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Volume 21
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Int. J. Mol. Sci., Volume 21, Issue 7 (April-1 2020) – 400 articles

Cover Story (view full-size image): Many integral membrane proteins, including ion channels, are modulated structurally and functionally by the surrounding lipids, but the molecular mechanisms behind it remain largely unknown. Here, we have reviewed the multiple alterations lipids cause on the prokaryotic KcsA, possibly the best studied ion channel undergoing lipid modulation. Interestingly, most such effects have in common the initial binding of anionic lipids to two key arginine residues located at non-annular lipid binding sites on the channel protein. Thus, processes as different as the inactivation of channel K+ currents or the assembly of clusters from individual KcsA channels depend on such lipid binding. View this paper.
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10 pages, 2401 KiB  
Article
Therapeutic Effects of Human Amniotic Epithelial Stem Cells in a Transgenic Mouse Model of Alzheimer’s Disease
by Ka Young Kim, Yoo-Hun Suh and Keun-A Chang
Int. J. Mol. Sci. 2020, 21(7), 2658; https://doi.org/10.3390/ijms21072658 - 10 Apr 2020
Cited by 21 | Viewed by 4379
Abstract
Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is characterized clinically by cognitive decline and pathologically by the development of amyloid plaques. AD is the most common cause of dementia among older people. However, there is currently no cure for AD. In this study, [...] Read more.
Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is characterized clinically by cognitive decline and pathologically by the development of amyloid plaques. AD is the most common cause of dementia among older people. However, there is currently no cure for AD. In this study, we aimed to elucidate the therapeutic effects of human amniotic epithelial stem cells (hAESCs) in a transgenic mouse model of AD. Tg2576 transgenic (Tg) mice underwent behavioral tests, namely the Morris water maze and Y-maze tests, to assess their cognitive function. In the Morris water maze test, hAESC-treated Tg mice exhibited significantly shorter escape latencies than vehicle-treated Tg mice. In the Y-maze test, hAESC-treated Tg mice exhibited significantly higher rate of spontaneous alteration than vehicle-treated Tg mice, while the total number of arm entries did not differ between the groups. Furthermore, Congo red staining revealed that hAESCs injection reduced the number of amyloid plaques present in the brains of Tg mice. Finally, beta-secretase (BACE) activity was significantly decreased in Tg mice at 60 min after hAESCs injection. In this study, we found that intracerebral injection of hAESCs alleviated cognitive impairment in a Tg2576 mouse model of AD. Our results indicate that hAESCs injection reduced amyloid plaques caused by reduced BACE activity. These results indicate that hAESCs may be a useful therapeutic agent for the treatment of AD-related memory impairment. Full article
(This article belongs to the Special Issue Applications of Mesenchymal Stem Cells in Neuroscience)
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Figure 1
<p>Experimental schemes. (<b>A</b>) Experimental scheme for the behavioral tests and intracerebral injections. (<b>B</b>) Stereotaxic coordinates of the intracerebral injection sites. The red arrows indicate the injected site of hAESCs (or vehicle).</p> Full article ">Figure 2
<p>Effects of hAESCs transplantation on cognitive deficits in Tg2576 Alzheimer’s disease transgenic mice. (<b>A</b>) The Morris water maze (MWM) test was performed 3 months after intracerebral injection. Training trials were performed on 6 consecutive days, and the escape latencies of the mice were recoded. (<b>B</b>) The MWM probe test was conducted 48 h after the final training trial. (<b>C</b>) Representative swim paths in the MWM. (<b>D</b>) The total number of arm entries in the Y-maze was recorded. (<b>E</b>) The rate of spontaneous alternation in the Y-maze was calculated. All data represent the mean ± standard error of the mean (<span class="html-italic">n</span> = 10–15 per group). Data from the MWM test were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons and data from the Y-maze test were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. WT-vehicle, <sup>*</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>**</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>***</sup> <span class="html-italic">p</span> &lt; 0.001; WT-hAESC, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; Tg-hAESC, <sup><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.05, <sup><span>$</span><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.01 compared to Tg-vehicle. ANOVA, analysis of variance; hAESCs, human amniotic epithelial stem cells; Tg, transgenic.</p> Full article ">Figure 2 Cont.
<p>Effects of hAESCs transplantation on cognitive deficits in Tg2576 Alzheimer’s disease transgenic mice. (<b>A</b>) The Morris water maze (MWM) test was performed 3 months after intracerebral injection. Training trials were performed on 6 consecutive days, and the escape latencies of the mice were recoded. (<b>B</b>) The MWM probe test was conducted 48 h after the final training trial. (<b>C</b>) Representative swim paths in the MWM. (<b>D</b>) The total number of arm entries in the Y-maze was recorded. (<b>E</b>) The rate of spontaneous alternation in the Y-maze was calculated. All data represent the mean ± standard error of the mean (<span class="html-italic">n</span> = 10–15 per group). Data from the MWM test were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons and data from the Y-maze test were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. WT-vehicle, <sup>*</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>**</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>***</sup> <span class="html-italic">p</span> &lt; 0.001; WT-hAESC, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; Tg-hAESC, <sup><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.05, <sup><span>$</span><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.01 compared to Tg-vehicle. ANOVA, analysis of variance; hAESCs, human amniotic epithelial stem cells; Tg, transgenic.</p> Full article ">Figure 3
<p>Effects of hAESCs injection on the number of amyloid plaques in the hippocampus and cortex of Tg2576 Alzheimer’s disease transgenic mice. (<b>A</b>) Hippocampal and cortical sections were stained with Congo red to detect amyloid plaques (<b>a</b>–<b>d</b>: upper panel, prefrontal cortex (PFC) and hippocampus (HP), <b>e</b>–<b>h</b>: lower panel, entorhinal cortex (EC), scale bar = 200 μm). The arrows indicate Congo red-stained amyloid plaques. Square with the dotted line contains enlarged images of the brain sections of Tg-vehicle mice (<b>i</b> &amp; <b>j</b>) (scale bar = 100 μm). (<b>B</b>) The number of plaques in the hippocampal and cortical regions of the Tg-vehicle and Tg-hAESC groups was counted. (<b>C</b>) BACE activity levels were analyzed 60 min after injection in the Tg-vehicle and Tg-hAESC groups. All data represent the mean ± standard error of the mean (<span class="html-italic">n</span> = 10–15 per group). All statistical analyses were performed using the unpaired t test. <sup>*</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>**</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>***</sup> <span class="html-italic">p</span> &lt; 0.001. BACE, beta-secretase; hAESC, human amniotic epithelial stem cells; Tg, transgenic.</p> Full article ">Figure 3 Cont.
<p>Effects of hAESCs injection on the number of amyloid plaques in the hippocampus and cortex of Tg2576 Alzheimer’s disease transgenic mice. (<b>A</b>) Hippocampal and cortical sections were stained with Congo red to detect amyloid plaques (<b>a</b>–<b>d</b>: upper panel, prefrontal cortex (PFC) and hippocampus (HP), <b>e</b>–<b>h</b>: lower panel, entorhinal cortex (EC), scale bar = 200 μm). The arrows indicate Congo red-stained amyloid plaques. Square with the dotted line contains enlarged images of the brain sections of Tg-vehicle mice (<b>i</b> &amp; <b>j</b>) (scale bar = 100 μm). (<b>B</b>) The number of plaques in the hippocampal and cortical regions of the Tg-vehicle and Tg-hAESC groups was counted. (<b>C</b>) BACE activity levels were analyzed 60 min after injection in the Tg-vehicle and Tg-hAESC groups. All data represent the mean ± standard error of the mean (<span class="html-italic">n</span> = 10–15 per group). All statistical analyses were performed using the unpaired t test. <sup>*</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>**</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>***</sup> <span class="html-italic">p</span> &lt; 0.001. BACE, beta-secretase; hAESC, human amniotic epithelial stem cells; Tg, transgenic.</p> Full article ">
19 pages, 1768 KiB  
Review
A Review of SARS-CoV-2 and the Ongoing Clinical Trials
by Yung-Fang Tu, Chian-Shiu Chien, Aliaksandr A. Yarmishyn, Yi-Ying Lin, Yung-Hung Luo, Yi-Tsung Lin, Wei-Yi Lai, De-Ming Yang, Shih-Jie Chou, Yi-Ping Yang, Mong-Lien Wang and Shih-Hwa Chiou
Int. J. Mol. Sci. 2020, 21(7), 2657; https://doi.org/10.3390/ijms21072657 - 10 Apr 2020
Cited by 520 | Viewed by 50242
Abstract
The sudden outbreak of 2019 novel coronavirus (2019-nCoV, later named SARS-CoV-2) in Wuhan, China, which rapidly grew into a global pandemic, marked the third introduction of a virulent coronavirus into the human society, affecting not only the healthcare system, but also the global [...] Read more.
The sudden outbreak of 2019 novel coronavirus (2019-nCoV, later named SARS-CoV-2) in Wuhan, China, which rapidly grew into a global pandemic, marked the third introduction of a virulent coronavirus into the human society, affecting not only the healthcare system, but also the global economy. Although our understanding of coronaviruses has undergone a huge leap after two precedents, the effective approaches to treatment and epidemiological control are still lacking. In this article, we present a succinct overview of the epidemiology, clinical features, and molecular characteristics of SARS-CoV-2. We summarize the current epidemiological and clinical data from the initial Wuhan studies, and emphasize several features of SARS-CoV-2, which differentiate it from SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), such as high variability of disease presentation. We systematize the current clinical trials that have been rapidly initiated after the outbreak of COVID-19 pandemic. Whereas the trials on SARS-CoV-2 genome-based specific vaccines and therapeutic antibodies are currently being tested, this solution is more long-term, as they require thorough testing of their safety. On the other hand, the repurposing of the existing therapeutic agents previously designed for other virus infections and pathologies happens to be the only practical approach as a rapid response measure to the emergent pandemic, as most of these agents have already been tested for their safety. These agents can be divided into two broad categories, those that can directly target the virus replication cycle, and those based on immunotherapy approaches either aimed to boost innate antiviral immune responses or alleviate damage induced by dysregulated inflammatory responses. The initial clinical studies revealed the promising therapeutic potential of several of such drugs, including favipiravir, a broad-spectrum antiviral drug that interferes with the viral replication, and hydroxychloroquine, the repurposed antimalarial drug that interferes with the virus endosomal entry pathway. We speculate that the current pandemic emergency will be a trigger for more systematic drug repurposing design approaches based on big data analysis. Full article
(This article belongs to the Section Molecular Microbiology)
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<p>Overview of symptomatic, radiological and laboratory characteristics of COVID-19.</p> Full article ">Figure 2
<p>Overview of the repurposed therapeutic drugs undergoing clinical trial against COVID-19 in the context of host pathways and virus replication mechanisms.</p> Full article ">
18 pages, 3603 KiB  
Article
A Novel Ruthenium Based Coordination Compound Against Pathogenic Bacteria
by Vishma Pratap Sur, Aninda Mazumdar, Pavel Kopel, Soumajit Mukherjee, Petr Vítek, Hana Michalkova, Markéta Vaculovičová and Amitava Moulick
Int. J. Mol. Sci. 2020, 21(7), 2656; https://doi.org/10.3390/ijms21072656 - 10 Apr 2020
Cited by 14 | Viewed by 4910
Abstract
The current epidemic of antibiotic-resistant infections urges to develop alternatives to less-effective antibiotics. To assess anti-bacterial potential, a novel coordinate compound (RU-S4) was synthesized using ruthenium-Schiff base-benzimidazole ligand, where ruthenium chloride was used as the central atom. RU-S4 was characterized by scanning electron [...] Read more.
The current epidemic of antibiotic-resistant infections urges to develop alternatives to less-effective antibiotics. To assess anti-bacterial potential, a novel coordinate compound (RU-S4) was synthesized using ruthenium-Schiff base-benzimidazole ligand, where ruthenium chloride was used as the central atom. RU-S4 was characterized by scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDS), and Raman spectroscopy. Antibacterial effect of RU-S4 was studied against Staphylococcus aureus (NCTC 8511), vancomycin-resistant Staphylococcus aureus (VRSA) (CCM 1767), methicillin-resistant Staphylococcus aureus (MRSA) (ST239: SCCmecIIIA), and hospital isolate Staphylococcus epidermidis. The antibacterial activity of RU-S4 was checked by growth curve analysis and the outcome was supported by optical microscopy imaging and fluorescence LIVE/DEAD cell imaging. In vivo (balb/c mice) infection model prepared with VRSA (CCM 1767) and treated with RU-S4. In our experimental conditions, all infected mice were cured. The interaction of coordination compound with bacterial cells were further confirmed by cryo-scanning electron microscope (Cryo-SEM). RU-S4 was completely non-toxic against mammalian cells and in mice and subsequently treated with synthesized RU-S4. Full article
(This article belongs to the Special Issue Staphylococcus aureus Infection: Pathogenesis, Antimicrobial Resistance and Diagnostics)
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Figure 1
<p>(<b>a</b>) benzimidazole ligand structure, (<b>b</b>) Schiff base ligand structure, (<b>c</b>) possible structure of RU-S4.</p> Full article ">Figure 2
<p>Scanning electron microscopy image for RU-S4 in different scales: (<b>a</b>) scale 500 nm, (<b>b</b>) scale 1 µm, (<b>c</b>) scale 2 µm.</p> Full article ">Figure 3
<p>Raman spectra and listed bands of Ru-S4 complex as obtained by the 514.5 nm excitation.</p> Full article ">Figure 4
<p>Bacterial growth curve and viability curve (<b>a</b>–<b>d</b>); The growth curve of VRSA (CCM 1767), MRSA (ST239: SCCmecIIIA), <span class="html-italic">S. aureus</span> (NCTC 8511), <span class="html-italic">Staphylococcus epidermidis</span> respectively (<b>e</b>–<b>h</b>); Viability percentage of VRSA (CCM 1767), MRSA (ST239: SCCmecIIIA), <span class="html-italic">S. aureus</span> (NCTC 8511), hospital sample <span class="html-italic">Staphylococcus epidermidis</span> respectively. Data represent the mean ± SD, <span class="html-italic">n</span> = 3.</p> Full article ">Figure 5
<p>Cytotoxicity for RU-S4 against the human cell line.</p> Full article ">Figure 6
<p>Percentage of hemolysis of blood cells treated with RU-S4 in a blood sample.</p> Full article ">Figure 7
<p>Optical microscopy image for RU-S4 treatment against <span class="html-italic">S. aureus</span> (NCTC 8511), MRSA (ST239: SCCmecIIIA), VRSA (CCM 1767), and untreated cells. No morphological changes were seen in untreated cells whereas the treated bacterial cells were ruptured and almost no visible structured cells can be seen. Scale bar is 5 µm.</p> Full article ">Figure 8
<p>LIVE/DEAD cell imaging for RU-S4 treatment against <span class="html-italic">S. aureus</span> (NCTC 8511), MRSA (ST239: SCCmecIIIA), and VRSA (CCM 1767). Green cells define living cells whether red cells stand for dead cells. Scale bar is 10 µm.</p> Full article ">Figure 9
<p>Scanning electron microscope (SEM) image: (<b>a</b>) left panel shows untreated coccus cells (VRSA) (CCM 1767), (<b>b</b>) right panel shows treated coccus (VRSA) (CCM 1767).</p> Full article ">Figure 10
<p>In vivo infection model preparation, treatment, and recovery: (<b>a</b>) after bacterial infection dose, (<b>b</b>) day 1 Infection initiation, (<b>c</b>) day 2 Infection and wound growth, (<b>d</b>) day 3 inflammation and swelling, (<b>e</b>) day 6 no significant changes were observed, (<b>f</b>) day 9 wound started to heal, (<b>g</b>) day 12 wound started to heal, (<b>h</b>) day 15 fully recovered.</p> Full article ">Figure 11
<p>In vivo experiment where MMP Sense fluoresces in infected mice not in control and with time intervals the fluorescence disappears due to the recovery.</p> Full article ">
6 pages, 227 KiB  
Editorial
Salicylic Acid Signalling in Plants
by Tibor Janda, Gabriella Szalai and Magda Pál
Int. J. Mol. Sci. 2020, 21(7), 2655; https://doi.org/10.3390/ijms21072655 - 10 Apr 2020
Cited by 65 | Viewed by 6606
Abstract
Ten articles published in the “Special Issue: Salicylic Acid Signalling in Plants” are summarized, in order to get a global picture about the mode of action of salicylic acid in plants, and about its interaction with other stress-signalling routes. Its ecological aspects and [...] Read more.
