represent 10 µm. Representative images were based on three independent experimental repeats.</p> Full article ">Figure 2
<p>Modifying LPS (O:9) to LPS (O:2). (<b>a</b>) Part of the wild-type O-antigen locus from <span class="html-italic">S.</span> Typhi ZH9 was modified using two test approaches: by deleting the majority of the <span class="html-italic">rfbE</span> cistron to generate <span class="html-italic">S.</span> Typhi ZH9PL2 or by replacing the <span class="html-italic">rfbE</span> cistron with a spacer DNA sequence to maintain the original reading frame to generate <span class="html-italic">S.</span> Typhi ZH9W. (<b>b</b>) Fluorescence microscopy images showing the parental <span class="html-italic">S.</span> Typhi ZH9 and derivative strains, ZH9PL2 and ZH9W, probed with anti-<span class="html-italic">S.</span> Typhi LPS (O:9) or anti-<span class="html-italic">S.</span> Paratyphi A LPS (O:2) monoclonal antibodies followed by Dylight 488 secondary antibodies; the left column images are phase contrast images and the right column images are immuno-fluorescence micrographs. Images were taken at 100× magnification. Scale bars represent 10 µm. Representative images based on three independent experimental repeats. (<b>c</b>) Silver-stained polyacrylamide gel of LPS extracts from the parental <span class="html-italic">S.</span> Typhi ZH9 and derivative strains, ZH9PL2 and ZH9W, indicating the short and long O-antigen chains. LPS = lipopolysaccharide; mAb = monoclonal antibody.</p> Full article ">Figure 3
<p>Converting flagellin and LPS in the final new strain, ZH9PA. (<b>a</b>) Fluorescence microscopy images showing the <span class="html-italic">S.</span> Typhi ZH9 derivative strain, ZH9PA, probed with anti-<span class="html-italic">S</span>. Typhi (H:d) or anti-<span class="html-italic">S</span>. Paratyphi A (H:a) flagellin antiserum and anti-<span class="html-italic">S.</span> Typhi (O:9) or anti-<span class="html-italic">S.</span> Paratyphi A (O:2) LPS mAbs; the left images are phase contrast images and right images are immuno-fluorescence micrographs. Images were taken at 100× magnification. Scale bars represent 10µm. Representative images based on three independent experimental repeats. (<b>b</b>) Western blots of membrane fractions probed with anti-<span class="html-italic">S.</span> Typhi (H:d) or anti-<span class="html-italic">S.</span> Paratyphi A (H:a) flagellin antisera using ZH9 or SPAV as positive controls, respectively. Purified flagellin proteins were also included as a positive control. (<b>c</b>) Dot blot probed with anti-<span class="html-italic">S.</span> Typhi and anti-<span class="html-italic">S.</span> Paratyphi A LPS mAbs. (<b>d</b>) Silver-stained polyacrylamide gel of LPS preparations from <span class="html-italic">S.</span> Typhi ZH9 and derivative strains, ZH9PA, indicating the short and long O-antigen chains. LPS = lipopolysaccharide; mAb = monoclonal antibody; SPAV = attenuated <span class="html-italic">S.</span> Paratyphi A.</p> Full article ">Figure 4
<p>Comparison of growth profiles. Bacteria were seeded into LB broth cultures at OD<sub>600 nm</sub> = 0.1 and grown for 24 h. At regular intervals, samples were taken and analysed by spectrophotometry or by titration on agar plates. Optical density (a measure of growth density) and bacterial titre were plotted for ZH9 (the parental strain) and ZH9PA (the modified strain). The late exponential growth phase (5 to 8 h) is shown in grey. (<b>a</b>) OD<sub>600 nm</sub> and CFU/mL measurements compared within each individual strain. (<b>b</b>) OD<sub>600 nm</sub> or CFU/mL measurements compared between both strains. Statistical comparisons were made using a two-way ANOVA, based on triplicate cultures in a single experiment. CFU = colony-forming units; ml = millilitres; nm = nanometres.</p> Full article ">Figure 5
<p>Anti-LPS IgG antibody responses following <span class="html-italic">in vivo</span> vaccination. (<b>a</b>) Specific IgG antibody responses against <span class="html-italic">S.</span> Typhi LPS (O:9). (<b>b</b>) Specific IgG antibody responses against <span class="html-italic">S.</span> Paratyphi A LPS (O:2). Antibody responses were evaluated by ELISA in Balb/c mouse serum at 35 or 42 days following subcutaneous vaccination with 10<sup>8</sup> CFU ZH9 (•), 10<sup>8</sup> CFU ZH9PA (♦) or a 1:1 mix of 0.5 × 10<sup>8</sup> CFU of ZH9 and 0.5 × 10<sup>8</sup> CFU of ZH9PA (Entervax™ basic formulation (▲)). Pre-vaccination (d0) samples were pooled across individual mice to generate the negative assay control (dotted line). Each data point represents an individual mouse, and data were pooled across three independent experiments. Mean values are represented by the horizontal bar. Statistical comparisons were made using a one-way ANOVA. ELISA = enzyme-linked immunosorbent assay; EPT = end point titre; IgG = immunoglobulin G; LPS = lipopolysaccharide; OD = optical density.</p> Full article ">
19 pages, 3301 KiB  
Article
Late Health Effects of Partial Body Irradiation Injury in a Minipig Model Are Associated with Changes in Systemic and Cardiac IGF-1 Signaling
by Bernadette Hritzo, Saeed Y. Aghdam, Betre Legesse, Amandeep Kaur, Maohua Cao, Marjan Boerma, Nabarun Chakraborty, George Dimitrov, Aarti Gautam, Rasha Hammamieh, William Wilkins, Alena Tsioplaya, Gregory P. Holmes-Hampton and Maria Moroni
Int. J. Mol. Sci. 2021, 22(6), 3286; https://doi.org/10.3390/ijms22063286 - 23 Mar 2021
Cited by 6 | Viewed by 3317
Abstract
Clinical, epidemiological, and experimental evidence demonstrate non-cancer, cardiovascular, and endocrine effects of ionizing radiation exposure including growth hormone deficiency, obesity, metabolic syndrome, diabetes, and hyperinsulinemia. Insulin-like growth factor-1 (IGF-1) signaling perturbations are implicated in development of cardiovascular disease and metabolic syndrome. The minipig [...] Read more.
Clinical, epidemiological, and experimental evidence demonstrate non-cancer, cardiovascular, and endocrine effects of ionizing radiation exposure including growth hormone deficiency, obesity, metabolic syndrome, diabetes, and hyperinsulinemia. Insulin-like growth factor-1 (IGF-1) signaling perturbations are implicated in development of cardiovascular disease and metabolic syndrome. The minipig is an emerging model for studying radiation effects given its high analogy to human anatomy and physiology. Here we use a minipig model to study late health effects of radiation by exposing male Göttingen minipigs to 1.9–2.0 Gy X-rays (lower limb tibias spared). Animals were monitored for 120 days following irradiation and blood counts, body weight, heart rate, clinical chemistry parameters, and circulating biomarkers were assessed longitudinally. Collagen deposition, histolopathology, IGF-1 signaling, and mRNA sequencing were evaluated in tissues. Our findings indicate a single exposure induced histopathological changes, attenuated circulating IGF-1, and disrupted cardiac IGF-1 signaling. Electrolytes, lipid profiles, liver and kidney markers, and heart rate and rhythm were also affected. In the heart, collagen deposition was significantly increased and transforming growth factor beta-1 (TGF-beta-1) was induced following irradiation; collagen deposition and fibrosis were also observed in the kidney of irradiated animals. Our findings show Göttingen minipigs are a suitable large animal model to study long-term effects of radiation exposure and radiation-induced inhibition of IGF-1 signaling may play a role in development of late organ injuries. Full article
(This article belongs to the Section Molecular Biology)
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Figure 1

Figure 1
<p>Evaluation of lymphocytes, white blood cells (WBCs), absolute neutrophil counts (ANCs), platelets, red blood cells (RBCs) and C-reactive protein (CRP) levels in irradiated animals. (<b>A</b>) Lymphocyte, (<b>B</b>) WBC, (<b>C</b>) ANC, (<b>D</b>) platelet, and (<b>E</b>) RBC counts were determined longitudinally in whole blood and CRP levels (<b>F</b>) were measured by ELISA in plasma collected throughout the study; values are reported as average +/− standard error of the mean (sem). Dashed line marks the threshold of severe cytopenia (0.5 × 10<sup>3</sup>/μL for neutrophils and 20 × 10<sup>3</sup>/μL for platelets).</p> Full article ">Figure 2
<p>Evaluation of body weight and heart rate in irradiated animals. (<b>A</b>) Body weight and (<b>B</b>) heart rate were measured throughout the study in minipigs exposed to PBI; heart rates were reported as averages +/− sem (<b>B</b>). Body weight measurements were compared to values from reference age-matched, non-irradiated animals provided by the vendor and plotted as a control (standard weight).</p> Full article ">Figure 3
<p>Analysis of fibrosis markers in heart and kidney samples of control and irradiated minipigs. (<b>A</b>) Histological analysis of collagen deposition in heart left ventricle (left) and kidney (right) sections using Masson’s Trichrome staining (blue staining). (<b>B</b>) Collagen deposition in the left ventricle was quantified using Sirius Red (red staining) supplemented with Fast Green to visualize cytoplasm; representative photomicrographs (20× magnification; 100 μm scale bar) are shown. (<b>C</b>) The percentage of left ventricular area occupied by collagen (field of view) was measured and averages are reported +/− sem (control: <span class="html-italic">n</span> = 3; PBI: <span class="html-italic">n</span> = 7). (<b>D</b>) TGF-beta-1 expression was assessed by western blot analysis in heart lysates of irradiated and control animals; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (<b>E</b>) Densitometric quantification was performed and values were normalized to GAPDH and presented as averages +/− sem.</p> Full article ">Figure 4
<p>Analysis of plasma IGF-1 levels in irradiated animals by ELISA. (<b>A</b>) Values are shown as averages +/− sem over time. (<b>B</b>) Data from individual animals were normalized to pre-irradiation levels and grouped by radiation dose; 1.9 Gy (<span class="html-italic">n</span> = 4 animals, individual measurements for a given animal are represented by data points of a single color: dark green, blue, green, or yellow) and (<b>C</b>) 2.0 Gy (<span class="html-italic">n</span> = 3 animals, individual measurements for a given animal are represented by data points of a single color: blue, green, or yellow).</p> Full article ">Figure 5
<p>Longitudinal analysis of clinical chemistry parameters in the plasma of irradiated minipigs. (<b>A</b>–<b>L</b>) Reference values for each parameter were obtained from the vendor from non-irradiated, age-matched animals and are represented by the broken red line; the solid black line marks the pre-irradiation level of each parameter. Values are reported over time as averages +/− sem.</p> Full article ">Figure 6
<p>Longitudinal analysis of plasma lipid profiles in irradiated animals. Cholesterol, HDL, LDL, and triglyceride levels from individual animals were normalized to pre-irradiation levels and plotted over time. The grey box represents the pre-irradiation value measured for each parameter.</p> Full article ">Figure 7
<p>Western blot analysis of IGF-1 signaling pathway and PPAR activation in heart lysates of irradiated and control minipigs. (<b>A</b>) Evaluation of IGF-1R, Akt, p44/42 and PPAR phosphorylation in heart lysates of irradiated and control minipigs by SDS-PAGE and western blot. (<b>B</b>–<b>E</b>) Densitometric quantification was performed and values were normalized to respective loading control and presented as averages +/− sem.</p> Full article ">Figure 8
<p>Schematic depiction of RNAseq analysis showing the key signaling nodes affected by irradiation in the hearts of minipigs. By mRNA sequencing and analysis, IGF-1 (<b>A</b>), TGF-beta (<b>B</b>), and PPAR-gamma (<b>C</b>) were identified as predicted upstream regulators in the heart of irradiated minipigs per z-score calculation. The oval and rectangular nodes represent candidate genes and corresponding regulators, respectively. The arrow-headed edges represent the relationships between the two nodes, and the red and green colors denote up- and down-regulated nodes, respectively.</p> Full article ">
18 pages, 32313 KiB  
Article
Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15
by Igor Ivanov, Alejandro Cruz, Alexander Zhuravlev, Almerinda Di Venere, Eleonora Nicolai, Sabine Stehling, José M. Lluch, Àngels González-Lafont and Hartmut Kuhn
Int. J. Mol. Sci. 2021, 22(6), 3285; https://doi.org/10.3390/ijms22063285 - 23 Mar 2021
Cited by 5 | Viewed by 2626
Abstract
Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit [...] Read more.
Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties. Full article
(This article belongs to the Special Issue Structural, Functional and Folding Strategies of Oligomeric Proteins)
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Figure 1

Figure 1
<p>(<b>A</b>) Overlay of crystal structures of rabbit ALOX15 (PDB entry 2P0M, conformer A, grey) and human ALOX15B (PDB entry 4NRE, brown). (<b>B</b>) Overlay of crystal structures of rabbit ALOX15 (PDB entry 2P0M, conformer A, grey) and the AA 12-lipoxygenating porcine ALOX15 catalytic domain (PDB entry 3RDE, brown). (<b>C</b>) Structural heterogeneity of rabbit ALOX15 and human ALOX15 B in salt-free aqueous solutions. (C-I) Native polyacrylamide gel electrophoresis (PAGE). For this analysis, the enzymes were prepared and maintained in 20 mM Tris-HCl buffer, pH 8.0 containing 130 mM NaCl. (C-II) Native PAGE. For this analysis, the enzyme was prepared and maintained in 20 mM Tris-HCl buffer, pH 8.0 that does not contain NaCl (desalted enzyme preparation eluted as a single peak by and size-exclusion chromatography). (C-III) Denaturing PAGE. For this analysis the enzyme was prepared and maintained in 20 mM Tris-HCl buffer, pH 8.0, but the running buffer contained 0.5% SDS.</p> Full article ">Figure 2
<p>Structural characterization of wild-type and mutant rabbit ALOX15. (<b>A</b>) Native PAGE electrophoresis of rabbit ALOX15 and its catalytic domain. (<b>B</b>) Effect of pH on structural heterogeneity of ALOX15. At pH 8.0 the two conformers (red labelled enzyme pools I and II) are better resolved when compared with pH 6.8. (<b>C</b>) Crystal structure of conformer A (no ligand at the active site). In this structure the side chain of His585 (H585) is localized on the protein surface and is accessible to the solvent. (<b>D</b>) Crystal structure of conformer B (ligand bound at the active site). Here the side chain of His585 (H585) is buried inside the protein between the side chains of Glu185 (E585) and Lys189 (K189). It is shielded from the solvent. (<b>E</b>) Elution order of the purified ALOX15 variants from the Resource Q (6 mL) column in anion exchange chromatography using a linear NaCl gradient. Absorbance of the column effluent at 280 nm (green, black, blue) and conductivity (red curve) were simultaneously recorded. (<b>F</b>) Size exclusion chromatography (left panel) of the purified recombinant proteins was carried in 20 mM Tris-HCl buffer and we estimated their hydrodynamic radii using different calibration proteins (right panel). The elution volume of the wild-type ALOX15 and the mutants is labelled with asterisk. (<b>G</b>) Native PAGE of rabbit ALOX15 mutants was performed at two different pH as described above for the wild-type ALOX15. (<b>H</b>) SDS-PAGE of the enzyme preparations (the numbers correspond to the protein samples that are present on panels B and G). (<b>I</b>) CD spectra of wild-type and mutant rabbit ALOX15. (<b>J</b>) Thermal stability of wild-type rabbit ALOX15 and its His585Glu (H585E) mutant. The relative intensity of the CD signal of ALOX solution was measured at 220 nm at different temperatures. (<b>K</b>) Fluorescence spectra of wild-type and mutant rabbit ALOX15. (<b>L</b>) The shift of the maximum of the fluorescence spectrum in the presence of different concentrations of GdnHCl was monitored by fluorescence steady state spectroscopy for wild-type rabbit ALOX15 and its His585Glu (H585E) mutant. The GdnHCl denaturation curves follow a 3-state transition model (ground state M ↔ intermediate sate I ↔ unfolded state U).</p> Full article ">Figure 3
<p>Contribution of Trp181 (W181) and His585 (H585) to the inter-monomer interface of rabbit ALOX15 and impact of mutations on the reaction kinetics of the enzyme. (<b>A</b>) Crystal structure of the rabbit ALOX15 heterodimer (PDB entry 2P0M) consisting of a ligand-free conformer A (brown) and a ligand-bound conformer B (grey). Inset: Amino acid residues contributing to the inter-monomer interface. (<b>B</b>) The two structures of the rabbit ALOX15. α2 Helix of conformer A (α2A) is strongly dislocated when compared with conformer B (α2B). (<b>C</b>) Reaction kinetics of wild-type rabbit ALOX15 and of two enzyme mutants. Linoleic (left panel) or arachidonic acid (right panel) oxygenation was assayed spectrophotometrically (increase in absorbance at 235 nm) at different substrate concentrations. For each measurement 56 nM LOX (final enzyme concentration) normalized to the iron content was used.</p> Full article ">Figure 4
<p>Structural consequences of Trp181Glu exchange on dimer formation and substrate alignment. (<b>A</b>) Inter-monomer interface of wild-type rabbit ALOX15. Secondary structural elements of conformer A are shown in mustard and those of conformer B are given in green. (<b>B</b>). Inter-monomer interface of the Trp181Glu mutant of rabbit ALOX15. Secondary structural elements of conformer A are shown in light blue and those of conformer B in purple. (<b>C</b>) Most representative binding mode of AA in WT-ALOX15 (dark green) and in Trp181Glu-ALOX15 (purple) dimers. WT-ALOX15 (green with a percentage of transparency) and Trp181Glu-ALOX15 (light blue with a percentage of transparency) backbones have been superimposed. The side-chains of some selected residues for WT-ALOX15 and Trp181Glu-ALOX15 dimers have been displayed in green and light blue, respectively. (<b>D</b>) Most representative binding mode of LA in WT-ALOX15 (dark green) and in Trp181Glu-ALOX15 (purple) dimers. WT-ALOX15 (green with a percentage of transparency) and Trp181Glu-ALOX15 (light blue with a percentage of transparency) backbones have been superimposed. The side-chains of some selected residues for WT-ALOX15 and Trp181Glu-ALOX15 dimers have been displayed in green and light blue, respectively. (<b>E</b>) C<sub>13</sub>-OH distance in the Trp181Glu-ALOX15-AA (in blue) and in the WT-ALOX15-AA (in green) complexes versus time. (<b>F</b>) C<sub>11</sub>-OH distance in the Trp181Glu-ALOX15-LA (in blue) and in the WT-ALOX15-LA (in green) complexes versus time.</p> Full article ">
14 pages, 2343 KiB  
Article
The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma
by Eugene Choi, Sung Jean Park, Gunhee Lee, Seung Kew Yoon, Minho Lee and Suk Kyeong Lee
Int. J. Mol. Sci. 2021, 22(6), 3284; https://doi.org/10.3390/ijms22063284 - 23 Mar 2021
Cited by 5 | Viewed by 3405
Abstract
Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using [...] Read more.
Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway. Full article
(This article belongs to the Special Issue Molecular Advances in Cancer Genetics)
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Figure 1
<p><span class="html-italic">GNAQ</span> T96S mutation. (<b>A</b>) Four hotspot mutations mapped in a schematic diagram of the GNAQ protein sequence (amino acids 1 to 359). Helical domain (beige), GTPase domain (brown), switch regions 1–3 (gray), and hotspot mutations (red triangles) are indicated. (<b>B</b>) The GNAQ sequence in the SK-Hep-1 cell line. The GNAQ sequence of SK-Hep-1 was analyzed and compared to the wild-type GNAQ reference sequence from the National Center for Biotechnology Information (NCBI; NM_002072.5). (<b>C</b>) Overexpression of the wild-type or the T96S mutant GNAQ. The control vector, pcGNAQ, or pcGNAQ T96S was transfected into SK-Hep-1 cells. After 24 h, a Western blot was performed to analyze the level of GNAQ protein using anti-Flag (left) or anti-GNAQ (right) antibodies in SK-Hep-1. Anti-α-tubulin antibody was used for normalization.</p> Full article ">Figure 2
<p>Effect of the GNAQ T96S mutation on cell proliferation, anchorage-independent growth, and migration. SK-Hep-1 cells were transfected with the control vector, pcGNAQ, or pcGNAQ T96S. (<b>A</b>) Effect of the GNAQ T96S mutation on cell growth. To measure SK-Hep-1 cell proliferation, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well immediately after transfection and every 24 h after transfection for three days. Absorbance at 540 nm was analyzed via SoftMax apparatus. Error bars indicate SD (<span class="html-italic">n</span> = 3). (<b>B</b>) Effect of the GNAQ T96S mutation on anchorage-independent cell growth. The cells were harvested 24 h after transfection and seeded in agar. Cells were cultured for three weeks in a 37 °C CO<sub>2</sub> incubator and then observed. Upper panels show pictures taken with a camera. Lower panels show images observed with microscope IX70 through a ×40 objective (left). Similar experiments were carried out three times independently. Each value represents the mean SD of all three experiments (right). (<b>C</b>) Transwell migration assay. The cells were harvested 24 h after transfection and seeded in the upper chamber. After 48 h, the migrated cells were stained and observed with a microscope IX70 through a ×200 objective (left). Similar experiments were carried out three times independently. Each value represents the mean SD of all three experiments (right).</p> Full article ">Figure 3
<p>Increased ERK signaling due to GNAQ T96S mutation. SK-Hep-1 cells were transfected with the control vector, pcGNAQ, or pcGNAQ T96S. Western blot was carried out using three independently transfected cell sets to detect total and phosphorylated forms of ERK. Anti-ERK (1:500), anti-pERK (1:500), and anti-GNAQ (1:500) antibodies were used. Comparable loading amounts were confirmed by detection with anti-α-tubulin (1:2000) antibody. Phosphorylated ERK levels were normalized by the total ERK levels in the plot shown at the lower panel.</p> Full article ">Figure 4
<p>Screening for the GNAQ T96S (rs753716491) mutation in 456 Korean patients. A single nucleotide polymorphism (SNP) assay was performed to determine the frequency of the GNAQ T96S mutation in Korean liver cancer patients. The cancer tissues of 456 Korean liver cancer patients were collected, and DNA was extracted. DNA samples were genotyped with an rs753716491 probe in the Taqman real-time polymerase chain reaction (PCR) system.</p> Full article ">Figure 5
<p>The complex structure (PDB ID: 4EKD) of GNAQ (tan) and RGS2 (green). The regions of Switches I–III are depicted in pink. Helix 2 of GNAQ is colored in orange. The dotted rectangles in the center are enlarged in the left or right panels. The left panel shows the hydrogen bonding network between the helical domain of GNAQ and RGS2. The identification of hydrogen bonding was calculated using the software University of California, San Francisco (UCSF) Chimera. The right panel shows the hydrophobic network between helix 2 and helix 4 of GNAQ. The side chain of T96 is depicted in red. The side-chain atoms of Tyr151 are shown in cyan. The solid-line arrows represent the distances between the methyl protons of T96 and the other atoms. The dotted line arrows show the distance between the side chain of Tyr151 and the side chain of Ala93 and Leu97. The distance unit, Å, is removed for clear presentation.</p> Full article ">
15 pages, 1125 KiB  
Review
Recent Advances Clarifying the Structure and Function of Plant Apyrases (Nucleoside Triphosphate Diphosphohydrolases)
by Greg Clark, Katherine A. Brown, Manas K. Tripathy and Stanley J. Roux
Int. J. Mol. Sci. 2021, 22(6), 3283; https://doi.org/10.3390/ijms22063283 - 23 Mar 2021
Cited by 16 | Viewed by 2953
Abstract
Studies implicating an important role for apyrase (NTPDase) enzymes in plant growth and development began appearing in the literature more than three decades ago. After early studies primarily in potato, Arabidopsis and legumes, especially important discoveries that advanced an understanding of the biochemistry, [...] Read more.
