International Journal of Molecular Sciences

14 pages, 2970 KiB

Open AccessArticle

Highly Efficient Removal of Uranium from an Aqueous Solution by a Novel Phosphonic Acid-Functionalized Magnetic Microsphere Adsorbent

by Jizhou Zhao, Peng Lu, Tengteng He, Jing Huang, Shiao Zhang, Yan Liu, Yun Wang, Cheng Meng and Dingzhong Yuan

Int. J. Mol. Sci. 2022, 23(24), 16227; https://doi.org/10.3390/ijms232416227 - 19 Dec 2022

Cited by 6 | Viewed by 2682

Abstract

The development of adsorption materials which can efficiently isolate and enrich uranium is of great scientific significance to sustainable development and environmental protection. In this work, a novel phosphonic acid-functionalized magnetic microsphere adsorbent Fe₃O₄/P (GMA-MBA)-PO₄ was developed by [...] Read more.

The development of adsorption materials which can efficiently isolate and enrich uranium is of great scientific significance to sustainable development and environmental protection. In this work, a novel phosphonic acid-functionalized magnetic microsphere adsorbent Fe₃O₄/P (GMA-MBA)-PO₄ was developed by functionalized Fe₃O₄/P (GMA-MBA) prepared by distill-precipitation polymerization with O-phosphoethanolamine. The adsorption process was endothermic, spontaneous and kinetically followed the pseudo second-order model. The maximum uranium adsorption capacity obtained from the Langmuir model was 333.33 mg g⁻¹ at 298 K. In addition, the adsorbent also had good acid resistance and superparamagnetic properties, which could be quickly separated by a magnetic field. XPS analysis showed that the adsorption of adsorbent mainly depended on the complexation of phosphonic acid group with uranium. This work offers a promising candidate for the application of magnetic adsorbents in the field of uranium separation and enrichment. Full article

(This article belongs to the Special Issue Adsorption Materials and Adsorption Behavior)

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The TEM images for (A) Fe3O4, (B) activated Fe3O4, (C) Fe3O4/P (GMA-MBA) and (D) magnetic adsorbent Fe3O4/P (GMA-MBA)-PO4. Full article ">Figure 2
Infrared spectra of (a) Fe3O4; (b) activated Fe3O4; (c) Fe3O4/P (GMA-MBA) and (d) Fe3O4/P (GMA-MBA)-PO4. Full article ">Figure 3
(A) XRD pattern of Fe3O4, and Fe3O4/P(GMA-MBA)-PO4; (B) VSM curve; (C) TGA curve and (D) XPS spectrum of Fe3O4/P(GMA-MBA)-PO4. Full article ">Figure 4
(A) Adsorption performance of Fe3O4/P (GMA-MBA) and Fe3O4/P (GMA-MBA)-PO4 on uranium under different pH values; (B) influence of contact time on uranium adsorption capacity (C0 = 100 mg L−1, T = 298 K, pH = 4.5, m/V = 0.4 g L−1); (C) effect of uranium concentration on adsorption capacity of Fe3O4/P (GMA-MBA) and Fe3O4/P (GMA-MBA)-PO4 (T = 180 min, pH = 4.5, T = 298 K, m/V = 0.4 g L−1); (D) thermodynamic image of U (VI) adsorbed by Fe3O4/P (GMA-MBA) and Fe3O4/P (GMA-MBA)-PO4 (C0 = 100 mg L−1, pH = 4.5, m/V = 0.4 g L−1). Full article ">Figure 5
Fe3O4/P(GMA-MBA)-PO4 was soaked in a HNO3 medium with pH 4.5 for 24 h before and after: (A) Infrared spectra; (B) VSM spectrum; (C) TEM images taken before immersion; (D) TEM images after immersion. Full article ">Figure 6
(A) P 2p map of Fe3O4/P (GMA-MBA)-PO4; (B) P 2p map of Fe3O4/P (GMA-MBA)-PO4-U; (C) O 1s map of Fe3O4/P (GMA-MBA)-PO4; (D) O 1s map of Fe3O4/P (GMA-MBA)-PO4-U. Full article ">Scheme 1
Proposed synthesis of the magnetic adsorbent Fe3O4/P (GMA-MBA)-PO4. Full article ">

25 pages, 852 KiB

Open AccessReview

Fatty Liver Disease-Alcoholic and Non-Alcoholic: Similar but Different

by Stephen D. H. Malnick, Pavel Alin, Marina Somin and Manuela G. Neuman

Int. J. Mol. Sci. 2022, 23(24), 16226; https://doi.org/10.3390/ijms232416226 - 19 Dec 2022

Cited by 34 | Viewed by 10403

Abstract

In alcohol-induced liver disease (ALD) and in non-alcoholic fatty liver disease (NAFLD), there are abnormal accumulations of fat in the liver. This phenomenon may be related to excessive alcohol consumption, as well as the combination of alcohol consumption and medications. There is an [...] Read more.

In alcohol-induced liver disease (ALD) and in non-alcoholic fatty liver disease (NAFLD), there are abnormal accumulations of fat in the liver. This phenomenon may be related to excessive alcohol consumption, as well as the combination of alcohol consumption and medications. There is an evolution from simple steatosis to steatohepatitis, fibrosis and cirrhosis leading to hepatocellular carcinoma (HCC). Hepatic pathology is very similar regarding non-alcoholic fatty liver disease (NAFLD) and ALD. Initially, there is lipid accumulation in parenchyma and progression to lobular inflammation. The morphological changes in the liver mitochondria, perivenular and perisinusoidal fibrosis, and hepatocellular ballooning, apoptosis and necrosis and accumulation of fibrosis may lead to the development of cirrhosis and HCC. Medical history of ethanol consumption, laboratory markers of chronic ethanol intake, AST/ALT ratio on the one hand and features of the metabolic syndrome on the other hand, may help in estimating the contribution of alcohol intake and the metabolic syndrome, respectively, to liver steatosis. Full article

(This article belongs to the Topic Clinical, Translational and Basic Research on Liver Diseases)

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14 pages, 3166 KiB

Open AccessArticle

Acting on the CFTR Membrane-Spanning Domains Interface Rescues Some Misfolded Mutants

by Nesrine Baatallah, Ahmad Elbahnsi, Benoit Chevalier, Solène Castanier, Jean-Paul Mornon, Iwona Pranke, Aleksander Edelman, Isabelle Sermet-Gaudelus, Isabelle Callebaut and Alexandre Hinzpeter

Int. J. Mol. Sci. 2022, 23(24), 16225; https://doi.org/10.3390/ijms232416225 - 19 Dec 2022

Cited by 3 | Viewed by 2306

Abstract

ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in [...] Read more.

ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in the MSD1 at the level of the membrane inner leaflet, containing both multiple mutations affecting folding and a binding site for pharmaco-chaperones that stabilize this region. This groove is also present in ABCB proteins, however it is covered by a short elbow helix, while in ABCC proteins it remains unprotected, due to a lower position of the elbow helix in the presence of the ABCC-specific lasso motif. Here, we identified a MSD1 second-site mutation located in the vicinity of the CFTR MSD1 groove that partially rescued the folding defect of cystic fibrosis causing mutations located within MSD1, while having no effect on the most frequent mutation, F508del, located within NBD1. A model of the mutated protein 3D structure suggests additional interaction between MSD1 and MSD2, strengthening the assembly at the level of the MSD intracellular loops. Altogether, these results provide insightful information in understanding key features of the folding and function of the CFTR protein in particular, and more generally, of type IV ABC transporters. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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13 pages, 698 KiB

Open AccessReview

Hypertriglyceridemia and Atherosclerotic Carotid Artery Stenosis

by Yoichi Miura and Hidenori Suzuki

Int. J. Mol. Sci. 2022, 23(24), 16224; https://doi.org/10.3390/ijms232416224 - 19 Dec 2022

Cited by 11 | Viewed by 3045

Abstract

Both fasting and non-fasting hypertriglyceridemia have emerged as residual risk factors for atherosclerotic disease. However, it is unclear whether hypertriglyceridemia increases the risks of the progression of carotid artery stenosis. Statins are well known to prevent carotid plaque progression and improve carotid plaque [...] Read more.

Both fasting and non-fasting hypertriglyceridemia have emerged as residual risk factors for atherosclerotic disease. However, it is unclear whether hypertriglyceridemia increases the risks of the progression of carotid artery stenosis. Statins are well known to prevent carotid plaque progression and improve carotid plaque instability. In addition, statin therapy is also known to reduce cerebrovascular events in patients with carotid artery stenosis and to improve clinical outcomes in patients undergoing revascularization procedures. On the other hand, there have been no randomized controlled trials showing that the combination of non-statin lipid-lowering drugs with statins has additional beneficial effects over statin monotherapy to prevent cerebrovascular events and stenosis progression in patients with carotid artery stenosis. In this article, the authors demonstrate the mechanisms of atherosclerosis formation associated with hypertriglyceridemia and the potential role of lipid-lowering drugs on carotid artery stenosis. The authors also review the articles reporting the relationships between hypertriglyceridemia and carotid artery stenosis. Full article

(This article belongs to the Special Issue Atherosclerosis: From Molecular Mechanisms, Pathophysiology to Novel Therapeutic Approaches)

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24 pages, 1200 KiB

Open AccessReview

Cardiac Sarcomere Signaling in Health and Disease

by Ashley A. Martin, Brian R. Thompson, Dongwoo Hahn, Addeli Bez Batti Angulski, Nora Hosny, Houda Cohen and Joseph M. Metzger

Int. J. Mol. Sci. 2022, 23(24), 16223; https://doi.org/10.3390/ijms232416223 - 19 Dec 2022

Cited by 12 | Viewed by 5878

Abstract

The cardiac sarcomere is a triumph of biological evolution wherein myriad contractile and regulatory proteins assemble into a quasi-crystalline lattice to serve as the central point upon which cardiac muscle contraction occurs. This review focuses on the many signaling components and mechanisms of [...] Read more.

The cardiac sarcomere is a triumph of biological evolution wherein myriad contractile and regulatory proteins assemble into a quasi-crystalline lattice to serve as the central point upon which cardiac muscle contraction occurs. This review focuses on the many signaling components and mechanisms of regulation that impact cardiac sarcomere function. We highlight the roles of the thick and thin filament, both as necessary structural and regulatory building blocks of the sarcomere as well as targets of functionally impactful modifications. Currently, a new focus emerging in the field is inter-myofilament signaling, and we discuss here the important mediators of this mechanism, including myosin-binding protein C and titin. As the understanding of sarcomere signaling advances, so do the methods with which it is studied. This is reviewed here through discussion of recent live muscle systems in which the sarcomere can be studied under intact, physiologically relevant conditions. Full article

(This article belongs to the Special Issue Structural, Chemical and Energetic Signals in Striated Muscle Function)

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11 pages, 1911 KiB

Open AccessArticle

Dysregulated Expression of Transposable Elements in TDP-43^M337V Human Motor Neurons That Recapitulate Amyotrophic Lateral Sclerosis In Vitro

by Braulio Valdebenito-Maturana, Matias Ignacio Rojas-Tapia, Mónica Carrasco and Juan Carlos Tapia

Int. J. Mol. Sci. 2022, 23(24), 16222; https://doi.org/10.3390/ijms232416222 - 19 Dec 2022

Cited by 3 | Viewed by 2400

Abstract

Amyotrophic lateral sclerosis (ALS) is a disease that progressively annihilates spinal cord motor neurons, causing severe motor decline and death. The disease is divided into familial and sporadic ALS. Mutations in the TAR DNA binding protein 43 (TDP-43) have been involved in the [...] Read more.