Ten articles published in the “Special Issue: Salicylic Acid Signalling in Plants” are summarized, in order to get a global picture about the mode of action of salicylic acid in plants, and about its interaction with other stress-signalling routes. Its ecological aspects and possible practical use are also discussed. Full article
(This article belongs to the Special Issue Salicylic Acid Signalling in Plants)
17 pages, 5636 KiB  
Article
The Interactions between the Antimicrobial Peptide P-113 and Living Candida albicans Cells Shed Light on Mechanisms of Antifungal Activity and Resistance
by Kuang-Ting Cheng, Chih-Lung Wu, Bak-Sau Yip, Ya-Han Chih, Kuang-Li Peng, Su-Ya Hsu, Hui-Yuan Yu and Jya-Wei Cheng
Int. J. Mol. Sci. 2020, 21(7), 2654; https://doi.org/10.3390/ijms21072654 - 10 Apr 2020
Cited by 23 | Viewed by 4241
Abstract
In the absence of proper immunity, such as in the case of acquired immune deficiency syndrome (AIDS) patients, Candida albicans, the most common human fungal pathogen, may cause mucosal and even life-threatening systemic infections. P-113 (AKRHHGYKRKFH), an antimicrobial peptide (AMP) derived from [...] Read more.
In the absence of proper immunity, such as in the case of acquired immune deficiency syndrome (AIDS) patients, Candida albicans, the most common human fungal pathogen, may cause mucosal and even life-threatening systemic infections. P-113 (AKRHHGYKRKFH), an antimicrobial peptide (AMP) derived from the human salivary protein histatin 5, shows good safety and efficacy profiles in gingivitis and human immunodeficiency virus (HIV) patients with oral candidiasis. However, little is known about how P-113 interacts with Candida albicans or its degradation by Candida-secreted proteases that contribute to the fungi’s resistance. Here, we use solution nuclear magnetic resonance (NMR) methods to elucidate the molecular mechanism of interactions between P-113 and living Candida albicans cells. Furthermore, we found that proteolytic cleavage of the C-terminus prevents the entry of P-113 into cells and that increasing the hydrophobicity of the peptide can significantly increase its antifungal activity. These results could help in the design of novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications. Full article
(This article belongs to the Special Issue Selected Papers from the 8th Asia-Pacific NMR Symposium (APNMR): Recent Advances in NMR Spectroscopy)
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Figure 1

Figure 1
<p><sup>1</sup>H-<sup>15</sup>N HSQC NMR spectra of 0.25 mM P-113 peptides presented in (<b>a</b>) broth only, (<b>b</b>) 10<sup>7</sup> colony-forming units (CFU)/mL of <span class="html-italic">Candida Albicans</span>, and (<b>c</b>) 10<sup>7</sup> CFU/mL of <span class="html-italic">C. albicans</span> + 0.5 mM pepstatin A at 301 K for 24 h. The chemical shifts of P-113 peptides moved dramatically after <span class="html-italic">C. albicans</span> titration. However, these shifts were inhibited by the protease inhibitor pepstatin A.</p> Full article ">Figure 2
<p>Strip plots representative of three-dimensional, amide-detected heteronuclear NMR spectra for backbone assignment. Inter- and intraresidual correlations of P-113 residues were obtained from correlating (<b>a</b>) an HNCA spectrum to (<b>b</b>) an HN(CO)CA spectrum. The blue line indicates the sequential walk. A segment of P-113 after degradation by <span class="html-italic">C. albicans</span> at 301 K is shown, depicting lost connectivity between the pairs of residues Y7-K8, K8-R9, and K10-F11.</p> Full article ">Figure 3
<p><sup>15</sup>N–<sup>13</sup>C CON spectra of the 0.25 mM P-113 peptides presented in (<b>a</b>) Sabouraud dextrose (SD) broth only, and (<b>b</b>) 10<sup>7</sup> CFU/mL of <span class="html-italic">C. albicans</span> at 301 K for 24 h. A comparison of the two spectra clearly shows that cross-peaks of Y7-R8, R8-K9, and R10-F11 were lost by degradation, indicating the cleavage sites. (<b>c</b>) <span class="html-italic">C. albicans</span> proteinase cleavage sites of P-113. Solid arrows indicate the cleavage sites.</p> Full article ">Figure 4
<p>Time-course analysis of 0.25 mM P-113 cleavage following interaction with 10<sup>7</sup> CFU/mL of <span class="html-italic">C. albicans</span> by HSQC spectroscopy (left panels), confocal laser microscopy (middle panels), and scanning electron microscopy (right panels). The bar corresponds to 5 μm. An overlay of <sup>1</sup>H-<sup>1</sup>5N HSQC spectra for 0.25 mM of P-113 in SD broth only (blue) and broth with <span class="html-italic">C. albicans</span> at 301 K at different time points (red) is shown. Perturbations of chemical shifts were observed over 5 h of incubation with <span class="html-italic">C. albicans</span> cells. Cell morphology did not change over the 5 h incubation period.</p> Full article ">Figure 5
<p><sup>1</sup>H-<sup>15</sup>N HSQC spectra for the different selectively unlabeled samples of P-113 treated with <span class="html-italic">C. albicans</span> at 301 K for 24 h. Blue peaks represent for uniformly <sup>15</sup>N-labeled P-113: (<b>a</b>) green peaks for histidine selectively unlabeled P-113; (<b>b</b>) brown peaks for arginine selectively unlabeled P-113; (<b>c</b>) red peaks for lysine selectively unlabeled P-113.</p> Full article ">Figure 6
<p>Time-dependent decrease in the intensity of <sup>15</sup>N-<sup>1</sup>H NMR signals of P-113 residues during incubation with <span class="html-italic">C. albicans</span>.</p> Full article ">Figure 7
<p>Time killing activity of FITC-labeled P-113 peptide and its FITC-labeled derivatives against <span class="html-italic">Candida albicans</span>. Cells were treated with 1× MIC of (<b>a</b>) P-113 (black circle), FITC-labeled N-terminal truncated P-113 (black square), FITC-labeled C-terminal truncated P-113 (black triangle), and (<b>b</b>) treated with FITC-labeled Bip-P-113 (white square), and FITC-labeled Dip-P-113 (white triangle). Killed cells (%) = (cell number in peptide-free control − cell number in sample)/(cell number in peptide-free control) × 100. Error bars represent the standard errors of the mean.</p> Full article ">Figure 8
<p>Localizations of FITC truncated P-113 peptides in <span class="html-italic">C. albicans</span>. Fluorescence microscopy of <span class="html-italic">C. albicans</span> (10<sup>7</sup> CFU/mL) incubated at 28 °C for 30 min with 50 μM of FITC-truncated P-113 or FITC only. The left panels show a bright field, the middle panels show FITC images, and the right panels show merged images. The bar corresponds to 5 μm.</p> Full article ">Figure 9
<p>Fluorescence of <span class="html-italic">C. albicans</span> FITC P-113, FITC N-terminal truncated P-113, and FITC C-terminal truncated P-113 groups determined by flow cytometry analysis and expressed as fold increase compared to the control group incubated with FITC only. <span class="html-italic">C. labicans</span> (5 × 10<sup>6</sup> CFU/mL) was incubated at 28 °C for 30 min with 50 μM of FITC P-113 or their derivatives. The results are presented as the means ± standard deviations (<span class="html-italic">n</span> = 3) of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001 compared to the control group; ### <span class="html-italic">p</span> &lt; 0.001 compared to P-113; ns = no significant difference.</p> Full article ">Figure 10
<p>(<b>a</b>) Localizations of FITC peptides in in <span class="html-italic">Candida albicans</span>. Confocal microscopy was used to visualize 10<sup>7</sup> CFU/mL of <span class="html-italic">C. albicans</span> were incubated at 28 °C for 5 min with 50 μM of FITC-conjugated peptides. The left panels show a bright field, the middle panels show FITC images, and the right panels show merged images. The bar corresponds to 5 μm. (<b>b</b>) Scanning electron microscopic micrographs of <span class="html-italic">C. albicans</span> only as a control and treated with Bip-P-113 and Dip-P-113 peptides (Bip = β-(4,4β-biphenyl)alanine; Dip = β-diphenylalanine). Each figure is magnified ×8000.</p> Full article ">Figure 11
<p>Model of the proposed interactions of P-113 and Bip-P-113 with <span class="html-italic">Candida albicans</span>. P-113 peptides recognize and bind to Ssa1/2 proteins and then translocate to the cytoplasm, leading to cell death. However, some of P-113 peptides are degraded by <span class="html-italic">C. albicans</span> secreted aspartic proteases (Saps). In contrast, Bip-P-113 accumulates in cell membrane and induces pore formation.</p> Full article ">
14 pages, 1852 KiB  
Review
Circulating Tumor Cell Clusters: United We Stand Divided We Fall
by Samuel Amintas, Aurélie Bedel, François Moreau-Gaudry, Julian Boutin, Louis Buscail, Jean-Philippe Merlio, Véronique Vendrely, Sandrine Dabernat and Etienne Buscail
Int. J. Mol. Sci. 2020, 21(7), 2653; https://doi.org/10.3390/ijms21072653 - 10 Apr 2020
Cited by 81 | Viewed by 6515
Abstract
The presence of circulating tumor cells (CTCs) and CTC clusters, also known as tumor microemboli, in biological fluids has long been described. Intensive research on single CTCs has made a significant contribution in understanding tumor invasion, metastasis tropism, and intra-tumor heterogeneity. Moreover, their [...] Read more.
The presence of circulating tumor cells (CTCs) and CTC clusters, also known as tumor microemboli, in biological fluids has long been described. Intensive research on single CTCs has made a significant contribution in understanding tumor invasion, metastasis tropism, and intra-tumor heterogeneity. Moreover, their being minimally invasive biomarkers has positioned them for diagnosis, prognosis, and recurrence monitoring tools. Initially, CTC clusters were out of focus, but major recent advances in the knowledge of their biogenesis and dissemination reposition them as critical actors in the pathophysiology of cancer, especially metastasis. Increasing evidence suggests that “united” CTCs, organized in clusters, resist better and carry stronger metastatic capacities than “divided” single CTCs. This review gathers recent insight on CTC cluster origin and dissemination. We will focus on their distinct molecular package necessary to resist multiple cell deaths that all circulating cells normally face. We will describe the molecular basis of their increased metastatic potential as compared to single CTCs. We will consider their clinical relevance as prognostic biomarkers. Finally, we will propose future directions for research and clinical applications in this promising topic in cancer. Full article
(This article belongs to the Special Issue Circulating Tumor Cells: Pathological, Molecular and Functional Characteristics)
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Figure 1
<p>Origin and dissemination of circulating tumor cell (CTC) clusters. Cell aggregates detach from the primary tumor site and metastases by cell jamming to produce homotypic monoclonal or polyclonal tumor clusters. Released cells can also interact with stromal or immune cells in the inflammatory peri-tumoral infiltrate forming heterotypic clusters. Heterotypic cluster formation can also occur in blood vessels by association with circulating immune cells, and possibly with platelets. Intravasation of CTC clusters can occur by invadopodia and macrophage lead or through leaky blood vessels common in tumor microenvironment. In the bloodstream, clustering strengthens CTCs by anoïkis resistance, shear stress resistance, and immune escape and enhances their stemness, resulting in boosted metastatic potential. After extravasation in tissue with favorable microenvironment conditions, clusters can form monoclonal or polyclonal metastasis depending on their initial nature. This figure was performed using free online Servier Medical Art at <a href="http://www.servier.com" target="_blank">www.servier.com</a>.</p> Full article ">Figure 2
<p>Molecular differences between CTC clusters and single CTCs. CTC clusters like single CTCs harbor the molecular hallmark of their primary tumor. By contrast, multiple pathways like cell–cell adhesion (desmosomes), stemness (surface markers and associated transcription factors), and proliferation (higher KI67) are up-regulated in CTC clusters. On the other hand, apoptosis and immune activation pathways like Major Histocompatibility Complex MHC type II presentation, T-cell activation, and Tumor Necrosis Factor TNF signaling are down-regulated. Epithelial to mesenchymal transition status of CTC clusters is crucial for single CTC migratory property but is debated for clusters. This figure was performed using free online Servier Medical Art at <a href="http://www.servier.com" target="_blank">www.servier.com</a>.</p> Full article ">Figure 3
<p>CTC clusters in clinical perspective. In the course of patient management, CTC cluster enumeration individualized from CTC detection might provide additional noninvasive information on patient prognosis, and might be a good companion biomarker of metastatic disease assessment. Further individual characterization of cells within CTC clusters will be helpful to measure intra-tumor heterogeneity and drug sensitivity/resistance. This figure was performed using free online Servier Medical Art at <a href="http://www.servier.com" target="_blank">www.servier.com</a>.</p> Full article ">
21 pages, 4622 KiB  
Article
Functional Analysis of IRF1 Reveals its Role in the Activation of the Type I IFN Pathway in Golden Pompano, Trachinotus ovatus (Linnaeus 1758)
by Ke-Cheng Zhu, Nan Zhang, Bao-Suo Liu, Liang Guo, Hua-Yang Guo, Shi-Gui Jiang and Dian-Chang Zhang
Int. J. Mol. Sci. 2020, 21(7), 2652; https://doi.org/10.3390/ijms21072652 - 10 Apr 2020
Cited by 13 | Viewed by 3410
Abstract
Interferon (IFN) regulatory factor 1 (IRF1), a transcription factor with a novel helix–turn–helix DNA-binding domain, plays a crucial role in innate immunity by regulating the type I IFN signaling pathway. However, the regulatory mechanism through which IRF1 regulates type I IFN in fish [...] Read more.