Studies implicating an important role for apyrase (NTPDase) enzymes in plant growth and development began appearing in the literature more than three decades ago. After early studies primarily in potato, Arabidopsis and legumes, especially important discoveries that advanced an understanding of the biochemistry, structure and function of these enzymes have been published in the last half-dozen years, revealing that they carry out key functions in diverse other plants. These recent discoveries about plant apyrases include, among others, novel findings on its crystal structures, its biochemistry, its roles in plant stress responses and its induction of major changes in gene expression when its expression is suppressed or enhanced. This review will describe and discuss these recent advances and the major questions about plant apyrases that remain unanswered. Full article
(This article belongs to the Special Issue New Horizons in Plant Cell Signaling)
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Graphical abstract

Graphical abstract
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<p>Models of selected plant APYs. Homology models of the potato (<b>A</b>) and pea PsNTP9 (<b>B</b>) apyrases are shown as ribbons colored with a rainbow spectrum. Both models are aligned with the crystal structure of the <span class="html-italic">V. unguiculata</span> APY crystal structure [<a href="#B41-ijms-22-03283" class="html-bibr">41</a>] show as a purple thread. Models and structural alignment were produced using the I-TASSER server [<a href="#B45-ijms-22-03283" class="html-bibr">45</a>,<a href="#B46-ijms-22-03283" class="html-bibr">46</a>,<a href="#B47-ijms-22-03283" class="html-bibr">47</a>].</p> Full article ">Figure 2
<p>Active site of <span class="html-italic">T. repens</span> APY in complex with AMP. Residues in the active site and AMP (labeled [AMP]501) are shown in ball-and-stick representation (white bonds, residues; gray bonds, AMP). Interactions between residues and AMP are shown as green dashed lines within a rendered molecular surface. The adenine base of AMP is shown sandwiched between Y303 and F360 stabilized by π-stacking interactions. Residue D307 is also displayed. The area occupied by this residue in the active site is postulated to influence substrate affinities and specificities (see text and ref. [<a href="#B41-ijms-22-03283" class="html-bibr">41</a>]). This image was created using coordinates from RSCB PDB entry 5U7V [<a href="#B41-ijms-22-03283" class="html-bibr">41</a>] with the NGL viewer [<a href="#B52-ijms-22-03283" class="html-bibr">52</a>].</p> Full article ">Figure 3
<p>Sequence alignments of the N-terminal regions of plant APYs. The multisequence alignment was generated using Clustal Omega [<a href="#B55-ijms-22-03283" class="html-bibr">55</a>]. TOPCONS webserver [<a href="#B54-ijms-22-03283" class="html-bibr">54</a>] predictions are shown in bold. The predicted single-pass outward-facing transmembrane helix in the AtAPY1 sequence is highlighted in green. Bold red indicates predicted secretion peptide sequences. Relative positions of these N-terminal sequences to the ACR1 sequence motif (green box) are shown, the latter of which is present in the globular portion of all homology models and crystal structures.</p> Full article ">
13 pages, 2922 KiB  
Article
Anti-Platelet Properties of Phenolic and Nonpolar Fractions Isolated from Various Organs of Elaeagnus rhamnoides (L.) A. Nelson in Whole Blood
by Bartosz Skalski, Joanna Rywaniak, Aleksandra Szustka, Jerzy Żuchowski, Anna Stochmal and Beata Olas
Int. J. Mol. Sci. 2021, 22(6), 3282; https://doi.org/10.3390/ijms22063282 - 23 Mar 2021
Cited by 11 | Viewed by 2390
Abstract
Sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) is a shrub growing in coastal areas. Its organs contain a range of bioactive substances including vitamins, fatty acids, various micro and macro elements, as well as phenolic compounds. Numerous studies of sea buckthorn have [...] Read more.
Sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) is a shrub growing in coastal areas. Its organs contain a range of bioactive substances including vitamins, fatty acids, various micro and macro elements, as well as phenolic compounds. Numerous studies of sea buckthorn have found it to have anticancer, anti-ulcer, hepatoprotective, antibacterial, and antiviral properties. Some studies suggest that it also affects the hemostasis system. The aim of the study was to determine the effect of six polyphenols rich and triterpenic acids rich fractions (A–F), taken from various organs of sea buckthorn, on the activation of blood platelets using whole blood, and to assess the effect of the tested fractions on platelet proteins: fraction A (polyphenols rich fraction from fruits), fraction B (triterpenic acids rich fraction from fruits), fraction C (polyphenols rich fraction from leaves), fraction D (triterpenic acids rich fraction from leaves), fraction E (polyphenols rich fraction from twigs), and fraction F (triterpenic acids rich fraction from twigs). Hemostasis parameters were determined using flow cytometry and T-TAS (Total Thrombus-formation Analysis System). Additionally, electrophoresis was performed under reducing and non-reducing conditions. Although all tested fractions inhibit platelet activation, the greatest anti-platelet activity was demonstrated by fraction A, which was rich in flavonol glycosides. In addition, none of the tested fractions (A–F) caused any changes in the platelet proteome, and their anti-platelet potential is not dependent on the P2Y12 receptor. Full article
(This article belongs to the Special Issue Flavonoids)
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<p>Effects of different plant fractions (5 and 50 µg/mL; 30 min) on expression of P-selectin on resting (<b>A</b>) or agonist-stimulated blood platelets: 10 µM ADP (adenosine diphosphate) (<b>B</b>), 20 µM ADP (<b>C</b>), and 10 µg/mL collagen (<b>D</b>) in whole blood samples. The blood platelets were distinguished based on the expression of CD61/PerCP. For each sample, 10,000 CD61-positive objects (blood platelets) were acquired. For the assessment of P-selectin expression, samples were labeled with fluorescently conjugated monoclonal antibody CD62P. Results are shown as the percentage of platelets expressing CD62P. Data represent mean ± SD of 6 healthy volunteers (each experiment performed in triplicate). * <span class="html-italic">p</span> &lt; 0.05 (vs. control platelets–blood platelets without tested fraction).</p> Full article ">Figure 1 Cont.
<p>Effects of different plant fractions (5 and 50 µg/mL; 30 min) on expression of P-selectin on resting (<b>A</b>) or agonist-stimulated blood platelets: 10 µM ADP (adenosine diphosphate) (<b>B</b>), 20 µM ADP (<b>C</b>), and 10 µg/mL collagen (<b>D</b>) in whole blood samples. The blood platelets were distinguished based on the expression of CD61/PerCP. For each sample, 10,000 CD61-positive objects (blood platelets) were acquired. For the assessment of P-selectin expression, samples were labeled with fluorescently conjugated monoclonal antibody CD62P. Results are shown as the percentage of platelets expressing CD62P. Data represent mean ± SD of 6 healthy volunteers (each experiment performed in triplicate). * <span class="html-italic">p</span> &lt; 0.05 (vs. control platelets–blood platelets without tested fraction).</p> Full article ">Figure 2
<p>Effects of different plant fractions (5 and 50 µg/mL; 30 min) on expression of the active form of GPIIb/IIIa on resting (<b>A</b>) or agonist-stimulated blood platelets: 10 µM ADP (<b>B</b>), 20 µM ADP (<b>C</b>), and 10 µg/mL collagen (<b>D</b>) in whole blood samples. The blood platelets were distinguished based on the expression of CD61. For each sample, 10,000 CD61-positive objects (blood platelets) were acquired. For the assessment of GPIIb/IIIa expression, samples were labeled with fluorescently conjugated monoclonal antibody PAC-1/FITC. Results are shown as the percentage of platelets binding PAC-1/FITC. Data represent mean ± SD of 6 healthy volunteers (each experiment performed in triplicate). * <span class="html-italic">p</span> &lt; 0.05 (vs. control platelets–blood platelets without tested fraction).</p> Full article ">Figure 2 Cont.
<p>Effects of different plant fractions (5 and 50 µg/mL; 30 min) on expression of the active form of GPIIb/IIIa on resting (<b>A</b>) or agonist-stimulated blood platelets: 10 µM ADP (<b>B</b>), 20 µM ADP (<b>C</b>), and 10 µg/mL collagen (<b>D</b>) in whole blood samples. The blood platelets were distinguished based on the expression of CD61. For each sample, 10,000 CD61-positive objects (blood platelets) were acquired. For the assessment of GPIIb/IIIa expression, samples were labeled with fluorescently conjugated monoclonal antibody PAC-1/FITC. Results are shown as the percentage of platelets binding PAC-1/FITC. Data represent mean ± SD of 6 healthy volunteers (each experiment performed in triplicate). * <span class="html-italic">p</span> &lt; 0.05 (vs. control platelets–blood platelets without tested fraction).</p> Full article ">Figure 3
<p>Effects of fraction A (concentration 5 and 50 µg/mL, incubation time—30 min) on the expression of P-selectin and the active form of GPIIb/IIIa on platelets stimulated by 10 µg/mL collagen in whole blood samples. Figure demonstrates selected diagrams.</p> Full article ">Figure 4
<p>Electrophoretic patterns of blood platelet proteome in the presence of different plant fractions (50 µg/mL; 30 min): reducing conditions (<b>a</b>), non-reducing conditions (<b>b</b>).</p> Full article ">Figure 5
<p>Effects of different plant fractions (50 µg/mL; 30 min) on vasodilator-stimulated phosphoprotein (VASP) phosphorylation in ADP—activated blood platelets. Data represent mean ± SD of 6 healthy volunteers (each experiment performed in triplicate).</p> Full article ">Figure 6
<p>Effects of different plant fractions (50 µg/mL; 30 min) on the T-TAS (Total Thrombus formation Analysis system) using the PL-chip (chip for analysis of platelet thrombus formation (primary hemostatic ability)) in whole blood samples (<b>a</b>). Whole blood samples were analyzed by the T-TAS at the shear rates of 1000 s<sup>−1</sup> on the PL-chips. Area under the curve (AUC<sub>10</sub>) in PL are shown as closed circles. Data represent mean ± SD of 6 healthy volunteers (each experiment performed in triplicate). * <span class="html-italic">p</span> &lt; 0.05 (vs. control sample–whole blood without tested fraction). <a href="#ijms-22-03282-f006" class="html-fig">Figure 6</a> (<b>b</b>) demonstrates selected diagram for fraction A.</p> Full article ">
16 pages, 2980 KiB  
Article
Loss of BOK Has a Minor Impact on Acetaminophen Overdose-Induced Liver Damage in Mice
by Samara Naim, Yuniel Fernandez-Marrero, Simone de Brot, Daniel Bachmann and Thomas Kaufmann
Int. J. Mol. Sci. 2021, 22(6), 3281; https://doi.org/10.3390/ijms22063281 - 23 Mar 2021
Cited by 2 | Viewed by 2847
Abstract
Acetaminophen (APAP) is one of the most commonly used analgesic and anti-pyretic drugs, and APAP intoxication is one of the main reasons for liver transplantation following liver failure in the Western world. While APAP poisoning ultimately leads to liver necrosis, various programmed cell [...] Read more.
Acetaminophen (APAP) is one of the most commonly used analgesic and anti-pyretic drugs, and APAP intoxication is one of the main reasons for liver transplantation following liver failure in the Western world. While APAP poisoning ultimately leads to liver necrosis, various programmed cell death modalities have been implicated, including ER stress-triggered apoptosis. The BCL-2 family member BOK (BCL-2-related ovarian killer) has been described to modulate the unfolded protein response and to promote chemical-induced liver injury. We therefore investigated the impact of the loss of BOK following APAP overdosing in mice. Surprisingly, we observed sex-dependent differences in the activation of the unfolded protein response (UPR) in both wildtype (WT) and Bok-/- mice, with increased activation of JNK in females compared with males. Loss of BOK led to a decrease in JNK activation and a reduced percentage of centrilobular necrosis in both sexes after APAP treatment; however, this protection was more pronounced in Bok-/- females. Nevertheless, serum ALT and AST levels of Bok-/- and WT mice were comparable, indicating that there was no major difference in the overall outcome of liver injury. We conclude that after APAP overdosing, loss of BOK affects initiating signaling steps linked to ER stress, but has a more minor impact on the outcome of liver necrosis. Furthermore, we observed sex-dependent differences that might be worthwhile to investigate. Full article
(This article belongs to the Section Molecular Pharmacology)
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<p>CYP2E1 mRNA but not protein expression is increased in <span class="html-italic">Bok<sup>-/-</sup></span> males at baseline. qPCR analysis revealed increased expression of CYP2E1 and CYP1A2 in primary hepatocytes derived from <span class="html-italic">Bok<sup>-/-</sup></span> male mice compared with wildtype (WT) (<b>A</b>,<b>B</b>). Expression of CYP2E1, but not CYP2A1 was significantly increased in whole liver lysates derived from <span class="html-italic">Bok<sup>-/-</sup></span> males compared with WT (<b>C</b>,<b>D</b>). In whole liver lysates of female WT and <span class="html-italic">Bok<sup>-/-</sup></span> mice CYP2E1 (<b>E</b>) and CYP1A2 (<b>F</b>) were expressed at similar levels. CYP2E1 protein levels were comparable in whole liver lysates of untreated WT and <span class="html-italic">Bok<sup>-/-</sup></span> mice (<b>G</b>,<b>H</b>). Results are depicted as a fold change compared to sex-matched WT (<b>A</b>–<b>F</b>) or as GAPDH normalized values (<b>H</b>). Data are represented as mean ± S.D. and are derived from 3 mice per group. *: <span class="html-italic">p</span> &lt; 0.05; n.s.: not significant.</p> Full article ">Figure 2
<p>P53 expression correlates with PUMA expression but not with the severity of liver damage. qPCR analysis showed that livers from WT and <span class="html-italic">Bok<sup>-/-</sup></span> mice expressed p53 at similar levels (<b>A</b>) and that PUMA was induced in WT and <span class="html-italic">Bok<sup>-/-</sup></span> mice after APAP with higher induction observed in WT mice (<b>B</b>). Expression of PUMA and p53 correlated in males (<b>C</b>) and females (<b>D</b>). P53 expression did not correlate with serum ALT levels in males (<b>E</b>) nor in females (<b>F</b>). qPCR results are represented as a fold change compared to sex- and genotype matched PBS controls (<b>A</b>,<b>B</b>). Data are represented as mean ± S.D. and are derived from 8 to 9 mice per group. ****: <span class="html-italic">p</span> &lt; 0.0001; n.s.: not significant.</p> Full article ">Figure 3
<p>Acetaminophen (APAP) induces ER stress signaling. Transcriptional induction of ATF4 in response to APAP was increased in males compared to females (<b>A</b>) and increased in <span class="html-italic">Bok<sup>-/-</sup></span> females compared to WT females (<b>A</b>). In all mice, BIM was induced transcriptionally with significantly increased induction in <span class="html-italic">Bok<sup>-/-</sup></span> females compared with <span class="html-italic">Bok<sup>-/-</sup></span> males and WT females (<b>B</b>). A Western blot of total liver lysates from males (<b>C</b>) and females (<b>D</b>) showed similar protein expression levels of BIM and CHOP in WT and <span class="html-italic">Bok<sup>-/-</sup></span> mice, except for higher BIM expression levels in <span class="html-italic">Bok<sup>-/-</sup></span> females (<b>C</b>–<b>F</b>). PUMA protein was more strongly induced by APAP in females than in males with similar expression levels between genotypes (<b>C</b>,<b>D</b>,<b>G</b>). BCL-2-related ovarian killer (BOK) was downregulated in WT females (<b>D</b>,<b>H</b>) but not in WT males (<b>C</b>,<b>H</b>). Results are depicted as a fold change compared to sex- and genotype matched PBS controls (<b>A</b>,<b>B</b>,<b>G</b>,<b>H</b>) or as GAPDH normalized values (<b>E</b>,<b>F</b>). Data are represented as mean ± S.D. and are derived from 8 to 9 mice per group. *: <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; n.s.: not significant.</p> Full article ">Figure 4
<p>Activation of JNK after APAP treatment. The JNK pathway was activated in livers of males (<b>A</b>) and females (<b>B</b>) of both genotypes after 5 h of APAP treatment. pJNK was significantly increased in WT females compared with WT males and reduced in both <span class="html-italic">Bok<sup>-/-</sup></span> females (<b>C</b>) and males (<b>D</b>). (<b>D</b>) shows data of male mice from (<b>C</b>) as close-up. Data are represented as mean ± S.D. and are derived from 7 to 8 mice per group. ***: <span class="html-italic">p</span> &lt; 0.001; n.s.: not significant.</p> Full article ">Figure 5
<p>5 h APAP treated livers do not display active effector caspases while PARP cleavage is detectable. No caspase-3 or -7 cleavage was detected in the total liver lysates of males (<b>A</b>) and females (<b>B</b>) while low degree PARP cleavage was seen after 5 h of APAP treatment. Liver lysate from DEN-treated mice was used as positive control for apoptosis induction [<a href="#B40-ijms-22-03281" class="html-bibr">40</a>]. Data are derived from 8 to 9 mice per group.</p> Full article ">Figure 6
<p>The impact of BOK on the severity of liver damage is moderate and sex dependent. ALT and AST levels were similar in WT and <span class="html-italic">Bok<sup>-/-</sup></span> males (<b>A</b>) and females (<b>B</b>) after 5 h APAP. ALT and AST levels did not significantly differ between males and females (<b>C</b>,<b>D</b>). Histologically, centrilobular necrosis was detected in WT and <span class="html-italic">Bok<sup>-/-</sup></span> mice. Haematoxylin and eosin stain (<b>E</b>). Scale bars = 1 mm. The extent of centrilobular necrosis (indicated as a mean percentage of affected centrilobular-portal vein distance) was higher in WT mice compared to <span class="html-italic">Bok<sup>-/-</sup></span> mice, with the lowest values in <span class="html-italic">Bok<sup>-/-</sup></span> females (<b>F</b>). Data are represented as mean ± S.D. and are derived from 8 mice per group. n.s.: not significant.</p> Full article ">
37 pages, 3229 KiB  
Review
Breast Cancer and the Other Non-Coding RNAs
by Dana Dvorská, Dušan Braný, Marcela Ňachajová, Erika Halašová and Zuzana Danková
Int. J. Mol. Sci. 2021, 22(6), 3280; https://doi.org/10.3390/ijms22063280 - 23 Mar 2021
Cited by 20 | Viewed by 4981
Abstract
Breast cancer is very heterogenous and the most common gynaecological cancer, with various factors affecting its development. While its impact on human lives and national health budgets is still rising in almost all global areas, many molecular mechanisms affecting its onset and development [...] Read more.
Breast cancer is very heterogenous and the most common gynaecological cancer, with various factors affecting its development. While its impact on human lives and national health budgets is still rising in almost all global areas, many molecular mechanisms affecting its onset and development remain unclear. Conventional treatments still prove inadequate in some aspects, and appropriate molecular therapeutic targets are required for improved outcomes. Recent scientific interest has therefore focused on the non-coding RNAs roles in tumour development and their potential as therapeutic targets. These RNAs comprise the majority of the human transcript and their broad action mechanisms range from gene silencing to chromatin remodelling. Many non-coding RNAs also have altered expression in breast cancer cell lines and tissues, and this is often connected with increased proliferation, a degraded extracellular environment, and higher endothelial to mesenchymal transition. Herein, we summarise the known abnormalities in the function and expression of long non-coding RNAs, Piwi interacting RNAs, small nucleolar RNAs and small nuclear RNAs in breast cancer, and how these abnormalities affect the development of this deadly disease. Finally, the use of RNA interference to suppress breast cancer growth is summarised. Full article
(This article belongs to the Special Issue Non-coding RNA (ncRNA) in Cancer)
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<p>Canonical and non-canonical miRNA biogenesis pathways. miRNAs are usually transcribed by polymerase II; but some are also transcribed by polymerase III as in those miRNAs from clusters spread within Alu repeats. The primary transcript (pri-miRNA) in canonical biogenesis has typical loop structure after transcription and it can it be hundreds of base pairs long. The pri-miRNA is recognised by DGCR8 protein and this combines with Drosha enzyme to form a micro-processor complex which removes the miRNA tails and cuts pri-miRNA into smaller precursor miRNA (pre-miRNA). Exportin-5 transports this to the cytoplasm through nucleopores and the pre-miRNA is recognised there by the large Dicer RNAse protein. Dicer cleaves the stem loop and forms the mature double-stranded miRNA molecule. This is then loaded into the Argonaute protein family, the passenger strand is degraded and the ‘miRNA-induced silencing complex (miRISC) is formed. The miRISC then bouns to its target mRNA sequence, usually at the mRNA 3′UTR region. This miRISC can inactivate mRNA by direct cleavage, or physically prevent ribosome sub-unit binding.This figure also depicts two non-canonical miRNA biogenesis pathways; (1) ‘mirtron’ miRNAs are produced from introns during mRNA splicing, and this biogenesis is Drosha independent. Branched pre-mirtrons are formed after splicing, de-branched by lariat debranching enzyme (Ldbr), enzymatically trimmed and folded into pre-miRNA hairpins. (2) in Dicer independent biogenesis, the miRNAs are loaded directly into Ago2 protein which cleaves target strands in the middle of its 3′arm, and mature miRNA is then generated by poly(A)-specific ribonuclease’(PARN) trimming. miR-451 is the one known representative of Dicer independent biogenesis, and this is the most abundant miRNA in erythrocytes.</p> Full article ">Figure 2
<p>Variability in Long-noncoding RNA biogenesis. (<b>a</b>) Long intergenic non-coding RNAs (lincRNAs) are transcribed by Polymerase II from regions between two protein coding genes, and they are usually capped, polyadenylated and spliced as in mRNA, but some undergo only terminal cleavage and premature termination. (<b>b</b>) Natural antisense transcripts (NAT) are synthesised by RNA polymerase II from the antisense strand of the protein coding gene. There are three NAT forms – Complete, Intron-overlapped and Exon-overlaped. (<b>c</b>) MALAT1/NEAT1 is cleaved by RNAseP after transcription. The U-A-U structure stabilises its 3′ end and inhibits further cleavage and mascRNA 3′-end products with unknown function are also created. (<b>d</b>) sno lncRNAs are products of intron excision. The snoRNP complex is formed on both ends, and this protects sequence from further degradation. sno lncRNAs lack both capping and polyadenylation. (<b>e</b>) SPA lncRNAs have snoRNP at their 5′ends and 3′-ends and are polyadenylated. They originate as a product of read-through transcription, and this is followed by multistep 5′end trimming and 3′end processing. (<b>f</b>) Circular intronic RNAs are products of excision of intron with consensus sequence (5′splice site is GU rich and branchpoint site is C rich), 3′ end is usually trimmed and debranched. (<b>g</b>) Finally, the circular RNAs (circRNAs) are products of circular back-slicing of the pre-mRNA exons. Edited from [<a href="#B26-ijms-22-03280" class="html-bibr">26</a>] with permission.</p> Full article ">Figure 3
<p>Primary and secondary biogenesis of piRNAs in model <span class="html-italic">Drosophila melanogaster</span> germline cells. The piRNA precursors can be transcribed from uni-strand and dual-strand piRNA clusters by polymerase II. Majority of these precursors are antisense (5′-3′) relative to transposon transcripts. Export from nucleus to processing sites is mediated by UAP-56 activity. The piRNA precursors are resolved by Armitage (armi) RNA helicase after export, and this leads to their unwinding. The 5′end processing is then mediated by the Zucchini mitochondria-associated nuclease (ZUC). ZUC action transforms the piRNA precursors into pre-piRNAs which are subsequently loaded into Piwi or AUB protein complexes. Here, fragments with Uracil bias at the 5′end are primarily selected. The overhanging 3′end is trimmed with 3′ to 5′ Nibbler exonuclease (Nib), Hen1 then methylates the 3′end and the piRNAs are then mature. piRNAs loaded into the Piwi protein are then involved in transcriptional gene silencing (TGS) in the nucleus. In contrast, the Aub–piRNA complex triggers the ping-pong amplification pathway by recognising and cleaving transposon mRNA. The product of this cleavage is converted into new sense oriented piRNA (secondary piRNA) which has a 10A bias, and this is subsequently loaded into the Ago3 protein complex and trimmed and methylated. The Ago3-piRNa complex similarly recognises and cleaves the anti-sense cluster transcript, and the product of this cleavage re-initiates the cycle. This provides one-cycle transposon sequence cleavage and simultaneous amplification of the piRNA sequence. The ping-pong amplification is therefore a mechanism of post-transcriptional gene silencing (PTSG).</p> Full article ">Figure 4
<p>siRNA action mechanism. Following delivery to the cytoplasm, the siRNA’s are directly loaded into the RISC complex or they undergo Dicer-mediated processing before loading in the RISC complex. Guide strand selection and passenger strand degradation depend on the several properties The guide strand has weaker binding at the 5′-end, is U-biased at that end and also has excess purines. The Ago/RISC complex then recognises the target mRNA and this is cleaved and degraded, or its translation is suppressed by sequestration in P-bodies. The presence of both individual siRNAs and those loaded in the Ago/RISC complex in the transfected cells’ nucleus has been noted, and there is also shuttling of this complex between cytoplasm and nucleus. The precise mechanisms of these actions require elucidation. Edited with permission from [<a href="#B209-ijms-22-03280" class="html-bibr">209</a>].</p> Full article ">Figure 5
<p><b>shRNA mechanism action</b>. shRNAs must be encoded in an appropriate expression vector for delivery to the nucleus for transcription. The shRNAs are transcribed by either RNA polymerase II or III, depending on the promoter driving their expression. The pri-shRNA primary transcript is recognised by the Drosha/DGCR8 complex and processed to precursor pre-shRNA. These shRNAs are then transported into the cytoplasm via Exportin 5, loaded into the Dicer/PRBT/PACT complex and processed to mature shRNAs. The shRNAs in the DICER complex then associates with the Ago/RISC complex, and this results in mRNA cleavage and degradation or suppression of mRNA translation. Edited with permission from [<a href="#B209-ijms-22-03280" class="html-bibr">209</a>].</p> Full article ">
60 pages, 63296 KiB  
Review
Articular Chondrocyte Phenotype Regulation through the Cytoskeleton and the Signaling Processes That Originate from or Converge on the Cytoskeleton: Towards a Novel Understanding of the Intersection between Actin Dynamics and Chondrogenic Function
by Jasmin C. Lauer, Mischa Selig, Melanie L. Hart, Bodo Kurz and Bernd Rolauffs
Int. J. Mol. Sci. 2021, 22(6), 3279; https://doi.org/10.3390/ijms22063279 - 23 Mar 2021
Cited by 47 | Viewed by 5985
Abstract
Numerous studies have assembled a complex picture, in which extracellular stimuli and intracellular signaling pathways modulate the chondrocyte phenotype. Because many diseases are mechanobiology-related, this review asked to what extent phenotype regulators control chondrocyte function through the cytoskeleton and cytoskeleton-regulating signaling processes. Such [...] Read more.
