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Volume 23
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Int. J. Mol. Sci., Volume 23, Issue 17 (September-1 2022) – 695 articles

Cover Story (view full-size image): Connexin-based channels represent the physiological substrate of direct intercellular communication in living organisms, allowing the coordination of cellular activities and controlling cell growth, differentiation, and maintenance of tissue homeostasis. In the central nervous system (CNS), homeostasis is closely linked to the delicate balance of the microenvironment in which different cellular components coexist. The specific connexin signature of nerve cellular components represents a critical factor in the delicate physio-pathological balance. Connexin dysregulation is emerging as a detrimental cellular response to stressful stimuli or as an attempt to counteract disease progression. As such, detailed analysis of the role of connexin-based channels is of strategic importance for developing novel therapeutic approaches for CNS disease. View this paper
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14 pages, 2284 KiB  
Article
Characterization of Two-Component System CitB Family in Salmonella Pullorum
by Xiamei Kang, Xiao Zhou, Yanting Tang, Zhijie Jiang, Jiaqi Chen, Muhammad Mohsin and Min Yue
Int. J. Mol. Sci. 2022, 23(17), 10201; https://doi.org/10.3390/ijms231710201 - 5 Sep 2022
Cited by 14 | Viewed by 3021
Abstract
Salmonella enterica, serovar Gallinarum, biovar Pullorum, is an avian-specific pathogen which has caused considerable economic losses to the poultry industry worldwide. Two-component systems (TCSs) play an essential role in obtaining nutrients, detecting the presence of neighboring bacteria and regulating the expression of virulence [...] Read more.
Salmonella enterica, serovar Gallinarum, biovar Pullorum, is an avian-specific pathogen which has caused considerable economic losses to the poultry industry worldwide. Two-component systems (TCSs) play an essential role in obtaining nutrients, detecting the presence of neighboring bacteria and regulating the expression of virulence factors. The genome analysis of S. Pullorum strain S06004 suggesting the carriage of 22 pairs of TCSs, which belong to five families named CitB, OmpR, NarL, Chemotaxis and LuxR. In the CitB family, three pairs of TCSs, namely CitA-CitB, DcuS-DcuR and DpiB-DpiA, remain unaddressed in S. Pullorum. To systematically investigate the function of the CitB family in S. Pullorum, four mutants, ΔcitAB (abbreviated as Δcit), ΔdcuSR (Δdcu), ΔdpiBA (Δdpi) and ΔcitABΔdcuSRΔdpiBA (Δ3), were made using the CRISPR/Cas9 system. The results demonstrated that the CitB family did not affect the growth of bacteria, the results of biochemical tests, invasion and proliferation in chicken macrophage HD-11 cells and the expression of fimbrial protein. But the mutants showed thicker biofilm formation, higher resistance to antimicrobial agents, enhanced tolerance to inhibition by egg albumen and increased virulence in chicken embryos. Moreover, the deletion of Dpi TCS was detrimental to survival after exposure to hyperosmotic and oxidative environments, as well as the long-term colonization of the small intestine of chickens. Collectively, we provided new knowledge regarding the possible role of the CitB family involved in the pathogenic processes of S. Pullorum. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Bacterial Communication and Their Control)
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Figure 1
<p>Deletion of TCSs of the CitB family did not affect the growth and the results of biochemical tests. (<b>A</b>–<b>C</b>) Growth curves of WT R51 and mutants under LB shaking conditions (<b>A</b>), LB static culture (<b>B</b>) and M9 minimal medium static culture (<b>C</b>) (a, aerobic conditions; an, anaerobic conditions). (<b>D</b>) The biochemical test results of WT and mutants.</p> Full article ">Figure 2
<p>Deletion of TCSs of the CitB family increased the biofilm formation and the antibacterial resistance to aminoglycoside drugs under anaerobic conditions. (<b>A</b>,<b>B</b>) Determination of biofilm formed by WT and mutants at 20 °C, 28 °C, 37 °C and 42 °C cultured in LB broth for 24 h under aerobic conditions (<b>A</b>) and anaerobic conditions (<b>B</b>). (<b>C</b>,<b>D</b>) Biofilm formation in different bile salts at 42 °C for 24 h under aerobic conditions (<b>C</b>) and anaerobic conditions (<b>D</b>). (<b>E</b>) MIC values of WT and mutants against the tested antimicrobial agents. The abbreviations of antimicrobial agents are listed in <a href="#sec4dot6-ijms-23-10201" class="html-sec">Section 4.6</a>. Orange-filled numbers indicate that the MIC values of the mutants are greater than those of the WT. Statistical significance of differences was evaluated by one-way ANOVA test (* <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).</p> Full article ">Figure 3
<p>Hyperosmotic, oxidative, acid and heat stress assays. Treatment conditions from left to right were 10% glucose solution for 30 min, 4 mM hydrogen peroxide solution for 30 min, NaCl solution with pH 3.5 for 30 min and 50 °C for 10 min, respectively. Statistical significance of differences was evaluated by one-way ANOVA test (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 4
<p>Deletion of TCSs of the CitB family increased the growth in egg albumen and virulence of the chicken embryo. (<b>A</b>) The survival of WT and mutants after 24 h cultured in egg white at 37 °C and the ratio calculated as CFU (colony forming units) at 24 h/CFU at 0 h. (<b>B</b>) Survival curves for chicken embryos infected with WT and mutants. Sixteen-day-old chicken embryos were inoculated, each strain with 50 CFU via allantoic cavity injection and monitored for five days until hatching out of the shell. (<b>C</b>,<b>D</b>) Bacterial loads in liver (<b>C</b>) and spleen (<b>D</b>) tissues of infected embryos 5 days post-infection. Statistical significance of differences was evaluated by one-way ANOVA test (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 5
<p>Deletion of Dpi TCS was detrimental to long-term colonization of the small intestine but did not affect the expression of the fimbrial protein. (<b>A</b>) Survival curves for chickens infected with WT and mutants. One-day-old chickens were inoculated, each strain with 10<sup>6</sup> CFU, by oral injection and monitored for 21 days. (<b>B</b>–<b>D</b>) Bacterial loads in the liver, spleen, small intestine tissues and faecal samples 3 (<b>B</b>), 7 (<b>C</b>) and 14 (<b>D</b>) days post-infection. (<b>E</b>) Western blotting analysis detected fimbrial protein expression of WT and mutants. The GAPDH was used as a reference. (<b>F</b>) The relative expression level of four fimbrial proteins in WT and mutants. The statistical significance of differences was evaluated by one-way ANOVA test (* <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
18 pages, 3544 KiB  
Article
Proteomics Profiling of Osteoporosis and Osteopenia Patients and Associated Network Analysis
by Mysoon M. Al-Ansari, Shereen M. Aleidi, Afshan Masood, Eman A. Alnehmi, Mai Abdel Jabar, Maha Almogren, Mohammed Alshaker, Hicham Benabdelkamel and Anas M. Abdel Rahman
Int. J. Mol. Sci. 2022, 23(17), 10200; https://doi.org/10.3390/ijms231710200 - 5 Sep 2022
Cited by 18 | Viewed by 3740
Abstract
Bone mass reduction due to an imbalance in osteogenesis and osteolysis is characterized by low bone mineral density (LBMD) and is clinically classified as osteopenia (ON) or osteoporosis (OP), which is more severe. Multiple biomarkers for diagnosing OP and its progression have been [...] Read more.
Bone mass reduction due to an imbalance in osteogenesis and osteolysis is characterized by low bone mineral density (LBMD) and is clinically classified as osteopenia (ON) or osteoporosis (OP), which is more severe. Multiple biomarkers for diagnosing OP and its progression have been reported; however, most of these lack specificity. This cohort study aimed to investigate sensitive and specific LBMD-associated protein biomarkers in patients diagnosed with ON and OP. A label-free liquid chromatography-mass spectrometry (LC-MS) proteomics approach was used to analyze serum samples. Patients’ proteomics profiles were filtered for potential confounding effects, such as age, sex, chronic diseases, and medication. A distinctive proteomics profile between the control, ON, and OP groups (Q2 = 0.7295, R2 = 0.9180) was identified, and significant dysregulation in a panel of proteins (n = 20) was common among the three groups. A comparison of these proteins showed that the levels of eight proteins were upregulated in ON, compared to those in the control and the OP groups, while the levels of eleven proteins were downregulated in the ON group compared to those in the control group. Interestingly, only one protein, myosin heavy chain 14 (MYH14), showed a linear increase from the control to the ON group, with the highest abundance in the OP group. A significant separation in the proteomics profile between the ON and OP groups (Q2 = 0.8760, R2 = 0.991) was also noted. Furthermore, a total of twenty-six proteins were found to be dysregulated between the ON and the OP groups, with fourteen upregulated and twelve downregulated proteins in the OP, compared to that in the ON group. Most of the identified dysregulated proteins were immunoglobulins, complement proteins, cytoskeletal proteins, coagulation factors, and various enzymes. Of these identified proteins, the highest area under the curve (AUC) in the receiver operating characteristic (ROC) analysis was related to three proteins (immunoglobulin Lambda constant 1 (IGLC1), RNA binding protein (MEX3B), and fibulin 1 (FBLN1)). Multiple reaction monitoring (MRM), LC-MS, was used to validate some of the identified proteins. A network pathway analysis of the differentially abundant proteins demonstrated dysregulation of inflammatory signaling pathways in the LBMD patients, including the tumor necrosis factor (TNF), toll-like receptor (TL4), and interferon-γ (IFNG) signaling pathways. These results reveal the existence of potentially sensitive protein biomarkers that could be used in further investigations of bone health and OP progression. Full article
(This article belongs to the Special Issue Molecular Advances in Osteoporosis Research)
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<p>Overall proteomics analysis and exclusion of confounders-associated proteins. (<b>A</b>) Determination of confounder-independent proteins from the overall detected proteins. (<b>B</b>) Venn diagram demonstrating overlap between confounder-independent proteins (medications, TD, Sex, and T2DM) (<span class="html-italic">n</span> = 212, 154, 122, and 123, respectively) using moderate t-test and considering fold change (FC 1.5) and cut-off <span class="html-italic">p</span>-value &lt; 0.05. A total of 68 proteins were identified as being significantly associated with LBMD, independent of the effect of confounders. Abbreviations: LBMD: low bone mineral density; TD: thyroid disease; T2DM: type 2 diabetes mellitus. * Two-way ANOVA with FDR-corrected <span class="html-italic">p</span>-value (FDRp) cutoff = 0.05.</p> Full article ">Figure 2
<p>Proteomics profiling between healthy control (Ctrl), osteopenic (ON), and osteoporotic (OP) patients. (<b>A</b>) Orthogonal partial least squares-discriminant analysis (OPLS-DA) cross-validation illustrates the significant differences between the three study groups (Ctrl, ON, OP) (Q<sup>2</sup> = 0.7295, and R<sup>2</sup> = 0.9180). (<b>B</b>) Venn diagram demonstrating the significantly dysregulated proteins between Ctrl vs. ON (<span class="html-italic">n</span> = 65), Ctrl vs. OP (<span class="html-italic">n</span> = 34), and ON vs. OP (<span class="html-italic">n</span> = 51), considering FC of 1.5 and <span class="html-italic">p</span>-value of 0.05. Also shown is the identification of common and significant proteins (<span class="html-italic">n</span> = 20) between the three study groups. (<b>C</b>) Levels of commonly dysregulated proteins (G20) among the three groups, where G8 were upregulated to the highest abundance in the ON group, compared to that in the control group and then downregulated in the OP group, compared to that in the ON group, while G11 was downregulated to the lowest abundance in ON, compared to that in control and then upregulated in OP compared to that in ON. (<b>D</b>) Heat map showing the identity and expression levels of the 20 significantly detected proteins among the three study groups and also those associated with fracture history (FH) (highlighted with an asterisk). Green and colors mean down and up-regulation, respectively.</p> Full article ">Figure 2 Cont.
<p>Proteomics profiling between healthy control (Ctrl), osteopenic (ON), and osteoporotic (OP) patients. (<b>A</b>) Orthogonal partial least squares-discriminant analysis (OPLS-DA) cross-validation illustrates the significant differences between the three study groups (Ctrl, ON, OP) (Q<sup>2</sup> = 0.7295, and R<sup>2</sup> = 0.9180). (<b>B</b>) Venn diagram demonstrating the significantly dysregulated proteins between Ctrl vs. ON (<span class="html-italic">n</span> = 65), Ctrl vs. OP (<span class="html-italic">n</span> = 34), and ON vs. OP (<span class="html-italic">n</span> = 51), considering FC of 1.5 and <span class="html-italic">p</span>-value of 0.05. Also shown is the identification of common and significant proteins (<span class="html-italic">n</span> = 20) between the three study groups. (<b>C</b>) Levels of commonly dysregulated proteins (G20) among the three groups, where G8 were upregulated to the highest abundance in the ON group, compared to that in the control group and then downregulated in the OP group, compared to that in the ON group, while G11 was downregulated to the lowest abundance in ON, compared to that in control and then upregulated in OP compared to that in ON. (<b>D</b>) Heat map showing the identity and expression levels of the 20 significantly detected proteins among the three study groups and also those associated with fracture history (FH) (highlighted with an asterisk). Green and colors mean down and up-regulation, respectively.</p> Full article ">Figure 3
<p>Proteomics profiling between ON and OP patients. (<b>A</b>) Orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot showing the relative separation between ON and OP groups (Q<sup>2</sup> = 0.876, and R<sup>2</sup> = 0.991) after excluding three outlier values detected using the random forest algorithm. (<b>B</b>) Volcano plot analysis of ON versus OP showing significantly dysregulated proteins (false discovery rate (FDR)-corrected <span class="html-italic">p</span>-value &lt; 0.05, and fold change (FC) &gt; 1.5 or &lt; 0.67). A total of (G49) proteins were found to be dysregulated (26 up-regulated and 23 down-regulated) in OP patients, compared to those in ON patients. (<b>C</b>) Venn diagram illustrating an overlap between the confounder’s independent proteins (<span class="html-italic">n</span> = 68) and the dysregulated proteins between the ON and OP groups (G49). A total of 26 proteins were significantly dysregulated (14 up-regulated and 12 down-regulated) in OP, compared to those in ON patients. (<b>D</b>) Heat map showing the expression and the identity of the dysregulated proteins between the ON and OP groups along with fracture history (FH)-associated proteins (highlighted with an asterisk). Green and colors mean down and up-regulation, respectively.</p> Full article ">Figure 3 Cont.
<p>Proteomics profiling between ON and OP patients. (<b>A</b>) Orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot showing the relative separation between ON and OP groups (Q<sup>2</sup> = 0.876, and R<sup>2</sup> = 0.991) after excluding three outlier values detected using the random forest algorithm. (<b>B</b>) Volcano plot analysis of ON versus OP showing significantly dysregulated proteins (false discovery rate (FDR)-corrected <span class="html-italic">p</span>-value &lt; 0.05, and fold change (FC) &gt; 1.5 or &lt; 0.67). A total of (G49) proteins were found to be dysregulated (26 up-regulated and 23 down-regulated) in OP patients, compared to those in ON patients. (<b>C</b>) Venn diagram illustrating an overlap between the confounder’s independent proteins (<span class="html-italic">n</span> = 68) and the dysregulated proteins between the ON and OP groups (G49). A total of 26 proteins were significantly dysregulated (14 up-regulated and 12 down-regulated) in OP, compared to those in ON patients. (<b>D</b>) Heat map showing the expression and the identity of the dysregulated proteins between the ON and OP groups along with fracture history (FH)-associated proteins (highlighted with an asterisk). Green and colors mean down and up-regulation, respectively.</p> Full article ">Figure 4
<p>Results of biomarker evaluation in ON and OP. (<b>A</b>) Exploratory ROC curve generated by the OPLS-DA model; AUC values were calculated by mathematical integration of the combination of 5, 10, 15, 25, 50, and 100 proteins. (<b>B</b>–<b>D</b>) Three proteins with the highest AUC: (<b>B</b>) IGLC1, AUC = 0.929; (<b>C</b>) MEX3B, AUC = 0.884; and (<b>D</b>) FBLNI, AUC = 0.883.</p> Full article ">Figure 5
<p>Validation of the expression of selected proteins in the ON and OP groups using multiple reaction monitoring (MRM). (<b>A</b>) Representative MRM chromatograms for protein signature peptides selected from the Skyline Software and confirmed using PeptideAtlas. (<b>B</b>) Scatter plots with bar-graph for the expressions of selected proteins. The differences between the study groups were evaluated using an unpaired <span class="html-italic">t</span>-test with significance set at <span class="html-italic">p</span>-value &lt;0.01 (denoted by **), &lt;0.001 (denoted by ***), and &lt;0.0001(denoted by ****).</p> Full article ">Figure 6
<p>Network analysis and biological pathways related to the significantly identified proteins in the study population. (<b>A</b>) Network pathway analysis of the significantly dysregulated proteins identified in the ON group, compared to those in the OP group revealed that they were related to the developmental disorder, hereditary disorder, and metabolic disease. The analysis also showed the involvement of the TNF and IFNG signaling pathways. (<b>B</b>) The top canonical pathways related to the significantly dysregulated proteins identified in the ON group, compared to those in the OP group.</p> Full article ">
18 pages, 3103 KiB  
Article
Tubular IKKβ Deletion Alleviates Acute Ischemic Kidney Injury and Facilitates Tissue Regeneration
by Eileen Dahlke, Toni Engmann, Yaman Anan, Robert Häsler, Giovanni Solinas and Franziska Theilig
Int. J. Mol. Sci. 2022, 23(17), 10199; https://doi.org/10.3390/ijms231710199 - 5 Sep 2022
Viewed by 2481
Abstract
Acute kidney injury (AKI) is a common renal injury leading to relevant morbidity and mortality worldwide. Most of the clinical cases of AKI are caused by ischemia reperfusion (I/R) injury with renal ischemia injury followed by reperfusion injury and activation of the innate [...] Read more.
Acute kidney injury (AKI) is a common renal injury leading to relevant morbidity and mortality worldwide. Most of the clinical cases of AKI are caused by ischemia reperfusion (I/R) injury with renal ischemia injury followed by reperfusion injury and activation of the innate immune response converging to NF-ĸB pathway induction. Despite the clear role of NF-ĸB in inflammation, it has recently been acknowledged that NF-ĸB may impact other cell functions. To identify NF-ĸB function with respect to metabolism, vascular function and oxidative stress after I/R injury and to decipher in detail the underlying mechanism, we generated a transgenic mouse model with targeted deletion of IKKβ along the tubule and applied I/R injury followed by its analysis after 2 and 14 days after I/R injury. Tubular IKKβ deletion ameliorated renal function and reduced tissue damage. RNAseq data together with immunohistochemical, biochemical and morphometric analysis demonstrated an ameliorated vascular organization and mRNA expression profile for increased angiogenesis in mice with tubular IKKβ deletion at 2 days after I/R injury. RNAseq and protein analysis indicate an ameliorated metabolism, oxidative species handling and timely-adapted cell proliferation and apoptosis as well as reduced fibrosis in mice with tubular IKKβ deletion at 14 days after I/R injury. In conclusion, mice with tubular IKKβ deletion upon I/R injury display improved renal function and reduced tissue damage and fibrosis in association with improved vascularization, metabolism, reactive species disposal and fine-tuned cell proliferation. Full article
(This article belongs to the Special Issue New Insights into the Molecular Mechanisms of Kidney Injury and Repair)
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Figure 1
<p>Genetic tubular IKKβ-deletion ameliorates I/R injury. (<b>a</b>) Scheme illustrating deletion of <span class="html-italic">IKKβ</span> gene under Pax8 promoter along the tubule; (<b>b</b>) semi-quantitative analysis of IKKβ mRNA expression derived from Exon 3 in tubular cells of control and IKKβ<sup>∆Tub</sup>. Arithmetic means ± SEM of <span class="html-italic">n</span> = 12 per group; * <span class="html-italic">p</span> &lt; 0.05, <span class="html-italic">t</span>-test; (<b>c</b>) timeline of experimental procedure; (<b>d</b>,<b>e</b>) plasma creatinine (<b>d</b>) and blood urea nitrogen (BUN; (<b>e</b>)) values of control and IKKβ<sup>∆Tub</sup> at baseline, 2 days and 14 days after I/R injury. Arithmetic means ± SEM of <span class="html-italic">n</span> = 3–11 per group; (<b>f</b>) semi-quantitative evaluation of tubulointerstitial injury. Arithmetic means ± SEM of <span class="html-italic">n</span> = 3–11 per group; (<b>g</b>) representative images of Masson trichrome stained kidney section of control and IKKβ<sup>∆Tub</sup> at baseline, 2 days and 14 days after I/R injury. Scale bar = 50 µm. Tubular necrosis (x), loss of brush border (*), eosinophilic debris (#) and cellular infiltration (§) and fibrotic areas (+) are shown; (<b>h</b>) representative electron microscopy images of control and IKKβ<sup>∆Tub</sup> at baseline, 2 days and 14 days after I/R injury. Scale bar = 2 µm. Reduced visibility of mitochondria (arrow), cellular debris (#), loss of brush border (arrow head) and basement membrane thickening (*) are encountered; (<b>i</b>) principal coordinate analyses between control and IKKβ<sup>∆Tub</sup> at baseline, 2 days and 14 days after I/R injury displayed as variation between samples; IKKβ<sup>∆Tub</sup> vs. control at baseline, at 2 days and 14 days after I/R injury based on filter criteria DESeq <span class="html-italic">p</span>-values &lt; 0.05; fold change &gt; 1.5. For (<b>d</b>–<b>f</b>) * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, Mann–Whitney–U test (if <span class="html-italic">n</span> &lt; 4 Lord test).</p> Full article ">Figure 2
<p>Tubular deletion of IKKβ alters baseline gene expression. (<b>a</b>) heatmaps of significantly increased mRNA of proteins important in signaling transduction and epithelial transport; (<b>b</b>) Heatmaps of significantly augmented mRNA expression levels coding for enzymes of metabolism (glycolysis, of lipid uptake, storage and biosynthesis) and mitochondria and mitochondrial uncoupling. IKKβ<sup>∆Tub</sup> vs. control based on genes, selected by the filter criteria DESeq <span class="html-italic">p</span>-values &lt; 0.05; fold change &gt; 1.5; (<b>c</b>,<b>d</b>) real-time PCR of Hk2 and Pfkfb3. Arithmetic means ± SEM of <span class="html-italic">n</span> = 4–5 per group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 using Lord test; (<b>e</b>) heatmaps of significantly altered genes known to ameliorate the extent of I/R injury. (#) marks the most significantly differentially altered mRNA expression level. All heatmaps show samples organized in columns and genes in rows, expression intensities are color-coded.</p> Full article ">Figure 3
<p>Tubular deletion of IKKβ induces angiogenesis. (<b>a</b>) representative images of semi-thin sections from control and IKKβ<sup>∆Tub</sup> at baseline, 2 days and 14 days after I/R injury. Scale bar = 50 µm; (<b>b</b>) morphometric analysis of tubule–capillary contact length in %. Arithmetic means ± SEM of <span class="html-italic">n</span> = 3–6 per group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 using a Mann–Whitney–U test (if <span class="html-italic">n</span> &lt; 4 Lord test). (<b>c</b>,<b>d</b>) Heatmaps of significantly altered mRNA of proteins known to affect angiogenesis at 2 days after I/R injury compared to baseline (<b>c</b>) and at 14 days after I/R injury compared to baseline (<b>d</b>). Filter criteria were DESeq <span class="html-italic">p</span>-values &lt; 0.05; fold change &gt; 1.5. All heatmaps show samples organized in columns and genes in rows, expression intensities are color-coded. The green stippled circle indicates mRNA of I/R groups used for relative expression in heatmaps by comparing only control with IKKβ<sup>∆Tub</sup> at 2 days (<b>c</b>) and 14 days (<b>d</b>) after I/R injury.</p> Full article ">Figure 4
<p>Tubular deletion of IKKβ affects glucose metabolism and reactive oxygen species handling after I/R injury. (<b>a</b>,<b>b</b>) heatmaps of significantly altered mRNA of proteins known to affect glucose metabolism at 2 days (<b>a</b>) and at 14 days (<b>b</b>) after I/R injury compared to baseline. Filter criteria were DESeq <span class="html-italic">p</span>-values &lt; 0.05; fold change &gt; 1.5. mRNA further analyzed are encircled. All heatmaps (<b>a</b>,<b>b</b>,<b>g</b>) show samples organized in columns and genes in rows, expression intensities are color-coded. The green stippled circle indicates mRNA of I/R groups used for relative expression in heatmaps by comparing only control with IKKβ<sup>∆Tub</sup> at 2 days (<b>a</b>) and 14 days (<b>b</b>) after I/R injury. (<b>c</b>) Representative images of SGLT2 stained kidney sections and Western blot analysis from control and IKKβ<sup>∆Tub</sup> at baseline, 2 days and 14 days after I/R injury. Magnification scale bar = 50 µm. Densitometrical evaluation is presented in % arithmetic means ± SEM of <span class="html-italic">n</span> = 3–5 per group. (<b>d</b>,<b>f</b>) real-time PCR of Hk3 (<b>d</b>), Fbp1 (<b>e</b>) and Pfkfb3 (<b>f</b>) mRNA expression of kidneys from control and IKKβ<sup>∆Tub</sup> at baseline, 2 days and 14 days after I/R injury. Arithmetic means ± SEM of <span class="html-italic">n</span> = 3–6 per group. (<b>g</b>) heatmaps of significantly altered mRNA from proteins known to improve detoxification and reactive oxygen species handling at 14 days after I/R injury compared to baseline. Filter criteria were DESeq <span class="html-italic">p</span>-values &lt; 0.05; fold change &gt; 1.5. The green stippled circle indicates mRNA of I/R group used for relative expression in heatmaps by comparing only control with IKKβ<sup>∆Tub</sup> at 14 days after I/R injury; (<b>h</b>) representative images of GSTA1 stained kidney sections and Western blot analysis from control and IKKβ<sup>∆Tub</sup> at baseline, 2 days and 14 days after I/R injury. Magnification scale bar = 50 µm. Densitometrical evaluation is presented in % arithmetic means ± SEM of <span class="html-italic">n</span> = 4–9 per group. For (<b>c</b>–<b>f</b>,<b>h</b>) * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 using Mann–Whitney–U test (if <span class="html-italic">n</span> &lt; 4 Lord test).</p> Full article ">Figure 5
<p>Tubular deletion of IKKβ affects proliferation, tissue regeneration and fibrosis. (<b>a</b>) assessment of Ki67-positive proliferating cells in control and IKKβ<sup>∆Tub</sup> at 2 days and 14 days after I/R injury showing the semi-quantitative evaluation and representative images. Scale bar = 50 µm. Arithmetic means ± SEM of <span class="html-italic">n</span> = 4–12 per group; (<b>b</b>,<b>c</b>) heatmaps of significantly altered mRNA for cell proliferation, apoptosis and fibrosis at 2 days (<b>b</b>) and at 14 days (<b>c</b>) after I/R injury. Filter criteria were DESeq <span class="html-italic">p</span>-values &lt; 0.05; fold change &gt; 1.5. All heatmaps show samples organized in columns and genes in rows, expression intensities are color-coded. (<b>d</b>,<b>e</b>) assessment of MMP9 (<b>d</b>) and Col3a1 (<b>e</b>) showing densitometric analysis of respective Western blots and representing images for each strain and time point. Scale bar = 50 µm. Arithmetic means ± SEM of <span class="html-italic">n</span> = 3–9 per group. The green stippled circle indicates mRNA of I/R groups used for relative expression in heatmaps by comparing only control with IKKβ<sup>∆Tub</sup> at 2 days (<b>c</b>) and 14 days (<b>d</b>) after I/R injury. For (<b>a</b>,<b>d</b>,<b>e</b>) * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, Mann–Whitney–U test (if <span class="html-italic">n</span> &lt; 4 Lord test).</p> Full article ">
27 pages, 6311 KiB  
Review
BODIPYs in PDT: A Journey through the Most Interesting Molecules Produced in the Last 10 Years
by Miryam Chiara Malacarne, Marzia Bruna Gariboldi and Enrico Caruso
Int. J. Mol. Sci. 2022, 23(17), 10198; https://doi.org/10.3390/ijms231710198 - 5 Sep 2022
Cited by 26 | Viewed by 3993
Abstract
Over the past 30 years, photodynamic therapy (PDT) has shown great development. In the clinical setting the few approved molecules belong almost exclusively to the porphyrin family; but in the scientific field, in recent years many researchers have been interested in other families [...] Read more.