Interferon (IFN) regulatory factor 1 (IRF1), a transcription factor with a novel helix–turn–helix DNA-binding domain, plays a crucial role in innate immunity by regulating the type I IFN signaling pathway. However, the regulatory mechanism through which IRF1 regulates type I IFN in fish is not yet elucidated. In the present study, IRF1 was characterized from golden pompano, Trachinotus ovatus (designated ToIRF1), and its immune function was identified to elucidate the transcriptional regulatory mechanism of ToIFNa3. The full-length complementary DNA (cDNA) of IRF1 is 1763 bp, including a 900-bp open reading frame (ORF) encoding a 299-amino-acid polypeptide. The putative protein sequence has 42.7–71.7% identity to fish IRF1 and possesses a representative conserved domain (a DNA-binding domain (DBD) at the N-terminus). The genomic DNA sequence of ToIRF1 consists of eight exons and seven introns. Moreover, ToIRF1 is constitutively expressed in all examined tissues, with higher levels being observed in immune-relevant tissues (whole blood, gill, and skin). Additionally, Cryptocaryon irritans challenge in vivo increases ToIRF1 expression in the skin as determined by Western blotting (WB); however, protein levels of ToIRF1 in the gill did not change significantly. The subcellular localization indicates that ToIRF1 is localized in the nucleus and cytoplasm with or without polyinosinic/polycytidylic acid (poly (I:C)) induction. Furthermore, overexpression of ToIRF1 or ToIFNa3 shows that ToIRF1 can notably activate ToIFNa3 and interferon signaling molecule expression. Promoter sequence analysis finds that several interferon stimulating response element (ISRE) binding sites are present in the promoter of ToIFNa3. Additionally, truncation, point mutation, and electrophoretic mobile shift (EMSA) assays confirmed that ToIRF1 M5 ISRE binding sites are functionally important for ToIFNa3 transcription. These results may help to illuminate the roles of teleost IRF1 in the transcriptional mechanisms of type I IFN in the immune process. Full article
(This article belongs to the Section Molecular Biology)
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Figure 1
<p>Amino-acid sequences of interferon (IFN) regulatory factor 1 (IRF1) homologs in vertebrates. The overline indicates the conserved DNA-binding domain (DBD) signature (amino acids (aa) 1–113), which contains six conserved tryptophan residues. It is also called the IRF domain (yellow underlay). Identical (asterisks) and similar (: or ∙) residues identified by the CLUSTAL W program are indicated. The Latin abbreviation and accession numbers are listed in <a href="#app1-ijms-21-02652" class="html-app">Table S1</a> (<a href="#app1-ijms-21-02652" class="html-app">Supplementary Materials</a>).</p> Full article ">Figure 2
<p>Evolutionary status, structure, and tissue expression of the <span class="html-italic">ToIRF1</span> gene. (<b>A</b>) Genome structure analysis of IRF1 genes according to the phylogenetic relationship. Lengths of exons and introns of each IRF1 gene are displayed proportionally. Different colored boxes and lines represent exons and introns, respectively. The identical colored boxes represent homologous sequences. (<b>B</b>) Gene transcription of ToIRF1 in various tissues. The 12 tissues are whole blood (Bl), gill (Gi), head-kidney (Ki), small intestine (In), stomach (St), brain (Br), male gonad (Mg), fin (Fi), female gonad (Fg), spleen (Sp), white muscle (Wm), and liver (Li). Different letters indicate significant differences.</p> Full article ">Figure 3
<p>Western blot analysis of ToIRF1 proteins in gill and skin after infection with <span class="html-italic">Cryptocaryon irritans</span> (0, 3, 6, 12 hpi, 1 d, 2 d, and 3 d) in <span class="html-italic">Trachinotus ovatus</span>. (<b>A</b>) Western blot analysis was used to detect ToIRF1 expression. The experiment was divided into two groups, the control and infection groups. Lines 1 and 3 indicate the protein levels of ToIRF1 in gill and skin, respectively. Lines 2 and 4 indicate the levels of reference protein. (<b>B</b>) The corresponding ratio of gray values of ToIRF1 and GAPDH proteins; bars on the same group with different letters are significantly different from one another (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 4
<p>Subcellular localization of ToIRF1 in pompano cells. golden pompano snout tissue (GPS) cells seeded onto microscopy cover glass in six-well plates were transfected with 2 μg of pEGFP-N3 or pEGFP-ToIRF1 plasmid, which were considered as the control (<b>A</b>) and experimental (<b>B</b>) group, respectively. After 24 h, the cells were stimulated with polyinosinic/polycytidylic acid (poly (I:C)) (5 µg/mL) for 12 h, and then the cells were fixed and subjected to confocal microscopy analysis. Green staining represents the ToIRF1 protein signal (<b>B</b>), and blue staining indicates the nucleus region. All experiments were repeated at least three times, with similar results.</p> Full article ">Figure 5
<p>Ectopic expression of ToIRF1 increased the expression of the <span class="html-italic">ToIFNɑ3</span> gene. (<b>A</b>) GPS cells were transfected with ToIRF1 and empty vector, and then cells were collected for RNA extraction and qRT-PCR. (<b>B</b>) Dual-luciferase activity was driven by the ToIFNa3-p1 sequence upon the transfection of pEGFP-ToIRF1 and pEGFP-N3 into GPS cells. All values are presented as the means ± SD (<span class="html-italic">n</span> = 3). Asterisks indicate that the values are significantly different from the individual controls (* <span class="html-italic">p</span> &lt; 0.05, and ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 6
<p>Overexpression of ToIRF1 altered the expression levels of interferon signaling molecules in GPS cells for 36 h. The expression levels of interferon signaling molecules, including <span class="html-italic">IFNa3</span>, <span class="html-italic">TRAF6</span>, <span class="html-italic">ISG15</span>, <span class="html-italic">Viperin1</span>, <span class="html-italic">Viperin2</span>, <span class="html-italic">Mavs</span>, and <span class="html-italic">MXI</span>, were examined using qRT-PCR analysis. The <span class="html-italic">EF-1α</span> gene was employed as an internal control. The messenger RNA (mRNA) expression level in GPS cells transfected with an empty vector was set as one-fold. Different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 7
<p>Activation of ToIRF1 in response to rIFNa3. (<b>A</b>) GPS cells were cultured in 6-cm culture dishes (2.5 × 10<sup>6</sup> cells/dish) overnight and then treated with rIFNa3 in a range of doses as indicated for 24 h. GPS cell extracts were used to detect IRF1 proteins by Western blot analysis. (<b>B</b>) The corresponding ratio of gray values of ToIRF1 and GAPDH proteins. All values are presented as the means ± SD (<span class="html-italic">n</span> = 3). Bars on the same group with different letters are significantly different from one another (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 8
<p>Promoter activity analysis of the <span class="html-italic">ToIFNa3</span> gene. The structure and transcriptional activity of <span class="html-italic">ToIFNa3</span> promoters. Five recombinant plasmids were constructed [<a href="#B28-ijms-21-02652" class="html-bibr">28</a>,<a href="#B29-ijms-21-02652" class="html-bibr">29</a>] and transfected with the transcription factor ToIRF1 into HEK 293T cells. Different colored boxes indicate ToIRF1 binding sites located in different truncation regions. All values are presented as the means ± SD (<span class="html-italic">n</span> = 3). Bars on the same group with different letters are significantly different from one another (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 9
<p>Effects of six mutants on <span class="html-italic">ToIFNa3-p2</span> promoter activity. Mutations of promoter sequences are according to Reference [<a href="#B28-ijms-21-02652" class="html-bibr">28</a>]. Data are presented as the means ± SD (<span class="html-italic">n</span> = 3). Different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 10
<p>Binding reactions of IRF1 and ToIFNa3 promoter. Biotin-labeled EMSA probes were incubated with lysates of HEK293T cells containing ToIRF1 protein. WT, wild-type probe; MT: mutated probe. 1, negative control; 2, positive control; 3, plus ToIFNa3-P2-WT5; 4, ToIFNa3-P2-WT5 plus ToIRF1-Flag; 5, plus ToIFNa3-P2-MT5; 6, ToIFNa3-P2-MT5 plus ToIRF1-Flag.</p> Full article ">
14 pages, 10401 KiB  
Article
The Role of Taste Receptor mTAS1R3 in Chemical Communication of Gametes
by Michaela Frolikova, Tereza Otcenaskova, Eliska Valasková, Pavla Postlerova, Romana Stopkova, Pavel Stopka and Katerina Komrskova
Int. J. Mol. Sci. 2020, 21(7), 2651; https://doi.org/10.3390/ijms21072651 - 10 Apr 2020
Cited by 9 | Viewed by 3230
Abstract
Fertilization is a multiple step process leading to the fusion of female and male gametes and the formation of a zygote. Besides direct gamete membrane interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves gamete communication via chemical [...] Read more.
Fertilization is a multiple step process leading to the fusion of female and male gametes and the formation of a zygote. Besides direct gamete membrane interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves gamete communication via chemical molecules triggering various signaling pathways. This work focuses on a mouse taste receptor, mTAS1R3, encoded by the Tas1r3 gene, as a potential receptor mediating chemical communication between gametes using the C57BL/6J lab mouse strain. In order to specify the role of mTAS1R3, we aimed to characterize its precise localization in testis and sperm using super resolution microscopy. The testis cryo-section, acrosome-intact sperm released from cauda epididymis and sperm which underwent the acrosome reaction (AR) were evaluated. The mTAS1R3 receptor was detected in late spermatids where the acrosome was being formed and in the acrosomal cap of acrosome intact sperm. AR is triggered in mice during sperm maturation in the female reproductive tract and by passing through the egg surroundings such as cumulus oophorus cells. This AR onset is independent of the extracellular matrix of the oocyte called zona pellucida. After AR, the relocation of mTAS1R3 to the equatorial segment was observed and the receptor remained exposed to the outer surroundings of the female reproductive tract, where its physiological ligand, the amino acid L-glutamate, naturally occurs. Therefore, we targeted the possible interaction in vitro between the mTAS1R3 and L-glutamate as a part of chemical communication between sperm and egg and used an anti-mTAS1R3-specific antibody to block it. We detected that the acrosome reacted spermatozoa showed a chemotactic response in the presence of L-glutamate during and after the AR, and it is likely that mTAS1R3 acted as its mediator. Full article
(This article belongs to the Special Issue Advances in Molecular Regulation of Spermatozoa Function)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The expression of <span class="html-italic">Tas1r3</span> and <span class="html-italic">Cd46</span> is highest in testicles as revealed by qPCR analysis of mRNA across 10 mouse tissues. Prostate (P), tongue (TON), liver (L), cauda epididymis (CAU), olfactory epithelia (OE), lymph tissue (NL), nasal-associated major preputial gland (PP), Vomeronasal organ (VNO), spleen (SP) and testis (T). Normalized to <span class="html-italic">Gapdh</span> (dashed red line), <span class="html-italic">n</span><sub>male</sub> = 5.</p> Full article ">Figure 2
<p>Localization of mTAS1R3 (green) and CD46 (red) in mouse sperm revealed by Confocal Microscopy and Structure Illumination Microscopy (SIM). During spermiogenesis (<b>a</b>) mTAS1R3 (green) and (<b>b</b>) CD46 (red) are localized in elongated and late spermatids with a formed acrosome (<b>c</b>) both proteins colocalize (yellow); for details, see enlarged area and white arrows pointing to the spermatids. (<b>d</b>) In epididymal sperm, mTAS1R3 (green) is present in the apical acrosome specifically in the acrosomal membranes and corresponds to (<b>e</b>) CD46 (red) localization. (<b>f</b>) The colocalization of both mTAS1R3 and CD46 pattern (yellow) is shown in acrosomal membranes defining the intact acrosome overlaying the nucleus (blue). (<b>g</b>) During the acrosome reaction mTAS1R3 (green) relocates into the equatorial segment, as well as (<b>h</b>) CD46 (red) and (<b>i</b>) their colocalization (yellow) is shown with nucleus (blue) overlay. (<b>j</b>) SIM imaging shows precise localization of mTAS1R3 (green) in the acrosomal membranes. (<b>k</b>) Huygens software was used for better visualization of mutual position of mTAS1R3 (green) and CD46 (red) in acrosomal cap area. White color shows the place of colocalization with CD46. CD46 was used as a marker of the acrosomal membranes. Scale bars represent (<b>a</b>–<b>c</b>) 80 μm, (<b>d</b>–<b>j</b>) 1 μm, (<b>k</b>) 2 μm.</p> Full article ">Figure 3
<p>Sperm chemotactic response to <span class="html-italic">L-glutamate</span>. (<b>a</b>) Special chemotactic chamber was developed for assessing sperm attraction to <span class="html-italic">L-glutamate</span>. Wells were filled with M2 medium containing spermatozoa (left) and with <span class="html-italic">L-glutamate</span> (right) and connected through the bridge. The black arrow represents the direction of sperm movement, whereas the orange triangle represents the concentration gradient of L-glutamate. SecureSeal imaging spacers are in grey. (<b>b</b>) Comparison of chemotactic response of acrosome-reacted sperm to <span class="html-italic">L-glutamate</span> in the presence or absence of a specific goat polyclonal anti-mouse mTAS1R3 antibody, n<sub>male</sub> = 5. The response to (<b>c,e</b>) acrosome-intact and (<b>d,f</b>) acrosome-reacted sperm to <span class="html-italic">L-glutamate</span> was analyzed in (<b>c,d</b>) 0.1 μM and (<b>e,f</b>) 500 μM concentrations.</p> Full article ">
13 pages, 1954 KiB  
Article
Karyopherin α-2 Mediates MDC1 Nuclear Import through a Functional Nuclear Localization Signal in the tBRCT Domain of MDC1
by Kamalakannan Radhakrishnan, Seon-Joo Park, Seok Won Kim, Gurusamy Hariharasudhan, Seo-Yeon Jeong, In Youb Chang and Jung-Hee Lee
Int. J. Mol. Sci. 2020, 21(7), 2650; https://doi.org/10.3390/ijms21072650 - 10 Apr 2020
Cited by 4 | Viewed by 3612
Abstract
Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin α-2 (KPNA2), a nucleocytoplasmic transport adaptor, and [...] Read more.
Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin α-2 (KPNA2), a nucleocytoplasmic transport adaptor, and showed that KPNA2 is necessary for MDC1 nuclear import. Thereafter, we identified a functional nuclear localization signal (NLS) between amino acid residues 1989–1994 of the two Breast Cancer 1 (BRCA1) carboxyl-terminal (tBRCT) domain of MDC1 and demonstrated disruption of this NLS impaired interaction between MDC1 and KPNA2 and reduced nuclear localization of MDC1. In KPNA2-depleted cells, the recruitment of MDC1, along with the downstream signaling p roteins Ring Finger Protein 8 (RNF8), 53BP1-binding protein 1 (53BP1), BRCA1, and Ring Finger Protein 168 (RNF168), to DNA damage sites was abolished. Additionally, KPNA2-depleted cells had a decreased rate of homologous recombination (HR) repair. Our data suggest that KPNA2-mediated MDC1 nuclear import is important for DDR signaling and DSB repair. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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Figure 1
<p>KPNA2 interacts with two BRCA1 carboxyl-terminal (tBRCT) domain of mediator of DNA damage checkpoint protein 1 (MDC1). (<b>A</b>,<b>B</b>) HeLa cells, with or without exposure to ionizing radiation (IR), and whole-cell lysates were subjected to immunoprecipitation using an anti-MDC1 (<b>A</b>) or anti-KPNA2 (<b>B</b>) antibodies, followed by Western blotting using antibodies against either KPNA2 or MDC1. (<b>C</b>,<b>D</b>) HA-MDC1 and GFP-KPNA2 were co-transfected into HEK293T cells and exposed to IR. Whole cell lysates were subjected to immunoprecipitation using anti-HA (<b>C</b>) or anti-GFP (<b>D</b>) antibodies, followed by Western blotting using antibodies against either HA or GFP. *, nonspecific band. **, IgG heavy chain band. (<b>E</b>) Schematic representation of domain structure of wild-type MDC1 and the various deletion mutations is shown. (<b>F</b>,<b>G</b>) GFP-KPNA2 were transfected into HEK293T cells along with indicated deletion mutants of MDC1 (<b>F</b>) or HA-MDC1-FHA and HA-MDC1-tBRCT (<b>G</b>). Cell lysates were immunoprecipitated using anti-HA antibody and analyzed by Western blotting using the indicated antibodies.</p> Full article ">Figure 2
<p>Impaired nuclear translocation of MDC1 in KPNA2-depleted cells. (<b>A</b>) Five siRNAs (KPNA2 siRNA #1 ~ KPNA2 siRNA #5), each designed against different region of the KPNA2 target gene, were transiently transfected into HeLa cells. Western blotting was carried out to determine expression levels of KPNA2 and MDC1 in control or KPNA2-depleted cells. α-tubulin was included as an internal control. (<b>B</b>) Control or KPNA2-depleted HeLa cells were irradiated with or without IR, and the cytosol and nuclear fractions were isolated. The cytosol and nuclear extracts were subjected to Western blotting using antibodies against MDC1 and KPNA2. Histone H3 and α-tubulin were used as positive controls for nuclear and cytoplasmic fraction, respectively. (<b>C</b>) Same cells, as described in (<b>B</b>), were fixed for immunofluorescence staining (IF) of MDC1. DAPI (4′,6-diamidino-2-phenylindole) was used for nuclear staining (left). Scale bar; 5 μm. Bars graph obtained from IF of control and KPNA2 siRNA transfected cells showing the percentage in different cellular localization (right).</p> Full article ">Figure 3
<p>Identification of functional NLS in MDC1. (<b>A</b>) A schematic representation of HA-tagged wild-type MDC1 (MDC1-WT) and NLS-deleted HA-MDC1 (MDC1-ΔNLS). Putative nuclear localization signal (NLS, PARERR) is shown. (<b>B</b>) GFP-KPNA2 were transfected into HEK293T cells along with HA-MDC1-WT or HA-MDC1-ΔNLS. Whole cell lysates were then subjected to immunoprecipitation using anti-HA antibody, followed by Western blotting using indicated antibodies. (<b>C</b>) HeLa cells were transfected with MDC1-WT or MDC1-ΔNLS, and the cytosol and nuclear fractions were isolated. The cytosol and nuclear extracts were subjected to Western blotting using indicated antibodies. (<b>D</b>,<b>E</b>) MDC1-WT or MDC1-ΔNLS-transfected HeLa cells were irradiated with IR and the number of IR-induced HA-MDC1 foci was calculated. Representative images (<b>D</b>) and quantification (<b>E</b>) of HA-MDC1 foci are shown. Scale bar; 5 μm. Data represent mean ± SD (<span class="html-italic">n</span> = 3), ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 4
<p>KPNA2 knockdown reduces IR-induced MDC1 foci formation. (<b>A</b>) Control and KPNA2-depleted HeLa cells were treated with or without exposure to 5 Gy of IR and fixed at the indicated time points. Immunostaining was performed using MDC1 antibody and Nuclei were stained DAPI (upper). The percent of cells containing &gt;10 nuclear MDC1 foci was then calculated (lower). Scale bar, 5 μm. (<b>B</b>) Control and KPNA2-depleted cells were transfected with GFP-KPNA2 and laser-microirradiated using 405 UV laser. The representative images were shown after laser-microirradiation. Scale bar; 5 μm. (<b>C</b>) Control or KPNA2-depleted HeLa cells were exposed to 5 Gy of IR for 1~3 h and immunostained with indicated antibodies. The representative images (upper) and percentage (lower) of cells containing &gt;5–10 nuclear RNF8, 53BP1, BRCA1, RNF168, or γ-H2AX foci were shown. Scale bar; 5 μm. Data are presented as means ± s.d. P value are based on two-tailed Student’s <span class="html-italic">t</span>-test: **<span class="html-italic">p</span> &lt; 0.01 ns, not significant.</p> Full article ">Figure 5
<p>KPNA2 promotes HR repair. (<b>A</b>) Control and KPNA2-depleted HeLa cells were exposed to the indicated dose of IR and assessed for colony forming ability. The cell viability of untreated cells is defined as 100%. Data presented as mean ± s.d. (<b>B</b>) Control and KPNA2-depleted HeLa cells were exposed to IR. After 24 h, immunostaining was performed using γ-H2AX antibody. The percentage of cells containing &gt;10 γ-H2AX foci was then calculated. Data are presented as means ± s.d. (<b>C</b>) Control and KPNA2-depleted HeLa cells were untreated or treated with 5 Gy γ-irradiation. At the indicated time points, cells were harvested to carry out comet assay under neutral condition. Comet images were captured using fluorescence microscopy, and comet tail moment was analyzed using Komet 5.5 analysis software. Representative comet images obtained at different time points are shown. Scale bar; 100 μm. Changes in the tail moments between control and KPNA2 knockdown cells after IR treatment are represented in histogram. Data presented as mean ± s.d. (<b>D</b>,<b>E</b>) DR-GFP-U2OS cells transfected with the indicated siRNA combinations. The level of endogenous KPNA2 and MDC1 were analyzed by Western blotting (left). The GFP-positive cells were measured by Fluorescence-activated cell sorting (FACS). Data represent mean ± s.d. (<span class="html-italic">n</span> = 3), ** <span class="html-italic">p</span> &lt; 0.01. ns, not significant.</p> Full article ">
13 pages, 3183 KiB  
Article
Combination of Arsenic Trioxide and Valproic Acid Efficiently Inhibits Growth of Lung Cancer Cells via G2/M-Phase Arrest and Apoptotic Cell Death
by Hyun Kyung Park, Bo Ram Han and Woo Hyun Park
Int. J. Mol. Sci. 2020, 21(7), 2649; https://doi.org/10.3390/ijms21072649 - 10 Apr 2020
Cited by 27 | Viewed by 3968
Abstract
Arsenic trioxide (ATO; As2O3) has anti-cancer effects in various solid tumors as well as hematological malignancy. Valproic acid (VPA), which is known to be a histone deacetylase inhibitor, has also anti-cancer properties in several cancer cells including lung cancer [...] Read more.
Arsenic trioxide (ATO; As2O3) has anti-cancer effects in various solid tumors as well as hematological malignancy. Valproic acid (VPA), which is known to be a histone deacetylase inhibitor, has also anti-cancer properties in several cancer cells including lung cancer cells. Combined treatment of ATO and VPA (ATO/VPA) could synergistically enhance anti-cancer effects and reduce ATO toxicity ATO. In this study, the combined anti-cancer effects of ATO and VPA (ATO/VPA) was investigated in NCI-H460 and NCI-H1299 lung cancer cells in vitro and in vivo. A combination of 3 μM ATO and 3 mM VPA (ATO/VPA) strongly inhibited the growths of both lung cancer cell types. DNA flow cytometry indicated that ATO/VPA significantly induced G2/M-phase arrest in both cell lines. In addition, ATO/VPA strongly increased the percentages of sub-G1 cells and annexin V-FITC positive cells in both cells. However, lactate dehydrogenase (LDH) release from cells was not increased in ATO/VPA-treated cells. In addition, ATO/VPA increased apoptosis in both cell types, accompanied by loss of mitochondrial membrane potential (MMP, ∆Ψm), activation of caspases, and cleavage of anti-poly ADP ribose polymerase-1. Moreover, a pan-caspase inhibitor, Z-VAD, significantly reduced apoptotic cell death induced by ATO/VPA. In the xenograft model, ATO/VPA synergistically inhibited growth of NCI-H460-derived xenograft tumors. In conclusion, the combination of ATO/VPA effectively inhibited the growth of lung cancer cells through G2/M-phase arrest and apoptotic cell death, and had a synergistic antitumor effect in vivo. Full article
(This article belongs to the Section Molecular Oncology)
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<p>Effects of arsenic trioxide (ATO) and valproic acid (VPA) on cell growth and necrotic cell death in NCI-H460 and NCI-H1299 cells. Exponentially growing cells were treated with the indicated doses of ATO or VPA for the indicated times. (<b>A</b>,<b>B</b>): Cellular growth changes in NCI-H460 and NCI-H1299 cells as assessed by MTT assays. (<b>C</b>–<b>F</b>): Necrotic cell death changes as assessed by LDH release and its activity. Lactate dehydrogenase (LDH) activity changes in ATO-treated NCI-H460 cells (<b>C</b>) and NCI-H1299 cells (<b>D</b>). (<b>E</b>,<b>F</b>): LDH activity changes in VPA-treated NCI-H460 cells (<b>E</b>) and NCI-H1299 cells (<b>F</b>). * <span class="html-italic">p</span> &lt; 0.05 as compared with the control group. (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 2
<p>Effects of ATO and VPA alone and in combination on cell morphology and cell cycle distributions. Exponentially growing NCI-H460 and NCI-H1299 cells were treated with 3 µM ATO and/or 3 mM VPA for 72 h. (<b>A</b>): Cell morphology changes were captured by an inverted microscope (40×). (<b>B</b>): Cell cycle distributions were measured by BD Accuri C6 flow cytometry (M1 regions show sub-G1 cells, M2: G1 phase, M3: S phase, M4: G2/M phase). (<b>C</b>): Percentages of G1, S, and G2/M phases in M2, M3, and M4 regions of <a href="#ijms-21-02649-f002" class="html-fig">Figure 2</a>B. (<b>D</b>): Percentages of sub-G1 cells in M1 regions of <a href="#ijms-21-02649-f002" class="html-fig">Figure 2</a>B. (<b>E</b>): LDH release in NCI-H460 and NCI-H1299 cells co-treated with ATO/VPA. * <span class="html-italic">p</span> &lt; 0.05 as compared with the control group. # <span class="html-italic">p</span> &lt; 0.05 as compared with cells treated with ATO or VPA. (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 3
<p>Effects of ATO and VPA alone and in combination on apoptosis and apoptosis-related proteins. (<b>A</b>): Exponentially growing NCI-H460 and NCI-H1299 cells were treated with 3 µM ATO and 3 mM VPA for 72 h. Annexin V-FITC/PI staining cells were measured by BD Accuri C6 flow cytometry. (<b>B</b>): Percentages of annexin V-FITC positive cells from <a href="#ijms-21-02649-f003" class="html-fig">Figure 3</a>A. (<b>C</b>): Expression levels of apoptosis-related proteins as analyzed by Western blot. NCI-H460 cells were treated with ATO and VPA alone and in combination, and NCI-H1299 cells were treated with ATO and VPA alone and in combination. * <span class="html-italic">p</span> &lt; 0.05 as compared with the control group. # <span class="html-italic">p</span> &lt; 0.05 as compared with cells treated with ATO or VPA. (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 4
<p>Effect of ATO and VPA alone and in combination on mitochondrial membrane potential (MMP) (∆Ψm). Exponentially growing cells were treated with 3 µM ATO and/or 3 mM VPA for 72 h. (<b>A</b>): Representative images of JC-1 (red and green) and DAPI (blue) in NCI-H1299 cells (100×). Red fluorescent images indicate higher MMP (∆Ψm) levels. Green fluorescent images show lower MMP (∆Ψm) levels. (<b>B</b>)and <b>C</b>: Graphs show the proportions of Rhodamine 123-negative (MMP (∆Ψm) loss) cells in NCI-H460 cells (<b>B</b>) and NCI-H1299 cells (<b>C</b>) as measured by BD Accuri C6 flow cytometry. * <span class="html-italic">p</span> &lt; 0.05 as compared with the control group. # <span class="html-italic">p</span> &lt; 0.05 as compared with cells treated with ATO or VPA. (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 5
<p>Effect of Z-VAD on cell death in ATO/VPA-treated lung cancer cells. Exponentially growing NCI-H460 and NCI-H1299 cells were co-treated with ATO/VPA in the presence or absence of 15 μM Z-VAD for 24 h. Sub-G1 and annexin V-stained cells were measured with BD Accuri C6 flow cytometry. (<b>A</b>): Percentages of sub-G1 cells. (<b>B</b>): LDH activity changes. (<b>C</b>): Percentages of annexin V-FITC positive cells. * <span class="html-italic">p</span> &lt; 0.05 as compared with the control group. # <span class="html-italic">p</span> &lt; 0.05 as compared with cells treated with ATO/VPA. (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 6
<p>The combination of ATO/VPA inhibits tumor growth in NCI-H460 xenografts from nude mice. (<b>A</b>): NCI-H460 cells (2 × 10<sup>6</sup>) were injected s.c. into flanks of 4 week-old female nude mice to establish tumors. Phosphate-buffered saline (PBS) (control), 5 mg/kg ATO, 400 mg/kg VPA, and ATO + VPA were injected i.p. every other day starting on day 10. (<b>B</b>,<b>C</b>): Body weight changes (<b>B</b>) and tumor volume changes (<b>C</b>) during 21 days after the first injection of drugs. (<b>D</b>): Images of representative tumors of PBS, ATO, ATO/VPA, and VPA injected mice.</p> Full article ">Figure 7
<p>Schematic diagram of ATO/VPA-induced cell growth inhibition in lung cancer cells.</p> Full article ">
46 pages, 2352 KiB  
Review
Multifaceted Role of PRDM Proteins in Human Cancer
by Amelia Casamassimi, Monica Rienzo, Erika Di Zazzo, Anna Sorrentino, Donatella Fiore, Maria Chiara Proto, Bruno Moncharmont, Patrizia Gazzerro, Maurizio Bifulco and Ciro Abbondanza
Int. J. Mol. Sci. 2020, 21(7), 2648; https://doi.org/10.3390/ijms21072648 - 10 Apr 2020
Cited by 44 | Viewed by 8219
Abstract
The PR/SET domain family (PRDM) comprise a family of genes whose protein products share a conserved N-terminal PR [PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1)] homologous domain structurally and functionally similar to the catalytic SET [...] Read more.