Numerous studies have assembled a complex picture, in which extracellular stimuli and intracellular signaling pathways modulate the chondrocyte phenotype. Because many diseases are mechanobiology-related, this review asked to what extent phenotype regulators control chondrocyte function through the cytoskeleton and cytoskeleton-regulating signaling processes. Such information would generate leverage for advanced articular cartilage repair. Serial passaging, pro-inflammatory cytokine signaling (TNF-α, IL-1α, IL-1β, IL-6, and IL-8), growth factors (TGF-α), and osteoarthritis not only induce dedifferentiation but also converge on RhoA/ROCK/Rac1/mDia1/mDia2/Cdc42 to promote actin polymerization/crosslinking for stress fiber (SF) formation. SF formation takes center stage in phenotype control, as both SF formation and SOX9 phosphorylation for COL2 expression are ROCK activity-dependent. Explaining how it is molecularly possible that dedifferentiation induces low COL2 expression but high SF formation, this review theorized that, in chondrocyte SOX9, phosphorylation by ROCK might effectively be sidelined in favor of other SF-promoting ROCK substrates, based on a differential ROCK affinity. In turn, actin depolymerization for redifferentiation would “free-up” ROCK to increase COL2 expression. Moreover, the actin cytoskeleton regulates COL1 expression, modulates COL2/aggrecan fragment generation, and mediates a fibrogenic/catabolic expression profile, highlighting that actin dynamics-regulating processes decisively control the chondrocyte phenotype. This suggests modulating the balance between actin polymerization/depolymerization for therapeutically controlling the chondrocyte phenotype. Full article
(This article belongs to the Special Issue The Future of Cartilage Repair in Complex Biological Situations)
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<p>Regulation of actin dynamics. The relevant signaling pathways and molecules for regulating actin depolymerization (top, green half circle) compared to actin polymerization and/or stress fiber (SF) formation (bottom, red half circle), including upstream signaling through pro-inflammatory cytokines and growth factors as well as downstream signaling to actin-modulating proteins, e.g., cofilin and gelsolin, are illustrated. The legend in the upper right corner details the symbols used; the dotted arrow termed “binding” describes the binding of a substance, which might cause either inhibition or activation. Details are specified in the text. Abbreviations: ADF: actin-depolymerizing factor, AIP1: actin-interacting protein 1, Arp2/3: actin-related protein 2/3, BMP-7: bone morphogenetic protein 7, CAP: cyclase-associated protein, CIN: chronophin, COL1: type I collagen, DEX: dexamethasone, EGF: epidermal growth factor, ERK: extracellular signal-regulated kinase, ERM: ezrin/radixin/moesin, FAK: focal adhesion kinase, FGF-1: fibroblast growth factor 1, FGF-2: fibroblast growth factor 2, GAPs: GTPase-activating proteins, GEFs: guanine-nucleotide exchange factors, G(M)-CSF: granulocyte(-macrophage) colony-stimulating factor, GSK-3: glycogen synthase kinase 3, HSP-27: heatshock protein 27, IL-1β: interleukin 1β, IL-6: interleukin 6, IL-8: interleukin 8, LIMK1/2: LIM kinase 1/2, MAPK: mitogen-activated protein kinase, MAPKAPK2: MAPK-activated protein kinase 2, MEK1/2 / MKK3/6: MAPK kinase 1/2 / 3/6, MLC: myosin light chain, MLCK: MLC kinase, MLCP: MLC phosphatase, MRCKα: myotonic dystrophy-related Cdc42-binding kinase α, MRTF: myocardin-related transcription factor, PAK1: p21-activated kinase, PI4P: phosphatidylinositol-4-phosphate, PIP2: phosphatidylinositol (4,5)-bisphosphate, PIP3: phosphatidylinositol (3,4,5)-trisphosphate, PI3K: phosphatidylinositol 3-kinase, PIP5KI: phosphatidylinositol 5-kinase type I, PKA/PKC/PKD: protein kinase A/C/D, PLCγ: phospholipase Cγ, PP1/PP2A/PP2B: protein phosphatase type 1/ 2A/ 2B, PTHrP: parathyroid hormone-related protein, ROCK: Rho-kinase, SOX9: SRY-box transcription factor 9, SSH: slingshot phosphatase, SZP: superficial zone protein, TESK1/2: testicular protein kinase 1/2, TGF-α/β1: transforming growth factor α/β1, TNF-α: tumor necrosis factor α, VASP: vasodilator-stimulated phosphoprotein, VEGF: vascular endothelial growth factor, YAP: Yes-associated protein.</p> Full article ">Figure 2
<p>Correlation between actin polymerization, dedifferentiation, and redifferentiation. Redifferentiation of dedifferentiated CHs induced by serial passaging depends on active SAPK and p38 MAPK, low active RhoA, and prevention of SFs, resulting in increased <span class="html-italic">SOX9</span> expression. The up and down arrows indicate an increase or decrease.</p> Full article ">Figure 3
<p>(<b>A</b>) Representative 40x images of primary hCHs with different morphologies and the F-actin distribution on day 1 of cultivation, acquired with an AxioObserver-Z1, Zeiss, Germany. Left side: CHs cultured on a glass substrate coated with fibronectin. The image was taken with oil immersion. Right side: a CH cultured on a tissue culture polystyrene substrate. Red: F-actin, blue: nucleus. Scale bar: 50 µm. (<b>B</b>) Schematic actin organization, FA area, spreading area, and motility related to the CH differentiation status. Differentiated and round CHs have a cortical F-actin spheroid that appears as a “ring” in 2D and punctate (freckled) actin in the cytoplasm [<a href="#B70-ijms-22-03279" class="html-bibr">70</a>,<a href="#B71-ijms-22-03279" class="html-bibr">71</a>,<a href="#B72-ijms-22-03279" class="html-bibr">72</a>,<a href="#B169-ijms-22-03279" class="html-bibr">169</a>,<a href="#B170-ijms-22-03279" class="html-bibr">170</a>,<a href="#B175-ijms-22-03279" class="html-bibr">175</a>,<a href="#B196-ijms-22-03279" class="html-bibr">196</a>], and have low total actin. (<b>C</b>) A high G-/F-actin ratio [<a href="#B71-ijms-22-03279" class="html-bibr">71</a>], little or no SFs [<a href="#B70-ijms-22-03279" class="html-bibr">70</a>,<a href="#B71-ijms-22-03279" class="html-bibr">71</a>,<a href="#B72-ijms-22-03279" class="html-bibr">72</a>,<a href="#B196-ijms-22-03279" class="html-bibr">196</a>], a small FA area, small spreading area, and low motility [<a href="#B170-ijms-22-03279" class="html-bibr">170</a>] characterize differentiated CHs. Dedifferentiated, fibroblastic CHs have prominent, thick SFs [<a href="#B70-ijms-22-03279" class="html-bibr">70</a>,<a href="#B71-ijms-22-03279" class="html-bibr">71</a>,<a href="#B72-ijms-22-03279" class="html-bibr">72</a>,<a href="#B170-ijms-22-03279" class="html-bibr">170</a>,<a href="#B175-ijms-22-03279" class="html-bibr">175</a>,<a href="#B196-ijms-22-03279" class="html-bibr">196</a>], a high total actin, a low G-/F-actin ratio [<a href="#B71-ijms-22-03279" class="html-bibr">71</a>], a large focal adhesion (FA) area, large spreading area, and high motility [<a href="#B170-ijms-22-03279" class="html-bibr">170</a>].</p> Full article ">Figure 4
<p>(<b>A</b>) Rounded vs. (<b>B</b>) spread morphologies of primary hCHs imaged in super-resolution using 3D structured illumination microscopy (3D-SIM) on a GE DeltaScan OMX SR microscope. Isolated primary AC hCHs from ankle joints were plated on a fibronectin (FN)-coated cover glass using standard 2D cell culture techniques, fixed with 4% paraformaldehyde, stained with ActinRed 555 Ready probes Reagent (ThermoFisher), and imaged. The maximum intensity projections of the volumetric image stacks are shown. Scale bars: 2 μm. The images are reprinted (adapted) with permission from [<a href="#B197-ijms-22-03279" class="html-bibr">197</a>]. Copyright (2020) American Chemical Society. We also received reprint permission from Scott T. Wood [<a href="#B197-ijms-22-03279" class="html-bibr">197</a>].</p> Full article ">Figure 5
<p>Cytoskeletal differences between healthy and osteoarthritis (OA) hCHs were detected for tubulin, vinculin, gelsolin, destrin, cofilin-1, and cofilin-2 [<a href="#B205-ijms-22-03279" class="html-bibr">205</a>]. Due to the increase in the latter and its higher actin assembly activity [<a href="#B206-ijms-22-03279" class="html-bibr">206</a>], presumably causing overall enhanced F-actin, results in elevated cell elastic moduli. The pro-inflammatory cytokine IL-1β increased F-actin in healthy pCHs that were cultured for 1 day [<a href="#B70-ijms-22-03279" class="html-bibr">70</a>], but also induced disassembly of tubulin, vimentin, vinculin, and actin in healthy and OA hCHs that were cultured for up to 2 weeks [<a href="#B199-ijms-22-03279" class="html-bibr">199</a>]. Abbreviations: IL-1β: interleukin-1β, OA: osteoarthritic, hCHs: human chondrocytes, pCHs: porcine chondrocytes. The up and down arrows indicate an increase or decrease.</p> Full article ">Figure 6
<p>Signaling of pro-inflammatory cytokines IL-1β, IL-6, and IL-8, causing alterations in the actin cytoskeleton by signaling through the RhoA/ROCK or Rac1 pathway. Abbreviations: IL-1β: interleukin-1β, IL-6: interleukin-6, IL-8, interleukin-8, ROCK: Rho-kinase.</p> Full article ">Figure 7
<p>Correlation between growth factor signaling, the actin cytoskeleton, and CCN2 expression in CHs. The actin cytoskeleton modulates FGF-2 and BMP signaling through the Rac1 pathway, affecting the expression of CCN2 that functions as a mechano-sensing regulator [<a href="#B238-ijms-22-03279" class="html-bibr">238</a>]. The CH actin cytoskeleton is regulated through the signaling of the growth factors FGF-2, IGF-1, TGF-β1, and TGF-α. The latter may induce stress fiber formation. Abbreviations: BMP: bone morphogenetic protein, CCN2: connective tissue growth factor, FGF-2: fibroblast growth factor 2, IGF-1: insulin growth factor I, TGF-α: transforming growth factor α, TGF-β1: transforming growth factor β1. Arrows indicate induction.</p> Full article ">Figure 8
<p>The TGF-β-induced stress fiber formation, cell stiffening, and OA-induced switch of the TGF-β receptors and downstream Smad signaling, causing terminal differentiation. During OA onset, increased TGF-β induces stress fiber formation through Rho GTPase signaling. Segregation of TGF-βRI and TGF-βRII through inhibitory cellular tension inhibits Smad3 phosphorylation, whereas a heteromeric complex formation of TGF-βRI and TGF-βRII through permissive cellular tension is required for Smad3 phosphorylation. Additionally, the receptor switches from ALK5/TGF-βRI and downstream Smad2/3 signaling, mediating COL2 production, to ALK1/TGF-βRI and downstream Smad1/5/8 signaling, mediating MMP-13 expression. Complex formation of Smad3 with Runx2 inhibits terminal differentiation, whereas the Smad1–Runx2 complex induces terminal differentiation in CHs. Abbreviations: COLII: type II collagen, MMP-13: matrix metalloproteinase 13, TGF-β: transforming growth factor β, TGF-βRI: transforming growth factor β receptor I, TGF-βRII: transforming growth factor β receptor II. The up arrow indicates an increase.</p> Full article ">Figure 9
<p>Correlation between cytoskeletal stiffness and SF amount vs. SF thickness and distribution. SF amount assembled of actin and myosin (with myosin having a higher impact) have a greater effect on global cell stiffness than SF thickness and alignment. The bold arrow indicates a larger effect than the regular arrow in the output section. The up arrow indicates an increase.</p> Full article ">Figure 10
<p>The specific range of actin polymerization causes TGF-β-induced superficial zone protein (SZP) production that results in joint lubrication and health.</p> Full article ">Figure 11
<p>Effects of OA, growth factors, and serial passaging on stress fiber formation and the subsequent effects on TGF-β receptor function and CH phenotype. Abbreviations: ACAN: aggrecan, ADAMTS-5: a disintegrin and metalloproteinase with thrombospondin motifs 5, COL1: type I collagen, COL2: type II collagen, COL3: type II collagen, COL10: type X collagen, ERK: extracellular signal-regulated kinase, (s)GAG: (sulfated) glycosaminoglycan, JNK: JUN N-terminal kinase, MEK: mitogen-activated kinase kinase 1/2, MRTF: myocardin-related transcription factor, NO: nitric oxide, OA: osteoarthritis, PKC: protein kinase C, TGF-βRI: transforming growth factor β receptor I, TNF-α: tumor necrosis factor α, IL-1β: interleukin-1β, MMP-3: matrix metalloproteinase 3, MMP-13: matrix metalloproteinase 13, SOX4: SRY-box transcription factor 4. The up and down arrows indicate an increase or decrease.</p> Full article ">Figure 12
<p>Regulation of <span class="html-italic">SOX9</span> mRNA expression and SOX9 phosphorylation. (<b>A</b>) mRNA expression of <span class="html-italic">SOX9</span> is induced through IL-1β, TGF-α or -β, FGF-1 and -2, DEX, and PTHrP in specific conditions. (<b>B</b>) Phosphorylation at S64 by PKA is induced through BMP and TGF signaling. (<b>C</b>) Phosphorylation at S181 is performed by ROCK and PKA; the latter is induced through BMP, TGF, and PTHrP signaling. (<b>D</b>) Phosphorylation at S211 is performed by PKA and p38 MAPK as well as mediated through TGF-β1 and Smad2/3 signaling. (<b>E</b>) Phosphorylation at T236 by GSK-3 is regulated through PI3K/Akt signaling. Signaling pathways and kinases involved in SOX9 phosphorylation are in part listed by [<a href="#B301-ijms-22-03279" class="html-bibr">301</a>]. Details are specified in the text. Abbreviations: BMP: bone morphogenetic protein, DEX: dexamethasone, ERK: extracellular signal-regulated kinase, FGF-1: fibroblast growth factor 1, FGF-2: fibroblast growth factor 2, GSK-3: glycogen synthase kinase 3, IL-1β: interleukin 1β, MAPK: mitogen-activated protein kinase, MEK: mitogen-activated protein kinase 1/2, PI3K: phosphatidylinositol 3-kinase, PKA: protein kinase A, PTHrP: parathyroid hormone-related protein, ROCK: Rho-kinase, SF: stress fiber, SOX9: SRY-box transcription factor 9, TGF-α: transforming growth factor α, TGF-β: transforming growth factor β, TGFR1: transforming growth factor receptor 1, YAP: Yes-associated protein.</p> Full article ">Figure 13
<p>Relationship of the balance of actin polymerization and depolymerization with chondrocyte phenotype. Increased actin polymerization is associated with inducing a dedifferentiated, fibrogenic phenotype (red; indicated with by the “-“ symbol), whereas actin depolymerization is associated with regaining a chondrogenic phenotype (green; indicated with the “+” symbol).</p> Full article ">Figure 14
<p>CH phenotype regulation, e.g., in OA or through serial passaging, pro-inflammatory cytokines, and/or growth factors control CH function through SF formation and the actin-regulating signaling pathways RhoA/ROCK/Rac1/mDia1/mDia2/Cdc42. SF formation in CHs induces a dedifferentiated fibroblastic morphology, a fibrogenic (increased <span class="html-italic">COL1</span> and <span class="html-italic">COL3</span> expression) and catabolic (formation of COL2 and aggrecan fragments) phenotype, and increased <span class="html-italic">COL1</span> expression, induced by the nuclear localization of MRTF. This review theorized that in CHs phosphorylation of SOX9 (and subsequently increased <span class="html-italic">COL2</span> expression as chondrogenic phenotype marker) by ROCK is effectively sidelined in favor of other SF promoting ROCK substrates (LIMK2, ezrin, and MLC), based on a differential affinity of various ROCK substrates, explaining how it is molecularly possible that dedifferentiation induces low <span class="html-italic">COL2</span> expression but high SF formation. Abbreviations: COL1: type I collagen, COL2: type II collagen, COL3: type III collagen, IL-1α: interleukin 1α, IL-1β: interleukin 1β, IL-6: interleukin 6, IL-8: interleukin 8, MRTF: myocardin-related transcription factor, ROCK: Rho-kinase, SOX9: SRY-box transcription factor 9, TGF-α: transforming growth factor α, TNF-α: tumor necrosis factor α. The up and down arrows indicate an increase or decrease.</p> Full article ">
28 pages, 7509 KiB  
Article
Utilizing an Animal Model to Identify Brain Neurodegeneration-Related Biomarkers in Aging
by Ming-Hui Yang, Yi-Ming Arthur Chen, Shan-Chen Tu, Pei-Ling Chi, Kuo-Pin Chuang, Chin-Chuan Chang, Chiang-Hsuan Lee, Yi-Ling Chen, Che-Hsin Lee, Cheng-Hui Yuan and Yu-Chang Tyan
Int. J. Mol. Sci. 2021, 22(6), 3278; https://doi.org/10.3390/ijms22063278 - 23 Mar 2021
Cited by 2 | Viewed by 3835
Abstract
Glycine N-methyltransferase (GNMT) regulates S-adenosylmethionine (SAMe), a methyl donor in methylation. Over-expressed SAMe may cause neurogenic capacity reduction and memory impairment. GNMT knockout mice (GNMT-KO) was applied as an experimental model to evaluate its effect on neurons. In this study, proteins from brain [...] Read more.
