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Curr. Issues Mol. Biol., Volume 45, Issue 3 (March 2023) – 58 articles

Cover Story (view full-size image): Influenza viruses cause highly contagious respiratory infections and seasonal flu epidemics. The emergence of drug-resistant strains highlights groundbreaking for new antiviral therapeutic approaches. In this study, we provide the first biochemical evidence that 18-hydroxyferruginol (1) and 18-oxoferruginol (2) from Torreya nucifera can inhibit influenza virus infection by modulating the PI3K-Akt and ERK signaling pathways. The consequence of these events is the inhibition of viral RNP export from the nucleus and the disruption of influenza virus replication. Our findings suggest that these abietane diterpenoids have potential as novel antiviral candidates for the development of effective influenza therapies. View this paper
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15 pages, 4608 KiB  
Article
Sucrose Transporter StSUT2 Affects Potato Plants Growth, Flowering Time, and Tuber Yield
by Hui-Ling Gong, Jin-Bao Liu, Clement Igiraneza and Leonce Dusengemungu
Curr. Issues Mol. Biol. 2023, 45(3), 2629-2643; https://doi.org/10.3390/cimb45030172 - 22 Mar 2023
Cited by 6 | Viewed by 2269
Abstract
Background: Sucrose transporters (SUTs) mediate sucrose phloem loading in source tissue and sucrose unloading into sink tissue in potatoes and higher plants, thus playing a crucial role in plant growth and development. In potatoes, the physiological function of the sucrose transporters StSUT1 and [...] Read more.
Background: Sucrose transporters (SUTs) mediate sucrose phloem loading in source tissue and sucrose unloading into sink tissue in potatoes and higher plants, thus playing a crucial role in plant growth and development. In potatoes, the physiological function of the sucrose transporters StSUT1 and StSUT4 has been clarified, whereas the physiological role of StSUT2 is not yet fully understood. Methods and Results: This study analyzed the relative expression of StSUT2 compared to that of StSUT1 and StSUT4 in different tissues from potatoes and its impact on different physiological characteristics by using StSUT2-RNA interference lines. Here, we report a negative effect of StSUT2-RNA interference on plant height, fresh weight, internodes number, leaf area, flowering time, and tuber yield. However, our data indicate that StSUT2 is not involved in carbohydrate accumulation in potato leaves and tubers. In addition, the data of the RNA-seq between the StSUT2-RNA interference line and WT showed that 152 genes were differentially expressed, of which 128 genes were upregulated and 24 genes were downregulated, and the GO and KEGG analyses revealed that differentially expressed genes were mainly related to cell wall composition metabolism. Conclusions: Thus, StSUT2 functions in potato plant growth, flowering time, and tuber yield without affecting carbohydrate accumulation in the leaves and tubers but may be involved in cell wall composition metabolism. Full article
(This article belongs to the Special Issue Functional Genomics and Comparative Genomics Analysis in Plants)
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<p>An illustration of the summarized experimental procedure used to analyze potato RNAi-plants.</p> Full article ">Figure 2
<p>Relative expression of StSUT1 (<b>A</b>), StSUT2 (<b>B</b>), and StSUT4 (<b>C</b>) in different tissues of potato plants by qPCR. According to Duncan’s multiple range test, the same letters are not significantly different at <span class="html-italic">p</span> ≤ 0.05.</p> Full article ">Figure 2 Cont.
<p>Relative expression of StSUT1 (<b>A</b>), StSUT2 (<b>B</b>), and StSUT4 (<b>C</b>) in different tissues of potato plants by qPCR. According to Duncan’s multiple range test, the same letters are not significantly different at <span class="html-italic">p</span> ≤ 0.05.</p> Full article ">Figure 3
<p>StSUT2 expression level in the leaf of StSUT2-RNA interference lines. According to Duncan’s multiple range test, the same letters are not significantly different at <span class="html-italic">p</span> ≤ 0.05.</p> Full article ">Figure 4
<p>The phenotype of StSUT2-RNA interference plants (<b>A</b>) and its plants height (<b>B</b>) and fresh weight of the aerial (<b>C</b>) and underground parts (<b>D</b>). Plants were monitored within 4 weeks, 6 weeks, and 8 weeks of time duration. “*” means the difference is statistically significant between RNAi lines and WT.</p> Full article ">Figure 4 Cont.
<p>The phenotype of StSUT2-RNA interference plants (<b>A</b>) and its plants height (<b>B</b>) and fresh weight of the aerial (<b>C</b>) and underground parts (<b>D</b>). Plants were monitored within 4 weeks, 6 weeks, and 8 weeks of time duration. “*” means the difference is statistically significant between RNAi lines and WT.</p> Full article ">Figure 5
<p>StSUT2-RNAi interference negatively affects the internode numbers (<b>A</b>) and leaf area (<b>B</b>). Leaf samples were taken at the third, fourth, and fifth leaves from the top of the plants. “*” means the difference is statistically significant between RNAi lines and WT.</p> Full article ">Figure 6
<p>Flowering time (<b>A</b>) and the number of leaves at the flowering time (<b>B</b>) are significantly affected by StSUT2-RNAi. “*” means the difference is statistically significant between RNAi lines and WT.</p> Full article ">Figure 7
<p>Effects of StSUT2-RNAi on potato tuber yield (<b>A</b>), number (<b>B</b>), and size (<b>C</b>). “*” means the difference is statistically significant between RNAi lines and WT.</p> Full article ">Figure 7 Cont.
<p>Effects of StSUT2-RNAi on potato tuber yield (<b>A</b>), number (<b>B</b>), and size (<b>C</b>). “*” means the difference is statistically significant between RNAi lines and WT.</p> Full article ">Figure 8
<p>Volcano plot of differentially expressed gene distribution.</p> Full article ">Figure 9
<p>The enriched KEGG pathway of differentially expressed genes.</p> Full article ">Figure 10
<p>The most enriched GO terms of differentially expressed genes.</p> Full article ">
20 pages, 1640 KiB  
Review
Origin and Emergence of Microglia in the CNS—An Interesting (Hi)story of an Eccentric Cell
by Iasonas Dermitzakis, Maria Eleni Manthou, Soultana Meditskou, Marie-Ève Tremblay, Steven Petratos, Lida Zoupi, Marina Boziki, Evangelia Kesidou, Constantina Simeonidou and Paschalis Theotokis
Curr. Issues Mol. Biol. 2023, 45(3), 2609-2628; https://doi.org/10.3390/cimb45030171 - 22 Mar 2023
Cited by 19 | Viewed by 7188
Abstract
Microglia belong to tissue-resident macrophages of the central nervous system (CNS), representing the primary innate immune cells. This cell type constitutes ~7% of non-neuronal cells in the mammalian brain and has a variety of biological roles integral to homeostasis and pathophysiology from the [...] Read more.
Microglia belong to tissue-resident macrophages of the central nervous system (CNS), representing the primary innate immune cells. This cell type constitutes ~7% of non-neuronal cells in the mammalian brain and has a variety of biological roles integral to homeostasis and pathophysiology from the late embryonic to adult brain. Its unique identity that distinguishes its “glial” features from tissue-resident macrophages resides in the fact that once entering the CNS, it is perennially exposed to a unique environment following the formation of the blood–brain barrier. Additionally, tissue-resident macrophage progenies derive from various peripheral sites that exhibit hematopoietic potential, and this has resulted in interpretation issues surrounding their origin. Intensive research endeavors have intended to track microglial progenitors during development and disease. The current review provides a corpus of recent evidence in an attempt to disentangle the birthplace of microglia from the progenitor state and underlies the molecular elements that drive microgliogenesis. Furthermore, it caters towards tracking the lineage spatiotemporally during embryonic development and outlining microglial repopulation in the mature CNS. This collection of data can potentially shed light on the therapeutic potential of microglia for CNS perturbations across various levels of severity. Full article
(This article belongs to the Special Issue Molecular Mechanisms in Demyelinating Disorders of the Central Nervous System)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Timeline view of microglial origin since their discovery by Río Hortega.</p> Full article ">Figure 2
<p>Microgliogenesis at a glance. The primitive erythro-myeloid progenitors (EMPs; early, c−MYB−independent, CSF−1R<sup>+</sup> EMPs) arise from the yolk sac (YS) as early as embryonic day 7.5 (E7.5). These cells give rise to CD45<sup>+</sup> c−kit<sub>lo</sub> CX<sub>3</sub>CR1<sup>−</sup> immature (A1) cells that develop into CD45<sup>+</sup> c−kit<sup>−</sup> CX<sub>3</sub>CR1<sup>+</sup> (A2) cells. The early differentiation of microglial progenitors is regulated by the expression of RUNX1, PU.1, and IRF8. The invasion of progenitors into the neural tube begins at E9.5 through blood circulation and is followed by proliferation and terminal differentiation. As the blood–brain barrier becomes impermeable to small molecules at E14.5, the microglia invasion may be prevented. The transformation of immature microglia into ramified (mature form) occurs between the second and third postnatal weeks. The migration, proliferation, and terminal differentiation of microglia are also orchestrated from the depicted molecular cues. Light blue arrow timeline represents prenatal period, dark blue arrow timeline refers to postnatal days.</p> Full article ">
12 pages, 2214 KiB  
Case Report
Stroke-Associating Acute Limb Ischemia Due to the Rupture of a Hydatid Cyst
by Mihaela Lungu, Violeta Diana Oprea, Andrei Lucian Zaharia, Bianca Stan, Laura Rebegea, Dan Iulian Mocanu, Eva Maria Elkan, Elena Niculet and Ana Croitoru
Curr. Issues Mol. Biol. 2023, 45(3), 2597-2608; https://doi.org/10.3390/cimb45030170 - 22 Mar 2023
Viewed by 2357
Abstract
(1) Background: Hydatidosis, or human cystic echinococcosis, is a zoonotic disease. Endemic in some areas, recently it has an increasing incidence in wider regions, determined by population migration. Clinical features depend on the localization and level of infection: asymptomatic or with signs related [...] Read more.
(1) Background: Hydatidosis, or human cystic echinococcosis, is a zoonotic disease. Endemic in some areas, recently it has an increasing incidence in wider regions, determined by population migration. Clinical features depend on the localization and level of infection: asymptomatic or with signs related to hypersensitivity, organic functional deficiencies, expanding mass effects, cyst infection and sudden death. In rare cases, the rupture of a hydatid cyst causes emboli formation by the residual laminated membrane. (2) Methods: We performed an extensive literature review, starting from the case of a 25-year-old patient presenting with neurologic symptoms relevant for acute stroke, associating right upper limb ischemia. (3) Results: Imaging investigations revealed the source of the emboli as the rupture of a hydatid cyst, the patient presenting multiple pericardial and mediastinal localizations. Cerebral imaging confirmed an acute left occipital ischemic lesion, with complete recovery of the neurological deficit after therapy, while surgery for acute brachial artery ischemia had a favorable postoperative evolution. Specific anthelmintic therapy was initiated. An extensive literature review using available databases revealed the scarcity of data on embolism as a consequence of cyst rupture, highlighting the significant risk of clinicians overlooking this possible etiology. (4) Conclusions: An associated allergic reaction should raise the hypothesis of a hydatid cyst rupture as a cause of any level acute ischemic lesion. Full article
(This article belongs to the Special Issue Pathophysiology and Molecular Mechanisms of Acute Stroke)
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Figure 1
<p>Cerebral imaging: (<b>a</b>) native brain CT aspect: left occipital ischemic hypodense lesion; (<b>b</b>) brain MRI aspect: left occipital ischemia.</p> Full article ">Figure 2
<p>Contrast and native CT of thorax and abdomen shows multiple (<b>a</b>) mediastinal and (<b>b</b>) pericardial hydatid cysts.</p> Full article ">Figure 3
<p>Histopathological examination of the hydatid cyst membrane collected from the brachial artery: (<b>a</b>) HE × 40: fibrin clot enclosing fragments of the anhistous membrane; (<b>b</b>) HE × 100: anhistous membrane fragment embedded in fibrin-blood clot; (<b>c</b>) HE × 40: anhistous membrane fragment; (<b>d</b>) Histopathological examination HE × 40: anhistous membrane fragment and blood clot.</p> Full article ">Figure 4
<p>Histopathological examination: HE × 40: anhistous membrane fragment with associated dense inflammatory infiltrate.</p> Full article ">
17 pages, 2355 KiB  
Article
Alteration of Mesenchymal Stem Cells Isolated from Glioblastoma Multiforme under the Influence of Photodynamic Treatment
by Kalina Tumangelova-Yuzeir, Krassimir Minkin, Ivan Angelov, Ekaterina Ivanova-Todorova, Ekaterina Kurteva, Georgi Vasilev, Jeliazko Arabadjiev, Petar Karazapryanov, Kaloyan Gabrovski, Lidia Zaharieva, Tsanislava Genova and Dobroslav Kyurkchiev
Curr. Issues Mol. Biol. 2023, 45(3), 2580-2596; https://doi.org/10.3390/cimb45030169 - 21 Mar 2023
Cited by 2 | Viewed by 3436
Abstract
The central hypothesis for the development of glioblastoma multiforme (GBM) postulates that the tumor begins its development by transforming neural stem cells into cancer stem cells (CSC). Recently, it has become clear that another kind of stem cell, the mesenchymal stem cell (MSC), [...] Read more.
The central hypothesis for the development of glioblastoma multiforme (GBM) postulates that the tumor begins its development by transforming neural stem cells into cancer stem cells (CSC). Recently, it has become clear that another kind of stem cell, the mesenchymal stem cell (MSC), plays a role in the tumor stroma. Mesenchymal stem cells, along with their typical markers, can express neural markers and are capable of neural transdifferentiation. From this perspective, it is hypothesized that MSCs can give rise to CSC. In addition, MSCs suppress the immune cells through direct contact and secretory factors. Photodynamic therapy aims to selectively accumulate a photosensitizer in neoplastic cells, forming reactive oxygen species (ROS) upon irradiation, initiating death pathways. In our experiments, MSCs from 15 glioblastomas (GB-MSC) were isolated and cultured. The cells were treated with 5-ALA and irradiated. Flow cytometry and ELISA were used to detect the marker expression and soluble-factor secretion. The MSCs’ neural markers, Nestin, Sox2, and glial fibrillary acid protein (GFAP), were down-regulated, but the expression levels of the mesenchymal markers CD73, CD90, and CD105 were retained. The GB-MSCs also reduced their expression of PD-L1 and increased their secretion of PGE2. Our results give us grounds to speculate that the photodynamic impact on GB-MSCs reduces their capacity for neural transdifferentiation. Full article
(This article belongs to the Topic Porphyrinoids Photosensitizers for Biomedical Applications)
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<p>Photodynamic treatment of GB-MSCs. Schematic diagram of experimental steps and procedures.</p> Full article ">Figure 2
<p>Boxplot showing the change in the percentage of Nestin-positive GB-MSCs across paired samples of control cell cultures, cell cultures treated with light only, and cell cultures exposed to 5-ALA and light. Nestin expression was evaluated using multi-color flow cytometry, and every dot on the box plot represents the percentage values of all Nestin-positive GBM-MSCs in that particular cell culture.</p> Full article ">Figure 3
<p>Boxplot showing the change in the percentage of GFAP-positive GB-MSCs across paired samples of control cell cultures, cell cultures treated with light only, and cell cultures exposed to 5-ALA and light. The GFAP expression was evaluated using multi-color flow cytometry, and every dot on the box plot represents the percentage values of all GFAP-positive GBM-MSCs in that particular cell culture.</p> Full article ">Figure 4
<p>Boxplot showing the change in MFI levels of GFAP expression by GB-MSCs across paired samples of control cell cultures, cell cultures treated with light only, and cell cultures exposed to 5-ALA and light. The MFI GFAP expression was evaluated using multi-color flow cytometry, and every dot on the box plot represents the values of all GFAP-MFIs in that particular cell culture.</p> Full article ">Figure 5
<p>Boxplot showing the change in the percentage of Sox2-positive GB-MSCs across paired samples of control cell cultures, cell cultures treated with light only, and cell cultures exposed to 5-ALA and light. The Sox2 expression was evaluated using multi-color flow cytometry, and every dot on the box plot represents the percentage values of all Sox2-positive GBM-MSCs in that particular cell culture.</p> Full article ">Figure 6
<p>Boxplot showing the change in the percentage of PD-L1-positive GB-MSCs across paired samples of control cell cultures, cell cultures treated with light only, and cell cultures exposed to 5-ALA and light. The PD-L1 expression was evaluated using multi-color flow cytometry, and every dot on the box plot represents the percentage values of all the PD-L1 positive GBM-MSCs in that particular cell culture.</p> Full article ">Figure 7
<p>Boxplot showing the change in the levels of PGE2 across paired samples of control cell cultures, cell cultures treated with light only, and cell cultures exposed to 5-ALA and light. The effect of the PGE2 secretion on the protein levels was evaluated using ELISA Kit for human PGE2. Every dot on the box plot represents the levels of PGE2 in pg/mL produced by GBM-MSCs in that particular cell culture.</p> Full article ">Figure 8
<p>Radar plot summarizing the previous results of the observed percentages of Nestin, GFAP, PD-L1, and Sox2-positive GB-MSCs and the mean fluorescence intensity (MFI) of expression of Nestin, GFAP, PD-L1, and Sox2 markers at single cellular levels in GB-MSCs. Features’ values are mean-centered and divided by one standard deviation for better visual generalization. The punctured line depicts the feature values among control GB-MSCs, and the bold line depicts GB-MSCs exposed to 5-ALA and light. The radar plot demonstrates the decreased percentages of Nestin, GFAP, and PD-L1 expression by GB-MSCs treated with 5-ALA and light and the lack of change in the Nestin, GFAP, and PD-L1 MFI, with no alteration in the percentage and MFI of Sox2 expression.</p> Full article ">Figure 9
<p>The summary of the results obtained for the effect of PDT on GB-MSCs. The PDT leads to the down-regulation of markers associated predominantly with neural differentiation (Nestin, Sox-2, and GFAP). The markers associated predominantly with the mesenchymal phenotype (CD73, CD90, CD105, and CD44) retain their expression. According to the substances engaged with immunomodulation, PD-1L decreases its expression, but PGE2 demonstrates elevated secretion levels.</p> Full article ">
19 pages, 3948 KiB  
Article
The Effect of a Diet Enriched with Jerusalem artichoke, Inulin, and Fluoxetine on Cognitive Functions, Neurogenesis, and the Composition of the Intestinal Microbiota in Mice
by Aleksandra Szewczyk, Marta Andres-Mach, Mirosław Zagaja, Agnieszka Kaczmarczyk-Ziemba, Maciej Maj and Joanna Szala-Rycaj
Curr. Issues Mol. Biol. 2023, 45(3), 2561-2579; https://doi.org/10.3390/cimb45030168 - 21 Mar 2023
Cited by 5 | Viewed by 3963
Abstract
The aim of the study was to assess the effect of long-term administration of natural prebiotics: Jerusalem artichoke (topinambur, TPB) and inulin (INU) as well as one of the most popular antidepressants, fluoxetine (FLU), on the proliferation of neural stem cells, learning and [...] Read more.