The PR/SET domain family (PRDM) comprise a family of genes whose protein products share a conserved N-terminal PR [PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1)] homologous domain structurally and functionally similar to the catalytic SET [Su(var)3-9, enhancer-of-zeste and trithorax] domain of histone methyltransferases (HMTs). These genes are involved in epigenetic regulation of gene expression through their intrinsic HMTase activity or via interactions with other chromatin modifying enzymes. In this way they control a broad spectrum of biological processes, including proliferation and differentiation control, cell cycle progression, and maintenance of immune cell homeostasis. In cancer, tumor-specific dysfunctions of PRDM genes alter their expression by genetic and/or epigenetic modifications. A common characteristic of most PRDM genes is to encode for two main molecular variants with or without the PR domain. They are generated by either alternative splicing or alternative use of different promoters and play opposite roles, particularly in cancer where their imbalance can be often observed. In this scenario, PRDM proteins are involved in cancer onset, invasion, and metastasis and their altered expression is related to poor prognosis and clinical outcome. These functions strongly suggest their potential use in cancer management as diagnostic or prognostic tools and as new targets of therapeutic intervention. Full article
(This article belongs to the Special Issue Zinc-Finger Proteins in Health and Disease: Focus on PRDMs)
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Figure 1

Figure 1
<p>PRDM proteins contribution in the mechanisms related to invasiveness and metastasis. This scheme illustrates the proposed molecular mechanisms involving some PRDMs during invasion and metastasis. (<b>A</b>) Higher PRDM1 expression is detected in estrogen receptor alpha (ERα)-negative breast cancer cells and primary breast tumors. Mechanistically, Bcl-2, induced by RelB, interacts with and activates Ras in the mitochondrial membrane. In turn, Ras induces the expression of PRDM1/BLIMP1, which downregulates ERα gene expression by direct binding to its promoter, thus promoting a reduction in the levels of E-cadherin and γ-catenin and a corresponding increase in the migratory phenotype of breast cancer cells. The lymphocyte lineage-restricted transcription factor Aiolos negatively regulates PRDM1 and p66<sup>Shc</sup> transcription; in addition, loss of PRDM1 expression reduces the expression of p66<sup>Shc</sup>. Thus, the absence of PRDM1 protein promotes cancer cell invasion and at the same time confers anoikis resistance to the cancer cell. (<b>B</b>) TGF-β1 promotes PRDM1/BLIMP1 gene transcription via c-Raf and AP-1 pathway. Blimp1, in turn, by reducing the expression of BMP-5, induces the expression of SNAI1, the epithelial–mesenchymal transition (EMT) master regulator. (<b>C</b>) The miR-23b downmodulation and the ErbB2/p130Cas/MAPK axis activation increases the expression of the transcriptional repressor PRDM1/Blimp1, thus mediating cell invasion. (<b>D</b>) PRDM3 synergizes with FOS in expression regulation of gene products controlling cell invasion. PRDM4 mediates cell invasion by interacting with YAP at <span class="html-italic">ITGB2</span> gene promoter. PRDM13 upregulates DLC1 and ARHGAP30 proteins thus inhibiting cell invasion. (<b>E</b>) PRDM2 controls the expression of several genes involved in EMT, with vimentin being the most significantly regulated gene. PRDM16 inhibits EMT by repressing the transcription of <span class="html-italic">MUC4</span>. (<b>F</b>) The miR-424→cdc42→prdm14 axis controls cell invasion. In particular, miR-424 knockdown induces expression of Cdc42 that in turn positively regulates PRDM14 through the activation of Pak1 and Stat5. PRDM14 promotes cell migration by regulating the expression level of matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinases (TIMP). Knockdown of PRDM14 reduced cancer stem cell phenotypes via miR-125a-3p and Fyn expression regulation in pancreatic cancer (see text for additional details).</p> Full article ">Figure 2
<p>PRDM proteins action in the regulation of apoptosis genes expression. Although the precise and direct involvement of PRDMs in apoptosis is not completely unravelled, it is established that they are able to control the expression of several genes participating in this biological process, like <span class="html-italic">BCL-XL</span>, <span class="html-italic">BCL2</span>, and <span class="html-italic">TP53</span> among the others. This scheme illustrates the regulation of apoptotic genes by PRDMs where a direct link was demonstrated (see text for additional details).</p> Full article ">Figure 3
<p>PRDM proteins participation in signal transduction pathways, proliferation, and gene expression regulation. PRDM proteins play a pivotal role in the transduction of signals that control cell proliferation and differentiation. (<b>A</b>) PRDM1 and PRDM5 antagonize the Wnt/β-catenin pathway. PRDM1 reduces the expression of <span class="html-italic">DKK1</span> while PRDM5 forms a chromatin complex with CBP, TCF, and β-catenin that prevents Wnt target gene expression. (<b>B</b>) PRDM2/RIZ1 counteracts the insulin-like growth factor-1 (IGF-1) receptor and the downstream signaling component ERK1/2 and AKT. PRDM8 suppresses the PI3K/AKT/mTOR signaling cascade through the regulation of nucleosome assembly protein 1-like 1 (NAP1L1). (<b>C</b>) The <span class="html-italic">PRDM2</span> gene product, PRDM2a/RIZ1, is a downstream effector of estrogen action and is related to estrogen-regulated cancer cell proliferation. ERα modulates the PRDM2/RIZ isoforms intracellular concentration ratio, by an indirect and selective decrease of RIZ1 expression and a transcriptional activation of RIZ2. (<b>D</b>) TGF-β signaling plays important roles in cytostasis and normal epithelium differentiation, and alterations in TGF-β signaling have been identified in many malignancies. MEL1/PRDM16 interacts with SKI and inhibits TGF-β signaling by stabilizing the inactive Smad3-SKI complex on the promoter of TGF-β target genes. PRDM3 negatively regulates TGF-β signaling through binding and inactivating SMAD3 proteins. (E) PRDM14 binds an intron of <span class="html-italic">NOTCH1</span> gene and modifies the chromatin structure (H3K4me3) allowing access of the RAG recombinase complex. RAG deletes part of the <span class="html-italic">NOTCH1</span> promoter and consequently a truncated, ligand-independent Notch1 protein is produced. (<b>F</b>) PRDM3 through its first zinc finger domain, associates and inhibits JNK activity, thus protecting cells from stress-induced cell death that is dependent on JNK activation. Otherwise, PRDM5 upregulates JNK expression. (<b>G</b>) PRDM11 represses the oncogenes Fos and Jun that are frequently induced by aberrant growth factor signaling or oncogenic activation of MAP kinase signaling, such as constitutively active RAS. PRDM3 downregulates <span class="html-italic">SERPIN-B2</span> gene that might play an important role in enhancing cell proliferation by preventing protection of Rb proteolysis and/or in the suppression of cell differentiation. PRDM13 inhibits cell proliferation by upregulating INCA1, a CDK inhibitor and ADAMTS12, a novel antitumor protease that modulates the extracellular signal-regulated kinase signaling pathway. (<b>H</b>) Hypoxia-induced miR-214 inhibits <span class="html-italic">PRDM16</span> expression, thus promoting both cell proliferation and migration and enhancing the Warburg effect.</p> Full article ">Figure 4
<p>Involvement of PRDM proteins in double-strand break (DSB) DNA repair. Many insults are responsible for DNA double-strand breaks (DSBs) that impair DNA replication and proper chromosome segregation. DSB repair system disfunction is frequently observed in cancer, thus rendering cells prone to transformation. PRDM2/RIZ1 and PRDM9 are implied in the DSB repair complex, which is essential for ensuring accurate DNA repair and maintenance of genomic integrity. First, PARP is recruited at the DSB where it catalyzes the formation of poly (ADP-ribose) chains, facilitating the docking of the MRN complex to the DSB. The MRN complex, with its nuclease activity and DNA binding capability, is involved in the initial processing of DSBs. Subsequently, ataxia telangiectasia mutated (ATM) kinase induces the recruitment of the mH2A1.2/RIZ1 complex at DSB sites. PRDM2/RIZ1 induces the H3K9me2 and in that way enables a dynamic switch in chromatin conformation. Finally, the mH2A1.2/RIZ1 module recruits BRCA1. PRDM9 also affects the DSB initiation and repair, thus allowing genetic exchange between chromosomes.</p> Full article ">Figure 5
<p>PRDM activity in cancer stemness. The figure summarizes the mechanisms regulated by some PRDMs and possibly involved in cancer stemness (see text for detailed description).</p> Full article ">
3 pages, 184 KiB  
Reply
Reply to Comments: Using the Cardio-Ankle Vascular Index (CAVI) or the Mathematical Correction Form (CAVI0) in Clinical Practice
by Bart Spronck, Alexander Jurko, Michal Mestanik, Alberto P. Avolio and Ingrid Tonhajzerova
Int. J. Mol. Sci. 2020, 21(7), 2647; https://doi.org/10.3390/ijms21072647 - 10 Apr 2020
Cited by 3 | Viewed by 2443
Abstract
We read with great interest Alizargar et al [...] Full article
(This article belongs to the Special Issue Endothelial Dysfunction: Pathophysiology and Molecular Mechanisms)
12 pages, 2987 KiB  
Article
CD44 Can Compensate for IgSF11 Deficiency by Associating with the Scaffold Protein PSD-95 during Osteoclast Differentiation
by Hyunsoo Kim, Noriko Takegahara, Matthew C. Walsh and Yongwon Choi
Int. J. Mol. Sci. 2020, 21(7), 2646; https://doi.org/10.3390/ijms21072646 - 10 Apr 2020
Cited by 6 | Viewed by 3203
Abstract
Differentiation of osteoclasts, which are specialized multinucleated macrophages capable of bone resorption, is driven primarily by receptor activator of NF-κB ligand (RANKL). Additional signaling from cell surface receptors, such as cell adhesion molecules (CAMs), is also required for osteoclast maturation. Previously, we have [...] Read more.