Glycine N-methyltransferase (GNMT) regulates S-adenosylmethionine (SAMe), a methyl donor in methylation. Over-expressed SAMe may cause neurogenic capacity reduction and memory impairment. GNMT knockout mice (GNMT-KO) was applied as an experimental model to evaluate its effect on neurons. In this study, proteins from brain tissues were studied using proteomic approaches, Haemotoxylin and Eosin staining, immunohistochemistry, Western blotting, and ingenuity pathway analysis. The expression of Receptor-interacting protein 1(RIPK1) and Caspase 3 were up-regulated and activity-dependent neuroprotective protein (ADNP) was down-regulated in GNMT-KO mice regardless of the age. Besides, proteins related to neuropathology, such as excitatory amino acid transporter 2, calcium/calmodulin-dependent protein kinase type II subunit alpha, and Cu-Zn superoxide dismutase were found only in the group of aged wild-type mice; 4-aminobutyrate amino transferase, limbic system-associated membrane protein, sodium- and chloride-dependent GABA transporter 3 and ProSAAS were found only in the group of young GNMT-KO mice and are related to function of neurons; serum albumin and Rho GDP dissociation inhibitor 1 were found only in the group of aged GNMT-KO mice and are connected to neurodegenerative disorders. With proteomic analyses, a pathway involving Gonadotropin-releasing hormone (GnRH) signal was found to be associated with aging. The GnRH pathway could provide additional information on the mechanism of aging and non-aging related neurodegeneration, and these protein markers may be served in developing future therapeutic treatments to ameliorate aging and prevent diseases. Full article
(This article belongs to the Special Issue Peripheral Biomarkers in Neurodegenerative Diseases 2.0)
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<p>Hematoxylin and eosin staining histopathological examination of the brains from (<b>A</b>) WY, (<b>B</b>) WO, (<b>C</b>) GY, and (<b>D</b>) GO mice. The sizes and shapes of Purkinje cells located in the ganglion cell layer were different (200X). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>−/−</sup> mice (GO).</p> Full article ">Figure 2
<p>The results of immunohistochemistry staining of SMP30of the brain samples from (<b>A</b>) WY, (<b>B</b>) WO, (<b>C</b>) GY, and (<b>D</b>) GO mice. The cell density of the granular layer of the cerebellar medulla is denser and the staining are more concentrated in the samples of wild-type (Wt) mice (200X). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>−/−</sup> mice (GO).</p> Full article ">Figure 3
<p>(<b>A</b>) Brain lysates from WY, WO, GY, and GO mice were collected, and levels of SMP30 were analyzed by Western blotting. (<b>B</b>) Beta-actin was used to normalize Western blot data (<span class="html-italic">n</span> = 8). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>-/-</sup> mice (GO).</p> Full article ">Figure 4
<p>Immunohistochemistry (IHC) of activity-dependent neuroprotective protein (ADNP)using mouse brain samples from (<b>A</b>) WY, (<b>B</b>) WO, (<b>C</b>) GY, and (<b>D</b>) GO mice. The nerve fibers, located in the molecular layer of the cerebellar medulla, are more densely distributed and have a longer length in WY mice (100X). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>−/−</sup> mice (GO).</p> Full article ">Figure 5
<p>(<b>A</b>) Brain lysates from WY, WO, GY, and GO mice were collected, and levels of ADNP were analyzed by Western blotting. (<b>B</b>) Beta-actin was used to normalize Western blot data (<span class="html-italic">n</span> = 8, *** <span class="html-italic">p</span> &lt; 0.001). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>−/−</sup> mice (GO).</p> Full article ">Figure 6
<p>Immunohistochemistry (IHC) of RIPK1 using mouse brain samples from (<b>A</b>) WY, (<b>B</b>) WO, (<b>C</b>) GY, and (<b>D</b>) GO mice. The cell morphology of Purkinje cells in WY is intact, which is not the case for the WO and GY group. The cell morphology of Purkinje cells in GO is incomplete and fragmented (200X). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>−/−</sup> mice (GO).</p> Full article ">Figure 7
<p>(<b>A</b>) Brain lysates from Wt, GY, and GO mice were collected, and levels of RIPK1 were analyzed by Western blotting. The RIPK1 expression was down-regulated or non-detectable in some WY and WO mice; thus, the WY and WO mice were combined into one group as Wt. (<b>B</b>) Beta-actin was used to normalize Western blot data (<span class="html-italic">n</span> = 8, * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.005). Abbreviations: wild-type mice (Wt); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>−/−</sup> mice (GO).</p> Full article ">Figure 8
<p>Immunohistochemistry (IHC) of Caspase 3 using mouse brain samples from (<b>A</b>) WY, (<b>B</b>) WO, (<b>C</b>) GY, and (<b>D</b>) GO mice. The staining is located in the granular layer of the cerebellar medulla (200X). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>−/−</sup> mice (GO).</p> Full article ">Figure 9
<p>(<b>A</b>) Brain lysates from WY, WO, GY, and GO mice were collected, and levels of Caspase 3 were analyzed by Western blotting. (<b>B</b>) Beta-actin was used to normalize Western blot data (<span class="html-italic">n</span> = 8). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>−/−</sup> mice (GO).</p> Full article ">Figure 10
<p>The Mascot results indicate that 198 proteins were unique for the groups of WY mice. Compared with the WY mice, 16, 27, and 25 proteins were unique for the groups of WO, GY, and GO mice, respectively. After deducting the proteins found from the WY mice and common proteins, there are only eight, nine, and eight unique proteins in the groups of WO, GY, and GO mice. Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT<sup>−/−</sup> mice (GY); old GNMT<sup>−/−</sup> mice (GO).</p> Full article ">Figure 11
<p>The protein-protein interaction pathways were illustrated. Eight proteins identified from the Ingenuity Pathway Analysis (IPA) were PAK1, DNM1, DNM2, DNM3, GNAI2, GNAS, CAMK2A, and CAMK2B, which were inter-connected with other proteins.</p> Full article ">
24 pages, 4409 KiB  
Review
Development of Two-Dimensional Nanomaterials Based Electrochemical Biosensors on Enhancing the Analysis of Food Toxicants
by Iruthayapandi Selestin Raja, Mohan Vedhanayagam, Desingh Raj Preeth, Chuntae Kim, Jong Hun Lee and Dong Wook Han
Int. J. Mol. Sci. 2021, 22(6), 3277; https://doi.org/10.3390/ijms22063277 - 23 Mar 2021
Cited by 20 | Viewed by 4320
Abstract
In recent times, food safety has become a topic of debate as the foodborne diseases triggered by chemical and biological contaminants affect human health and the food industry’s profits. Though conventional analytical instrumentation-based food sensors are available, the consumers did not appreciate them [...] Read more.
In recent times, food safety has become a topic of debate as the foodborne diseases triggered by chemical and biological contaminants affect human health and the food industry’s profits. Though conventional analytical instrumentation-based food sensors are available, the consumers did not appreciate them because of the drawbacks of complexity, greater number of analysis steps, expensive enzymes, and lack of portability. Hence, designing easy-to-use tests for the rapid analysis of food contaminants has become essential in the food industry. Under this context, electrochemical biosensors have received attention among researchers as they bear the advantages of operational simplicity, portability, stability, easy miniaturization, and low cost. Two-dimensional (2D) nanomaterials have a larger surface area to volume compared to other dimensional nanomaterials. Hence, researchers nowadays are inclined to develop 2D nanomaterials-based electrochemical biosensors to significantly improve the sensor’s sensitivity, selectivity, and reproducibility while measuring the food toxicants. In the present review, we compile the contribution of 2D nanomaterials in electrochemical biosensors to test the food toxicants and discuss the future directions in the field. Further, we describe the types of food toxicity, methodologies quantifying food analytes, how the electrochemical food sensor works, and the general biomedical properties of 2D nanomaterials. Full article
(This article belongs to the Special Issue Multidisciplinary Investigations of Nanoparticle Synthesis and Analysis for Possible Biomedical and Food Technology Applications)
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Full article ">Figure 1
<p>Ensuring food safety through electrochemical analyses of food toxic analytes from different sources has been shown schematically.</p> Full article ">Figure 2
<p>(<b>A</b>) Schematic illustration of a typical electrochemical food sensor and its various components, including bioreceptors, transducer, and signal processor [<a href="#B68-ijms-22-03277" class="html-bibr">68</a>]. The structure of two-dimensional nanomaterials used to fabricate the electrode surfaces in biosensors has been shown [<a href="#B69-ijms-22-03277" class="html-bibr">69</a>]. (<b>B</b>) Different types of electrochemical techniques have been presented [<a href="#B51-ijms-22-03277" class="html-bibr">51</a>].</p> Full article ">Figure 3
<p>(<b>A</b>) Demonstration of step-by-step fabrication of the GO-based biosensor (antibody-Au NPs-polypyrrole/electrochemically reduced GO-screen printed carbon electrode) and (<b>B</b>) electrochemical immunosensing of the system employed for the detection of mycotoxins through DPV signals [<a href="#B135-ijms-22-03277" class="html-bibr">135</a>].</p> Full article ">Figure 4
<p>(<b>A</b>) Schematic demonstration of fabrication, biofunctionalization, and sensing of the LIG immunosensor. LIG is processed onto a polyimide sheet to create the working electrode, and subsequently, the electrode is passivated with lacquer. SEM image of the LIG surface is shown. The Salmonella antibodies are immobilized on the working electrode via carbodiimide cross-linking chemistry (EDC/NHS) to detect Salmonella microbes. (<b>B</b>) The linear calibration curve of charge transfer resistance change (ΔR<sub>ct</sub>) vs. <span class="html-italic">S. enterica</span> concentrations (generated from Nyquist plots of impedance spectra) in chicken broth. (<b>C</b>) ΔR<sub>ct</sub> vs. different interferent bacterial species (10<sup>4</sup> CFU mL<sup>−1</sup>) to show the specificity of the immunosensor. (<b>D</b>) Shelf-life test to investigate the stability of the immunosensors for seven days. All the data shown as mean ± SD, <span class="html-italic">n</span> = 3. * means significantly difference (<span class="html-italic">p</span> &lt; 0.05) [<a href="#B137-ijms-22-03277" class="html-bibr">137</a>].</p> Full article ">Figure 5
<p>Schematic diagram demonstrating the preparation of food detection system containing electrochemically reduced graphene oxide/cyclodextrin modified glassy carbon electrode (E-rGO/CDs/GCE) to quantify the amount of imidacloprid (IDP) in test solution [<a href="#B142-ijms-22-03277" class="html-bibr">142</a>]. CE—counter electrode, RE—reference electrode, and WE—working electrode.</p> Full article ">Figure 6
<p>(<b>A</b>) Schematic diagram illustrating the detection mechanism of an rGO-based biosensor. SNAP-25-GFP peptide is immobilized on the rGO surface, which is previously conjugated with pyrenebutyric acid. The target BoNT-LcAs specifically cleave SNAP-25-GFP molecules, detaching them from rGO/Au electrode surface. The detection of enzymatic activity decreases the hindrance of redox probes transfer on electrodes resulting in increased electrochemical currents. (<b>B</b>) TEM image of rGO flakes and (<b>C</b>) rGO sheets with ripples and wrinkles. (<b>D</b>) Raman spectra of GO and rGO. (<b>E</b>) Specificity testing of control buffer and fresh BoNT-LcA, heated BoNT-LcA, and fresh BoNT-LcA at the concentration of 1 ng mL<sup>−1</sup>. (<b>F</b>) Relative DPV peak current change (ΔI%) for the same samples [<a href="#B143-ijms-22-03277" class="html-bibr">143</a>].</p> Full article ">Figure 7
<p>(<b>A</b>) Schematic of the proposed molecular packing structure and (<b>B</b>) HRTEM image of YbMoSe<sub>2</sub>. (<b>C</b>) Nyquist plot demonstrating the electrochemical performance of bare, MoSe<sub>2</sub>, and YbMoSe<sub>2</sub> glassy carbon electrodes in 5 mM ferricyanide system in 0.1 M of KCl. The inset shows an equivalent circuit model (R<sub>ct</sub>—charge transfer resistance; C<sub>dl</sub>—double-layer capacitance; R<sub>s</sub>—solution resistance; W—Warburg impedance). (<b>D</b>) CV of bare GCE (a), MoSe<sub>2</sub>/GCE (b), and YbMoSe<sub>2</sub>/GCE (c) with 0.29 mM diphenylamine in N<sub>2</sub> purged buffer at 50 mV s<sup>−1</sup> [<a href="#B146-ijms-22-03277" class="html-bibr">146</a>].</p> Full article ">
20 pages, 6581 KiB  
Review
New Insights into the Mammalian Egg Zona Pellucida
by Carla Moros-Nicolás, Pascale Chevret, María Jiménez-Movilla, Blanca Algarra, Paula Cots-Rodríguez, Leopoldo González-Brusi, Manuel Avilés and Mª José Izquierdo-Rico
Int. J. Mol. Sci. 2021, 22(6), 3276; https://doi.org/10.3390/ijms22063276 - 23 Mar 2021
Cited by 24 | Viewed by 8123
Abstract
Mammalian oocytes are surrounded by an extracellular coat called the zona pellucida (ZP), which, from an evolutionary point of view, is the most ancient of the coats that envelope vertebrate oocytes and conceptuses. This matrix separates the oocyte from cumulus cells and is [...] Read more.
Mammalian oocytes are surrounded by an extracellular coat called the zona pellucida (ZP), which, from an evolutionary point of view, is the most ancient of the coats that envelope vertebrate oocytes and conceptuses. This matrix separates the oocyte from cumulus cells and is responsible for species-specific recognition between gametes, preventing polyspermy and protecting the preimplantation embryo. The ZP is a dynamic structure that shows different properties before and after fertilization. Until very recently, mammalian ZP was believed to be composed of only three glycoproteins, ZP1, ZP2 and ZP3, as first described in mouse. However, studies have revealed that this composition is not necessarily applicable to other mammals. Such differences can be explained by an analysis of the molecular evolution of the ZP gene family, during which ZP genes have suffered pseudogenization and duplication events that have resulted in differing models of ZP protein composition. The many discoveries made in recent years related to ZP composition and evolution suggest that a compilation would be useful. Moreover, this review analyses ZP biosynthesis, the role of each ZP protein in different mammalian species and how these proteins may interact among themselves and with other proteins present in the oviductal lumen. Full article
(This article belongs to the Special Issue Molecular and Physiological Regulation of Mammalian Oocyte and Embryo Development)
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<p>The zona pellucida (ZP) of rabbit (<b>A</b>) and bovine (<b>C</b>) oocytes captured by conventional Hoffmann inverted microscopy appears very similar. The same zona pellucida captured by polarized light microscopy (<b>B</b>,<b>D</b>) reveals a different multilaminar structure and distinctly birefringence, and the meiotic spindle is easily appreciated (arrowhead). Scale bars 50 µm.</p> Full article ">Figure 2
<p>Zona pellucida composition in mammals. The ZP is composed of three, four, seven, or eight proteins. In house mouse (<span class="html-italic">Mus musculus</span>), the ZP is composed by ZP1, ZP2, and ZP3. In pig, cow, dolphin, dog, and fox, the ZP is formed by ZP2, ZP3, and ZP4. In most of mammals, the ZP is formed by four proteins: ZP1, ZP2, ZP3, and ZP4. In marsupials, two scenarios are found: four proteins in South American marsupials (ZP1, ZP2, ZP3-1b, and ZP3-1c) or seven proteins in Australasian marsupials (ZP1, ZP2, ZP3-1a, ZP3-1b, ZP3-1c, ZP4, ZPAX). In monotremes, the ZP is composed of eight ZP proteins: ZPY, ZP1 ZP2, ZP3-1a, ZP3-1b, ZP3-2, ZP4, ZPAX. In eutherian mammals ZP3-1c is written as ZP3 in order not to complicate the current nomenclature.</p> Full article ">Figure 3
<p>Evolution of ZP genes in mammals. Six ZP genes were probably present in the common ancestor. The gain and the loss of a gene in the phylogeny are indicated respectively by a plus or minus sign with the name of the gene concerned.</p> Full article ">Figure 4
<p>Ultrastructural analysis of the endoplasmic reticulum in the mice oocytes during the folliculogenesis. (<b>A</b>) Bilaminar primary ovarian follicle, semithin sections (right panel) and transmission electron microscope (panel left) immunostaining with anti-PDI antibody. The circular structure was specifically labelled with the anti-PDI antibody. (<b>B</b>) Multilaminar primary ovarian follicle, semithin sections (right panel), and transmission electron microscope (left panel) immunostaining with anti-PDI antibody. Note the presence of small dark vesicles (arrows) distributed throughout ooplasm specifically labelled with ER marker (Jiménez-Movilla M, Avilés M, Castells MT, Ballesta. Ultrastructural analysis of the endoplasmic reticulum in the mice oocytes during the folliculogenesis. First International Congress of Histology and Tissue Engineering, Spain, 2005).</p> Full article ">Figure 5
<p>Electron microscopical analysis of oocyte organelles involved in protein trafficking. (<b>A</b>) Human prophase I oocyte immunolabeled with anti-human ZP3 antibody. The Golgi apparatus showed a moderate reactivity. (<b>B</b>) Inmatured mice oocytes. Multivesicular aggregates (MVA) consisted of multiple vesicles embedded in an amorphous material, and the majority of them were found in close proximity to the oolemma. (<b>C</b>) Human prophase I oocyte immunolabeled with anti-human ZP3 antibody. Lysosomes like structures were strongly reactive (arrows) M: Mitochondria.</p> Full article ">
31 pages, 6580 KiB  
Article
Neonatal Mesenchymal Stem Cell Treatment Improves Myelination Impaired by Global Perinatal Asphyxia in Rats
by Andrea Tapia-Bustos, Carolyne Lespay-Rebolledo, Valentina Vío, Ronald Pérez-Lobos, Emmanuel Casanova-Ortiz, Fernando Ezquer, Mario Herrera-Marschitz and Paola Morales
Int. J. Mol. Sci. 2021, 22(6), 3275; https://doi.org/10.3390/ijms22063275 - 23 Mar 2021
Cited by 6 | Viewed by 3592
Abstract
The effect of perinatal asphyxia (PA) on oligodendrocyte (OL), neuroinflammation, and cell viability was evaluated in telencephalon of rats at postnatal day (P)1, 7, and 14, a period characterized by a spur of neuronal networking, evaluating the effect of mesenchymal stem cell (MSCs)-treatment. [...] Read more.
The effect of perinatal asphyxia (PA) on oligodendrocyte (OL), neuroinflammation, and cell viability was evaluated in telencephalon of rats at postnatal day (P)1, 7, and 14, a period characterized by a spur of neuronal networking, evaluating the effect of mesenchymal stem cell (MSCs)-treatment. The issue was investigated with a rat model of global PA, mimicking a clinical risk occurring under labor. PA was induced by immersing fetus-containing uterine horns into a water bath for 21 min (AS), using sibling-caesarean-delivered fetuses (CS) as controls. Two hours after delivery, AS and CS neonates were injected with either 5 μL of vehicle (10% plasma) or 5 × 104 MSCs into the lateral ventricle. Samples were assayed for myelin-basic protein (MBP) levels; Olig-1/Olig-2 transcriptional factors; Gglial phenotype; neuroinflammation, and delayed cell death. The main effects were observed at P7, including: (i) A decrease of MBP-immunoreactivity in external capsule, corpus callosum, cingulum, but not in fimbriae of hippocampus; (ii) an increase of Olig-1-mRNA levels; (iii) an increase of IL-6-mRNA, but not in protein levels; (iv) an increase in cell death, including OLs; and (v) MSCs treatment prevented the effect of PA on myelination, OLs number, and cell death. The present findings show that PA induces regional- and developmental-dependent changes on myelination and OLs maturation. Neonatal MSCs treatment improves survival of mature OLs and myelination in telencephalic white matter. Full article
(This article belongs to the Special Issue Brain Hypoxia: Mechanisms of Resilience and Tolerance)
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<p>Effect of perinatal asphyxia (PA) and neonatal development on myelination at P1, P7, and P14, measured in external capsule (<b>A</b>); corpus callosum (<b>B</b>); cingulum (<b>C</b>) and fimbriae of hippocampus (<b>D</b>) of rat neonates. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red) and DAPI (blue; nuclei)-positive cells in external capsule (1A); corpus callosum (1B); cingulum (1C); and fimbriae of hippocampus (1D) from control (CS) and asphyxia-exposed (AS) rat neonates. Microphotographs show MBP, indicating both myelinated fibers and mature oligodendrocytes (OLs). Scale bar: 20 μm. At P1, no MBP immunoreactivity was observed in any of the analyzed regions and experimental conditions. The density of MBP increased significantly along development. At P7, the density of MBP fibers (white head arrows) was low, letting us visualize individual mature OL (white arrows). In corpus callosum and fimbriae of hippocampus some individual OL can also be seen, showing long and branched processes. In AS, there was a decrease in the density of MBP in external capsule, corpus callosum, and cingulum compared to that in CS. No differences could be seen in fimbriae of hippocampus at P7. At P14 a dense network of MBP fibers can be seen in all regions, but no independent OLs soma can be distinguished.</p> Full article ">Figure 1 Cont.
<p>Effect of perinatal asphyxia (PA) and neonatal development on myelination at P1, P7, and P14, measured in external capsule (<b>A</b>); corpus callosum (<b>B</b>); cingulum (<b>C</b>) and fimbriae of hippocampus (<b>D</b>) of rat neonates. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red) and DAPI (blue; nuclei)-positive cells in external capsule (1A); corpus callosum (1B); cingulum (1C); and fimbriae of hippocampus (1D) from control (CS) and asphyxia-exposed (AS) rat neonates. Microphotographs show MBP, indicating both myelinated fibers and mature oligodendrocytes (OLs). Scale bar: 20 μm. At P1, no MBP immunoreactivity was observed in any of the analyzed regions and experimental conditions. The density of MBP increased significantly along development. At P7, the density of MBP fibers (white head arrows) was low, letting us visualize individual mature OL (white arrows). In corpus callosum and fimbriae of hippocampus some individual OL can also be seen, showing long and branched processes. In AS, there was a decrease in the density of MBP in external capsule, corpus callosum, and cingulum compared to that in CS. No differences could be seen in fimbriae of hippocampus at P7. At P14 a dense network of MBP fibers can be seen in all regions, but no independent OLs soma can be distinguished.</p> Full article ">Figure 2
<p>Effect of perinatal asphyxia (PA) on glial cells at P7, measured in external capsule (<b>A</b>); corpus callosum (<b>B</b>); cingulum (<b>C</b>); and fimbriae of hippocampus (<b>D</b>) of rat neonates. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red), glial fibrillary acidic protein (GFAP; red), ionized calcium binding adaptor molecule 1 (Iba-1; green) and DAPI (blue; nuclei)-positive cells in external capsule (2A); corpus callosum (2B); cingulum (2C); and fimbriae of hippocampus (2D), from control (CS) and asphyxia exposed (AS) rat neonates. White arrows show mature oligodendrocyte (OL), astrocyte, and microglia phenotype. Scale bar: 20 μm. At P7, the number of MBP-DAPI+ cells/mm<sup>3</sup> decreased after PA in external capsule, corpus callosum, and cingulum, but not in fimbriae of the hippocampus.</p> Full article ">Figure 2 Cont.
<p>Effect of perinatal asphyxia (PA) on glial cells at P7, measured in external capsule (<b>A</b>); corpus callosum (<b>B</b>); cingulum (<b>C</b>); and fimbriae of hippocampus (<b>D</b>) of rat neonates. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red), glial fibrillary acidic protein (GFAP; red), ionized calcium binding adaptor molecule 1 (Iba-1; green) and DAPI (blue; nuclei)-positive cells in external capsule (2A); corpus callosum (2B); cingulum (2C); and fimbriae of hippocampus (2D), from control (CS) and asphyxia exposed (AS) rat neonates. White arrows show mature oligodendrocyte (OL), astrocyte, and microglia phenotype. Scale bar: 20 μm. At P7, the number of MBP-DAPI+ cells/mm<sup>3</sup> decreased after PA in external capsule, corpus callosum, and cingulum, but not in fimbriae of the hippocampus.</p> Full article ">Figure 3
<p>Effect of MSCs treatment on cell death induced by perinatal asphyxia (PA), measured at P7 in external capsule (<b>A</b>); corpus callosum (<b>B</b>); and cingulum (<b>C</b>) of rat neonates. Representative microphotographs obtained by confocal microscopy showing TUNEL (green), DAPI (nuclei, blue)-positive cells in external capsule (3A); corpus callosum (3B); and cingulum (3C) from vehicle- and MSCs-treated control (CS) and asphyxia-exposed (AS) neonates. Scale bar: 20 μm. (<b>A</b>–<b>C</b>). The number of TUNEL-DAPI cell/mm<sup>3</sup> is increased when comparing vehicle-treated AS versus CS neonates, but the number of TUNEL-DAPI cell/mm<sup>3</sup> is decreased in MSCs- versus vehicle-treated AS rat neonates in all evaluated regions.</p> Full article ">Figure 3 Cont.
<p>Effect of MSCs treatment on cell death induced by perinatal asphyxia (PA), measured at P7 in external capsule (<b>A</b>); corpus callosum (<b>B</b>); and cingulum (<b>C</b>) of rat neonates. Representative microphotographs obtained by confocal microscopy showing TUNEL (green), DAPI (nuclei, blue)-positive cells in external capsule (3A); corpus callosum (3B); and cingulum (3C) from vehicle- and MSCs-treated control (CS) and asphyxia-exposed (AS) neonates. Scale bar: 20 μm. (<b>A</b>–<b>C</b>). The number of TUNEL-DAPI cell/mm<sup>3</sup> is increased when comparing vehicle-treated AS versus CS neonates, but the number of TUNEL-DAPI cell/mm<sup>3</sup> is decreased in MSCs- versus vehicle-treated AS rat neonates in all evaluated regions.</p> Full article ">Figure 4
<p>Effect of MSCs treatment on myelination and mature oligodendrocyte (OLs) injury induced by perinatal asphyxia (PA), measured at P7 in external capsule (<b>A</b>), and cingulum (<b>B</b>) of control (CS) and (AS) rats. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red) and DAPI (blue; nuclei)-positive cells in external capsule (4A); and cingulum (4B) from control (CS) and asphyxia-exposed (AS) rat neonates, including vehicle and MSCs treated groups. Microphotographs show MBP, indicating myelinated fibers (white head arrows) and mature oligodendrocytes (OL) (white arrows). Scale bar: 20 μm. The density of MBP and number of MBP-DAPI cells/mm<sup>3</sup> is increased in MSCs- versus vehicle-treated AS neonates in all evaluated regions.</p> Full article ">
31 pages, 5795 KiB  
Article
Behavioral Alterations and Decreased Number of Parvalbumin-Positive Interneurons in Wistar Rats after Maternal Immune Activation by Lipopolysaccharide: Sex Matters
by Iveta Vojtechova, Kristyna Maleninska, Viera Kutna, Ondrej Klovrza, Klara Tuckova, Tomas Petrasek and Ales Stuchlik
Int. J. Mol. Sci. 2021, 22(6), 3274; https://doi.org/10.3390/ijms22063274 - 23 Mar 2021
Cited by 22 | Viewed by 4247
Abstract
Maternal immune activation (MIA) during pregnancy represents an important environmental factor in the etiology of schizophrenia and autism spectrum disorders (ASD). Our goal was to investigate the impacts of MIA on the brain and behavior of adolescent and adult offspring, as a rat [...] Read more.