Amyotrophic lateral sclerosis (ALS) is a disease that progressively annihilates spinal cord motor neurons, causing severe motor decline and death. The disease is divided into familial and sporadic ALS. Mutations in the TAR DNA binding protein 43 (TDP-43) have been involved in the pathological emergence and progression of ALS, although the molecular mechanisms eliciting the disease are unknown. Transposable elements (TEs) and DNA sequences capable of transposing within the genome become dysregulated and transcribed in the presence of TDP-43 mutations. We performed RNA-Seq in human motor neurons (iMNs) derived from induced pluripotent stem cells (iPSCs) from TDP-43 wild-type—iMNs-TDP-43^WT—and mutant—iMNs-TDP-43^M337V—genotypes at 7 and 14 DIV, and, with state-of-the-art bioinformatic tools, analyzed whether TDP-43^M337V alters both gene expression and TE activity. Our results show that TDP-43^M337V induced global changes in the gene expression and TEs levels at all in vitro stages studied. Interestingly, many genetic pathways overlapped with that of the TEs activity, suggesting that TEs control the expression of several genes. TEs correlated with genes that played key roles in the extracellular matrix and RNA processing: all the regulatory pathways affected in ALS. Thus, the loss of TE regulation is present in TDP-43 mutations and is a critical determinant of the disease in human motor neurons. Overall, our results support the evidence that indicates TEs are critical regulatory sequences contributing to ALS neurodegeneration. Full article

(This article belongs to the Special Issue Molecular Research on Neurodegenerative Diseases 3.0)

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PCA analysis of RNA-Seq libraries for genes and TEs (A) PCAs corresponding to genes (left) and TEs (right) at 7 DIV (top panels). (B) 14 DIV (bottom panels) results are shown. iMNs TDP-43WT and iMNs TDP-43 M337V samples are shown in blue and red, respectively. Despite the inherent expression variance, samples can be clustered as indicated by PCAs. Full article ">Figure 2
Differential expression of genes and TEs in iMNs TDP-43WT and TDP-43M337V. Volcano plots showing all genes (left) and TEs (right) differentially expressed (DE) at 7 DIV (top panel) and 14 DIV (lower panel). Up-regulated and down-regulated genes and TEs are shown in red and blue circles; gray circles represent genes and TEs that did not meet the overall expression threshold (|log2(fold change)|≥ 2; adjusted p-value ≤ 0.05; see <a href="#sec4-ijms-23-16222" class="html-sec">Section 4</a>). Full article ">Figure 3
Biological process enrichment analysis of differentially expressed genes in iMNs TDP-43M337V. Top 20 enriched biological processes associated with differentially expressed (DE) genes, down-regulated (blue) and up-regulated (red) at each time point are shown. Each list goes in descending order according to their statistical significance. The dot size corresponds to the number of genes associated with the respective biological process (“Intersection size”). Full article ">Figure 4
Pathway enrichment analysis for genes associated with differentially expressed TEs iMNs TDP-43M337V. (A) A Venn diagram is shown to indicate the number of gene-TE pairs associated with each time point: 7 (green), and 14 DIV (red). (B) Top 20 enriched biological processes of genes positively associated with TEs at 7 DIV. (C) Top 20 enriched biological processes of genes positively associated with TEs at 14 DIV. Each list goes in descending order according to their statistical significance. The dot size corresponds to the number of genes associated with the respective biological process (“Intersection size”; see <a href="#sec4-ijms-23-16222" class="html-sec">Section 4</a>). Full article ">

3 pages, 200 KiB

Open AccessEditorial

Telocytes and Other Interstitial Cells 2.0: From Structure to Function

by Sanda Maria Cretoiu

Int. J. Mol. Sci. 2022, 23(24), 16221; https://doi.org/10.3390/ijms232416221 - 19 Dec 2022

Cited by 2 | Viewed by 1614

Abstract

Interstitial cells are often seen as those cells that fill the space between parenchymal cells, responsible for fulfilling the function of an organ [...] Full article

(This article belongs to the Special Issue Telocytes and Other Interstitial Cells 2.0: From Structure to Function)

19 pages, 3918 KiB

Open AccessArticle

Molecular Mechanisms Underlying Metabolic Resistance to Cyflumetofen and Bifenthrin in Tetranychus urticae Koch on Cowpea

by Zhenxiu Liu, Fuxing Wu, Weikang Liang, Lijuan Zhou and Jiguang Huang

Int. J. Mol. Sci. 2022, 23(24), 16220; https://doi.org/10.3390/ijms232416220 - 19 Dec 2022

Cited by 6 | Viewed by 2202

Abstract

Tetranychus urticae Koch (T. urticae) is one of the most tremendous herbivores due to its polyphagous characteristics, and is resistant to most acaricides. In this study, enzyme-linked immunosorbent assay (ELISA), transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) were carried out [...] Read more.

Tetranychus urticae Koch (T. urticae) is one of the most tremendous herbivores due to its polyphagous characteristics, and is resistant to most acaricides. In this study, enzyme-linked immunosorbent assay (ELISA), transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) were carried out to analyze the mechanisms of T. urticae metabolic resistance to cyflumetofen and bifenthrin on cowpea. The enzyme activity of UDP-glucuronosyltransferases (UGTs) and carboxylesterases (CarEs) in the cyflumetofen-resistant (R_cfm) strain significantly decreased, while that of cytochrome P450 monooxygenases (P450s) significantly increased. Meanwhile, the activities of glutathione-S-transferases (GSTs), CarEs and P450s in the bifenthrin-resistant (R_bft) strain were significantly higher than those in the susceptible strain (Lab_SS). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses, in the R_cfm mite strain, two carboxyl/cholinesterase (CCE) genes and two P450 genes were upregulated and one gene was downregulated, namely CYP392E7; in the R_bft mite strain, eleven CCE, nine UGT, two P450, four GST and three ABC genes were upregulated, while four CCE and three P450 genes were downregulated. Additionally, 94 differentially expressed genes (DEGs) were common to the two resistant groups. Specifically, TuCCE46 and TuCCE70 were upregulated in both resistant groups. Furthermore, the qRT-PCR validation data were consistent with those from the transcriptome sequencing analysis. Specifically, TuCCE46 (3.37-fold) was significantly upregulated in the R_cfm strain, while in the R_bft strain, TeturUGT22 (5.29-fold), teturUGT58p (1.74-fold), CYP392A11 (2.89-fold) and TuGSTd15 (5.12-fold) were significantly upregulated and TuCCE01 (0.13-fold) and CYP392A2p (0.07-fold) were significantly downregulated. Our study indicates that TuCCE46 might play the most important role in resistance to cyflumetofen, and TuCCE01, teturUGT58p, teturUGT22, CYP392A11, TuGSTd15, TuGSTm09 and TuABCG-13 were prominent in the resistance to bifenthrin. These findings provide further insight into the critical genes involved in the metabolic resistance of T. urticae to cyflumetofen and bifenthrin. Full article

(This article belongs to the Section Molecular Biology)

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21 pages, 798 KiB

Open AccessReview

Mitochondrial Aging and Senolytic Natural Products with Protective Potential

by Andrea Deledda, Emanuele Giordano, Fernanda Velluzzi, Giovanna Flore, Sara Franceschelli, Lorenza Speranza and Patrizio Ripari

Int. J. Mol. Sci. 2022, 23(24), 16219; https://doi.org/10.3390/ijms232416219 - 19 Dec 2022

Cited by 13 | Viewed by 11660

Abstract

Living organisms do not disregard the laws of thermodynamics and must therefore consume energy for their survival. In this way, cellular energy exchanges, which aim above all at the production of ATP, a fundamental molecule used by the cell for its metabolisms, favor [...] Read more.

Living organisms do not disregard the laws of thermodynamics and must therefore consume energy for their survival. In this way, cellular energy exchanges, which aim above all at the production of ATP, a fundamental molecule used by the cell for its metabolisms, favor the formation of waste products that, if not properly disposed of, can contribute to cellular aging and damage. Numerous genes have been linked to aging, with some favoring it (gerontogenes) and others blocking it (longevity pathways). Animal model studies have shown that calorie restriction (CR) may promote longevity pathways, but given the difficult application of CR in humans, research is investigating the use of CR-mimetic substances capable of producing the same effect. These include some phytonutrients such as oleuropein, hydroxytyrosol, epigallo-catechin-gallate, fisetin, quercetin, and curcumin and minerals such as magnesium and selenium. Some of them also have senolytic effects, which promote the apoptosis of defective cells that accumulate over the years (senescent cells) and disrupt normal metabolism. In this article, we review the properties of these natural elements that can promote a longer and healthier life. Full article

(This article belongs to the Special Issue Correlation between Nutrition, Oxidative Stress and Disease 3.0)

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17 pages, 3831 KiB

Open AccessArticle

CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

by Trygve A. H. Strømsnes, Sebastian E. Schmidke, Mitra Azad, Øyvind Singstad, Idun M. Grønsberg, Roy A. Dalmo and Arinze S. Okoli

Int. J. Mol. Sci. 2022, 23(24), 16218; https://doi.org/10.3390/ijms232416218 - 19 Dec 2022

Cited by 4 | Viewed by 4101

Abstract

Finfish production has seen over three-fold increase in the past 30 years (1990–2020), and Atlantic salmon (A. salmon; salmo salar) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one [...] Read more.

Finfish production has seen over three-fold increase in the past 30 years (1990–2020), and Atlantic salmon (A. salmon; salmo salar) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci—cr2 and mmp9 of A. salmon—were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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sgRNA analysis and in vitro cleavage assay for cr2 and mmp9 genes. (a) sgRNA analysis on 2% agarose gel. UD: undenatured sgRNA, D: denatured sgRNA. Single bands detected for the denatured sgRNAs highlighted the purity of the products, and multiple bands in the undenatured sgRNA resulted from the presence of RNA secondary structures; (b) gel shows duplicate lanes for each sample. Lanes 1–6: experimental samples, lane 7: no Cas9 control, lane 8: no sgRNA control; (c) lane 1: no Cas9 control, lane 2: no sgRNA control, lanes 3–5: experimental samples. Cleavage products at expected band sizes were detected for all samples containing the sgRNA + Cas9 + gDNA. No product at the expected band size was observed for all the control samples. Full article ">Figure 2
Ribonucleoprotein (RNP) and plasmid (px458) transfection of (a) ASK-1 and (b) CHSE-214 cells. Vertical panels show cells transfected with (i) RNP (sgRNA:Cas9-EGFP complex); (ii) Cas9-EGFP protein control; (iii) plasmid (px458); (iv) sham control. Horizontal panels show pictures of cells taken with (1) fluorescent channel; (2) light channel; (3) overlay of the light and fluorescent panels. Pictures were taken at day 7 after RNP electroporation (1600 V, 10 ms, 3 pulses) and day 2 after px458 electroporation (1200 V, 20 ms, 2 pulses). Strong adherence of Cas9-EGFP was observed on the cell surface of CHSE-214, and it was difficult to entirely wash off with PBS, explaining the high background fluorescence in the Cas9-EGFP- and RNP-transfected CHSE-214 cells. Magnification X20. (Fluorescent pictures of transfected SHK-1 cells are not shown). Full article ">Figure 3
Analyses of mutations in the target regions of cr2 and mmp9 genes due to the applied CRISPR/Cas9 strategies. (a) T7E1 assay of the cr2 RNP-transfected CHSE-214 cells. Cleaved fragments corresponding to T7 endonuclease 1 digestion of gDNA extract from the RNP-transfected cells are indicated with white arrows in lanes 3 and 4. Similar results were obtained in RNP-transfected ASK-1 and SHK-1 cells for both cr2 and mmp9. (b) Representative Sanger sequencing chromatogram for the target region of cr2 (upper left) and mmp9 (upper right) in ASK-1 cells, either wild-type (WT) or edited with CRISPR/Cas9. The binding regions are represented by the sgRNA rectangular bar, and the edited regions are boxed in dashed lines. (c) Representative output of DECODRE analysis of a cr2 RNP-transfected- (lower left) and an mmp9 RNP-transfected (lower right) ASK-1 cell. Indel type and editing frequency are indicated, as well as the aligned sequences of the wild-type and edited samples. The sgRNA including the PAM sequences (red and green bar) highlights the exact target sequence and Cas9 cut site. Boxed dashed lines indicate the exact site of the observed indel mutations. Full article ">Figure 4
Isolation and clonal expansion of single cell lines following FACS or puromycin selection of positively transfected cells. (a) Micrographs of FACS-isolated single cell-derived clonal ASK-1 cell line at day 14, day 18 and day 27 following FACS (magnification 20× for day 14 and day 18; 5× for day 27); (b) micrograph of FACS-isolated single cell-derived clonal CHSE-214 cell line at day 4 after FACS (magnification 10×); (c) micrograph of puromycin-selected SHK-1 cells at day 20 after puromycin treatment (magnification 10×). Full article ">Figure 5
Targeted regions of interest for (A) cr2 and (B) mmp9 indicated by their respective sgRNAs and flanked by primers used in amplifying these regions. Full article ">Figure 5 Cont.
Targeted regions of interest for (A) cr2 and (B) mmp9 indicated by their respective sgRNAs and flanked by primers used in amplifying these regions. Full article ">