Over the past 30 years, photodynamic therapy (PDT) has shown great development. In the clinical setting the few approved molecules belong almost exclusively to the porphyrin family; but in the scientific field, in recent years many researchers have been interested in other families of photosensitizers, among which BODIPY has shown particular interest. BODIPY is the acronym for 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene, and is a family of molecules well-known for their properties in the field of imaging. In order for these molecules to be used in PDT, a structural modification is necessary which involves the introduction of heavy atoms, such as bromine and iodine, in the beta positions of the pyrrole ring; this change favors the intersystem crossing, and increases the 1O2 yield. This mini review focused on a series of structural changes made to BODIPYs to further increase 1O2 production and bioavailability by improving cell targeting or photoactivity efficiency. Full article
(This article belongs to the Special Issue Materials for Photobiology)
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<p>Schematic PDT Treatment: photodynamic strategy involves photosensitizer (PS) administration (local or systemic injection). Light irradiation is applied depending on the drug-light interval (time necessary to drug accumulation within the tumor). The activation of PS leads to the generation of <sup>1</sup>O<sub>2</sub> and ROS that cause cancer cell death through apoptosis, necrosis or autophagy.</p> Full article ">Figure 2
<p>Chemical structures of compounds <b>2</b> and <b>8</b> [<a href="#B86-ijms-23-10198" class="html-bibr">86</a>].</p> Full article ">Figure 3
<p>Chemical structures of compounds <b>3</b> and <b>4</b> [<a href="#B87-ijms-23-10198" class="html-bibr">87</a>].</p> Full article ">Figure 4
<p>Chemical structures of compound <b>3</b> [<a href="#B89-ijms-23-10198" class="html-bibr">89</a>].</p> Full article ">Figure 5
<p>Chemical structures of compounds <b>Br<sub>2</sub>BOAc</b> and <b>I<sub>2</sub>BOAc</b> [<a href="#B90-ijms-23-10198" class="html-bibr">90</a>,<a href="#B91-ijms-23-10198" class="html-bibr">91</a>].</p> Full article ">Figure 6
<p>Chemical structures of compounds <b>6</b> (<b>PYR</b>) and <b>7</b> (<b>EPO</b>) [<a href="#B93-ijms-23-10198" class="html-bibr">93</a>].</p> Full article ">Figure 7
<p>Chemical structures of compounds <b>4</b> (<b>BDPI</b>) and <b>6</b> (<b>BBDPI</b>) [<a href="#B102-ijms-23-10198" class="html-bibr">102</a>].</p> Full article ">Figure 8
<p>Chemical structures of compounds <b>2a</b>–<b>d</b> [<a href="#B103-ijms-23-10198" class="html-bibr">103</a>].</p> Full article ">Figure 9
<p>Chemical structures of compound <b>4</b> [<a href="#B68-ijms-23-10198" class="html-bibr">68</a>].</p> Full article ">Figure 10
<p>Chemical structures of compound <b>BDILa</b> [<a href="#B112-ijms-23-10198" class="html-bibr">112</a>].</p> Full article ">Figure 11
<p>Chemical structures of compound <b>BDPI-Lyso</b> [<a href="#B115-ijms-23-10198" class="html-bibr">115</a>].</p> Full article ">Figure 12
<p>Chemical structures of compound <b>MBDP</b> [<a href="#B119-ijms-23-10198" class="html-bibr">119</a>].</p> Full article ">Figure 13
<p>Chemical structures of compound <b>10</b> [<a href="#B121-ijms-23-10198" class="html-bibr">121</a>].</p> Full article ">Figure 14
<p>Chemical structures of compound <b>3</b> [<a href="#B122-ijms-23-10198" class="html-bibr">122</a>,<a href="#B123-ijms-23-10198" class="html-bibr">123</a>].</p> Full article ">Figure 15
<p>Chemical structures of compound <b>p(GEMA-co-BODIPYMA)-2I</b> [<a href="#B111-ijms-23-10198" class="html-bibr">111</a>].</p> Full article ">Figure 16
<p>Chemical structures of compound <b>PTUBDP</b> [<a href="#B129-ijms-23-10198" class="html-bibr">129</a>].</p> Full article ">Figure 17
<p>Chemical structures of compound <b>8</b> [<a href="#B131-ijms-23-10198" class="html-bibr">131</a>].</p> Full article ">Figure 18
<p>Chemical structures of compound <b>1</b> (<b>IY-IY-PDT</b>) [<a href="#B135-ijms-23-10198" class="html-bibr">135</a>].</p> Full article ">Figure 19
<p>Chemical structures of compound <b>3b</b> [<a href="#B138-ijms-23-10198" class="html-bibr">138</a>].</p> Full article ">Figure 20
<p>Chemical structures of compound <b>8a</b> [<a href="#B139-ijms-23-10198" class="html-bibr">139</a>].</p> Full article ">Figure 21
<p>Chemical structures of compound <b>7</b> [<a href="#B140-ijms-23-10198" class="html-bibr">140</a>].</p> Full article ">
13 pages, 1544 KiB  
Article
The Ambivalence of Connexin43 Gap Peptides in Cardioprotection of the Isolated Heart against Ischemic Injury
by Aleksander Tank Falck, Bjarte Aarmo Lund, David Johansen, Trine Lund and Kirsti Ytrehus
Int. J. Mol. Sci. 2022, 23(17), 10197; https://doi.org/10.3390/ijms231710197 - 5 Sep 2022
Cited by 3 | Viewed by 2091
Abstract
The present study investigates infarct-reducing effects of blocking ischemia-induced opening of connexin43 hemichannels using peptides Gap19, Gap26 or Gap27. Cardioprotection by ischemic preconditioning (IPC) and Gap peptides was compared, and combined treatment was tested in isolated, perfused male rat hearts using function and [...] Read more.
The present study investigates infarct-reducing effects of blocking ischemia-induced opening of connexin43 hemichannels using peptides Gap19, Gap26 or Gap27. Cardioprotection by ischemic preconditioning (IPC) and Gap peptides was compared, and combined treatment was tested in isolated, perfused male rat hearts using function and infarct size after global ischemia, high-resolution respirometry of isolated mitochondrial and peptide binding kinetics as endpoints. The Gap peptides reduced infarct size significantly when given prior to ischemia plus at reperfusion (Gap19 76.2 ± 2.7, Gap26 72.9 ± 5.8 and Gap27 71.9 ± 5.8% of untreated control infarcts, mean ± SEM). Cardioprotection was lost when Gap26, but not Gap27 or Gap19, was combined with triggering IPC (IPC 73.4 ± 5.5, Gap19-IPC 60.9 ± 5.1, Gap26-IPC 109.6 ± 7.8, Gap27-IPC 56.3 ± 8.0% of untreated control infarct). Binding stability of peptide Gap26 to its specific extracellular loop sequence (EL2) of connexin43 was stronger than Gap27 to its corresponding loop EL1 (dissociation rate constant Kd 0.061 ± 0.004 vs. 0.0043 ± 0.0001 s−1, mean ± SD). Mitochondria from IPC hearts showed slightly but significantly reduced respiratory control ratio (RCR). In vitro addition of Gap peptides did not significantly alter respiration. If transient hemichannel activity is part of the IPC triggering event, inhibition of IPC triggering stimuli might limit the use of cardioprotective Gap peptides. Full article
(This article belongs to the Special Issue Novel Molecular Targets in Cardiovascular Diseases 2.0)
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<p>Infarct size in % of untreated control infarcts. Isolated buffer perfused hearts were subjected to global ischemia followed by reperfusion. Infarct size relative to the total ventricular volume was measured and results expressed in % of untreated control infarcts. (<b>a</b>) Controls subjected to ischemia-reperfusion with no treatment and hearts treated with Gap19, Gap 26 or Gap 27 (0.05 µM) before ischemia initially at reperfusion. (<b>b</b>) Hearts subjected to ischemic preconditioning IPC prior to 30 min ischemia-reperfusion with no treatment or with Gap19, Gap 26 or Gap 27 (0.05 µM) added to the buffer only during the PC treatment. Open symbols represent individual hearts, closed symbols are group mean ± SEM, * <span class="html-italic">p</span> &lt; 0.05 vs. control.</p> Full article ">Figure 2
<p>(<b>a</b>–<b>c</b>) Heart function in isolated hearts subjected to global ischemia followed by reperfusion with or without IPC and/or Gap peptide treatment: (<b>a</b>) Left ventricle developed pressure (LVDP) in control hearts (closed symbols) and hearts subjected to ischemic preconditioning (IPC) (open symbols) followed by 30 min ischemia and 120 min reperfusion * <span class="html-italic">p</span> &lt; 0.05 vs. control. (<b>b</b>) Postischemic recovery (after 30 min ischemia and 120 min reperfusion) of LVDP as % initial preischemic values. Untreated hearts (controls) and hearts treated with Gap peptides 0.5 uM before and after 30 min ischemia are shown as open bars. Ischemic preconditioning (IPC) treated hearts are shown with closed bars. * <span class="html-italic">p</span> &lt; 0.05 comparing IPC with hearts without IPC treatment. (<b>c</b>) End diastolic pressure (EDP, mmHg) prior to ischemia and at the end of 120 min reperfusion in the eight test groups. Endpoint contracture was significantly reduced by IPC in all groups. * <span class="html-italic">p</span> &lt; 0.05 compared with postischemic hearts not treated with IPC. End diastolic pressure was significantly higher in IPC Gap26 treated hearts compared to IPC Gap27 treated hearts δ <span class="html-italic">p</span> &lt; 0.05. (<b>d</b>,<b>e</b>) Coronary flow: (<b>d</b>) with/without Gap peptides given prior to 30 min ischemia and initially during reperfusion. * <span class="html-italic">p</span> &lt; 0.05 by ANOVA comparing groups at the same timepoint, Gap19 vs. no Gap peptide prior to ischemia and Gap peptides vs. control at reperfusion. (<b>e</b>) Coronary flow with/without Gap peptides given during triggering IPC. * <span class="html-italic">p</span> &lt; 0.05 by ANOVA, Gap19 vs. no Gap peptide prior to IPC, Gap26 vs. no Gap peptide at reperfusion).</p> Full article ">Figure 3
<p>Subsarcolemmal mitochondrial respiratory control ration (RCR) and phosphate/oxygen (P/O) ratio. (<b>a</b>) Respiratory control ratio (RCR) as max O<sub>2</sub> flux (OXPHOS) at saturating [ADP] divided by state 2 leak respiration (glutamate—maleate as substrates). * <span class="html-italic">p</span> &lt; 0.05 by ANOVA compared to mitochondria from hearts not subjected to IPC. (<b>b</b>) Phosphate/oxygen (P/O) ratio as µmol ADP added divided by µmol oxygen used (in the presence of glutamate—maleate). The grouped bars represent the presence of increasing concentrations of Gap peptides (0 and 0.5, 5, 50 µM as indicated) in the respiration chambers: Gap19 (G19), Gap26 (G26), Gap27 (G27).</p> Full article ">Figure 4
<p>Biophysical characterization of Gap26 and Gap27 peptide binding. Gap peptides were injected over NTA surfaces with captured EL1 (<b>a</b>) and EL2 (<b>b</b>) on RadA-scaffolds. (<b>a</b>) RadA-EL1 with Gap27 (0.16–20 µM) and (<b>b</b>) RadA-EL2 with Gap26 (3–50 µM) showed clear differences in off-rates. The fit to a 1:1 binding model are shown as dashed lines. Colors indicate the concentrations of the injections in the order, from lowest concentration to highest: blue–orange–green–red.</p> Full article ">
16 pages, 3451 KiB  
Article
Osteogenic Efficacy of Human Trophoblasts-Derived Conditioned Medium on Mesenchymal Stem Cells
by Yoon-Young Go, Chan-Mi Lee, Sung-Won Chae and Jae-Jun Song
Int. J. Mol. Sci. 2022, 23(17), 10196; https://doi.org/10.3390/ijms231710196 - 5 Sep 2022
Cited by 2 | Viewed by 2473
Abstract
Trophoblasts play an important role in the regulation of the development and function of the placenta. Our recent study demonstrated the skin regeneration capacity of trophoblast-derived extracellular vesicles (EV). Here, we aimed to determine the potential of trophoblast-derived conditioned medium (TB-CM) in enhancing [...] Read more.
Trophoblasts play an important role in the regulation of the development and function of the placenta. Our recent study demonstrated the skin regeneration capacity of trophoblast-derived extracellular vesicles (EV). Here, we aimed to determine the potential of trophoblast-derived conditioned medium (TB-CM) in enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We found that TB-CM promoted the osteogenic differentiation of MSCs in a dose-dependent manner. Furthermore, it inhibited adipogenesis of MSCs. We also found that the primary trophoblast-derived conditioned medium (PTB-CM) significantly enhanced the proliferation and osteogenic differentiation of human MSCs. Our study demonstrated the regulatory mechanisms underlying the TB-CM-induced osteogenesis in MSCs. An upregulation of genes associated with cytokines/chemokines was observed. The treatment of MSCs with TB-CM stimulated osteogenesis by activating several biological processes, such as mitogen-activated protein kinase (MAPK) and bone morphogenetic protein 2 (BMP2) signaling. This study demonstrated the proliferative and osteogenic efficacies of the trophoblast-derived secretomes, suggesting their potential for use in clinical interventions for bone regeneration and treatment. Full article
(This article belongs to the Special Issue Stem Cell Activation in Adult Organism 2023)
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<p>Trophoblast-derived conditioned medium (TB-CM) induces the osteogenic differentiation of mesenchymal stem cells (MSCs). Human MSCs were cultured in varying concentrations of TB-CM in an osteogenic induction medium (OIM) for 14 days. (<b>A</b>) In the early stages of osteogenesis (days 3 and 7), the cells were harvested, and the alkaline phosphatase (ALP) activity of each cell lysate was determined and normalized by protein concentration. <span class="html-italic">p</span>-values were obtained using the <span class="html-italic">t</span>-test, compared to the OIM control. (<b>B</b>) Quantification of calcium deposition on the cell surface was performed on day 14. <span class="html-italic">p</span>-values were obtained using the <span class="html-italic">t</span>-test, compared to the OIM control. (<b>C</b>) During the osteogenic differentiation of MSCs, the relative mRNA expression levels of <span class="html-italic">IBSP</span>, <span class="html-italic">ALP</span>, <span class="html-italic">RUNX2</span>, <span class="html-italic">OSX</span>, <span class="html-italic">OCN</span>, and <span class="html-italic">OPN</span> were determined using quantitative reverse-transcription polymerase chain reaction (<span class="html-italic">IBSP</span> and <span class="html-italic">ALP</span>: day 3, <span class="html-italic">RUNX2</span> and <span class="html-italic">OSX</span>: day 7, <span class="html-italic">OCN</span> and <span class="html-italic">OPN</span>: day 14). <span class="html-italic">p</span>-values were obtained using the <span class="html-italic">t</span>-test and then compared to those of the GM control. (<b>D</b>) The matrix mineralization of the MSCs was evaluated by alizarin red S staining, followed by determining the extent of destaining in each culture (arbitrary unit, AU). Representative images of OIM and OIM + 30% TB-CM were taken under a light microscope. Scale bar: 100 μm. Data are presented as mean ± standard deviation (SD) (n = 3); * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 2
<p>Trophoblast-derived conditioned medium (TB-CM) inhibits adipogenesis of mesenchymal stem cells (MSCs). (<b>A</b>) Oil red O staining showing the production of lipid droplets after the adipogenic differentiation of human MSCs upon culturing for 14 days in the adipogenic induction medium (AIM). The inhibitory effect of 50% TB-CM on the adipogenesis of MSCs was determined by the quantification of stained cells, as represented in the graph (AU, arbitary unit; *** <span class="html-italic">p</span> &lt; 0.001). (<b>B</b>) The relative expression levels of genes encoding for PPAR-g, adiponectin, and leptin in the adipogenic MSCs treated with 10, 20, and 50% TB-CM for 14 days were determined by performing a quantitative reverse transcription-polymerase chain reaction. Data are represented as mean ± SD. <span class="html-italic">p</span>-values were obtained using analysis of variance; * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 3
<p>Effect of regenerative ability of primary trophoblast-derived conditioned medium (PTB-CM) on mesenchymal stem cells (MSCs). (<b>A</b>) Representative images of human placenta tissue and isolated primary trophoblasts (in vitro cultivation for 72 h). Expression of cytokeratin 7 (red, intracellular marker of human placental villous trophoblasts) was observed as immunofluorescence obtained by nuclear counterstaining with DAPI (blue). (<b>B</b>) Conditioned medium obtained from the in vitro-cultured primary trophoblasts was collected every day for 15 days, followed by determining the size and concentration of nanoparticles by performing nanoparticle tracking analysis in the early culture period. <span class="html-italic">p</span>-values were obtained using Student’s <span class="html-italic">t</span>-test, compared to the day 2 data; * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01. (<b>C</b>) Human MSCs (in passage 9) were treated with varying concentrations of PTB-CM for 24 h. Representative images of MSCs treated with 60% PTB-CM and that of the control group were shown, and the cells were quantified to determine the proliferation-promoting effect of PTB-CM. <span class="html-italic">p</span>-values were obtained using a <span class="html-italic">t</span>-test, compared to that of the untreated cells. (<b>D</b>) The proliferation rate of MSCs was compared with the treatment of 30% MSCs-, PTB-, and TSC (trophoblasts stem cells)-derived CM under the same experimental condition. <span class="html-italic">p</span>-values were obtained using a <span class="html-italic">t</span>-test, compared to the untreated or MSC-CM treated cells. (<b>E</b>) MSCs were cultured in a serum-free medium, with an indicated concentration of PTB-CM. PTB-CM was used to divide into early (days 1–7) and late (days 8–14) time collected medium. CCK8 analysis determined the effect of PTB-CM in cell proliferation, indicating an increase in the percentage of cells after PTB-CM supplementation (early, red; late, blue). <span class="html-italic">p</span>-values were obtained using a <span class="html-italic">t</span>-test, compared to the serum-free control. (<b>F</b>) On days 7 and 14 of culturing the osteoblasts in OIM, the effect of PTB-CM on osteogenesis was evaluated by determining the ALP activity, calcium deposition, and <span class="html-italic">RUNX2</span> gene expression. <span class="html-italic">p</span>-values were obtained using a <span class="html-italic">t</span>-test, compared to the OIM group. Data are represented as mean ± SD; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>Enrichment analysis of GO terms and KEGG pathways in the TB-CM-treated mesenchymal stem cells (MSCs) during osteogenesis. MSCs were treated with 30% TB-CM under OIM conditions. RNA-seq was performed to identify the differentially expressed genes (DEGs) in the treated MSCs. (<b>A</b>) The significantly upregulated and downregulated DEGs are represented in the form of a volcano plot (<span class="html-italic">p</span> &lt; 0.05, FC ≥ 1.5). (<b>B</b>) DEGs were assigned to three GO categories: biological processes (red bars), molecular functions (blue bars), and cellular components (green bars). The enrichment scores of the GO terms are shown as −log<sub>10</sub> (<span class="html-italic">p</span>-value). (<b>C</b>) The ten most significant pathways of DEGs in TB-CM-treated MSCs are presented using a bar chart with enrichment scores shown as {−log<sub>10</sub> (<span class="html-italic">p</span>-value)}. (<b>D</b>) Biological processes and pathways of DEGs in the TB-CM-treated MSCs were analyzed using the ClueGO bioinformatics tool. The most significant GO terms and pathways per functional group are shown in bold and in the same color. Node size depicts the level of statistical significance (<span class="html-italic">p</span> &lt; 0.05). Consistently analyzed terms, including cytokine-mediated signaling pathways and MAPK cascades, are indicated by red circles. TB-CM, trophoblast-derived conditioned medium; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase.</p> Full article ">Figure 5
<p>TB-CM induces significant transcriptome alteration in HNDF cells during migration. (<b>A</b>) Proteins associated with the MAPK pathway, such as ERK, p-ERK, p38, p-p38, JNK, and p-JNK, were evaluated for their expression levels in the TB-CM-treated mesenchymal stem cells (MSCs) by performing western blot analysis. The expression levels AKT and p-AKT in MSCs treated with TB-CM were also detected using western blot analysis. A quantitative analysis of the western blot was performed, which showed significant expression levels of protein, as presented in the histogram. <span class="html-italic">p</span>-values were obtained using a <span class="html-italic">t</span>-test, compared to that of the GM group. (<b>B</b>) The relative expression levels of BMP2 and GREM1 encoding genes were analyzed both in the TB-CM-treated and control MSCs. A reverse transcription-polymerase chain reaction was performed to confirm the increase in the expression levels of <span class="html-italic">CXCL</span> (<b>C</b>)-, <span class="html-italic">CCL</span> (<b>D</b>)-, and <span class="html-italic">MMP</span> (<b>E</b>)-associated genes in the TB-CM-treated MSCs. Data have been indicated as mean ± SD; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001, compared to the corresponding control.</p> Full article ">
11 pages, 1428 KiB  
Article
Circulating Ageing Neutrophils as a Marker of Asymptomatic Polyvascular Atherosclerosis in Statin-Naïve Patients without Established Cardiovascular Disease
by Vadim Genkel, Ilya Dolgushin, Irina Baturina, Albina Savochkina, Karina Nikushkina, Anna Minasova, Lubov Pykhova, Veronika Sumerkina, Alla Kuznetsova and Igor Shaposhnik
Int. J. Mol. Sci. 2022, 23(17), 10195; https://doi.org/10.3390/ijms231710195 - 5 Sep 2022
Cited by 1 | Viewed by 2243
Abstract
Background: Current data on the possible involvement of aging neutrophils in atherogenesis are limited. This study aimed to research the diagnostic value of aging neutrophils in their relation to subclinical atherosclerosis in statin-naïve patients without established atherosclerotic cardiovascular diseases (ASCVD). Methods: The study [...] Read more.