Maternal immune activation (MIA) during pregnancy represents an important environmental factor in the etiology of schizophrenia and autism spectrum disorders (ASD). Our goal was to investigate the impacts of MIA on the brain and behavior of adolescent and adult offspring, as a rat model of these neurodevelopmental disorders. We injected bacterial lipopolysaccharide (LPS, 1 mg/kg) to pregnant Wistar dams from gestational day 7, every other day, up to delivery. Behavior of the offspring was examined in a comprehensive battery of tasks at postnatal days P45 and P90. Several brain parameters were analyzed at P28. The results showed that prenatal immune activation caused social and communication impairments in the adult offspring of both sexes; males were affected already in adolescence. MIA also caused prepulse inhibition deficit in females and increased the startle reaction in males. Anxiety and hypolocomotion were apparent in LPS-affected males and females. In the 28-day-old LPS offspring, we found enlargement of the brain and decreased numbers of parvalbumin-positive interneurons in the frontal cortex in both sexes. To conclude, our data indicate that sex of the offspring plays a crucial role in the development of the MIA-induced behavioral alterations, whereas changes in the brain apparent in young animals are sex-independent. Full article
(This article belongs to the Special Issue Neuroinflammation: The Pathogenic Mechanism of Neurological Disorders)
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<p>Brain morphology in P28-old rats. (<b>a</b>) Areas of four different brain sections were larger in both males and females of lipopolysaccharide (LPS)-treated dams compared to controls, as three-way analysis of variance with repeated measures (3wANOVA-RM) confirmed. In addition, males had larger brain area than females. (<b>b</b>) Cortical thickness in the LPS-exposed offspring was not significantly different from the control offspring, as it was shown by the two-way ANOVA (2wANOVA), but there was the sex effect, as males had thicker cortex than females. (<b>c</b>,<b>d</b>) Ventricles area (<b>c</b>) and dorsal hippocampal area (<b>d</b>) shown as a percentage of whole brain area, both analyzed by 2wANOVA, were not significantly affected by prenatal exposure to LPS. Scale bars show 2 mm. In the graphs in panels (<b>b</b>–<b>d</b>), each boxplot shows an average value for the left and right hemispheres. The boxplots show median, first and third quartile and minimum and maximum values; the dots show individual values. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001, ns = no significance. cc = corpus callosum; ctx = cortex; hipp = hippocampus.</p> Full article ">Figure 2
<p>Parvalbumin-positive (PV+) interneurons and microglia in P28-old rats. (<b>a</b>) Number of PV+ interneurons was decreased in the upper part of the frontal cortex of both hemispheres (area 1), with the higher difference found in the left hemisphere, in both males and females prenatally exposed to LPS, as 2wANOVA analyses showed. Moreover, there was a sex difference with males showing higher density of PV+ interneurons, specifically in the left hemisphere. The graph shows averaged values from left and right hemispheres separately, and for an average of both. (<b>b</b>,<b>c</b>) The difference between groups in the number of PV+ interneurons located in lower part of the frontal cortex (area 2) (<b>b</b>), as well as in the dorsal hippocampus (<b>c</b>) was not significant, according to 2wANOVA. However, males in general showed lower number of PV+ interneurons in the dorsal hippocampus compared to females, more profound in CA1-3 areas than the dentate gyrus. (<b>d</b>) The number of microglia in the dentate gyrus of the dorsal hippocampus was also not affected by prenatal experience with LPS, according to 2wANOVA. The boxplots show median, first and third quartile, and minimum and maximum values; the dots show individual values. * <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, ns = no significance. ctx = cortex; DG = dentate gyrus; hipp = hippocampus.</p> Full article ">Figure 3
<p>Social behavior in P45 and P90 rats. (<b>a</b>) Duration of non-anogenital social contact was lower in males of LPS-treated dams than males of control dams at P45 as well as P90, as shown by 3wANOVA-RM. However, LPS females differed from control females neither at P45 nor at P90. In addition, sex difference measured by 2wANOVA for P45 showed that males participated in non-anogenital social contacts for longer time than females in adolescence, but not in adulthood. (<b>b</b>) Duration of anogenital exploration was not shown to be affected by LPS. Adult animals spent more time by anogenital exploration than adolescents, as shown by 3wANOVA-RM. The boxplots show median, first and third quartile, and minimum and maximum values; the dots show individual values. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>Ultrasonic vocalization (USV) in P45 and P90 rats. (<b>a</b>) In adolescence, a distinct effect of LPS on males and females was found in total number of calls. 2wANOVA revealed that LPS males emitted less vocalizations compared to control males, but LPS females emitted a higher amount of vocalization compared to control females. In adulthood, the difference was not present. In addition, males emitted a higher total number of calls than females. Adult animals produced much more vocalizations than adolescents. (<b>b</b>) Trill-like USV was not affected by LPS in any age. However, males emitted a higher number of trill-like elements than females, and also adult rats had a higher number of trill-like elements in comparison to adolescents. (<b>c</b>,<b>d</b>) Average duration of a simple call did not differ between groups. However, adult females prenatally exposed to LPS showed a shorter average duration of a composite call than control females, as 2wANOVA revealed. 3wANOVA-RM also revealed longer average duration of simple calls and composite calls in males in comparison to females. Average duration of simple calls was significantly higher in adult animals than adolescents; in the composite calls, the same tendency was present as a strong trend. (<b>e</b>) The proportion of simple versus composite calls was changed in LPS-exposed rats, as shown by 3wANOVA-RM and 2wANOVA. LPS-exposed rats of both sexes, especially at P90, spent shorter time by emission of composite calls than simple calls, compared to controls. In addition, P45 males spent a longer time emitting the composite calls, relative to the simple calls than P45 females. (<b>f</b>) Examples of analyzed types of USV: simple calls, trill-like elements, composite calls. The boxplots show median, first and third quartile, and minimum and maximum values; the dots show individual values. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>The beam walking test in P45 and P90 rats. LPS-exposed rats of both sexes spent less time by crossing the wide or narrow beam compared to controls at both ages, according to 3wANOVA-RM. Males took more time to cross the narrow beam than females. Adult rats spent less time traversing the wide beam than adolescent rats. The upper panel show data averaged only for whole-beam crossings. The boxplots show median, first and third quartile, and minimum and maximum values; the dots show individual values. In the x axes of the lower panels, the numbers “1/4” and ”1/2” mean training phase (0.5 or 1 m, respectively, of the beam) and ”1” means the test phase (the whole 2-m long beam). The values shown in the lower panels indicate means ± S.E.M. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p>Non-social behavior in P45 and P90 rats. (<b>a</b>) In the open field test (OF), the rats of both sexes prenatally exposed to LPS spent less time in the center of the arena in comparison to saline-exposed rats, as revealed by 3wANOVA-RM with simple effects and 2wANOVA, but only in adulthood, not in adolescence. (<b>b</b>) Total walked distance measured in OF was changed differently at P45 and P90 by prenatal exposure to LPS. In adolescence, LPS males did not differ from control males; in contrast, LPS females walked a longer distance than control females. In adulthood, both LPS males and females tended to walk a shorter distance than the control animals. In addition, males showed decreased locomotor activity compared to females as well as adults compared to adolescents. The results were evaluated by 3wANOVA-RM and 2wANOVA. (<b>c</b>,<b>d</b>) In the elevated plus maze test (EPM), the proportion of open arm visits to total arm visits (<b>c</b>) and frequency of risk assessment behavior (<b>d</b>) were not significantly affected by LPS administration to pregnant dams. However, sex differences were found by 3wANOVA-RM: males had lower open arm visits ratio and also lower risk assessment behavior compared to females. Similar result was found in adult rats in comparison to adolescents. (<b>e</b>) Activity in EPM was not changed by LPS experience at P45, but it slightly decreased at P90 in LPS males as well as females, as shown by 3wANOVA-RM and as a trend by 2wANOVA. In males, the activity was also decreased in comparison to females. (<b>f</b>) LPS-exposed animals showed a higher startle response to strong acoustic stimuli (120 dB), compared to controls, according to 2wANOVA. The difference was more manifested in males. Moreover, the analysis revealed a higher startle response in males compared to females. (<b>g</b>) Prepulse inhibition deficit was found by 2wANOVA and <span class="html-italic">t</span>-tests in LPS females, especially after strong stimulus with 80-dB prepulse (PP), but not in LPS males. The boxplots show median, first and third quartile, and minimum and maximum values; the dots show individual values. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 7
<p>(<b>a</b>) The design of the experiment. In rat females, estrous cycle was determined for approximately one week. Estral females were mated with a male for 24 h. During pregnancy, females received six subcutaneous (s.c.) injections of lipopolysaccharide (LPS) at a dose of 1 mg/kg, or 0.9% saline solution (control), from embryonic day E7 to E21 (delivery), every other day. The offspring of both sexes were used for brain analysis at postnatal day P28 (weaning) or for the behavioral testing in adolescence (P45), which was repeated in adulthood (P90). (<b>b</b>) The structure of a bacterial LPS; according to [<a href="#B36-ijms-22-03274" class="html-bibr">36</a>].</p> Full article ">Figure A1
<p>Pregnancy success. Weight gain of LPS- and saline-treated females in comparison to non-pregnant females. LPS administration did not negatively affect gaining of weight.</p> Full article ">Figure A2
<p>The beam walking test. In general, the number of footslips was very low in all animals, and the difference between LPS-exposed and control rats was revealed neither at P45, nor at P90, by the negative binomial model with log estimates values. In addition, sex differences were not significant.</p> Full article ">
17 pages, 4049 KiB  
Article
Regulatory Role of Sugars on the Settlement Inducing Activity of a Conspecific Cue in Pacific Oyster Crassostrea gigas
by Mary Grace Sedanza, Hee-Jin Kim, Xerxes Seposo, Asami Yoshida, Kenichi Yamaguchi and Cyril Glenn Satuito
Int. J. Mol. Sci. 2021, 22(6), 3273; https://doi.org/10.3390/ijms22063273 - 23 Mar 2021
Cited by 5 | Viewed by 3310
Abstract
This study evaluated the larval settlement inducing effect of sugars and a conspecific cue from adult shell extract of Crassostrea gigas. To understand how the presence of different chemical cues regulate settlement behavior, oyster larvae were exposed to 12 types of sugars, [...] Read more.
This study evaluated the larval settlement inducing effect of sugars and a conspecific cue from adult shell extract of Crassostrea gigas. To understand how the presence of different chemical cues regulate settlement behavior, oyster larvae were exposed to 12 types of sugars, shell extract-coated and non-coated surfaces, and under varied sugar exposure times. Lectin-glycan interaction effects on settlement and its localization on oyster larval tissues were investigated. The results showed that the conspecific cue elicited a positive concentration dependent settlement inducing trend. Sugars in the absence of a conspecific cue, C. gigas adult shell extract, did not promote settlement. Whereas, in the presence of the cue, showed varied effects, most of which were found inhibitory at different concentrations. Sugar treated larvae exposed for 2 h showed significant settlement inhibition in the presence of a conspecific cue. Neu5Ac, as well as GlcNAc sugars, showed a similar interaction trend with wheat germ agglutinin (WGA) lectin. WGA-FITC conjugate showed positive binding on the foot, velum, and mantle when exposed to GlcNAc sugars. This study suggests that a WGA lectin-like receptor and its endogenous ligand are both found in the larval chemoreceptors and the shell Ethylenediaminetetraacetic acid (EDTA) extract that may complementarily work together to allow the oyster larva greater selectivity during site selection. Full article
(This article belongs to the Special Issue Natural Compounds with Cancer-Selective Toxicity)
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<p>Settlement percentages of <span class="html-italic">C. gigas</span> larvae on different substrates coated with varying amounts of CgSE after 24 h. Asterisks (*) denote significant differences in amount coated on different substrates, using 0 µg as the baseline, determined via quasi-binomial glm (<span class="html-italic">p</span> &lt; 0.05, <span class="html-italic">n</span> = 6, using different batches of larvae).</p> Full article ">Figure 2
<p>Settlement percentages of sugar treated <span class="html-italic">C. gigas</span> larvae exposed to different mono- and di-saccharides at different concentrations (10<sup>−10</sup>, 10<sup>−8</sup>, 10<sup>−6</sup>, 10<sup>−4</sup> M). Settlement percentages in (<b>A</b>) non-coated surfaces and (<b>B</b>) CgSE-coated surfaces. Untreated oysters in filtered seawater (C-FSW) and untreated oysters exposed to shell extract only (C-CgSE) served as control. CgSE (50 µg) was applied to all coated surfaces. A sugar-specific statistical analysis was performed. Asterisks (*) indicate significantly different with respect to untreated oysters exposed to FSW alone (C-FSW, <a href="#ijms-22-03273-f002" class="html-fig">Figure 2</a>A) and CgSE alone (C-CgSE, <a href="#ijms-22-03273-f002" class="html-fig">Figure 2</a>B), determined via quasi-binomial glm (<span class="html-italic">p</span> &lt; 0.05). Missing bars in the figure indicate no settlement. Data are the means (SEM) of six replicates using different batches of larvae.</p> Full article ">Figure 3
<p>Settlement percentages of <span class="html-italic">C. gigas</span> larvae in response to different sugars and exposure times (0.25, 2, and 24 h) in the presence of CgSE. Following each exposure time, the larvae were thoroughly rinsed with filtered seawater and were incubated for 24 h in CgSE-coated wells. Larvae treated to a 24 h sugar exposure period was continuously immersed in CgSE-coated wells. Asterisks (*) indicate significantly inhibiting groups, determined via quasi-binomial glm (α= 0.05, <span class="html-italic">n</span> = 6, using different batches of larvae).</p> Full article ">Figure 4
<p>Settlement percentages in CgSE-treated GF/C filter papers when exposed to: WGA-binding sugars, (<b>A</b>) GlcNAc and (<b>B</b>) Neu5Ac, under varying concentrations of sugar treatment alone (shaded bars), and under WGA-GlcNAc or WGA-Neu5Ac mixture treatments (striped bars), for 2 h prior to assay. Adsorbed CgSE on GF/C filter papers alone served as a control (unshaded bars). Asterisks (*) denote a significant inhibiting effect on settlement, compared with other treatments determined via quasi-binomial glm (<span class="html-italic">p</span> &lt; 0.05). Data are means (SEM) of three replicates.</p> Full article ">Figure 5
<p>WGA binding to GlcNAc moieties on the mantle, foot, and velum tissues of <span class="html-italic">C. gigas</span> larvae stain green under epifluorescence view at 100× magnification and 200× magnification (plate <b>D</b> inset only). (<b>A</b>,<b>B</b>) GlcNAc sugar treated larvae at 10<sup>−4</sup> M show weakly stained mantle, foot, and velum tissues. (<b>C</b>,<b>D</b>) GlcNAc sugar treated oyster larva at 10<sup>−10</sup> M shows an intense binding stain on the mantle, velum, and foot tissues, as well as its cilia (inset photo). (<b>E</b>,<b>F</b>) Untreated oyster larva (positive control) with WGA-FITC conjugated lectin shows binding stain on the mantle and foot tissues. (<b>G</b>,<b>H</b>) Untreated larvae (negative control) without WGA-FITC staining indicates that the larvae do not exhibit autofluorescence. Abbreviations: f = foot, m = mantle, v = velum. Scale bar = 100 µm.</p> Full article ">
41 pages, 2234 KiB  
Review
A Mitocentric View of the Main Bacterial and Parasitic Infectious Diseases in the Pediatric Population
by Sonia Romero-Cordero, Richard Kirwan, Antoni Noguera-Julian, Francesc Cardellach, Clàudia Fortuny and Constanza Morén
Int. J. Mol. Sci. 2021, 22(6), 3272; https://doi.org/10.3390/ijms22063272 - 23 Mar 2021
Cited by 3 | Viewed by 3970
Abstract
Infectious diseases occur worldwide with great frequency in both adults and children. Both infections and their treatments trigger mitochondrial interactions at multiple levels: (i) incorporation of damaged or mutated proteins to the complexes of the electron transport chain, (ii) mitochondrial genome (depletion, deletions, [...] Read more.
Infectious diseases occur worldwide with great frequency in both adults and children. Both infections and their treatments trigger mitochondrial interactions at multiple levels: (i) incorporation of damaged or mutated proteins to the complexes of the electron transport chain, (ii) mitochondrial genome (depletion, deletions, and point mutations) and mitochondrial dynamics (fusion and fission), (iii) membrane potential, (iv) apoptotic regulation, (v) generation of reactive oxygen species, among others. Such alterations may result in serious adverse clinical events with great impact on children’s quality of life, even resulting in death. As such, bacterial agents are frequently associated with loss of mitochondrial membrane potential and cytochrome c release, ultimately leading to mitochondrial apoptosis by activation of caspases-3 and -9. Using Rayyan QCRI software for systematic reviews, we explore the association between mitochondrial alterations and pediatric infections including (i) bacterial: M. tuberculosis, E. cloacae, P. mirabilis, E. coli, S. enterica, S. aureus, S. pneumoniae, N. meningitidis and (ii) parasitic: P. falciparum. We analyze how these pediatric infections and their treatments may lead to mitochondrial deterioration in this especially vulnerable population, with the intention of improving both the understanding of these diseases and their management in clinical practice. Full article
(This article belongs to the Special Issue Mitochondrial Function and Communication)
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<p>Simplified general summary of the main mitochondrial metabolic pathways. ADP, adenosine diphosphate; ATP, adenosine triphosphate; I, complex I; II, complex II; III, complex III; IV, complex IV; CoQ, coenzyme Q; CytC, cytochrome C; FADH, flavin and adenine dinucleotide; H+, proton; NADH, nicotinamide adenine dinucleotide hydrogen; OXPHOS, oxidative phosphorylation system; TCA, tricarboxylic acid and V, V complex.</p> Full article ">Figure 2
<p>Mitochondrial respiratory chain and oxidative phosphorylation system in the mitochondria. ADP, adenosine diphosphate; ATP, adenosine triphosphate; I, complex I; II, complex II; III, complex III; IV, complex IV; V, complex V; CoQ, coenzyme Q; CytC, cytochrome C; e, electrons; FADH, flavin and adenine dinucleotide; H+, proton; NADH, nicotinamide adenine dinucleotide hydrogen and OXPHOS, oxidative phosphorylation system.</p> Full article ">Figure 3
<p>Mitochondrial anaerobiosis state during mitochondrial dysfunction. ADP, adenosine diphosphate; ATP, adenosine triphosphate; I, complex I; II, complex II; III, complex III; IV, complex IV; V, complex V; CoQ, coenzyme Q; CytC, cytochrome C; FADH, flavin and adenine dinucleotide hydrogen; H+, proton; NADH, nicotinamide adenine dinucleotide hydrogen; OXPHOS, oxidative phosphorylation system; TCA, tricarboxylic acid.</p> Full article ">Figure 4
<p>Types of caspases: classification and main functions. Initiator caspases, including CARD [<a href="#B17-ijms-22-03272" class="html-bibr">17</a>] and DED [<a href="#B18-ijms-22-03272" class="html-bibr">18</a>], and effector caspases, including procaspases, as well as their functions are represented. CARD, caspase activation and recruitment domain; DED, death effector domain.</p> Full article ">Figure 5
<p>Replication stages in malaria infection. The sporozoites enter the circulation after the mosquito’s bite. They are then transported through the blood to hepatocytes in the liver, initiating the exo-red cell cycle. There, they rapidly multiply within hepatocytes by multiple cycles of asexual division and transform into merozoites that enter the bloodstream and leave the liver. Merozoites invade red blood cells, initiating the erythrocytic cycle. As the nucleus begins to divide, the trophozoite is now called a developing schizont. The mature schizont contains merozoites that are released into the bloodstream. Although many merozoites are destroyed by the immune system, others immediately invade red blood cells, in which a new cycle of erythrocytic schizogony begins. After several generations of erythrocytes, male and female gametocytes develop from some merozoites (sexual cycle). With the union of the gametes, the egg is generated in the mosquito’s intestine. The egg is mobile and will give rise to an oocyst that will divide again and give sporozoites.</p> Full article ">
14 pages, 2327 KiB  
Article
Characterisation of Novel Angiogenic and Potent Anti-Inflammatory Effects of Micro-Fragmented Adipose Tissue
by Baoqiang Guo, Xenia Sawkulycz, Nima Heidari, Ralph Rogers, Donghui Liu and Mark Slevin
Int. J. Mol. Sci. 2021, 22(6), 3271; https://doi.org/10.3390/ijms22063271 - 23 Mar 2021
Cited by 12 | Viewed by 3408
Abstract
Adipose tissue and more specifically micro-fragmented adipose tissue (MFAT) obtained from liposuction has recently been shown to possess interesting medicinal properties whereby its application supports pain reduction and may enhance tissue regeneration particularly in osteoarthritis. Here we have characterised samples of MFAT produced [...] Read more.