19 pages, 728 KiB

Open AccessReview

Extracellular Vesicles as Biomarkers in Liver Disease

by Rocío Muñoz-Hernández, Ángela Rojas, Sheila Gato, Javier Gallego, Antonio Gil-Gómez, María José Castro, Javier Ampuero and Manuel Romero-Gómez

Int. J. Mol. Sci. 2022, 23(24), 16217; https://doi.org/10.3390/ijms232416217 - 19 Dec 2022

Cited by 16 | Viewed by 3734

Abstract

Extracellular vesicles (EVs) are membrane-derived vesicles released by a variety of cell types, including hepatocytes, hepatic stellate cells, and immune cells in normal and pathological conditions. Depending on their biogenesis, there is a complex repertoire of EVs that differ in size and origin. [...] Read more.

Extracellular vesicles (EVs) are membrane-derived vesicles released by a variety of cell types, including hepatocytes, hepatic stellate cells, and immune cells in normal and pathological conditions. Depending on their biogenesis, there is a complex repertoire of EVs that differ in size and origin. EVs can carry lipids, proteins, coding and non-coding RNAs, and mitochondrial DNA causing alterations to the recipient cells, functioning as intercellular mediators of cell–cell communication (auto-, para-, juxta-, or even endocrine). Nevertheless, many questions remain unanswered in relation to the function of EVs under physiological and pathological conditions. The development and optimization of methods for EV isolation are crucial for characterizing their biological functions, as well as their potential as a treatment option in the clinic. In this manuscript, we will comprehensively review the results from different studies that investigated the role of hepatic EVs during liver diseases, including non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic liver disease, fibrosis, and hepatocellular carcinoma. In general, the identification of patients with early-stage liver disease leads to better therapeutic interventions and optimal management. Although more light needs to be shed on the mechanisms of EVs, their use for early diagnosis, follow-up, and prognosis has come into the focus of research as a high-potential source of ‘liquid biopsies’, since they can be found in almost all biological fluids. The use of EVs as new targets or nanovectors in drug delivery systems for liver disease therapy is also summarized. Full article

(This article belongs to the Special Issue Molecular Biomarkers in Cancer and Metabolic Disease 2.0)

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17 pages, 9897 KiB

Open AccessArticle

iADRGSE: A Graph-Embedding and Self-Attention Encoding for Identifying Adverse Drug Reaction in the Earlier Phase of Drug Development

by Xiang Cheng, Meiling Cheng, Liyi Yu and Xuan Xiao

Int. J. Mol. Sci. 2022, 23(24), 16216; https://doi.org/10.3390/ijms232416216 - 19 Dec 2022

Cited by 5 | Viewed by 3358

Abstract

Adverse drug reactions (ADRs) are a major issue to be addressed by the pharmaceutical industry. Early and accurate detection of potential ADRs contributes to enhancing drug safety and reducing financial expenses. The majority of the approaches that have been employed to identify ADRs [...] Read more.

Adverse drug reactions (ADRs) are a major issue to be addressed by the pharmaceutical industry. Early and accurate detection of potential ADRs contributes to enhancing drug safety and reducing financial expenses. The majority of the approaches that have been employed to identify ADRs are limited to determining whether a drug exhibits an ADR, rather than identifying the exact type of ADR. By introducing the “multi-level feature-fusion deep-learning model”, a new predictor, called iADRGSE, has been developed, which can be used to identify adverse drug reactions at the early stage of drug discovery. iADRGSE integrates a self-attentive module and a graph-network module that can extract one-dimensional sub-structure sequence information and two-dimensional chemical-structure graph information of drug molecules. As a demonstration, cross-validation and independent testing were performed with iADRGSE on a dataset of ADRs classified into 27 categories, based on SOC (system organ classification). In addition, experiments comparing iADRGSE with approaches such as NPF were conducted on the OMOP dataset, using the jackknife test method. Experiments show that iADRGSE was superior to existing state-of-the-art predictors. Full article

(This article belongs to the Special Issue The Development and Application of Computational Models for Identifying Disease Markers)

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Model performance with different hyperparameter settings. (a)The performance of models under different number of heads. (b) The performance of models under different L2-Regularization. (c) The performance of models under different dropout. (d) The performance of models under different Learning rate. Full article ">Figure 2
Results of the comparison method and iADRGSE on the OMOP dataset. Full article ">Figure 3
Drug and ADR association graph, <img alt="Ijms 23 16216 i001" src="/ijms/ijms-23-16216/article_deploy/html/images/ijms-23-16216-i001.png"/> represents the drug node, while <img alt="Ijms 23 16216 i002" src="/ijms/ijms-23-16216/article_deploy/html/images/ijms-23-16216-i002.png"/> represents the adverse reaction node; the more drugs connected to the ADR, the larger the node of the ADR; the line connecting the drug node and the ADR node indicates that the drug has these adverse reactions, and the thickened line indicates the potential adverse reactions of the drug. Full article ">Figure 4
Number of drugs with one or more ADR types in the ADRs benchmark dataset. Full article ">Figure 5
Sample distribution plot: horizontal axis represents the sample size, vertical axis represents the 27 ADR labels; <img alt="Ijms 23 16216 i003" src="/ijms/ijms-23-16216/article_deploy/html/images/ijms-23-16216-i003.png"/> represents positive samples, while <img alt="Ijms 23 16216 i004" src="/ijms/ijms-23-16216/article_deploy/html/images/ijms-23-16216-i004.png"/> is negative samples. Full article ">Figure 6
Label co-occurrence diagram, referring to the simultaneous occurrence of two labels; the green circle represents the label, and the size of the circle is the frequency of that ADR label; the red line connecting the two circles represents the simultaneous occurrence of these two ADR labels; the color shade of the edges indicates the number of times this group of labels appears; the darker the color, the more often this group of labels appears. Full article ">Figure 7
iADRGSE framework. (a) Graph channel. We perform the RDKit tool to convert the drug SMILES into chemical structure graphs and feed them into a pretrained GIN network, to learn graph-based structural information. (b) Sequence channel. The preprocessing unit utilizes Open Babel software to generate molecular substructure sequences from the SMILES of drugs. Then, the substructure sequences are represented as one-dimensional sequence vectors through the embedding layer. Next, the correlation information of each substructure is extracted further, using the encoder unit with a multi-head self-attention mechanism. Finally, the feed-forward unit (a multi-fully connected layer) receives encoded data from the upper layer to obtain the final sequence-based structural information of drugs. (c) Prediction module. These two types of structural information are concatenated and then mapped to the size of the labels, through an affine transformation for multi-label prediction. Full article ">

3 pages, 189 KiB

Open AccessEditorial

MicroRNA, Insulin Resistance, and Metabolic Disorders

by Wan Lee

Int. J. Mol. Sci. 2022, 23(24), 16215; https://doi.org/10.3390/ijms232416215 - 19 Dec 2022

Cited by 6 | Viewed by 1935

Abstract

Insulin resistance is a significant health problem worldwide that contributes to a number of disorders, including type 2 diabetes and metabolic syndrome [...] Full article

(This article belongs to the Special Issue MicroRNA, Insulin Resistance, and Metabolic Disorders)

20 pages, 1686 KiB

Open AccessArticle

DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy

by Magy Sallam, Mohamed Mysara, Mohammed Abderrafi Benotmane, Radia Tamarat, Susana Constantino Rosa Santos, Anne P. G. Crijns, Daan Spoor, Filip Van Nieuwerburgh, Dieter Deforce, Sarah Baatout, Pieter-Jan Guns, An Aerts and Raghda Ramadan

Int. J. Mol. Sci. 2022, 23(24), 16214; https://doi.org/10.3390/ijms232416214 - 19 Dec 2022

Cited by 6 | Viewed by 3015 | Correction

Abstract

Radiation-Induced CardioVascular Disease (RICVD) is an important concern in thoracic radiotherapy with complex underlying pathophysiology. Recently, we proposed DNA methylation as a possible mechanism contributing to RICVD. The current study investigates DNA methylation in heart-irradiated rats and radiotherapy-treated breast cancer (BC) patients. Rats [...] Read more.

Radiation-Induced CardioVascular Disease (RICVD) is an important concern in thoracic radiotherapy with complex underlying pathophysiology. Recently, we proposed DNA methylation as a possible mechanism contributing to RICVD. The current study investigates DNA methylation in heart-irradiated rats and radiotherapy-treated breast cancer (BC) patients. Rats received fractionated whole heart X-irradiation (0, 0.92, 6.9 and 27.6 Gy total doses) and blood was collected after 1.5, 3, 7 and 12 months. Global and gene-specific methylation of the samples were evaluated; and gene expression of selected differentially methylated regions (DMRs) was validated in rat and BC patient blood. In rats receiving an absorbed dose of 27.6 Gy, DNA methylation alterations were detected up to 7 months with differential expression of cardiac-relevant DMRs. Of those, SLMAP showed increased expression at 1.5 months, which correlated with hypomethylation. Furthermore, E2F6 inversely correlated with a decreased global longitudinal strain. In BC patients, E2F6 and SLMAP exhibited differential expression directly and 6 months after radiotherapy, respectively. This study describes a systemic radiation fingerprint at the DNA methylation level, elucidating a possible association of DNA methylation to RICVD pathophysiology, to be validated in future mechanistic studies. Full article

(This article belongs to the Special Issue Advances in Radiation Toxicity)