The aim of the study was to assess the effect of long-term administration of natural prebiotics: Jerusalem artichoke (topinambur, TPB) and inulin (INU) as well as one of the most popular antidepressants, fluoxetine (FLU), on the proliferation of neural stem cells, learning and memory functions, and the composition of the intestinal microbiota in mice. Cognitive functions were assessed using the Morris Water Maze (MWM)Test. Cells were counted using a confocal microscope and ImageJ software. We performed 16S rRNA sequencing to assess changes in the gut microbiome of the mice. The obtained results showed that the 10-week supplementation with TPB (250 mg/kg) and INU (66 mg/kg) stimulates the growth of probiotic bacteria, does not affect the learning and memory process, and does not disturb the proliferation of neural stem cells in the tested animals. Based on this data, we can assume that both TPB and INU seem to be safe for the proper course of neurogenesis. However, 2-week administration of FLU confirmed an inhibitory impact on Lactobacillus growth and negatively affected behavioral function and neurogenesis in healthy animals. The above studies suggest that the natural prebiotics TPB and INU, as natural supplements, may have the potential to enrich the diversity of intestinal microbiota, which may be beneficial for the BGM axis, cognitive functions, and neurogenesis. Full article
(This article belongs to the Special Issue Interstitial Cells of Cajal and Nervous System in Aging and Oxidative Stress)
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<p>Schematic illustration of the experimental design.</p> Full article ">Figure 2
<p>Scheme of areas of the hippocampal DG designated for quantitative analysis of neurogenesis. A—BrdU positive cells; ML—molecular layer; GCL—granule cell layer; SGZ—subgranular zone.</p> Full article ">Figure 3
<p>Effect of TPB, INU, and FLU on the body weight of mice. TPB—topinambur; INU—inulin; FLU—fluoxetine. Each bar represents the mean for seven mice; error bars are S.E.M.</p> Full article ">Figure 4
<p>The impact of TPB, INU, and FLU on escape latency (<b>A</b>), distance (<b>B</b>), and the total average percentage of time spent in the W-channel (<b>C</b>). TPB—topinambur; INU—inulin; FLU—fluoxetine. Each bar represents the mean for seven mice; error bars are S.E.M. (* <span class="html-italic">p</span> &lt; 0.05; <span class="html-italic">n</span> = 7).</p> Full article ">Figure 5
<p>The recordings of the paths of directional flow in the W-channel for one selected animal from each study group. (<b>A</b>) control group; (<b>B</b>) TPB (Topinambur); (<b>C</b>) INU (Inulin); (<b>D</b>) FLU (Fluoxetine). The recording was made using a camera and a computer system with the Video Mot2 System software by TSE System.</p> Full article ">Figure 6
<p>Quantitative analysis of the neurogenesis process after long-term treatment with TPB, INU, and FLU in mice. The effect of long-term treatment with TPB, INU, and FLU on the total newborn cells (<b>A</b>), newborn neurons (<b>B</b>), and newborn astrocytes (<b>C</b>) in the dentate subgranular zone of treated mice. The numbers of cells represent an estimate of the total number of positively labeled cells in the subgranular zone in both hemispheres. TPB—topinambur; INU—inulin; FLU—fluoxetine. Each bar represents the mean for five mice; error bars are S.E.M. (* <span class="html-italic">p</span> &lt; 0.05,** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001; <span class="html-italic">n</span> = 5).</p> Full article ">Figure 7
<p>Alpha diversity indices calculated for different treatment groups. (<b>A</b>)—Chao1 index; (<b>B</b>)—Shannon index. The results were analyzed using the Kruskal–Wallis test. Data are presented as the mean ± SE.</p> Full article ">Figure 8
<p>Relative abundance of bacterial phyla identified in tested groups of samples.</p> Full article ">Figure 9
<p>(<b>A</b>) Relative abundance of bacterial genera identified in at least two tested groups of samples with the abundance above 1%. (<b>B</b>) Relative abundance of <span class="html-italic">Lactobacillus</span>.</p> Full article ">Figure 10
<p>Principal–coordinate analysis (PCoA) showing a two–dimensional ordination of tested microbiota profiles grouped according to the treatment.</p> Full article ">Figure 11
<p>LEfSe analysis of the gut microbiota profiles grouped according to the treatment.</p> Full article ">
12 pages, 537 KiB  
Brief Report
ParticleChromo3D+: A Web Server for ParticleChromo3D Algorithm for 3D Chromosome Structure Reconstruction
by David Vadnais and Oluwatosin Oluwadare
Curr. Issues Mol. Biol. 2023, 45(3), 2549-2560; https://doi.org/10.3390/cimb45030167 - 17 Mar 2023
Cited by 1 | Viewed by 1600
Abstract
Understanding the three-dimensional (3D) structure of chromatin is invaluable for researching how it functions. One way to gather this information is the chromosome conformation capture (3C) technique and its follow-up technique Hi-C. Here, we present ParticleChromo3D+, a containerized web-based genome structure reconstruction server/tool [...] Read more.
Understanding the three-dimensional (3D) structure of chromatin is invaluable for researching how it functions. One way to gather this information is the chromosome conformation capture (3C) technique and its follow-up technique Hi-C. Here, we present ParticleChromo3D+, a containerized web-based genome structure reconstruction server/tool that provides researchers with a portable and accurate tool for analyses. Additionally, ParticleChromo3D+ provides a more user-friendly way to access its capabilities via a graphical user interface (GUI). ParticleChromo3D+ can save time for researchers by increasing the accessibility of genome reconstruction, easing usage pain points, and offloading computational processing/installation time. Full article
(This article belongs to the Collection Feature Papers in Current Issues in Molecular Biology)
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<p>ParticleChromo3D [<a href="#B18-cimb-45-00167" class="html-bibr">18</a>] Process Diagram.</p> Full article ">Figure 2
<p>Comparison of REST, SOAP, gRPC, and WSDL searches worldwide.</p> Full article ">Figure 3
<p>Standard expected use case.</p> Full article ">Figure 4
<p>Graphical user interface screenshot for the ParticleChromo3D+ webserver.</p> Full article ">Figure 5
<p>Runtime box plots by chromosome.</p> Full article ">Figure 6
<p>SCC box plots by chromosome.</p> Full article ">Figure 7
<p>PCC box plots by chromosome.</p> Full article ">
16 pages, 832 KiB  
Article
Significant Association of Estrogen Receptor-β Isoforms and Coactivators in Breast Cancer Subtypes
by Young Choi and Simcha Pollack
Curr. Issues Mol. Biol. 2023, 45(3), 2533-2548; https://doi.org/10.3390/cimb45030166 - 17 Mar 2023
Viewed by 2139
Abstract
Nuclear receptor coregulators are the principal regulators of Estrogen Receptor (ER)-mediated transcription. ERβ, an ER subtype first identified in 1996, is associated with poor outcomes in breast cancer (BCa) subtypes, and the coexpression of the ERβ1 isoform and AIB-1 and TIF-2 coactivators in [...] Read more.
Nuclear receptor coregulators are the principal regulators of Estrogen Receptor (ER)-mediated transcription. ERβ, an ER subtype first identified in 1996, is associated with poor outcomes in breast cancer (BCa) subtypes, and the coexpression of the ERβ1 isoform and AIB-1 and TIF-2 coactivators in BCa-associated myofibroblasts is associated with high-grade BCa. We aimed to identify the specific coactivators that are involved in the progression of ERβ-expressing BCa. ERβ isoforms, coactivators, and prognostic markers were tested using standard immunohistochemistry. AIB-1, TIF-2, NF-kB, p-c-Jun, and/or cyclin D1 were differentially correlated with ERβ isoform expression in the BCa subtypes and subgroups. The coexpression of the ERβ5 and/or ERβ1 isoforms and the coactivators were found to be correlated with a high expression of P53, Ki-67, and Her2/neu and large-sized and/or high-grade tumors in BCa. Our study supports the notion that ERβ isoforms and coactivators seemingly coregulate the proliferation and progression of BCa and may provide insight into the potential therapeutic uses of the coactivators in BCa. Full article
(This article belongs to the Special Issue Advanced Molecular Solutions for Cancer Therapy)
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<p>Immunohistochemistry stains of ERβ1 expression and coactivators in infiltrating duct carcinoma: (<b>A</b>) H &amp; E staining of infiltrating duct carcinoma; (<b>B</b>) Erβ1 expression in the nuclei of benign epithelial cells and myoepithelial cells, stromal cells, and lymphocytes; (<b>C</b>) ERβ1 expression in the nuclei of neoplastic epithelial cells of infiltrating carcinoma and stromal cells and lymphocytes (immunohistochemistry staining using polyclonal ERβ1 385p/AR385-10R antibody); (<b>D</b>) ERα is expressed only in the nuclei of epithelial cells (original magnification 20×); immunohistochemistry stains of (<b>E</b>) AIB-1, (<b>F</b>) TIF-2, (<b>G</b>) SRC-1, and (<b>H</b>) p-c-Jun coactivators showing a positive nuclear reaction in the infiltrating carcinoma (original magnification 40×).</p> Full article ">
12 pages, 1665 KiB  
Article
Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants
by Laura A. E. Van Poelvoorde, François E. Dufrasne, Steven Van Gucht, Xavier Saelens and Nancy H. C. Roosens
Curr. Issues Mol. Biol. 2023, 45(3), 2521-2532; https://doi.org/10.3390/cimb45030165 - 17 Mar 2023
Cited by 7 | Viewed by 2207
Abstract
The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 [...] Read more.
The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance. Full article
(This article belongs to the Section Molecular Microbiology)
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Figure 1

Figure 1
<p><b>Visualisation of NA segment with the position of the E119V mutation that is linked to antiviral resistance in A(H3N2) influenza viruses</b>. NA contains three major functional domains, namely the signal anchor that contains the transmembrane domain, and the stalk domain and catalytic domain [<a href="#B39-cimb-45-00165" class="html-bibr">39</a>]. The stalk domain is believed to position NA in respect to its receptor and the length varies within and across NA subtypes [<a href="#B40-cimb-45-00165" class="html-bibr">40</a>,<a href="#B41-cimb-45-00165" class="html-bibr">41</a>,<a href="#B42-cimb-45-00165" class="html-bibr">42</a>,<a href="#B43-cimb-45-00165" class="html-bibr">43</a>,<a href="#B44-cimb-45-00165" class="html-bibr">44</a>,<a href="#B45-cimb-45-00165" class="html-bibr">45</a>,<a href="#B46-cimb-45-00165" class="html-bibr">46</a>]. The catalytic domain is responsible for the viral release and it removes the cell surface receptor to prevent interparticle aggregation [<a href="#B47-cimb-45-00165" class="html-bibr">47</a>,<a href="#B48-cimb-45-00165" class="html-bibr">48</a>,<a href="#B49-cimb-45-00165" class="html-bibr">49</a>,<a href="#B50-cimb-45-00165" class="html-bibr">50</a>,<a href="#B51-cimb-45-00165" class="html-bibr">51</a>,<a href="#B52-cimb-45-00165" class="html-bibr">52</a>]. This contributes to the assembly process by tethering the stalk to the membrane in a tetrameric conformation. The modification introduced by reverse genetics allows a substitution of a glutamate-to-valine mutation in NA at residue 119 (red line). Therefore, the NA with E119V may accommodate a water molecule, which would interfere with the binding of oseltamivir to the active site. Moreover, the E119V mutation affected the optimal orientation of oseltamivir within the active site and also affected its overall conformation [<a href="#B11-cimb-45-00165" class="html-bibr">11</a>,<a href="#B53-cimb-45-00165" class="html-bibr">53</a>]. The primers and probes bind with the sequence and the two probes will allow the discrimination between glutamate (E, FAM in blue) and valine (V, HEX in green) at position 119.</p> Full article ">Figure 2
<p><b>QuantaSoft 2D view of results before (A) and after (B) optimisation of quenchers and thermal gradient.</b> The negative droplets are shown in black; droplets containing the FAM-labelled E119 wild type are blue and the HEX-labelled V119 mutant are green. Droplets containing both fluorescent probes are orange.</p> Full article ">Figure 3
<p><b>ddPCR results of the different proportions of the wild-type virus and the mutant virus.</b> The grey line represents the theoretical percentages of the mixes, while the blue line is the observed results. The red line represents the linear trend line of the obtained results.</p> Full article ">Figure 4
<p><b>Inhibitory activity of oseltamivir on mixed populations of NAI-susceptible and -resistant recombinant influenza H3N2 viruses with the E119V-NA mutation</b>. To compare the results, the fold change is used, which means that the IC50 of the tested virus is compared to the control. The fold changes at 10% and 100% are indicated by a red line. When the fold change is below 10, there is normal inhibition. When the fold change is between 10 and 100, there is reduced inhibition. If above 100, the fold change indicates highly reduced inhibition [<a href="#B20-cimb-45-00165" class="html-bibr">20</a>].</p> Full article ">
16 pages, 3821 KiB  
Article
Mutant K-Ras in Pancreatic Cancer: An Insight on the Role of Wild-Type N-Ras and K-Ras-Dependent Cell Cycle Regulation
by Robert Ferguson, Karen Aughton, Anthony Evans, Victoria Shaw, Jane Armstrong, Adam Ware, Laura Bennett, Eithne Costello and William Greenhalf
Curr. Issues Mol. Biol. 2023, 45(3), 2505-2520; https://doi.org/10.3390/cimb45030164 - 17 Mar 2023
Cited by 2 | Viewed by 2703
Abstract
The development of K-Ras independence may explain the failure of targeted therapy for pancreatic cancer (PC). In this paper, active N as well as K-Ras was shown in all human cell lines tested. In a cell line dependent on mutant K-Ras, it was [...] Read more.