Background: Current data on the possible involvement of aging neutrophils in atherogenesis are limited. This study aimed to research the diagnostic value of aging neutrophils in their relation to subclinical atherosclerosis in statin-naïve patients without established atherosclerotic cardiovascular diseases (ASCVD). Methods: The study was carried out on 151 statin-naïve patients aged 40–64 years old without ASCVD. All patients underwent duplex scanning of the carotid arteries, lower limb arteries and abdominal aorta. Phenotyping and differentiation of neutrophil subpopulations were performed through flow cytometry (Navios 6/2, Beckman Coulter, USA). Results: The number of CD62LloCXCR4hi-neutrophils is known to be significantly higher in patients with subclinical atherosclerosis compared with patients without atherosclerosis (p = 0.006). An increase in the number of CD62LloCXCR4hi-neutrophils above cut-off values makes it possible to predict atherosclerosis in at least one vascular bed with sensitivity of 35.4–50.5% and specificity of 80.0–92.1%, in two vascular beds with sensitivity of 44.7–84.4% and specificity of 80.8–33.3%. Conclusion: In statin-naïve patients 40–64 years old without established ASCVD with subclinical atherosclerosis, there is an increase in circulating CD62LloCXCR4hi-neutrophils. It was also concluded that the increase in the number of circulating CD62LloCXCR4hi-neutrophils demonstrated moderate diagnostic efficiency (AUC 0.617–0.656) in relation to the detection of subclinical atherosclerosis, including polyvascular atherosclerosis. Full article
(This article belongs to the Special Issue Neutrophil in Cell Biology and Diseases)
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<p>Study design.</p> Full article ">Figure 2
<p>Number of circulating aging neutrophils according to the presence of atherosclerotic plaque in at least one vascular bed (<b>A</b>) or the number of affected vascular beds (<b>B</b>).</p> Full article ">Figure 3
<p>ROC curves demonstrating the diagnostic value of relative and absolute CD62L<sup>lo</sup>CXCR4<sup>hi</sup>-neutrophils counts in relation to the detection of atherosclerosis in one (<b>A</b>,<b>B</b>) and two vascular beds (<b>C</b>,<b>D</b>).</p> Full article ">
21 pages, 6661 KiB  
Article
Proteomic Analysis of HCC-1954 and MCF-7 Cell Lines Highlights Crosstalk between αv and β1 Integrins, E-Cadherin and HER-2
by Denise de Abreu Pereira, Vanessa Sandim, Thais F. B. Fernandes, Vitor Hugo Almeida, Murilo Ramos Rocha, Ronaldo J. F. C. do Amaral, Maria Isabel D. Rossi, Dário Eluan Kalume and Russolina B. Zingali
Int. J. Mol. Sci. 2022, 23(17), 10194; https://doi.org/10.3390/ijms231710194 - 5 Sep 2022
Cited by 6 | Viewed by 3910
Abstract
Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is [...] Read more.
Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, β1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and β1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. β1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and β1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane β1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of β1 integrin modulation and its effect on the mechanism of trastuzumab resistance. Full article
(This article belongs to the Section Molecular Oncology)
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<p>HER-2 protein expression analysis in HCC-1954 and MCF-7 cell lines. (<b>A</b>) Immunofluorescence of HER-2 in HCC-1954 and in MCF-7 cell lines. (<b>B</b>) Western blot of HER-2 in total extract (TE), flow-through (FT) and membrane-enriched (ME) fractions of HCC-1954 and MCF-7 cell lines. Raw data of Western blot membrane are shown in <a href="#app1-ijms-23-10194" class="html-app">Supplementary Figure S9A</a>.</p> Full article ">Figure 2
<p>Proteomic data Analysis (<b>A</b>) Interactive Venn diagram of the 343 proteins identified and quantified with a 95% confidence level (ANOVA <span class="html-italic">p</span> ≤ 0.05) in the Progenesis QI analysis of our label-free proteomic data of HCC-1954 and MCF-7 ME fractions. (<b>B</b>) Volcano plots of all proteins from HCC-1954 and MCF-7 ME fractions. Proteins with increased fold change ratio are indicated by black circles, whereas the grey circles denote those proteins presenting a decreased fold change ratio (Data are available in <a href="#app1-ijms-23-10194" class="html-app">Supplementary Table S2</a>). (<b>C</b>) GO Panther classification of increased proteins with a relative fold change higher than 2 (0.5 &lt; FC &gt; 2) for the ratio HCC-1954 ME/MCF-7 ME in the Protein class and (<b>D</b>) Pathway Classification.</p> Full article ">Figure 3
<p>Proteomaps and top pathway analysis of increased proteins with a relative fold change higher than 2 (0.5 &lt; FC &gt; 2) for the ratio HCC-1954 ME/MCF-7 ME. (<b>A</b>,<b>C</b>) show the biological processes analysed for HCC-1954 and MCF-7, respectively. (<b>B</b>,<b>D</b>) show the proteins classified in each case. (<b>E</b>) Top pathways of ME fraction protein classification from HCC-1954 and MCF-7 performed with the Kegg Mapper platform. Interaction networks of proteins increased (<b>F</b>) in HCC-1954 ME and (<b>G</b>) in MCF-7 ME. HCC-1954 with a PPI enrichment <span class="html-italic">p</span>-value of &lt; 1.0 × 10<sup>−16</sup> and MCF-7 with a PPI enrichment <span class="html-italic">p</span>-value of 4.48 × 10<sup>−5</sup>, respectively.</p> Full article ">Figure 4
<p>Integrin phenotypes and E-cadherin protein expression of HCC-1954 and MCF-7 human breast cancer cell lines. Histograms shows the expression of CD49b, CD51, CD29, and CD51/CD61 (<b>A</b>–<b>D</b>) in HCC-1954 and (<b>E</b>–<b>H</b>) in MCF-7 cell lines. Dotted grey lines are unstained controls. (<b>I</b>) shows the median fluorescence intensity (MFI) of three independent experiments. (<b>J</b>) Immunofluorescence of E-cadherin in HCC-1954 and MCF-7 cell lines. (<b>K</b>) Western blot of E-cadherin in total extract (TE), flow-through (FT) and membrane-enriched (ME) fractions of HCC-1954 and MCF-7 cell lines. (<b>L</b>) Western blot quantification of three independent experiments. <span class="html-italic">p</span>-value &lt; 0.05 (*); &lt;0.01 (**); &lt;0.001 (***). Raw data of Western blot membrane are shown in <a href="#app1-ijms-23-10194" class="html-app">Supplementary Figure S9B</a>.</p> Full article ">Figure 5
<p>β1 Integrin expression in the HCC-1954, BT-474, and MCF-7 cell lines treated (T) or not treated (NT) with trastuzumab. (<b>A</b>) Western blot of β1 integrin showing different expression levels in HCC-1954, BT-474, and MCF-7 cell lines in not-treated (NT) and treated (T) with 20 µg/mL of trastuzumab for 72 h. (<b>B</b>) Western blot quantification of β1 integrin in three independent experiments. Bar chart shows quantification of protein levels compared to the control in each condition. Actin was used as the load control. Error bars show standard deviation, ** <span class="html-italic">p</span>  &lt; 0.01. Raw data of Western blot membrane are shown in <a href="#app1-ijms-23-10194" class="html-app">Supplementary Figure S9C</a>.</p> Full article ">Figure 6
<p>Trastuzumab effect on HCC-1954 and BT-474 migration potential. (<b>A</b>–<b>D</b>) Images of HCC-1954 cells and (<b>E</b>–<b>H</b>) BT-474 cells. (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>) when the scratch/wound was performed, and (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>) 24 h after. (<b>I</b>) Quantification of the gap (%). (NT) not-treated and (T) treated. These results show an enhanced migration of HCC-1954 cells with treatment, as evidenced by a smaller gap after 24 h compared to not-treated cells. On the other hand, treated BT-474 cells presented a larger gap after 24 h compared to not-treated cells. Scale bars correspond to 100 µm. <span class="html-italic">p</span>-value &lt; 0.05 (*); &lt;0.01 (**).</p> Full article ">
19 pages, 3869 KiB  
Article
From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action
by Laura Furia, Simone Pelicci, Mirco Scanarini, Pier Giuseppe Pelicci and Mario Faretta
Int. J. Mol. Sci. 2022, 23(17), 10193; https://doi.org/10.3390/ijms231710193 - 5 Sep 2022
Cited by 4 | Viewed by 2456
Abstract
53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a [...] Read more.
53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e., exposure to ionizing radiation. The localization of 53BP1 protein constitutes a key to decipher the network of activities exerted in response to stress. We present here an automated-microscopy for image cytometry protocol to analyze the evolution of the DDR, and to demonstrate how 53BP1 moved from damaged sites, where the well-known interaction with the DSB marker γH2A.X takes place, to nucleoplasm, interacting with p53, and enhancing the transcriptional regulation of the guardian of the genome protein. Molecular interactions have been quantitatively described and spatiotemporally localized at the highest spatial resolution by a simultaneous analysis of the impairment of the cell-cycle progression. Thanks to the high statistical sampling of the presented protocol, we provide a detailed quantitative description of the molecular events following the DSBs formation. Single-Molecule Localization Microscopy (SMLM) Analysis finally confirmed the p53–53BP1 interaction on the tens of nanometers scale during the distinct phases of the response. Full article
(This article belongs to the Special Issue Radiation Damage in Biomolecules and Cells 2.0)
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<p>Image cytometry analysis of the cell-cycle progression after X-ray irradiation. (<b>A</b>) Pulse and chase analysis applied to control and irradiated cells after a pulse of EdU followed by washing and chasing with fresh medium. At time 0-h, only actively replicating cells incorporate (EdU+) the synthetic analogue during the 20 min of incubation. The evolution of the different populations was then followed by measurement of the DNA content (see Material and Methods). (<b>B</b>) Histograms report the DNA content distribution of the entire cell population (<span class="html-italic">n</span> &gt; 5000). Dot plots report the expression profile of p53, p21, and KI67 in relation to the DNA content at the indicated time after the irradiation. Reported data refer to a representative experiment.</p> Full article ">Figure 2
<p>Image cytometry analysis of the IR-induced foci kinetics after X-ray irradiation. (<b>A</b>) After cell identification, γH2A.X (left) and 53BP1 (right) signals were segmented in each detected nucleus to locate and count foci, and measure their integrated intensity per nucleus (i.e., sum of the intensity of all the foci present in a nucleus) and their average size. (<b>B</b>) Statistical analysis of the foci intensity distribution. The entire population of the indicated foci (<span class="html-italic">n</span> &gt; 10<sup>5</sup>) was analyzed as an independent entity without considering the cell of origin. The relative distribution of the two parameters reveals the kinetics of recruitment of 53BP1 protein into γH2A.X foci. Reported data refer to a representative experiment.</p> Full article ">Figure 3
<p>Image cytometry analysis of the IR-induced 53BP1 content and foci kinetics after X-ray irradiation. Analysis of protein content per nucleus, and of the fraction of the 53BP1 intensity localized in foci versus the total fluorescence of the nucleus in relation to the DNA content at the indicated time-points.</p> Full article ">Figure 4
<p>Cell-cycle analysis of the checkpoint protein content and DDR-related parameters after 5Gy irradiation. Statistical analysis of the indicated parameters calculated by targeting selected subpopulations according to their cell-cycle position at the irradiation (EdU+ and −) and at the selected time-point (DNA content: 2N, midN, 4N). The adopted regions are indicated in <a href="#ijms-23-10193-f001" class="html-fig">Figure 1</a>. At the later time-points, some cell-cycle fractions were not indicated due to their low representativeness (number of events less than 500) caused by the cell-cycle arrest.</p> Full article ">Figure 5
<p>Image cytometry analysis of protein–protein interaction after X-ray irradiation detected a by Proximity Ligation Assay (PLA). (<b>A</b>) The dot-plots report the bivariate distribution of DNA content and of the number of interaction spots detected by a PLA assay between the indicated proteins. (<b>B</b>) PLA-spot population analysis (independent from the cell of origin) in relation to the position of IR foci. In the dot-plots on the right, only PLA-spots–IR-foci residing within a distance of 1 μm were considered. (<b>C</b>) Analysis of p53-, 53BP1-, and γH2A.X-related parameters according to the intensity of the 53BP1–p53 interaction. The cell population was subdivided according to the value of the median of the number of PLA spots per cell distribution.</p> Full article ">Figure 6
<p>Three-dimensional high-resolution confocal microscopy of 53BP1-p53 putative complex. Samples stained for the detection of 53BP1–p53 PLA spots were analyzed according to the described image cytometry procedure to select a PLA-enriched high-p53 expression phenotype. Cells were relocated (an exemplificative cell of interest is reported (white square) in the widefield images in the upper row; scale bar, 25 microns) to perform the high-resolution 3D analysis in confocal imaging. Pictures show conventional and lateral views of a selected slice (left) and 3D maximum intensity projections from different angles (right) at the indicated time-points for a representative cell.</p> Full article ">Figure 7
<p>Analysis of single molecule colocalization between 53BP1 and p53. Analysis of representative dSTORM images of MCF10A nuclei acquired upon labeling of (<b>A</b>,<b>E</b>) 53BP1 (red) and p53 (green). (<b>B</b>) Shown are (from left to right) the dual-color STORM image ROI at different spatial resolutions (10 nm and 50 nm) and the map of the colocalized fraction recovered by local ICCS. (<b>C</b>) ROI spatial correlation functions recovered by ICCS. (<b>D</b>) Colocalized fraction (fICCS) extracted from ICCS analysis at different timepoints. (data are mean ± s.d. of the mean values of fICCS calculated on each cell of NT, 6 h, 24 h, and 48 h; <span class="html-italic">n</span> = 50). The ICCS plot shows the cross-correlation function (black squares) and the red (red circles) and green (green triangles) channel autocorrelation functions along with the corresponding fits (solid lines). (<b>E</b>) A.M.I.CO image analysis of p53 spot distribution at 53BP1 foci (cyan) on representative dual color dSTORM image of MCF10A nucleus. (<b>F</b>) Total distribution of the number of 53BP1 foci containing the number of p53 spots at 6 h and 24 h. Scale bar: 3 μm. Scale bar ROI: 1 μm.</p> Full article ">
18 pages, 4938 KiB  
Article
Advantage of Dimethyl Sulfoxide in the Fabrication of Binder-Free Layered Double Hydroxides Electrodes: Impacts of Physical Parameters on the Crystalline Domain and Electrochemical Performance
by Gayi Nyongombe, Guy L. Kabongo, Luyanda L. Noto and Mokhotjwa S. Dhlamini
Int. J. Mol. Sci. 2022, 23(17), 10192; https://doi.org/10.3390/ijms231710192 - 5 Sep 2022
Cited by 1 | Viewed by 1719
Abstract
The electrode fabrication stage is a crucial step in the design of supercapacitors. The latter involves the binder generally for adhesive purposes. The binder is electrochemically dormant and has weak interactions, leading to isolating the active material and conductive additive and then compromising [...] Read more.
The electrode fabrication stage is a crucial step in the design of supercapacitors. The latter involves the binder generally for adhesive purposes. The binder is electrochemically dormant and has weak interactions, leading to isolating the active material and conductive additive and then compromising the electrochemical performance. Designing binder-free electrodes is a practical way to improve the electrochemical performance of supercapacitors. However, most of the methods developed for the fabrication of binder-free LDH electrodes do not accommodate LDH materials prepared via the co-precipitation or ions exchange routes. Herein, we developed a novel method to fabricate binder-free LDH electrodes which accommodates LDH materials from other synthesis routes. The induced impacts of various physical parameters such as the temperature and time applied during the fabrication process on the crystalline domain and electrochemical performances of all the binder-free LDH electrodes were studied. The electrochemical analysis showed that the electrode prepared at 200 °C-1 h exhibited the best electrochemical performance compared to its counterparts. A specific capacitance of 3050.95 Fg−1 at 10 mVs−1 was achieved by it, while its Rct value was 0.68 Ω. Moreover, it retained 97% of capacitance after 5000 cycles at 120 mVs−1. The XRD and FTIR studies demonstrated that its excellent electrochemical performance was due to its crystalline domain which had held an important amount of water than other electrodes. The as-developed method proved to be reliable and advantageous due to its simplicity and cost-effectiveness. Full article
(This article belongs to the Special Issue Polymeric Hybrid Nanomaterials for Biomedical and Energy Applications)
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<p>XRD patterns of the as-synthesized LDH used as electrode active material.</p> Full article ">Figure 2
<p>(<b>a</b>) Schematic illustration of the fabrication process of binder-free LDH electrodes; (<b>b</b>) the image of the as-fabricated binder-free LDH electrode; (<b>c</b>) XRD patterns of the LDH-100-1h, LDH-100-1h30, LDH-100-2h, LDH-150-1h, and LDH-200-1h electrodes; and (<b>d</b>) FTIR spectra of the LDH-100-1h, LDH-100-1h30, LDH-100-2h, LDH-150-1h, and LDH-200-1h electrodes.</p> Full article ">Figure 3
<p>FESEM images of (<b>a</b>) the as-synthesized LDH used as electrode active material; (<b>b</b>–<b>f</b>) the LDH-100-1h, LDH-100-1h30, LDH-100-2h, LDH-150-1h, and LDH-200-1h electrodes.</p> Full article ">Figure 4
<p>(<b>a</b>–<b>e</b>) FESEM images of the LDH-100-1h, LDH-100-1h30, LDH-100-2h, LDH-150-1h, and LDH-200-1h electrodes at a different magnification.</p> Full article ">Figure 5
<p>(<b>a</b>) Comparative CV curves for the LDH-100-1h, LDH-100-1h30, and LDH-100-2h electrodes at a scan rate of 10 mVs<sup>−1</sup>; (<b>b</b>) comparative CV curves for the LDH-100-2h, LDH-150-1h, and LDH-200-1h electrodes at a scan rate of 10 mVs<sup>−1</sup>.</p> Full article ">Figure 6
<p>CV curves for the (<b>a</b>) LDH-100-1h, (<b>b</b>) LDH-100-1h30, (<b>c</b>) LDH-100-2h, (<b>d</b>) LDH-150-1h, and (<b>e</b>) LDH-200-1h electrodes, respectively at various scan rates.</p> Full article ">Figure 7
<p>Dependence of the anodic peak current (ipa) on the square root of the scan rate for the (<b>a</b>) LDH-100-1h, (<b>b</b>) LDH-100-1h30, (<b>c</b>) LDH-100-2h, (<b>d</b>) LDH-150-1h, and (<b>e</b>) LDH-200-1h electrodes, respectively.</p> Full article ">Figure 8
<p>Linear relationship related to various potential levels of i/V1/2 against V1/2 for the (<b>a</b>) LDH-100-1h, (<b>b</b>) LDH-100-1h30, (<b>c</b>) LDH-100-2h, (<b>d</b>) LDH-150-1h, and (<b>e</b>) LDH-200-1h electrodes, respectively; (<b>f</b>) contribution fractions of the surface capacitance and diffusion-controlled processes for the LDH-100-1h, LDH-100-1h30, LDH-100-2h, LDH-150-1h, and LDH-200-1h electrodes, respectively, at the scan rate of 10 mVs<sup>−1</sup>.</p> Full article ">Figure 9
<p>(<b>a</b>) Nyquist plots for the LDH-100-1h, LDH-100-1h30, and LDH-100-2h electrodes; and (<b>b</b>) Nyquist plots for the LDH-100-2h, LDH-150-1h, and LDH-200-1h electrodes.</p> Full article ">Figure 10
<p>(<b>a</b>) Specific capacitances of the LDH-100-1h, LDH-100-1h30, LDH-100-2h, LDH-150-1h, and LDH-200-1h electrodes at various scan rates; (<b>b</b>) plot of capacitance retention against cycle number (insert: cyclic voltammogram of the LDH-200-1h electrode at 120 mVs<sup>−1</sup>).</p> Full article ">
13 pages, 1015 KiB  
Review
Targeting Persistent Neuroinflammation after Hypoxic-Ischemic Encephalopathy—Is Exendin-4 the Answer?
by Kelly Q. Zhou, Simerdeep K. Dhillon, Laura Bennet, Alistair J. Gunn and Joanne O. Davidson
Int. J. Mol. Sci. 2022, 23(17), 10191; https://doi.org/10.3390/ijms231710191 - 5 Sep 2022
Cited by 11 | Viewed by 3182
Abstract
Hypoxic-ischemic encephalopathy is brain injury resulting from the loss of oxygen and blood supply around the time of birth. It is associated with a high risk of death or disability. The only approved treatment is therapeutic hypothermia. Therapeutic hypothermia has consistently been shown [...] Read more.
Hypoxic-ischemic encephalopathy is brain injury resulting from the loss of oxygen and blood supply around the time of birth. It is associated with a high risk of death or disability. The only approved treatment is therapeutic hypothermia. Therapeutic hypothermia has consistently been shown to significantly reduce the risk of death and disability in infants with hypoxic-ischemic encephalopathy. However, approximately 29% of infants treated with therapeutic hypothermia still develop disability. Recent preclinical and clinical studies have shown that there is still persistent neuroinflammation even after treating with therapeutic hypothermia, which may contribute to the deficits seen in infants despite treatment. This suggests that potentially targeting this persistent neuroinflammation would have an additive benefit in addition to therapeutic hypothermia. A potential additive treatment is Exendin-4, which is a glucagon-like peptide 1 receptor agonist. Preclinical data from various in vitro and in vivo disease models have shown that Exendin-4 has anti-inflammatory, mitochondrial protective, anti-apoptotic, anti-oxidative and neurotrophic effects. Although preclinical studies of the effect of Exendin-4 in perinatal hypoxic-ischemic brain injury are limited, a seminal study in neonatal mice showed that Exendin-4 had promising neuroprotective effects. Further studies on Exendin-4 neuroprotection for perinatal hypoxic-ischemic brain injury, including in large animal translational models are warranted to better understand its safety, window of opportunity and effectiveness as an adjunct with therapeutic hypothermia. Full article
(This article belongs to the Special Issue Novel Anti-inflammatory Molecules)
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<p>The evolution of perinatal hypoxic-ischemic brain injury and the effects of the current treatment (therapeutic hypothermia) and potential treatment (Exendin-4).</p> Full article ">Figure 2
<p>A schematic diagram showing the anti-inflammatory effect of Exendin-4. ↓: decrease.</p> Full article ">
19 pages, 590 KiB  
Article
Thermodynamic Analysis of the Solubility of Isoniazid in (PEG 200 + Water) Cosolvent Mixtures from 278.15 K to 318.15 K
by Daniela Baracaldo-Santamaría, Carlos Alberto Calderon-Ospina, Claudia Patricia Ortiz, Rossember Edén Cardenas-Torres, Fleming Martinez and Daniel Ricardo Delgado
Int. J. Mol. Sci. 2022, 23(17), 10190; https://doi.org/10.3390/ijms231710190 - 5 Sep 2022
Cited by 7 | Viewed by 2390
Abstract
The solubility of drugs in cosolvent systems of pharmaceutical interest is of great importance for understanding and optimizing a large number of processes. Here, we report the solubility of isoniazid in nine (PEG 200 + water) cosolvent mixtures at nine temperatures (278.15, 283.15, [...] Read more.