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Figure 1
Percentage of 5 mC (%) after fractionated irradiation of 0, 0.04, 0.3 and 1.2 Gy resulting in total irradiation dose of 0, 0.92, 6.9 and 27.6 Gy as measured by MethylFlash Global DNA Methylation (5 mC) ELISA Easy Kit at 1.5, 3, 7 and 12 months after irradiation. Plotted values represent group means ± standard error of mean (SEM) with the number of rats per group indicated per bar. Statistical analysis was performed using SPSS generalized linear model module and multiple comparison correction was performed using least significant difference (LSD) (** = p-value < 0.01, *** < 0.001). Full article ">Figure 2
Significant hyper- (pale grey) and hypo- (dark grey) methylated DMR counts (p-value < 0.05) identified by SureSelect MethylSeq in rats receiving 27.6 Gy FI relative to sham-irradiated rats at 1.5 and 7 months after irradiation. Full article ">Figure 3
Pathway analysis of significant DMRs (p-value < 0.05). Statistically significant KEGG pathways (p-value < 0.05, Q-value < 0.25) were visualized by STRING-db and Cytoscape, respectively. Full article ">Figure 4
mRNA expression levels of SLMAP (A), LDLR (B), ITPR2 (C), E2F6 (D) and PTPN2 (E) in the blood of rats receiving either sham irradiation (0 Gy) or fractionated irradiation of 0.92, 6.9 and 27.6 Gy and sampled after 1.5, 3, 7 and 12 months. Data are presented as log fold change normalized to PHLPP1 (* = p-value < 0.05, ** < 0.01, *** < 0.001). Number of rats per group is indicated atop their respective bars. Plotted values represent group means ± standard error of mean (SEM). Statistical analysis was performed by SPSS General linear model and generalized linear models for data following normal and non-normal distribution, respectively. Multiple comparison correction was performed using Fisher’s LSD. Full article ">Figure 5
Mean log fold change of SLMAP (A), LDLR (B), ITPR2 (C), E2F6 (D), PTPN (E) and CX43 (GJA1) (F) expression in the blood of right- (n = 9) and left-sided (n = 16) BC patients sampled at diagnosis (V0), immediately after radiotherapy (V1) and 6 months after radiotherapy (V2). Data are presented as mean log fold changes in gene expression normalized to TBP ± SEM. Displayed significance values were calculated using observed log expression fold changes (* = p-value < 0.05). Statistical analysis was performed using SPSS generalized estimating equations module and multiple comparison correction was performed using LSD. Full article ">

22 pages, 8717 KiB

Open AccessArticle

An Isochroman Analog of CD3254 and Allyl-, Isochroman-Analogs of NEt-TMN Prove to Be More Potent Retinoid-X-Receptor (RXR) Selective Agonists Than Bexarotene

by Peter W. Jurutka, Orsola di Martino, Sabeeha Reshi, Sanchita Mallick, Michael A. Sausedo, Grant A. Moen, Isaac J. Lee, Dominic J. Ivan, Tyler D. Krall, Samuel J. Peoples, Anthony Perez, Lucas Tromba, Anh Le, Iraj Khadka, Ryan Petros, Brianna M. Savage, Eleine Salama, Jakline Salama, Joseph W. Ziller, Youngbin Noh, Ming-Yue Lee, Wei Liu, John S. Welch, Pamela A. Marshall and Carl E. Wagner Show full author list Hide full author list

Int. J. Mol. Sci. 2022, 23(24), 16213; https://doi.org/10.3390/ijms232416213 - 19 Dec 2022

Cited by 6 | Viewed by 3032

Abstract

Bexarotene is an FDA-approved drug for the treatment of cutaneous T-cell lymphoma (CTCL); however, its use provokes or disrupts other retinoid-X-receptor (RXR)-dependent nuclear receptor pathways and thereby incites side effects including hypothyroidism and raised triglycerides. Two novel bexarotene analogs, as well as three [...] Read more.

Bexarotene is an FDA-approved drug for the treatment of cutaneous T-cell lymphoma (CTCL); however, its use provokes or disrupts other retinoid-X-receptor (RXR)-dependent nuclear receptor pathways and thereby incites side effects including hypothyroidism and raised triglycerides. Two novel bexarotene analogs, as well as three unique CD3254 analogs and thirteen novel NEt-TMN analogs, were synthesized and characterized for their ability to induce RXR agonism in comparison to bexarotene (1). Several analogs in all three groups possessed an isochroman ring substitution for the bexarotene aliphatic group. Analogs were modeled for RXR binding affinity, and EC₅₀ as well as IC₅₀ values were established for all analogs in a KMT2A-MLLT3 leukemia cell line. All analogs were assessed for liver-X-receptor (LXR) activity in an LXRE system to gauge the potential for the compounds to provoke raised triglycerides by increasing LXR activity, as well as to drive LXRE-mediated transcription of brain ApoE expression as a marker for potential therapeutic use in neurodegenerative disorders. Preliminary results suggest these compounds display a broad spectrum of off-target activities. However, many of the novel compounds were observed to be more potent than 1. While some RXR agonists cross-signal the retinoic acid receptor (RAR), many of the rexinoids in this work displayed reduced RAR activity. The isochroman group did not appear to substantially reduce RXR activity on its own. The results of this study reveal that modifying potent, selective rexinoids like bexarotene, CD3254, and NEt-TMN can provide rexinoids with increased RXR selectivity, decreased potential for cross-signaling, and improved anti-proliferative characteristics in leukemia models compared to 1. Full article

(This article belongs to the Special Issue Novel Strategies in the Development of New Therapies, Drug Substances and Drug Carriers 2.0)

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15 pages, 1406 KiB

Open AccessArticle

New Wolbachia pipientis Genotype Increasing Heat Stress Resistance of Drosophila melanogaster Host Is Characterized by a Large Chromosomal Inversion

by Aleksandra E. Korenskaia, Olga D. Shishkina, Alexandra I. Klimenko, Olga V. Andreenkova, Margarita A. Bobrovskikh, Natalja V. Shatskaya, Gennady V. Vasiliev and Nataly E. Gruntenko

Int. J. Mol. Sci. 2022, 23(24), 16212; https://doi.org/10.3390/ijms232416212 - 19 Dec 2022

Cited by 4 | Viewed by 2324

Abstract

The maternally transmitted endocellular bacteria Wolbachia is a well-known symbiont of insects, demonstrating both negative and positive effects on host fitness. The previously found Wolbachia strain wMelPlus is characterized by a positive effect on the stress-resistance of its host Drosophila melanogaster, under [...] Read more.

The maternally transmitted endocellular bacteria Wolbachia is a well-known symbiont of insects, demonstrating both negative and positive effects on host fitness. The previously found Wolbachia strain wMelPlus is characterized by a positive effect on the stress-resistance of its host Drosophila melanogaster, under heat stress conditions. This investigation is dedicated to studying the genomic underpinnings of such an effect. We sequenced two closely related Wolbachia strains, wMelPlus and wMelCS¹¹², assembled their complete genomes, and performed comparative genomic analysis engaging available Wolbachia genomes from the wMel and wMelCS groups. Despite the two strains under study sharing very close gene-composition, we discovered a large (>1/6 of total genome) chromosomal inversion in wMelPlus, spanning through the region that includes the area of the inversion earlier found in the wMel group of Wolbachia genotypes. A number of genes in unique inversion blocks of wMelPlus were identified that might be involved in the induction of a stress-resistant phenotype in the host. We hypothesize that such an inversion could rearrange established genetic regulatory-networks, causing the observed effects of such a complex fly phenotype as a modulation of heat stress resistance. Based on our findings, we propose that wMelPlus be distinguished as a separate genotype of the wMelCS group, named wMelCS3. Full article

(This article belongs to the Special Issue Host-Microbe Interaction 2022)

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18 pages, 20119 KiB

Open AccessArticle

Effects of Cardiac Stem Cell on Postinfarction Arrhythmogenic Substrate

by Ángel Arenal, Gonzalo R. Ríos-Muñoz, Alejandro Carta-Bergaz, Pablo M. Ruiz-Hernández, Esther Pérez-David, Verónica Crisóstomo, Gerard Loughlin, Ricardo Sanz-Ruiz, Javier Fernández-Portales, Alejandra Acosta, Claudia Báez-Díaz, Virginia Blanco-Blázquez, María J. Ledesma-Carbayo, Miriam Pareja, María E. Fernández-Santos, Francisco M. Sánchez-Margallo, Javier G. Casado and Francisco Fernández-Avilés

Int. J. Mol. Sci. 2022, 23(24), 16211; https://doi.org/10.3390/ijms232416211 - 19 Dec 2022

Cited by 2 | Viewed by 2650

Abstract

Clinical data suggest that cardiosphere-derived cells (CDCs) could modify post-infarction scar and ventricular remodeling and reduce the incidence of ventricular tachycardia (VT). This paper assesses the effect of CDCs on VT substrate in a pig model of postinfarction monomorphic VT. We studied the [...] Read more.

Clinical data suggest that cardiosphere-derived cells (CDCs) could modify post-infarction scar and ventricular remodeling and reduce the incidence of ventricular tachycardia (VT). This paper assesses the effect of CDCs on VT substrate in a pig model of postinfarction monomorphic VT. We studied the effect of CDCs on the electrophysiological properties and histological structure of dense scar and heterogeneous tissue (HT). Optical mapping and histological evaluation were performed 16 weeks after the induction of a myocardial infarction by transient occlusion of the left anterior descending (LAD) artery in 21 pigs. Four weeks after LAD occlusion, pigs were randomized to receive intracoronary plus trans-myocardial CDCs (IC+TM group, n: 10) or to a control group. Optical mapping (OM) showed an action potential duration (APD) gradient between HT and normal tissue in both groups. CDCs increased conduction velocity (53 ± 5 vs. 45 ± 6 cm/s, p < 0.01), prolonged APD (280 ± 30 ms vs. 220 ± 40 ms, p < 0.01) and decreased APD dispersion in the HT. During OM, a VT was induced in one and seven of the IC+TM and control hearts (p = 0.03), respectively; five of these VTs had their critical isthmus located in intra-scar HT found adjacent to the coronary arteries. Histological evaluation of HT revealed less fibrosis (p < 0.01), lower density of myofibroblasts (p = 0.001), and higher density of connexin-43 in the IC+TM group. Scar and left ventricular volumes did not show differences between groups. Allogeneic CDCs early after myocardial infarction can modify the structure and electrophysiology of post-infarction scar. These findings pave the way for novel therapeutic properties of CDCs. Full article

(This article belongs to the Special Issue New Insights into Cardiovascular Diseases in Basic Research)

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15 pages, 1468 KiB

Open AccessArticle

The Association of PLAUR Genotype and Soluble suPAR Serum Level with COVID-19-Related Lung Damage Severity

by Ludmila A. Nekrasova, Anna A. Shmakova, Larisa M. Samokhodskaya, Karina I. Kirillova, Simona S. Stoyanova, Elena A. Mershina, Galina B. Nazarova, Kseniya A. Rubina, Ekaterina V. Semina and Armais A. Kamalov

Int. J. Mol. Sci. 2022, 23(24), 16210; https://doi.org/10.3390/ijms232416210 - 19 Dec 2022

Cited by 7 | Viewed by 2236

Abstract

Uncovering the risk factors for acute respiratory disease coronavirus 2019 (COVID-19) severity may help to provide a valuable tool for early patient stratification and proper treatment implementation, improving the patient outcome and lowering the burden on the healthcare system. Here we report the [...] Read more.