The development of K-Ras independence may explain the failure of targeted therapy for pancreatic cancer (PC). In this paper, active N as well as K-Ras was shown in all human cell lines tested. In a cell line dependent on mutant K-Ras, it was shown that depleting K-Ras reduced total Ras activity, while cell lines described as independent had no significant decline in total Ras activity. The knockdown of N-Ras showed it had an important role in controlling the relative level of oxidative metabolism, but only K-Ras depletion caused a decrease in G2 cyclins. Proteasome inhibition reversed this, and other targets of APC/c were also decreased by K-Ras depletion. K-Ras depletion did not cause an increase in ubiquitinated G2 cyclins but instead caused exit from the G2 phase to slow relative to completion of the S-phase, suggesting that the mutant K-Ras may inhibit APC/c prior to anaphase and stabilise G2 cyclins independently of this. We propose that, during tumorigenesis, cancer cells expressing wild-type N-Ras protein are selected because the protein protects cancer cells from the deleterious effects of the cell cycle-independent induction of cyclins by mutant K-Ras. Mutation independence results when N-Ras activity becomes adequate to drive cell division, even in cells where K-Ras is inhibited. Full article
(This article belongs to the Special Issue New Sight: Molecular Research in Pancreatic Cancer)
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Figure 1

Figure 1
<p>Total and active Ras isoforms in cancer cell lines: (<b>a</b>) Western blot analysis of total Ras in K-Ras mutation addicted (Suit-2) and independent cell lines (Panc1, BXPC3, HeLa). For each cell line, the upper panels show all Ras protein isotypes, and the lower panel shows active Ras isolated by pulldown with Raf, followed by panels of the total levels of each individual isotype. Cells were treated as shown with K-Ras, siRNA, or non-targeting control as indicated (RF = RISC Free, NT = Non-Targeting, KD = Knock Down). K-Ras depletion only depletes active Ras in the mutant Ras-dependent cell line Suit-2. (<b>b</b>) Western blot analysis of Suit-2 showing the presence of N-Ras in the upper panels and active N-Ras in the lower panel. Cells were treated as shown with K-Ras, siRNA, or non-targeting control. (<b>c</b>) Total Ras isoforms (left) or active Ras isolated by Raf pulldown (right). Cell line name is shown above the blot.</p> Full article ">Figure 2
<p>Effect of K-Ras depletion on G2 cyclins and other APC/c targets. (<b>a</b>) Western blot analysis of Suit-2 cells treated with K-Ras, N-Ras, or non-targeting siRNA showing the levels of cyclins. (<b>b</b>) Other targets of the APC/c complex and their levels following siRNA treatment.</p> Full article ">Figure 3
<p>Cell cycle analysis following K-Ras depletion: (<b>a</b>) FACS analysis of the cell cycle using PI staining showing reduced S-phase but little change in G2 level. (<b>b</b>) An example of Edu analysis of the cell cycle showing that K-Ras depletion increases the percentage of unlabelled cells in G2. (<b>c</b>,<b>d</b>) Graphs showing how unlabelled G2 cells change with length of Edu labelling. (<b>c</b>) After 18 h of labelling, nearly 40% of K-Ras-depleted G2 cells are still unlabelled; however, this declines with labelling time as labelled cells enter from S-phase and unlabelled cells move towards G1. Without K-Ras depletion, the proportion of unlabelled cells seem to be stable (therefore, any entry of cells from S-phase is compensated for by disproportionate exit of the G2 of labelled cells compared to unlabelled). (<b>d</b>) With K-Ras depletion, the percentage of total unlabelled cells that are in G2 remains almost constant, suggesting that unlabelled cells are leaving G2 and entering G1 at a slower rate than unlabelled G1 cells are entering S-phase. Without K-Ras depletion, the unlabelled cells from G2 accumulate in G1 over time, suggesting that cells are exiting G2 at an equivalent or greater rate than cells are leaving G1.</p> Full article ">Figure 4
<p>Effect of K-Ras depletion on G2 cyclins with or without Bortezomib treatment: (<b>a</b>) Western blot showing the effect of K-Ras depletion on total cyclin B in Suit-2 cells and how bortezomib treatment alters the effect. (<b>b</b>) Western blot showing the effect of bortezomib and siRNA in combination.</p> Full article ">Figure 5
<p>Effect of K-Ras depletion on G2 cyclin transcription. Graph showing an example of the decrease observed in G2 cyclin transcript when K-Ras or cyclin D is depleted. Cyclin B transcript levels after depletion with K-Ras are significantly lower than in non-targeting controls and after depletion with cyclin D (<span class="html-italic">p</span> &lt; 0.001, Mann–Whitney U test, standard deviation used as error bar). Cyclin A levels are also significantly lower after depletion of K-Ras than with no depletion; however, there was an equivalent decrease after cyclin D depletion.</p> Full article ">Figure 6
<p>Effects of K-Ras and N-Ras depletion on oxidative phosphorylation: (<b>a</b>) Representative Sea Horse analysis showing oxygen consumption rate (OCR) measured over time with the treatments shown. Oligomycin reduced oxygen consumption to just that due to proton leak, while FCCP uncoupled mitochondria resulting in maximal oxygen consumption. K-Ras knockdown caused an increase in uncoupled oxygen consumption while the N-Ras knockdown caused a reduction in maximal and basal oxygen consumption as well as a decrease in proton leak from mitochondria. Knockdown of N-Ras with K-Ras restores basal oxygen consumption with a smaller increase in maximal oxygen consumption and no reduction in proton leak. (<b>b</b>) The same experiment but showing Extra Cellular Acidification Rate (ECAR). K-Ras knockdown causes a reduction in ECAR, and N-Ras knockdown alone causes a smaller ECAR reduction, which is equivalent to the ECAR reduction seen when N-Ras and K-Ras are knocked down. (<b>c</b>) OCR/ECR is a measure of relative levels of respiration and glycolysis. K-Ras knockdown causes an increase in relative levels of respiration. Repeated experiments have showed this to be significantly greater than the increase seen with both K-Ras and N-Ras knockdown. N-Ras knockdown alone had little overall effect (<b>d</b>). (<b>e</b>) When OCR/ECAR is adjusted to the maximum increase in active mitochondrial activity (the difference from maximal to proton leak oxygen consumption), N-Ras knockdown was shown to reduce mitochondrial efficiency (Mann–Whitney U test, <span class="html-italic">p</span> &lt; 0.001, standard deviation used as error bar); moreover, K-Ras knockdown had less effect on efficiency and the combination of K-Ras with N-Ras knockdown restored mitochondrial efficiency.</p> Full article ">Figure 7
<p>Model of a compensatory mechanism of wild-type Ras expression in cells with mutant K-Ras: (1) Mutant K-Ras causes reduced mitochondrial activity (possibly by reducing the number of mitochondria) and promotes the production of G2 cyclins by inhibition of the proteolysis of an unknown transcriptional activator (x) of cyclin B1. (2) Wild-type (or mutant) Ras will increase transcription of G2 cyclins via G1 cyclins in a cell cycle-dependent fashion. EGFR can activate this process but can also increase G1 cyclins by directly binding to their promoters, compensating for the non-cyclic levels linked to the mutant protein. Furthermore, based on results from N-Ras knockdown, the wild type (N-Ras) increases glycolysis and the efficiency of respiration. (3) G2 cyclins will be degraded via the APC/c complex, therefore, in the absence of mutant Ras, the level of the G2 cyclins will be limited by the proportion of cells in G2. (4) Bortezomib inhibits the proteasome, causing stabilisation of ubiquitinated G2 cyclins, reducing the production of ubiquitinated protein by restricting entry to (and exit from) G2.</p> Full article ">
14 pages, 1978 KiB  
Article
Effects of Large Extracellular Vesicles from Kidney Cancer Patients on the Growth and Environment of Renal Cell Carcinoma Xenografts in a Mouse Model
by Matthieu Ferragu, Luisa Vergori, Vincent Le Corre, Sarah Bellal, Maria del Carmen Martinez and Pierre Bigot
Curr. Issues Mol. Biol. 2023, 45(3), 2491-2504; https://doi.org/10.3390/cimb45030163 - 17 Mar 2023
Viewed by 2416
Abstract
Plasma membrane-derived vesicles, also referred to as large extracellular vesicles (lEVs), are implicated in several pathophysiological situations, including cancer. However, to date, no studies have evaluated the effects of lEVs isolated from patients with renal cancer on the development of their tumors. In [...] Read more.
Plasma membrane-derived vesicles, also referred to as large extracellular vesicles (lEVs), are implicated in several pathophysiological situations, including cancer. However, to date, no studies have evaluated the effects of lEVs isolated from patients with renal cancer on the development of their tumors. In this study, we investigated the effects of three types of lEVs on the growth and peritumoral environment of xenograft clear cell renal cell carcinoma in a mouse model. Xenograft cancer cells were derived from patients’ nephrectomy specimens. Three types of lEVs were obtained from pre-nephrectomy patient blood (cEV), the supernatant of primary cancer cell culture (sEV) and from blood from individuals with no medical history of cancer (iEV). Xenograft volume was measured after nine weeks of growth. Xenografts were then removed, and the expression of CD31 and Ki67 were evaluated. We also measured the expression of MMP2 and Ca9 in the native mouse kidney. lEVs from kidney cancer patients (cEV and sEV) tend to increase the size of xenografts, a factor that is related to an increase in vascularization and tumor cell proliferation. cEV also altered organs that were distant from the xenograft. These results suggest that lEVs in cancer patients are involved in both tumor growth and cancer progression. Full article
(This article belongs to the Special Issue Advances in Molecular Pathogenesis Regulation in Cancer)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Characteristics and growth of primary culture cell line. Cells isolated from the patients have a renal and cancerous phenotype. Their proliferation was stable and continuous over time. (<b>A</b>) Confocal microscopy images of cells from patients 1, 3 and 4. (<b>B</b>) Proliferation of all primary culture lines as a function of time. Confluence at T0 is normalized to 1 on the graph and then measured as a function of time by the Incucyte <sup>®</sup> live Cell Analysis software.</p> Full article ">Figure 2
<p>Measurement and comparison of xenograft volume. lEVs isolated from the patients’ bloodstream (cEV) and from the primary culture supernatant (sEV) altered the peritumoral environment. (<b>A</b>) Representative images of xenograft volumes measured by ultrasound in the different groups after 9 weeks of growth. (<b>B</b>) Box plot of tumor volume in the different groups after 9 weeks of growth. Results are expressed in cubic millimeters (mm<sup>3</sup>). The analysis was performed by a Kruskal–Wallis test on GraphPad Prism8 software.</p> Full article ">Figure 3
<p>Evaluation of CD31 and Ki67 index in xenograft by confocal microscopy (objective ×20). (<b>A</b>) Representative images of CD31 labeling of the xenograft. Blue fluorescence represents DAPI (nuclei), and green fluorescence represents endothelial marker CD31 labeling. (<b>B</b>) Quantification: results are expressed in arbitrary immunofluorescence units (AU). (<b>C</b>) Representative images of nuclear labeling of xenograft by Ki67. Blue fluorescence represents DAPI (nuclei), and red fluorescence represents the marker of proliferation Ki-67. (<b>D</b>) Quantification: results are expressed as immunofluorescence index per nucleus (%Ki67 + nuclei). Scale bar = 50 µm. Data processing coupled with an ANOVA test was performed on GraphPad Prism8 software. Data are expressed with a box plot, (n = 5), * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 4
<p>Western blot analysis of Ca9 and MMP2 expression in mouse kidney. lEVs isolated from the bloodstream of patients (cEV) altered the native mouse kidney. Quantification and comparison of Ca9 and MMP2 proteins. Results are expressed as Ca9/β-Actin ratio. (<b>A</b>) Representative Western blot images of kidneys from mice 1, 2 and 3 of 5 in different groups. Line 1 MMP2: 70 kDa; line 2 Ca9: 54 kDa; line 3 β-Actin: 43 kDa. First and last lines correspond to protein ladders (Thermo Fisher Scientific, Waltham, MA, USA). (<b>B</b>) Ca9/β–actin and (<b>C</b>) MMP2/β–actin ratios (n = 5). Data processing, coupled with an ANOVA test, was performed on GraphPad Prism8 software. Data are expressed with a box plot (n = 5), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
17 pages, 5707 KiB  
Article
The In Vitro and In Vivo Anticancer Effect of Photomed for Photodynamic Therapy: Comparison with Photofrin and Radachlorin
by Jieun Kim, Johyun Kim, Heewon Yoon, Yoon-Jee Chae, Kiyon Rhew and Ji-Eun Chang
Curr. Issues Mol. Biol. 2023, 45(3), 2474-2490; https://doi.org/10.3390/cimb45030162 - 17 Mar 2023
Cited by 10 | Viewed by 2359
Abstract
To overcome the limitation of conventional cancer treatments, photodynamic therapy (PDT) has been introduced as another treatment option. PDT provides a non-invasive, non-surgical way with reduced toxicity. To improve the antitumor efficacy of PDT, we synthesized a novel photosensitizer, a 3-substituted methyl pyropheophorbide-a [...] Read more.
To overcome the limitation of conventional cancer treatments, photodynamic therapy (PDT) has been introduced as another treatment option. PDT provides a non-invasive, non-surgical way with reduced toxicity. To improve the antitumor efficacy of PDT, we synthesized a novel photosensitizer, a 3-substituted methyl pyropheophorbide-a derivative (Photomed). The purpose of the study was to evaluate the antitumor effect of PDT with Photomed comparing with the clinically approved photosensitizers Photofrin and Radachlorin. The cytotoxicity assay against SCC VII cells (murine squamous cell carcinoma) was performed to determine whether Photomed is safe without PDT and whether Photomed is effective against cancer cells with PDT. An in vivo anticancer efficacy study was also performed using SCC VII tumor-bearing mice. The mice were divided into small-tumor and large-tumor groups to identify whether Photomed-induced PDT is effective for not only small tumors but also large tumors. From in vitro and in vivo studies, Photomed was confirmed to be (1) a safe photosensitizer without laser irradiation, (2) the most effective photosensitizer with PDT against cancers compared to Photofrin and Radachlorin and (3) effective with PDT in treating not only small tumors but also large tumors. In conclusion, Photomed may contribute as a novel, potential photosensitizer for use in PDT cancer treatment. Full article
(This article belongs to the Topic Porphyrinoids Photosensitizers for Biomedical Applications)
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Figure 1

Figure 1
<p>(<b>A</b>) Synthesis of Photomed. (a) <span class="html-italic">t</span>-BuNH<sub>2</sub>BH<sub>3</sub>, CH<sub>2</sub>Cl<sub>2</sub>, for 12 h, at room temperature, under nitrogen. (b) Acetyl acetone, Zn(OAc)<sub>2</sub>, for 1 h at 60 °C, for 1.5 h at 110 °C, 6 N HCl for 20 min. (c) Hydrazine hydrate, EtOH, CH<sub>2</sub>Cl<sub>2</sub>, for 2 h, at room temperature. (<b>B</b>) Structures of Photofrin [<a href="#B24-cimb-45-00162" class="html-bibr">24</a>] and Radachlorin: chlorin e6 (I), chlorin p6 (II) and purpurin (III) [<a href="#B23-cimb-45-00162" class="html-bibr">23</a>].</p> Full article ">Figure 2
<p>Singlet oxygen photogeneration assay. PBS, 1 μM of methylene blue or photosensitizers (Photofrin, Radachlorin and Photomed) were added to 50 μM of 1,3-diphenylisobenzofuran (DPBF) solution and irradiated (5 mW/cm<sup>2</sup>, 400 s) with the proper wavelength (630 nm for Photofrin and 662 nm for Radachlorin and Photomed) of PDT lasers. Remaining DPBF was measured at 418 nm absorbance. Data are presented as means ± SD (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 3
<p>Scene of the in vitro PDT experiment. After 4 or 24 h of incubation with various concentrations (0, 0.1, 0.2 0.25, 0.4 and 0.5 μM) of photosensitizers under dark conditions, the cells were irradiated (5 mW/cm<sup>2</sup>, 400 s) with the proper wavelength (630 nm for Photofrin and 662 nm for Radachlorin and Photomed) of PDT lasers. Every in vitro irradiation was performed in the clean bench under dark conditions.</p> Full article ">Figure 4
<p>In vitro viability of SCC VII cells after various concentrations (0, 0.1, 0.2 0.25, 0.4 and 0.5 μM) of Photofrin, Radachlorin and Photomed treatment. (<b>a</b>) 4 h incubation of photosensitizers followed by no irradiation. (<b>b</b>) 4 h incubation of photosensitizers followed by light irradiation (PDT). (<b>c</b>) 24 h incubation of photosensitizers followed by no irradiation. (<b>d</b>) 24 h incubation of photosensitizers followed by light irradiation (PDT). Data are presented as means ± SD (<span class="html-italic">n</span> = 3). ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, Photofrin vs. Photomed. ## <span class="html-italic">p</span> &lt; 0.01, ### <span class="html-italic">p</span> &lt; 0.001, Radachlorin vs. Photomed.</p> Full article ">Figure 5
<p>Fluorescence microscope analysis of apoptotic cells induced by PDT. Amounts of 0.5 μM of Photofrin, Radachlorin and Photomed were treated for 24 h followed by no irradiation or light irradiation (5 mW/cm<sup>2</sup>, 400 s). Hoechst 33342 staining (blue, first column) indicating nuclei and Annexin V-FITC staining (green, second column) indicating apoptotic cells by PDT. Magnification 100×.</p> Full article ">Figure 6
<p>Scene of the in vivo PDT experiment. Tumor-bearing mouse models were established by subcutaneously injecting SCC VII cells to BALB/C nude mice. Three hours after intravenous injection (on day 0 and 7) of 1 mg/kg of photosensitizers, tumors were irradiated (400 mW/cm<sup>2</sup>, 500 s) with the proper wavelength (630 nm for Photofrin and 662 nm for Radachlorin and Photomed) of PDT lasers (on day 0 and 7). Every in vivo irradiation was performed on the heating pad set at 37 °C and under anesthesia.</p> Full article ">Figure 7
<p>Tumor volume and body weight monitoring of SCC VII tumor-bearing mice after double (on days 0 and 7, red arrows) administration of PBS, Photofrin, Radachlorin and Photomed followed by light irradiation (400 mW/cm<sup>2</sup>, 500 s) (on days 0 and 7, red arrows) 3 h after each injection. (<b>a</b>,<b>d</b>) Small-tumor group (tumor size ranging from 100 to 200 mm<sup>3</sup>) (<span class="html-italic">n</span> = 5). (<b>b</b>,<b>e</b>) Large-tumor group (tumor size ranging from 200 to 300 mm<sup>3</sup>) (<span class="html-italic">n</span> = 5). (<b>c</b>,<b>f</b>) Small- and large-tumor combined group (tumor size ranging from 100 to 300 mm<sup>3</sup>) (<span class="html-italic">n</span> = 10). The tumor volume (mm<sup>3</sup>) was calculated using the following formula; (length × width<sup>2</sup>)/2. Data are presented as means ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, PBS vs. Photomed. # <span class="html-italic">p</span> &lt; 0.05, ## <span class="html-italic">p</span> &lt; 0.01, ### <span class="html-italic">p</span> &lt; 0.001, Radachlorin vs. Photomed. + <span class="html-italic">p</span> &lt; 0.05, ++ <span class="html-italic">p</span> &lt; 0.01, +++ <span class="html-italic">p</span> &lt; 0.001, Photofrin vs. Photomed.</p> Full article ">Figure 8
<p>The representative tumor surface changes from SCC VII tumor-bearing mice before and after the first PDT with PBS, Photofrin, Radachlorin and Photomed.</p> Full article ">Figure 9
<p>The representative histological analysis of tumor tissues from SCC VII tumor-bearing mice on day 10, after double (on days 0 and 7) intravenous injection of PBS, Photofrin, Radachlorin and Photomed with light irradiation (on days 0 and 7) 3 h after each administration (M: mitotic area, N: necrotic area). The scale bar in images represents 100 μm.</p> Full article ">
13 pages, 789 KiB  
Review
Pesticidal Toxicity of Phosphine and Its Interaction with Other Pest Control Treatments
by Saad M. Alzahrani and Paul R. Ebert
Curr. Issues Mol. Biol. 2023, 45(3), 2461-2473; https://doi.org/10.3390/cimb45030161 - 17 Mar 2023
Cited by 9 | Viewed by 3212
Abstract
Phosphine is the most widely used fumigant for stored grains due to a lack of better alternatives, all of which have serious shortcomings that restrict their use. The extensive use of phosphine has led to the development of resistance among insect pests of [...] Read more.