The solubility of drugs in cosolvent systems of pharmaceutical interest is of great importance for understanding and optimizing a large number of processes. Here, we report the solubility of isoniazid in nine (PEG 200 + water) cosolvent mixtures at nine temperatures (278.15, 283.15, 288.15, 293.15, 298.15, 303.15, 308.15, and 318.15 K) determined by UV–vis spectrophotometry. From the solubility data, the thermodynamic solution, mixing, and transfer functions were calculated in addition to performing the enthalpy–entropy compensation analysis. The solubility of isoniazid depends on the concentration of PEG 200 (positive cosolvent effect) and temperature (endothermic process) reaching its maximum solubility in pure PEG 200 at 318.15 K and the lowest solubility in pure water at 278.15 K. The solution process is favored by the solution entropy and according to the enthalpy–entropy compensation analysis it is driven by entropy in mixtures rich in water and by enthalpy in mixtures rich in PEG 200. Full article
(This article belongs to the Collection Feature Papers in Molecular Pharmacology)
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<p>Molecular structure of isoniazid.</p> Full article ">Figure 2
<p>Molar fraction of isoniazid (10<math display="inline"><semantics> <msup> <mrow/> <mn>3</mn> </msup> </semantics></math><math display="inline"><semantics> <msub> <mi>x</mi> <mn>3</mn> </msub> </semantics></math>) depending on the cosolvent composition (mass fraction) free of solute. □: 278.15 K; ■: 283.15 K; ∘: 288.15 K; •: 293.15 K; ▲: 298.15 K; Δ: 303.15 K; ♦: 308.15 K; ⋄: 313.15 K, and +: 318.15 K.</p> Full article ">Figure 3
<p>Molar fraction of isoniazid (10<math display="inline"><semantics> <msup> <mrow/> <mn>3</mn> </msup> </semantics></math><math display="inline"><semantics> <msub> <mi>x</mi> <mn>3</mn> </msub> </semantics></math>) depending on the solubility parameter of the cosolvent mixture free of solute. □: 278.15 K; ■: 283.15 K; ∘: 288.15 K; •: 293.15 K; ▲: 298.15 K; Δ: 303.15 K; ♦: 308.15 K; ⋄: 313.15 K, and +: 318.15 K.</p> Full article ">Figure 4
<p>Molecular structure of the INH analog.</p> Full article ">Figure 5
<p>DSC Spectra of Isoniazid.</p> Full article ">Figure 6
<p>Experimental solubility of isoniazid in pure water □: Gong et al. [<a href="#B12-ijms-23-10190" class="html-bibr">12</a>]; ■: this work.</p> Full article ">Figure 7
<p>van ’t Hoff plot, for isoniazid (3) in (PEG 200 (1) + water (2)) cosolvent mixtures at some cosolvent mixtures, ♦: <math display="inline"><semantics> <mrow> <msub> <mi>w</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>0.0</mn> </mrow> </semantics></math>; ▲: <math display="inline"><semantics> <mrow> <msub> <mi>w</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>0.2</mn> </mrow> </semantics></math>; ■: <math display="inline"><semantics> <mrow> <msub> <mi>w</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>0.4</mn> </mrow> </semantics></math>; ⋄: <math display="inline"><semantics> <mrow> <msub> <mi>w</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>0.6</mn> </mrow> </semantics></math>; △: <math display="inline"><semantics> <mrow> <msub> <mi>w</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>0.8</mn> </mrow> </semantics></math>; ∘: <math display="inline"><semantics> <mrow> <msub> <mi>w</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>0.9</mn> </mrow> </semantics></math>; □: <math display="inline"><semantics> <mrow> <msub> <mi>w</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>1.0</mn> </mrow> </semantics></math>.</p> Full article ">Figure 8
<p>Relation between enthalpy (<math display="inline"><semantics> <mrow> <msub> <mo>Δ</mo> <mi>soln</mi> </msub> <msup> <mi>H</mi> <mi>o</mi> </msup> </mrow> </semantics></math>) and entropy (<math display="inline"><semantics> <mrow> <msub> <mi>T</mi> <mi>hm</mi> </msub> <msub> <mo>Δ</mo> <mi>soln</mi> </msub> <msup> <mi>S</mi> <mi>o</mi> </msup> </mrow> </semantics></math>) in terms of the process of isoniazid (3) solution in (PEG 200 (1) + water (2)) cosolvent mixtures at 297.6 K. The isoenergetic curves for <math display="inline"><semantics> <mrow> <msub> <mo>Δ</mo> <mi>soln</mi> </msub> <msup> <mi>G</mi> <mi>o</mi> </msup> </mrow> </semantics></math> are represented by dotted lines.</p> Full article ">Figure 9
<p>Relation between enthalpy (<math display="inline"><semantics> <mrow> <msub> <mo>Δ</mo> <mi>tr</mi> </msub> <msup> <mi>H</mi> <mi>o</mi> </msup> </mrow> </semantics></math>) and entropy (<math display="inline"><semantics> <mrow> <msub> <mi>T</mi> <mi>hm</mi> </msub> <msub> <mo>Δ</mo> <mi>tr</mi> </msub> <msup> <mi>S</mi> <mi>o</mi> </msup> </mrow> </semantics></math>) of the process transfer of isoniazid (3) in (PEG 200 (1) + water (2)) cosolvent mixtures at 297.6 K. The isoenergetic curves for <math display="inline"><semantics> <mrow> <msub> <mo>Δ</mo> <mi>mix</mi> </msub> <msup> <mi>G</mi> <mi>o</mi> </msup> </mrow> </semantics></math> are represented by dotted lines.</p> Full article ">Figure 10
<p>Relation between enthalpy (<math display="inline"><semantics> <mrow> <msub> <mo>Δ</mo> <mi>mix</mi> </msub> <msup> <mi>H</mi> <mi>o</mi> </msup> </mrow> </semantics></math>) and entropy (<math display="inline"><semantics> <mrow> <msub> <mi>T</mi> <mi>hm</mi> </msub> <msub> <mo>Δ</mo> <mi>mix</mi> </msub> <msup> <mi>S</mi> <mi>o</mi> </msup> </mrow> </semantics></math>) of the process mixing of isoniazid (3) in (PEG 200 (1) + water (2)) cosolvent mixtures at 297.6 K. The isoenergetic curves for <math display="inline"><semantics> <mrow> <msub> <mo>Δ</mo> <mi>mix</mi> </msub> <msup> <mi>G</mi> <mi>o</mi> </msup> </mrow> </semantics></math> are represented by dotted lines.</p> Full article ">Figure 11
<p>Enthalpy–entropy compensation plot for the solubility of isoniazid (3) in (PEG 200 (1) + water (2)) mixtures at <math display="inline"><semantics> <msub> <mi>T</mi> <mrow> <mi>h</mi> <mi>m</mi> </mrow> </msub> </semantics></math> = 297.6 K.</p> Full article ">Figure 12
<p>Enthalpy–entropy compensation plot for the solubility of isoniazid (3) in (PEG 200 (1) + water (2)) mixtures at <math display="inline"><semantics> <msub> <mi>T</mi> <mrow> <mi>h</mi> <mi>m</mi> </mrow> </msub> </semantics></math> = 297.6 K.</p> Full article ">Figure 13
<p>Gibbs energy of transfer of isoniazid (3) from neat water to (PEG 200 (1) + water (2)) cosolvent mixtures at <span class="html-italic">T</span> = 298.15 K.</p> Full article ">Figure 14
<p><math display="inline"><semantics> <mrow> <mi>δ</mi> <msub> <mi>x</mi> <mrow> <mn>1</mn> <mo>,</mo> <mn>3</mn> </mrow> </msub> </mrow> </semantics></math> values for the isoniazid (3) (PEG 200 (1) + water (2)) cosolvent mixtures at 298.15 K.</p> Full article ">
17 pages, 1045 KiB  
Review
Thalassemia Intermedia: Chelator or Not?
by Yen-Chien Lee, Chi-Tai Yen, Yen-Ling Lee and Rong-Jane Chen
Int. J. Mol. Sci. 2022, 23(17), 10189; https://doi.org/10.3390/ijms231710189 - 5 Sep 2022
Cited by 5 | Viewed by 5489
Abstract
Thalassemia is the most common genetic disorder worldwide. Thalassemia intermedia (TI) is non-transfusion-dependent thalassemia (NTDT), which includes β-TI hemoglobin, E/β-thalassemia and hemoglobin H (HbH) disease. Due to the availability of iron chelation therapy, the life expectancy of thalassemia major (TM) patients is now [...] Read more.
Thalassemia is the most common genetic disorder worldwide. Thalassemia intermedia (TI) is non-transfusion-dependent thalassemia (NTDT), which includes β-TI hemoglobin, E/β-thalassemia and hemoglobin H (HbH) disease. Due to the availability of iron chelation therapy, the life expectancy of thalassemia major (TM) patients is now close to that of TI patients. Iron overload is noted in TI due to the increasing iron absorption from the intestine. Questions are raised regarding the relationship between iron chelation therapy and decreased patient morbidity/mortality, as well as the starting threshold for chelation therapy. Searching all the available articles up to 12 August 2022, iron-chelation-related TI was reviewed. In addition to splenectomized patients, osteoporosis was the most common morbidity among TI cases. Most study designs related to ferritin level and morbidities were cross-sectional and most were from the same Italian study groups. Intervention studies of iron chelation therapy included a subgroup of TI that required regular transfusion. Liver iron concentration (LIC) ≥ 5 mg/g/dw measured by MRI and ferritin level > 300 ng/mL were suggested as indicators to start iron chelation therapy, and iron chelation therapy was suggested to be stopped at a ferritin level ≤ 300 ng/mL. No studies showed improved overall survival rates by iron chelation therapy. TI morbidities and mortalities cannot be explained by iron overload alone. Hypoxemia and hemolysis may play a role. Head-to-head studies comparing different treatment methods, including hydroxyurea, fetal hemoglobin-inducing agents, hypertransfusion as well as iron chelation therapy are needed for TI, hopefully separating β-TI and HbH disease. In addition, the target hemoglobin level should be determined for β-TI and HbH disease. Full article
(This article belongs to the Collection Feature Papers in Molecular Genetics and Genomics)
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<p>The regulation of hepcidin in thalassemia intermedia.</p> Full article ">Figure 2
<p>Traditionally, iron is stored in the reticuloendothelial system (<b>A</b>), but in thalassemia intermedia, iron is stored in hepatocytes (<b>B</b>).</p> Full article ">
18 pages, 4148 KiB  
Article
Leonurine Reduces Oxidative Stress and Provides Neuroprotection against Ischemic Injury via Modulating Oxidative and NO/NOS Pathway
by Ziteng Deng, Jiao Li, Xiaoquan Tang, Dan Li, Yazhou Wang, Shengxi Wu, Kai Fan and Yunfei Ma
Int. J. Mol. Sci. 2022, 23(17), 10188; https://doi.org/10.3390/ijms231710188 - 5 Sep 2022
Cited by 24 | Viewed by 3034
Abstract
Leonurine (Leo) has been found to have neuroprotective effects against cerebral ischemic injury. However, the exact molecular mechanism underlying its neuroprotective ability remains unclear. The aim of the present study was to investigate whether Leo could provide protection through the nitric oxide (NO)/nitric [...] Read more.
Leonurine (Leo) has been found to have neuroprotective effects against cerebral ischemic injury. However, the exact molecular mechanism underlying its neuroprotective ability remains unclear. The aim of the present study was to investigate whether Leo could provide protection through the nitric oxide (NO)/nitric oxide synthase (NOS) pathway. We firstly explored the effects of NO/NOS signaling on oxidative stress and apoptosis in in vivo and in vitro models of cerebral ischemia. Further, we evaluated the protective effects of Leo against oxygen and glucose deprivation (OGD)-induced oxidative stress and apoptosis in PC12 cells. We found that the rats showed anxiety-like behavior, and the morphology and number of neurons were changed in a model of photochemically induced cerebral ischemia. Both in vivo and in vitro results show that the activity of superoxide dismutase (SOD) and glutathione (GSH) contents were decreased after ischemia, and reactive oxygen species (ROS) and malondialdehyde (MDA) levels were increased, indicating that cerebral ischemia induced oxidative stress and neuronal damage. Moreover, the contents of NO, total NOS, constitutive NOS (cNOS) and inducible NOS (iNOS) were increased after ischemia in rat and PC12 cells. Treatment with L-nitroarginine methyl ester (L-NAME), a nonselective NOS inhibitor, could reverse the change in NO/NOS expression and abolish these detrimental effects of ischemia. Leo treatment decreased ROS and MDA levels and increased the activity of SOD and GSH contents in PC12 cells exposed to OGD. Furthermore, Leo reduced NO/NOS production and cell apoptosis, decreased Bax expression and increased Bcl-2 levels in OGD-treated PC12 cells. All the data suggest that Leo protected against oxidative stress and neuronal apoptosis in cerebral ischemia by inhibiting the NO/NOS system. Our findings indicate that Leo could be a potential agent for the intervention of ischemic stroke and highlighted the NO/NOS-mediated oxidative stress signaling. Full article
(This article belongs to the Topic Antioxidants and Oxidative Stress in Brain Health)
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<p>Behaviour of rats after cerebral ischemia and L-NAME treatment according to the OFT. (<b>A</b>) Rat behaviour and physical appearance in each group after ischemic induction. (<b>B</b>) The trajectory of rats in the OFT. (<b>C</b>) The duration of staying in the inner and outer zone in the OFT. Data are presented as mean ± SEM (<span class="html-italic">n</span> = 6 per group). ** indicates that the difference between the Is and the CON is extremely significant (<span class="html-italic">p</span> &lt; 0.01); # indicates that the difference between the L-NAME and the Is is significant (<span class="html-italic">p</span> &lt; 0.05). CON: control group; Sham: sham-operated control group; Is: ischemic brain stroke group; L-NAME: pretreatment <span class="html-small-caps">L</span>-nitroarginine methyl ester; (L-NAME) + ischemic brain stroke group; the same below in vivo.</p> Full article ">Figure 2
<p>Oxidative stress levels in serum and brain tissue of rats at 24 h after ischemic induction. Effect of ischemic induction on the content of SOD (<b>A</b>), MDA (<b>B</b>) and GSH (<b>C</b>) in serum and brain tissue, and the content of ROS in brain tissue (<b>D</b>). Data are presented as mean ± SEM (<span class="html-italic">n</span> = 6 per group). * or ** indicates that the difference between the Is and the CON is significant (<span class="html-italic">p</span> &lt; 0.05) or extremely significant (<span class="html-italic">p</span> &lt; 0.01); # or ## indicates that the difference between the L-NAME and the Is is significant (<span class="html-italic">p</span> &lt; 0.05) or extremely significant (<span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 3
<p>Expression of NO/NOS in serum and brain tissue. (<b>A</b>) The content of NO and NOS/cNOS/iNOS in serum at 24 h after ischemic induction in each group. (<b>B</b>) The content of NO and NOS/cNOS/iNOS in brain tissue at 2 h, 6 h, 12 h and 24 h after ischemic induction. (<b>C</b>) The effects of L-NAME on the highest expression of NO and NOS/cNOS/iNOS (at 24 h after ischemic induction) in brain tissue. Data are presented as mean ± SEM (<span class="html-italic">n</span> = 6 per group). * or ** indicates that the difference between the Is and the CON is significant (<span class="html-italic">p</span> &lt; 0.05) or extremely significant (<span class="html-italic">p</span> &lt; 0.01); # or ## indicates that the difference between the L-NAME and the Is is significant (<span class="html-italic">p</span> &lt; 0.05) or extremely significant (<span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 4
<p>Effect of cerebral ischemia and L-NAME on cortical neurons of rats (<b>A</b>) Cortical morphology as depicted by Nissl staining, 20× on the left and 40× on the right. (<b>B</b>) Distribution of NeuN-positive neurons in the cerebral cortices by immunohistochemistry staining. (<b>C</b>) Cell density of NeuN-positive neurons in the cerebral cortices. Data are presented as mean ± SEM (<span class="html-italic">n</span> = 6 per group). ** indicates that the difference between the Is and the CON is extremely significant (<span class="html-italic">p</span> &lt; 0.01); ## indicates that the difference between the L-NAME and the Is is extremely significant (<span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 5
<p>Protein expression of Bax and Bcl-2 at 24 h after cerebral ischemia. (<b>A</b>) Protein expression of Bax and Bcl-2 in each group by Western blot analysis. (<b>B</b>) The ratio of Bax/Bcl-2 in each group. Image J analysis of Bax (<b>C</b>) and Bcl-2 (<b>D</b>) levels. The experiments were performed in triplicate. Data are presented as mean ± SEM (<span class="html-italic">n</span> = 6 per group). ** indicates that the difference between the Is and the CON is extremely significant (<span class="html-italic">p</span> &lt; 0.01); # or ## indicates that the difference between the L-NAME and the Is is significant (<span class="html-italic">p</span> &lt; 0.05) or extremely significant (<span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 6
<p>Effects of leonurine on oxidative stress factors in OGD-induced PC12 cells. The fluorescence value of ROS (<b>A</b>,<b>B</b>) and the contents of SOD (<b>C</b>), MDA (<b>D</b>) and GSH (<b>E</b>) in each group after OGD for 2 h. Data are presented as mean ± SEM. ** indicates that the difference between the OGD and the CON is extremely significant (<span class="html-italic">p</span> &lt; 0.01); # or ## indicates significant (<span class="html-italic">p</span> &lt; 0.05) or extremely significant (<span class="html-italic">p</span> &lt; 0.01) difference compared with the OGD. CON: control group; OGD: OGD model group; L-NAME: the L-NAME group (1 mmol/L); LEO-LOW: low dose of leonurine (50 μg/mL); LEO-MID: middle dose of leonurine (100 μg/mL); and LEO-HIGH: high dose of leonurine (200 μg/mL). The same below in vitro.</p> Full article ">Figure 7
<p>Effects of leonurine on NO and NOS expression levels in OGD-induced PC12 cells. The expressions of NO and NOS/cNOS/iNOS at different times after OGD (<b>A</b>). Effects of L-NAME and different doses of leonurine on the highest expression of NO at 24 h (<b>B</b>) and NOS at 12 h (<b>C</b>)/cNOS at 12 h (<b>D</b>)/iNOS at 6 h (<b>E</b>). Data are presented as mean ± SEM. * or ** indicates that the difference between the OGD and the CON is significant (<span class="html-italic">p</span> &lt; 0.05) or extremely significant (<span class="html-italic">p</span> &lt; 0.01); # or ## indicates that significant (<span class="html-italic">p</span> &lt; 0.05) or extremely significant (<span class="html-italic">p</span> &lt; 0.01) difference compared with the OGD.</p> Full article ">Figure 8
<p>Effects of leonurine on the apoptosis of OGD-induced PC12 cells. (<b>A</b>) The apoptosis of OGD-induced PC12 cells by AO/EB staining. (<b>B</b>) OGD-induced PC12 cells at different stages of apoptosis of each group by Annexin V/PI and the percentage of apoptotic cells in each group by flow cytometry. (<b>C</b>) Data are presented as mean ± SEM. ** indicates that the difference between the OGD and the CON is extremely significant (<span class="html-italic">p</span> &lt; 0.01); ## indicates that extremely significant (<span class="html-italic">p</span> &lt; 0.01) difference compared with the OGD.</p> Full article ">Figure 9
<p>Effects of leonurine on levels of Bax and Bcl-2 after OGD. (<b>A</b>) Protein expression of Bax and Bcl-2 in each group at 24 h after OGD by Western blot analysis. (<b>B</b>) The ratio of Bax/Bcl-2 in each group. Image J analysis of Bax (<b>C</b>) and Bcl-2 (<b>D</b>) levels. The experiments were performed in triplicate. Data are presented as mean ± SEM. ** indicates that the difference between the OGD and the CON is extremely significant (<span class="html-italic">p</span> &lt; 0.01); # or ## indicates that significant (<span class="html-italic">p</span> &lt; 0.05) or extremely significant (<span class="html-italic">p</span> &lt; 0.01) difference compared with the OGD.</p> Full article ">
12 pages, 4155 KiB  
Article
Quantitative Chemical Exchange Saturation Transfer Imaging of Amide Proton Transfer Differentiates between Cerebellopontine Angle Schwannoma and Meningioma: Preliminary Results
by Hirofumi Koike, Minoru Morikawa, Hideki Ishimaru, Reiko Ideguchi, Masataka Uetani, Takeshi Hiu, Takayuki Matsuo and Mitsuharu Miyoshi
Int. J. Mol. Sci. 2022, 23(17), 10187; https://doi.org/10.3390/ijms231710187 - 5 Sep 2022
Cited by 7 | Viewed by 2532
Abstract
Vestibular schwannomas are the most common tumor at the common cerebellopontine angle, followed by meningiomas. Differentiation of these tumors is critical because of the different surgical approaches required for treatment. Recent studies have demonstrated the utility of amide proton transfer (APT)-chemical exchange saturation [...] Read more.
Vestibular schwannomas are the most common tumor at the common cerebellopontine angle, followed by meningiomas. Differentiation of these tumors is critical because of the different surgical approaches required for treatment. Recent studies have demonstrated the utility of amide proton transfer (APT)-chemical exchange saturation transfer (CEST) imaging in evaluating malignant brain tumors. However, APT imaging has not been applied in benign tumors. Here, we explored the potential of APT in differentiating between schwannomas and meningiomas at the cerebellopontine angle. We retrospectively evaluated nine patients with schwannoma and nine patients with meningioma who underwent APT-CEST MRI from November 2020 to April 2022 pre-operation. All 18 tumors were histologically diagnosed. There was a significant difference in magnetization transfer ratio asymmetry (MTRasym) values (0.033 ± 0.012 vs. 0.021 ± 0.004; p = 0.007) between the schwannoma and meningioma groups. Receiver operative curve analysis showed that MTRasym values clearly differentiated between the schwannoma and meningioma groups. At an MTRasym value threshold of 0.024, the diagnostic sensitivity, specificity, positive predictive value, and negative predictive values for MTRasym were 88.9%, 77.8%, 80.0%, and 87.5%, respectively. Our results demonstrated the ability of MTRasym values on APT-CEST imaging to discriminate patients with schwannomas from patients with meningiomas. Full article
(This article belongs to the Section Molecular Oncology)
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<p>Imaging from a 43-year-old man with a schwannoma with T2 hyperintensity. (<b>a</b>) MRI, T2 sequence, axial section, showing heterogeneous high intensity in a schwannoma in the right cerebellopontine angle (CPA) (white arrow). (<b>b</b>) MRI, 3D contrast-enhanced T1 sequence, axial section, showing heterogeneous enhancement in a schwannoma in the right CPA (white arrow). (<b>c</b>) MRI, APT-CEST scout image, axial section, showing the ROI (blue circle) in a schwannoma in the right CPA. (<b>d</b>) MRI, APT-CEST sequence, axial section, showing high SI in a schwannoma in the right CPA (white arrow).</p> Full article ">Figure 2
<p>Imaging from a 66-year-old woman with a schwannoma with T2 low intensity. (<b>a</b>) MRI, T2 sequence, axial section, showing homogeneous low intensity in a schwannoma in the left CPA (white arrow). (<b>b</b>) MRI, 3D contrast-enhanced T1 sequence, axial section, showing homogeneous enhancement in a schwannoma in the left CPA (white arrow). (<b>c</b>) MRI, APT-CEST scout image, axial section, showing the ROI (blue circle) in a schwannoma in the left CPA. (<b>d</b>) MRI, APT-CEST sequence, axial section, showing low SI in a schwannoma in the left CPA (white arrow).</p> Full article ">Figure 3
<p>Imaging from a 78-year-old woman with a meningioma. (<b>a</b>) MRI, T2 sequence, axial section, showing homogeneous low intensity in a meningioma in the left CPA (white arrow). (<b>b</b>) MRI, 3D contrast-enhanced T1 sequence, axial section, showing homogeneous enhancement in a meningioma in the left CPA (white arrow). (<b>c</b>) MRI, APT-CEST scout image, axial section, showing the ROI (blue circle) in a meningioma in the left CPA. (<b>d</b>) MRI, APT-CEST sequence, axial section, showing low SI in a meningioma in the left CPA (white arrow).</p> Full article ">Figure 4
<p>ROC analysis. ROC analyses demonstrated moderate discriminatory power for mean MTR<sub>asym</sub> to differentiate between patients in the schwannoma group and meningioma group. When an AUC value of &lt;0.024 was used as the threshold for diagnosis, the sensitivity, specificity, positive predictive value, and negative predictive value were 88.9%, 77.8%, 80.0%, and 87.5%, respectively.</p> Full article ">
12 pages, 945 KiB  
Review
Antenatal Glucocorticoid Administration Promotes Cardiac Structure and Energy Metabolism Maturation in Preterm Fetuses
by Kenzo Sakurai, Yuko Takeba, Yosuke Osada, Masanori Mizuno, Yoshimitsu Tsuzuki, Kentaro Aso, Keisuke Kida, Yuki Ohta, Masanori Ootaki, Taroh Iiri, Isamu Hokuto, Naoki Shimizu and Naoki Matsumoto
Int. J. Mol. Sci. 2022, 23(17), 10186; https://doi.org/10.3390/ijms231710186 - 5 Sep 2022
Cited by 2 | Viewed by 3024
Abstract
Although the rate of preterm birth has increased in recent decades, a number of preterm infants have escaped death due to improvements in perinatal and neonatal care. Antenatal glucocorticoid (GC) therapy has significantly contributed to progression in lung maturation; however, its potential effects [...] Read more.
Although the rate of preterm birth has increased in recent decades, a number of preterm infants have escaped death due to improvements in perinatal and neonatal care. Antenatal glucocorticoid (GC) therapy has significantly contributed to progression in lung maturation; however, its potential effects on other organs remain controversial. Furthermore, the effects of antenatal GC therapy on the fetal heart show both pros and cons. Translational research in animal models indicates that constant fetal exposure to antenatal GC administration is sufficient for lung maturation. We have established a premature fetal rat model to investigate immature cardiopulmonary functions in the lungs and heart, including the effects of antenatal GC administration. In this review, we explain the mechanisms of antenatal GC actions on the heart in the fetus compared to those in the neonate. Antenatal GCs may contribute to premature heart maturation by accelerating cardiomyocyte proliferation, angiogenesis, energy production, and sarcoplasmic reticulum function. Additionally, this review specifically focuses on fetal heart growth with antenatal GC administration in experimental animal models. Moreover, knowledge regarding antenatal GC administration in experimental animal models can be coupled with that from developmental biology, with the potential for the generation of functional cells and tissues that could be used for regenerative medical purposes in the future. Full article
(This article belongs to the Special Issue Post-transcriptional Gene Expression Regulation in Eukaryotic Energy Metabolism)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Schematic diagram of cell proliferation and angiogenesis. Pregnant rats are administered glucocorticoids (GCs) subcutaneously for two days, on gestational days 17 and 19, and fetuses are delivered by cesarean section. On histological analysis, the cross-sectional area of the myocardium is smaller in 19-day-old fetuses than in neonates. Physiological hypertrophy is induced by cardiac cell proliferation and angiogenesis. Antenatal GC administration enhances phosphatidylinositol-3 kinase (PI3K), which activates Akt-1. Moreover, Akt-1 accelerates both phosphorylated glycogen synthase kinase-3β (p-GSK-3β) and vascular endothelial growth factor (VEGF) expression. Subsequently, p-GSK-3β contributes to the activation of cardiomyocyte proliferation factors, β-catenin, Yes-associated protein (Yap), and GATA-4. Accordingly, cell proliferation markers, Ki-67 and cyclin D1, are increased in the nuclei of fetal cardiomyocytes. GATA binding protein, GATA.</p> Full article ">Figure 2
<p>Schematic diagram of energy production and acceleration of cardiac contraction. Energy metabolism in the fetal heart is relatively dependent on anaerobic glycolysis. α-Enolase converts glucose into pyruvate. Energy released during glycolysis is used to make ATP, and antenatal glucocorticoid (GC) administration accelerates these pathways. Furthermore, antenatal GC administration induces creatine kinase (CK) protein production by promoting myofibrillar-bound M isoenzyme CK (CK-M) and mitochondrial CK (Mi-CK) gene expression in the mitochondria. Increased CK-M and Mi-CK enzymes contribute to increased ATP synthesis. Cardiac energy metabolism is essential for normal cardiac contractile function. GATA-4 has not only involvement in cell proliferation but also in binding the cells to the troponin T promoter. Antenatal GC increases the expression of GATA-4, troponin T, SERCA2a, and phospholamban. Cardiac contraction is increased with the increase in intracellular Ca<sup>2+</sup> concentration and troponin T expression. SERCA2a plays a crucial role in the regulation of the intracellular Ca<sup>2+</sup> concentration in the SR. Ca<sup>2+</sup> enters myocytes during an action potential through voltage-gated Ca<sup>2+</sup> channels in the sarcolemma. This Ca<sup>2+</sup> influx triggers the release of Ca<sup>2+</sup> from the SR by RyR2, which increases the intracellular Ca<sup>2+</sup> concentration, resulting in myocardial contraction. Sarco-endoplasmic reticulum Ca-ATPase, SERCA; sarcoendoplasmic reticulum, SR; ryanodine receptor, RyR.</p> Full article ">
15 pages, 3971 KiB  
Article
The MKK2a Gene Involved in the MAPK Signaling Cascades Enhances Populus Salt Tolerance
by Jiali Wang, Zimou Sun, Caihui Chen and Meng Xu
Int. J. Mol. Sci. 2022, 23(17), 10185; https://doi.org/10.3390/ijms231710185 - 5 Sep 2022
Cited by 11 | Viewed by 2198
Abstract
Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction modules, which transmit environmental signals in plant cells through stepwise phosphorylation and play indispensable roles in a wide range of physiological and biochemical processes. Here, we isolated and characterized a gene encoding MKK2 [...] Read more.
Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction modules, which transmit environmental signals in plant cells through stepwise phosphorylation and play indispensable roles in a wide range of physiological and biochemical processes. Here, we isolated and characterized a gene encoding MKK2 protein from poplar through the rapid amplification of cDNA ends (RACE). The full-length PeMKK2a gene was 1571 bp, including a 1068 bp open reading frame (ORF) encoding 355 amino acids, and the putative PeMKK2a protein belongs to the PKc_like (protein kinase domain) family (70–336 amino acids) in the PKc_MAPKK_plant subfamily and contains 62 sites of possible phosphorylation and two conserved domains, DLK and S/T-xxxxx-S/T. Detailed information about its gene structure, sequence similarities, subcellular localization, and transcript profiles under salt-stress conditions was revealed. Transgenic poplar lines overexpressing PeMKK2a exhibited higher activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) than non-transgenic poplar under salt stress conditions. These results will provide insight into the roles of MAPK signaling cascades in poplar response to salt stress. Full article
(This article belongs to the Special Issue Advanced Research in Plant Responses to Environmental Stresses 2.0)
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<p>(<b>a</b>) Exon and intron composition of <span class="html-italic">PeMKK2a</span> and <span class="html-italic">PtMKKs</span>. (The * represents the <span class="html-italic">MKK2</span> gene cloned from “Nanlin 895”). (<b>b</b>) Multiple alignment of amino acid sequences of MKK2 proteins in different plant species. The black background indicates conserved regions, and the red background indicates specific conserved structural domains. (Here, only part of the sequence is shown.) (<b>c</b>) Nucleotide and deduced amino acid sequences of <span class="html-italic">PeMKK2a</span>.</p> Full article ">Figure 2
<p>(<b>a</b>) Phylogenetic relationships of <span class="html-italic">A. thaliana</span> and <span class="html-italic">P. trichocarpa</span> MKK proteins. (<b>b</b>) Hierarchical clustering of the expression profiles of 10 <span class="html-italic">MAPKK</span> gene family members for different development periods and tissues in <span class="html-italic">P. trichocarpa</span> (CK, stem segment; WR1, one−week−old roots; WR2, two−week−old roots; RT, root tips; ST, stem tips).</p> Full article ">Figure 3
<p>Expression pattern of <span class="html-italic">PeMKK2a</span> under salt stress. “Nanlin895” were exposed to salt stress at 100 mM and 300 mM for 0 h, 2 h, 6 h, 12 h, 24 h, and 72 h. Samples were taken for quantitative analysis. (<b>a</b>) Relative expression level in root. (<b>b</b>) Relative expression level in stem. (<b>c</b>) Relative expression level in leaf.</p> Full article ">Figure 4
<p>Subcellular localization of the PeMKK2a protein in poplar protoplast. Chlorophyll autofluorescence (auto), green fluorescence protein (GFP), merged and bright images are shown. (Scale bar 10 μm. The 35::GFP fusion was used as a positive protein control).</p> Full article ">Figure 5
<p>(<b>a</b>) Genomic DNA-level detection. (<b>b</b>) The expression level of <span class="html-italic">PeMKK2a</span> in eight transgenic lines and the CK, according to qRT-PCR (CK, non-transgenic poplar; T2, T4, T21, T27, T30, T37, T46, and T52, transgenic lines). (<b>c</b>) Phenotypic observations of transgenic plants under salt stress. (Unstressed poplar plants constituted the positive control. All the treatments included three biological replicates).</p> Full article ">Figure 6
<p>SOD activity of transgenic and non-transgenic poplar plants subjected to different NaCl concentrations. Note: (<b>a</b>) without NaCl; (<b>b</b>) 100 mM NaCl; (<b>c</b>) 200 mM NaCl; and (<b>d</b>) 300 mM NaCl (The “*” above the histogram indicates significance. “*” indicates <span class="html-italic">p</span> &lt; 0.05, “**” indicates <span class="html-italic">p</span> &lt; 0.01, and “***” indicates <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 7
<p>CAT activity of transgenic and non-transgenic poplar plants subjected to different NaCl concentrations. Note: (<b>a</b>) without NaCl; (<b>b</b>) 100 mM NaCl; (<b>c</b>) 200 mM NaCl; and (<b>d</b>) 300 mM NaCl (The “*” above the histogram indicates significance. “*” indicates <span class="html-italic">p</span> &lt; 0.05, “**” indicates <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 8
<p>POD activity of transgenic and non-transgenic poplar plants subjected to different NaCl concentrations. Note: (<b>a</b>) without NaCl; (<b>b</b>) 100 mM NaCl; (<b>c</b>) 200 mM NaCl; and (<b>d</b>) 300 mM NaCl (The “*” above the histogram indicates significance. “*” indicates <span class="html-italic">p</span> &lt; 0.05, “**” indicates <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">
15 pages, 2323 KiB  
Article
Systemic Beta-Hydroxybutyrate Affects BDNF and Autophagy into the Retina of Diabetic Mice
by Maria Consiglia Trotta, Carlo Gesualdo, Hildegard Herman, Sami Gharbia, Cornel Balta, Caterina Claudia Lepre, Marina Russo, Annalisa Itro, Giovanbattista D’Amico, Luisa Peluso, Iacopo Panarese, Gorizio Pieretti, Giuseppe Ferraro, Francesca Simonelli, Michele D’Amico, Settimio Rossi and Anca Hermenean
Int. J. Mol. Sci. 2022, 23(17), 10184; https://doi.org/10.3390/ijms231710184 - 5 Sep 2022
Cited by 13 | Viewed by 3316
Abstract
Background: Diabetic retinopathy (DR) is a neurovascular disease, characterized by a deficiency of brain-derived neurotrophic factor (BDNF), a regulator of autophagy. Beta-hydroxybutyrate (BHB), previously reported as a protective agent in DR, has been associated with BDNF promotion. Here, we investigated whether systemic BHB [...] Read more.
Background: Diabetic retinopathy (DR) is a neurovascular disease, characterized by a deficiency of brain-derived neurotrophic factor (BDNF), a regulator of autophagy. Beta-hydroxybutyrate (BHB), previously reported as a protective agent in DR, has been associated with BDNF promotion. Here, we investigated whether systemic BHB affects the retinal levels of BDNF and local autophagy in diabetic mice with retinopathy; Methods: C57BL/6J mice were administered with intraperitoneal (i.p.) streptozotocin (STZ) (75 mg/kg) injection to develop diabetes. After 2 weeks, they received i.p. injections of BHB (25–50–100 mg/kg) twice a week for 10 weeks. Retinal samples were collected in order to perform immunofluorescence, Western blotting, and ELISA analysis; Results: BHB 50 mg/kg and 100 mg/kg significantly improved retinal BDNF levels (p < 0.01) in diabetic mice. This improvement was negatively associated with autophagosome–lysosome formations (marked by LC3B and ATG14) and to higher levels of connexin 43 (p < 0.01), a marker of cell integrity. Moreover, BHB administration significantly reduced M1 microglial activation and autophagy (p < 0.01); Conclusions: The systemic administration of BHB in mice with DR improves the retinal levels of BDNF, with the consequent reduction of the abnormal microglial autophagy. This leads to retinal cell safety through connexin 43 restoration. Full article
(This article belongs to the Special Issue Novel Insights in Retinal Diseases Pathophysiology and Therapies)
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<p>Retinal levels of BDNF (<b>A</b>), PI3K (<b>B</b>), LC3B (<b>C</b>), and strength of association between BDNF and LC3B (<b>D</b>) in non-diabetic mice (CTRL), diabetic mice (STZ), and diabetic mice treated with BHB 25 mg/kg (BHB 25), 50 mg/kg (BHB 50), and 100 mg/kg (BHB 100). BDNF (pg/mL) and LC3B (densitometric units, DU) levels are expressed as mean ± SD of N = 8 retinas per group. ** <span class="html-italic">p</span> &lt; 0.01 vs. CTRL; °° <span class="html-italic">p</span> &lt; 0.01 vs. STZ.</p> Full article ">Figure 2
<p>Retinal ATG14 levels (<b>A</b>) and their strength of association with BDNF (<b>B</b>) in non-diabetic mice (CTRL), diabetic mice (STZ), and diabetic mice treated with BHB 25 mg/kg (BHB 25), 50 mg/kg (BHB 50), and 100 mg/kg (BHB 100). ATG14 levels (ng/mL) are expressed as mean ± SD of N = 8 retinas per group. ** <span class="html-italic">p</span> &lt; 0.01 vs. CTRL; °° <span class="html-italic">p</span> &lt; 0.01 vs. STZ.</p> Full article ">Figure 3
<p>Retinal Cnx43 levels (<b>A</b>) and their strength of association with LC3B and ATG14 (<b>B</b>) in non-diabetic mice (CTRL), diabetic mice (STZ), and diabetic mice treated with BHB 25 mg/kg (BHB 25), 50 mg/kg (BHB 50), and 100 mg/kg (BHB 100). Cnx43 levels (ng/mL) are expressed as mean ± SD of N = 8 retinas per group. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 vs. CTRL; °° <span class="html-italic">p</span> &lt; 0.01 vs. STZ.</p> Full article ">Figure 4
<p>Retinal Iba1 levels (<b>A</b>) and their strength of association with BDNF (<b>B</b>) in non-diabetic mice (CTRL), diabetic mice (STZ), and diabetic mice treated with BHB 25 mg/kg (BHB 25), 50 mg/kg (BHB 50), and 100 mg/kg (BHB 100). Iba1 levels (densitometric units, DU) are expressed as mean ± SD of N = 8 retinas per group. ** <span class="html-italic">p</span> &lt; 0.01 vs. CTRL; °° <span class="html-italic">p</span> &lt; 0.01 vs. STZ.</p> Full article ">Figure 5
<p>Detection of LC3b immunolabeling in Iba1-positive microglia (<b>A</b>) and relative graph (<b>B</b>) in non-diabetic mice (CTRL), diabetic mice (STZ), and diabetic mice treated with BHB 25 mg/kg (BHB 25), 50 mg/kg (BHB 50), and 100 mg/kg (BHB 100). (<b>A</b>) Sagittal retina sections of visible retinal pigment epithelium (RPE) and outer nuclear layer (ONL), showing the higher co-localization (arrow) of Iba1 (red) and LC3B (green) in the ONL of STZ and BHB 25 groups. Magnification ×63. (<b>B</b>) Graph expressing the percentage (%) of LC3B-positive Iba1 cells on total Iba1 cells counted. The results are reported as mean ± SD of N = 8 retinas per group. ** <span class="html-italic">p</span> &lt; 0.01 vs. CTRL; °° <span class="html-italic">p</span> &lt; 0.01 vs. STZ.</p> Full article ">Figure 5 Cont.
<p>Detection of LC3b immunolabeling in Iba1-positive microglia (<b>A</b>) and relative graph (<b>B</b>) in non-diabetic mice (CTRL), diabetic mice (STZ), and diabetic mice treated with BHB 25 mg/kg (BHB 25), 50 mg/kg (BHB 50), and 100 mg/kg (BHB 100). (<b>A</b>) Sagittal retina sections of visible retinal pigment epithelium (RPE) and outer nuclear layer (ONL), showing the higher co-localization (arrow) of Iba1 (red) and LC3B (green) in the ONL of STZ and BHB 25 groups. Magnification ×63. (<b>B</b>) Graph expressing the percentage (%) of LC3B-positive Iba1 cells on total Iba1 cells counted. The results are reported as mean ± SD of N = 8 retinas per group. ** <span class="html-italic">p</span> &lt; 0.01 vs. CTRL; °° <span class="html-italic">p</span> &lt; 0.01 vs. STZ.</p> Full article ">Figure 6
<p>Retinal levels of BDNF (<b>A</b>) and Cnx43 (<b>B</b>) in non-diabetic mice (CTRL), diabetic mice treated with PTX (1 µg/100 µL) (PTX) and diabetic mice treated with both PTX and BHB 25 mg/kg (PTX-BHB 25), 50 mg/kg (PTX-BHB 50), and 100 mg/kg (PTX-BHB 100). BDNF (pg/mL) and Cnx43 (ng/mL) levels are expressed as mean ± SD of N = 8 retinas per group. ** <span class="html-italic">p</span> &lt; 0.01 vs. CTRL.</p> Full article ">
19 pages, 6621 KiB  
Article
Genome-Wide Characterization of PIN Auxin Efflux Carrier Gene Family in Mikania micrantha
by Lihua Chen, Minling Cai, Minghao Chen, Weiqian Ke, Yanru Pan, Jundong Huang, Junjie Zhang and Changlian Peng
Int. J. Mol. Sci. 2022, 23(17), 10183; https://doi.org/10.3390/ijms231710183 - 5 Sep 2022
Cited by 6 | Viewed by 2353
Abstract
Mikania micrantha, recognized as one of the world’s top 10 pernicious weeds, is a rapidly spreading tropical vine that has invaded the coastal areas of South China, causing serious economic losses and environmental damage. Rapid stem growth is an important feature of [...] Read more.
Mikania micrantha, recognized as one of the world’s top 10 pernicious weeds, is a rapidly spreading tropical vine that has invaded the coastal areas of South China, causing serious economic losses and environmental damage. Rapid stem growth is an important feature of M. micrantha which may be related to its greater number of genes involved in auxin signaling and transport pathways and its ability to synthesize more auxin under adverse conditions to promote or maintain stem growth. Plant growth and development is closely connected to the regulation of endogenous hormones, especially the polar transport and asymmetric distribution of auxin. The PIN-FORMED (PIN) auxin efflux carrier gene family plays a key role in the polar transport of auxin and then regulates the growth of different plant tissues, which could indicate that the rapid growth of M. micrantha is closely related to this PIN-dependent auxin regulation. In this study, 11 PIN genes were identified and the phylogenetic relationship and structural compositions of the gene family in M. micrantha were analyzed by employing multiple bioinformatic methods. The phylogenetic analysis indicated that the PIN proteins could be divided into five distinct clades. The structural analysis revealed that three putative types of PIN (canonical, noncanonical and semi-canonical) exist among the proteins according to the length and the composition of the hydrophilic domain. The majority of the PINs were involved in the process of axillary bud differentiation and stem response under abiotic stress, indicating that M. micrantha may regulate its growth, development and stress response by regulating PIN expression in the axillary bud and stem, which may help explain its strong growth ability and environmental adaptability. Our study emphasized the structural features and stress response patterns of the PIN gene family and provided useful insights for further study into the molecular mechanism of auxin-regulated growth and control in M. micrantha. Full article
(This article belongs to the Special Issue Environmental Stress and Plants 2.0)
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<p>Phylogenetic analysis of PIN auxin transporter in <span class="html-italic">M. micrantha</span>, <span class="html-italic">A. thaliana</span>, <span class="html-italic">B. oleracea</span> and <span class="html-italic">V. vinifera</span>. The 43 PIN proteins from four plant species could be classified into six distinct clades (group I–VI) and differentiated in color. <span class="html-italic">M. micrantha</span>, <span class="html-italic">A. thaliana</span>, <span class="html-italic">B. oleracea</span> and <span class="html-italic">V. vinifera</span> are distinguished by solid circle shapes in red, yellow, blue and green, respectively.</p> Full article ">Figure 2
<p>Chromosomal localization of PIN genes in <span class="html-italic">M. micrantha</span>. (<b>a</b>) The genome visualization tool MapChart was employed to analyze the <span class="html-italic">M. micrantha</span> genome. <span class="html-italic">M. micrantha</span> chromosomes were arranged in blocks. The 11 PIN genes were mapped by locus. (<b>b</b>) Three pairs of segmental duplicated genes E3N88_14925/E3N88_15091, E3N88_45195/E3N88_40533, E3N88_03858/E3N88_03888 are shown with dotted arrows.</p> Full article ">Figure 3
<p>Exon–intron organization of PIN genes in <span class="html-italic">M. micrantha</span>, <span class="html-italic">A. thaliana</span>, <span class="html-italic">B. oleracea</span> and <span class="html-italic">V. vinifera</span>. Green boxes represent exons, untranslated regions (UTRs) are indicated by yellow boxes, and black lines represent introns. The lengths of the boxes and lines are scaled based on gene length. The exon and intron sizes can be estimated using the scale at the bottom. <span class="html-italic">M. micrantha</span>, <span class="html-italic">A. thaliana</span>, <span class="html-italic">B. oleracea</span> and <span class="html-italic">V. vinifera</span> are distinguished by solid circle shapes in red, yellow, blue and green, respectively.</p> Full article ">Figure 4
<p>Transmembrane analysis of PIN proteins in <span class="html-italic">M. micrantha</span>. The red peaks represent the predicted transmembrane domain and blue color line indicate the central hydrophobic loop of proteins.</p> Full article ">Figure 5
<p>Conserved motifs analysis of PINs in <span class="html-italic">M. micrantha</span>, <span class="html-italic">A. thaliana</span>, <span class="html-italic">B. oleracea</span> and <span class="html-italic">V. vinifera</span>. Ten predicted motifs are shown by different colored boxes, and motif sizes are demonstrated by the scale on the right. <span class="html-italic">M. micrantha</span>, <span class="html-italic">A. thaliana</span>, <span class="html-italic">B. oleracea</span> and <span class="html-italic">V. vinifera</span> are distinguished by solid circle shapes in red, yellow, blue and green, respectively.</p> Full article ">Figure 6
<p>Multiple sequence alignment of PIN proteins in <span class="html-italic">M. micrantha</span>. Identical (100%), conservative (≥75%) and block (≥50%) of similar amino acid residues are shaded in deep blue, pink and light green, respectively. The black line indicates hydrophobic domain, the red line indicates hydrophilic domain. The motifs that constitute the highly conserved canonical regions HC1-HC4 within the common central hydrophilic region are shown as blue, orange, yellow and green rectangles, respectively.</p> Full article ">Figure 7
<p>Analysis of cis-acting regulatory elements (CAREs) of the PIN gene family. (<b>a</b>) Putative CAREs of the PIN gene family in <span class="html-italic">M. micrantha</span>. Twelve cis-acting regulatory elements commonly found in PIN genes are shown with different colored squares, respectively. (<b>b</b>) Number of response-related CAREs of the PIN gene family in three vines.</p> Full article ">Figure 8
<p>The PIN genes expression pattern in different <span class="html-italic">M. micrantha</span> tissues (root, stem, leaf and flower). (<b>a</b>) Expression heatmaps of PIN genes in different tissues. The FPKM values were transformed to log2 (value + 1). The color scale is shown at the right, and higher expression levels are shown in red. (<b>b</b>) Relative expressions of PIN genes in different tissues. The histogram represents the relative expression level of PIN genes in different tissues compared with the expression level in stem, which was normalized to a value of 1. The error bars represent the standard errors (SEs) of three to seven biological replicates.</p> Full article ">Figure 9
<p>Expression heatmaps of PIN family genes under different treatments. (<b>a</b>) The expression of PIN family genes under low-light treatment. (FL = full light (100%), LL = low light (30%)). (<b>b</b>) The expression of PIN family genes of <span class="html-italic">M. micrantha</span> in defoliation treatment (CK = undefoliated samples, TR = defoliated samples). (<b>c</b>) The expression of PIN family genes of <span class="html-italic">M. micrantha</span> after CPPU or GIC treatments (CK = water application, CPPU5 and CPPU 75 = 5 ppm and 75 ppm of N-(2-Chloro-4-pyridyl)-N′-phenylurea, and GIC5 and GIC75 = 5 ppm and 75 ppm mixture of gibberellic acid, indole-3-acetic acid, and CPPU). The FPKM values were transformed to log2 (value + 1). The color scale is shown at the right, and higher expression levels are shown in red.</p> Full article ">
18 pages, 8859 KiB  
Article
Ectopic Expression of AeNAC83, a NAC Transcription Factor from Abelmoschus esculentus, Inhibits Growth and Confers Tolerance to Salt Stress in Arabidopsis
by Xuan Zhao, Tingting Wu, Shixian Guo, Junling Hu and Yihua Zhan
Int. J. Mol. Sci. 2022, 23(17), 10182; https://doi.org/10.3390/ijms231710182 - 5 Sep 2022
Cited by 10 | Viewed by 2322
Abstract
NAC transcription factors play crucial roles in plant growth, development and stress responses. Previously, we preliminarily identified that the transcription factor AeNAC83 gene was significantly up-regulated under salt stress in okra (Abelmoschus esculentus). Herein, we cloned the nuclear-localized AeNAC83 from okra [...] Read more.