Uncovering the risk factors for acute respiratory disease coronavirus 2019 (COVID-19) severity may help to provide a valuable tool for early patient stratification and proper treatment implementation, improving the patient outcome and lowering the burden on the healthcare system. Here we report the results of a single-center retrospective cohort study on 151 severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected symptomatic hospitalized adult patients. We assessed the association of several blood test measurements, soluble urokinase receptor (uPAR) serum level and specific single nucleotide polymorphisms of ACE (I/D), NOS3 (rs2070744, rs1799983), SERPINE1 (rs1799768), PLAU (rs2227564) and PLAUR (rs344781, rs2302524) genes, with the disease severity classified by the percentage of lung involvement on computerized tomography scans. Our findings reveal that the T/C genotype of PLAUR rs2302524 was independently associated with a less severe lung damage (odds ratio 0.258 [0.071–0.811]). Along with high C-reactive protein, fibrinogen and soluble uPAR serum levels turned out to be independently associated with more severe lung damage in COVID-19 patients. The identified factors may be further employed as predictors of a possibly severe COVID-19 clinical course. Full article

(This article belongs to the Special Issue Molecular and Genetic Aspects of SARS-CoV-2 Infection and COVID-19 Disease 2.0)

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Figure 1
COVID-19-related lung damage severity in cohort study. (A) Density plot of the percentage of lung involvement in the study cohort in male (M, blue dashed line) and female (F, red solid line) groups. (B) Box plot of the percentage of lung involvement in the study cohort in male (M, blue) and female (F, red) groups, p = 0.193, t-test. (C) Box plot of the percentage of lung involvement in the study cohort among different age groups, p = 0.229, ANOVA. Black dots represent outliers. Full article ">Figure 2
Correlation plots for the blood test parameters and lung damage severity (% of lung involvement). The Spearman correlation coefficients (R) and the corresponding p-values (p) are shown. The red line represents the linear regression fit, and the grey area represents the 95% confidence intervals. APTT, activated partial thromboplastin time; CRP, C-reactive protein. Full article ">Figure 3
Empirical receiver operating characteristic (ROC) curves of the blood test parameters in predicting lung damage > 25%. The areas under the ROC curves (AUC) [95% confidence intervals] are provided for each parameter. The dashed line represents the chance line. APTT, activated partial thromboplastin time; CRP, C-reactive protein; FPR, the false positive rate; TPR, the true positive rate. Full article ">Figure 4
Forest plot of multivariable logistic regression model for factors associated with COVID-19-related lung damage severity. The horizontal lines correspond to odds ratio [95% confidence intervals] of “severe” vs. “mild” lung damage for each variable included in the association study. CRP, C-reactive protein. * p < 0.05; ** p < 0.01. Full article ">

3 pages, 200 KiB

Open AccessEditorial

Special Issue on “Enzymes as Biocatalysts: Current Research Trends and Applications”

by Sérgio F. Sousa

Int. J. Mol. Sci. 2022, 23(24), 16209; https://doi.org/10.3390/ijms232416209 - 19 Dec 2022

Cited by 2 | Viewed by 1921

Abstract

Enzymes are able to catalyze a wide diversity of chemical reactions in nature, and they do it at an amazing level [...] Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

14 pages, 2272 KiB

Open AccessArticle

Febrile-Range Hyperthermia Can Prevent Toxic Effects of Neutrophil Extracellular Traps on Mesenchymal Stem Cells

by Caren Linnemann, Andreas K. Nussler, Tina Histing and Sabrina Ehnert

Int. J. Mol. Sci. 2022, 23(24), 16208; https://doi.org/10.3390/ijms232416208 - 19 Dec 2022

Cited by 5 | Viewed by 2085

Abstract

Fracture healing is characterized by an inflammatory phase directly after fracture which has a strong impact on the healing outcome. Neutrophils are strong contributors here and can release neutrophil extracellular traps (NETs). NETs are found after trauma, originally thought to capture pathogens. However, [...] Read more.

Fracture healing is characterized by an inflammatory phase directly after fracture which has a strong impact on the healing outcome. Neutrophils are strong contributors here and can release neutrophil extracellular traps (NETs). NETs are found after trauma, originally thought to capture pathogens. However, they can lead to tissue damage and impede wound healing processes. Their role in fracture healing remains unclear. In this study, the effect of isolated NETs on the function of bone-forming mesenchymal stem cells (SCP-1 cells) was examined. NETs were isolated from stimulated healthy neutrophils and viability, migration, and differentiation of SCP-1 cells were analyzed after the addition of NETs. NETs severely impaired the viability of SCP-1 cells, induced necrosis and already nontoxic concentrations reduced migration significantly. Short-term incubation with NETs had a persistent negative effect on osteogenic differentiation, as measured by AP activity and matrix formation. The addition of DNase or protease inhibitors failed to reverse the negative effect of NETs, whereas a short febrile-range temperature treatment successfully reduced the toxicity and membrane destruction. Thus, the possible modification of the negative effects of NETs in fracture hematomas could be an interesting new target to improve bone healing, particularly in patients with chronic diseases such as diabetes. Full article

(This article belongs to the Special Issue Neutrophil Extracellular Traps (NETs) in Immunity and Diseases)

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Figure 1
NETs are toxic to SCP-1 cells. SCP-1 cells were incubated with different concentrations of NETs for 48 h. (A) Mitochondrial activity was determined by Resazurin conversion. (B) Total protein content was determined by SRB staining. (C) Percental LDH release as a marker for cell death. (D) % of total Calcein released into the supernatant after NETs (0.5 ng/μL) treatment. Mann–Whitney U-test. (E) Live (Calcein-AM)/Dead (PI) staining of SCP-1 cells after 22 h. Scale bar 200 μm. N = 3, n = 3. * p < 0.05, ** p < 0.01, **** p < 0.0001 as determined by the Kruskal–Wallis test. Full article ">Figure 2
NETs impaired the migration of SCP-1 cells. The migration of SCP-1 cells was analyzed after 45 h of incubation with NETs. (A) Migration to a cell-free area. (B) Exemplary images of migration of SCP-1 cells after 45 h. Cells were stained with SRB. White circles highlight the cell-free area at time point 0 h. Original magnification 2×, scale bar 1000 μm. N = 3, n = 3. * p < 0.05, *** p < 0.001, **** p < 0.0001, as determined by the Kruskal–Wallis test. Full article ">Figure 3
NETs impaired the differentiation of SCP-1 cells. SCP-1 cells were pre-incubated for 48 h with different concentrations of NETs, then osteogenically differentiated for 28 days. (A) Mitochondrial activity was determined by Resazurin conversion. (B) Total protein content was determined by SRB staining. (C) AP activity was determined photometrically. (D) Matrix mineralization was determined by Alizarin Red staining on day 28 of differentiation. (E) Exemplary images of Alizarin Red staining on day 28. Original magnification 2×, scale bar 2000 μm. N = 4, n = 3. Median ± 95% CI. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by two-way ANOVA. Full article ">Figure 4
DNase did not prevent NETs toxicity. SCP-1 cells were incubated with NETs and DNase for 48 h. (A) Mitochondrial activity was determined by Resazurin conversion. (B) Total protein content was determined by SRB staining. (C) Effectiveness of DNA removal was analyzed by PCR. N = 3, n = 3. ** p < 0.01, *** p < 0.001, **** p < 0.0001, as determined by the Kruskal-Wallis test. Full article ">Figure 5
Inhibition of proteases or histones did not prevent NETs toxicity. (A,B) SCP-1 cells were incubated with 0.5 ng/μL of NETs and 0.5 μg/mL of Proteinase K. After 48 h, mitochondrial activity (A) and total protein content (B) were determined. (C,D) SCP-1 cells were incubated with 0.5 ng/μL of NETs and either 5 μg/mL of Leupeptin or 1 μg/mL of Pepstatin A. Mitochondrial activity (C) and total protein content (D) were determined. (E) The DNA content of NETs correlates with the protein content N = 3, n = 3. Effect of histone antibodies or heparin (50 μg/mL) treatment (F–H): (F) mitochondrial activity (resazurin conversion) values; (G) total protein content (SRB staining); and (H) LDH release as indicator for cell death (in relation to lysed cells). N = 2–3, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, as determined by the Kruskal–Wallis test. Full article ">Figure 6
40 °C is sufficient to block NETs toxicity. NETs were incubated at the indicated temperatures before addition to SCP-1 cells for 48 h. (A) Mitochondrial activity determined by Resazurin conversion measurement. (B) Total protein content determined by SRB staining. (C) Percental LDH release as a marker for cell death. (D) Release of Calcein into the SCP-1 cell culture supernatant after 22 h of incubation, pos. Ctrl is Triton-X-100-treated cells. (E) Calcein-AM/PI staining of SCP-1 cells after 22 h. Scale bar 200 μm. N = 3, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by the Kruskal–Wallis test. Full article ">

20 pages, 7803 KiB

Open AccessArticle

Pharmaceutical Development of Nanostructured Vesicular Hydrogel Formulations of Rifampicin for Wound Healing

by Chantal M. Wallenwein, Verena Weigel, Götz Hofhaus, Namrata Dhakal, Wolfgang Schatton, Svetlana Gelperina, Florian K. Groeber-Becker, Jennifer Dressman and Matthias G. Wacker

Int. J. Mol. Sci. 2022, 23(24), 16207; https://doi.org/10.3390/ijms232416207 - 19 Dec 2022

Cited by 7 | Viewed by 3081

Abstract

Chronic wounds exhibit elevated levels of inflammatory cytokines, resulting in the release of proteolytic enzymes which delay wound-healing processes. In recent years, rifampicin has gained significant attention in the treatment of chronic wounds due to an interesting combination of antibacterial and anti-inflammatory effects. [...] Read more.

Chronic wounds exhibit elevated levels of inflammatory cytokines, resulting in the release of proteolytic enzymes which delay wound-healing processes. In recent years, rifampicin has gained significant attention in the treatment of chronic wounds due to an interesting combination of antibacterial and anti-inflammatory effects. Unfortunately, rifampicin is sensitive to hydrolysis and oxidation. As a result, no topical drug product for wound-healing applications has been approved. To address this medical need two nanostructured hydrogel formulations of rifampicin were developed. The liposomal vesicles were embedded into hydroxypropyl methylcellulose (HPMC) gel or a combination of hyaluronic acid and marine collagen. To protect rifampicin from degradation in aqueous environments, a freeze-drying method was developed. Before freeze-drying, two well-defined hydrogel preparations were obtained. After freeze-drying, the visual appearance, chemical stability, residual moisture content, and redispersion time of both preparations were within acceptable limits. However, the morphological characterization revealed an increase in the vesicle size for collagen–hyaluronic acid hydrogel. This was confirmed by subsequent release studies. Interactions of marine collagen with phosphatidylcholine were held responsible for this effect. The HPMC hydrogel formulation remained stable over 6 months of storage. Moving forward, this product fulfills all criteria to be evaluated in preclinical and clinical studies. Full article

(This article belongs to the Special Issue Nanostructured Formulations in Pharmaceutical, Medical and Cosmetic Applications 2.0)

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Full article ">Figure 1
Comparison of three different formulation approaches using various celluloses before and after freeze-drying with (A) presenting the physicochemical characteristics including particle size (left axis, light green and blue bars), zeta potential (left axis, dark green and blue bars), and PDI (green and blue line) and (B) indicating the drug content (Mean ± SD, n = 3). Full article ">Figure 2
Batch reproducibility of HPMC gel manufacture with (A) indicating the reproducibility of five batches regarding their physicochemical characteristics including particle size (left axis, light green bars), zeta potential (left axis, dark green bars), and PDI (green line) and (B) presenting the drug content of different batches (light green bars) after freeze-drying (Mean ± SD, n = 5). Full article ">Figure 3
Representative examples of the freeze-dried collagen–hyaluronic acid hydrogels with rifampicin-loaded liposomes before (A) and after (B) process optimization together with the process records. After a first process optimization, the configuration (C) did not lead to a well-defined product irrespective of the addition of cryoprotectants and lyoprotectants. After further process optimization, a well-defined cake (B) was achieved. The final process parameters are also presented (D). Full article ">Figure 4
Visualization of the vesicles embedded in the HPMC gel before freeze-drying (A) and after redispersion of the freeze-dried product (B) using Cryo-TEM. The micrographs of the liposomal collagen–hyaluronic acid gel before freeze-drying and after redispersion of the lyophilized cake are presented in (C,D), respectively. Full article ">Figure 5
TEM micrographs of the collagen–hyaluronic acid hydrogel obtained in the dried state show a 4800-fold (left) and 6800-fold magnification (right). Full article ">Figure 6
Comparison of physical (A,C) stability including particle size (left axis, light green, blue and red bars), zeta potential (left axis, dark green, blue and red bars), and PDI (green, blue, and red line) and chemical (B,D) stability of HPMC gel (A,B) and hyaluronic acid plus collagen gel (C,D) over 6 months (mean values ± SD, n = 3). Full article ">Figure 7
Comparative release study with the two nanostructured hydrogels and the liposomal dispersion (mean ± SD, n = 3). Full article ">Figure 8
Comparison of the viscosity of the nanostructured HPMC gel before and after freeze-drying at two different temperatures (25 °C and 32 °C) (mean ± SD, n = 3). Full article ">Figure 9
Investigation of the in vitro skin irritation of the rifampicin-loaded liposomes and the nanostructured HPMC gel with rifampicin in comparison to a rifampicin solution. As a control, the vehicles without rifampicin were also examined. Full article ">