Phosphine is the most widely used fumigant for stored grains due to a lack of better alternatives, all of which have serious shortcomings that restrict their use. The extensive use of phosphine has led to the development of resistance among insect pests of grain, which threatens its status as a reliable fumigant. Understanding the mode of action of phosphine as well as its resistance mechanisms provides insight that may lead to improved phosphine efficacy and pest control strategies. The mechanisms of action in phosphine vary from disrupting metabolism and oxidative stress to neurotoxicity. Phosphine resistance is genetically inherited and is mediated by the mitochondrial dihydrolipoamide dehydrogenase complex. In this regard, laboratory studies have revealed treatments that synergistically enhance phosphine toxicity that may be used to suppress resistance development and enhance efficacy. Here, we discuss the reported phosphine modes of action, mechanisms of resistance and interactions with other treatments. Full article
(This article belongs to the Collection Feature Papers in Current Issues in Molecular Biology)
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Figure 1
<p>A stylized representation of a non-linear response to phosphine. The days required to achieve 99.0% mortality are plotted on the <span class="html-italic">y</span>-axis, whereas the concentration of phosphine to which the insects were exposed is plotted on the <span class="html-italic">x</span>-axis. For context, a typical linear dose response curve as observed for typical pesticides is shown as a faint dashed line. The bold dashed lines represent fast and slow modes of action of phosphine that, when combined, result in the observed non-linear response [<a href="#B26-cimb-45-00161" class="html-bibr">26</a>].</p> Full article ">Figure 2
<p>Ionizing radiation results in physical damage to DNA as well as high levels of oxidative stress. Phosphine also causes significant oxidative stress and has been shown to damage DNA [<a href="#B106-cimb-45-00161" class="html-bibr">106</a>]. A <span class="html-italic">C. elegans</span> strain that is resistant to radiation induced apoptosis does not show cross-resistance to phosphine. This indicates that the superficial similarity between the modes of action of phosphine and ionizing radiation is not reflected in a shared antiapoptotic resistance mechanism.</p> Full article ">
9 pages, 277 KiB  
Brief Report
Ubiquitin Is Not a Blood Biomarker of an Early Cognitive Decline in the Polish Elderly
by Oliwia McFarlane, Mariusz Kozakiewicz, Milena Wojciechowska and Kornelia Kędziora-Kornatowska
Curr. Issues Mol. Biol. 2023, 45(3), 2452-2460; https://doi.org/10.3390/cimb45030160 - 16 Mar 2023
Cited by 3 | Viewed by 1556
Abstract
Together with development of new pharmaceutical interventions, as well as the introduction of the concept of initial dementia phase, the demand for early diagnosis has been growing. Research on potential blood biomarkers, amazingly attractive, mainly due to the facility of deriving the material, [...] Read more.
Together with development of new pharmaceutical interventions, as well as the introduction of the concept of initial dementia phase, the demand for early diagnosis has been growing. Research on potential blood biomarkers, amazingly attractive, mainly due to the facility of deriving the material, has provided ambiguous results throughout. The existence of an association between ubiquitin and Alzheimer’s disease pathology suggests that it could be a potential neurodegeneration biomarker. The present study aims to identify and assess the relationship between ubiquitin with regard to the adequacy as a biomarker of an initial dementia and cognitive decline in the elderly. Method: The study sample was composed of 230 participants: 109 women and 121 men aged 65 and older. The relationships of plasma ubiquitin levels with cognitive performance, gender, and age were analyzed. The assessments were performed in three groups of cognitive functioning level: cognitively normal, mild cognitive impairment, and mild dementia, of which the subjects were divided with the Mini-Mental State Examination (MMSE). Results: No significant disparities in plasma ubiquitin levels for various levels of cognitive functioning were identified. Significantly higher plasma ubiquitin levels in women were found in comparison to men. No significant differences were found in ubiquitin concentrations based on age. Results suggest that ubiquitin does not meet the requirements for qualification as a blood biomarker of an early cognitive decline. In order to thoroughly evaluate the potential of research on ubiquitin in connection to an early neurodegenerative process, further studies are needed. Full article
(This article belongs to the Special Issue Aging, Age-Related Changes in the Brain and the Progression of Alzheimer’s Disease)
8 pages, 1474 KiB  
Communication
Immunohistochemical Analysis of Spermatogenesis in Patients with SARS-CoV-2 Invasion in Different Age Groups
by Grigory A. Demyashkin, Evgeniya Kogan, Tatiana Demura, Dmitry Boldyrev, Matvey Vadyukhin, Vladimir Schekin, Peter Shegay and Andrey Kaprin
Curr. Issues Mol. Biol. 2023, 45(3), 2444-2451; https://doi.org/10.3390/cimb45030159 - 16 Mar 2023
Cited by 2 | Viewed by 2954
Abstract
Based on studies that focused on the effect of SARS-CoV-2 on human tissues, not only pulmonary invasion was revealed, but also impaired testicular function. Thus, the study of the mechanisms of influence of SARS-CoV-2 on spermatogenesis is still relevant. Of particular interest is [...] Read more.
Based on studies that focused on the effect of SARS-CoV-2 on human tissues, not only pulmonary invasion was revealed, but also impaired testicular function. Thus, the study of the mechanisms of influence of SARS-CoV-2 on spermatogenesis is still relevant. Of particular interest is the study of pathomorphological changes in men of different age groups. The purpose of this study was to evaluate immunohistochemical changes in spermatogenesis during SARS-CoV-2 invasion in different age groups. In our study, for the first time, a cohort of COVID-19-positive patients of different age groups was collected, and the following were conducted––confocal microscopy of the testicles and immunohistochemical evaluation of spermatogenesis disorders in SARS-CoV-2 invasion with antibodies to the spike protein, the nucleocapsid protein of the SARS-CoV-2 virus, and angiotensin convertase type 2. An IHC study and confocal microscopy of testicular autopsies from COVID-19-positive patients revealed an increase in the number of S-protein- and nucleocapsid-positively stained spermatogenic cells, which indicates SARS-CoV-2 invasion into them. A correlation was found between the number of ACE2-positive germ cells and the degree of hypospermatogenesis, and in the group of patients with confirmed coronavirus infection older than 45 years, the decrease in spermatogenic function was more pronounced than in the cohort of young people. Thus, our study found a decrease in both spermatogenic and endocrine (Leydig cells) testicular functions in patients with COVID-19 infection. In the elderly, these changes were significantly higher than in the group of young patients. Full article
(This article belongs to the Section Molecular Medicine)
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<p>Morphological picture of a testicle fragment from a man with a confirmed novel coronavirus infection. Stained with hematoxylin and eosin, magn. ×100.</p> Full article ">Figure 2
<p>Immunohistochemical pattern of germinal epithelium in patients with confirmed novel coronavirus infection. Stained with hematoxylin, magn. ×200.</p> Full article ">Figure 3
<p>Patient P.; 51 years old, FISH study by confocal microscopy. SARS-CoV-2 invasion into germ cells. Visualization with secondary antibodies conjugated with Alexa Fluor 488 (green glow), DAPI core staining (blue glow). Magn. ×200.</p> Full article ">
13 pages, 17664 KiB  
Article
Increased Yield of Extracellular Vesicles after Cytochalasin B Treatment and Vortexing
by Sirina V. Kurbangaleeva, Valeriia Y. Syromiatnikova, Angelina E. Prokopeva, Aleksey M. Rogov, Artur A. Khannanov, Albert A. Rizvanov and Marina O. Gomzikova
Curr. Issues Mol. Biol. 2023, 45(3), 2431-2443; https://doi.org/10.3390/cimb45030158 - 15 Mar 2023
Cited by 6 | Viewed by 2609
Abstract
Extracellular vesicles (EVs) are promising therapeutic instruments and vectors for therapeutics delivery. In order to increase the yield of EVs, a method of inducing EVs release using cytochalasin B is being actively developed. In this work, we compared the yield of naturally occurring [...] Read more.
Extracellular vesicles (EVs) are promising therapeutic instruments and vectors for therapeutics delivery. In order to increase the yield of EVs, a method of inducing EVs release using cytochalasin B is being actively developed. In this work, we compared the yield of naturally occurring extracellular vesicles and cytochalasin B-induced membrane vesicles (CIMVs) from mesenchymal stem cells (MSCs). In order to maintain accuracy in the comparative analysis, the same culture was used for the isolation of EVs and CIMVs: conditioned medium was used for EVs isolation and cells were harvested for CIMVs production. The pellets obtained after centrifugation 2300× g, 10,000× g and 100,000× g were analyzed using scanning electron microscopy analysis (SEM), flow cytometry, the bicinchoninic acid assay, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). We found that the use of cytochalasin B treatment and vortexing resulted in the production of a more homogeneous population of membrane vesicles with a median diameter greater than that of EVs. We found that EVs-like particles remained in the FBS, despite overnight ultracentrifugation, which introduced a significant inaccuracy in the calculation of the EVs yield. Therefore, we cultivated cells in a serum-free medium for the subsequent isolation of EVs. We observed that the number of CIMVs significantly exceeded the number of EVs after each step of centrifugation (2300× g, 10,000× g and 100,000× g) by up to 5, 9, and 20 times, respectively. Full article
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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<p>Scheme of the experiments. EVs and CIMVs were isolated from the same MSCs culture. Created with BioRender.com.</p> Full article ">Figure 2
<p>Analysis of morphology (<b>A</b>) and size distribution (<b>B</b>) of vesicles obtained during EVs and CIMVs isolation after centrifugation 2300× <span class="html-italic">g</span>, 10,000× <span class="html-italic">g</span>, and 100,000× <span class="html-italic">g</span>. Scale bar—1 µm.</p> Full article ">Figure 3
<p>Average diameter of mouse EVs and CIMVs centrifuged at 2300× <span class="html-italic">g</span>, 10,000× <span class="html-italic">g</span>, and 100,000× <span class="html-italic">g</span>. Method—dynamic light scattering. The data represent mean ± SD. ns—not statistically significant. (**)—level of significance <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 4
<p>Analysis of EVs and CIMVs yield. (<b>A</b>) Average number of EVs and CIMV after centrifugation 2300× <span class="html-italic">g</span>, 10,000× <span class="html-italic">g</span>, and 100,000× <span class="html-italic">g</span>. Flow cytometry method. (<b>B</b>) Total protein concentrations of EVs and CIMVs after centrifugation 2300× <span class="html-italic">g</span>, 10,000× <span class="html-italic">g</span>, and 100,000× <span class="html-italic">g</span>. Bicinchoninic acid method. The data represent mean ± SD. ns—not statistically significant. (*)—level of significance <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p>Analysis of the number of EVs isolated from conditioned medium and the number of EVs-like particles isolated from the EVs-depleted medium. NTA method.</p> Full article ">Figure 6
<p>Analysis of EVs and CIMVs yields. Average number of EVs and CIMV after centrifugation 2300× <span class="html-italic">g</span>, 10,000× <span class="html-italic">g</span>, and 100,000× <span class="html-italic">g</span>. NTA method.</p> Full article ">
9 pages, 1236 KiB  
Case Report
A Novel Nonsense Pathogenic TTN Variant Identified in a Patient with Severe Dilated Cardiomyopathy
by Caterina Micolonghi, Marco Fabiani, Erika Pagannone, Camilla Savio, Marta Ricci, Silvia Caroselli, Vittoria Gambioli, Beatrice Musumeci, Aldo Germani, Giacomo Tini, Camillo Autore, Antonio Pizzuti, Vincenzo Visco, Speranza Rubattu, Simona Petrucci and Maria Piane
Curr. Issues Mol. Biol. 2023, 45(3), 2422-2430; https://doi.org/10.3390/cimb45030157 - 15 Mar 2023
Cited by 2 | Viewed by 2473
Abstract
Both genetic and environmental factors contribute to the development of dilated cardiomyopathy. Among the genes involved, TTN mutations, including truncated variants, explain 25% of DCM cases. We performed genetic counseling and analysis on a 57-year-old woman diagnosed with severe DCM and presenting relevant [...] Read more.
Both genetic and environmental factors contribute to the development of dilated cardiomyopathy. Among the genes involved, TTN mutations, including truncated variants, explain 25% of DCM cases. We performed genetic counseling and analysis on a 57-year-old woman diagnosed with severe DCM and presenting relevant acquired risk factors for DCM (hypertension, diabetes, smoking habit, and/or previous alcohol and cocaine abuse) and with a family history of both DCM and sudden cardiac death. The left ventricular systolic function, as assessed by standard echocardiography, was 20%. The genetic analysis performed using TruSight Cardio panel, including 174 genes related to cardiac genetic diseases, revealed a novel nonsense TTN variant (TTN:c.103591A > T, p.Lys34531*), falling within the M-band region of the titin protein. This region is known for its important role in maintaining the structure of the sarcomere and in promoting sarcomerogenesis. The identified variant was classified as likely pathogenic based on ACMG criteria. The current results support the need of genetic analysis in the presence of a family history, even when relevant acquired risk factors for DCM may have contributed to the severity of the disease. Full article
(This article belongs to the Special Issue Focus on Molecular Basis of Cardiac Diseases)
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<p>(<b>A</b>) Pedigree of the family showing the phenotypes of the affected relatives and the genotype of the proband. Male is represented by a square; female is represented by a circle. (<b>B</b>) Multi-gene panel analysis. NGS and Sanger sequencing of the proband showing the c.103591A &gt; T (p.Lys34531*) in <span class="html-italic">TTN</span> on genomic DNA. The identified pathogenic variant in <span class="html-italic">TTN</span> is visualized by Integrative Genome Viewer (IGV) software. Ref Seq (Reference sequencing) used for variant annotation: NM_001267550.2.</p> Full article ">Figure 2
<p>Echocardiographic and electrocardiographic images of the proband (III:1). (<b>A</b>) Two-dimensional transthoracic echocardiogram diastolic frames of parasternal long-axis view and (<b>B</b>) 4-chamber view showing dilated and globular-shaped left ventricle, with septal thinning. (<b>C</b>) Electrocardiogram showing left anterior hemiblock and slight ST segment depression from V4 to V6. LV—left ventricle; RV—right ventricle; LA—left atrium; RA—right atrium; AO—aorta; IVS—interventricular septum.</p> Full article ">
13 pages, 3196 KiB  
Article
Evaluation of Some Benzo[g]Quinazoline Derivatives as Antiviral Agents against Human Rotavirus Wa Strain: Biological Screening and Docking Study
by Hatem A. Abuelizz, Ahmed H. Bakheit, Mohamed Marzouk, Waled M. El-Senousy, Mohamed M. Abdellatif, Gamal A. E. Mostafa and Rashad Al-Salahi
Curr. Issues Mol. Biol. 2023, 45(3), 2409-2421; https://doi.org/10.3390/cimb45030156 - 14 Mar 2023
Cited by 3 | Viewed by 2548
Abstract
Globally, rotavirus (RV) is the most common cause of acute gastroenteritis in infants and toddlers; however, there are currently no agents available that are tailored to treat rotavirus infection in particular. Improved and widespread immunization programs are being implemented worldwide to reduce rotavirus [...] Read more.