Adipose tissue and more specifically micro-fragmented adipose tissue (MFAT) obtained from liposuction has recently been shown to possess interesting medicinal properties whereby its application supports pain reduction and may enhance tissue regeneration particularly in osteoarthritis. Here we have characterised samples of MFAT produced using the Lipogems® International Spa system from eight volunteer individuals in order to understand the critical biological mechanisms through which they act. A variation was found in the MFAT cluster size between individual samples and this translated into a similar variation in the ability of purified mesenchymal stem cells (MSCs) to form colony-forming units. Almost all of the isolated cells were CD105/CD90/CD45+ indicating stemness. An analysis of the secretions of cytokines from MFAT samples in a culture using targeted arrays and an enzyme-linked immunosorbent assay (ELISA) showed a long-term specific and significant expression of proteins associated with anti-inflammation (e.g., interleukin-1 receptor alpha (Il-1Rα) antagonist), pro-regeneration (e.g., hepatocyte growth factor), anti-scarring and pro-angiogenesis (e.g., transforming growth factor beta 1 and 2 (TGFβ1/2) and anti-bacterial (e.g., chemokine C-X-C motif ligand-9 (CXCL-9). Angiogenesis and angiogenic signalling were notably increased in primary bovine aortic endothelial cells (BAEC) to a different extent in each individual sample of the conditioned medium whilst a direct capacity of the conditioned medium to block inflammation induced by lipopolysaccharides was shown. This work characterises the biological mechanisms through which a strong, long-lasting, and potentially beneficial effect can be observed regarding pain reduction, protection and regeneration in osteoarthritic joints treated with MFAT. Full article
(This article belongs to the Special Issue Adipose Stem Cells 3.0)
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<p>Micro-fragmented adipose tissue (MFAT) indicated most of the clusters were medium sized. (<b>A</b>); MFAT image of clusters seen under the microscope (×40). (<b>B</b>); The number of medium sized clusters (0.3–0.75 mm in diameter) from MFAT of six patients was significantly higher than the smaller sized clusters (&lt;0.3 mm) and large sized clusters (&gt;0.75 mm) (<span class="html-italic">p</span> &lt; 0.01) in a 10 field measured at ×40 magnification per sample.</p> Full article ">Figure 2
<p>The numbers of fibroblastoid colony-forming unit (CFU-F) colonies of 0.25 mL of MFAT from the six patients in <a href="#ijms-22-03271-f001" class="html-fig">Figure 1</a> indicated a large variation. A total of 0.25 mL MFAT was digested with collagenase in MEMα and then cultured in a MEMα complete medium for 10 days, stained with Giemsa and fixed with 100% methanol. (<b>A</b>); shows colonies observed with the naked eye. (<b>B</b>); the mean numbers of colonies ± SD. (<b>C</b>,<b>D</b>) show the magnified appearance of an individual colony under microscopy (×40).</p> Full article ">Figure 3
<p>Phenotype of passage 4 of AD-MSCs from patients’ MFAT. Almost all of the cells (&gt;98%) indicated CD90+CD45-, CD73+CD45-, CD105+CD45- showing stem cell/ mesenchymal stem cell (MSC) origin and capacity. One sample only shown here.</p> Full article ">Figure 4
<p>Cytokines and growth factors in the MFAT condition medium in a serum free culture for one day and five days. (<b>A</b>)<b>;</b> shows an example of a heat map of 32 factors after a serum free culture of MFAT (carried out on 7/8 of the donors). With multiplex analysis, 50 µL of the MFAT serum free culture condition medium was detected for 32 cytokines and growth factors. Other heat maps and raw data are shown in <a href="#app1-ijms-22-03271" class="html-app">Supplementary Tables S1 and S2</a>. (<b>B</b>); There was a significant increase in IL-1Ra between one day and five-day culturing in serum free MEMα (<span class="html-italic">p</span> &lt; 0.05 *). (<b>C</b>); There was a significant increase in CXCL9 between one day and five-day culturing in serum free MEMα (<span class="html-italic">p</span> &lt; 0.05 *). (<b>D</b>); The expression of HGF was found but this remained stable between one day and five-day culturing (<span class="html-italic">p</span> &gt; 0.05 *). (<b>E</b>); MIF indicated significantly higher in five-day culturing than in one day cultures (<span class="html-italic">p</span> &lt; 0.05). (<b>F</b>–<b>G</b>); TGFβ1-3 secretion significantly increased in the MFAT serum free culture condition medium on day five (&lt;0.01 **). Similarly, TGFβ3 secretion significantly increased in the MFAT serum free culture condition medium on day five as well (&lt;0.01 **).</p> Full article ">Figure 4 Cont.
<p>Cytokines and growth factors in the MFAT condition medium in a serum free culture for one day and five days. (<b>A</b>)<b>;</b> shows an example of a heat map of 32 factors after a serum free culture of MFAT (carried out on 7/8 of the donors). With multiplex analysis, 50 µL of the MFAT serum free culture condition medium was detected for 32 cytokines and growth factors. Other heat maps and raw data are shown in <a href="#app1-ijms-22-03271" class="html-app">Supplementary Tables S1 and S2</a>. (<b>B</b>); There was a significant increase in IL-1Ra between one day and five-day culturing in serum free MEMα (<span class="html-italic">p</span> &lt; 0.05 *). (<b>C</b>); There was a significant increase in CXCL9 between one day and five-day culturing in serum free MEMα (<span class="html-italic">p</span> &lt; 0.05 *). (<b>D</b>); The expression of HGF was found but this remained stable between one day and five-day culturing (<span class="html-italic">p</span> &gt; 0.05 *). (<b>E</b>); MIF indicated significantly higher in five-day culturing than in one day cultures (<span class="html-italic">p</span> &lt; 0.05). (<b>F</b>–<b>G</b>); TGFβ1-3 secretion significantly increased in the MFAT serum free culture condition medium on day five (&lt;0.01 **). Similarly, TGFβ3 secretion significantly increased in the MFAT serum free culture condition medium on day five as well (&lt;0.01 **).</p> Full article ">Figure 5
<p>The pro-angiogenic effect of the MFAT serum free culture condition medium collected at day one and day five. The day five conditioned medium indicated a higher pro-angiogenic effect on the production of tube-like structures in BAEC than day one (<span class="html-italic">p</span> &lt; 0.05) (<b>A</b>,<b>B</b>). Western blotting in (<b>C</b>,<b>D</b>) shows the increased notable relative p-ERK expression in the conditioned medium of MFAT cultures after 24 h with a reduction in the day five conditioned medium. Lanes: (individual samples labelled as L) 1: Negative control (DEM, basal medium), 2: Positive control (EBM2, completed medium), 3: L1 24 h, 4: L2 24 h, 5: L3 24 h, 6: L4 24 h, 7: L5 24 h, 8: L1 five days, 9: L2 five days, 10: L3 five days, 11: L4 five days, 12: L5 five days.</p> Full article ">Figure 6
<p>The inhibitory effect of the MFAT condition medium on the LPS mediated cytokines secretion MFAT conditioned medium indicated a significant inhibition on LPS (10 ng/mL) mediated IL-1ß1 secretion (<span class="html-italic">p</span> &lt; 0.01 **). (<b>A</b>); the MFAT condition medium indicated a significant inhibition of LPS (10 ng/mL) mediated IL-1β (<span class="html-italic">p</span> &lt; 0.01 **). (<b>B</b>); Il-6 expression. n = 5 samples tested from the day five conditioned medium of MFAT cultures by an ELISA.</p> Full article ">
16 pages, 2859 KiB  
Article
Chemically and Green Synthesized ZnO Nanoparticles Alter Key Immunological Molecules in Common Carp (Cyprinus carpio) Skin Mucus
by Ghasem Rashidian, Carlo C. Lazado, Heba H. Mahboub, Ramin Mohammadi-Aloucheh, Marko D. Prokić, Hend S. Nada and Caterina Faggio
Int. J. Mol. Sci. 2021, 22(6), 3270; https://doi.org/10.3390/ijms22063270 - 23 Mar 2021
Cited by 76 | Viewed by 4926
Abstract
This study was conducted to compare the effects of commercially available (C) and green synthesized (GS) Zinc oxide nanoparticles (ZnO-NPs) on immunological responses of common carp (Cyprinus carpio) skin mucus. GS ZnO-NPs were generated using Thymus pubescent and characterized by UV–vis [...] Read more.
This study was conducted to compare the effects of commercially available (C) and green synthesized (GS) Zinc oxide nanoparticles (ZnO-NPs) on immunological responses of common carp (Cyprinus carpio) skin mucus. GS ZnO-NPs were generated using Thymus pubescent and characterized by UV–vis diffuse reflectance spectroscopy (DRS), Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), scanning electron microscope (SEM), and energy-dispersive X-ray spectroscopy (EDX). Fish (n = 150) were randomly allocated into five groups in triplicate and received a waterborne concentration of 0% (control), 25%, and 50% of LC50 96 h of commercially available (C1 and C2) and green synthesized ZnO-NPs (GS1 and GS2) for 21 days. Results from XRD displayed ZnO-NPs with 58 nm in size and UV-vis DRS, EDX, and FT-IR analysis showed that some functional groups from plant extract bonded to the surface of NPs. The SEM images showed that ZnO-NPs have conical morphology. Acute toxicity study showed a higher dose of LC5096h for green synthesized ZnO-NPs (78.9 mg.L−1) compared to the commercial source (59.95 mg.L−1). The highest activity of lysozyme and alternative complement activity (ACH50) were found in control and GS1 groups. A significant decrease in alkaline phosphatase activity (ALP) was found in C1 and C2 groups compared to other treatments. Protease activity (P) was significantly decreased in the C2 group compared to the control and GS groups. Total immunoglobulin (total Ig) content was the highest in the control. In addition, total Ig in the GS1 group was higher than GS2. The exposure to ZnO-NPs lowered total protein content in all experimental groups when compared to control. Present findings revealed lower induced immunosuppressive effects by green synthesized ZnO-NPs on key parameters of fish skin mucus. Full article
(This article belongs to the Special Issue Fish Mucosal Physiology and Immunology)
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<p>X-ray powder diffraction (XRD) patterns of commercial (C) and green synthesized (GS) ZnO-NPs.</p> Full article ">Figure 2
<p>Energy-dispersive X-ray spectroscopy (EDX) spectra of commercial (C) and green synthesized (GS) ZnO-NPs.</p> Full article ">Figure 3
<p>Scanning electron microscope (SEM) image of green synthesized ZnO-NPs.</p> Full article ">Figure 4
<p>Fourier-transform infrared spectroscopy (FTIR) spectra of <span class="html-italic">Thymus pubescent</span> extract, commercial (C) and green synthesized (GS) ZnO-NPs.</p> Full article ">Figure 5
<p>UV–vis diffuse reflectance spectroscopy (DRS) spectra of commercial (C) and green synthesized (GS) ZnO-NPs.</p> Full article ">Figure 6
<p>The cumulative mortality of common carp exposed to different levels of commercial and green synthesized ZnO-NPs for 96 h (30 fish for each concentration).</p> Full article ">Figure 7
<p>Correspondence analysis ordination plot and position of the five analyzed groups (control, GS1, GS2, C1, and C2) to the analyzed immune parameters.</p> Full article ">Figure 8
<p>Fabrication scheme of ZnO-NPs from Zinc nitrate using leaf extract of plant <span class="html-italic">Thymus pubescens.</span></p> Full article ">
15 pages, 4542 KiB  
Article
Cardiac Protective Effect of Kirenol against Doxorubicin-Induced Cardiac Hypertrophy in H9c2 Cells through Nrf2 Signaling via PI3K/AKT Pathways
by Abdullah M. Alzahrani, Peramaiyan Rajendran, Vishnu Priya Veeraraghavan and Hamza Hanieh
Int. J. Mol. Sci. 2021, 22(6), 3269; https://doi.org/10.3390/ijms22063269 - 23 Mar 2021
Cited by 25 | Viewed by 4026
Abstract
Kirenol (KRL) is a biologically active substance extracted from Herba Siegesbeckiae. This natural type of diterpenoid has been widely adopted for its important anti-inflammatory and anti-rheumatic properties. Despite several studies claiming the benefits of KRL, its cardiac effects have not yet been clarified. [...] Read more.
Kirenol (KRL) is a biologically active substance extracted from Herba Siegesbeckiae. This natural type of diterpenoid has been widely adopted for its important anti-inflammatory and anti-rheumatic properties. Despite several studies claiming the benefits of KRL, its cardiac effects have not yet been clarified. Cardiotoxicity remains a key concern associated with the long-term administration of doxorubicin (DOX). The generation of reactive oxygen species (ROS) causes oxidative stress, significantly contributing to DOX-induced cardiac damage. The purpose of the current study is to investigate the cardio-protective effects of KRL against apoptosis in H9c2 cells induced by DOX. The analysis of cellular apoptosis was performed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining assay and measuring the modulation in the expression levels of proteins involved in apoptosis and Nrf2 signaling, the oxidative stress markers. Furthermore, Western blotting was used to determine cell survival. KRL treatment, with Nrf2 upregulation and activation, accompanied by activation of PI3K/AKT, could prevent the administration of DOX to induce cardiac oxidative stress, remodeling, and other effects. Additionally, the diterpenoid enhanced the activation of Bcl2 and Bcl-xL, while suppressing apoptosis marker proteins. As a result, KRL is considered a potential agent against hypertrophy resulting from cardiac deterioration. The study results show that KRL not only activates the IGF-IR-dependent p-PI3K/p-AKT and Nrf2 signaling pathway, but also suppresses caspase-dependent apoptosis. Full article
(This article belongs to the Special Issue Biomolecular Mediators in Cardiomyopathies)
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<p>Kirenol (KRL)-attenuated DOX-induced cytotoxicity in H9c2 cardiac cells. (<b>A</b>) Chemical structure of kirenol. (<b>B</b>,<b>C</b>) Effect of KRL on the viability of H9c2 cardiomyocytes examined by an MTT assay. H9c2 cells were treated with increasing concentrations of KRL (2.5–20 μmol) for 24 and 48 h, respectively. Cell viability (%) was measured as follows: (A570 of treated cells/A570 of untreated cells) × 100. (<b>D</b>,<b>E</b>) The cell viability with increasing concentrations of DOX (0.1–1 μmol) for 24 and 48 h, respectively. (<b>F</b>,<b>G</b>) The effects of KRL along with DOX on the cell viability of H9c2 cells were determined by MTT assay. H9c2 cells were cultured in serum free media for 3 h followed by the treatment with KRL for 2 h before or after DOX treatment, respectively. Data are represented as the mean ± SD of triplicate values (<span class="html-italic">n</span> = 3) and * <span class="html-italic">p</span> &lt; 0.05 represents significant variations compared with the control. # <span class="html-italic">p</span> &lt; 0.05 represents significant variations as compared to DOX alone and KRL with DOX treatment groups.</p> Full article ">Figure 2
<p>Effects of KRL on DOX-induced hypertrophy. H9c2 cells were treated for 24 h with 15 µM of KRL, 2 h before DOX (0.25 µmol) treatment. (<b>A</b>) Representative Western blots showing changes in the protein levels of ANP and BNP. (<b>B</b>) KRL is illustrated to downregulate MMP9 and MMP2. Western blot analysis was performed to determine the total protein MMP9 and MMp2 levels in the total extract by including β-actin as an internal loading control. (<b>C</b>) H9c2 cells were treated for 24 h with 10 and 15 µM of KRL, 2 h before the DOX (0.25 µmol) treatment. The cells then underwent actin filament staining to observe the changes in the surface area of the cardiomyocytes. Scale bar indicated 100µ m at 20× magnification. Data are represented as the mean ± SD of triplicate values (<span class="html-italic">n</span> = 3) and * <span class="html-italic">p</span> &lt; 0.05 represents significant variations compared with the control. # <span class="html-italic">p</span> &lt; 0.05 represents significant variations as compared to DOX alone and KRL with DOX treatment groups.</p> Full article ">Figure 3
<p>KRL enhances cell survival mechanism. H9c2 cells were cultured in serum-free media for 3 h, followed by treatment with KRL for 2 h before or after DOX treatment, respectively. (<b>A</b>,<b>B</b>) KRL activates the IGF1R-mediated survival pathway in H9c2 cells. The expression of p-IGF1R, PI3K, p-PI3K, AKT, and p-AKT was analyzed by Western blotting. β-actin was used as the internal control. (<b>C</b>) Representative Western blots showing the changes in MAPK signaling proteins (pP38 and pJNK) in H9c2 cells. Data are represented as the mean ± SD of triplicate values (<span class="html-italic">n</span> = 3) and * <span class="html-italic">p</span> &lt; 0.05 represents significant variations compared with the control. # <span class="html-italic">p</span> &lt; 0.05 represents significant variations as compared to DOX alone and KRL with DOX treatment groups.</p> Full article ">Figure 4
<p>KRL prevents DOX-induced cardiac oxidative stress. (<b>A</b>) Cells were treated with KRL (15 μmol) for 2 h prior to DOX (0.25 µmol) stimulation for 24 h. Changes in 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (4-HNE) protein levels of mitochondria were monitored by Western blotting. (<b>B</b>) The activation of total Nrf2, HO-1, and NQO-1 was examined by Western blotting. Data are represented as the mean ± SD of triplicate values (<span class="html-italic">n</span> = 3) and * <span class="html-italic">p</span> &lt; 0.05 represents significant variations compared with the control. # <span class="html-italic">p</span> &lt; 0.05 represents significant variations as compared to DOX alone and KRL with DOX treatment groups.</p> Full article ">Figure 5
<p>KRL enhances NRf2 translocation in DOX-treated H9c2 cells. (<b>A</b>) Changes in Nrf2 levels were determined in nuclear and cytosolic fractions of cells. Relative changes in protein intensities were quantified using Image Studio Lite software and presented as a histogram, with the control set at one-fold. (<b>B</b>) Cells subjected to reactive oxygen species (ROS) inhibitor treatment (1 mM NAC for 60 min) before KRL treatment for 24 h. The expression of total Nrf2 was analyzed by Western blotting. (<b>C</b>) Cells were pre-treated with KRL (15 μmol) for 2 h and then stimulated with or without DOX (0.25 µmol) for 24 h. Immunofluorescence staining was performed to detect the nuclear localization of Nrf2. Following incubation with primary antibody (anti-Nrf2) and conjugated secondary antibody, cells were stained with DAPI (1 μg/mL) for 5 min. The subcellular localization of Nrf2 in all conditions was visualized under fluorescence microscopy. Scale bar indicated 100µ m at 20× magnification. (<b>D</b>) Indicated Nrf2 immunofluorescence intensity levels of Control and treated H9c2 cells. Data are represented as the mean ± SD of triplicate values (<span class="html-italic">n</span> = 3) and * <span class="html-italic">p</span> &lt; 0.05 represents significant variations compared with the control. # <span class="html-italic">p</span> &lt; 0.05 represents significant variations as compared to DOX alone and KRL with DOX treatment groups.</p> Full article ">Figure 6
<p>KRL inhibits DOX-induced apoptosis in cardiomyocytes. (<b>A</b>) Cells were pretreated with 15 µmol of KRL for 2 h followed by DOX (0.25 µmol for 24 h). The expression levels of anti-apoptotic Bcl2 and Bcl-xL and apoptotic proteins cleaved caspase-3 activation and PARP cleavage were measured by the Western blot method. (<b>B</b>) Apoptotic nuclei were detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and the nuclei were detected by DAPI staining, showing modulations in apoptotic levels with DOX against and with KRL. Scale bar indicated 100 µm at 20× magnification. Data are represented as the mean ± SD of triplicate values (<span class="html-italic">n</span> = 3) and * <span class="html-italic">p</span> &lt; 0.05 represents significant variations compared with the control. # <span class="html-italic">p</span> &lt; 0.05 represents significant variations as compared to DOX alone and KRL with DOX treatment groups.</p> Full article ">
12 pages, 1941 KiB  
Article
Vaporized Hydrogen Peroxide and Ozone Gas Synergistically Reduce Prion Infectivity on Stainless Steel Wire
by Hideyuki Hara, Junji Chida, Agriani Dini Pasiana, Keiji Uchiyama, Yutaka Kikuchi, Tomoko Naito, Yuichi Takahashi, Junji Yamamura, Hisashi Kuromatsu and Suehiro Sakaguchi
Int. J. Mol. Sci. 2021, 22(6), 3268; https://doi.org/10.3390/ijms22063268 - 23 Mar 2021
Cited by 2 | Viewed by 2760
Abstract
Prions are infectious agents causing prion diseases, which include Creutzfeldt–Jakob disease (CJD) in humans. Several cases have been reported to be transmitted through medical instruments that were used for preclinical CJD patients, raising public health concerns on iatrogenic transmissions of the disease. Since [...] Read more.
Prions are infectious agents causing prion diseases, which include Creutzfeldt–Jakob disease (CJD) in humans. Several cases have been reported to be transmitted through medical instruments that were used for preclinical CJD patients, raising public health concerns on iatrogenic transmissions of the disease. Since preclinical CJD patients are currently difficult to identify, medical instruments need to be adequately sterilized so as not to transmit the disease. In this study, we investigated the sterilizing activity of two oxidizing agents, ozone gas and vaporized hydrogen peroxide, against prions fixed on stainless steel wires using a mouse bioassay. Mice intracerebrally implanted with prion-contaminated stainless steel wires treated with ozone gas or vaporized hydrogen peroxide developed prion disease later than those implanted with control prion-contaminated stainless steel wires, indicating that ozone gas and vaporized hydrogen peroxide could reduce prion infectivity on wires. Incubation times were further elongated in mice implanted with prion-contaminated stainless steel wires treated with ozone gas-mixed vaporized hydrogen peroxide, indicating that ozone gas mixed with vaporized hydrogen peroxide reduces prions on these wires more potently than ozone gas or vaporized hydrogen peroxide. These results suggest that ozone gas mixed with vaporized hydrogen peroxide might be more useful for prion sterilization than ozone gas or vaporized hydrogen peroxide alone. Full article
(This article belongs to the Special Issue Prions and Prion Diseases 2.0)
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<p>Ozone gas and vaporized hydrogen peroxide synergistically reduce prion infectivity of stainless steel wires. (<b>A</b>) Schematic representations of the sterilization protocols for ozone gas mixed with vaporized hydrogen peroxide, ozone gas alone, and vaporized hydrogen peroxide alone in the ET (endotoxin) mode. For sterilization with ozone gas mixed with vaporized hydrogen peroxide, RML prion-contaminated stainless steel wires were pre-treated by injection of 25,000 ppm ozone gas for 3 min and followed by 6 cycles of a 10 min sterilization process, which comprises injection of 45% hydrogen peroxide followed by injection of 25,000 ppm ozone gas and 3% hydrogen peroxide 5 min later. For the treatment with ozone gas alone or vaporized hydrogen peroxide alone, water was used instead of hydrogen peroxide or ozone gas, respectively. The sterilization process of each mode is terminated by injection of air. (<b>B</b>) The percentage of symptom-free mice after intracerebral inoculation with 1% brain homogenate (1% BH) from RML-infected, diseased mice and intracerebral implantation with gas-unexposed, ozone gas-exposed, vaporized hydrogen peroxide-exposed, and ozone gas mixed with vaporized hydrogen peroxide-exposed, RML prion-contaminated wires. O<sub>3</sub>, ozone; H<sub>2</sub>O<sub>2</sub>, hydrogen peroxide; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 2
<p>Western blotting for PrP<sup>Sc</sup> in the brains of mice implanted with various gas-treated, RML prion-contaminated stainless steel wires. Brain homogenates from mice uninfected with RML prions (<span class="html-italic">n</span> = 4), implanted with gas-untreated, RML prion-contaminated stainless steel wires (<span class="html-italic">n</span> = 6), ozone gas-treated, RML prion-contaminated stainless steel wires (<span class="html-italic">n</span> = 6), vaporized hydrogen peroxide-treated, RML prion-contaminated wires (<span class="html-italic">n</span> = 5), and ozone gas mixed with vaporized hydrogen peroxide-treated, RML prion-contaminated wires (<span class="html-italic">n</span> = 7) were treated with or without proteinase K (PK) and subjected to Western blotting with 6D11 anti-PrP antibody. β-actin is an internal control for Western blotting. O<sub>3</sub>, ozone; H<sub>2</sub>O<sub>2</sub>, hydrogen peroxide.</p> Full article ">Figure 3
<p>Brain pathologies in mice implanted with various gas-treated, RML prion-contaminated stainless steel wires. Representative pictures of HE-stained (<b>A</b>) and PrP<sup>Sc</sup>-stained (<b>B</b>) brain sections from mice uninfected with RML prions (<span class="html-italic">n</span> = 4, 10-week old), implanted with gas-untreated, RML prion-contaminated stainless steel wires (<span class="html-italic">n</span> = 4), ozone gas-treated, RML prion-contaminated stainless steel wires (<span class="html-italic">n</span> = 4), vaporized hydrogen peroxide-treated, RML prion-contaminated wires (<span class="html-italic">n</span> = 4), and ozone gas mixed with vaporized hydrogen peroxide-treated, RML prion-contaminated wires (<span class="html-italic">n</span> = 3) are shown. O<sub>3</sub>, ozone; H<sub>2</sub>O<sub>2</sub>, hydrogen peroxide. Cx, cerebral cortex; Hp, hippocampus; Th, thalamus; Cb, cerebellum. Scale bar, 100 μm.</p> Full article ">Figure 4
<p>Sterilization of RML prions fixed on stainless steel wires in different sterilization modes. (<b>A</b>) Schematic representations of the sterilization protocols of ozone gas mixed with vaporized hydrogen peroxide in the standard, the short, and the long modes. RML prions-fixed wires were pre-treated by injection of 25,000 ppm ozone gas for 3 min and followed by 2 cycles of a 14-min sterilization process comprising injection of 45% hydrogen peroxide followed by injection of 25,000 ppm ozone gas and 3% hydrogen peroxide 5 min later in the standard mode, a 7-min sterilization process comprising injection of 45% hydrogen peroxide followed by injection of 25,000 ppm ozone gas and 3% hydrogen peroxide 5 min later in the short mode, and a 19.5-min sterilization process comprising injection of 45% hydrogen peroxide followed by injection of 25,000 ppm ozone gas and 45% hydrogen peroxide 6.5 min later in the long mode. The sterilization process of each mode was terminated by injection of air. (<b>B</b>) The percentage of symptom-free mice after intracerebral implantation with RML prion-contaminated stainless steel wires exposed to ozone gas mixed with vaporized hydrogen peroxide-exposed in the standard, the short, and the long modes. O<sub>3</sub>, ozone; H<sub>2</sub>O<sub>2</sub>, hydrogen peroxide; * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; n.s., not significant.</p> Full article ">
32 pages, 1731 KiB  
Review
Secreted Extracellular Vesicle Molecular Cargo as a Novel Liquid Biopsy Diagnostics of Central Nervous System Diseases
by Sara Monteiro-Reis, Carina Carvalho-Maia, Genevieve Bart, Seppo J. Vainio, Juliana Pedro, Eunice R. Silva, Goreti Sales, Rui Henrique and Carmen Jerónimo
Int. J. Mol. Sci. 2021, 22(6), 3267; https://doi.org/10.3390/ijms22063267 - 23 Mar 2021
Cited by 15 | Viewed by 4248
Abstract
Secreted extracellular vesicles (EVs) are heterogeneous cell-derived membranous granules which carry a large diversity of molecules and participate in intercellular communication by transferring these molecules to target cells by endocytosis. In the last decade, EVs’ role in several pathological conditions, from etiology to [...] Read more.