13 pages, 5373 KiB

Open AccessArticle

Effect of Novel AKT Inhibitor Vevorisertib as Single Agent and in Combination with Sorafenib on Hepatocellular Carcinoma in a Cirrhotic Rat Model

by Keerthi Kurma, Ayca Zeybek Kuyucu, Gaël S. Roth, Nathalie Sturm, Marion Mercey-Ressejac, Giovanni Abbadessa, Yi Yu, Herve Lerat, Patrice N. Marche, Thomas Decaens and Zuzana Macek Jilkova

Int. J. Mol. Sci. 2022, 23(24), 16206; https://doi.org/10.3390/ijms232416206 - 19 Dec 2022

Cited by 8 | Viewed by 2577

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. The AKT pathway is often activated in HCC cases, and a longer exposure to tyrosine kinase inhibitors such as sorafenib may lead to over-activation of the AKT pathway, leading to [...] Read more.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. The AKT pathway is often activated in HCC cases, and a longer exposure to tyrosine kinase inhibitors such as sorafenib may lead to over-activation of the AKT pathway, leading to HCC resistance. Here, we studied the efficacy of a new generation of allosteric AKT inhibitor, vevorisertib, alone or in combination with sorafenib. To identify specific adverse effects related to the background of cirrhosis, we used a diethylnitrosamine (DEN)-induced cirrhotic rat model. Vevorisertib was tested in vitro on Hep3B, HepG2, HuH7 and PLC/PRF cell lines. Rats were treated weekly with intra-peritoneal injections of DEN for 14 weeks to obtain cirrhosis with fully developed HCC. After that, rats were randomized into four groups (n = 7/group): control, sorafenib, vevorisertib and the combination of vevorisertib + sorafenib, and treated for 6 weeks. Tumor progression was followed by MRI. We demonstrated that the vevorisertib is a highly potent treatment, blocking the phosphorylation of AKT. The tumor progression in the rat liver was significantly reduced by treatment with vevorisertib + sorafenib (49.4%) compared to the control group (158.8%, p < 0.0001). Tumor size, tumor number and tumor cell proliferation were significantly reduced in both the vevorisertib group and vevorisertib + sorafenib groups compared to the control group. Sirius red staining showed an improvement in liver fibrosis by vevorisertib and the combination treatment. Moreover, vevorisertib + sorafenib treatment was associated with a normalization in the liver vasculature. Altogether, vevorisertib as a single agent and its combination with sorafenib exerted a strong suppression of tumor progression and improved liver fibrosis. Thus, results provide a rationale for testing vevorisertib in clinical settings and confirm the importance of targeting AKT in HCC. Full article

(This article belongs to the Special Issue Toward a New Era in the Management of Hepatocellular Carcinoma: Novel Perspective on Therapeutic Options and Biomarkers)

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12 pages, 2287 KiB

Open AccessArticle

Receptor-Independent Anti-Ferroptotic Activity of TrkB Modulators

by Md. Jakaria, Abdel A. Belaidi, Adam Southon, Krista A. Dent, Darius J. R. Lane, Ashley I. Bush and Scott Ayton

Int. J. Mol. Sci. 2022, 23(24), 16205; https://doi.org/10.3390/ijms232416205 - 19 Dec 2022

Cited by 5 | Viewed by 2734

Abstract

Dysregulated brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signalling is implicated in several neurodegenerative diseases, including Alzheimer’s disease. A failure of neurotrophic support may participate in neurodegenerative mechanisms, such as ferroptosis, which has likewise been implicated in this disease class. The current [...] Read more.

Dysregulated brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signalling is implicated in several neurodegenerative diseases, including Alzheimer’s disease. A failure of neurotrophic support may participate in neurodegenerative mechanisms, such as ferroptosis, which has likewise been implicated in this disease class. The current study investigated whether modulators of TrkB signalling affect ferroptosis. Cell viability, C11 BODIPY, and cell-free oxidation assays were used to observe the impact of TrkB modulators, and an immunoblot assay was used to detect TrkB expression. TrkB modulators such as agonist BDNF, antagonist ANA-12, and inhibitor K252a did not affect RSL3-induced ferroptosis sensitivity in primary cortical neurons expressing detectable TrkB receptors. Several other modulators of the TrkB receptor, including agonist 7,8-DHF, activator phenelzine sulphate, and inhibitor GNF-5837, conferred protection against a range of ferroptosis inducers in several immortalised neuronal and non-neuronal cell lines, such as N27 and HT-1080 cells. We found these immortalised cell lines lack detectable TrkB receptor expression, so the anti-ferroptotic activity of these TrkB modulators was most likely due to their inherent radical-trapping antioxidant properties, which should be considered when interpreting their experimental findings. These modulators or their variants could be potential anti-ferroptotic therapeutics for various diseases. Full article

(This article belongs to the Special Issue Iron Metabolism in Health and Disease)

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Activity of TrkB modulators against ferroptosis in mature primary cortical neurons. Immunoblot shows TrkB expression in mature primary cortical neurons (a). GNF-5837 inhibits (while other TrkB modulators do not affect) RSL3-induced ferroptosis in primary cortical neurons (b–e). Cells were co-treated with ferroptosis inducer RSL3 and tested compounds for 24 h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data points signify mean percentage survival relative to untreated controls ± SEM, n = 9–10 from 2–3 independent experiments. p values were calculated using the two-way ANOVA (Dunnett’s multiple comparisons test, (c) and the unpaired t-test (d), and the significant level is noted as **** p < 0.0001 (vs. control). Full article ">Figure 2
Both 7,8-DHF and phenelzine protect ferroptosis in several immortalised cell lines. Efficacy of 7,8-DHF, Lip-1, and DFP against erastin- and RSL3-induced ferroptosis in N27 cells (a,b). Like Lip-1 and DFP, 7,8-DHF significantly prevents ferroptosis in HT-1080 and U-251 cells (c,d). Efficacy of phenelzine against erastin- and RSL3-induced ferroptosis in HT-1080 and N27 cells (e,f). Cells were co-treated with ferroptosis inducers and tested compounds for 24 h. Cell viability was measured by MTT assay. Data points signify mean percentage survival relative to untreated controls ± SEM, n = 12–20 from 3–5 independent experiments. p values were calculated using the two-way ANOVA (Šídák’s multiple comparisons test), and the significant levels are noted as ** p < 0.01 and **** p < 0.0001 (vs. no compound). Full article ">Figure 3
Both 7,8-DHF and phenelzine protect against ferroptosis and have RTA properties. Both 7,8-DHF and phenelzine attenuate FINO2- and tBH-induced toxicity (a,b). RTA properties of 7,8-DHF, phenelzine and Lip-1 in cell-free ABTS assay (c). Cells were co-treated with ferroptosis inducers and tested compounds for 24 h, except for tBH, which had a 4 h co-treatment. Cell viability assay was measured by MTT assay. Data points signify mean percentage survival relative to untreated controls ± SEM, n = 9–16 from 3–4 independent experiments. P values were calculated using the two-way ANOVA (Dunnett’s multiple comparisons test), and the significant level is noted as **** p < 0.0001 (vs. control). Full article ">Figure 4
GNF-5837 protects ferroptosis in several immortalised cell lines. Comparative anti-ferroptotic activity between GNF-5837 and Lip-1 for erastin- and RSL3-induced ferroptosis following 24 h incubation in different cell lines: N27, HT-22, and HT-1080 cells (a–f). Cell viability was measured by MTT assay. Data points signify mean percentage survival relative to untreated controls ± SEM, n = 11–16 from 3–4 independent experiments. Full article ">Figure 5
GNF-5837 protects against ferroptosis likely via its RTA property. Toxicity assay upon 48 h treatment with GNF-5837 in different cell lines (a). GNF-5837 protects against toxicity induced by FINO2, tBH, and iron in N27 cells (b). Like Lip-1, GNF-5837 significantly protects against lipid peroxidation in the C11 BODIPY assay caused by erastin, BSO, and RSL3 in N27 cells (c). RTA property of GNF-5837 and Lip-1 against lipid peroxidation in cell-free MDA assay (d). Cells were co-treated with ferroptosis inducers and tested compounds for 24 h, except for tBH-induced ferroptosis, which had a 4 h co-treatment, and MTT assay measured cell viability. Lipid peroxidation in C11 BODIPY assay is expressed as the ratio of green to red C11 BODIPY fluorescence. Data points signify mean percentage survival relative to respective controls ± SEM (n = 36 for (a), n = 9–12 for (b,c), and n = 6 for (d) from 3–4 independent experiments). P values were calculated using the one-way ANOVA (Tukey’s multiple comparisons test for (b)) and two-way ANOVA (Dunnett’s multiple comparisons test for (c)), and the significant level is noted as **** p < 0.0001 (vs. inducers/control). Full article ">

15 pages, 4992 KiB

Open AccessArticle

Growth of GaN Thin Films Using Plasma Enhanced Atomic Layer Deposition: Effect of Ammonia-Containing Plasma Power on Residual Oxygen Capture

by Shicong Jiang, Wan-Yu Wu, Fangbin Ren, Chia-Hsun Hsu, Xiaoying Zhang, Peng Gao, Dong-Sing Wuu, Chien-Jung Huang, Shui-Yang Lien and Wenzhang Zhu

Int. J. Mol. Sci. 2022, 23(24), 16204; https://doi.org/10.3390/ijms232416204 - 19 Dec 2022

Cited by 3 | Viewed by 2686

Abstract

In recent years, the application of (In, Al, Ga)N materials in photovoltaic devices has attracted much attention. Like InGaN, it is a direct band gap material with high absorption at the band edge, suitable for high efficiency photovoltaic devices. Nonetheless, it is important [...] Read more.

In recent years, the application of (In, Al, Ga)N materials in photovoltaic devices has attracted much attention. Like InGaN, it is a direct band gap material with high absorption at the band edge, suitable for high efficiency photovoltaic devices. Nonetheless, it is important to deposit high-quality GaN material as a foundation. Plasma-enhanced atomic layer deposition (PEALD) combines the advantages of the ALD process with the use of plasma and is often used to deposit thin films with different needs. However, residual oxygen during growth has always been an unavoidable issue affecting the quality of the resulting film, especially in growing gallium nitride (GaN) films. In this study, the NH₃-containing plasma was used to capture the oxygen absorbed on the growing surface to improve the quality of GaN films. By diagnosing the plasma, NH₂, NH, and H radicals controlled by the plasma power has a strong influence not only on the oxygen content in growing GaN films but also on the growth rate, crystallinity, and surface roughness. The NH and NH₂ radicals contribute to the growth of GaN films while the H radicals selectively dissociate Ga-OH bonds on the film surface and etch the grown films. At high plasma power, the GaN film with the lowest Ga-O bond ratio has a saturated growth rate, a better crystallinity, a rougher surface, and a lower bandgap. In addition, the deposition mechanism of GaN thin films prepared with a trimethylgallium metal source and NH₃/Ar plasma PEALD involving oxygen participation or not is also discussed in the study. Full article

(This article belongs to the Special Issue Advancements in Solar Cells and Materials for Photovoltaics)

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7 pages, 1029 KiB

Open AccessCommunication

Usnic Acid-Mediated Exchange of Protons for Divalent Metal Cations across Lipid Membranes: Relevance to Mitochondrial Uncoupling

by Tatyana I. Rokitskaya, Alexander M. Arutyunyan, Ljudmila S. Khailova, Alisa D. Kataeva, Alexander M. Firsov, Elena A. Kotova and Yuri N. Antonenko

Int. J. Mol. Sci. 2022, 23(24), 16203; https://doi.org/10.3390/ijms232416203 - 19 Dec 2022

Viewed by 1838

Abstract

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic [...] Read more.