Globally, rotavirus (RV) is the most common cause of acute gastroenteritis in infants and toddlers; however, there are currently no agents available that are tailored to treat rotavirus infection in particular. Improved and widespread immunization programs are being implemented worldwide to reduce rotavirus morbidity and mortality. Despite certain immunizations, there are no licensed antivirals that can attack rotavirus in hosts. Benzoquinazolines, chemical components synthesized in our laboratory, were developed as antiviral agents, and showed good activity against herpes simplex, coxsackievirus B4 and hepatitis A and C. In this research project, an in vitro investigation of the effectiveness of benzoquinazoline derivatives 1–16 against human rotavirus Wa strains was carried out. All compounds exhibited antiviral activity, however compounds 1–3, 9 and 16 showed the greatest activity (reduction percentages ranged from 50 to 66%). In-silico molecular docking of highly active compounds, which were selected after studying the biological activity of all investigated of benzo[g]quinazolines compounds, was implemented into the protein’s putative binding site to establish an optimal orientation for binding. As a result, compounds 1, 3, 9, and 16 are promising anti-rotavirus Wa strains that lead with Outer Capsid protein VP4 inhibition. Full article
(This article belongs to the Special Issue Drugs: Mechanisms of Action, Molecular Targets and Biological Activities)
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<p>The target benzo[g]quinazolines <b>1</b>–<b>16</b>.</p> Full article ">Figure 2
<p>Structural modification on benzoquinazolines. Increase in the activity accompanied by inserting methyl group at position 3; Replacing C = S by S-Bn substituted as in case compound <b>9</b> resulted in increasing the activity; Replacing C = S by -NH-NH<sub>2</sub> as in case compound <b>16</b> resulted in increasing the activity in regards to compounds <b>14</b> and <b>15</b>; Presence of electron donating/withdrawing groups produced a variety of activity as in <b>8, 6</b> and <b>9</b>.</p> Full article ">Figure 3
<p>Predicted binding pocket of the Human rotavirus strain Wa by MOE-2015.10 a co-crystallized ligand atom that is present in the pocket.</p> Full article ">Figure 4
<p>Human rotavirus ligand interaction diagram (2DWR). Active site residues: polar residues pink, hydrophobic residues green, acidic residues red, basic residues blue. Green and blue arrows represent hydrogen bonding to sidechain and backbone atoms. (<b>A</b>,<b>B</b>): 2D and 3D for interactions of compound <b>1;</b> (<b>C</b>,<b>D</b>): 2D and 3D for interactions of compound <b>3;</b> (<b>E</b>,<b>F</b>): 2D and 3D for interactions of compound <b>9;</b> (<b>G</b>,<b>H</b>): 2D and 3D for interactions of compound <b>16</b>.</p> Full article ">Figure 4 Cont.
<p>Human rotavirus ligand interaction diagram (2DWR). Active site residues: polar residues pink, hydrophobic residues green, acidic residues red, basic residues blue. Green and blue arrows represent hydrogen bonding to sidechain and backbone atoms. (<b>A</b>,<b>B</b>): 2D and 3D for interactions of compound <b>1;</b> (<b>C</b>,<b>D</b>): 2D and 3D for interactions of compound <b>3;</b> (<b>E</b>,<b>F</b>): 2D and 3D for interactions of compound <b>9;</b> (<b>G</b>,<b>H</b>): 2D and 3D for interactions of compound <b>16</b>.</p> Full article ">Figure 5
<p>Interactions between ligands and proteins. (<b>A</b>) Surface representation of the Human rotavirus strain Wa (PDB ID: 2DWR), in three-dimensional representation, with an overlay of studied compounds (<b>1</b>, <b>3</b>, <b>9</b>, and <b>16</b>) at the active site of the enzyme (polar, hydrophobic, and exposed surfaces colored as pink, green, and red, respectively); (<b>B</b>) Predicted binding poses of docked ligands (sticks) with an overlay, only residues located within a 5Å radius of bound ligands are displayed (lines). The hydrogen bond is shown by black dashed lines.</p> Full article ">
16 pages, 6632 KiB  
Article
The Potential Inhibitory Role of Acetyl-L-Carnitine on Proliferation, Migration, and Gene Expression in HepG2 and HT29 Human Adenocarcinoma Cell Lines
by Sarah Albogami
Curr. Issues Mol. Biol. 2023, 45(3), 2393-2408; https://doi.org/10.3390/cimb45030155 - 14 Mar 2023
Cited by 2 | Viewed by 3468
Abstract
Malignancies of the liver and colon are the most prevalent forms of digestive system cancer globally. Chemotherapy, one of the most significant treatments, has severe side effects. Chemoprevention using natural or synthetic medications can potentially reduce cancer severity. Acetyl-L-carnitine (ALC) is an acetylated [...] Read more.
Malignancies of the liver and colon are the most prevalent forms of digestive system cancer globally. Chemotherapy, one of the most significant treatments, has severe side effects. Chemoprevention using natural or synthetic medications can potentially reduce cancer severity. Acetyl-L-carnitine (ALC) is an acetylated derivative of carnitine essential for intermediate metabolism in most tissues. This study aimed to investigate the effects of ALC on the proliferation, migration, and gene expression of human liver (HepG2) and colorectal (HT29) adenocarcinoma cell lines. The cell viability and half maximal inhibitory concentration of both cancer cell lines were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Wound healing after treatment was assessed using a migration assay. Morphological changes were imaged using brightfield and fluorescence microscopy. Post treatment, apoptotic DNA was detected using a DNA fragmentation assay. The relative mRNA expressions of matrix metallopeptidase 9 (MMP9) and vascular endothelial growth factor (VEGF) were evaluated using RT-PCR. The results showed that ALC treatment affects the wound-healing ability of HepG2 and HT29 cell lines. Changes in nuclear morphology were detected under fluorescent microscopy. ALC also downregulates the expression levels of MMP9 and VEGF in HepG2 and HT29 cell lines. Our results indicate that the anticancer action of ALC is likely mediated by a decrease in adhesion, migration, and invasion. Full article
(This article belongs to the Special Issue Adhesion, Metastasis and Inhibition of Cancer Cells)
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Graphical abstract

Graphical abstract
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<p>Dose–response curve in HepG2, and HT29 cells after treatment with acetyl-L-carnitine (ALC) at 24, 48, and 72 h. (<b>A</b>–<b>C</b>) HepG2 cell line, (<b>D</b>–<b>F</b>) HT29 cell line. Both cell lines were treated with 0, 0.5, 5, 10, 15, 30, and 60 μM/mL at 24, 48, 72 h. The viability was measured at different time points for each cell line. The point where the red dashed line meets the green dashed line is the absolute IC<sub>50</sub>. The concentration was transformed to log concentration and normalized to percentage, then the mean of six repeats was plotted by nonlinear regression. The error bars correspond to standard deviation (SD).</p> Full article ">Figure 2
<p>The effect of ALC treatment on wound closure in HepG2 cells at 0, 24, 48, and 72 h. (<b>A</b>) Control HepG2 cells, (<b>B</b>) ALC-treated HepG2 cells. HepG2 cells were treated with their IC<sub>50</sub> of ALC, and brightfield images were acquired at 0, 24, 48, and 72 h using an inverted microscope with a 10× objective lens. (<b>C</b>) A summary of the wound-healing assay’s findings. ImageJ was used to measure the wound closure percentage. Two-way ANOVA analysis was performed to compare the wound closure percentage for control vs. treated cells. The error bars correspond to SD, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 3
<p>The effect of ALC treatment on wound closure in HT29 cells at 0, 24, 48, and 72 h. (<b>A</b>) Control HT29 cells, (<b>B</b>) ALC-treated HT29 cells. HT29 cells were treated with their IC<sub>50</sub> concentrations of ALC, and brightfield images were acquired at 0, 24, 48, and 72 h using an inverted microscope with a 10× objective lens. (<b>C</b>) A summary of the wound-healing assay’s findings. ImageJ was used to measure the wound closure percentage. Two-way ANOVA analysis was performed to compare the wound closure percentage for control vs. treated cells. The error bars correspond to SD, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 4
<p>The effect of ALC treatment on cell morphology and DNA fragmentation in HepG2 and HT29 cells. (<b>A</b>) HepG2 cells and (<b>B</b>) HT29 cells. Cells were treated with their IC<sub>50</sub> concentrations for 48 h and images of cells were captured under a brightfield microscope. Apoptotic cells are marked by arrows. Gel electrophoresis of apoptotic DNA collected from each cell was performed after treatment with ALC at IC<sub>50</sub> concentrations for 48 h, (<b>1</b>) Kb DNA ladder, (<b>2</b>) control cell, (<b>3</b>) cell treated with ALC. Apoptotic DNA fragmentation is marked by rectangles.</p> Full article ">Figure 5
<p>Fluorescent microscopy examination to evaluate apoptotic nucleus morphological changes in HepG2 cells after ALC treatment compared to control. (<b>A</b>) control HepG2 cells, (<b>B</b>) ALC-treated HepG2 cells. HepG2 cells were treated with their IC<sub>50</sub> concentration of ALC for 48 h and stained with DAPI and propidium iodide (PI). Brightfield images were acquired under fluorescence microscopy with appropriate filters for PI and DAPI. ImageJ was used to overlay images. Rectangles show some cells after being magnified to illustrate the apoptotic effect of ALC on the nucleus.</p> Full article ">Figure 6
<p>Fluorescent microscopy examination to evaluate apoptotic nucleus morphological changes in HT29 cells after ALC treatment compared to control. (<b>A</b>) control HT29 cell line, (<b>B</b>) ALC-treated HT29 cell line. HT29 cell lines were treated with their IC<sub>50</sub> concentration of ALC for 48 h and stained with DAPI and PI. Images were acquired under fluorescence microscopy with appropriate filters for PI and DAPI. ImageJ was used to overlay images. Rectangles show some cells after being magnified to illustrate the apoptotic effect of ALC on the nucleus.</p> Full article ">Figure 7
<p>Apoptotic cells (%) under fluorescent microscopy in HepG2 and HT29 cells after ALC treatment compared to control. (<b>A</b>) HpG2 cells, (<b>B</b>) HT29 cells. The proportion of apoptotic cells in each cell line after the treatments were determined using inverted fluorescence microscopy. An unpaired <span class="html-italic">t</span>-test analysis was used to compare the apoptotic % between treated and control in each cell line. Data expressed as means ± SE of three experiments * <span class="html-italic">p</span> &lt; 0.05 compared to control.</p> Full article ">Figure 8
<p>The mRNA expression of MMPs and VEGF in HepG2 and HT29 cells after ALC treatment. (<b>A</b>) MMPs mRNA expression levels in HepG2, (<b>B</b>) MMPs mRNA expression levels in HT29, (<b>C</b>) VEGF mRNA expression levels in HepG2, and (<b>D</b>) VEGF mRNA expression levels in HT29. The expression levels of all genes were normalized to that of the β-actin gene. An unpaired <span class="html-italic">t-</span>test analysis was used to compare the expression of each gene compared to the control. Data expressed as means ± SE of three experiments. * <span class="html-italic">p</span> &lt; 0.05 compared to control.</p> Full article ">
12 pages, 4031 KiB  
Article
A Feedback Loop between TGF-β1 and ATG5 Mediated by miR-122-5p Regulates Fibrosis and EMT in Human Trabecular Meshwork Cells
by Munmun Chakraborthy and Aparna Rao
Curr. Issues Mol. Biol. 2023, 45(3), 2381-2392; https://doi.org/10.3390/cimb45030154 - 13 Mar 2023
Cited by 3 | Viewed by 2103
Abstract
Autophagy is a cell’s evolutionary conserved process for degrading and recycling cellular proteins and removing damaged organelles. There has been an increasing interest in identifying the basic cellular mechanism of autophagy and its implications in health and illness during the last decade. Many [...] Read more.
Autophagy is a cell’s evolutionary conserved process for degrading and recycling cellular proteins and removing damaged organelles. There has been an increasing interest in identifying the basic cellular mechanism of autophagy and its implications in health and illness during the last decade. Many proteinopathies such as Alzheimer’s and Huntington’s disease are reported to be associated with impaired autophagy. The functional significance of autophagy in exfoliation syndrome/exfoliation glaucoma (XFS/XFG), remains unknown though it is presumed to be impaired autophagy to be responsible for the aggregopathy characteristic of this disease. In the current study we have shown that autophagy or ATG5 is enhanced in response to TGF-β1 in human trabecular meshwork (HTM) cells and TGF-β1 induced autophagy is necessary for increased expression of profibrotic proteins and epithelial to mesenchymal (EMT) through Smad3 that lead to aggregopathy. Inhibition of ATG5 by siRNA mediated knockdown reduced profibrotic and EMT markers and increased protein aggregates in the presence of TGF-β1 stimulation. The miR-122-5p, which was increased upon TGF exposure, was also reduced upon ATG5 inhibition. We thus conclude that TGF-β1 induces autophagy in primary HTM cells and a positive feedback loop exists between TGF-β1 and ATG5 that regulated TGF downstream effects mainly mediated by Smad3 signaling with miR-122-5p also playing a role. Full article
(This article belongs to the Collection Feature Papers in Current Issues in Molecular Biology)
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<p>TGF-β1 induced ATG5 activation in human trabecular meshwork (HTM cells). TGF-β1 (10 ng/mL) applied to primary HTM cells for 48–72 h, increased relative mRNA expression of (<b>A</b>) <span class="html-italic">ATG-5</span> and (<b>B</b>) LC3-II. It also increased levels of (<b>C</b>) ATG-5 proteins and lipidation of LC3 II, (<b>D</b>,<b>E</b>) Densitometry analysis of bands. The results represent the averages of three separate tests conducted. The error bars represent the standard deviation (*—<span class="html-italic">p</span> &lt; 0.05, **—<span class="html-italic">p</span> &lt; 0.01, by <span class="html-italic">t</span>-test).</p> Full article ">Figure 2
<p>TGF-induced fibrosis and EMT is regulated by ATG5. (<b>A</b>) The protein levels of ATG-5, LC3-I, LC3-II, SMA, vimentin, fibronectin, and fibrillin in whole-cell lysates of siRNA-mediated knockdown HTM cells with (10 ng/mL) and without TGF-β1. (<b>B</b>–<b>G</b>), relative quantification of the proteins obtained using densitometry analysis of the bands. The error bars represent the standard deviation (*—<span class="html-italic">p</span> &lt; 0.05, **—<span class="html-italic">p</span> &lt; 0.01, ***—<span class="html-italic">p</span> &lt; 0.001, by <span class="html-italic">t</span>-test).</p> Full article ">Figure 3
<p>Apoptosis is regulated by ATG5 in HTM cells. HTM cells were treated with TGF-β1 (10 ng/mL) and siRNA and the total number of apoptotic cells was calculated by flow cytometry. (<b>A</b>–<b>D</b>) The total number of apoptotic cells in TGF-β1 treated and siRNA knockdown HTM cells.</p> Full article ">Figure 4
<p><span class="html-italic">ATG5</span> regulates TGF-β1 induced effects in HTM cells via Smad3 signaling. HTM primary cells treated with 2.5 µM Smad3 inhibitor (SIS3) and with (10 ng/mL) or without TGF-β1 showed a relatively lower expression of autophagy markers, (<b>A</b>) ATG-5, and (<b>B</b>) LC3-II as compared to time-matched controls. (<b>C</b>) Western blot analysis of ATG-5 and LC3-I/II proteins shows a similar trend, (<b>D</b>,<b>E</b>) densitometry analysis of the bands, (<b>F</b>) Levels of Smad3 in ATG5 inhibited cells and (<b>G</b>) densitometry analysis of Smad3 bands. The error bars represent standard deviation (*—<span class="html-italic">p</span> &lt; 0.05, **—<span class="html-italic">p</span> &lt; 0.01, ****—<span class="html-italic">p</span> &lt; 0.0001, by <span class="html-italic">t</span>-test).</p> Full article ">Figure 5
<p>ATG5-TGF crosstalk may be mediated by miR-122-5p. Relative miRNA expression levels of, miR-424-5p, miR-124-3p, and miR-122-5p in (<b>A</b>) Transforming growth factor, TGF-β1 (10 ng/mL) treated human trabecular meshwork (HTM) cells and (<b>B</b>) siRNA mediated knockdown HTM cells with (10 ng/mL) and without TGF-β1 exposure. The error bars represent standard deviation ***—<span class="html-italic">p</span> &lt; 0.001, by one-way ANOVA).</p> Full article ">Figure 6
<p>Electron microscopic appearance of protein aggregates. Control (<b>a</b>) and 10 ng/mL TGF-β1 treated cells (<b>b</b>) were observed under electron microscope. One-µm thin slices show protein aggregate localization (indicated by white arrows, (<b>b</b>) in 72 h, 10 ng/mL TGF-β1 treated cells (original magnification ×50,000).</p> Full article ">Figure 7
<p>Transforming growth factor, TGF induced ATG5 activation plays a crucial role in preventing TGF induced aggregate formation in HTM cells. Amyloid fibrils were found around and closer to the nucleus and oligomer 11 was spread throughout in siATG5 cells exposed to 10 ng/mL TGF-β1. Magnification 20×, Bar = 50 µM.</p> Full article ">
9 pages, 2186 KiB  
Article
Early Fruit Development Regulation-Related Genes Concordantly Expressed with TCP Transcription Factors in Tomato (Solanum lycopersicum)
by Sherif Edris, Aala A. Abulfaraj, Rania M. Makki, Salah Abo-Aba, Mardi M. Algandaby, Jamal Sabir, Robert K. Jansen, Fotouh M. El Domyati and Ahmed Bahieldin
Curr. Issues Mol. Biol. 2023, 45(3), 2372-2380; https://doi.org/10.3390/cimb45030153 - 13 Mar 2023
Cited by 3 | Viewed by 2311
Abstract
The tomato (Solanum lycopersicum L.) is considered one of the most important vegetable crops globally, both agronomically and economically; however, its fruit development regulation network is still unclear. The transcription factors serve as master regulators, activating many genes and/or metabolic pathways throughout [...] Read more.