Secreted extracellular vesicles (EVs) are heterogeneous cell-derived membranous granules which carry a large diversity of molecules and participate in intercellular communication by transferring these molecules to target cells by endocytosis. In the last decade, EVs’ role in several pathological conditions, from etiology to disease progression or therapy evasion, has been consolidated, including in central nervous system (CNS)-related disorders. For this review, we performed a systematic search of original works published, reporting the presence of molecular components expressed in the CNS via EVs, which have been purified from plasma, serum or cerebrospinal fluid. Our aim is to provide a list of molecular EV components that have been identified from both nonpathological conditions and the most common CNS-related disorders. We discuss the methods used to isolate and enrich EVs from specific CNS-cells and the relevance of its components in each disease context. Full article
(This article belongs to the Special Issue Role and Dynamics of Extracellular Vesicles in Central Nervous System Diseases)
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<p>Flow diagram representing a summary of the conducted methodology for this review.</p> Full article ">Figure 2
<p>Schematic representation of the brain microenvironment, its main cellular components and how the brain–blood-barrier permeability allows for extracellular vesicles to reach circulation. Created with BioRender.com.</p> Full article ">Figure 3
<p>Schematic representation of the use of extracellular vesicles in liquid biopsies for identification of specific central nervous system (CNS)-related pathological conditions. Created with BioRender.com (accessed on 20 March 2021).</p> Full article ">
19 pages, 4196 KiB  
Article
An Arabidopsis Oxalyl-CoA Decarboxylase, AtOXC, Is Important for Oxalate Catabolism in Plants
by Justin Foster, Ninghui Cheng, Vincent Paris, Lingfei Wang, Jin Wang, Xiaoqiang Wang and Paul A. Nakata
Int. J. Mol. Sci. 2021, 22(6), 3266; https://doi.org/10.3390/ijms22063266 - 23 Mar 2021
Cited by 9 | Viewed by 3263
Abstract
Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well-studied enzyme capable of [...] Read more.
Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well-studied enzyme capable of degrading oxalate, but not all plants possess this activity. Recently, acyl-activating enzyme 3 (AAE3), encoding an oxalyl-CoA synthetase, was identified in Arabidopsis. This enzyme has been proposed to catalyze the first step in an alternative pathway of oxalate degradation. Since this initial discovery, this enzyme and proposed pathway have been found to be important to other plants and yeast as well. In this study, we identify, in Arabidopsis, an oxalyl-CoA decarboxylase (AtOXC) that is capable of catalyzing the second step in this proposed pathway of oxalate catabolism. This enzyme breaks down oxalyl-CoA, the product of AtAAE3, into formyl-CoA and CO2. AtOXC:GFP localization suggested that this enzyme functions within the cytosol of the cell. An Atoxc knock-down mutant showed a reduction in the ability to degrade oxalate into CO2. This reduction in AtOXC activity resulted in an increase in the accumulation of oxalate and the enzyme substrate, oxalyl-CoA. Size exclusion studies suggest that the enzyme functions as a dimer. Computer modeling of the AtOXC enzyme structure identified amino acids of predicted importance in co-factor binding and catalysis. Overall, these results suggest that AtOXC catalyzes the second step in this alternative pathway of oxalate catabolism. Full article
(This article belongs to the Section Molecular Plant Sciences)
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<p>Proposed pathway of oxalate catabolism. AtAAE3, acyl-activating enzyme 3, which possesses an oxalyl-CoA synthetase activity [<a href="#B21-ijms-22-03266" class="html-bibr">21</a>], AtOXC, oxalyl-CoA decarboxylase (this study), and enzymes catalyzing the last two steps remain to be determined.</p> Full article ">Figure 2
<p>Comparison of the predicted amino acid sequences of OXC from plants and bacteria. Sequence alignment of OXCs from <span class="html-italic">Arabidopsis thaliana</span>, <span class="html-italic">Zea mays</span>, <span class="html-italic">Oxalobacter formigenes</span>, and <span class="html-italic">E. coli</span>. The secondary structure elements observed in the AtOXC modeled structure are shown above the alignment. Conserved residues are highlighted.</p> Full article ">Figure 3
<p>Biochemical analysis of AtOXC. (<b>A</b>) SDS-PAGE gel of nickel-affinity-purified His-AtOXC protein (<b>left</b>) and molecular weight markers (<b>right</b>). (<b>B</b>) HPLC analysis of enzyme reaction mix without and with added AtOXC. (<b>C</b>) Size exclusion chromatography of AtOXC using a Superdex 200 Increase 10/300 GL column. A dimeric form of AtOXC was detected during gel filtration using an ÄKTA purifier (GE Healthcare).</p> Full article ">Figure 4
<p>Molecular modeling of AtOXC. (<b>A</b>) A modeled structure of AtOXC docked with cofactors ThDP and Mg<sup>2+</sup> ion, and activator ADP. Both ThDP (<b>green</b>) and ADP (<b>red</b>) are shown as stick models, and Mg<sup>2+</sup> ion is shown as a sphere model in green. (<b>B</b>) Binding site of cofactors ThDP and Mg<sup>2+</sup> ion. ThDP is shown as an orange stick model. Selected protein residues within the binding pocket are labeled and shown as stick models in cyan for residues from one subunit and yellow for residues from other subunit, and Mg<sup>2+</sup> ion is shown as a gray sphere model. (<b>C</b>) Dimeric model of AtOXC. The two monomers are shown in green and cyan, respectively.</p> Full article ">Figure 5
<p><span class="html-italic">AtOXC</span> expression in plants. <span class="html-italic">AtOXC</span>::Gus staining in 1-week-old seedlings (<b>A</b>), primary root (<b>B</b>), and lateral root (<b>C</b>). <span class="html-italic">AtOXC</span>::Gus staining in 2-week-old seedlings (<b>D</b>), primary root (<b>E</b>), and lateral roots (<b>F</b>,<b>G</b>). <span class="html-italic">AtOXC</span>::Gus staining in mature leaves (<b>H</b>,<b>I</b>), stem (<b>J</b>), and flower (<b>K</b>). (<b>L</b>) qRT-PCR analysis of <span class="html-italic">AtOXC</span> expression in different tissues of Arabidopsis. Ubiquitin 10 (<span class="html-italic">UBQ10)</span> was used as an internal control. Student’s <span class="html-italic">t-</span>test, <span class="html-italic">n</span> = 6, ** <span class="html-italic">p</span> &lt; 0.01, indicating a significant difference between various tissues vs. roots.</p> Full article ">Figure 6
<p>Subcellular localization of AtOXC-GFP. AtOXC-GFP expression in leaves of <span class="html-italic">A. thaliana</span> (<b>left</b>), chloroplast autofluorescence (<b>middle</b>), and merge of AtOXC-GFP and autofluorescence (<b>right</b>). Bar = 10 µm.</p> Full article ">Figure 7
<p>Measurement of oxalate degradation to CO<sub>2</sub>. (<b>A</b>) Relative <span class="html-italic">AtOXC</span> transcript levels in leaves of <span class="html-italic">AtOXC</span> knock-down mutant compared to WT as measured by qRT-PCR. (<b>B</b>) Radiolabeled CO<sub>2</sub> measurements. WT and <span class="html-italic">AtOXC</span> knock-down leaf pieces were fed with 2.5 µCi of [<sup>14</sup>C]-oxalate along with 300 µM non-labeled oxalate. The <sup>14</sup>CO<sub>2</sub> evolved was captured using 1 M KOH and the relative radioactivity was measured.</p> Full article ">Figure 8
<p>Assessment of oxalate and oxalyl-CoA accumulation in seeds. (<b>A</b>) Comparison of the calcium oxalate crystal phenotypes in seeds from WT and AtOXC knock-down plants. Crystals are bright spots denoted by arrows. Bar = 200 µm. (<b>B</b>) Total oxalate in seeds from WT and AtOXC knock-down plants. (<b>C</b>) Assessment of oxalyl-CoA accumulation in WT and AtOXC knock-down plants.</p> Full article ">Figure 9
<p>Response to exogenous application of oxalate. (<b>A</b>) WT and <span class="html-italic">AtOXC</span> knock-down mutant phenotypes in response to external oxalate exposure. (<b>B</b>) Accumulation of oxalyl-CoA accumulation in WT and <span class="html-italic">AtOXC</span> knock-down mutant leaves after external oxalate exposure.</p> Full article ">
26 pages, 1199 KiB  
Review
The Role of Innate and Adaptive Immune Cells in Skeletal Muscle Regeneration
by Natalia Ziemkiewicz, Genevieve Hilliard, Nicholas A. Pullen and Koyal Garg
Int. J. Mol. Sci. 2021, 22(6), 3265; https://doi.org/10.3390/ijms22063265 - 23 Mar 2021
Cited by 52 | Viewed by 9741
Abstract
Skeletal muscle regeneration is highly dependent on the inflammatory response. A wide variety of innate and adaptive immune cells orchestrate the complex process of muscle repair. This review provides information about the various types of immune cells and biomolecules that have been shown [...] Read more.
Skeletal muscle regeneration is highly dependent on the inflammatory response. A wide variety of innate and adaptive immune cells orchestrate the complex process of muscle repair. This review provides information about the various types of immune cells and biomolecules that have been shown to mediate muscle regeneration following injury and degenerative diseases. Recently developed cell and drug-based immunomodulatory strategies are highlighted. An improved understanding of the immune response to injured and diseased skeletal muscle will be essential for the development of therapeutic strategies. Full article
(This article belongs to the Section Biochemistry)
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<p>Skeletal muscle regeneration is dependent on the inflammatory response. Following acute injuries, the pro-inflammatory cells support satellite cell proliferation, while anti-inflammatory cells support differentiation (<b>top</b>). In chronic injuries, persistent inflammation impairs satellite cell activity resulting in muscle wasting and fibrosis (<b>bottom</b>). Adapted from [<a href="#B7-ijms-22-03265" class="html-bibr">7</a>].</p> Full article ">Figure 2
<p>Inflammatory cells of the innate and adaptive immune system participate in the process of muscle regeneration and repair. Blue arrows indicate immune cells recruiting each other through the secretion of various soluble mediators.</p> Full article ">
33 pages, 1435 KiB  
Review
Genetics of Azoospermia
by Francesca Cioppi, Viktoria Rosta and Csilla Krausz
Int. J. Mol. Sci. 2021, 22(6), 3264; https://doi.org/10.3390/ijms22063264 - 23 Mar 2021
Cited by 74 | Viewed by 12529
Abstract
Azoospermia affects 1% of men, and it can be due to: (i) hypothalamic-pituitary dysfunction, (ii) primary quantitative spermatogenic disturbances, (iii) urogenital duct obstruction. Known genetic factors contribute to all these categories, and genetic testing is part of the routine diagnostic workup of azoospermic [...] Read more.
Azoospermia affects 1% of men, and it can be due to: (i) hypothalamic-pituitary dysfunction, (ii) primary quantitative spermatogenic disturbances, (iii) urogenital duct obstruction. Known genetic factors contribute to all these categories, and genetic testing is part of the routine diagnostic workup of azoospermic men. The diagnostic yield of genetic tests in azoospermia is different in the different etiological categories, with the highest in Congenital Bilateral Absence of Vas Deferens (90%) and the lowest in Non-Obstructive Azoospermia (NOA) due to primary testicular failure (~30%). Whole-Exome Sequencing allowed the discovery of an increasing number of monogenic defects of NOA with a current list of 38 candidate genes. These genes are of potential clinical relevance for future gene panel-based screening. We classified these genes according to the associated-testicular histology underlying the NOA phenotype. The validation and the discovery of novel NOA genes will radically improve patient management. Interestingly, approximately 37% of candidate genes are shared in human male and female gonadal failure, implying that genetic counselling should be extended also to female family members of NOA patients. Full article
(This article belongs to the Special Issue Novel Physiology and Molecular Pathology of Reproduction, Novel Treatments of Infertility)
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<p>Diagnostic yield of genetic testing in azoospermia with different etiology: (<b>a</b>) Congenital Hypogonadotropic Hypogonadism; (<b>b</b>) Non-Obstructive Azoospermia due to primary testicular failure, after the exclusion of all know acquired causes; (<b>c</b>) Congenital Bilateral Absence of Vas Deferens. Abbreviations: AZF—Azoospermia Factor Region; CBAVD—Congenital Bilateral Absence of Vas Deferens; CHH—Congenital Hypogonadotropic Hypogonadism; NOA—Non-Obstructive Azoospermia; * See Reviews [<a href="#B7-ijms-22-03264" class="html-bibr">7</a>,<a href="#B8-ijms-22-03264" class="html-bibr">8</a>]; ** 47,XXY Klinefelter syndrome, 46,XX male syndrome, Yq’-‘; *** See articles [<a href="#B9-ijms-22-03264" class="html-bibr">9</a>,<a href="#B10-ijms-22-03264" class="html-bibr">10</a>,<a href="#B11-ijms-22-03264" class="html-bibr">11</a>].</p> Full article ">Figure 2
<p>Semen phenotype and TESE outcomes of the different types of AZF microdeletion. Abbreviations: AZF—Azoospermia Factor Region; Cen—centromere; PAR1—Pseudoautosomal Region 1; PAR2—Pseudoautosomal Region 2; <span class="html-italic">SRY</span>—Sex-determining Region Y gene; TESE—Testicular Sperm Extraction.</p> Full article ">
26 pages, 9347 KiB  
Review
A Review of Ex Vivo X-ray Microfocus Computed Tomography-Based Characterization of the Cardiovascular System
by Lisa Leyssens, Camille Pestiaux and Greet Kerckhofs
Int. J. Mol. Sci. 2021, 22(6), 3263; https://doi.org/10.3390/ijms22063263 - 23 Mar 2021
Cited by 14 | Viewed by 4951
Abstract
Cardiovascular malformations and diseases are common but complex and often not yet fully understood. To better understand the effects of structural and microstructural changes of the heart and the vasculature on their proper functioning, a detailed characterization of the microstructure is crucial. In [...] Read more.
Cardiovascular malformations and diseases are common but complex and often not yet fully understood. To better understand the effects of structural and microstructural changes of the heart and the vasculature on their proper functioning, a detailed characterization of the microstructure is crucial. In vivo imaging approaches are noninvasive and allow visualizing the heart and the vasculature in 3D. However, their spatial image resolution is often too limited for microstructural analyses, and hence, ex vivo imaging is preferred for this purpose. Ex vivo X-ray microfocus computed tomography (microCT) is a rapidly emerging high-resolution 3D structural imaging technique often used for the assessment of calcified tissues. Contrast-enhanced microCT (CE-CT) or phase-contrast microCT (PC-CT) improve this technique by additionally allowing the distinction of different low X-ray-absorbing soft tissues. In this review, we present the strengths of ex vivo microCT, CE-CT and PC-CT for quantitative 3D imaging of the structure and/or microstructure of the heart, the vasculature and their substructures in healthy and diseased state. We also discuss their current limitations, mainly with regard to the contrasting methods and the tissue preparation. Full article
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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<p>Schematic representation of the structure of this review. In <a href="#sec2-ijms-22-03263" class="html-sec">Section 2</a>, we provide a general description of X-ray microfocus computed tomography (microCT), contrast-enhanced microCT (CE-CT) and phase-contrast microCT (PC-CT). The third section describes the use of microCT for imaging the heart with three different parts: (<b>A</b>) the whole heart, (<b>B</b>) the morphometrical assessment of the myocardium (adapted from Reference [<a href="#B31-ijms-22-03263" class="html-bibr">31</a>]) and (<b>C</b>) the heart valves. <a href="#sec4-ijms-22-03263" class="html-sec">Section 4</a> focuses on (<b>D</b>) the spatial distribution and morphometrics of the vasculature (adapted from Reference [<a href="#B5-ijms-22-03263" class="html-bibr">5</a>]) and (<b>E</b>) the vessel wall microstructure. We conclude with the current limitations and future perspectives.</p> Full article ">Figure 2
<p>Whole-heart CE-CT imaging (<a href="#app1-ijms-22-03263" class="html-app">Appendix A</a>). Mouse heart stained with 3.5% (<span class="html-italic">wt/v</span>) Hafnium-substituted Wells-Dawson polyoxometalate (Hf-WD POM) (<b>A</b>–<b>D</b>,<b>K</b>), rinsed in phosphate-buffered saline (PBS) and then stained with 3.5% isotonic Lugol’s iodine (<b>E</b>–<b>H</b>,<b>J</b>). (<b>A</b>,<b>F</b>) 3D renderings with the heart valves in orange and (<b>B</b>–<b>E</b>,<b>G</b>–<b>J</b>) orthogonal slices. (<b>K</b>,<b>L</b>) A zoom-in on the tricuspid valve after Hf-WD POM and isotonic Lugol’s iodine staining, respectively. Arrows indicate blood vessels, and asterisks indicate the tricuspid valve. Scale bars are 1 mm (<b>A</b>,<b>B</b>,<b>D</b>–<b>G</b>,<b>I</b>,<b>J</b>), 0.3 mm (<b>C</b>,<b>H</b>,<b>K</b>,<b>L</b>) and 0.2 mm (<b>M</b>,<b>N</b>).</p> Full article ">Figure 3
<p>Extraction of the fiber orientation in rat hearts based on PC-CT<b>.</b> First column: quantification of the orientation of myocyte aggregates in (<b>A</b>) WKY and (<b>B</b>) LAD hearts: ventricular helical angle maps in four chambers view. White dashed lines were used for additional illustrations. Last three columns: collagen segmentation in high-resolution images. (<b>C</b>–<b>G</b>) Representative PC-CT image slices from subvolumes in the left ventricular septum of the WKY, SHR and ISO hearts, respectively. (<b>D</b>–<b>H</b>) 3D rendering of collagen segmentation in the same subvolumes, visually showing the increase in density and change in shape and distribution around the tissue. Scar tissues can be seen in the ISO specimen. ISO: isoproterenol-treated rats, LAD: left anterior descending artery ligation model, SHR: spontaneously hypertensive rat and WKY: Wistar Kyoto rat model (adapted from and with kind permission from Reference [<a href="#B31-ijms-22-03263" class="html-bibr">31</a>], licensed under the Creative Commons Attribution 4.0 International License [<a href="#B95-ijms-22-03263" class="html-bibr">95</a>]).</p> Full article ">Figure 4
<p>MicroCT imaging of fresh calcified aortic valves (<span class="html-italic">n</span> = 3) from human patients that were explanted during aortic valve replacement (<a href="#app1-ijms-22-03263" class="html-app">Appendix A</a>). (<b>A</b>,<b>C</b>) Cross-sectional 2D microCT image (no contrast enhancement) of samples 1.2 and 2.3, respectively, after the region of interest (ROI) selection. (<b>B</b>,<b>D</b>) 3D rendering of samples 1.2 and 2.3, respectively; calcifications are white and soft tissue red. (<b>E</b>) Volume fraction of the calcifications in the entire valve. Semiautomatic segmentation of the soft tissue and the different densities of calcification were done based on greyscale differences. Scale bars represent 1 mm.</p> Full article ">Figure 5
<p>Microfil-perfused rat kidney vasculature in progressive ischemia/reperfusion (I/R)-induced renal injury. (<b>A</b>) Representative CE-CT renderings of the sham control and I/R days 14, 21 and 56 (2D cross-sectional images in the transversal (I), coronal (II) and sagittal (III) planes, as well as 3D volume renderings). CE-CT-based quantification of (<b>B</b>) the vascular branching points, (<b>C</b>) mean vessel tortuosity and (<b>D</b>) mean vessel diameter in the sham control and I/R days 14, 21 and 56 for the 4th- (Aa. interlobulares) and 5th (afferent arterioles)-order branching points. Progressive rarefaction of the functional vessels and continuous shrinkage of the fibrotic kidneys, as well as an increased vessel tortuosity over time, can be seen. Scale bars are 200 µm. ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001 (adapted from and with kind permission from Reference [<a href="#B5-ijms-22-03263" class="html-bibr">5</a>]).</p> Full article ">Figure 6
<p>Bone marrow vasculature of the tibial metaphysis stained with Hf-WD POM. (<b>A</b>) Young (YNG), (<b>B</b>) old and (<b>C</b>) high-fat diet mice (HFD). Scale bars represent 250 µm. Quantification of the (<b>D</b>) volume fraction of the blood vessels in the medular open volume, (<b>E</b>) average blood vessel thickness, (<b>F</b>) blood vessel density, (<b>G</b>) total number of branches and (<b>H</b>) average branch length. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01 (adapted from and with kind permission from Reference [<a href="#B78-ijms-22-03263" class="html-bibr">78</a>] with permission from Elsevier).</p> Full article ">Figure 7
<p>Abdominal rat aorta stained with Hf-WD POM (<a href="#app1-ijms-22-03263" class="html-app">Appendix A</a>). (<b>A</b>) Cross-sectional 2D CE-CT slice, (<b>B</b>) ROI selection and segmentation of aortic wall from background with an indication of the substructures, (<b>C</b>) segmentation of the media and adventitia and elastic fibers on a 2D ROI slice and (<b>D</b>) a 3D view of the segmentation on the ROI of the aortic wall. Scale bars are 100 µm (<b>A</b>,<b>D</b>) and 50 µm (<b>B</b>,<b>C</b>).</p> Full article ">Figure 8
<p>Paraffin-embedded rat common carotid artery without CESA. Major arterial substructures are readily identifiable in the vessel wall. (<b>A</b>) Virtual slice extracted from an X-ray tomogram of an intact rat common carotid artery (yellow box indicates the magnified region in panel (<b>B</b>). (<b>B</b>) Major arterial substructures. (<b>C</b>) Rendering showing the output of the segmentation process that enables the medial and adventitial layers to be virtually dissected (adapted from and with kind permission from Reference [<a href="#B113-ijms-22-03263" class="html-bibr">113</a>]—licensed under the Creative Commons Attribution 4.0 International License [<a href="#B95-ijms-22-03263" class="html-bibr">95</a>]).</p> Full article ">
17 pages, 1449 KiB  
Review
CircRNA—Protein Interactions in Muscle Development and Diseases
by Shuailong Zheng, Xujia Zhang, Emmanuel Odame, Xiaoli Xu, Yuan Chen, Jiangfeng Ye, Helin Zhou, Dinghui Dai, Bismark Kyei, Siyuan Zhan, Jiaxue Cao, Jiazhong Guo, Tao Zhong, Linjie Wang, Li Li and Hongping Zhang
Int. J. Mol. Sci. 2021, 22(6), 3262; https://doi.org/10.3390/ijms22063262 - 23 Mar 2021
Cited by 46 | Viewed by 6345
Abstract
Circular RNA (circRNA) is a kind of novel endogenous noncoding RNA formed through back-splicing of mRNA precursor. The biogenesis, degradation, nucleus–cytoplasm transport, location, and even translation of circRNA are controlled by RNA-binding proteins (RBPs). Therefore, circRNAs and the chaperoned RBPs play critical roles [...] Read more.