NAC transcription factors play crucial roles in plant growth, development and stress responses. Previously, we preliminarily identified that the transcription factor AeNAC83 gene was significantly up-regulated under salt stress in okra (Abelmoschus esculentus). Herein, we cloned the nuclear-localized AeNAC83 from okra and identified its possible role in salt stress response and plant growth. The down-regulation of AeNAC83 caused by virus-induced gene silencing enhanced plant sensitivity to salt stress and increased the biomass accumulation of okra seedlings. Meanwhile, AeNAC83-overexpression Arabidopsis lines improved salt tolerance and exhibited many altered phenotypes, including small rosette, short primary roots, and promoted crown roots and root hairs. RNA-seq showed numerous genes at the transcriptional level that changed significantly in the AeNAC83-overexpression transgenic and the wild Arabidopsis with or without NaCl treatment, respectively. The expression of most phenylpropanoid and flavonoid biosynthesis-related genes was largely induced by salt stress. While genes encoding key proteins involved in photosynthesis were almost declined dramatically in AeNAC83-overexpression transgenic plants, and NaCl treatment further resulted in the down-regulation of these genes. Furthermore, DEGs encoding various plant hormone signal pathways were also identified. These results indicate that AeNAC83 is involved in resistance to salt stress and plant growth. Full article
(This article belongs to the Special Issue Research on Plant Genomics and Breeding)
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<p>Characterization of AeNAC83: (<b>A</b>) Multiple sequence alignment of AeNAC83 and its homologous NAC proteins. The NAC domain with five conserved regions (a–e) are indicated by red lines; (<b>B</b>) Phylogenetic analysis of AeNAC83 with its homologous NAC proteins.</p> Full article ">Figure 2
<p>Subcellular localization and expression pattern of AeNAC83: (<b>A</b>) Subcellular localization of AeNAC83 in <span class="html-italic">N. benthamiana</span>. <span class="html-italic">N. benthamiana</span> cells were transformed with 35Sp:: <span class="html-italic">AeNAC83</span>:GFP or pCAMBIA1300-GFP. After incubating for 48 h, the transformed cells were observed under a confocal microscope. The photographs were taken under detecting GFP fluorescence, bright field, and in combination (merge), respectively. Empty vector (pCAMBIA1300-GFP) was used as a control; (<b>B</b>) Expression of <span class="html-italic">AeNAC83</span> in okra seedlings after 300 mM NaCl treatment by qRT-PCR. Total RNA for expression analysis was isolated from leaves of two-week-old seedlings after 300 mM NaCl treatment for 1, 3, 5, and 7 days. Data are presented as mean ± SD (<span class="html-italic">n</span> = 3). Different letters denote significant differences at <span class="html-italic">p</span> &lt; 0.05, using ANOVA and Duncan’s multiple tests.</p> Full article ">Figure 3
<p>Phenotype analysis of <span class="html-italic">AeNAC83</span>-silenced okra plants produced by VIGS under salt stress. The <span class="html-italic">Agrobacterium tumefaciens</span> GV3101 cell culture harboring the pTRV2 or <span class="html-italic">AeNAC83</span>-pTRV2 together with pTRV1 were mixed with a ratio of 1:1 and syringe-infiltrated into okra cotyledons. At 25 d post-inoculation (dpi), the leaves were used for gene expression assay and 300 mM NaCl treatment for 7 days. (<b>A</b>) Expression analysis of <span class="html-italic">AeNAC83</span> in <span class="html-italic">AeNAC83</span>-silenced okra seedlings by qRT-PCR. (<b>B</b>) Images of plant phenotype. (<b>C</b>) The leaf fresh weight. (<b>D</b>) Total chlorophyll content. Data are presented as mean ± SD (<span class="html-italic">n</span> = 10). Different letters denote significant differences at <span class="html-italic">p</span> &lt; 0.05, using ANOVA and Duncan’s multiple tests.</p> Full article ">Figure 4
<p>Roles of AeNAC83 in salt tolerance and in growth in <span class="html-italic">Arabidopsis</span> transgenic plants. Four-day-old seedlings of wild-type (WT) and two transgenic lines (OX3 and OX7) were transplanted on 1/2 MS medium supplemented with 0, 120, or 150 mM NaCl for 10 days. (<b>A</b>) Semi-quantitative analysis of <span class="html-italic">AeNAC83</span> gene expression in wild-type (WT) and <span class="html-italic">AeNAC83</span>-overexpression transgenic plants. The first bands show <span class="html-italic">AeNAC83</span> gene expression (30 cycles) and the bands below show <span class="html-italic">AtActin</span> gene expression (26 cycles) used as internal control. (<b>B</b>) Phenotypes of seedlings on 1/2 MS medium supplemented with 0, 120, or 150 mM NaCl, respectively. (<b>C</b>,<b>D</b>) Fresh weight and primary root length of seedlings at the end of the experiment in (<b>B</b>). Data are presented as mean ± SD (<span class="html-italic">n</span> = 10). Different letters denote significant differences at <span class="html-italic">p</span> &lt; 0.05, using ANOVA and Duncan’s multiple tests. (<b>E</b>) Root phenotype of seedlings on 1/2 MS medium without NaCl treatment. The red arrows indicated the crown root.</p> Full article ">Figure 5
<p>Transcriptional variations in <span class="html-italic">Arabidopsis</span> wild-type (WT) and transgenic plant (OX3) under NaCl treatment: (<b>A</b>) Expression profiles of the DEGs under NaCl treatment were shown by a heatmap; (<b>B</b>) Significance analysis of the DEGs in different comparisons by volcanoplots; (<b>C</b>) The number of up- and down-regulated genes in different comparisons; (<b>D</b>) Venn diagrams showed the proportions of the up- and down-regulated genes in four comparisons. WT-CK, OX3-CK: WT and OX3 grown under optimum conditions; WT-N, OX3-N: WT and OX3 were subjected to salt stress. Three replicates for each treatment.</p> Full article ">Figure 6
<p>The top 20 enriched KEGG pathways enrichment analysis of DEGs in the four comparison groups. (<b>A</b>–<b>D</b>) KEGG pathway enrichment analysis of DEGs in the WT-CK vs. OX3-CK, WT-CK vs. WT-N, OX3-CK vs. OX3-N, and WT-N vs. OX3-N comparisons, respectively.</p> Full article ">Figure 7
<p>Transcript abundance changes of the phenylpropanoid and flavonoid biosynthesis pathway-related DEGs in <span class="html-italic">Arabidopsis</span> wild type (WT) and transgenic plant (OX3) under NaCl treatment. (<b>A</b>) Heat map of DEGs in phenylpropanoid biosynthesis pathway. The log2-transformed FPKM values of DEGs were used to generate the diagram. 4CL, 4-coumarate-CoA ligase; C4H, cinnamate-4-hydroxylase; F5H, ferulate-5-hydroxylase; CCR, cinnamoyl-CoA reductase; CCoAOMT, caffeoyl-CoA O-methyltransferase; COMT, caffeic acid 3-O-methyltransferase; HCT, hydroxyl cinnamoyl transferase; CAD, cinnamyl-alcohol dehydrogenase; SGT, scopoletin glucosyltransferase; CSE, caffeoylshikimate esterase; CGT, coniferyl-alcohol glucosyltransferase; β-G, beta-glucosidase; POD, peroxidase. (<b>B</b>) Gene expression of DEGs in flavonoid biosynthesis pathway. DFR: dihydroflavonol 4-reductase; F3H: flavanone 3-hydroxylase; CYP73A: trans-cinnamate 4-monooxygenase; ANS: anthocyanidin synthase; FLS: flavonol synthase; CHS: chalcone synthase; PGT1: phlorizin synthase; CHI: chalcone isomerase; HCT: shikimate O-hydroxycinnamoyltransferase. * indicate statistical significance based on two-tailed Student’s <span class="html-italic">t</span>-test at <span class="html-italic">p</span>-values &lt; 0.05.</p> Full article ">Figure 8
<p>Transcript abundance changes of photosynthesis pathway-related DEGs in <span class="html-italic">Arabidopsis</span> wild-type (WT) and transgenic plant (OX3) under NaCl treatment. (<b>A</b>) Photosynthesis pathway (ko00195). Different letters indicated the different subunits of photosynthetic complexes. (<b>B</b>) Heat map of DEGs in photosynthesis pathway. The log2-transformed FPKM values of DEGs were used to generate the diagram.</p> Full article ">Figure 9
<p>Transcript abundance changes of the various hormone signal transduction pathways-related DEGs in <span class="html-italic">Arabidopsis</span> wild-type (WT) and transgenic plant (OX3) under NaCl treatment. (<b>A</b>) Plant hormone signal transduction pathways. (<b>B</b>) Heat map of DEGs in various hormone signal transduction pathways. The log2-transformed FPKM values of DEGs were used to generate the diagram.</p> Full article ">
13 pages, 1634 KiB  
Article
Imidacloprid Impairs Glutamatergic Synaptic Plasticity and Desensitizes Mechanosensitive, Nociceptive, and Photogenic Response of Drosophila melanogaster by Mediating Oxidative Stress, Which Could Be Rescued by Osthole
by Chuan-Hsiu Liu, Mei-Ying Chen, Jack Cheng, Tsai-Ni Chuang, Hsin-Ping Liu and Wei-Yong Lin
Int. J. Mol. Sci. 2022, 23(17), 10181; https://doi.org/10.3390/ijms231710181 - 5 Sep 2022
Cited by 1 | Viewed by 2138
Abstract
Background: Imidacloprid (IMD) is a widely used neonicotinoid-targeting insect nicotine acetylcholine receptors (nAChRs). However, off-target effects raise environmental concerns, including the IMD’s impairment of the memory of honeybees and rodents. Although the down-regulation of inotropic glutamate receptor (iGluR) was proposed as the cause, [...] Read more.
Background: Imidacloprid (IMD) is a widely used neonicotinoid-targeting insect nicotine acetylcholine receptors (nAChRs). However, off-target effects raise environmental concerns, including the IMD’s impairment of the memory of honeybees and rodents. Although the down-regulation of inotropic glutamate receptor (iGluR) was proposed as the cause, whether IMD directly manipulates the activation or inhibition of iGluR is unknown. Using electrophysiological recording on fruit fly neuromuscular junction (NMJ), we found that IMD of 0.125 and 12.5 mg/L did not activate glutamate receptors nor inhibit the glutamate-triggered depolarization of the glutamatergic synapse. However, chronic IMD treatment attenuated short-term facilitation (STF) of NMJ by more than 20%. Moreover, by behavioral assays, we found that IMD desensitized the fruit flies’ response to mechanosensitive, nociceptive, and photogenic stimuli. Finally, the treatment of the antioxidant osthole rescued the chronic IMD-induced phenotypes. We clarified that IMD is neither agonist nor antagonist of glutamate receptors, but chronic treatment with environmental-relevant concentrations impairs glutamatergic plasticity of the NMJ of fruit flies and interferes with the sensory response by mediating oxidative stress. Full article
(This article belongs to the Special Issue Pesticides Exposure and Toxicity)
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<p>The selection of the dose of IMD and the acute application on the glutamatergic neuromuscular junction (NMJ) of the fruit fly: (<b>A</b>) survival of fruit flies receiving different doses of IMD; (<b>B</b>) the dose of 0.125 mg/L did not affect the body weight of the flies; (<b>C</b>) the representative voltage recording showing the puff of glutamate on NMJ triggered a dose-dependent synaptic voltage change; (<b>D</b>) the representative voltage recording showing two doses of IMD puff did not trigger voltage change of NMJ nor decrease the amplitude of the following glutamate puff; (<b>E</b>) the quantification of (<b>D</b>); (<b>F</b>) the representative voltage recording showing two doses of IMD did not antagonize glutamate puff; (<b>G</b>) the quantification of (<b>F</b>). The error bar stands for the standard error of the mean. The red arrows stand for the puff of chemicals on NMJ. ∆V<sub>M</sub>, voltage change of the membrane potential; IMD, imidacloprid; Glu, glutamate. n.s., not significant.</p> Full article ">Figure 2
<p>Chronic IMD treatment attenuated the short-term facilitation (STF) of the glutamatergic neuromuscular junction (NMJ) of the fruit fly: (<b>A</b>) the representative recording showing the evoked currents by five consecutive electric pulses of the frequency of 25 Hz; (<b>B</b>) the quantification of (<b>A</b>); (<b>C</b>) the representative recording showing the evoked currents by electric pulse with increasing frequency from 0.5 to 20 Hz; (<b>D</b>) the quantification of (<b>C</b>). The error bar stands for the standard error of the mean. The *, **, or *** denotes the <span class="html-italic">p</span>-value &lt; 0.05, 0.01, or 0.001 for Two-way ANOVA with Tukey’s multiple comparison test. IMD, imidacloprid of 0.125 mg/L.</p> Full article ">Figure 3
<p>Chronic IMD treatment evoked behavioral phenotypes of the fruit fly. IMD caused alteration of the mechanosensory response: (<b>A</b>) scheme showing touch score counting; (<b>B</b>) the touch score; (<b>C</b>) gene expression of the mechanosensory pathway; IMD decreased the nociceptive response; (<b>D</b>) diagram showing the nociception assay; (<b>E</b>) the nociception behavior of male and female flies; (<b>F</b>) gene expression of the nociception pathway; IMD causes alteration of the light perception; (<b>G</b>) the representative waveform of electroretinogram (ERG) upon light on-off stimuli; (<b>H</b>) the quantification of ERG waveform; (<b>I</b>) gene expression of the phototransduction pathway. Error bar stands for standard error of the mean. The *, **, or *** denotes the <span class="html-italic">p</span>-value &lt; 0.05, 0.01, or 0.001 for Student’s <span class="html-italic">t</span>-test; IMD, imidacloprid of 0.125 mg/L; ∆V<sub>R</sub>, voltage change of electroretinogram; RPA, receptor potential amplitude. The operational definition of ∆V<sub>R</sub>, RPA, on-transient, and off-transient is illustrated in (<b>G</b>).</p> Full article ">Figure 4
<p>The antioxidant osthole (OST) rescued the phenotypes of the IMD-treated fruit fly: (<b>A</b>) differentially expressed genes of the oxidative stress response pathway; (<b>B</b>) the rescued 25 Hz short-term facilitation (STF) of the neuromuscular junction; (<b>C</b>) the rescued retinal ∆V<sub>R</sub> and (<b>D</b>) RPA; (<b>E</b>) the rescued mechanosensory perception; (<b>F</b>) the rescued nociceptive perception. Error bar stands for standard error of the mean. The *, **, or *** denotes the <span class="html-italic">p</span>-value &lt; 0.05, 0.01, or 0.001 and n.s. for not significant of Student’s <span class="html-italic">t</span>-test (<b>A</b>); two-way ANOVA with Tukey’s multiple comparison test (<b>B</b>); and one-way ANOVA with Tukey’s multiple comparison test (<b>C</b>–<b>F</b>); IMD, imidacloprid of 0.125 mg/L; OST, osthole of 6 μg/mL; ∆V<sub>R</sub>, voltage change of electroretinogram; RPA, receptor potential amplitude.</p> Full article ">
20 pages, 42635 KiB  
Article
Mechano-Transduction Boosts the Aging Effects in Human Erythrocytes Submitted to Mechanical Stimulation
by Simone Dinarelli, Giovanni Longo, Antonio Francioso, Luciana Mosca and Marco Girasole
Int. J. Mol. Sci. 2022, 23(17), 10180; https://doi.org/10.3390/ijms231710180 - 5 Sep 2022
Cited by 4 | Viewed by 1775
Abstract
Erythrocytes’ aging and mechano-transduction are fundamental cellular pathways that determine the red blood cells’ (RBCs) behavior and function. The aging pattern can be influenced, in morphological, biochemical, and metabolic terms by the environmental conditions. In this paper, we studied the effect of a [...] Read more.
Erythrocytes’ aging and mechano-transduction are fundamental cellular pathways that determine the red blood cells’ (RBCs) behavior and function. The aging pattern can be influenced, in morphological, biochemical, and metabolic terms by the environmental conditions. In this paper, we studied the effect of a moderate mechanical stimulation applied through external shaking during the RBCs aging and revealed a strong acceleration of the aging pattern induced by such stimulation. Moreover, we evaluated the behavior of the main cellular effectors and resources in the presence of drugs (diamide) or of specific inhibitors of the mechano-transduction (probenecid, carbenoxolone, and glibenclamide). This approach provided the first evidence of a direct cross-correlation between aging and mechano-transduction and permitted an evaluation of the overall metabolic regulation and of the insurgence of specific morphological features, such as micro-vesicles and roughness alterations. Overall, for the first time the present data provided a schematic to understand the integration of distinct complex patterns in a comprehensive view of the cell and of its interactions with the environment. Mechano-transduction produces structural effects that are correlated with the stimulation and the strength of the environmental stimulation is paramount to effectively activate and trigger the biological cascades initiated by the mechano-sensing. Full article
(This article belongs to the Special Issue Roles of Erythrocytes in Human Health and Disease)
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<p>Percentage of cell lysis (<b>a</b>) and position of the Soret Peak (<b>b</b>) for static (red circles) and shaken (blue empty squares) samples. The overall stress experienced by the mechanically stimulated cells is higher with respect to the static ones at aging times, approximately, longer than 4 days.</p> Full article ">Figure 2
<p>Histograms reporting the GSH/GSGG ratio (<b>a</b>) and the intracellular ATP content (<b>b</b>) for static (orange bars) and mechanically stimulated (black bars) samples. The energy consumption is higher in the shaken samples with respect to the static ones. In terms of reducing power, this difference is significant already after 1 day of ageing, while in the ATP trend, this difference becomes evident after 4 days of ageing. The ATP concentrations are shown in log scale.</p> Full article ">Figure 3
<p>Results of the rejuvenation procedure on static (panel (<b>a</b>)) and mechanically stimulated (panel (<b>b</b>)) samples. The red squares represent the intracellular ATP content prior to the rejuvenation procedure, while the blue dots represent the intracellular ATP content immediately after the procedure. Insets are shown inside the graphs, to better highlight the ATP increment during the reload. The ATP concentrations are shown in log scale.</p> Full article ">Figure 4
<p>Hemoglobin re-oxidation kinetics after rejuvenation performed at the second (<b>a</b>); eighth (<b>b</b>), and eleventh day (<b>c</b>) for static controls (red squares) and mechanically stimulated (blue circles) samples. The Soret peak in shaken cells always occurs at shorter wavelengths (i.e., is more oxidated) than in static controls. This indicates that Hb re-oxidation is always faster for mechanically stimulated cells, suggesting that the shaken RBCs experience a higher stress condition.</p> Full article ">Figure 5
<p>Morphological evolution and volume variation as a function of the RBC aging. Panels (<b>a</b>,<b>b</b>) show the morphological evolution in absence (<b>a</b>) or presence (<b>b</b>) of mechanical stimulation. The red, light blue, and black columns represent the percentage of altered, non-biconcave, shapes observed in the samples. The data arise from a count of at least 1000 cells per sample performed with a conventional optical microscope. Therefore, these analyses do not have enough sensitivity on the nanoscale to appreciate the details of the cell membrane but these data allow the analysis of a statistically significant dataset composed of a high number of cells. Panel (<b>c</b>) shows the decreasing trend of the mean cell volume, as measured using AFM imaging, for static controls (red circle) and mechanically stimulated cells (open square) as function of the aging. It is worth noting that, for experimental reasons, the optical and the AFM images were collected on alternating days, in such a way that the comparison between these techniques must be performed in term of the overall observed trends.</p> Full article ">Figure 6
<p>Roughness trend measured for static controls (red circles) and for mechanically stimulated sample (blue squares). The faster decreasing trend in shaken cells indicates a larger disengagement of the cell skeleton from the overlying membrane over time.</p> Full article ">Figure 7
<p>Typical AFM images of the RBCs’ morphology for static controls (upper panels) and mechanically stimulated samples (lower panels) after three (<b>a</b>,<b>d</b>), six (<b>b</b>,<b>e</b>), and nine days (<b>c</b>,<b>f</b>) of aging. Altered morphologies can be observed more often, especially at the longer times, in mechanically stimulated samples than in static controls.</p> Full article ">Figure 8
<p>Typical high resolution images of the plasma membrane for static controls (upper panels) and mechanically stimulated samples (lower panels) after one (<b>a</b>,<b>e</b>), three (<b>b</b>,<b>f</b>), six (<b>c</b>,<b>g</b>), and nine days (<b>d</b>,<b>h</b>) of aging. The data suggest that the occurrence of features such as micro-vesicles and other membrane markers of aging arises sooner in the mechanically-stimulated samples.</p> Full article ">Figure 9
<p>Panels (<b>a</b>,<b>b</b>) report, respectively, the percentage of cell lysis in static controls (<b>a</b>) and in mechanically stimulated cells (<b>b</b>) in the presence and absence of DIAM, CARB, GLIB, and PROB. Moreover, in panels (<b>c</b>,<b>d</b>) we compared the ratio of the lysis curves (shown in <a href="#ijms-23-10180-f002" class="html-fig">Figure 2</a>a,b) for CTR/PROB (<b>c</b>) and for CTR/DIAM (<b>d</b>). The ratio has been performed both in static and in shaken conditions and is reported in the Y axis of (<b>c</b>,<b>d</b>). If the mechanical stimulus would provide a specific increase of cell lysis, this would result in systematic differences in the shaken vs. static trends. On contrary, the data show that mechanical stimulus always determines an increase in cell lysis (<b>a</b> vs. <b>b</b>) but this increasing is not mechanical stimulus-dependent as the two trends in the presence or absence of mechanical stimulus are equivalent (<b>c</b>,<b>d</b>).</p> Full article ">Figure 10
<p>Intracellular ATP measured at various aging times in static (<b>a</b>,<b>c</b>,<b>e</b>) and mechanically stimulated (<b>b</b>,<b>d</b>,<b>f</b>) samples in the presence and absence of drugs that interfere with the mechano-transduction. Respectively, DIAM (<b>a</b>,<b>b</b>); GLIB (<b>c</b>,<b>d</b>), and PROB (<b>e</b>,<b>f</b>) and their specific controls are shown. The data evidence that the drugs have no significant effect in static conditions, while, in the presence of mechanical stimulation, n higher amount of ATP can be measured, at least during the critical aging phase when higher consumption of resource is required (i.e., the fifth day). Data analogous to PROB were measured for CARB. The ATP concentrations are shown in log scale.</p> Full article ">
16 pages, 5517 KiB  
Review
Retinal Ganglion Cell Survival and Axon Regeneration after Optic Nerve Injury: Role of Inflammation and Other Factors
by Kimberly A. Wong and Larry I. Benowitz
Int. J. Mol. Sci. 2022, 23(17), 10179; https://doi.org/10.3390/ijms231710179 - 5 Sep 2022
Cited by 31 | Viewed by 5063
Abstract
The optic nerve, like most pathways in the mature central nervous system, cannot regenerate if injured, and within days, retinal ganglion cells (RGCs), the neurons that extend axons through the optic nerve, begin to die. Thus, there are few clinical options to improve [...] Read more.
The optic nerve, like most pathways in the mature central nervous system, cannot regenerate if injured, and within days, retinal ganglion cells (RGCs), the neurons that extend axons through the optic nerve, begin to die. Thus, there are few clinical options to improve vision after traumatic or ischemic optic nerve injury or in neurodegenerative diseases such as glaucoma, dominant optic neuropathy, or optic pathway gliomas. Research over the past two decades has identified several strategies to enable RGCs to regenerate axons the entire length of the optic nerve, in some cases leading to modest reinnervation of di- and mesencephalic visual relay centers. This review primarily focuses on the role of the innate immune system in improving RGC survival and axon regeneration, and its synergy with manipulations of signal transduction pathways, transcription factors, and cell-extrinsic suppressors of axon growth. Research in this field provides hope that clinically effective strategies to improve vision in patients with currently untreatable losses could become a reality in 5–10 years. Full article
(This article belongs to the Special Issue Optic Neuropathies: Current and Future Strategies for Optic Nerve Protection and Repair)
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<p>Inflammation-induced optic nerve regeneration and the role of Oncomodulin (Ocm). (<b>a</b>) Negative control: Absence of axon regeneration following optic nerve injury (asterisk: site of optic nerve crush injury). (<b>b</b>) Intraocular inflammation induced by injury to the lens (shown here) or by other means cited in the text enables retinal ganglion cells (RGCs) to regenerate axons past the injury site. (<b>c</b>) Intraocular inflammation is associated with a rapid infiltration of Gr-1-positive neutrophils (red) that express high levels of Ocm (green). (<b>d</b>) P1, a peptide antagonist of Ocm based on the N-terminus of the protein, suppresses Zymosan-induced regeneration. (<b>e</b>) Slow-release polymer beads containing the cAMP analog CPT-cAMP induce minimal regeneration after optic nerve injury. (<b>f</b>) Extensive regeneration with slow-release beads containing Ocm + CPT-cAMP [<a href="#B5-ijms-23-10179" class="html-bibr">5</a>,<a href="#B6-ijms-23-10179" class="html-bibr">6</a>,<a href="#B13-ijms-23-10179" class="html-bibr">13</a>].</p> Full article ">Figure 2
<p>Macrophage-derived SDF1 complements the effects of Ocm (previous page). (<b>a</b>) SDF1 (red) is highly expressed in F4/80 macrophages that infiltrate the vitreous by 24 h after intraocular injection of Zymosan. (<b>b</b>) Among multiple trophic factors tested in dissociated adult retinal cell cultures, SDF1 is the only factor that enhances the effects of Ocm (combined with co-factors mannose and CPT-cAMP). B: Brain-derived neurotrophic factor (BDNF); C: ciliary neurotrophic factor (CNTF); G: glial cell-derived trophic factor (GDNF); L: leukemia inhibitory factor (LIF); T: tumor-necrosis factor (TNF); F: fibroblast growth factor-2; I: insulin-like growth factor 2 (IGF2). *** <span class="html-italic">p</span> &lt; 0.001. (<b>c</b>) SDF1 combined with Ocm and CPT-cAMP induces similar levels of regeneration as Zymosan. (<b>d</b>) SDF1 alters the response of different RGC populations to PTEN deletion. aRGCs, the population that extends axons in response to Pten deletion, are identified by virtue of expressing GFP from the Kcng4 promoter. Axons arising from all RGCs, whether aRGCs or non-aRGCs, are labeled with CTB. Top row: In response to SDF1 alone, regenerating GFP-negative axons all arise from non-aRGCs. Middle row: Pten deletion alone induces regeneration primarily from aRGCs (note extensive overlap of GFP and CTB in last panel). Bottom row: Combining SDF1 and Pten deletion suppresses regeneration from aRGCs while strongly increasing overall levels of regeneration from non-αRGCs. (<b>e</b>) Schematic illustration showing the response of different RGC populations to various treatments. Top row: AAV2 expressing anti-Pten shRNA (AAV2-shPten) induces axon growth primarily from aRGCs (green cells). AAV2-shLuciferase virus (AAV2-shLuc) has no effects. Middle row: Either recombinant SDF1 (rSDF1) or AAV2 expressing SDF1 (AAV2-SDF1) induces moderate axon growth primarily from non-aRGCs (middle left: orange cells). When combined with Pten deletion, SDF1 induces non-aRGCs to regenerate lengthy axons but prevents aRGCs from responding to Pten deletion (middle right). Bottom row: Zymosan elevates levels of neutrophil-derived Ocm, macrophage-derived SDF1, and other factors, stimulating regeneration from both α- and non-αRGCs (bottom left); Pten deletion combined with Zymosan strongly augments outgrowth from both subtypes (bottom right) [<a href="#B8-ijms-23-10179" class="html-bibr">8</a>]. Asterisks show lesion site.</p> Full article ">Figure 3
<p>CNTF gene therapy induces optic nerve regeneration by augmenting inflammation and chemokine CCL5. (<b>a</b>) Recombinant CNTF protein (1 µg) does not induce appreciable regeneration. (<b>b</b>,<b>c</b>) Unlike a control adeno-associated protein expressing green fluorescent protein (AAV2-GFP), AAV2 expressing CNTF induces appreciable levels. (<b>d</b>,<b>e</b>) Whereas mice heterozygous for the chemokine 2 receptor (CCR2<sup>+/−</sup>) regenerate axons in response to CNTF gene therapy (<b>d</b>), homozygous null mice show a strongly diminished response (<b>e</b>). (<b>f</b>,<b>g</b>) CRISPR-Cas9 mediated deletion of the chemokine 5 receptor CCR5. Mice received intraocular injection of an adeno-associated virus expressing Cas9 driven by the synuclein-g promoter plus a second virus expressing either GFP ((<b>f</b>), control) or a small guide RNA directed to CCR5 (<b>g</b>), which nearly eliminated regeneration induced by CNTF gene therapy. (<b>h</b>) Recombinant CCL5 induces nearly as much regeneration as CNTF gene therapy. Asterisks indicate lesion site [<a href="#B22-ijms-23-10179" class="html-bibr">22</a>].</p> Full article ">Figure 4
<p>Synergy between intraocular inflammation and deleting receptors for cell-extrinsic suppressors of axon growth. (<b>a</b>–<b>f</b>) Longitudinal sections through the optic nerves of wild-type mice (WT) or mice lacking all 3 isoforms of the Nogo receptor (NgR123<sup>−/−</sup>) or of NgR1 and 3 plus PTPs, a receptor that mediates inhibitory effects of chondroitin sulfate proteoglycans (CSPGs). As indicated, mice either underwent optic nerve injury alone or with intraocular inflammation following intraocular injection of zymosan. (<b>a’</b>–<b>f’</b>) Regions of optic nerves shown at greater magnification in the corresponding panels below. Note the dramatic increase in regeneration when combining intraocular inflammation with deletion of receptors for the inhibitory molecules associated with myelin and CSPGs. Asterisks show lesion site [<a href="#B40-ijms-23-10179" class="html-bibr">40</a>].</p> Full article ">Figure 5
<p>Injury-induced pathways contributing to RGC death after optic nerve injury. Encircled question marks (?) indicate as yet unknown relationships. Up arrows indicate increases.</p> Full article ">Figure 6
<p>Phagocytic microglia/monocytes alter the local environment of the injury site and modulate axon regeneration. (<b>a</b>) Single confocal planes with orthogonal views of myelin basic protein (MBP) debris inside CR3+ cells. Images are from 0.2 and 0.4 mm distal to the injury site at 14 DPI [<a href="#B44-ijms-23-10179" class="html-bibr">44</a>]. (<b>b</b>) CR3+ microglia and monocytes expand within the site of injury (dotted line) from 1 to 5 days after crush, resulting in a progressive clearance of MBP from the distal optic nerve, allowing for uninhibited axon regeneration [<a href="#B44-ijms-23-10179" class="html-bibr">44</a>]. (<b>c</b>) Treatment with CSF1R inhibitor, PLX5622 (PLX) results in efficient clearance of IBA1+ microglia from the retina, which is maintained by 14 days after ONI (14d post-ONI). (<b>d</b>) Microglia deletion enhances the number of GAP43+ (green) axons regenerating past the crush site (dotted line). Right: Quantitation of regenerating axons 0.5 mm distal to the injury site (☐); mean ± s.e.m, n = optic nerves, **, <span class="html-italic">p</span> ≤ 0.01 by <span class="html-italic">t</span>-test [<a href="#B44-ijms-23-10179" class="html-bibr">44</a>].</p> Full article ">Figure 7
<p>Full-length optic nerve regeneration. (<b>a</b>) Intraocular inflammation combined with CPT-cAMP and virally mediated PTEN deletion enables RGCs to regenerate axons the full length of the optic nerve in 8–10 weeks. Axons are labeled by intraocular injection of cholera toxin B fragment (CTB, red). (<b>b</b>) Control optic nerve in case treated with intraocular Zymosan and a control virus. (<b>c</b>–<b>e</b>) Enlargements of area within rectangle show overlap in immunostaining fibers for CTB (<b>c</b>, red) and the growth-associated protein GAP43 (<b>d</b>, green). Asterisks show lesion site [<a href="#B79-ijms-23-10179" class="html-bibr">79</a>].</p> Full article ">
15 pages, 2373 KiB  
Article
DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus
by Tao Liu, Ying Xu, Xiaojie Wang, Qing Ye, Zhenzhen Liu, Zhufeng Zhang, Jilin Liu, Yudong Yang, Xu Peng and Nan Peng
Int. J. Mol. Sci. 2022, 23(17), 10178; https://doi.org/10.3390/ijms231710178 - 5 Sep 2022
Cited by 1 | Viewed by 2118
Abstract
CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the [...] Read more.
CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor. Full article
(This article belongs to the Special Issue CRISPR-Cas in Genomic Manipulation and Antimicrobial Resistance)
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Figure 1
<p><b>Mutations at the leader-proximal end reduce spacer acquisition.</b> (<b>A</b>) Diagram of the type I-A CRISPR-Cas system in <span class="html-italic">S. islandicus</span> REY15A. The 206 bp leader sequences are identical in CRISPR locus1 and locus2 but located inverted. The type I-A <span class="html-italic">cas</span> genes and most of the CRISPR arrays were deleted in the ΔIA-locus2. Simultaneously, mutations identical to the sequences in <a href="#ijms-23-10178-f001" class="html-fig">Figure 1</a>B were introduced into the leader of CRISPR locus2 to generate Mut1–4 mutant strains. Repeats are marked as black diamonds, and spacers are marked as green rectangles. (<b>B</b>) PCR amplification of the leader proximal regions of both CRISPR loci before (upper lanes) and after <span class="html-italic">csa3a</span> overexpression (carrying the <span class="html-italic">csa3a</span>-overexpression plasmid, pCsa3a) in WT or mutated strains (Mut1–4). For WT::pCsa3a and Mut4::pCsa3a, there are four main bands at both locus1 and locus2. For Mut1::pCsa3a and Mut2::pCsa3a, there are four main bands at locus1 but only two main bands at locus2. For Mut3::pCsa3a, there are four main bands at locus1 but three main bands at locus2. The bands corresponding to PCR products of the expanded arrays are indicated as blue arrows, and the black arrow indicates the parental bands. The sizes of the bands are indicated on the left. This result represents three independent spacer acquisition analyses.</p> Full article ">Figure 2
<p><b>Cas1 binds the stem-loop structure at the leader-proximal end</b>. (<b>A</b>) Sequence of the leader-proximal end in the subtype I-A CRISPR-Cas system of <span class="html-italic">S. islandicus</span> REY15A. The stem-loop structure is indicated by arrows. Numbers below indicate the locations of the leader sequence relative to the first repeat. (<b>B</b>) EMSA analysis of the binding between the Cas1 or Cas2 proteins and the 5′-end FAM fluorescent labeled leader-proximal probe. The probe sequences are shown in (<b>A</b>), and the reaction mixtures containing 0.3 μM probe and increasing concentration of Cas1 (0.6 μM and 1.2 μM) or Cas2 (3.3 μM and 6.7 μM) were loaded on the 12% native PAGE gel. (<b>C</b>) EMSA analysis of the effect of Cas2 and Cas2 with spacer on Cas1 binding to the 5′-end FAM fluorescent labeled leader-proximal probe. (<b>D</b>) EMSA analysis of Cas1 binding to different probes. These probes were generated through annealing of 5′-end FAM-labelled sense strand oligonucleotide (S) with unlabeled anti-sense strand oligonucleotide (<b>A</b>) at 1:5, 1:1 or 5:1 ratio, respectively. “S” indicates 5′-end FAM-labelled sense strand oligonucleotide, and “Mut1” indicates mutated 5′-end FAM-labelled sense strand oligonucleotide (the sequence is shown in <a href="#ijms-23-10178-f001" class="html-fig">Figure 1</a>B).</p> Full article ">Figure 3
<p><b>Cas1 non-specifically binds to stem-loop structured ssDNA.</b> (<b>A</b>,<b>B</b>) EMSA analysis of Cas1 (0.6 μM and 1.2 μM) binding to the wildtype and the mutated probes (0.3 μM). These probes were generated through annealing of 5′-end FAM-labelled sense strand oligonucleotides and complementary unlabeled anti-sense strand oligonucleotides at 5:1 molar ratio, respectively. The sequences of Mut1 and Mut3 are shown in <a href="#ijms-23-10178-f001" class="html-fig">Figure 1</a>B. MutS carries two mutated, and complementary, sequences at the stem region which occur individually in Mut1 and Mut3. The sequence of newST is completely different from the wildtype, but carries a similar stem-loop structure (7 bp stem with 4 nt loop).</p> Full article ">Figure 4
<p><b>Csa3a interacts with Cas1 to modulate Cas1 integrase activity.</b> (<b>A</b>) His-tag pull-down assay to analyze the interaction between Csa3a and Cas1. SDS-PAGE analysis of purified recombinant Csa3a-His, GST, GST-Cas1, and Ni-NTA column elution fractions of Csa3a-His incubated with GST or GST-Cas1. GST-Cas1 incubated with Ni-NTA beads was used as the control. Standard protein molecular mass (kDa) markers (M) are indicated on the left. (<b>B</b>) Cross-linking mass spectrometry to determine the interaction between Csa3a and Cas1. Inter-molecular self-links are indicated as orange drops. Cross-links between Csa3a and Cas1 are indicated as cyan lines. The red vertical line indicates the predicted nuclease active site E137 of Cas1 in <span class="html-italic">S. islandicus</span>; wHTH domain indicates the winged Helix-Turn-Helix domain. (<b>C</b>) EMSA analysis of Csa3a (0.6 μM, 1.2 μM, and 2.4 μM), Cas1 (0.6 μM and 1.2 μM), and 1.2 μM Cas1 with increasing amounts of Csa3a (0.6 μM, 1.2 μM, and 2.4 μM) binding to the wildtype probe. Well shift and super shifts appearing when Csa3a was added into the reaction. (<b>D</b>,<b>E</b>) in vitro integration of prespacers into the supercoiled plasmid using a 39 bp 5′-end FAM-labeled dsDNA as the prespacer. Addition of Cas1, Csa3a and the distal motif DNA (Csa3a-binding DNA) as a competitor are indicated above the gel images. Samples were separated by 1.5% agarose gel and visualized by EtBr staining (<b>D</b>) or fluorescent imaging (<b>E</b>). C: concatemer; N: nicked, L: linear; S: supercoiled; P: 5′-end FAM-labeled prespacer; I1, I2 and I3: integrated products.</p> Full article ">Figure 5
<p><b>Leader motifs disassociate Csa3a-Cas1 interaction for efficient CRISPR adaptation.</b> (<b>A</b>) EMSA assay to determine the competition effect of leader-proximal motif on the interaction between Csa3a and Cas1. Diagram of the Csa3a binding distal motif (−143~−94) and the Cas1 binding proximal motif (−25~+7) in the leader. Numbers are relative to the first nucleotide of the repeat sequence. The leader is shown in grey, and the CRISPR repeat is shown in green, followed by a spacer shown in orange. Lanes 1–3: 5′-end FAM-labeled proximal motif ssDNA with stem-loop structure (P1) was incubated with increasing amounts of Cas1. Lanes 4–6: 5′-end HEX-labeled distal motif dsDNA (P2) was incubated with increasing amounts of Csa3a. Lanes 7–8: P2 was incubated with Csa3a and increasing amounts of Cas1. Lanes 9–10: increasing amounts of P1 were added as the competitor for Cas1 binding. (<b>B</b>) PCR amplification for newly integrated spacers in both CRISPR loci after <span class="html-italic">csa3a</span> overexpression in ΔIA_locus2 strain (control) and ΔIA_distal (mutations introduced into the distal motif in the leader of locus2) strain. Diagram of the CRISPR loci of ΔIA_locus2 and ΔIA_distal is shown above. L1 and L2 represent the identical leaders of locus1 and locus2, respectively. Expanded bands relative to the new repeat-spacer units are indicated by arrows. (<b>C</b>) Proportion of new spacers obtained by analysing high-throughput sequencing of PCR products of both loci inΔIA_locus2 and ΔIA_distal strains. Error bars: standard derivations of three independent experiments. Statistical significance: n.s., non-significance; *** <span class="html-italic">p</span> &lt; 0.001; two-way ANOVA and Dunnett.</p> Full article ">Figure 6
<p><b>A proposal for Cas1 binding DNA structure and modulation of integrase activity by a CRISPR regulator.</b> Csa3a can form a complex with Cas1 to inhibit the integrase activity but not the DNA binding activity of Cas1. The Csa3a-Cas1 complex can bind to some sites with a specific stem-loop structure in the genome. When Csa3a-Cas1 complex binds to the sites outside the CRISPR array, the integrase activity is repressed to avoid atypical spacer integration. Only when the complex encounters the leader does the integrase activity recover by Cas1 binding to the stem-loop structure at the proximal end and Csa3a binding to the distal motif.</p> Full article ">
23 pages, 6658 KiB  
Article
In Vitro Characterization of Renal Drug Transporter Activity in Kidney Cancer
by Pedro Caetano-Pinto, Nathanil Justian, Maria Dib, Jana Fischer, Maryna Somova, Martin Burchardt and Ingmar Wolff
Int. J. Mol. Sci. 2022, 23(17), 10177; https://doi.org/10.3390/ijms231710177 - 5 Sep 2022
Cited by 6 | Viewed by 3441
Abstract
The activity of drug transporters is central to the secretory function of the kidneys and a defining feature of renal proximal tubule epithelial cells (RPTECs). The expression, regulation, and function of these membrane-bound proteins is well understood under normal renal physiological conditions. However, [...] Read more.
The activity of drug transporters is central to the secretory function of the kidneys and a defining feature of renal proximal tubule epithelial cells (RPTECs). The expression, regulation, and function of these membrane-bound proteins is well understood under normal renal physiological conditions. However, the impact of drug transporters on the pathophysiology of kidney cancer is still elusive. In the present study, we employed different renal cell carcinoma (RCC) cell lines and a prototypical non-malignant RPTEC cell line to characterize the activity, expression, and potential regulatory mechanisms of relevant renal drug transporters in RCC in vitro. An analysis of the uptake and efflux activity, the expression of drug transporters, and the evaluation of cisplatin cytotoxicity under the effects of methylation or epidermal growth factor receptor (EGFR) inhibition showed that the RCC cells retained substantial drug transport activity. In RCC cells, P-glycoprotein was localized in the nucleus and its pharmacological inhibition enhanced cisplatin toxicity in non-malignant RPTECs. On the other hand, methylation inhibition enhanced cisplatin toxicity by upregulating the organic cation uptake activity in RCC cells. Differential effects of methylation and EGFR were observed in transporter expression, showing regulatory heterogeneity in these cells. Interestingly, the non-malignant RPTEC cell line that was used lacked the machinery responsible for organic cation transport, which reiterates the functional losses that renal cells undergo in vitro. Full article
(This article belongs to the Special Issue Overcoming Biological Barriers: Importance of Membrane Transporters in Homeostasis, Disease, and Disease Treatment)
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<p><b>Organic cation transport activity.</b> Uptake of ASP<sup>+</sup> in the presence or absence of OCT inhibitor imipramine. Functional data derived from fluorescent assays were fitted according to Michaelis–Menten kinetics to evaluate the drug transport activity in RPTEC-TERT1 cells (<b>A</b>), CAKI-1 cells (<b>B</b>), and 786-O cells (<b>C</b>). The reduced uptake of ASP<sup>+</sup> after inhibition indicated the presence of active organic cation uptake transport. Functional data were obtained from a minimum of two independent assays, performed in triplicate.</p> Full article ">Figure 2
<p><b>P-glycoprotein drug transport activity.</b> Uptake of calcein-AM in the presence or absence of the P-gp inhibitor valspodar. Functional data derived from fluorescent assays were fitted according to Michaelis–Menten kinetics to evaluate the drug transport activity in RPTEC-TERT1 cells (<b>A</b>), CAKI-1 cells (<b>B</b>), and 786-O cells (<b>C</b>). An increase in calcein retention after inhibition indicated the presence of P-gp-mediated efflux activity. Functional data were obtained from a minimum of two independent assays, performed in triplicate.</p> Full article ">Figure 3
<p><b>Breast cancer resistance protein drug transport activity.</b> Uptake of Hoechst33342 in the presence or absence of the BCRP inhibitor KO143. Functional data derived from fluorescent assays were fitted according to Michaelis–Menten kinetics to evaluate the drug transport activity in RPTEC-TERT1 cells (<b>A</b>), CAKI-1 cells (<b>B</b>), and 786-O cells (<b>C</b>). An increase in Hoechst33342 retention after inhibition indicated the presence of BCRP-mediated efflux activity. Functional data were obtained from a minimum of two independent assays, performed in triplicate.</p> Full article ">Figure 4
<p><b>Gene expression of key renal drug transporters in non-malignant and malignant RPTECs after treatment with methylation or EGFR inhibitors.</b> Expression is depicted as absolute, with 2<sup>−ΔCt</sup> values expressed in a log10 scale (<b>A</b>,<b>C</b>,<b>E</b>) and as relative, with 2<sup>−ΔΔCt</sup> values (fold) depicted in a linear scale (<b>B</b>,<b>D</b>,<b>F</b>). OCT2 was not found to be expressed in any of the cell lines tested and its expression was recovered in CAKI-1 cells (<b>C</b>) after decitabine exposure. Results represent data collected from three independent experiments. (* <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>Gene expression of relevant drug transporters in the kidney after treatment with methylation or EGFR inhibitors</b>. Expression is depicted as absolute, with 2<sup>−ΔCt</sup> values expressed in a log10 scale. RPTEC-TERT1 cells (<b>A</b>) did not express MATE1 and MATE2 transporters; in CAKI-1 cells (<b>B</b>), MATE1 and MATE2 were sensitive to CTX exposure; and in 786-O cells (<b>C</b>), MATE1 was absent. Results represent data collected from three independent experiments.</p> Full article ">Figure 6
<p><b>Immunofluorescent localization of BCRP and OCT1 in non-tumor and RCC cell lines.</b> In RPTEC-TERT1 cells (<b>A</b>), CAKI-1 cells (<b>B</b>), and 786-O cells (<b>C</b>), BCRP expression was seemingly dispersed through the cytoplasm and in the vicinity of the nuclear region, with no predominant localization observed in the membrane. Similar to BCRP, OCT1 expression in RPTEC-TERT1 cells (<b>D</b>), CAKI-1 cells (<b>E</b>), and 786-O cells (<b>F</b>) was dispersed in the cells. All panels represent a composite image, including a nuclear tracer (Hoeschst33342, blue), f-actin (Alexa488, green), and BCRP or OCT1 (Alexa555, red). Amplified sections in the right bottom corner of the panels represent the nuclear and respective transporter staining. All images presented were acquired with a magnification of 60×. The bottom-left panel in each image represents the amplified detail of the respective image without the phalloidin staining.</p> Full article ">Figure 7
<p><b>Immunofluorescent localization of P-gp in non-tumor and RCC cell lines.</b> In RPTEC-TERT1 cells (<b>A</b>), P-gp expression was seen dispersed throughout the cytoplasm, with no predominant localization in particular cellular structures. In CAKI-1 (<b>B</b>) and 786-O (<b>C</b>) cells, P-gp expression was seemingly confined to the nuclear region of the cells, which was evident from the co-localization of the nuclear tracer (blue) and the P-gp stain (red). Panels <b>A1</b>, <b>B1</b>, and <b>C1</b> represent a composite image, including a nuclear tracer (Hoechst33342, blue), f-actin (Alexa488, green), and P-gp (Alexa555, red). Panels <b>A2</b>, <b>B2</b>, and <b>C2</b> represent a composite image of P-gp and f-actin. Panels <b>A3</b>, <b>B3</b>, and <b>C3</b> represent a composite image of P-gp and the nuclear staining. The images in panels <b>A1</b>, <b>B1</b>, and <b>C1</b> were acquired with a magnification of 20× to provide an overview of cell density and monolayer growth. The images in panels <b>A2,A3</b>, <b>B2,B3</b>, and <b>C2,C3</b> were acquired with a magnification of 60× to capture the cellular morphology in detail.</p> Full article ">Figure 8
<p><b>Effects of methylation and EGFR inhibition on the toxicity of cisplatin</b>. In RPTEC-TERT1 (<b>A</b>) and CAKI-1 (<b>B</b>) cells, decitabine enhanced toxicity, while in 786-O cells (<b>C</b>), no effect was observed.</p> Full article ">Figure 9
<p><b>Accumulation of ASP<sup>+</sup> and Hoecht33342 after decitabine or CTX treatment.</b> In RPTEC-TERT1 cells, ASP<sup>+</sup> (<b>A</b>) uptake was upregulated after decitabine treatment and Hoechst33342 (<b>B</b>) retention was reduced after CTX exposure. In CAKI-1 cells, decitabine yielded an increased ASP<sup>+</sup> uptake (<b>C</b>) while CTX increased Hoechst33342 retention (<b>D</b>). In 786-O cells, decitabine reduced ASP<sup>+</sup> uptake (<b>E</b>) and Hoechst33342 retention (<b>F</b>). The accumulation of fluorescent substrates after treatment with decitabine or CTX was low as opposed to the untreated samples. Therefore, the relative activity scale (y-axis) was set at 0.5-fold to demonstrate the differences. All assays represent a minimum of three independent assays performed in triplicate.</p> Full article ">Figure 10
<p><b>Effects of P-gp inhibition on cisplatin toxicity.</b> The inhibitor valspodar reduced the cisplatin toxicity in RPTEC-TERT1 cells (<b>A</b>), but had no effect on CAKI-1 (<b>B</b>) and 786-O (<b>C</b>) cells. Similar effects were observed with the fluorescently labeled apoptosis marker annexin V, after a 200 µM cisplatin exposure. Annexin V fluorescence increased in RPTEC-TERT1 cells after cisplatin treatment (<b>D</b>,<b>G2</b>) relative to the untreated cells (<b>G1</b>), an effect that was absent when cisplatin was co-exposed with the P-gp inhibitor valspodar (<b>G3</b>). Annexin V fluorescence was not affected by cisplatin in CAKI-1 (<b>E</b>) and 786-O (<b>F</b>) cells. The concentration of 200 µM was selected in order to induce limited toxicity and prevent excessive cell death. Images in panels G1–3 were acquired with a magnification of 20×.</p> Full article ">Figure A1
<p><b>Gene expression of key and relevant renal drug transporters of primary RPTECs after treatment with methylation or EGFR inhibitors.</b> Expression is depicted as absolute, with 2<sup>−ΔCt</sup> values expressed in a log10 scale (<b>A</b>,<b>C</b>) and as relative, with 2<sup>−ΔΔCt</sup> values (fold) depicted in a linear scale (<b>B</b>). Results represent data collected from three independent experiments. (* <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure A2
<p><b>Uptake and efflux transport activities in HRPTECs.</b> Uptake of ASP<sup>+</sup> in the presence or absence of the OCT inhibitor imipramine (<b>A</b>). Uptake of calcein-AM in the presence or absence of the P-gp inhibitor valspodar (<b>B</b>). Uptake of Hoechst33342 in the presence or absence of the BCRP inhibitor KO143 (<b>C</b>). Functional data derived from fluorescent assays were fitted according to Michaelis–Menten kinetics to evaluate the activity. Functional data were obtained from a minimum of two independent assays, performed in triplicate.</p> Full article ">Figure A3
<p><b>In CAKI-1 cells, acidic pH reduces ASP<sup>+</sup> uptake and reduces cisplatin toxicity.</b> (<b>A</b>): Imipramine inhibition of ASP<sup>+</sup> was performed after 30 min of pre-incubation with Krebs buffer (pH = 6). (<b>B</b>): Cisplatin toxicity after pre-incubation with culture media (pH = 6) and subsequent drug exposure and recovery. Assays were performed as described in <a href="#sec4dot2-ijms-23-10177" class="html-sec">Section 4.2</a> and <a href="#sec4dot3-ijms-23-10177" class="html-sec">Section 4.3</a> except for the pH levels in the Krebs buffer and MEM culture media, which were adjusted to 6.</p> Full article ">Figure A4
<p><b>Accumulation of calcein after decitabine or CTX treatment in non-malignant and malignant RPTEC models</b>. No tangible differences were observed in calcein retention after pre-treatment with the inhibitors in non-malignant cells (<b>A</b>) or RCC cell lines CAKI-1(<b>B</b>) and 786-O (<b>C</b>).</p> Full article ">Figure A5
<p><b>EGFR gene expression in</b><b>non-malignant and malignant RPTEC models.</b> Relative to the expression of the housekeeping (reference) gene ACTB, EGFR expression was upregulated in tumor cells (CAKI-1 and 786-O) when compared to the non-malignant cells (RPTEC-TERT1) and primary RPTECs. Expression is depicted as absolute, with 2<sup>−ΔCt</sup> values expressed in a linear scale.</p> Full article ">
14 pages, 3211 KiB  
Article
Increased Lipids in Chlamydomonas reinhardtii by Multiple Regulations of DOF, LACS2, and CIS1
by Bin Jia, Jianbo Yin, Xiaolian Li, Yingling Li, Xingcai Yang, Chengxiang Lan and Ying Huang
Int. J. Mol. Sci. 2022, 23(17), 10176; https://doi.org/10.3390/ijms231710176 - 5 Sep 2022
Cited by 9 | Viewed by 2725
Abstract
Microalgal lipids are essential for biofuel and dietary supplement production. Lipid engineering for higher production has been studied for years. However, due to the complexity of lipid metabolism, single-gene engineering gradually encounters bottlenecks. Multiple gene regulation is more beneficial to boosting lipid accumulation [...] Read more.