The tomato (Solanum lycopersicum L.) is considered one of the most important vegetable crops globally, both agronomically and economically; however, its fruit development regulation network is still unclear. The transcription factors serve as master regulators, activating many genes and/or metabolic pathways throughout the entire plant life cycle. In this study, we identified the transcription factors that are coordinated with TCP gene family regulation in early fruit development by making use of the high-throughput sequencing of RNA (RNAseq) technique. A total of 23 TCP-encoding genes were found to be regulated at various stages during the growth of the fruit. The expression patterns of five TCPs were consistent with those of other transcription factors and genes. There are two unique subgroups of this larger family: class I and class II TCPs. Others were directly associated with the growth and/or ripening of fruit, while others were involved in the production of the hormone auxin. Moreover, it was discovered that TCP18 had an expression pattern that was similar to that of the ethylene-responsive transcription factor 4 (ERF4). Tomato fruit set and overall development are under the direction of a gene called auxin response factor 5 (ARF5). TCP15 revealed an expression that was in sync with this gene. This study provides insight into the potential processes that help in acquiring superior fruit qualities by accelerating fruit growth and ripening. Full article
(This article belongs to the Special Issue Functional Genomics and Comparative Genomics Analysis in Plants)
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<p>The concordant expression between <span class="html-italic">TCPs</span> and other genes related to fruit development across the time of early fruit development (0-12D) in tomato cultivar Chico III. Clusters were upregulated at five (cluster 1), four (cluster 2), three (cluster 3), two (cluster 4), and one (cluster 5) time point.</p> Full article ">Figure 2
<p>Gene networking generated from transcriptome-based cluster analysis for the <span class="html-italic">TCP</span> genes (in red, blue, or lilac) concordantly expressed with other genes (in green) in tomato cultivar Chico III. Black lines indicate interactions based on the transcriptomic results of the present study, while red lines indicate interactions based on the yeast one-hybrid results of the Parapunov [<a href="#B9-cimb-45-00153" class="html-bibr">9</a>]. Solid lines indicate direct connections, while the dashed line indicates intermediate connection(s).</p> Full article ">Figure 3
<p>Expression profiles of the four <span class="html-italic">TPC</span> genes, namely, <span class="html-italic">TPC9</span>, <span class="html-italic">TPC12</span>, <span class="html-italic">TPC15</span>, and <span class="html-italic">TPC18</span> during early fruit development stages in tomato cultivar Chico III.</p> Full article ">Figure 4
<p>Regulation of fruit development and ripening is mutually driven by <span class="html-italic">TCP9</span> and <span class="html-italic">ZFP2</span> in tomato, respectively. <span class="html-italic">TCP9</span> possibly drives the expression of the <span class="html-italic">AO3</span> gene towards ABA biosynthesis, while <span class="html-italic">ZFP2</span> drives the suppression of the <span class="html-italic">CNR</span> gene towards ethylene production. Modulation between ABA and ethylene is also driven by <span class="html-italic">ZFP2</span>. Regulation of genes encoding <span class="html-italic">TCP12</span>, <span class="html-italic">TCP15</span>, and <span class="html-italic">TCP18</span> is also shown, along with two concordantly expressed genes, namely, <span class="html-italic">ERF4</span> and <span class="html-italic">ARF5</span>.</p> Full article ">
21 pages, 1548 KiB  
Review
Natural Products for the Treatment of Pulmonary Hypertension: Mechanism, Progress, and Future Opportunities
by Zuomei Zeng, Xinyue Wang, Lidan Cui, Hongjuan Wang, Jian Guo and Yucai Chen
Curr. Issues Mol. Biol. 2023, 45(3), 2351-2371; https://doi.org/10.3390/cimb45030152 - 13 Mar 2023
Cited by 4 | Viewed by 4808
Abstract
Pulmonary hypertension (PH) is a lethal disease due to the remodeling of pulmonary vessels. Its pathophysiological characteristics include increased pulmonary arterial pressure and pulmonary vascular resistance, leading to right heart failure and death. The pathological mechanism of PH is complex and includes inflammation, [...] Read more.
Pulmonary hypertension (PH) is a lethal disease due to the remodeling of pulmonary vessels. Its pathophysiological characteristics include increased pulmonary arterial pressure and pulmonary vascular resistance, leading to right heart failure and death. The pathological mechanism of PH is complex and includes inflammation, oxidative stress, vasoconstriction/diastolic imbalance, genetic factors, and ion channel abnormalities. Currently, many clinical drugs for the treatment of PH mainly play their role by relaxing pulmonary arteries, and the treatment effect is limited. Recent studies have shown that various natural products have unique therapeutic advantages for PH with complex pathological mechanisms owing to their multitarget characteristics and low toxicity. This review summarizes the main natural products and their pharmacological mechanisms in PH treatment to provide a useful reference for future research and development of new anti-PH drugs and their mechanisms. Full article
(This article belongs to the Topic Blood Physiology: Molecular Mechanisms of Vascular Wall Functioning, 2nd Volume)
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<p>Some deeply studied natural products can play a role in the treatment of pulmonary hypertension by improving inflammatory responses, inhibiting oxidative stress, reducing apoptotic resistance, and regulating abnormal ion channels and collagen deposition, which can effectively improve pulmonary vascular remodeling and right ventricular hypertrophy. Abbreviations: PH, pulmonary hypertension; NF-κB, nuclear factor-κB; and ROS, reactive oxygen species.</p> Full article ">Figure 2
<p>Chemical structures of natural products for the treatment of pulmonary hypertension. (<b>A</b>) Tanshinone IIA; (<b>B</b>) salvianolic acid A; (<b>C</b>) magnesium lithospermate B; (<b>D</b>) tetramethylpyrazine; (<b>E</b>) resveratrol; (<b>F</b>) baicalin; (<b>G</b>) baicalein; (<b>H</b>) puerarin; (<b>I</b>) genistein; (<b>J</b>) astragaloside IV; and (<b>K</b>) curcumin.</p> Full article ">Figure 3
<p>Pulmonary vessels are affected by hypoxia, genetics, and many factors, leading to pulmonary vasoconstriction, vascular remodeling, thrombosis, and right ventricular hypertrophy through inflammation, oxidative stress, apoptotic resistance, ion channel abnormalities, and collagen deposition pathways. A variety of natural products can be used to treat pulmonary hypertension through these mechanisms. Abbreviations: TRPC1, 6: transient receptor potential 1,6; SOCE: store-operated Ca<sup>2+</sup> entry; PKG-PPAR-γ: hypoxia-inhibited protein kinase G-peroxisome proliferator-activated receptor-γ; BMPR2: bone morphogenetic protein type 2 receptor; BMP9: bone morphogenetic protein 9; EndMT: endothelial-to-mesenchymal transition; ROS: reactive oxygen species; TGFβ1: transforming growth factor-β1; Nrf2/HO-1: NF-E2-related factor 2/heme oxygenase-1; NOX: NADPH oxidases; ERK: extracellular signal-regulated kinase; VPO1: vascular peroxidase-1; HIF-1α: hypoxia-inducible factor-1α; NF-κB: nuclear factor-kappa B; PCNA: proliferating cell nuclear antigen; PI3K/Akt: phosphoinositide-3-kinase/protein kinase B; CaSRs: calcium-sensing receptors; VEGF: vascular endothelial growth factor; iNOS: inducible nitric oxide synthase; PKG-1: cGMP-dependent protein kinases 1; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; eNOS: endothelial nitric oxide synthase; SphK1/S1P: sphingosine kinase 1/sphingosine-1-phosphate; MAPK: mitogen-activated protein kinase; MMP-9: matrix metalloproteinase-9; Bcl-2: B cell lymphoma-2; Bax: BCL-2-associated X; NLRP3: nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing-3; NO: nitric oxide; IL-1β: interleukin-1β; ECM: extracellular matrix.</p> Full article ">
13 pages, 1629 KiB  
Article
Transcriptomic Establishment of Pig Macrophage Polarization Signatures
by Jing Li, Teng Yuan, Anjing Zhang, Peidong Yang, Li He, Keren Long, Chuang Tang, Li Chen, Mingzhou Li and Lu Lu
Curr. Issues Mol. Biol. 2023, 45(3), 2338-2350; https://doi.org/10.3390/cimb45030151 - 12 Mar 2023
Viewed by 3154
Abstract
Macrophages are the foremost controllers of innate and acquired immunity, playing important roles in tissue homeostasis, vasculogenesis, and congenital metabolism. In vitro macrophages are crucial models for understanding the regulatory mechanism of immune responses and the diagnosis or treatment of a variety of [...] Read more.
Macrophages are the foremost controllers of innate and acquired immunity, playing important roles in tissue homeostasis, vasculogenesis, and congenital metabolism. In vitro macrophages are crucial models for understanding the regulatory mechanism of immune responses and the diagnosis or treatment of a variety of diseases. Pigs are the most important agricultural animals and valuable animal models for preclinical studies, but there is no unified method for porcine macrophage isolation and differentiation at present; no systematic study has compared porcine macrophages obtained by different methods. In the current study, we obtained two M1 macrophages (M1_IFNγ + LPS, and M1_GM-CSF) and two M2 macrophages (M2_IL4 + IL10, and M2_M-CSF), and compared the transcriptomic profiles between and within macrophage phenotypes. We observed the transcriptional differences either between or within phenotypes. Porcine M1 and M2 macrophages have consistent gene signatures with human and mouse macrophage phenotypes, respectively. Moreover, we performed GSEA analysis to attribute the prognostic value of our macrophage signatures in discriminating various pathogen infections. Our study provided a framework to guide the interrogation of macrophage phenotypes in the context of health and disease. The approach described here could be used to propose new biomarkers for diagnosis in diverse clinical settings including porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), Toxoplasma gondii (T. gondii), porcine circovirus type 2 (PCV2), Haemophilus parasuis serovar 4 (HPS4), Mycoplasma hyopneumoniae (Mhp), Streptococcus suis serotype 2 (SS2), and LPS from Salmonella enterica serotype minnesota Re 595. Full article
(This article belongs to the Topic Animal Models of Human Disease)
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<p>Transcriptomic comparison of porcine macrophage phenotypes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) versus M2 (M1_IFNγ + LPS, and M1_GM-CSF). (<b>A</b>) PCA clustering M1_IFNγ + LPS (<span class="html-italic">n</span> = 4), M1_GM-CSF (<span class="html-italic">n</span> = 4), M2_IL4 + IL10 (<span class="html-italic">n</span> = 3), M2_M-CSF (<span class="html-italic">n</span> = 3); (<b>B</b>) Top enriched GO biological processes of 730 DEG genes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) and M2 (M2_IL4 + IL10, and M2_M-CSF); (<b>C</b>) Heatmap exhibiting 49 classical macrophage marker genes expression profile among M1_IFNγ + LPS, M1_GM-CSF, M2_IL4 + IL10, and M2_M-CSF; (<b>D</b>) The DEGs between M1 and M2 were studied by using the String online tool. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree.</p> Full article ">Figure 2
<p>Comparison of transcriptomics in different phenotypes of porcine macrophages. (<b>A</b>) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method, M1_IFNγ + LPS, and M1_GM-CSF; (<b>B</b>) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method M2_IL4 + IL10 and M2_M-CSF; (<b>C</b>) GO pathway enrichment analysis of two polarized macrophages, M1_IFNγ + LPS and M1_GM-CSF; (<b>D</b>) GO pathway enrichment analysis of two polarized macrophages, M2_IL4 + IL10 and M2_M-CSF; (<b>E</b>) The DEGs were studied by using the String online tool, M1_IFNγ + LPS, and M1_GM-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 1; (<b>F</b>) The DEGs are studied by using the String online tool, M2_IL4 + IL10, and M2_M-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 2.</p> Full article ">Figure 3
<p>Validation of M1_IFNγ + LPS, M1_GMCSF, M2_M-CSF, and M2_IL4 + IL10 signatures. (<b>A</b>) SS2-infected macrophages were enriched in M1_IFNγ + LPS based on GSEA (Data source: Microarray analysis); (<b>B</b>) PRRSV-infected macrophages were enriched in M1_GM-CSF based on GSEA (Data source: RNA-seq); (<b>C</b>). <span class="html-italic">T. gondii</span>-infected macrophages were enriched in M2_IL4 + IL10 based on GSEA (Data source: RNA-seq); (<b>D</b>) <span class="html-italic">T. gondii Me49</span>-infected macrophages were enriched in M2_M-CSF based on GSEA (Data source: RNA-seq).</p> Full article ">
12 pages, 1759 KiB  
Article
Effect of Atorvastatin on Angiogenesis-Related Genes VEGF-A, HGF and IGF-1 and the Modulation of PI3K/AKT/mTOR Transcripts in Bone-Marrow-Derived Mesenchymal Stem Cells
by Adriana Adamičková, Nikola Chomaničová, Andrea Gažová, Juraj Maďarič, Zdenko Červenák, Simona Valášková, Matúš Adamička and Jan Kyselovic
Curr. Issues Mol. Biol. 2023, 45(3), 2326-2337; https://doi.org/10.3390/cimb45030150 - 10 Mar 2023
Cited by 7 | Viewed by 2197
Abstract
Stem cell transplantation represents a unique therapeutic tool in tissue engineering and regenerative medicine. However, it was shown that the post-injection survival of stem cells is poor, warranting a more comprehensive understanding of activated regenerative pathways. Numerous studies indicate that statins improve the [...] Read more.
Stem cell transplantation represents a unique therapeutic tool in tissue engineering and regenerative medicine. However, it was shown that the post-injection survival of stem cells is poor, warranting a more comprehensive understanding of activated regenerative pathways. Numerous studies indicate that statins improve the therapeutic efficacy of stem cells in regenerative medicine. In the present study, we investigated the effect of the most widely prescribed statin, atorvastatin, on the characteristics and properties of bone-marrow-derived mesenchymal stem cells (BM-MSCs) cultured in vitro. We found that atorvastatin did not decrease the viability of BM-MSCs, nor did it change the expression of MSC cell surface markers. Atorvastatin upregulated the mRNA expression levels of VEGF-A and HGF, whereas the mRNA expression level of IGF-1 was decreased. In addition, the PI3K/AKT signaling pathway was modulated by atorvastatin as indicated by the high mRNA expression levels of PI3K and AKT. Moreover, our data revealed the upregulation of mTOR mRNA levels; however, no change was observed in the BAX and BCL-2 transcripts. We propose that atorvastatin benefits BM-MSC treatment due to its ability to upregulate angiogenesis-related genes expression and transcripts of the PI3K/AKT/mTOR pathway. Full article
(This article belongs to the Special Issue Mesenchymal Stem Cells (MSC), Exosomes, and Other MSC Products in Research and Clinics)
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<p>Morphology of human BM-MSCs and flow cytometry analysis of BM-MSCs expanded to passage three under normoxic conditions. (<b>A</b>) BM-MCSs exhibit an elongated fibroblast-like morphology (10× magnification). (<b>B</b>) BM-MSCs expressed characteristic mesenchymal stem cell markers (CD73, CD90, CD105) while the non-MSC markers (CD14, CD20, CD34 and CD45) were not detected. BM-MSC, bone-marrow-derived mesenchymal stem cell.</p> Full article ">Figure 2
<p>(<b>A</b>) Flow cytometry analysis of viability with propidium iodide staining in control (CTR), 0.1% DMSO and atorvastatin pre-treatment group (ATO) of BM-MSCs. Data are presented as the mean ± standard deviation (n = 3). (<b>B</b>) Quantifying cultured BM-MSCs by flow cytometry (expression of MSC-positive markers CD73, CD90 and CD105 and MSC-negative markers CD14, CD20, CD34 and CD45) in CTR, 0.1% DMSO and ATO group of BM-MSCs. Data are presented as the mean ± standard deviation (n = 3). (<b>C</b>) Fluorescence microscopy of BM-MSCs cultured with 0.1% DMSO and ATO after 48 h and stained for vimentin (green). Cell nuclei were counterstained with DAPI (blue) (scale bar 100 µm).</p> Full article ">Figure 3
<p>Effect of atorvastatin on (<b>A</b>) <span class="html-italic">VEGF-A</span>, (<b>B</b>) <span class="html-italic">HGF</span>, (<b>C</b>) <span class="html-italic">FGF-1R</span>, (<b>D</b>) <span class="html-italic">IGF-1</span>, (<b>E</b>) <span class="html-italic">IGF-2</span> and (<b>F</b>) <span class="html-italic">IGF-1R</span> mRNA expression in BM-MSCs. Data are presented as mean ± standard deviation of three experiments from different donors (n = 3) performed in triplicate (* <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.001 compared to 0.1% DMSO group).</p> Full article ">Figure 4
<p>Effect of atorvastatin on (<b>A</b>) <span class="html-italic">AKT1</span>, (<b>B</b>) <span class="html-italic">PI3KCA</span>, (<b>C</b>) <span class="html-italic">mTOR</span>, (<b>D</b>) <span class="html-italic">BAX</span>, (<b>E</b>) <span class="html-italic">BCL-2</span> and (<b>F</b>) <span class="html-italic">HMGCR</span> mRNA expression in BM-MSCs. Data are presented as the mean ± standard deviation of three experiments from different donors (n = 3) performed in triplicate (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01 compared to 0.1% DMSO group).</p> Full article ">
17 pages, 3838 KiB  
Article
Change in Long Non-Coding RNA Expression Profile Related to the Antagonistic Effect of Clostridium perfringens Type C on Piglet Spleen
by Zunqiang Yan, Pengfei Wang, Qiaoli Yang, Xiaoli Gao, Shuangbao Gun and Xiaoyu Huang
Curr. Issues Mol. Biol. 2023, 45(3), 2309-2325; https://doi.org/10.3390/cimb45030149 - 9 Mar 2023
Cited by 2 | Viewed by 1904
Abstract
LncRNAs play important roles in resisting bacterial infection via host immune and inflammation responses. Clostridium perfringens (C. perfringens) type C is one of the main bacteria causing piglet diarrhea diseases, leading to major economic losses in the pig industry worldwide. In [...] Read more.