Circular RNA (circRNA) is a kind of novel endogenous noncoding RNA formed through back-splicing of mRNA precursor. The biogenesis, degradation, nucleus–cytoplasm transport, location, and even translation of circRNA are controlled by RNA-binding proteins (RBPs). Therefore, circRNAs and the chaperoned RBPs play critical roles in biological functions that significantly contribute to normal animal development and disease. In this review, we systematically characterize the possible molecular mechanism of circRNA–protein interactions, summarize the latest research on circRNA–protein interactions in muscle development and myocardial disease, and discuss the future application of circRNA in treating muscle diseases. Finally, we provide several valid prediction methods and experimental verification approaches. Our review reveals the significance of circRNAs and their protein chaperones and provides a reference for further study in this field. Full article
(This article belongs to the Section Molecular Biophysics)
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<p>The biogenesis and regulating mechanisms of circular RNA (circRNA). (<b>A</b>–<b>E</b>) CircRNAs are formed by back-splicing into three major types of circRNA. (<b>F</b>–<b>I</b>) Various regulation mechanisms of circRNAs. (<b>J</b>) CircRNA can generate pseudogene by reverse transcription. (<b>K</b>) CircRNA can play a role as a biological molecular marker. (<b>L</b>) CircRNAs can interact with RBPs. IRES: internal ribosome entry site; m<sup>6</sup>A: N<sup>6</sup>-methyladenosine; U1 snRNP: U1 small nuclear ribonucleoprotein.</p> Full article ">Figure 2
<p>The interactions between circRNAs and RNA-binding proteins (RBPs). (<b>A</b>,<b>B</b>) RBPs regulate the synthesis and degradation of circRNA. (<b>C</b>) RBPs are involved in the modification and editing of circRNA. (<b>D</b>) RBPs participate in the transportation of circRNA. (<b>E</b>) RBPs control the translation of circRNA. (<b>F</b>) CircRNAs can serve as RBP supermolecular sponges. (<b>G</b>) CircRNAs participate in the transportation of RBPs. (<b>H</b>) CircRNAs serve as decoys for RBPs. (<b>I</b>) CircRNAs function as scaffolds for RBPs. m<sup>6</sup>A: N<sup>6</sup>-methyladenosine.</p> Full article ">Figure 3
<p>CircRNA–protein interactions regulating muscle development and diseases. (<b>A</b>) PURA and PURB inhibit the transcription of <span class="html-italic">MHC</span>, and circsamd4 promotes myogenesis by binding PURA and PURB. (<b>B</b>) circAMOTL1 induces AKT1 phosphorylation and pAKT1 nuclear transport by combining AKT1 and PDK1. (<b>C</b>) FUS negatively regulates VEGF-A expression, and circFndc3b promotes VEGF-A expression by binding to FUS. (<b>D</b>) circNFIX acts as a protein scaffold to enhance the binding of Ybx1 and Nedd41, induce ubiquitination degradation of Ybx1, and inhibit cyclin A2 and cyclin B1. (<b>E</b>) circYAP can inhibit cardiac fibrosis by acting as a protein scaffold to promote TMP4 and ACTG complexes’ formation. TSS: transcription start site.</p> Full article ">
18 pages, 1584 KiB  
Review
Single-Cell Transcriptomics Supports a Role of CHD8 in Autism
by Anke Hoffmann and Dietmar Spengler
Int. J. Mol. Sci. 2021, 22(6), 3261; https://doi.org/10.3390/ijms22063261 - 23 Mar 2021
Cited by 12 | Viewed by 4381
Abstract
Chromodomain helicase domain 8 (CHD8) is one of the most frequently mutated and most penetrant genes in the autism spectrum disorder (ASD). Individuals with CHD8 mutations show leading symptoms of autism, macrocephaly, and facial dysmorphisms. The molecular and cellular mechanisms underpinning [...] Read more.
Chromodomain helicase domain 8 (CHD8) is one of the most frequently mutated and most penetrant genes in the autism spectrum disorder (ASD). Individuals with CHD8 mutations show leading symptoms of autism, macrocephaly, and facial dysmorphisms. The molecular and cellular mechanisms underpinning the early onset and development of these symptoms are still poorly understood and prevent timely and more efficient therapies of patients. Progress in this area will require an understanding of “when, why and how cells deviate from their normal trajectories”. High-throughput single-cell RNA sequencing (sc-RNAseq) directly quantifies information-bearing RNA molecules that enact each cell’s biological identity. Here, we discuss recent insights from sc-RNAseq of CRISPR/Cas9-editing of Chd8/CHD8 during mouse neocorticogenesis and human cerebral organoids. Given that the deregulation of the balance between excitation and inhibition (E/I balance) in cortical and subcortical circuits is thought to represent a major etiopathogenetic mechanism in ASD, we focus on the question of whether, and to what degree, results from current sc-RNAseq studies support this hypothesis. Beyond that, we discuss the pros and cons of these approaches and further steps to be taken to harvest the full potential of these transformative techniques. Full article
(This article belongs to the Special Issue The Genetic and Mechanistic Basis of Human Neurodevelopmental Disorders)
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<p>The frequency of disruptive de novo variants, including protein truncation variants and missense variants with an MPC (missense badness, PolyPhen-2, constraint) score ≥1 in autism spectrum disorder (ASD)-ascertained and neurodevelopmental delay (NDD)-ascertained cohorts is shown for the 102-associated genes. Fifty-three genes (orange circles) with a higher frequency in ASD are designated ASD-predominant (ASD<sub>P</sub>), while 49 genes with a higher frequency in NDD (light blue circles) are designated NDD-predominant (NDD<sub>P</sub>). The high confidence risk gene <span class="html-italic">CHD8</span> (chromodomain helicase domain 8), boxed in orange, top-ranks among ASD-predominant genes. Graphic adapted from [<a href="#B8-ijms-22-03261" class="html-bibr">8</a>], attribution license 5004260244019.</p> Full article ">Figure 2
<p>Schematic representation of <span class="html-italic">CHD8</span>. The signature motif of the entire CHD family is an N-terminal tandem chromodomain (green boxes) responsible for chromatin binding. The central SNF2-family ATPase domain consists of two lobes (light beige and beige box), with each containing two tandem RecA-like folds parts known as DExx and HELIC. The ATPase domain uses ATP hydrolysis to guide toward translocation down the DNA minor groove. The C-terminus contains functional motifs, such as SANT (light blue) or BRK (blue) domains. SANT domains support association with histone tails, while the BRK domain is also found in several SWI/SNF complexes. The localization of the <span class="html-italic">CHD8</span> loss-of-function mutations <span class="html-italic">S62X</span> and <span class="html-italic">E1114X</span> (see <a href="#sec5dot5-ijms-22-03261" class="html-sec">Section 5.5</a>) are highlighted by red arrows. Drawing is not to scale and refers to the long form of CHD8. Schematic adapted from [<a href="#B21-ijms-22-03261" class="html-bibr">21</a>], attribution CC BY.</p> Full article ">Figure 3
<p>Mid fetal spatio-temporal coexpression network in the human prefrontal cortex. The analysis comprised weeks 13 to 19 post conception. High confidence ASD risk genes (<span class="html-italic">hcASD seed gene</span>) are marked in black with <span class="html-italic">CHD8</span> boxed in orange. Probable ASD risk genes (<span class="html-italic">pASD gene</span>) are shown in grey, and the top 20 genes (<span class="html-italic">Top 20 gene</span>) best correlated with each hcASD gene in white. The lines (edges) represent coexpression correlations ≥0.7; positive correlations are shown in red and negative correlations are shown in blue. Graphic adapted from [<a href="#B51-ijms-22-03261" class="html-bibr">51</a>], attribution license 5004710196206.</p> Full article ">Figure 4
<p>Schematic of in vivo Perturb-Seq analysis of mouse corticogenesis. (<b>A</b>) The lateral ventricles of Cas9<sup>+/</sup><sup>−</sup> embryos were infected at E12.5 with lentiviral particles that contained a lentiviral guide RNA (gRNA) library targeting ASD/NDD risk genes. (<b>B</b>) ASD/NDD risk genes were gene-edited (“knocked-out”) in infected neural progenitor cells. These mutations were passed on to their progeny, including cells of the cortical lineage that form the upper and lower layer of the cortex. (<b>C</b>) At postnatal day 7, cortices were dissected and used for single-cell sequencing. (<b>D</b>) Gene expression modules were affected in a manner dependent on the individual gene perturbation and the specific cell type. Scheme adapted from [<a href="#B59-ijms-22-03261" class="html-bibr">59</a>], attribution license 5004760017864.</p> Full article ">
19 pages, 1002 KiB  
Review
Maintenance of Cell Wall Integrity under High Salinity
by Jianwei Liu, Wei Zhang, Shujie Long and Chunzhao Zhao
Int. J. Mol. Sci. 2021, 22(6), 3260; https://doi.org/10.3390/ijms22063260 - 23 Mar 2021
Cited by 57 | Viewed by 5466
Abstract
Cell wall biosynthesis is a complex biological process in plants. In the rapidly growing cells or in the plants that encounter a variety of environmental stresses, the compositions and the structure of cell wall can be dynamically changed. To constantly monitor cell wall [...] Read more.
Cell wall biosynthesis is a complex biological process in plants. In the rapidly growing cells or in the plants that encounter a variety of environmental stresses, the compositions and the structure of cell wall can be dynamically changed. To constantly monitor cell wall status, plants have evolved cell wall integrity (CWI) maintenance system, which allows rapid cell growth and improved adaptation of plants to adverse environmental conditions without the perturbation of cell wall organization. Salt stress is one of the abiotic stresses that can severely disrupt CWI, and studies have shown that the ability of plants to sense and maintain CWI is important for salt tolerance. In this review, we highlight the roles of CWI in salt tolerance and the mechanisms underlying the maintenance of CWI under salt stress. The unsolved questions regarding the association between the CWI and salt tolerance are discussed. Full article
(This article belongs to the Special Issue Molecular Aspects of Plant Salinity Stress and Tolerance)
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<p>Sensing and maintenance of cell wall integrity under salt stress. Salt stress-induced cell wall changes are proposed to be sensed by multiple receptor-like kinases, including FER, THE1, MIK2, FEI1/2, and WAK1/2. As one of the most important cell wall integrity (CWI) sensors, FER may function alone or together with LRX3/4/5-RALF22/23 module to perceive the perturbation of CWI caused by high salinity. The AHA2-mediated acidification of the apoplastic pH increases the affinity of LRXs with RALFs, while the alkaline state in the apoplast promotes the binding of RALFs with FER. FER and probably also other cell wall sensors convert salt-triggered cell wall signals to multiple intracellular signals, including Ca<sup>2+</sup>, ROS, abscisic acid (ABA), jasmonic acid (JA), and MPKs, which in turn regulate the expression of salt stress-responsive genes in the nucleus. Salt stress can alter the redox status in the apoplast, and RbohD/F-mediated production of the apoplastic H<sub>2</sub>O<sub>2</sub> may affect the cross-linking of cell wall polymers and activate H<sub>2</sub>O<sub>2</sub> sensor HPCA1. Glycosyl inositol phosphorylceramide (GIPC) sphingolipids participate in the sensing of extracellular salt by directly binding to sodium ions. Cell wall biosynthesis- and modification-related components, including pectin methyl esterases (PMEs), PME inhibitors (PMEIs), and cellulose synthase (CesA), are involved in the regulation of salt tolerance in plants. Upon initial exposure to salt stress, cortical microtubules are depolymerized and cellulose synthase complex (CSC) together with its companions CSI1 and CC1/2 are internalized into small CesA compartments/microtubule-associated CesA compartments (smaCCs/MASCs). At the growth recovery stage after salt application, FER is probably required for the regulation of the reassembly of cortical microtubules and the relocation of CSCs to the plasma membrane to synthesize cellulose, which subsequently enhances the adaptation of plants to salt stress. Solid lines represent direct regulations, and dashed lines represent in-direct or potential regulations.</p> Full article ">
24 pages, 2344 KiB  
Review
Structure and Function of Ion Channels Regulating Sperm Motility—An Overview
by Karolina Nowicka-Bauer and Monika Szymczak-Cendlak
Int. J. Mol. Sci. 2021, 22(6), 3259; https://doi.org/10.3390/ijms22063259 - 23 Mar 2021
Cited by 45 | Viewed by 5364
Abstract
Sperm motility is linked to the activation of signaling pathways that trigger movement. These pathways are mainly dependent on Ca2+, which acts as a secondary messenger. The maintenance of adequate Ca2+ concentrations is possible thanks to proper concentrations of other [...] Read more.
Sperm motility is linked to the activation of signaling pathways that trigger movement. These pathways are mainly dependent on Ca2+, which acts as a secondary messenger. The maintenance of adequate Ca2+ concentrations is possible thanks to proper concentrations of other ions, such as K+ and Na+, among others, that modulate plasma membrane potential and the intracellular pH. Like in every cell, ion homeostasis in spermatozoa is ensured by a vast spectrum of ion channels supported by the work of ion pumps and transporters. To achieve success in fertilization, sperm ion channels have to be sensitive to various external and internal factors. This sensitivity is provided by specific channel structures. In addition, novel sperm-specific channels or isoforms have been found with compositions that increase the chance of fertilization. Notably, the most significant sperm ion channel is the cation channel of sperm (CatSper), which is a sperm-specific Ca2+ channel required for the hyperactivation of sperm motility. The role of other ion channels in the spermatozoa, such as voltage-gated Ca2+ channels (VGCCs), Ca2+-activated Cl-channels (CaCCs), SLO K+ channels or voltage-gated H+ channels (VGHCs), is to ensure the activation and modulation of CatSper. As the activation of sperm motility differs among metazoa, different ion channels may participate; however, knowledge regarding these channels is still scarce. In the present review, the roles and structures of the most important known ion channels are described in regard to regulation of sperm motility in animals. Full article
(This article belongs to the Special Issue Ion Channels in Sperm Physiology 2.0)
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<p>Voltage-gated Ca<sup>2+</sup> channel (VGCC) structure scheme. (<b>a</b>) The topology of the α1 subunit is made up of four homologous domains that each consist of six transmembrane α helices (TM1–6). TM4 from each homologous domain serves as the voltage sensor moving outward and rotates under the influence of the electric field, thereby initiating a conformational change that opens the respective pore. TM5, TM6, and the loop between them (P-loop) from each domain form a pore. The C-terminal tail contains a Ca<sup>2+</sup> binding domain (CBD) and in some types of VGCCs a site for calmodulin (calcium-modulated protein; CaM) binding. The binding of Ca<sup>2+</sup> to CBD or via CaM inactivates the channels. (<b>b</b>) A schematic presentation of the VGCC subunits (α1, α2δ, β, and γ) with their spatial localizations. (<b>c</b>) Overview of the types of VGCCs in relation to Vm-dependent activation – high voltage activation (HVA) and low voltage activation (LVA) (based on References [<a href="#B26-ijms-22-03259" class="html-bibr">26</a>,<a href="#B27-ijms-22-03259" class="html-bibr">27</a>]).</p> Full article ">Figure 2
<p>Topology of a store-operated Ca<sup>2+</sup> channel (SOCC) created by ORAI1. Each ORAI protein has four TMs. TM2 and TM3 create a pore. There are two sites for STIM1 binding at the N- and C-termini. The interaction between STIM1 and ORAI activates the channel and the release of Ca<sup>2+</sup> from the endoplasmic reticulum (ER). The binding of Ca<sup>2+</sup> by the Ca<sup>2+</sup> binding domain (CBD) localized on the central loop inactivates the channel [<a href="#B51-ijms-22-03259" class="html-bibr">51</a>]. Additionally, it can also be inactivated upon CaM binding [<a href="#B52-ijms-22-03259" class="html-bibr">52</a>].</p> Full article ">Figure 3
<p>A topological and spatial structure of CatSper. (<b>a</b>) The α1 subunit created by CatSper1. Like most voltage-gated channels, each α subunit contains six transmembrane domains (TM1–TM6) creating two physiologically distinctive regions, namely the voltage-sensing domain (VSD; TM1–4) and pore-forming region (TM5–6). Each TM4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors (reviewed in Reference [<a href="#B57-ijms-22-03259" class="html-bibr">57</a>]). Voltage slopes move TM4, resulting in conformational changes that open and close the channel pore [<a href="#B64-ijms-22-03259" class="html-bibr">64</a>]. Additionally, a short and hydrophobic cyclic structure linking TM5–6 contains a conserved homologous amino acid sequence (T × D × W), which selectively permits Ca<sup>2+</sup> influx. The N-terminus of CatSper 1 contains a specific histidine-rich region that might be involved in the pH regulation of CatSper activity. (<b>b</b>) The topological localizations of all auxiliary subunits are not randomly organized. The auxiliary CatSperβ subunit has two predicted TMs that are separated by a large (ca. 1000 amino acids) extracellular loop [<a href="#B64-ijms-22-03259" class="html-bibr">64</a>], whereas CatSperγ, CatSperδ, and CatSperε feature only one TM. Brown et al. [<a href="#B69-ijms-22-03259" class="html-bibr">69</a>] suggested that CatSperζ is a late evolutionary adaptation to maximize fertilization success inside the female mammalian reproductive tract. The predicted topology of Hwang et al. [<a href="#B62-ijms-22-03259" class="html-bibr">62</a>] situates the CatSperζ and EFCAB9 subunits as a cytoplasm complex that is located just below the CatSper 1–4 subunits. This complex interacts with the channel pore as a gatekeeper. The increase in pH<sub>i</sub> causes Ca<sup>2+</sup> binding to highly conserved EF-hands of EFCAB9, leading to dissociation of the EFCAB9-CatSperζ complex and full activation of the channel. Accordingly, EFCAB9-CatSperζ appears to be responsible for both modulation of the channel activity and organization of the CatSper domains [<a href="#B62-ijms-22-03259" class="html-bibr">62</a>]. The scheme has been prepared based on Reference [<a href="#B62-ijms-22-03259" class="html-bibr">62</a>].</p> Full article ">Figure 4
<p>A simplified topology of the TMEM16A monomer. Each monomer has 10 TMs. The ion conduction pore of TMEM16A is formed by TMs three to seven in each subunit, and thus the CaCC features two pores [<a href="#B96-ijms-22-03259" class="html-bibr">96</a>,<a href="#B97-ijms-22-03259" class="html-bibr">97</a>]. As summarized in a review of Ji et al. [<a href="#B97-ijms-22-03259" class="html-bibr">97</a>], the activation of TMEM16A is gated by two main mechanisms: voltage (Vm) and low concentrations of Ca<sup>2+</sup> (&lt;600 nM) via the EEEEEAVK motif in the TM2–TM3 loop. Contreras-Vite et al. [<a href="#B98-ijms-22-03259" class="html-bibr">98</a>] proposed a gating mechanism model where TMEM16A is directly activated by the Vm-dependent binding of two Ca<sup>2+</sup> ions coupled by a Vm-dependent binding of one external Cl<sup>−</sup> ion. The scheme was prepared based on Reference [<a href="#B97-ijms-22-03259" class="html-bibr">97</a>].</p> Full article ">Figure 5
<p>SLO1 structure scheme. (<b>a</b>) A topology of the α subunit. Each α subunit consists of seven (0–6) TMs, where TM4 is a typical voltage-sensing domain (VSD). An extracellular loop between TM5 and TM6 forms the pore. The N-tail is located extracellularly but the C-end is a long tail containing the RCK1 (regulator of K<sup>+</sup> conductance 1) and RKC2 domains [<a href="#B135-ijms-22-03259" class="html-bibr">135</a>]. The structural difference between SLO1 and SLO3 is that there are “Ca<sup>2+</sup>-bowl” structures within the RKC domains of SLO1, making the channel sensitive to [Ca<sup>2+</sup>]<sub>i</sub>. (<b>b</b>) In the tetrameric structure of the channel, the cytoplasmic C-termini creates a gating ring. According to the literature, SLO1 has five auxiliary subunits: one β subunit (with two transmembrane domains) and four Leucine-rich repeat-containing membrane proteins (LRRCs, also named γ subunits), LRRC26, LRCC52, LRRC55, and LRRC38, which modulate SLO1 sensitivity to Vm and [Ca<sup>2+</sup>]<sub>i</sub> (revised by Reference [<a href="#B144-ijms-22-03259" class="html-bibr">144</a>]). In murine testes and spermatozoa, two auxiliary subunits of the SLO3 channel have been identified: Lrrc52 and Lrrc26. Both of them are involved in the regulation of SLO3, and the expression of Lrrc52 is critically dependent on the presence of SLO3 [<a href="#B143-ijms-22-03259" class="html-bibr">143</a>]. The schemes are adapted from References [<a href="#B136-ijms-22-03259" class="html-bibr">136</a>,<a href="#B144-ijms-22-03259" class="html-bibr">144</a>].</p> Full article ">Figure 6
<p>A structure of a voltage-gated Na<sup>+</sup> channel (VGNC) based on a SCN2A isoform. (<b>a</b>) The α subunit is created by four repeat domains (RD1–RD4) that each have six TMs. Classically, TM1–TM4 of each domain form a VSD where TM4 acts as a positively charged sensor. During depolarization, TM4 is believed to move toward the extracellular surface, allowing the channel to become permeable to ions. Na<sup>+</sup> is transported inside a cell through a pore (P-loop) formed between TM5 and TM6 of each RD. The RDs are connected with long intracytoplasmic loops with sites for protein phosphorylation via PKA and PKC [<a href="#B157-ijms-22-03259" class="html-bibr">157</a>]. The cytoplasmic loop between RD3 and RD4 contains an “h” (I × F × M sequence) motif, which stands for a hydrophobic triad of amino acids, namely, isoleucine, phenylalanine, and methionine (I1488, F1489, and M1490). The IFM motif is involved in the inactivation of VGNC, serving as a hydrophobic latch for a hinged lid formed by the loop between RD3 and RD4 [<a href="#B159-ijms-22-03259" class="html-bibr">159</a>]. Phosphorylation in the RD1/RD2 and RD3/RD4 loops modulates the channel inactivation (adapted from Reference [<a href="#B157-ijms-22-03259" class="html-bibr">157</a>], revised in Reference [<a href="#B160-ijms-22-03259" class="html-bibr">160</a>]). (<b>b</b>) A cartoon of VGNC created by the pore-forming α subunit and the two auxiliary β subunits.</p> Full article ">Figure 7
<p>A voltage-gated H<sup>+</sup> channel (VGHC) and its structure. (<b>a</b>) A VGHC monomer is created by four TMs which in classical voltage-gated channels comprise VSD. Accordingly, VGHCs do not possess a pore-forming domain (TM5-TM6) and the extrusion of H<sup>+</sup> ions probably takes place via a water wire spanning the VSD [<a href="#B168-ijms-22-03259" class="html-bibr">168</a>]. According to Boonamnaj et al. [<a href="#B169-ijms-22-03259" class="html-bibr">169</a>], in VGHC dimers, C-terminal tails interact by forming a coiled structure that stabilizes the channel. Sites of phosphorylation in the N-termini may enhance the selectivity of the channel. (<b>b</b>) A dimeric structure of a VGHC. As the VGHC has no pore-forming domains, H<sup>+</sup> diffuses through each monomer.</p> Full article ">
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