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn²⁺ > Mn²⁺ > Mg²⁺ > Ca²⁺ >> Cu²⁺. Remarkably, Cu²⁺ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations. Full article

(This article belongs to the Special Issue Mitochondrial Ion Channels and Exchangers in Cellular Pathophysiology)

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(a) Effect of magnesium ions (20 µM) on the induction of electrical current through BLM by usnic acid (UA, 20 µM) and the inhibitory effect of copper ions (20 µM); (b) dependence of the UA-mediated electrical current through BLM on the concentration of calcium, magnesium, manganese, copper and zinc ions. The solution was 10 mM Tris, 10 mM MES, 10 mM β-alanine and 10 mM KCl with a pH of 6.0. BLM voltage was 30 mV. BLM was created from DPhPC. The data in panel (b) are represented as the mean ± SE, with n = 3. Full article ">Figure 2
Effect of divalent metal cations (1 mM each) on usnic acid-mediated proton fluxes through liposomes loaded with the pH probe pyranine. The concentration of usnic acid was 30 µM. The inner liposomal pH was estimated from the pyranine fluorescence intensities measured at 505 nm upon excitation at 455 nm. Lasalocid A (1 µM) was added at the end of each track to equilibrate the pH. Lipid concentration was 20 µg/mL. Other conditions: see Materials and Methods section. At the zero-time moment, divalent metal cations were added in the form of chlorides at a concentration of 1 mM. The proton flux was stimulated by an alkaline pH shift from pH 6 to pH 8 at t = 75 s by the addition of the previously determined aliquot of KOH. The measurements were made at 15 °C in order to decrease the unspecific proton leak in liposomes. Full article ">Figure 3
Efflux of Ca2+ or Zn2+ from Ca2+- or Zn2+-loaded liposomes induced by usnic acid. Panel (a) displays time courses of the cation efflux from the liposomes, panel (b) shows a histogram of the cation efflux after 10 minutes of the efflux. The data were normalized to the maximal fluorescence level achieved after the addition of 100 nM ionomycin. The concentrations of Ca2+ or Zn2+ ions were estimated from the Fluo-5N fluorescence intensities measured at 520 nm upon excitation at 495 nm. Lipid concentration was 20 µg/mL; T = 25 °C. Other conditions: see Materials and Methods section. Full article ">Figure 4
CD (a) and absorbance (b) spectra of UA (50 µM) in the absence (black curve) and in the presence of 1 mM calcium (pink), magnesium (blue), zinc (green), manganese (red) and copper (cyan) ions in methanol. The concentration of CuSO4 was 0.1 mM. Full article ">

16 pages, 2855 KiB

Open AccessArticle

Genome-Wide Identification of the CER Gene Family and Significant Features in Climate Adaptation of Castanea mollissima

by Shuqing Zhao, Xinghua Nie, Xueqing Liu, Biyao Wang, Song Liu, Ling Qin and Yu Xing

Int. J. Mol. Sci. 2022, 23(24), 16202; https://doi.org/10.3390/ijms232416202 - 19 Dec 2022

Cited by 4 | Viewed by 2417

Abstract

The plant cuticle is the outermost layer of the aerial organs and an important barrier against biotic and abiotic stresses. The climate varies greatly between the north and south of China, with large differences in temperature and humidity, but Chinese chestnut is found [...] Read more.

The plant cuticle is the outermost layer of the aerial organs and an important barrier against biotic and abiotic stresses. The climate varies greatly between the north and south of China, with large differences in temperature and humidity, but Chinese chestnut is found in both regions. This study investigated the relationship between the wax layer of chestnut leaves and environmental adaptation. Firstly, semi-thin sections were used to verify that there is a significant difference in the thickness of the epicuticular wax layer between wild chestnut leaves in northwest and southeast China. Secondly, a whole-genome selective sweep was used to resequence wild chestnut samples from two typical regional populations, and significant genetic divergence was identified between the two populations in the CmCER1-1, CmCER1-5 and CmCER3 genes. Thirty-four CER genes were identified in the whole chestnut genome, and a series of predictive analyses were performed on the identified CmCER genes. The expression patterns of CmCER genes were classified into three trends—upregulation, upregulation followed by downregulation and continuous downregulation—when chestnut seedlings were treated with drought stress. Analysis of cultivars from two resource beds in Beijing and Liyang showed that the wax layer of the northern variety was thicker than that of the southern variety. For the Y-2 (Castanea mollissima genome sequencing material) cultivar, there were significant differences in the expression of CmCER1-1, CmCER1-5 and CmCER3 between the southern variety and the northern one-year-grafted variety. Therefore, this study suggests that the CER family genes play a role in environmental adaptations in chestnut, laying the foundation for further exploration of CmCER genes. It also demonstrates the importance of studying the adaptation of Chinese chestnut wax biosynthesis to the southern and northern environments. Full article

(This article belongs to the Special Issue Genomics-Based Tree Breeding to Improve Resilience in the Face of Global Climate Change)

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(a) Comparison of differences in leaf epicuticular wax layer thickness between northern and southern wild samples. Error bars indicate standard errors. ‘****’ on error bar indicates a significant difference between the two groups at p < 0.0001. (b) Characterization of the anatomical structures of Gansu and Tianmu Mountains samples. The length of the scale is 20μm. The complete semi-thin section is shown in <a href="#app1-ijms-23-16202" class="html-app">Figure S1</a>. (c) Leaf epicuticular wax layer thickness analysis of Gansu and Tianmu Mountains samples. Error bars indicate standard errors. ‘****’ on error bar indicates a significant difference between the two groups at p < 0.0001. Full article ">Figure 2
(a) Genomic landscape of the population differentiation in groups I (Gansu) and II (Tianmu Mountains) of wild Chinese chestnut, as measured by the fixation index (FST). (b) Sliding window analysis for further presentation. Full article ">Figure 3
Phylogenetic trees of 17 CERs in A. thaliana and 34 CERs in C. mollissima. MEGA11 was applied to construct the maximum-likelihood (ML) tree using the WAG with Freqs. (+F) model with 1000 bootstrap replicates. The number on the branch indicates the bootstrap value. The different groups are color-coded as indicated in the top left panel. Full article ">Figure 4
Distribution of conserved motifs in CmCERs. Individual motifs are indicated in different-colored horizontal bars below each figure. Numbers on the X-axis indicate amino-acid positions of the proteins. Full article ">Figure 5
Chromosomal location of CmCERs. Full article ">Figure 6
Cis-regulatory element analysis on CmCER genes. Full article ">Figure 7
(a) Expression levels of CmCER genes in drought-treated Chinese chestnut seedlings. (b) Comparison of differences in leaf epicuticular wax layer thickness between northern and southern varieties in the Beijing and Liyang germplasm repositories. Repository 1: Chestnut Experiment Station in the Huairou District of Beijing, China. Repository 2: Liyang Longtan Linchang. Error bars indicate standard errors. ‘**’ on the error bar indicates a significant difference between the two groups at p < 0.01. (c) Results of semi-thin sections of wild Y-2 leaves in Hubei and grafted Y-2 leaves in the north. Error bars indicate standard errors. ‘*’ on the error bar indicates a significant difference between the two groups at p < 0.05. The length of the scale is 20μm. The complete semi-thin section is shown in <a href="#app1-ijms-23-16202" class="html-app">Figure S2</a>. (d) Expression of CmCER1-1, CmCER1-5 and CmCER3 in grafted Y-2 varieties in the north and wild Y-2 varieties in the south. Error bars indicate standard errors. ‘*’ or ‘**’ on the error bar indicates a significant difference between the two groups at p < 0.05 or p < 0.01, respectively. Full article ">

19 pages, 577 KiB

Open AccessReview

Dysmenorrhoea: Can Medicinal Cannabis Bring New Hope for a Collective Group of Women Suffering in Pain, Globally?

by Amelia Seifalian, Julian Kenyon and Vik Khullar

Int. J. Mol. Sci. 2022, 23(24), 16201; https://doi.org/10.3390/ijms232416201 - 19 Dec 2022

Cited by 5 | Viewed by 5718

Abstract

Dysmenorrhoea effects up to 90% of women of reproductive age, with medical management options including over-the-counter analgesia or hormonal contraception. There has been a recent surge in medicinal cannabis research and its analgesic properties. This paper aims to critically investigate the current research [...] Read more.

Dysmenorrhoea effects up to 90% of women of reproductive age, with medical management options including over-the-counter analgesia or hormonal contraception. There has been a recent surge in medicinal cannabis research and its analgesic properties. This paper aims to critically investigate the current research of medicinal cannabis for pain relief and to discuss its potential application to treat dysmenorrhoea. Relevant keywords, including medicinal cannabis, pain, cannabinoids, tetrahydrocannabinol, dysmenorrhoea, and clinical trial, have been searched in the PubMed, EMBASE, MEDLINE, Google Scholar, Cochrane Library (Wiley) databases and a clinical trial website (clinicaltrials.gov). To identify the relevant studies for this paper, 84 papers were reviewed and 20 were discarded as irrelevant. This review critically evaluated cannabis-based medicines and their mechanism and properties in relation to pain relief. It also tabulated all clinical trials carried out investigating medicinal cannabis for pain relief and highlighted the side effects. In addition, the safety and toxicology of medicinal cannabis and barriers to use are highlighted. Two-thirds of the clinical trials summarised confirmed positive analgesic outcomes, with major side effects reported as nausea, drowsiness, and dry mouth. In conclusion, medicinal cannabis has promising applications in the management of dysmenorrhoea. The global medical cannabis market size was valued at USD 11.0 billion in 2021 and is expected to expand at a compound annual growth rate (CAGR) of 21.06% from 2022 to 2030. This will encourage academic as well as the pharmaceutical and medical device industries to study the application of medical cannabis in unmet clinical disorders. Full article

(This article belongs to the Special Issue Cannabis sativa: From Plants to Humans)

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20 pages, 1941 KiB

Open AccessReview

Plant Disease Resistance-Related Signaling Pathways: Recent Progress and Future Prospects

by Li-Na Ding, Yue-Tao Li, Yuan-Zhen Wu, Teng Li, Rui Geng, Jun Cao, Wei Zhang and Xiao-Li Tan

Int. J. Mol. Sci. 2022, 23(24), 16200; https://doi.org/10.3390/ijms232416200 - 19 Dec 2022

Cited by 113 | Viewed by 14124

Abstract

Plant–pathogen interactions induce a signal transmission series that stimulates the plant’s host defense system against pathogens and this, in turn, leads to disease resistance responses. Plant innate immunity mainly includes two lines of the defense system, called pathogen-associated molecular pattern-triggered immunity (PTI) and [...] Read more.