LncRNAs play important roles in resisting bacterial infection via host immune and inflammation responses. Clostridium perfringens (C. perfringens) type C is one of the main bacteria causing piglet diarrhea diseases, leading to major economic losses in the pig industry worldwide. In our previous studies, piglets resistant (SR) and susceptible (SS) to C. perfringens type C were identified based on differences in host immune capacity and total diarrhea scores. In this paper, the RNA-Seq data of the spleen were comprehensively reanalyzed to investigate antagonistic lncRNAs. Thus, 14 lncRNAs and 89 mRNAs were differentially expressed (DE) between the SR and SS groups compared to the control (SC) group. GO term enrichment, KEGG pathway enrichment and lncRNA-mRNA interactions were analyzed to identify four key lncRNA targeted genes via MAPK and NF-κB pathways to regulate cytokine genes (such as TNF-α and IL-6) against C. perfringens type C infection. The RT-qPCR results for six selected DE lncRNAs and mRNAs are consistent with the RNA-Seq data. This study analyzed the expression profiling of lncRNAs in the spleen of antagonistic and sensitive piglets and found four key lncRNAs against C. perfringens type C infection. The identification of antagonistic lncRNAs can facilitate investigations into the molecular mechanisms underlying resistance to diarrhea in piglets. Full article
(This article belongs to the Special Issue Studying the Function of RNAs Using Omics Approaches)
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<p>Analysis of DE lncRNAs and mRNAs in the spleen. Venn diagram showing DE lncRNAs (<b>A</b>) and mRNAs (<b>B</b>) among SR vs. SC, SS vs. SC and SR vs. SC groups. Volcano plot presenting DE lncRNAs (<b>C</b>) and mRNAs (<b>D</b>) between SR and SC groups.</p> Full article ">Figure 2
<p>Distributions of DE lncRNAs and mRNAs in pig chromosome. The <span class="html-italic">x</span>-axis indicates different chromosomes, and the <span class="html-italic">y</span>-axis indicates the number of lncRNAs or mRNAs.</p> Full article ">Figure 3
<p>Hierarchical heat map showing the expression values for lncRNAs (<b>A</b>) and mRNAs (<b>B</b>).</p> Full article ">Figure 4
<p>Genomic features of the different subtypes of lncRNAs. (<b>A</b>)Transcript length, (<b>B</b>) expression level, (<b>C</b>) exon count and (<b>D</b>) ORF length of different subtypes of lncRNAs were compared using the Kolmogorov–Smirnov test. Each region of the box plot indicates the maximum, upper quartile, median, lower quartile and minimum from top to bottom.</p> Full article ">Figure 5
<p>The secondary structures of 10 lncRNAs. (<b>A</b>) LNC_001595; (<b>B</b>) ALDBSSCT0000003048; (<b>C</b>) LNC_000191; (<b>D</b>) ALDBSSCT0000009442; (<b>E</b>) LNC_001065; (<b>F</b>) ALDBSSCT0000006918; (<b>G</b>) LNC_000263; (<b>H</b>) LNC_002009; (<b>I</b>) ALDBSSCT0000007366; (<b>J</b>) LNC_000042.</p> Full article ">Figure 6
<p>GO term analysis of the biological functions of the identified lncRNA target genes.</p> Full article ">Figure 7
<p>The top 20 KEGG pathways of lncRNA target genes. The <span class="html-italic">x</span>-axis indicates the gene ratio, and the <span class="html-italic">y</span>-axis indicates the name of the KEGG pathway. The size of the dot indicates the number of target genes, and the color of the dot indicates different <span class="html-italic">p</span> values.</p> Full article ">Figure 8
<p>Validation of the reliability of RNA-Seq data and four gene expression levels. Data are presented as Mean ± SE with three duplicates. One-way ANOVA was performed to calculate statistical significance followed by Duncan’s test. Different asterisks above bars indicate significant differences (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 9
<p>Relative expression of <span class="html-italic">LNC_001595</span> in different tissues. The results are shown as Mean ± SE with three duplicates. One-way ANOVA was conducted to calculate statistical significance followed by Duncan’s test. Different asterisks above bars indicate significant differences (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 10
<p>Cytokine gene expression levels (<b>A</b>) and concentrations (<b>B</b>) in the spleen. Data are displayed as Mean ± SE with three duplicates. One-way ANOVA was performed to calculate statistical significance followed by Duncan’s test. Different asterisks above bars indicate significant differences (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 11
<p>A diagram illustrating the potential lncRNAs, target genes and cytokines.</p> Full article ">
13 pages, 3255 KiB  
Article
The Insulin Receptor Substrate 2 Mediates the Action of Insulin on HeLa Cell Migration via the PI3K/Akt Signaling Pathway
by Anabel Martínez Báez, Ivone Castro Romero, Lilia Chihu Amparan, Jose Ramos Castañeda and Guadalupe Ayala
Curr. Issues Mol. Biol. 2023, 45(3), 2296-2308; https://doi.org/10.3390/cimb45030148 - 9 Mar 2023
Cited by 6 | Viewed by 2449
Abstract
Insulin signaling plays an important role in the development and progression of cancer since it is involved in proliferation and migration processes. It has been shown that the A isoform of the insulin receptor (IR-A) is often overexpressed, and its stimulation induces changes [...] Read more.
Insulin signaling plays an important role in the development and progression of cancer since it is involved in proliferation and migration processes. It has been shown that the A isoform of the insulin receptor (IR-A) is often overexpressed, and its stimulation induces changes in the expression of the insulin receptor substrates (IRS-1 and IRS-2), which are expressed differently in the different types of cancer. We study the participation of the insulin substrates IRS-1 and IRS-2 in the insulin signaling pathway in response to insulin and their involvement in the proliferation and migration of the cervical cancer cell line. Our results showed that under basal conditions, the IR-A isoform was predominantly expressed. Stimulation of HeLa cells with 50 nM insulin led to the phosphorylation of IR-A, showing a statistically significant increase at 30 min (p ≤ 0.05). Stimulation of HeLa cells with insulin induces PI3K and AKT phosphorylation through the activation of IRS2, but not IRS1. While PI3K reached the highest level at 30 min after treatment (p ≤ 0.05), AKT had the highest levels from 15 min (p ≤ 0.05) and remained constant for 6 h. ERK1 and ERK2 expression was also observed, but only ERK2 was phosphorylated in a time-dependent manner, reaching a maximum peak 5 min after insulin stimulation. Although no effect on cell proliferation was observed, insulin stimulation of HeLa cells markedly promoted cell migration. Full article
(This article belongs to the Special Issue Adhesion, Metastasis and Inhibition of Cancer Cells)
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<p>Insulin receptor (IR) isoforms are differentially expressed in HeLa cells. The mRNA levels of IR isoforms and IRS1/2 genes were analyzed in HeLa and MCF7 cell lines. Total RNA was purified, and mRNA levels were analyzed by RT-PCR with specific primers. (<b>A</b>) The 600 and 636 bp fragments correspond to isoforms A and B of the insulin receptor, respectively. Amplified fragments of 763 and 116 bp correspond to IRS-1 and IRS-2, respectively. The amplified fragment of 451bp corresponds to GAPDH (control). (<b>B</b>) Densitometric analysis of IRA, IRB, IRS1, IRS2, and GAPDH mRNA levels. The graph represents the mean ± SEM of three independent experiments (<span class="html-italic">n</span> = 3). ** <span class="html-italic">p</span> &lt; 0.01 compared to IRA.</p> Full article ">Figure 2
<p>Effect of insulin on the proliferation of HeLa cells with MTS assay. HeLa cells were treated with different insulin doses at different times of stimulation. The histograms represent the mean value ± standard error of the mean (SEM) of optic density values. The control group is cells without treatment. The graphs represent the mean ± SEM of three independent experiments (<span class="html-italic">n</span> = 6).</p> Full article ">Figure 3
<p>Insulin induces the phosphorylation of IR and IRS2 but not IRS1 in HeLa cells. Protein extracts from HeLa cells stimulated with 50 nM insulin were used to evaluate IR, IRS1, phospho-IRS1, IRS2, and phospho IRS2 by WB. (<b>A</b>) Densitometric analysis of phospho-IR and actin protein levels. The first bar is the control group without treatment. One-way ANOVA was performed, followed by the Tukey post hoc test to compare the treated groups against the control group (100%). * <span class="html-italic">p</span> &lt; 0.05. All experiments have been performed in three independent experiments in triplicate, and experimental data were expressed as mean ± standard deviation (SD). (<b>B</b>) Cells were lysed with RIPA. IRS2 protein was immunoprecipitated, and immunoblot analyses were performed to identify the indicated proteins from IP or cell lysates. (<b>C</b>) HeLa cells were stimulated in the absence (control) or presence of 50 nM insulin for the indicated time. MCF7 and MDA-MB-231 cells were used as positive and negative controls, respectively. IRS1 and phospho-IRS1 protein levels were detected by immunoblot analysis. ϐ-Actin was used as a control for protein degradation.</p> Full article ">Figure 4
<p>Cervical cancer cells express higher phosphorylation levels of PI3K/Akt1 than ERK1/2 in response to insulin treatment. (<b>A</b>) PI3K, phospho PI3K. (<b>B</b>) Akt1, phospho Akt1. (<b>C</b>) ERK1/2 and phospho ERK1/2 protein levels were analyzed by WB in whole-cell lysates from HeLa cells stimulated with 50 nM insulin. Data are representative of 3 independent experiments. Graphics show densitometric analysis of total levels and phosphorylated protein. The first bar is the control group without treatment. All experiments were performed in three independent experiments in triplicate, and experimental data were expressed as mean ± standard deviation (SD). * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 compared to control (taken as 100%).</p> Full article ">Figure 5
<p>Insulin treatment increases migration of HeLa cells. HeLa cells were stimulated with different concentrations of insulin in the presence of mitomycin C and with DMEM medium without SFB; cells were treated with mitomycin C in the control group. Cell migration was evaluated by quantifying the reduction in the open area (a lower percentage of the open area means a higher percentage of cell migration). The histograms represent the mean value ± standard error of the mean (SEM) of the percentage of the open area of three independent experiments in triplicate. One-way ANOVA was performed, followed by the Tukey post hoc test to compare the treated groups against the control group. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
12 pages, 2905 KiB  
Article
Abietane Diterpenoids Isolated from Torreya nucifera Disrupt Replication of Influenza Virus by Blocking the Phosphatidylinositol-3-Kinase (PI3K)-Akt and ERK Signaling Pathway
by Jaehoon Bae, Hyung-Jun Kwon, Ji Sun Park, Jinseok Jung, Young Bae Ryu, Woo Sik Kim, Ju Huck Lee, Jae-Ho Jeong, Jae Sung Lim, Woo Song Lee and Su-Jin Park
Curr. Issues Mol. Biol. 2023, 45(3), 2284-2295; https://doi.org/10.3390/cimb45030147 - 9 Mar 2023
Cited by 4 | Viewed by 2419
Abstract
Although vaccines and antiviral drugs are available, influenza viruses continue to pose a significant threat to vulnerable populations globally. With the emergence of drug-resistant strains, there is a growing need for novel antiviral therapeutic approaches. We found that 18-hydroxyferruginol (1) and [...] Read more.
Although vaccines and antiviral drugs are available, influenza viruses continue to pose a significant threat to vulnerable populations globally. With the emergence of drug-resistant strains, there is a growing need for novel antiviral therapeutic approaches. We found that 18-hydroxyferruginol (1) and 18-oxoferruginol (2) isolated from Torreya nucifera exhibited strong anti-influenza activity, with 50% inhibitory concentration values of 13.6 and 18.3 μM against H1N1, 12.8 and 10.8 μM against H9N2, and 29.2 μM (only compound 2) against H3N2 in the post-treatment assay, respectively. During the viral replication stages, the two compounds demonstrated stronger inhibition of viral RNA and protein in the late stages (12–18 h) than in the early stages (3–6 h). Moreover, both compounds inhibited PI3K-Akt signaling, which participates in viral replication during the later stages of infection. The ERK signaling pathway is also related to viral replication and was substantially inhibited by the two compounds. In particular, the inhibition of PI3K-Akt signaling by these compounds inhibited viral replication by sabotaging influenza ribonucleoprotein nucleus-to-cytoplasm export. These data indicate that compounds 1 and 2 could potentially reduce viral RNA and viral protein levels by inhibiting the PI3K-Akt signaling pathway. Our results suggest that abietane diterpenoids isolated from T. nucifera may be potent antiviral candidates for new influenza therapies. Full article
(This article belongs to the Special Issue Molecular and Real-World Evidence Research of Respiratory Diseases and Infections)
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<p>Effects of 18-hydroxyferruginol (<b>1</b>) and 18-oxoferruginol (<b>2</b>) on influenza viral RNA levels (M gene). (<b>A</b>) The structures of 18-hydroxyferruginol (<b>1</b>) and 18-oxoferruginol (<b>2</b>). (<b>B</b>) MDCK cells were infected with influenza viruses (0.001 MOI) for 1 h, viruses were removed, and cells were treated with 0.5% DMSO, 20 μM 18-hydroxyferruginol (<b>1</b>), 20 μM 18-oxoferruginol (<b>2</b>), or 10 μM oseltamivir for 3, 6, 12, and 18 h. Total RNA was extracted after virus infection, and the level of the M gene was measured. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 2
<p>18-Hydroxyferruginol (<b>1</b>) and 18-oxoferruginol (<b>2</b>) inhibit influenza-virus-induced activation of the PI3K-Akt signaling pathway. (<b>A</b>) MDCK cells were infected with H1N1, H9N2, or H3N2 at 0.001 MOI for 1 h and then incubated with 0.5% DMSO, 50 μM LY294002, 20 μM 18-hydroxyferruginol (<b>1</b>), or 20 μM 18-oxoferruginol (<b>2</b>) for 3, 6, 12, and 18 h. p-Akt and NS1 levels were measured using Western blot analysis. (<b>B</b>) The band densities of p-Akt and NS1 proteins was quantified using ImageJ software. Phosphorylated Akt and NS1 protein accumulations were normalized by the GAPDH level. (<b>C</b>) After infecting them with influenza viruses, MDCK cells were treated with 50 μM LY294002, 20 μM 18-hydroxyferruginol (<b>1</b>), or 20 μM 18-oxoferruginol (<b>2</b>). Total RNA was extracted 10 h after influenza virus infection, and the level of intracellular influenza viral RNA (NP gene) was measured. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>18-Hydroxyferruginol (<b>1</b>) and 18-oxoferruginol (<b>2</b>) inhibit the export of the influenza virus RNP complex from the nucleus to the cytoplasm. MDCK cells were infected with H1N1 at 0.001 MOI in the presence of 0.5% DMSO, 50 μM LY294002, 20 μM 18-hydroxyferruginol (<b>1</b>), or 20 μM 18-oxoferruginol (<b>2</b>). After 10 h, the cells were fixed in 4% paraformaldehyde. After blocking, the cells were incubated with the anti-NP antibody (green). Propidium iodide was used as a nuclear counterstain (red). Scale bar = 10 µm.</p> Full article ">Figure 4
<p>18-Hydroxyferruginol (<b>1</b>) and 18-oxoferruginol (<b>2</b>) inhibit influenza-virus-induced ERK phosphorylation. MDCK cells were infected with H1N1, H9N2, or H3N2 at 0.001 MOI for 1 h and incubated with DMSO (0.5%), LY294002 (50 μM), PD98059 (50 μM), 18-hydroxyferruginol (<b>1</b>) (20 μM), or 18-oxoferruginol (<b>2</b>) (20 μM) for 10 h. (<b>A</b>) p-MEK1/2, p-ERK, and NP levels were measured by Western blot analysis. (<b>B</b>) The band density was quantified by using ImageJ software. Phosphorylated ERK1/2, MEK1/2, and NP protein accumulations were normalized by the GAPDH level. (<b>C</b>) After infecting them with influenza viruses, MDCK cells were treated with 50 μM PD98059, 20 μM 18-hydroxyferruginol (<b>1</b>), or 20 μM 18-oxoferruginol (<b>2</b>). Total RNA was extracted 10 h after influenza virus infection, and the level of intracellular influenza viral RNA (NP gene) was measured. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. (<b>D</b>) Schematic depiction of 18-hydroxyferruginol (<b>1</b>) and 18-oxoferruginol (<b>2</b>) actions on PI3K-Akt and ERK signaling pathways and influenza replication.</p> Full article ">
18 pages, 1142 KiB  
Review
NOTCH Signaling in Osteosarcoma
by Zhenhao Zhang, Wei Wu and Zengwu Shao
Curr. Issues Mol. Biol. 2023, 45(3), 2266-2283; https://doi.org/10.3390/cimb45030146 - 8 Mar 2023
Cited by 9 | Viewed by 2835
Abstract
The combination of neoadjuvant chemotherapy and surgery has been promoted for the treatment of osteosarcoma; however, the local recurrence and lung metastasis rates remain high. Therefore, it is crucial to explore new therapeutic targets and strategies that are more effective. The NOTCH pathway [...] Read more.