Differentiation of osteoclasts, which are specialized multinucleated macrophages capable of bone resorption, is driven primarily by receptor activator of NF-κB ligand (RANKL). Additional signaling from cell surface receptors, such as cell adhesion molecules (CAMs), is also required for osteoclast maturation. Previously, we have demonstrated that immunoglobulin superfamily 11 (IgSF11), a member of the immunoglobulin-CAM (IgCAM) family, plays an important role in osteoclast differentiation through association with the scaffold protein postsynaptic density protein 95 (PSD-95). Here, we demonstrate that the osteoclast-expressed CAM CD44 can compensate for IgSF11 deficiency when cell–cell interaction conditions are suboptimal by associating with PSD-95. Impaired osteoclast differentiation in IgSF11-deficient (IgSF11−/−) cultures was rescued by antibody-mediated stimulation of CD44 or by treatment with low-molecular-weight hyaluronan (LMW-HA), a CD44 ligand. Biochemical analysis revealed that PSD-95, which is required for osteoclast differentiation, associates with CD44 in osteoclasts regardless of the presence or absence of IgSF11. RNAi-mediated knockdown of PSD-95 abrogated the effects of either CD44 stimulation or LMW-HA treatment on osteoclast differentiation, suggesting that CD44, similar to IgSF11, is functionally associated with PSD-95 during osteoclast differentiation. Taken together, these results reveal that CD44 can compensate for IgSF11 deficiency in osteoclasts through association with PSD-95. Full article
(This article belongs to the Special Issue Osteoclast Multinucleation Mechanisms)
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Figure 1
<p>Blocking CD44 inhibits osteoclast differentiation of IgSF11-deficient cells in high-cell-density cultures. High-cell-density cultures of IgSF11<sup>+/+</sup> and IgSF11<sup>−/−</sup> BMMs were treated with the indicated doses of soluble antibodies (<b>A</b>) anti-E-cadherin, (<b>B</b>) anti-CD9, (<b>C</b>) anti-CD44, and (<b>D</b>) anti-CD47, and cultured with M-CSF + RANKL for three days to induce osteoclast differentiation. On day three, cells were fixed and stained with TRAP (left). TRAP<sup>+</sup> multinucleated cells (three nuclei or more per cell) were counted and the frequency of TRAP<sup>+</sup> multinucleated cells is shown (right). Scale bars represent 100 μm. Data are shown as the mean ± S.D. *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 2
<p>Stimulation of CD44 rescues impaired IgSF11-deficient osteoclast differentiation. IgSF11<sup>+/+</sup> and IgSF11<sup>−/−</sup> BMMs were (<b>A</b>) seeded on plate-bound control IgG or anti-CD44 at low cell density or (<b>B</b>) treated with LMW HA at low cell density, and cultured with M-CSF + RANKL for three days to induce osteoclast differentiation. On day three, cells were fixed and stained with TRAP (left). TRAP<sup>+</sup> multinucleated cells (three nuclei or more per cell) were counted and the frequency of TRAP<sup>+</sup> multinucleated cells is shown (right). Scale bars represent 100 μm. Data are shown as the mean ± S.D. * <span class="html-italic">p</span> &lt; 0.05, **** <span class="html-italic">p</span> &lt; 0.0001, ns; not significant.</p> Full article ">Figure 3
<p>Stimulation of IgSF11 exerts a pro-osteoclastogenic effect in a CD44-independent manner. CD44<sup>+/+</sup> and CD44<sup>−/−</sup> BMMs were seeded on plate-bound control IgG or IgSF11-Fc at (<b>A</b>) high cell density, or (<b>B</b>) low cell density, and cultured with M-CSF + RANKL for three days to induce osteoclast differentiation. On day three, cells were fixed and stained with TRAP (left). TRAP<sup>+</sup> multinucleated cells (three nuclei or more per cell) were counted and the frequency of TRAP<sup>+</sup> multinucleated cells is shown (right). Scale bars represent 100 μm. Data are shown as the mean ± S.D. **** <span class="html-italic">p</span> &lt; 0.0001, ns; not significant.</p> Full article ">Figure 4
<p>PSD-95 is required for CD44-mediated osteoclast differentiation. (<b>A</b>) Coimmunoprecipitation (Co-IP) assay. IgSF11<sup>+/+</sup> and IgSF11<sup>−/−</sup> pre-osteoclasts were lysed and the lysates were immunoprecipitated with anti-CD44 antibody. Western blotting (WB) was performed with the indicated antibodies. Input shows amount of proteins in the lysates. (<b>B</b>) Effect of knockdown of PSD-95 on CD44 stimulation-induced osteoclast differentiation. Wild-type BMMs retrovirally transduced with the indicated shRNAs (EV, empty vector control) were cultured with M-CSF + RANKL in the presence of the indicated stimuli (LMW HA and/or plate bound anti-CD44) for three days. On day three, cells were fixed and stained for TRAP (top). Relative expression of PSD-95 was determined by Q-PCR (bottom left). TRAP<sup>+</sup> multinucleated cells (three nuclei or more per cell) were counted and the frequency of TRAP<sup>+</sup> multinucleated cells is shown (bottom right). Scale bars represent 100 μm. Data are shown as the mean ± S.D. *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
22 pages, 406 KiB  
Review
Promising Perspectives for Detection, Identification, and Quantification of Plant Pathogenic Fungi and Oomycetes through Targeting Mitochondrial DNA
by Tomasz Kulik, Katarzyna Bilska and Maciej Żelechowski
Int. J. Mol. Sci. 2020, 21(7), 2645; https://doi.org/10.3390/ijms21072645 - 10 Apr 2020
Cited by 30 | Viewed by 4398
Abstract
Fungi and oomycetes encompass many pathogens affecting crops worldwide. Their effective control requires screening pathogens across the local and international trade networks along with the monitoring of pathogen inocula in the field. Fundamentals to all of these concerns are their efficient detection, identification, [...] Read more.
Fungi and oomycetes encompass many pathogens affecting crops worldwide. Their effective control requires screening pathogens across the local and international trade networks along with the monitoring of pathogen inocula in the field. Fundamentals to all of these concerns are their efficient detection, identification, and quantification. The use of molecular markers showed the best promise in the field of plant pathogen diagnostics. However, despite the unquestionable benefits of DNA-based methods, two significant limitations are associated with their use. The first limitation concerns the insufficient level of sensitivity due to the very low and uneven distribution of pathogens in plant material. The second limitation pertains to the inability of widely used diagnostic assays to detect cryptic species. Targeting mtDNA appears to provide a solution to these challenges. Its high copy number in microbial cells makes mtDNA an attractive target for developing highly sensitive assays. In addition, previous studies on different pathogen taxa indicated that mitogenome sequence variation could improve cryptic species delimitation accuracy. This review sheds light on the potential application of mtDNA for pathogen diagnostics. This paper covers a brief description of qPCR and DNA barcoding as two major strategies enabling the diagnostics of plant pathogenic fungi and oomycetes. Both strategies are discussed along with the potential use of mtDNA, including their strengths and weaknesses. Full article
(This article belongs to the Section Molecular Microbiology)
23 pages, 1628 KiB  
Article
Novel Urethane-Dimethacrylate Monomers and Compositions for Use as Matrices in Dental Restorative Materials
by Izabela M. Barszczewska-Rybarek, Marta W. Chrószcz and Grzegorz Chladek
Int. J. Mol. Sci. 2020, 21(7), 2644; https://doi.org/10.3390/ijms21072644 - 10 Apr 2020
Cited by 42 | Viewed by 4429
Abstract
In this study, novel urethane-dimethacrylate monomers were synthesized from 1,3-bis(1-isocyanato-1-methylethyl)benzene (MEBDI) and oligoethylene glycols monomethacrylates, containing one to three oxyethylene groups. They can potentially be utilized as matrices in dental restorative materials. The obtained monomers were used to prepare four new formulations. Two [...] Read more.
In this study, novel urethane-dimethacrylate monomers were synthesized from 1,3-bis(1-isocyanato-1-methylethyl)benzene (MEBDI) and oligoethylene glycols monomethacrylates, containing one to three oxyethylene groups. They can potentially be utilized as matrices in dental restorative materials. The obtained monomers were used to prepare four new formulations. Two of them were solely composed of the MEBDI-based monomers. In a second pair, a monomer based on triethylene glycol monomethacrylate, used in 20 wt.%, was replaced with triethylene glycol dimethacrylate (TEGDMA), a reactive diluent typically used in dental materials. For comparison purposes, two formulations, using typical dental dimethacrylates (bisphenol A glycerolate dimethacrylate (Bis-GMA), urethane-dimethacrylate (UDMA) and TEGDMA) were prepared. The monomers and mixtures were tested for the viscosity and density. The homopolymers and copolymers, obtained via photopolymerization, were tested for the degree of conversion, polymerization shrinkage, water sorption and solubility, hardness, flexural strength and modulus. The newly developed formulations achieved promising physico-chemical and mechanical characteristics so as to be suitable for applications as dental composite matrices. A combination of the MEBDI-based urethane-dimethacrylates with TEGDMA resulted in copolymers with a high degree of conversion, low polymerization shrinkage, low water sorption and water solubility, and good mechanical properties. These parameters showed an improvement in relation to currently used dental formulations. Full article
(This article belongs to the Special Issue Recent Advances in Dental Materials and Biomaterials)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The representative FTIR spectra of the DM: (<b>a</b>) monomer and (<b>b</b>) polymer.</p> Full article ">Figure 2
<p>Schematic representation of sample groups tested in this study.</p> Full article ">Scheme 1
<p>The chemical structure of typical dental dimethacrylates.</p> Full article ">Scheme 2
<p>The synthesis route and chemical structure of the MEBDI-based urethane-dimethacrylates synthesized in this study (HM – <span class="html-italic">n</span> = 1, DM – <span class="html-italic">n</span> = 2 and TM – <span class="html-italic">n</span> = 3).</p> Full article ">
15 pages, 2212 KiB  
Article
Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells
by Bingen G. Monasterio, Noemi Jiménez-Rojo, Aritz B. García-Arribas, Howard Riezman, Félix M. Goñi and Alicia Alonso
Int. J. Mol. Sci. 2020, 21(7), 2643; https://doi.org/10.3390/ijms21072643 - 10 Apr 2020
Cited by 7 | Viewed by 4163
Abstract
This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so [...] Read more.
This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4–6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30–32 C atoms in their acyl chains but were relatively poor in those containing 34–40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes. Full article
(This article belongs to the Special Issue Lipid-Protein and Protein-Protein Interactions in Membranes)
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Figure 1
<p>Plasma membrane preparations from CHO cells. (<b>A</b>), giant plasma membrane vesicles (GPMV) or blebs. (<b>B</b>), PM patches. Bar 10 μm.</p> Full article ">Figure 2
<p>GPMV (bleb) formation and Laurdan GP measurements at 20 °C. (<b>A</b>), GPMV formation from a CHO cell (average GP value 0.44). (<b>B</b>), Generalized polarization plot of image A. (<b>C</b>), Laurdan staining of GPMV derived from CHO cells. (<b>D</b>), Generalized polarization plot of image C. (<b>E</b>), Laurdan emission spectrum of CHO cell blebs. Bar 10 µm.</p> Full article ">Figure 3
<p>Laurdan staining of plasma membrane patches (at 20 °C). (<b>A</b>), Laurdan staining of a CHO cell plasma membrane patch. (<b>B</b>), Generalized polarization plot of image A. (<b>C</b>) Laurdan emission spectrum of CHO cell PM patches.</p> Full article ">Figure 4
<p>Laurdan GP measurements. (<b>A</b>), Laurdan fluorescence emission spectra. Red, whole CHO cells; blue, SUV formed from CHO cell lipid extract; black, GPMV (blebs) from CHO cells; green, PM patches from CHO cells. Spectra retrieved at 40 °C. (<b>B</b>), Laurdan GP values obtained from microscopy images such as shown in <a href="#ijms-21-02643-f001" class="html-fig">Figure 1</a>, 2A, 2C, 3A, (at 20 °C, <span class="html-italic">n</span> = 150, value = mean + SD). Statistically significant differences were calculated with ANOVA and Student’s t-test. Significance: (*) <span class="html-italic">p</span> &lt; 0.05; (***) <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>Atomic Force Microscopy (AFM) measurements. (<b>A</b>), CHO cell plasma membrane patch over polylysine-coated mica. (<b>B</b>), Topographic image of the cross-section indicated by the blue line in 5A. (<b>C</b>), Breakthrough forces distribution of CHO cell plasma membranes (PM) patches. (<b>D</b>). Breakthrough forces distribution of CHO cell blebs. (<b>E</b>). Comparison of breakthrough forces, data extracted from experiments as in C, D. Whole lipid extract breakthrough value was obtained from supported planar bilayers formed with CHO cell lipid extracts. (<span class="html-italic">n</span>= at least 160, value = mean + SD) Statistically significant differences were calculated with ANOVA and Student´s t-test. Significance: (**) <span class="html-italic">p</span> &lt; 0.01; (***) <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p>Mass spectroscopy lipidomic analysis of whole cells and plasma membrane preparations. (<b>A</b>) Total phospholipids; (<b>B</b>) Short, long and very long glycerophospholipids; (<b>C</b>) Total cholesterol; (<b>D</b>) Phospholipid saturation level (DB = double bond); (<b>E</b>) Phosphatidylcholine distribution according to chain length; (<b>F</b>) Phosphatidylethanolamine distribution according to chain length. Bras: solid black, whole cells treated for GPMV preparation; striped, GPMV (blebs); empty, cells treated for PM patch preparation; dotted PM patches. Significance: (*) <span class="html-italic">p</span> &lt; 0.0 (**); <span class="html-italic">p</span> &lt; 0.01; (***) <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
15 pages, 4771 KiB  
Article
Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease
by Stefan Hintze, Sarah Limmer, Paulina Dabrowska-Schlepp, Birgit Berg, Nicola Krieghoff, Andreas Busch, Andreas Schaaf, Peter Meinke and Benedikt Schoser
Int. J. Mol. Sci. 2020, 21(7), 2642; https://doi.org/10.3390/ijms21072642 - 10 Apr 2020
Cited by 13 | Viewed by 4780
Abstract
Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and [...] Read more.
Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa−/− mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients. Full article
(This article belongs to the Special Issue Pathophysiology, Molecular Mechanism and Therapeutic Strategies of Lysosomal Storage Disorders (LSD))
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Characterization of moss-GAA variants in comparison to alglucosidase alfa: (<b>A</b>) modifications (GlcAc = <span class="html-italic">N</span>-Acetylglucosamine; Gal = Galactose; Man = Mannose; Neu5Ac = <span class="html-italic">N</span>-Acetylneuraminic acid), (<b>B</b>) reducing SDS-PAGE with Coomassie staining (left panel) and western blot (right panel), (<b>C</b>) specific enzyme activity, (<b>D</b>) total glycan profile via HILIC-HPLC. The red arrows in (<b>D</b>) correspond to the glycan structures in (<b>A</b>). The three major peaks are NaNaF, NaNa, and NaAF.</p> Full article ">Figure 2
<p>Uptake and activity of rhGAA in immortalized <span class="html-italic">Gaa</span><sup>−/−</sup> mouse myoblasts. (<b>A</b>) GAA activity assay of <span class="html-italic">Gaa</span><sup>−/−</sup> mouse myoblasts before and after treatment with all tested rhGAA variants. GAA activity is displayed in pmol/h/mg (total protein). (<b>B</b>) Immunofluorescence staining of <span class="html-italic">Gaa</span><sup>−/−</sup> mouse myoblasts for GAA and the lysosomal marker LAMP1 before and after treatment with all tested rhGAA variants. Scale bar 10 µm.</p> Full article ">Figure 3
<p>Uptake and activity of rhGAA in immortalized <span class="html-italic">Gaa</span><sup>−/−</sup> mouse myotubes. (<b>A</b>) GAA activity assay of isolated <span class="html-italic">Gaa</span><sup>−/−</sup> mouse myotubes before and after treatment with all tested rhGAA variants. GAA activity is displayed in pmol/h/mg (total protein). (<b>B</b>) Immunofluorescence staining of <span class="html-italic">Gaa</span><sup>−/−</sup> mouse myotubes for GAA and the lysosomal marker LAMP1 before and after treatment with all tested rhGAA variants. Scale bar 10 µm.</p> Full article ">Figure 4
<p>Uptake and activity of rhGAA in human myoblasts. (<b>A</b>) Immunofluorescence staining of Pompe patient primary myoblasts (example patient-1) for GAA and the lysosomal marker LAMP1 before and after treatment with all tested rhGAA variants. Scale bar 10 µm. (<b>B</b>) GAA activity assay of Pompe patient primary myoblasts before and after treatment with all tested rhGAA variants. GAA activity is displayed in pmol/h/mg (total protein). (<b>C</b>) PAS staining of Pompe patient primary myoblasts (example patient-1) before and after treatment with all tested rhGAA variants. Scale bar 40 µm.</p> Full article ">Figure 5
<p>Uptake and activity of rhGAA in human myotubes. (<b>A</b>) Immunofluorescence staining of Pompe patient myotubes (example patient-1) for GAA and the lysosomal marker LAMP1 before and after treatment with all tested rhGAA variants. Scale bar 10 µm. (<b>B</b>) GAA activity assay of Pompe patient myotubes before and after treatment with all tested rhGAA variants. GAA activity is displayed in pmol/h/mg (total protein). (<b>C</b>) PAS staining of Pompe patient myotubes (example patient-1) before and after treatment with all tested rhGAA variants. Scale bar 40 µm.</p> Full article ">Figure 5 Cont.