Microalgal lipids are essential for biofuel and dietary supplement production. Lipid engineering for higher production has been studied for years. However, due to the complexity of lipid metabolism, single-gene engineering gradually encounters bottlenecks. Multiple gene regulation is more beneficial to boosting lipid accumulation and further clarifying the complex regulatory mechanism of lipid biosynthesis in the homeostasis of lipids, carbohydrates, and protein metabolism. Here, three lipid-related genes, DOF, LACS2, and CIS, were co-regulated in Chlamydomonas reinhartii by two circles of transformation to overexpress DOF and knock down LACS2 and CIS simultaneously. With the multiple regulations of these genes, the intracellular lipids and FA content increased by 142% and 52%, respectively, compared with CC849, whereas the starch and protein contents decreased by 45% and 24%. Transcriptomic analysis showed that genes in TAG and FA biosynthesis were up-regulated, and genes in starch and protein metabolism were down-regulated. This revealed that more carbon precursor fluxes from starch and protein metabolism were redirected towards lipid synthesis pathways. These results showed that regulating genes in various metabolisms contributed to carbon flux redirection and significantly improved intracellular lipids, demonstrating the potential of multiple gene regulation strategies and providing possible candidates for lipid overproduction in microalgae. Full article
(This article belongs to the Section Molecular Biology)
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<p>The mRNA expression patterns of <span class="html-italic">crdof, cracs2,</span> and <span class="html-italic">cis1</span> in the DLC-4 and WT when strains were treated with HS (HS) and without HS (NH). (<b>A</b>) The expression levels of LACS2. (<b>B</b>) The expression levels of DOF. (<b>C</b>) The expression levels of CIS1. Statistical significance (one-way ANOVA) is represented by asterisks (*, **, and *** indicate a difference at <span class="html-italic">p</span> ≤ 0.05, <span class="html-italic">p</span> ≤ 0.01. and <span class="html-italic">p</span> ≤ 0.005, respectively). (<b>D</b>) Growth curves of the DLC-4 and WT treated with heat shock (HDLC-4 and HWT) or without heat shock (DLC-4 and WT) at the mid-log phase. Error bars depict SD.</p> Full article ">Figure 2
<p>Intracellular neutral lipid contents in the DLC-4 and WT when they were treated with HS (HDLC and HWT) or without HS (DLC and WT) at the mid-log phase. (<b>A</b>) The lipid profile of strains from day 4 to day 8. Results are shown as the fold changes of the fluorescence intensities of strain DLC-4 compared with those of the WT strain after BODIFY staining. Error bars depict SD. (<b>B</b>) The distribution of intracellular neutral lipids in the DLC-4 and WT. Lipid droplets were stained by BODIFY505/515 and visualized by a confocal microscope. Each scale bar indicates 10 μm.</p> Full article ">Figure 3
<p>Analysis of the TAG in the DLC-4 and WT when they were treated with HS (HDLC and HWT) or without HS (DLC and WT). (<b>A</b>): Lipids were separated on a TLC plate and visualized with iodine. The lipids from strains in the nitrogen-replete and nitrogen-deprived conditions are shown separately in the left and right panels. (<b>B</b>): The TAG content of the DLC-4 and WT separated from TLC. +N: nitrogen-replete condition, −N: nitrogen-deprived condition. (<b>C</b>): The TAG content of DLC-4 and WT under nitrogen-deprived conditions when strains were treated with HS (HS) and without HS (NH). TAG was stained by BODIPY and measured by a microplate reader. Statistical significance (one-way ANOVA) is represented by asterisks (*, **, and *** indicate a difference at <span class="html-italic">p</span> ≤ 0.05, <span class="html-italic">p</span> ≤ 0.01, and <span class="html-italic">p</span> ≤ 0.005, respectively). Error bars depict SD.</p> Full article ">Figure 4
<p>Total FA and lipid profiles in the DLC-4 and WT. (<b>A</b>) Total FAs of the DLC-4 and WT when strains were treated with HS (HS) and without HS (NH). (<b>B</b>) The profiles of the total lipids in the DLC-4 and WT when strains were treated with HS. (<b>C</b>) The profiles of the total lipids in the DLC-4 and WT when strains were treated without HS. Statistical significance (one-way ANOVA) is represented by asterisks (* and ** indicate a difference at <span class="html-italic">p</span> ≤ 0.05 and <span class="html-italic">p</span> ≤ 0.01, respectively). Error bars depict SD.</p> Full article ">Figure 5
<p>KEGG pathway classification and enrichment of DEGs from DLC vs. WT. (<b>A</b>) KEGG pathway classification of DEGs. (<b>B</b>) KEGG enrichment of DEGs involved in the lipid metabolism. The Q value ranges from 0 to 0.3. A Q value closer to zero suggests more considerable enrichment. Pathways with Q values ≤ 0.05 were defined as significantly enriched pathways for the DEGs. (<b>C</b>) The clustering heatmap of DEGs related to lipid metabolism. For the clustering heatmap, normalized counts were rescaled between −3 and 3; clustering was based on Pearson correlation.</p> Full article ">Figure 6
<p>The qRT-PCR analysis of the expression levels of key genes involved in lipid metabolism in the DLC-4 and WT when strains were treated with HS (HS) and without HS (NH). Statistical significance (one-way ANOVA) is represented by asterisks (*, **, and *** indicate a difference at <span class="html-italic">p</span> ≤ 0.05, <span class="html-italic">p</span> ≤ 0.01, and <span class="html-italic">p</span> ≤ 0.005, respectively). Error bars depict SD.</p> Full article ">Figure 7
<p>Schematic representation of the multiple regulations of DOF, LACS2, and CIS1.</p> Full article ">
17 pages, 1853 KiB  
Review
CRISPR-Based Genome Editing and Its Applications in Woody Plants
by Tian Min, Delight Hwarari, Dong’ao Li, Ali Movahedi and Liming Yang
Int. J. Mol. Sci. 2022, 23(17), 10175; https://doi.org/10.3390/ijms231710175 - 5 Sep 2022
Cited by 16 | Viewed by 4819
Abstract
CRISPR/Cas-based genome editing technology provides straightforward, proficient, and multifunctional ways for the site-directed modification of organism genomes and genes. The application of CRISPR-based technology in plants has a vast potential value in gene function research, germplasm innovation, and genetic improvement. The complexity of [...] Read more.
CRISPR/Cas-based genome editing technology provides straightforward, proficient, and multifunctional ways for the site-directed modification of organism genomes and genes. The application of CRISPR-based technology in plants has a vast potential value in gene function research, germplasm innovation, and genetic improvement. The complexity of woody plants genome may pose significant challenges in the application and expansion of various new editing techniques, such as Cas9, 12, 13, and 14 effectors, base editing, particularly for timberland species with a long life span, huge genome, and ploidy. Therefore, many novel optimisms have been drawn to molecular breeding research based on woody plants. This review summarizes the recent development of CRISPR/Cas applications for essential traits, including wood properties, flowering, biological stress, abiotic stress, growth, and development in woody plants. We outlined the current problems and future development trends of this technology in germplasm and the improvement of products in woody plants. Full article
(This article belongs to the Special Issue New Advance on Functional Genomics and Genome Editing in Plant)
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<p>Representative schematic diagrams of CRISPR/Cas editing. (<b>A</b>) Cas9 applies the PAM and sgRNA to cleave the target DNA and produce a DBS, which is repaired by NHEJ or HDR; (<b>B</b>) Cas12a uses a RuvC domain under the guidance of crRNA without the participation of tracrRNA to cleave dsDNA, producing a sticky end; (<b>C</b>) Cas13a targets RNA in the nucleus; (<b>D</b>) CRISPR/Cas14a targets ssDNA cleavage under the direction of sgRNA, and does not require a PAM sequence, producing SSB; (<b>E</b>) CBE system compliments the single base replacement of the target site C-T (G-A), cleaves a single target locus, and produces a staggering cut; (<b>F</b>) ABE system compliments a single base substitution of A-G (T-C), cleaving at the targeted loci, and displaces the DNA fragment, leaving a staggering end in dsDNA (Provided by <a href="http://BioRender.com" target="_blank">BioRender.com</a>; accessed on 14 March 2022).</p> Full article ">Figure 2
<p>CRISPR/Cas system delivery technology, transferring foreign recombinant DNA by the CRISPR/Cas9. From the left side, (1) agrobacterium tumefaciens, (2) gene gun, (3) virus vector, (4) nanoparticles, (5) PEG, and (6) liposome carrying the targeted gene into the plant cell, which is further cultured (Provided by <a href="http://BioRender.com" target="_blank">BioRender.com</a>; accessed on 14 March 2022).</p> Full article ">Figure 3
<p>Features of CRISPR-based genome editing in woody plants (Provided by Microsoft PowerPoint 2019).</p> Full article ">
18 pages, 4594 KiB  
Article
Distress-Mediated Remodeling of Cardiac Connexin-43 in a Novel Cell Model for Arrhythmogenic Heart Diseases
by Carl-Mattheis Wahl, Constanze Schmidt, Markus Hecker and Nina D. Ullrich
Int. J. Mol. Sci. 2022, 23(17), 10174; https://doi.org/10.3390/ijms231710174 - 5 Sep 2022
Cited by 10 | Viewed by 10954
Abstract
Gap junctions and their expression pattern are essential to robust function of intercellular communication and electrical propagation in cardiomyocytes. In healthy myocytes, the main cardiac gap junction protein connexin-43 (Cx43) is located at the intercalated disc providing a clear direction of signal spreading [...] Read more.
Gap junctions and their expression pattern are essential to robust function of intercellular communication and electrical propagation in cardiomyocytes. In healthy myocytes, the main cardiac gap junction protein connexin-43 (Cx43) is located at the intercalated disc providing a clear direction of signal spreading across the cardiac tissue. Dislocation of Cx43 to lateral membranes has been detected in numerous cardiac diseases leading to slowed conduction and high propensity for the development of arrhythmias. At the cellular level, arrhythmogenic diseases are associated with elevated levels of oxidative distress and gap junction remodeling affecting especially the amount and sarcolemmal distribution of Cx43 expression. So far, a mechanistic link between sustained oxidative distress and altered Cx43 expression has not yet been identified. Here, we propose a novel cell model based on murine induced-pluripotent stem cell-derived cardiomyocytes to investigate subcellular signaling pathways linking cardiomyocyte distress with gap junction remodeling. We tested the new hypothesis that chronic distress, induced by rapid pacing, leads to increased reactive oxygen species, which promotes expression of a micro-RNA, miR-1, specific for the control of Cx43. Our data demonstrate that Cx43 expression is highly sensitive to oxidative distress, leading to reduced expression. This effect can be efficiently prevented by the glutathione peroxidase mimetic ebselen. Moreover, Cx43 expression is tightly regulated by miR-1, which is activated by tachypacing-induced oxidative distress. In light of the high arrhythmogenic potential of altered Cx43 expression, we propose miR-1 as a novel target for pharmacological interventions to prevent the maladaptive remodeling processes during chronic distress in the heart. Full article
(This article belongs to the Special Issue New Insights into Cardiovascular Diseases in Basic Research)
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<p>Live imaging of ROS production in HL-1 cells and iPSC-CMs at different conditions. (<b>A</b>) Single confocal images recorded during a time-course experiment of imaging ROS-dependent oxidation of H<sub>2</sub>DCF to DCF in unstimulated HL-1 cells. (<b>B</b>) Time-dependent increase in DCF fluorescence triggered by TBHP perfusion of HL-1 cells. (<b>C</b>) Time course of the average DCF fluorescence during imaging of control (black) and TBHP-treated (red) HL-1 cells (<span class="html-italic">n</span> = 3). (<b>D</b>) Single confocal images of unstimulated iPSC-CMs during a time course recording. (<b>E</b>) Time-dependent increase in DCF fluorescence triggered by TBHP perfusion of iPSC-CMs. (<b>F</b>) Time course of the average DCF fluorescence during imaging of control (black) and TBHP-treated (red) iPSC-CMs (<span class="html-italic">n</span> = 3). The red bar indicates the time of TBHP perfusion (<span class="html-italic">n</span> numbers of repeated experiments).</p> Full article ">Figure 2
<p>Sample traces of Ca<sup>2+</sup> transients (black) and cell shortening (grey) in iPSC-CMs during spontaneous activity. (<b>A</b>) Ca<sup>2+</sup> transients and contractions were simultaneously measured by fura-2 and edge detection using the IonOptix setup. (<b>B</b>) Sample line scan image and corresponding line profile (black) of iPSC-CMs during electrical stimulation at 1 Hz and tachypaced at 4 Hz (<b>C</b>). Ca<sup>2+</sup> transients were measured with fluo-4 using a LSCM. Stimulation frequencies are indicated by red arrows in the top traces.</p> Full article ">Figure 3
<p>Tachypacing-induced ROS generation in iPSC-CMs. (<b>A</b>) Outline of the ROS imaging experiment indicating the four different phases. (<b>B</b>) Confocal images of cells loaded with the ROS-indicator H<sub>2</sub>DCF were taken immediately after pacing of the cells for 24 h. (<b>C</b>) Statistical analysis of the mean fluorescence intensities of all images and conditions recorded in phase 4a (Ctrl: <span class="html-italic">n</span> = 5, 1 Hz: <span class="html-italic">n</span> = 5, 4 Hz: <span class="html-italic">n</span> = 5, 4 Hz + ebselen, E: <span class="html-italic">n</span> = 3), normalized to Ctrl. (<b>D</b>) Experimental protocol. (<b>E</b>) Sample images taken at the beginning (<span class="html-italic">t</span> = 0) and end (<span class="html-italic">t</span> = 50 s) of ROS imaging. (<b>F</b>) Mean traces of the time-dependent increase in DCF fluorescence intensity after phase 4b. (<b>G</b>) Slope calculated from the data shown in (<b>F</b>) (<span class="html-italic">n</span> = 3 in all four conditions), normalized to Ctrl. Please note that a negative slope, as seen in one sample of the ebselen-treated cells, can be explained as a slight bleaching effect on the fluorescent dye during repetitive image acquisition. Please note: due to different acquisition modes, the fluorescence intensity cannot be compared between (<b>B</b>,<b>E</b>) (<span class="html-italic">n</span> numbers of repeated experiments; ns—no significant difference; * statistically significant difference).</p> Full article ">Figure 4
<p>Effect of tachypacing on Cx43 expression and miR-1 levels in iPSC-CMs. Top traces show the experimental protocol. (<b>A</b>) Representative Western blot and analysis of total Cx43 protein abundance in control (Ctrl, 1 Hz) and tachypaced cells after 24 h and 48 h of electrical stimulation, respectively, with statistical analysis of Cx43 protein normalized to GAPDH and Ctrl. (<b>B</b>) Relative miR-1 expression by Ctrl and tachypaced iPSC-CMs after 48 h, normalized to Ctrl. (<b>C</b>) Same experiment as in (<b>B</b>) but performed in the presence of 1 μM ebselen (<span class="html-italic">n</span> numbers of repeated experiments are indicated in the columns; ns—no significant difference; * statistically significant difference).</p> Full article ">Figure 5
<p>Effect of antimiR-1 on Cx43 expression in iPSC-CMs. (<b>A</b>) Representative confocal images of Cx43 immunofluorescence analysis in iPSC-CMs incubated with either scrambled control antimiR (scr-miR) or different concentrations of the miR-1-neutralizing antisense oligonucleotide (antimiR-1). (<b>B</b>) Statistical summary of the immunofluorescence analysis. Data are normalized to exposure to the scrambled control anti-miR (Ctrl) (<span class="html-italic">n</span> numbers of analyzed images of 3 individual experiments are indicated in the columns). (<b>C</b>) Representative Western blot and analysis of total Cx43 protein showing relative increase in total Cx43 protein expression by neutralization of miR-1 using 50 nM anti-miR-1. Data are normalized to GAPDH and Ctrl (<span class="html-italic">n</span> numbers of repeated experiments are indicated in the columns; * statistically significant difference).</p> Full article ">Figure 6
<p>Fluorescence recovery after photobleaching (FRAP) of calcein in iPSC-CMs. (<b>A</b>) The upper trace and scheme show the experimental protocol, underneath representative confocal sample images are shown for scrambled control oligonucleotide-treated iPSC-CMs (Control), anti-miR-1-treated cells and Cx43-overexpressing cells (Cx43-OE). Images were recorded at 4 different time points during the recordings. (<b>B</b>) Time course of FRAP for the three different conditions. The green bar and arrow indicate the time of photobleaching (Control: <span class="html-italic">n</span> = 40, anti-miR-1: <span class="html-italic">n</span> = 31, Cx43-OE: <span class="html-italic">n</span> = 23; <span class="html-italic">n</span> measurements from 3 different cultures).</p> Full article ">Figure 7
<p>Summary of the proposed signaling cascade of chronic tachypacing-induced Cx43 expression regulation: tachy-stimulation leads to oxidative distress or peroxide stress (designated as ROS) possibly by the indicated enzymes or signaling pathways (e.g., NOX-2, NOX-4, TGF-β, mitochondria, grey arrows). The resulting increase in peroxides (H<sub>2</sub>O<sub>2</sub>, lipid hydroperoxides) triggers enhanced miR-1 expression, presumably by transcriptional upregulation of miR-1. miR-1 leads to enhanced degradation of Cx43 mRNA, resulting in reduced Cx43 protein expression and gap junction formation at the membrane. Putative parallel influences of ROS and/or miR-1 especially on the excitation-contraction coupling mechanism, are indicated in orange. <span class="html-italic">Abbreviations:</span> IP<sub>3</sub>R: IP<sub>3</sub>-receptor, LTCC: L-type Ca<sup>2+</sup> channel, M: mitochondria, N: nucleus, Na<sub>v</sub>1.5: voltage-dependent Na<sup>+</sup> channel, NOX-2/4: NADPH oxidases 2 and 4, PLB: phospholamban, RyR: ryanodine receptor, SERCA: SR Ca<sup>2+</sup> ATPase, SR: sarcoplasmic reticulum, TGF-β: transforming growth factor beta, TT: transverse tubules.</p> Full article ">
21 pages, 1663 KiB  
Review
Maternal Supplementation of Probiotics, Prebiotics or Postbiotics to Prevent Offspring Metabolic Syndrome: The Gap between Preclinical Results and Clinical Translation
by Ying-Hua Huang, You-Lin Tain and Chien-Ning Hsu
Int. J. Mol. Sci. 2022, 23(17), 10173; https://doi.org/10.3390/ijms231710173 - 5 Sep 2022
Cited by 7 | Viewed by 4691
Abstract
Metabolic syndrome (MetS) is an extremely prevalent complex trait and it can originate in early life. This concept is now being termed the developmental origins of health and disease (DOHaD). Increasing evidence supports that disturbance of gut microbiota influences various risk factors of [...] Read more.
Metabolic syndrome (MetS) is an extremely prevalent complex trait and it can originate in early life. This concept is now being termed the developmental origins of health and disease (DOHaD). Increasing evidence supports that disturbance of gut microbiota influences various risk factors of MetS. The DOHaD theory provides an innovative strategy to prevent MetS through early intervention (i.e., reprogramming). In this review, we summarize the existing literature that supports how environmental cues induced MetS of developmental origins and the interplay between gut microbiota and other fundamental underlying mechanisms. We also present an overview of experimental animal models addressing implementation of gut microbiota-targeted reprogramming interventions to avert the programming of MetS. Even with growing evidence from animal studies supporting the uses of gut microbiota-targeted therapies start before birth to protect against MetS of developmental origins, their effects on pregnant women are still unknown and these results require further clinical translation. Full article
(This article belongs to the Special Issue New Approaches for the Management of Metabolic Syndrome and Related Health Alterations: A Focus in Probiotics, Parabiotics and Postbiotics)
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<p>A schematic depiction delineating early-life environmental cues that may cause the developmental programming in different organ systems leading to MetS and associated disorders in adult life.</p> Full article ">Figure 2
<p>A summary of the currently available reprogramming interventions for metabolic syndrome of developmental origins.</p> Full article ">
27 pages, 1836 KiB  
Review
Connexins and Glucose Metabolism in Cancer
by Jennifer C. Jones and Thomas M. Bodenstine
Int. J. Mol. Sci. 2022, 23(17), 10172; https://doi.org/10.3390/ijms231710172 - 5 Sep 2022
Cited by 9 | Viewed by 4633
Abstract
Connexins are a family of transmembrane proteins that regulate diverse cellular functions. Originally characterized for their ability to mediate direct intercellular communication through the formation of highly regulated membrane channels, their functions have been extended to the exchange of molecules with the extracellular [...] Read more.
Connexins are a family of transmembrane proteins that regulate diverse cellular functions. Originally characterized for their ability to mediate direct intercellular communication through the formation of highly regulated membrane channels, their functions have been extended to the exchange of molecules with the extracellular environment, and the ability to modulate numerous channel-independent effects on processes such as motility and survival. Notably, connexins have been implicated in cancer biology for their context-dependent roles that can both promote or suppress cancer cell function. Moreover, connexins are able to mediate many aspects of cellular metabolism including the intercellular coupling of nutrients and signaling molecules. During cancer progression, changes to substrate utilization occur to support energy production and biomass accumulation. This results in metabolic plasticity that promotes cell survival and proliferation, and can impact therapeutic resistance. Significant progress has been made in our understanding of connexin and cancer biology, however, delineating the roles these multi-faceted proteins play in metabolic adaptation of cancer cells is just beginning. Glucose represents a major carbon substrate for energy production, nucleotide synthesis, carbohydrate modifications and generation of biosynthetic intermediates. While cancer cells often exhibit a dependence on glycolytic metabolism for survival, cellular reprogramming of metabolic pathways is common when blood perfusion is limited in growing tumors. These metabolic changes drive aggressive phenotypes through the acquisition of functional traits. Connections between glucose metabolism and connexin function in cancer cells and the surrounding stroma are now apparent, however much remains to be discovered regarding these relationships. This review discusses the existing evidence in this area and highlights directions for continued investigation. Full article
(This article belongs to the Special Issue Metabolism Signaling and Gene Regulation in Human Health)
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<p>Canonical role of connexins in gap junctional intercellular communication. (<b>A</b>) Connexin proteins contain four transmembrane domains (TD), intracellular facing N- and C-terminal regions, two extracellular loops (EL) and one cytoplasmic loop (CL). C-terminal tail and CL are sites of post-translational modifications that affect many aspects of connexin biology. Six connexins form a hexameric channel known as a connexon which forms a highly regulated aqueous pore. (<b>B</b>) Connexons are inserted into the plasma membrane and connexons on opposing cell membranes connect to form a gap junction, capable of direct transfer of small molecules and ions. Interactions between ELs are important for formation of gap junctions while other domains mediate different aspects of channel permeability and gating properties. (<b>C</b>) Connexons composed of a single type of connexin are termed homomeric while connexons formed from multiple connexins are heteromeric. Gap junctions formed between the same homomeric channels are termed homotypic while connexon docking of differing homomeric channels, or between heteromeric channels are termed heterotypic. The multitude of permutations of these combinations affect the type and directionality of substrate transfer between cells.</p> Full article ">Figure 2
<p>Role of gap junctions during cancer progression. (<b>A</b>) GJIC is an essential part of cellular and tissue homeostasis in healthy cells. (<b>B</b>) During tumor initiation, loss of GJIC in cancer cells and downregulation of connexin expression is commonly observed. However, the functions of connexins and gap junctions can suppress or facilitate tumor growth. (<b>C</b>) During the multi-step process of metastasis, GJIC has been shown to facilitate interactions with other cells (e.g., endothelial cells) to promote the steps of the metastatic cascade. (<b>D</b>) Upon reaching a secondary site, adaptation to a new microenvironment must occur and evidence has shown that GJIC between metastatic cells and stromal cells of these sites can promote survival and outgrowth, completing the process of metastasis.</p> Full article ">Figure 3
<p>Examples of relationships between glucose availability, metabolism and connexin function. (<b>A</b>) Gap junctions allow for exchange of metabolites with a tumor. As tumors grow, regions of hypoxia develop depending on tumor vasculature and angiogenesis. Active GJIC allows for the transfer of glucose and bicarbonate from perfused regions to hypoxic areas. Additionally, metabolites such as lactate can be shuttled to normoxic areas for utilization and reduced buildup, collectively normalizing metabolism in the absence of an integrated blood supply, Refs. [<a href="#B156-ijms-23-10172" class="html-bibr">156</a>,<a href="#B157-ijms-23-10172" class="html-bibr">157</a>,<a href="#B158-ijms-23-10172" class="html-bibr">158</a>]. (<b>B</b>) In a process referred to as the reverse Warburg effect, cancer cells induce glycolytic metabolism in stromal cells such as cancer associated fibroblasts (CAFs). This in turn leads to stimulation of oxidative metabolism in cancer cells through the transfer of metabolites. In some models this effect has been shown to be dependent on unidirectional transfer through GJIC, Ref. [<a href="#B162-ijms-23-10172" class="html-bibr">162</a>]. (<b>C</b>) Connexin expression has been shown capable of controlling metabolic enzymes within cancer cells that affect glycolysis. Although much remains to be determined, many of these effects are mediated through channel-independent functions of connexins. Metabolic reprogramming, or inhibition of this process, affects the adaptability of cancer cells to metabolic changes in the microenvironment, Refs. [<a href="#B179-ijms-23-10172" class="html-bibr">179</a>,<a href="#B180-ijms-23-10172" class="html-bibr">180</a>,<a href="#B181-ijms-23-10172" class="html-bibr">181</a>]. (<b>D</b>) Adaptation to reduced glucose availability can lead to increased connexin expression, membrane localization and GJIC with associated increases in phenotypic qualities such as survival and invasion, Refs. [<a href="#B224-ijms-23-10172" class="html-bibr">224</a>,<a href="#B225-ijms-23-10172" class="html-bibr">225</a>]. Abbreviations: Glu, glucose; HCO<sub>3</sub><sup>−</sup>, bicarbonate; Lac, lactate; CAC, citric acid cycle. References correspond to related material discussed in this article.</p> Full article ">Figure 4
<p>Emerging connections between connexin regulation and glucose metabolism. Data for some relationships is limited in cancer and requires continued research to better define the significance of these associations, which are likely to exhibit interdependence.</p> Full article ">
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