The combination of neoadjuvant chemotherapy and surgery has been promoted for the treatment of osteosarcoma; however, the local recurrence and lung metastasis rates remain high. Therefore, it is crucial to explore new therapeutic targets and strategies that are more effective. The NOTCH pathway is not only involved in normal embryonic development but also plays an important role in the development of cancers. The expression level and signaling functional status of the NOTCH pathway vary in different histological types of cancer as well as in the same type of cancer from different patients, reflecting the distinct roles of the Notch pathway in tumorigenesis. Studies have reported abnormal activation of the NOTCH signaling pathway in most clinical specimens of osteosarcoma, which is closely related to a poor prognosis. Similarly, studies have reported that NOTCH signaling affected the biological behavior of osteosarcoma through various molecular mechanisms. NOTCH-targeted therapy has shown potential for the treatment of osteosarcoma in clinical research. After the introduction of the composition and biological functions of the NOTCH signaling pathway, the review paper discussed the clinical significance of dysfunction in osteosarcoma. Then the paper reviewed the recent relevant research progress made both in the cell lines and in the animal models of osteosarcoma. Finally, the paper explored the potential of the clinical application of NOTCH-targeted therapy for the treatment of osteosarcoma. Full article
(This article belongs to the Special Issue Targeting Tumor Microenvironment for Cancer Therapy)
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<p>The Notch signaling pathway.</p> Full article ">Figure 2
<p>Effect of the NOTCH signaling pathway on osteosarcoma.</p> Full article ">
18 pages, 2663 KiB  
Article
Identification of a miRNA Panel with a Potential Determinant Role in Patients Suffering from Periodontitis
by Oana Baru, Lajos Raduly, Cecilia Bica, Paul Chiroi, Liviuta Budisan, Nikolay Mehterov, Cristina Ciocan, Laura Ancuta Pop, Smaranda Buduru, Cornelia Braicu, Mandra Badea and Ioana Berindan-Neagoe
Curr. Issues Mol. Biol. 2023, 45(3), 2248-2265; https://doi.org/10.3390/cimb45030145 - 8 Mar 2023
Cited by 3 | Viewed by 2007
Abstract
In recent years, the role of microRNA (miRNA) in post-transcriptional gene regulation has advanced and supports strong evidence related to their important role in the regulation of a wide range of fundamental biological processes. Our study focuses on identifying specific alterations of miRNA [...] Read more.
In recent years, the role of microRNA (miRNA) in post-transcriptional gene regulation has advanced and supports strong evidence related to their important role in the regulation of a wide range of fundamental biological processes. Our study focuses on identifying specific alterations of miRNA patterns in periodontitis compared with healthy subjects. In the present study, we mapped the major miRNAs altered in patients with periodontitis (n = 3) compared with healthy subjects (n = 5), using microarray technology followed by a validation step by qRT-PCR and Ingenuity Pathways Analysis. Compared to healthy subjects, 159 differentially expressed miRNAs were identified among periodontitis patients, of which 89 were downregulated, and 70 were upregulated, considering a fold change of ±1.5 as the cut-off value and p ≤ 0.05. Key angiogenic miRNAs (miR-191-3p, miR-221-3p, miR-224-5p, miR-1228-3p) were further validated on a separate cohort of patients with periodontitis versus healthy controls by qRT-PCR, confirming the microarray data. Our findings indicate a periodontitis-specific miRNA expression pattern representing an essential issue for testing new potential diagnostic or prognostic biomarkers for periodontal disease. The identified miRNA profile in periodontal gingival tissue was linked to angiogenesis, with an important molecular mechanism that orchestrates cell fate. Full article
(This article belongs to the Special Issue Studying the Function of RNAs Using Omics Approaches)
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<p><b>Heatmap of the miRNA expression profile of periodontitis patients (n = 3) compared with healthy subjects (n = 5).</b> The extracted RNA from gingival tissue was fluorescently labeled, hybridized and analyzed by miRNA microarray. The obtained data were processed by Gene Spring software based on the hierarchical clustering of the analyzed samples. Red or green colors indicate differentially up- or downregulated miRNAs using FC ± 1.5 and <span class="html-italic">p</span> &lt; 0.05 as cut-off values.</p> Full article ">Figure 2
<p>Venn diagrams of (<b>A</b>) downregulated and (<b>B</b>) upregulated angiogenesis and EMT overlapping miRNA signature from gingival samples with periodontal diseases compared to controls. The miRNA gene list was downloaded from the NCBI (The National Center for Biotechnology Information) website by searching the Gene module for “Homo sapiens and angiogenesis” and “Homo sapiens and epithelial to mesenchymal transition”.</p> Full article ">Figure 3
<p><b>Representative IPA network to predict the targets of altered miRNAs</b> (<b>A</b>) N1: Neurological Disease, Organismal Injury and Abnormalities, Psychological Disorders; (<b>B</b>) N2: Gene Expression, Organismal Injury and Abnormalities, Reproductive System Disease; (<b>C</b>) N3: Glomerular Injury, Inflammatory Disease, Inflammatory Response; (<b>D</b>) N4: Cellular Development, Cellular Movement, Protein Synthesis. The red color of molecules represents upregulated transcripts, whereas the green color represents downregulated transcripts in periodontal gingival tissue. The intensity of each color shows the strength of regulation. The solid arrows represent experimentally examined associations. A dashed line depicts the experimentally proven interactions. Solid feedback circular line with a hand is related to autoregulation.</p> Full article ">Figure 4
<p>The expression level and ROC curve of tested miRNAs: (<b>A</b>) miR-191-3p, (<b>B</b>) miR-221-3p, (<b>C</b>) miR-224-5p, (<b>D</b>) miR-1228-3p. ROC curves for (<b>E</b>) miR-191-3p, (<b>F</b>) miR-221-3p, (<b>G</b>) miR-224-5p, (<b>H</b>) miR-1228-3p (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 5
<p><b>Multiple ROC curves for the analyzed miRNA were generated using the online tool CombiROC</b>, for different combinations: Combo I: miR-191-3p + miR-1228-3p, Combo II: miR-224-5p + miR-1228-3p, Combo III: miR-191-3p + miR-224-3p + miR-1228.</p> Full article ">Figure 6
<p><b>Functional Analysis of miRNAs Using DIANA-miRPath v3.0 interface</b>. (<b>A</b>) Heatmap created directly from the DIANA-miRPath v3.0 interface. The heatmap depicts the level of enrichment in GO categories of miR-191-3p, miR-221-3p, miR-224-5p and miR-1228-3p. (<b>B</b>) Venn Diagram of main miRNA targets related to hippo signaling. (<b>C</b>) protein network generated based on the main targets connected to miR-221-3p. (<b>D</b>) Protein network generated based on the main targets related to miR-224-5p.</p> Full article ">
18 pages, 6770 KiB  
Article
Terpene-Containing Analogues of Glitazars as Potential Therapeutic Agents for Metabolic Syndrome
by Mikhail E. Blokhin, Sergey O. Kuranov, Mikhail V. Khvostov, Vladislav V. Fomenko, Olga A. Luzina, Natalia A. Zhukova, Cham Elhajjar, Tatiana G. Tolstikova and Nariman F. Salakhutdinov
Curr. Issues Mol. Biol. 2023, 45(3), 2230-2247; https://doi.org/10.3390/cimb45030144 - 8 Mar 2023
Cited by 2 | Viewed by 1970
Abstract
Metabolic syndrome is a complex of abnormalities involving impaired glucose and lipid metabolism, which needs effective pharmacotherapy. One way to reduce lipid and glucose levels associated with this pathology is the simultaneous activation of nuclear PPAR-alpha and gamma. For this purpose, we synthesized [...] Read more.
Metabolic syndrome is a complex of abnormalities involving impaired glucose and lipid metabolism, which needs effective pharmacotherapy. One way to reduce lipid and glucose levels associated with this pathology is the simultaneous activation of nuclear PPAR-alpha and gamma. For this purpose, we synthesized a number of potential agonists based on the pharmacophore fragment of glitazars with the inclusion of mono- or diterpenic moiety in the molecular structure. The study of their pharmacological activity in mice with obesity and type 2 diabetes mellitus (C57Bl/6Ay) revealed one substance that was capable of reducing the triglyceride levels in the liver and adipose tissue of mice by enhancing their catabolism and expressing a hypoglycemic effect connected with the sensitization of mice tissue to insulin. It has also been shown to have no toxic effects on the liver. Full article
(This article belongs to the Special Issue Bioactive Compounds of Natural Products on Metabolic Disorders and Complications)
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<p>Glitazars–dual PPAR agonists.</p> Full article ">Figure 2
<p>Dihydrobetulonic acid amide <b>BM-249</b>.</p> Full article ">Figure 3
<p>Body weight change during the experiment. * <span class="html-italic">p</span> &lt; 0.05 compared to the AY mice.</p> Full article ">Figure 4
<p>OGTT results. The test conducted after 14 days of AY mice treatment by compound <b>9a</b>. Doses: <b>9a</b>–30 mg/kg, MF–250 mg/kg. * <span class="html-italic">p</span> &lt; 0.05 compared to the AY mice.</p> Full article ">Figure 5
<p>(<b>A</b>) OGTT results. The test conducted after 28 days of AY mice treatment using compound <b>9a</b>. (<b>B</b>) AUC calculated according to the OGTT data after 28 days of AY mice treatment using compound <b>9a</b>. Doses: <b>9a</b>–30 mg/kg, MF–250 mg/kg. * <span class="html-italic">p</span> &lt; 0.05 compared to the AY mice.</p> Full article ">Figure 5 Cont.
<p>(<b>A</b>) OGTT results. The test conducted after 28 days of AY mice treatment using compound <b>9a</b>. (<b>B</b>) AUC calculated according to the OGTT data after 28 days of AY mice treatment using compound <b>9a</b>. Doses: <b>9a</b>–30 mg/kg, MF–250 mg/kg. * <span class="html-italic">p</span> &lt; 0.05 compared to the AY mice.</p> Full article ">Figure 6
<p>Blood glucose levels in ITT performed 29 days after the beginning of the experiment. * <span class="html-italic">p</span> &lt; 0.05 compared to the AY mice.</p> Full article ">Figure 7
<p>Body temperature of the mice at the end of the experiment. * <span class="html-italic">p</span> &lt; 0.05 compared to the AY mice.</p> Full article ">Figure 8
<p>Histological evaluation of the liver in mice after 4 weeks of experiment. (<b>A</b>) AY mice treated by compound <b>9a</b> at a dose of 30 mg/kg, (<b>B</b>) AY mice (untreated), (<b>C</b>) AY mice treated by metformin at a dose of 250 mg/kg, (<b>D</b>) C57Bl/6 (healthy control). Hematoxylin and eosin staining, magnification ×200.</p> Full article ">Figure 9
<p>Histological evaluation of pancreas in mice after 4 weeks of experiment. (<b>A</b>) AY mice treated by compound <b>9a</b> at a dose of 30 mg/kg, (<b>B</b>) AY mice (untreated), (<b>C</b>) AY mice treated by metformin at a dose of 250 mg/kg, (<b>D</b>) C57Bl/6 (healthy control). Hematoxylin and eosin staining, magnification ×100.</p> Full article ">Figure 10
<p>Histological evaluation of brown fat in mice after 4 weeks of experiment. (<b>A</b>) AY mice treated by compound <b>9a</b> at a dose of 30 mg/kg, (<b>B</b>) AY mice (untreated), (<b>C</b>) AY mice treated by metformin at a dose of 250 mg/kg, (<b>D</b>) C57Bl/6 (healthy control). Hematoxylin and eosin staining, magnification ×100.</p> Full article ">Figure 10 Cont.
<p>Histological evaluation of brown fat in mice after 4 weeks of experiment. (<b>A</b>) AY mice treated by compound <b>9a</b> at a dose of 30 mg/kg, (<b>B</b>) AY mice (untreated), (<b>C</b>) AY mice treated by metformin at a dose of 250 mg/kg, (<b>D</b>) C57Bl/6 (healthy control). Hematoxylin and eosin staining, magnification ×100.</p> Full article ">Figure 11
<p>Histological evaluation of brown fat in mice after 4 weeks of experiment. (<b>A</b>) AY mice treated by compound <b>9a</b> at a dose of 30 mg/kg, (<b>B</b>) AY mice (untreated), (<b>C</b>) AY mice treated by metformin at a dose of 250 mg/kg, (<b>D</b>) C57Bl/6 (healthy control). Hematoxylin and eosin staining, magnification ×100.</p> Full article ">Figure 12
<p>Monoterpenoids and diterpenic acids.</p> Full article ">Scheme 1
<p>Synthesis of the target compounds.</p> Full article ">
17 pages, 678 KiB  
Article
Whole-Genome Analysis of Antimicrobial-Resistant Salmonella enterica Isolated from Duck Carcasses in Hanoi, Vietnam
by Trung Thanh Nguyen, Hoa Vinh Le, Ha Vu Thi Hai, Thanh Nguyen Tuan, Huong Minh Nguyen, Da Pham Xuan, Huyen Tran Thi Thanh and Hao Hong Le Thi
Curr. Issues Mol. Biol. 2023, 45(3), 2213-2229; https://doi.org/10.3390/cimb45030143 - 8 Mar 2023
Cited by 3 | Viewed by 2730
Abstract
Salmonella enterica is one of the most dangerous foodborne pathogens listed by the World Health Organization. In this study, whole-duck samples were collected at wet markets in five districts in Hanoi, Vietnam, in October 2019 to assess their Salmonella infection rates and evaluate [...] Read more.
Salmonella enterica is one of the most dangerous foodborne pathogens listed by the World Health Organization. In this study, whole-duck samples were collected at wet markets in five districts in Hanoi, Vietnam, in October 2019 to assess their Salmonella infection rates and evaluate the susceptibility of the isolated strains to antibiotics currently used in the prophylaxis and treatment of Salmonella infection. Based on the antibiotic resistance profiles, eight multidrug resistance strains were whole-genome-sequenced, and their antibiotic resistance genes, genotypes, multi-locus sequence-based typing (MLST), virulence factors, and plasmids were analyzed. The results of the antibiotic susceptibility test indicate that phenotypic resistance to tetracycline and cefazolin was the most common (82.4%, 28/34 samples). However, all isolates were susceptible to cefoxitin and meropenem. Among the eight sequenced strains, we identified 43 genes associated with resistance to multiple classes of antibiotics such as aminoglycoside, beta-lactam, chloramphenicol, lincosamide, quinolone, and tetracycline. Notably, all strains carried the blaCTX-M-55 gene, which confers resistance to third-generation antibiotics including cefotaxime, cefoperazone, ceftizoxime, and ceftazidime, as well as resistance genes of other broad-spectrum antibiotics used in clinical treatment such as gentamicin, tetracycline, chloramphenicol, and ampicillin. Forty-three different antibiotic resistance genes were predicted to be present in the isolated Salmonella strains’ genomes. In addition, three plasmids were predicted in two strains, 43_S11 and 60_S17. The sequenced genomes also indicated that all strains carried SPI-1, SPI-2, and SPI-3. These SPIs are composed of antimicrobial resistance gene clusters and thus represent a potential threat to public health management. Taken together, this study highlights the extent of multidrug-resistant Salmonella contamination in duck meat in Vietnam. Full article
(This article belongs to the Section Molecular Microbiology)
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<p>Antibiotic resistance profiles of duck carcass samples: cefazolin (CZ), cefoxitin (FOX), cefuroxime (CXM), ceftriaxone (CRO), ceftazidime (CAZ), cefotaxime (CTX), ciprofloxacin (CIP), trimethoprim (TMP), gentamicin (CN), tetracycline (TE), chloramphenicol (C), ampicillin (AMP), meropenem (MRP), nalidixic acid (NA), extended-spectrum beta-lactam (ESBL), AmpC β-lactamase (AmpC), and multidrug resistance (MDR).</p> Full article ">
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