<p>Uptake and activity of rhGAA in human myotubes. (<b>A</b>) Immunofluorescence staining of Pompe patient myotubes (example patient-1) for GAA and the lysosomal marker LAMP1 before and after treatment with all tested rhGAA variants. Scale bar 10 µm. (<b>B</b>) GAA activity assay of Pompe patient myotubes before and after treatment with all tested rhGAA variants. GAA activity is displayed in pmol/h/mg (total protein). (<b>C</b>) PAS staining of Pompe patient myotubes (example patient-1) before and after treatment with all tested rhGAA variants. Scale bar 40 µm.</p> Full article ">Figure 6
<p>Glycolytic measurements. Measurement of glycolysis has been performed in (<b>A</b>) immortalized <span class="html-italic">Gaa</span><sup>−/−</sup> mouse myoblasts (normalized to untreated <span class="html-italic">Gaa</span><sup>−/−</sup> mouse myoblasts); and (<b>B</b>) Pompe patient primary myoblasts (normalized to untreated patient myoblasts).</p> Full article ">
23 pages, 1285 KiB  
Review
Myocardium Metabolism in Physiological and Pathophysiological States: Implications of Epicardial Adipose Tissue and Potential Therapeutic Targets
by Nerea Gandoy-Fieiras, Jose Ramon Gonzalez-Juanatey and Sonia Eiras
Int. J. Mol. Sci. 2020, 21(7), 2641; https://doi.org/10.3390/ijms21072641 - 10 Apr 2020
Cited by 26 | Viewed by 5341
Abstract
The main energy substrate of adult cardiomyocytes for their contractility are the fatty acids. Its metabolism generates high ATP levels at the expense of high oxygen consumption in the mitochondria. Under low oxygen supply, they can get energy from other substrates, mainly glucose, [...] Read more.
The main energy substrate of adult cardiomyocytes for their contractility are the fatty acids. Its metabolism generates high ATP levels at the expense of high oxygen consumption in the mitochondria. Under low oxygen supply, they can get energy from other substrates, mainly glucose, lactate, ketone bodies, etc., but the mitochondrial dysfunction, in pathological conditions, reduces the oxidative metabolism. In consequence, fatty acids are stored into epicardial fat and its accumulation provokes inflammation, insulin resistance, and oxidative stress, which enhance the myocardium dysfunction. Some therapies focused on improvement the fatty acids entry into mitochondria have failed to demonstrate benefits on cardiovascular disorders. Oppositely, those therapies with effects on epicardial fat volume and inflammation might improve the oxidative metabolism of myocardium and might reduce the cardiovascular disease progression. This review aims at explain (a) the energy substrate adaptation of myocardium in physiological conditions, (b) the reduction of oxidative metabolism in pathological conditions and consequences on epicardial fat accumulation and insulin resistance, and (c) the reduction of cardiovascular outcomes after regulation by some therapies. Full article
(This article belongs to the Special Issue Adipogenesis and Adipose Tissue Metabolism)
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Figure 1
<p>Myocardium metabolism in pathological situations: In physiological conditions, fatty acids are mainly fuel of energy of cardiomyocytes. Obesity is associated with high fatty acids uptake that develops lipotoxicity and insulin resistance. It reduces the glucose uptake. However, during hypertrophic and ischemic situation, there is shift from fatty acids to glucose that is converted into lactate. This metabolism gets less ATP production, and there is mitochondrial dysfunction and ROS (reactive oxygen species) production.</p> Full article ">Figure 2
<p>Epicardial fat metabolism in physiological and pathological situations: The figure represents an adipocyte (yellow) and a blood vessel. Glucose is the main fuel of energy in adipocytes to be used in adipocyte metabolism. Glucose or fatty acids can be stored in triacylglycerides (TAG) by a lipogenesis process. Insulin, glucocorticoids, and growth hormone (GH) promote lipogenesis. TAG through lipolysis produce free fatty acids (FFA). Several factors contribute to lipolysis (fasting, cortisol, etc.). Pathological conditions, diabetes, and obesity promote lipogenesis because of free fatty acid (FFA) uptake from the blood through fatty acids transporter protein (FATP). However, systolic dysfunction due, in part, by the epinephrine increment, promotes lipolysis. Diastolic and ischemic situation will increase glucose uptake for producing lactate and H+ due to low oxygen levels.</p> Full article ">
17 pages, 2892 KiB  
Article
Histone Deacetylase TaHDT701 Functions in TaHDA6-TaHOS15 Complex to Regulate Wheat Defense Responses to Blumeria graminis f.sp. tritici
by Pengfei Zhi, Lingyao Kong, Jiao Liu, Xiaona Zhang, Xiaoyu Wang, Haoyu Li, Maokai Sun, Yan Li and Cheng Chang
Int. J. Mol. Sci. 2020, 21(7), 2640; https://doi.org/10.3390/ijms21072640 - 10 Apr 2020
Cited by 31 | Viewed by 3629
Abstract
Powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt) leads to severe economic losses in bread wheat (Triticum aestivum L.). To date, only a few epigenetic modulators have been revealed to regulate wheat powdery mildew resistance. In this study, [...] Read more.
Powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt) leads to severe economic losses in bread wheat (Triticum aestivum L.). To date, only a few epigenetic modulators have been revealed to regulate wheat powdery mildew resistance. In this study, the histone deacetylase 2 (HD2) type histone deacetylase TaHDT701 was identified as a negative regulator of wheat defense responses to Bgt. Using multiple approaches, we demonstrated that TaHDT701 associates with the RPD3 type histone deacetylase TaHDA6 and the WD40-repeat protein TaHOS15 to constitute a histone deacetylase complex, in which TaHDT701 could stabilize the TaHDA6-TaHOS15 association. Furthermore, knockdown of TaHDT701, TaHDA6, and TaHOS15 resulted in enhanced wheat powdery mildew resistance, suggesting that the TaHDT701-TaHDA6-TaHOS15 histone deacetylase complex negatively regulates wheat defense responses to Bgt. Moreover, chromatin immunoprecipitation assays revealed that TaHDT701 could function in concert with TaHOS15 to recruit TaHDA6 to the promoters of defense-related genes such as TaPR1, TaPR2, TaPR5, and TaWRKY45. In addition, silencing of TaHDT701, TaHDA6, and TaHOS15 resulted in the up-regulation of TaPR1, TaPR2, TaPR5, and TaWRKY45 accompanied with increased histone acetylation and methylation, as well as reduced nucleosome occupancy, at their promoters, suggesting that the TaHDT701-TaHDA6-TaHOS15 histone deacetylase complex suppresses wheat powdery mildew resistance by modulating chromatin state at defense-related genes. Full article
(This article belongs to the Special Issue Wheat and Barley: Acclimatization to Abiotic and Biotic Stress)
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Figure 1
<p><span class="html-italic">TaHDT701</span> is a negative regulator of bread wheat resistance to the powdery mildew pathogen <span class="html-italic">Blumeria graminis</span> f.sp. <span class="html-italic">tritici</span> (<span class="html-italic">Bgt</span>). (<b>A</b>) Expression profiles of <span class="html-italic">TaHDT701</span> in Yannong 999 leaves during the infection of <span class="html-italic">Bgt</span> virulent isolate E09. (<b>B</b>) Transcript abundance of <span class="html-italic">TaHDT701</span> in Yannong 999 leaves inoculated with barley stripe mosaic virus (BSMV)-<span class="html-italic">γ</span> and BSMV-<span class="html-italic">TaHDT701as.</span> (<b>C</b>) <span class="html-italic">Bgt</span> microcolony formation type on wheat plants infected with BSMV-<span class="html-italic">γ</span> and BSMV-<span class="html-italic">TaHDT701as.</span> Conidia that germinated and finally established microcolonies are indicated with black arrows, and conidia that germinated but failed to establish microcolonies are indicated with red arrows. Bar, 150 μm. (<b>D</b>) Statistical analysis of <span class="html-italic">Bgt</span> microcolony formation on BSMV-<span class="html-italic">γ</span> and BSMV-<span class="html-italic">TaHDT701as</span> wheat leaves. For each treatment, at least 500 <span class="html-italic">Bgt-</span>wheat interaction sites were separately counted. (<b>E</b>) Statistical analysis of <span class="html-italic">Bgt</span> haustorial formation in wheat epidermal cells bombarded with OE-<span class="html-italic">EV</span> or OE-<span class="html-italic">TaHDT701</span>. At least 50 cells were analyzed in one experiment. For (<b>A</b>), (<b>B</b>), (<b>D</b>), and (<b>E</b>), three independent biological replicates per treatment were statistically analyzed (t-test, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 2
<p>TaHDT701 interacts with TaHDA6 and TaHOS15. (<b>A</b>) Analysis of TaHDT701, TaHDA6, and TaHOS15 interaction in yeast-two hybrid. (<b>B</b>) In vitro glutathione S-transferase (GST) pull-down analysis of TaHDT701, TaHDA6, and TaHOS15 interaction. TaHDA6-His or TaHOS15-His was incubated with GST or GST-TaHDT701, and the GST resin-bound proteins were immunoblotted with the α-His antibody. (<b>C</b>) Luciferase complementation imaging (LCI) analysis of TaHDT701, TaHDA6, and TaHOS15 interaction. N- or C-terminal fragment of luciferase (LUC) (nLUC or cLUC) was fused with indicated proteins and indicated pairs were coexpressed in <span class="html-italic">N. benthamiana</span> through agro-infiltration.</p> Full article ">Figure 3
<p>TaHDT701 associates with TaHDA6 and TaHOS15 in a histone deacetylase complex in bread wheat. (<b>A</b>) Nuclear co-immunoprecipitation (co-IP) interaction analysis among TaHDT701, TaHDA6, and TaHOS15. Nuclear protein was extracted from the indicated BSMV-VIGS wheat leaves and subjected to immunoprecipitation with the α-TaHDA6 and α-TaHOS15 antibodies. The co-immunoprecipitation of TaHDT701, TaHDA6, and TaHOS15 was probed with the antibodies α-TaHDT701, α-TaHDA6, and α-TaHOS15. (<b>B</b>) Immunoprecipitation analysis of endogenous histone deacetylase activity in different background. Nuclear extracts were prepared from the indicated BSMV-VIGS leaves and immunoprecipitated with antibodies α-TaHDT701, α-TaHDA6, and α-TaHOS15. The immunoprecipitated complexes were examined for histone deacetylase activity, which was given as radioactivity (c.p.m.) of [<sup>3</sup>H]-acetate released from an acetylated histone H4 peptide. Data are means from three independent biological repeats. Significant differences were determined using Student’s t-test: ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 4
<p>Silencing of <span class="html-italic">TaHDT701</span>, <span class="html-italic">TaHDA6</span>, and <span class="html-italic">TaHOS15</span> compromises wheat susceptibility to <span class="html-italic">Bgt.</span> (<b>A</b>) Relative transcript abundance of <span class="html-italic">TaHDT701, TaHDA6,</span> and <span class="html-italic">TaHOS15</span> on wheat plants with different background. The expression level in BSMV-<span class="html-italic">γ</span> wheat leave at 0 hpi was set to 1. (<b>B</b>) <span class="html-italic">Bgt</span> microcolony formation type on wheat plants with different background. Conidia that germinated and finally established microcolonies are indicated with black arrows, and conidia that germinated but failed to establish microcolonies are indicated with red arrows. Bars = 100 μm. (<b>C</b>) Statistical analysis of <span class="html-italic">Bgt</span> microcolony formation on wheat plants with different background. For each treatment, at least 500 <span class="html-italic">Bgt-</span>wheat interaction sites were separately counted. (<b>D</b>) Statistical analysis of <span class="html-italic">Bgt</span> haustorial formation in wheat epidermal cells with different background. At least 100 cells were analyzed in one experiment. For (<b>A</b>), (<b>C</b>), and (<b>D</b>), three independent biological replicates per treatment were statistically analyzed (t-test, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 5
<p>Distribution of TaHDT701, TaHDA6, and TaHOS15 at chromatins of <span class="html-italic">TaPR1</span>, <span class="html-italic">TaPR2</span>, <span class="html-italic">TaPR5</span>, and <span class="html-italic">TaWRKY45</span> in different backgrounds. (<b>A</b>) Schematic diagram of <span class="html-italic">TaPR1</span>, <span class="html-italic">TaPR2</span>, <span class="html-italic">TaPR5</span>, and <span class="html-italic">TaWRKY45</span> genes. Fragments for chromatin immunoprecipitation (ChIP)-qPCR analysis were labeled with numbers. (<b>B</b>) The binding of TaHDT701 (upper panel), TaHDA6 (middle panel), and TaHOS15 (lower panel) on the promoter of <span class="html-italic">TaPR1</span>, <span class="html-italic">TaPR2</span>, <span class="html-italic">TaPR5</span>, and <span class="html-italic">TaWRKY45</span> in wheat protoplasts analyzed by ChIP-qPCR on wheat plants with different background. The antibodies used for immunoprecipitation are indicated on each graph. The fragments employed for ChIP-qPCR analysis are indicated in <a href="#ijms-21-02640-f005" class="html-fig">Figure 5</a>A. The data are means (±SE) from three independent biological repeats and were analyzed using Student’s t-test: ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 6