Plant–pathogen interactions induce a signal transmission series that stimulates the plant’s host defense system against pathogens and this, in turn, leads to disease resistance responses. Plant innate immunity mainly includes two lines of the defense system, called pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). There is extensive signal exchange and recognition in the process of triggering the plant immune signaling network. Plant messenger signaling molecules, such as calcium ions, reactive oxygen species, and nitric oxide, and plant hormone signaling molecules, such as salicylic acid, jasmonic acid, and ethylene, play key roles in inducing plant defense responses. In addition, heterotrimeric G proteins, the mitogen-activated protein kinase cascade, and non-coding RNAs (ncRNAs) play important roles in regulating disease resistance and the defense signal transduction network. This paper summarizes the status and progress in plant disease resistance and disease resistance signal transduction pathway research in recent years; discusses the complexities of, and interactions among, defense signal pathways; and forecasts future research prospects to provide new ideas for the prevention and control of plant diseases. Full article

(This article belongs to the Special Issue Cell Signaling in Model Plants 3.0)

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A model of immune responses in plant–pathogen interactions. A plant’s innate immune system consists of PTI and ETI. PTI induced through the recognition of PAMPs by PRRs can inhibit the growth of most pathogens. Then, downstream signaling pathways such as Ca2+ signaling, the MAPK cascade, and ROS signaling are activated. Some pathogens can release pathogenic effectors to interfere with PTI, causing susceptibility triggered by effectors. The disease R proteins with conserved NB-LRR can directly or indirectly identify specific effectors to trigger ETI, which often causes an HR at the infection site of the pathogen, and then inhibit the growth of the pathogens again. The SA and JA/ET signaling pathways are also involved in PTI and ETI activation and the resistance response to pathogen infections, thereby stimulating downstream transcription factors and initiating plant defense responses. Many ncRNAs play critical roles in PTI or ETI responses by regulating various biological processes, such as the MAPK cascade, the expression of signaling components, ROS production, plant hormone biosynthesis, and signaling. NB-LRR, nucleotide-binding leucine-rich repeat; PRRs, pattern recognition receptors; SA, salicylic acid; JA/ET, jasmonic acid/ethylene; HR, hypersensitive response; PCD, programmed cell death; SAR, systemic acquired resistance; R, resistance; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; PAMPs, pathogen-associated molecular pattern; PTI, PAMP-triggered immunity; ETI, effector-triggered immunity; ncRNAs, non-coding RNAs. The arrows indicate positive regulation, and open blocks indicate negative regulation. Full article ">Figure 2
Schematic representation of the plant defense signal transduction network. In the process of plant–pathogen interactions, a series of signals are triggered to induce plant defense responses. The complex and diverse signal pathways interact with each other and form signal transduction networks in plants. After the resistant host recognizes the elicitors produced by pathogens, it activates the signal transduction system, causing the release of Ca2+, an MAPK cascade reaction, and the activation of R genes. Ca2+ flowing into the cytoplasm can activate CaMs and CMLs, induce downstream NO synthesis, and then induce a primary immune response, including HR. Moreover, NO may regulate an HR/PCD through a synergistic effect with ROS. Besides being a signal to activate SAR, ROS can directly act as antibacterial effectors and enhance the structural resistance of the host. The interaction between the R gene and the avirulent gene (Avr) can stimulate a series of defense responses, such as the HR/PCD and SAR, which are induced by SA and give a strong resistance against some biotrophic pathogens. GDG1 is necessary for pathogen-induced SA biosynthesis, and its expression is regulated by EDS1 and PAD4. SA can also control the expression of GDG1, EDS1, and PAD4 through a positive feedback loop. The positive feedback regulation of GDG1 lies in the high level of SA accumulation. JA/ET signaling pathways mainly regulate plant resistance to necrotic pathogens and wounds. JA and ET also mediate the resistance induced by root microorganisms, which is called induced systemic resistance (ISR). SA inhibits the JA/ET pathway by activating NPRl, a positive regulatory gene of the SA pathway. ERF1 is located at the intersection of the JA and ET signaling pathway defense against pathogen infections and in wound responses. A JA signal can promote the interaction between JAZ and the SCFCOI1 ubiquitin ligase, resulting in the ubiquitination of the JAZ protein and degradation by the 26S proteasome, and then the activation of transcription factors such as MYC2 to induce JA responses. Ultimately, a series of downstream responses, such as the reinforcement of physical defensive structures, the production of secondary metabolites, the inhibition of growth pathogens by the induction of defensive proteins, the activation of the ROS scavenging system, and other disease resistance factors, are activated to fight against pathogen infection. CNGCs, cyclic nucleotide-gated channels; CaMs, calmodulins; CPKs, calcium-dependent protein kinases; GLR, glutamate receptor-like genes; NO, nitric oxide; POD, peroxidase; SOD, superoxide dismutase; CAT, catalase; APX, ascorbate peroxidase; GST, glutathione S-transferase; ROS, reactive oxygen species; RBOHD, respiratory burst oxidase homolog D; H2O2, hydrogen peroxide; O₂−, superoxide ion; OH, hydroxyl radical; BIK1, BOTRYTIS-INDUCED KINASE1; MAPK, mitogen-activated protein kinase; R-Avr, interaction between an avirulence (Avr) gene in the pathogen and the corresponding resistance (R) gene in the host; EDS1, enhanced disease susceptibility; PAD4, phytoalexin-deficient 4; NPR1, non-expresser of PR genes 1; GDG1, GH3-like defense gene 1; EIN2, ethylene-insensitive 2; ISR, induced systemic resistance; SCF, Skp/Cullin/F-box; COI1, coronatine-insensitive 1; LOX2, lipoxygenase 2; VSP2, vegetative storage protein 2; JAZ, jasmonate ZIM-domain; CTR1, copper transport protein 1; ETR1, ethylene receptor gene 1; ERF, ethylene response factor; PR, pathogenesis-related protein; PGIP, polygalacturonase-inhibitory protein; OXO, oxalate oxidase. The arrows indicate positive regulation, and open blocks indicate negative regulation. Dashed lines indicate possible or indirect interactions. Full article ">Figure 3
The function of SA in the formation of local resistance and SAR. Plants can accumulate a large amount of SA after being infected by many pathogens. In addition to the SA accumulated at the infection site, SA is also associated with the SAR of uninfected tissues. NPR1 is critical for SA-dependent PR gene expression and SAR. The inhibition of SA accumulation or biosynthesis will inhibit the formation of SAR. NahG is an inhibitor of SA synthesis, which can convert SA into inactive catechol. Therefore, the overexpression of this gene can inhibit SAR formation and PR gene expressions. INA and BTH are analogues of SA, which are also plant defense activators and can induce SAR and the expression of PR genes. MeSA is a derivative of SA, which can act as a mobile inducer of SAR and induce the expression of defense genes in adjacent plants. SAR, systemic acquired resistance; MeSA, methyl salicylate; BTH, benzothiadiazole; INA, 2,6-dichloroisonicotinic acid; NahG, salicylate hydroxylase; BSMT1, benzoic acid/salicylic acid carboxyl methyltransferases; MES, methylesterases. Full article ">

26 pages, 3074 KiB

Open AccessArticle

Relationship of Cognition and Alzheimer’s Disease with Gastrointestinal Tract Disorders: A Large-Scale Genetic Overlap and Mendelian Randomisation Analysis

by Emmanuel O. Adewuyi, Eleanor K. O’Brien, Tenielle Porter and Simon M. Laws

Int. J. Mol. Sci. 2022, 23(24), 16199; https://doi.org/10.3390/ijms232416199 - 19 Dec 2022

Cited by 16 | Viewed by 9258

Abstract

Emerging observational evidence suggests links between cognitive impairment and a range of gastrointestinal tract (GIT) disorders; however, the mechanisms underlying their relationships remain unclear. Leveraging large-scale genome-wide association studies’ summary statistics, we comprehensively assessed genetic overlap and potential causality of cognitive traits and [...] Read more.

Emerging observational evidence suggests links between cognitive impairment and a range of gastrointestinal tract (GIT) disorders; however, the mechanisms underlying their relationships remain unclear. Leveraging large-scale genome-wide association studies’ summary statistics, we comprehensively assessed genetic overlap and potential causality of cognitive traits and Alzheimer’s disease (AD) with several GIT disorders. We demonstrate a strong and highly significant inverse global genetic correlation between cognitive traits and GIT disorders—peptic ulcer disease (PUD), gastritis-duodenitis, diverticulosis, irritable bowel syndrome, and gastroesophageal reflux disease (GERD), but not inflammatory bowel disease (IBD). Further analysis detects 35 significant (p < 4.37 × 10⁻⁵) bivariate local genetic correlations between cognitive traits, AD, and GIT disorders (including IBD). Mendelian randomisation analysis suggests a risk-decreasing causality of educational attainment, intelligence, and other cognitive traits on PUD and GERD, but not IBD, and a putative association of GERD with cognitive function decline. Gene-based analysis reveals a significant gene-level genetic overlap of cognitive traits with AD and GIT disorders (IBD inclusive, p_{binomial-test} = 1.18 × 10^−3–2.20 × 10⁻¹⁶). Our study supports the protective roles of genetically-influenced educational attainments and other cognitive traits on the risk of GIT disorders and highlights a putative association of GERD with cognitive function decline. Findings from local genetic correlation analysis provide novel insights, indicating that the relationship of IBD with cognitive traits (and AD) will depend largely on their local effects across the genome. Full article

(This article belongs to the Special Issue Modulation of Microbiota-Gut-Brain Axis in the Disorders of Brain and Mind)

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17 pages, 5890 KiB

Open AccessReview

Anti-Inflammatory Drug Therapy in Chronic Subdural Hematoma: A Systematic Review and Meta-Analysis of Prospective Randomized, Double-Blind and Placebo-Controlled Trials

by Martin Vychopen, Erdem Güresir and Johannes Wach

Int. J. Mol. Sci. 2022, 23(24), 16198; https://doi.org/10.3390/ijms232416198 - 19 Dec 2022

Cited by 4 | Viewed by 2981

Abstract

Althoughanti-inflammatory drug therapy has been identified as potentially beneficial for patients suffering from chronic subdural hematoma (cSDH), contemporary literature presents contradictory results. In this meta-analysis, we aimed to investigate the impact of anti-inflammatory drug therapy on mortality and outcome. We searched for eligible [...] Read more.

Althoughanti-inflammatory drug therapy has been identified as potentially beneficial for patients suffering from chronic subdural hematoma (cSDH), contemporary literature presents contradictory results. In this meta-analysis, we aimed to investigate the impact of anti-inflammatory drug therapy on mortality and outcome. We searched for eligible randomized, placebo-controlled prospective trials (RTCs) on PubMed, Embase and Medline until July 2022. From 97 initially identified articles, five RTCs met the criteria and were included in our meta-analysis. Our results illustrate significantly lower rates of recurrent cSDH (OR: 0.35; 95% CI: 0.21–0.58, p = 0.0001) in patients undergoing anti-inflammatory therapy. In the subgroup of patients undergoing primary conservative treatment, anti-inflammatory therapy was associated with lower rates of “switch to surgery” cases (OR: 0.30; 95% CI: 0.14–0.63, p = 0.002). Despite these findings, anti-inflammatory drugs seemed to be associated with higher mortality rates in patients undergoing surgery (OR: 1.76; 95% CI: 1.03–3.01, p = 0.04), although in the case of primary conservative treatment, no effect on mortality has been observed (OR: 2.45; 95% CI: 0.35–17.15, p = 0.37). Further multicentric prospective randomized trials are needed to evaluate anti-inflammatory drugs as potentially suitable therapy for asymptomatic patients with cSDH to avoid the necessity of surgical hematoma evacuation on what are predominantly elderly, vulnerable, patients. Full article

(This article belongs to the Special Issue Pharmacological Strategies for Neuroinflammation in Brain Injury)

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Int. J. Mol. Sci., Volume 23, Issue 24 (December-2 2022) – 782 articles

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