<p>Silencing of <span class="html-italic">TaHDT701</span>, <span class="html-italic">TaHDA6</span>, and <span class="html-italic">TaHOS15</span> affects the histone H4K16Ac, nucleosome distribution and expression levels of <span class="html-italic">TaPR1</span>, <span class="html-italic">TaPR2</span>, <span class="html-italic">TaPR5</span>, and <span class="html-italic">TaWRKY45</span>. (<b>A</b>) ChIP-qPCR analysis of H4K16Ac at the promoter regions of <span class="html-italic">TaPR1</span>, <span class="html-italic">TaPR2</span>, <span class="html-italic">TaPR5</span>, and <span class="html-italic">TaWRKY45</span> on wheat plants with different background. Antibodies α-H4K16Ac were used for immunoprecipitation. Before ChIP-qPCR analysis, the wheat leaves with a typical BMSV symptom were inoculated with <span class="html-italic">Bgt</span> conidia for 0 and 24 h. The histone acetylation level in BSMV-<span class="html-italic">γ</span> wheat leaves was set to 1.0 at 0 hpi after normalization by histone H4 ChIP. The fragments employed for ChIP-qPCR analysis are indicated in <a href="#ijms-21-02640-f005" class="html-fig">Figure 5</a>A. (<b>B</b>) Micrococcal nuclease (MNase) analysis of nucleosome occupancy at promoter regions of <span class="html-italic">TaPR1</span>, <span class="html-italic">TaPR2</span>, <span class="html-italic">TaPR5</span>, and <span class="html-italic">TaWRKY45</span> on wheat plants with different background. The nucleosome occupancy levels in BSMV-<span class="html-italic">γ</span> wheat leaves at 0 hpi were set to 1.0. (<b>C</b>) RT-PCR analysis of <span class="html-italic">TaPR1</span>, <span class="html-italic">TaPR2</span>, <span class="html-italic">TaPR5</span> and <span class="html-italic">TaWRKY45</span> expression levels on wheat plants with different background. The expression levels in BSMV-<span class="html-italic">γ</span> wheat leaves at 0 hpi were set to 1.0. Three independent biological replicates per treatment were statistically analyzed (t-test, ** <span class="html-italic">p</span> &lt; 0.01). For (<b>A</b>), (<b>B</b>) and (<b>C</b>), three independent biological replicates per treatment were statistically analyzed (t-test, ** <span class="html-italic">p</span> &lt; 0.01). (<b>D</b>) A proposed model of the action of the TaHDT701-TaHDA6-TaHOS15 histone deacetylase complex in regulating wheat defense responses to <span class="html-italic">Bgt.</span> As shown in the left panel, the TaHDT701-TaHDA6-TaHOS15 histone deacetylase complex mediates histone deacetylation at the wheat defense-related genes such as <span class="html-italic">TaPR1</span>, <span class="html-italic">TaPR2</span>, <span class="html-italic">TaPR5</span> and <span class="html-italic">TaWRKY45</span>, which leads to the suppression of defense-related transcription and defense responses to <span class="html-italic">Bgt</span>. In the absence of the TaHDT701-TaHDA6-TaHOS15 histone deacetylase complex (shown in the right panel), chromatin at the defense-related genes is marked by the increased H4K16Ac, H3K9Ac, and H3K4me3, as well as the reduced nucleosome occupancy, thereby stimulating the defense-related transcription and defense responses to <span class="html-italic">Bgt</span>.</p> Full article ">
2 pages, 177 KiB  
Correction
Correction:Chlamydia psittaci PmpD-N Modulated Chicken Macrophage Function by Triggering Th2 Polarization and the TLR2/MyD88/NF-κB Signaling Pathway
by Jun Chu, Xiaohui Li, Guanggang Qu, Yihui Wang, Qiang Li, Yongxia Guo, Lei Hou, Jue Liu, Francis O. Eko and Cheng He
Int. J. Mol. Sci. 2020, 21(7), 2639; https://doi.org/10.3390/ijms21072639 - 10 Apr 2020
Cited by 2 | Viewed by 2094
Abstract
The authors would like to make the following corrections to their paper, published in the International Journal of Molecular Sciences [...] Full article
(This article belongs to the Special Issue Intracellular Organelle Rearrangement Induced by Pathogenic Infections)
3 pages, 820 KiB  
Correction
Correction: Wang, Y.T., et al. Selenium Nanoparticle Synthesized by Proteus mirabilis YC801: An Efficacious Pathway for Selenite Biotransformation and Detoxification. Int. J. Mol. Sci. 2018, 19, 3809
by Yuting Wang, Xian Shu, Jinyan Hou, Weili Lu, Weiwei Zhao, Shengwei Huang and Lifang Wu
Int. J. Mol. Sci. 2020, 21(7), 2638; https://doi.org/10.3390/ijms21072638 - 10 Apr 2020
Cited by 3 | Viewed by 2045
Abstract
The authors wish to make the following corrections to this paper [...] Full article
(This article belongs to the Section Biochemistry)
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<p>The FTIR spectrum of SeNPs synthesized by isolate YC801.</p> Full article ">Figure 7
<p>The FTIR spectrum of SeNPs synthesized by isolate YC801.</p> Full article ">
19 pages, 5045 KiB  
Article
Electrophoretic Deposition of Copper(II)–Chitosan Complexes for Antibacterial Coatings
by Muhammad Asim Akhtar, Kanwal Ilyas, Ivo Dlouhý, Filip Siska and Aldo R. Boccaccini
Int. J. Mol. Sci. 2020, 21(7), 2637; https://doi.org/10.3390/ijms21072637 - 10 Apr 2020
Cited by 43 | Viewed by 5663
Abstract
Bacterial infection associated with medical implants is a major threat to healthcare. This work reports the fabrication of Copper(II)–Chitosan (Cu(II)–CS) complex coatings deposited by electrophoretic deposition (EPD) as potential antibacterial candidate to combat microorganisms to reduce implant related infections. The successful deposition of [...] Read more.
Bacterial infection associated with medical implants is a major threat to healthcare. This work reports the fabrication of Copper(II)–Chitosan (Cu(II)–CS) complex coatings deposited by electrophoretic deposition (EPD) as potential antibacterial candidate to combat microorganisms to reduce implant related infections. The successful deposition of Cu(II)–CS complex coatings on stainless steel was confirmed by physicochemical characterizations. Morphological and elemental analyses by scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) spectroscopy verified the uniform distribution of copper in the Chitosan (CS) matrix. Moreover, homogeneous coatings without precipitation of metallic copper were confirmed by X-ray diffraction (XRD) spectroscopy and SEM micrographs. Controlled swelling behavior depicted the chelation of copper with polysaccharide chains that is key to the stability of Cu(II)–CS coatings. All investigated systems exhibited stable degradation rate in phosphate buffered saline (PBS)–lysozyme solution within seven days of incubation. The coatings presented higher mechanical properties with the increase in Cu(II) concentration. The crack-free coatings showed mildly hydrophobic behavior. Antibacterial assays were performed using both Gram-positive and Gram-negative bacteria. Outstanding antibacterial properties of the coatings were confirmed. After 24 h of incubation, cell studies of coatings confirms that up to a certain threshold concentration of Cu(II) were not cytotoxic to human osteoblast-like cells. Overall, our results show that uniform and homogeneous Cu(II)–CS coatings with good antibacterial and enhanced mechanical stability could be successfully deposited by EPD. Such antibiotic-free antibacterial coatings are potential candidates for biomedical implants. Full article
(This article belongs to the Special Issue Chitosan Functionalizations, Formulations and Composites)
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Graphical abstract
Full article ">Figure 1
<p>Scanning electron microscopy (SEM) morphologies of coatings (<b>A</b>) Chitosan (CS), (<b>B</b>) Copper(II)–Chitosan (Cu(II)–CS), and (<b>C</b>) cross-sectional image of Cu(II)–CS4, indicating a coating thickness of around 40μm.</p> Full article ">Figure 2
<p>EDX spectra of coatings: (<b>A</b>) SEM image of CS, (<b>B</b>) EDX pattern of CS, (<b>C</b>) SEM image of Cu(II)–CS4, (<b>D</b>) EDX pattern of Cu(II)–CS4, and (<b>E</b>) Cu mapping on Cu(II)–CS4 coating.</p> Full article ">Figure 3
<p>Fourier-transform infrared (FTIR) spectra of CS and Cu(II)–CS4 coatings. The relevent peaks are discussed in the text.</p> Full article ">Figure 4
<p>X-ray diffraction (XRD) patterns of CS and Cu(II)–CS4 coatings.</p> Full article ">Figure 5
<p>Overall variation of the hardness of the different coatings. Significantly different coatings from the CS coating are highlighted (* <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>SEM images of sctratchs on coatings at lower and higher loads (<b>A</b>,<b>B</b>) CS, (<b>C</b>,<b>D</b>) Cu(II)–CS1, (<b>E</b>,<b>F</b>) Cu(II)–CS2, (<b>G</b>,<b>H</b>) Cu(II)–CS3, and (<b>I</b>,<b>J</b>) Cu(II)–CS4 coatings.</p> Full article ">Figure 7
<p>Swelling behaviour of CS and Cu(II)–CS complex coatings with different Cu(II) concentrations with respect to time. Tests were carried out in phosphate buffered saline (PBS).</p> Full article ">Figure 8
<p>Degradation behaviour of CS and Cu(II)–CS coatings with different Cu(II) concentrations with respect to time in lysozyme–PBS solution.</p> Full article ">Figure 9
<p>(<b>A</b>) Average contact angle of water droplets on CS and Cu(II)–CS coatings measured at three timepoints (i.e., immediately after deposition, after 3 min and 5 min) and (<b>B</b>) profiles of water droplets on CS and Cu(II)–CS coatings immediately after deposition.</p> Full article ">Figure 10
<p>Bacterial growth as % of colonized area: (<b>A</b>) <span class="html-italic">Staphylococcus aureus</span> and (<b>B</b>) <span class="html-italic">Escherichia Coli</span>. (<b>C</b>) Optical Images of recultivated bacterial colonies on agar after 3 h of incubation for the different samples investigated.</p> Full article ">Figure 11
<p>(<b>A</b>) Graph representing MG-63 cell viability (WST-8 assay) on different samples investigated, (* <span class="html-italic">p</span> &lt; 0.05). (<b>B</b>) fluorescence microscope images showing the results of calcein-DAPI staining after 24 h of culture with CS and Cu(II)–CS coatings with different concentration of Cu(II).</p> Full article ">
14 pages, 3589 KiB  
Article
Underlying Ossification Phenotype in a Murine Model of Metastatic Synovial Sarcoma
by Matthew Kirkham, Austen Kalivas, Kaniz Fatema, Sarah Luelling, Brooke H. Dubansky, Benjamin Dubansky, Kevin B. Jones and Jared J. Barrott
Int. J. Mol. Sci. 2020, 21(7), 2636; https://doi.org/10.3390/ijms21072636 - 10 Apr 2020
Cited by 3 | Viewed by 3937
Abstract
Synovial sarcoma, an uncommon cancer, typically affects young adults. Survival rates range from 36% to 76%, decreasing significantly when metastases are present. Synovial sarcomas form in soft tissues, often near bones, with about 10% demonstrating ossification in the tumor. The literature is inconclusive [...] Read more.
Synovial sarcoma, an uncommon cancer, typically affects young adults. Survival rates range from 36% to 76%, decreasing significantly when metastases are present. Synovial sarcomas form in soft tissues, often near bones, with about 10% demonstrating ossification in the tumor. The literature is inconclusive on whether the presence of ossification portends a worse prognosis. To this end, we analyzed our genetic mouse models of synovial sarcoma to determine the extent of ossification in the tumors and its relationship with morbidity. We noted higher ossification within our metastatic mouse model of synovial sarcoma. Not only did we observe ossification within the tumors at a frequency of 7%, but an even higher frequency, 72%, of bone reactivity was detected by radiography. An enrichment of bone development genes was associated with primary tumors, even in the absence of an ossification phenotype. In spite of the ossification being intricately linked with the metastatic model, the presence of ossification was not associated with a faster or worse morbidity in the mice. Our conclusion is that both metastasis and ossification are dependent on time, but that they are independent of one another. Full article
(This article belongs to the Special Issue Cancer Cell Reprogramming)
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<p>Histological representation of ossification in mouse synovial sarcoma. (<b>a</b>,<b>b</b>) 10× and 40× insert of synovial sarcoma with ossified tumor matrix (OT) exhibiting calcium salt crystals (purple) embedded in osteoid (pink). The unossified tumor matrix (UT) contains sarcoma cells that are more epithelioid in appearance, and two osteoclast-like multinucleated giant cells are seen at the interface of the ossified matrix (<b>c</b>,<b>d</b>) 10× and 40× insert of synovial sarcoma with a more extensively calcified matrix interfaced with spindle-shaped tumor cells that are loosely packed in a fibrous stroma. (<b>e</b>,<b>f</b>) 10× and 40× insert of synovial sarcoma with uncalcified osteoid matrix interfaced with a dense population of spindle-shaped tumor cells embedded in a fibrous stroma. Scale bars in the 10× images = 100 μm, and scale bars in the 40× images = 20 μm. OT = Ossified Tumor, UT = Unossified Tumor, Arrow = lacunae, Arrowhead = calcium salt deposits of ossified tumor matrix, Chevron = osteoclast-like multinucleated giant cells, Star = artificial space at the interface of ossified and unossified tumor matrix.</p> Full article ">Figure 2
<p>Prevalence of ossification within mouse synovial sarcomas. Representative bar graphs of: (<b>a</b>) the percentage of mice showing evidence of ossification among heterozygous (<span class="html-italic">n</span> = 251) and homozygous (<span class="html-italic">n</span> = 212) phenotypes of <span class="html-italic">hSS1</span> and <span class="html-italic">hSS2</span>. Only 7.1% of total mice exhibited ossification through palpitation; (<b>b</b>) the overall survival of heterozygous and homozygous genotypes in weeks, demonstrating heterozygous phenotypes to have a higher survival rate than homozygous genotypes; and (<b>c</b>) the Kaplan Meier Survival curve of heterozygous and homozygous genotypes. Shaded area represents 90% CI. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 3
<p>Radiographic imaging of metastatic and nonmetastatic mice with the development of ossifying tumors, showing bone inflammation (red arrows) and interaction or lack of interaction for both groups. The control demonstrates the lack of ossification in the healthy mouse model, and the pie charts indicate the proportionality of reactivity in nonmetastatic mice (30%) and metastatic mice (72%).</p> Full article ">Figure 4
<p>Gene expression of the ossification genes found upregulated in mouse and human metastatic synovial sarcoma. (<b>a</b>) Heatmap showing RNA expression and the upregulation (red) or downregulation (blue) of bone development genes in <span class="html-italic">Pten</span> loss-induced tumors comparing nonmetastatic to metastatic (left) and metastatic to muscle tissue (right); (<b>b</b>) 46 genes were analyzed and determined to be statistically different between metastatic and normal muscle tissue by an adjusted <span class="html-italic">p</span>-value threshold of &lt; 0.05. Venn diagram representing statistically significant bone development genes from nonmetastatic vs. metastatic samples (orange, 26 genes) and muscle vs. metastatic samples (blue, 46 genes) showing an overlap of 20 genes of interest between groups; (<b>c</b>) Comparison of nonmetastatic to metastatic human synovial sarcoma sample expression of <span class="html-italic">PTHLH</span>, showing the spread and mean (bar) of each group. Statistical significance was found with regards to the upregulation of <span class="html-italic">PTHLH</span> in metastatic tumors in these patients. <span class="html-italic">p</span>-value = 0.039; (<b>d</b>) After fluorescence-activated cell sorting, NanoString sequencing was performed to identify the gene expression for secreted proteins involved in ossification that were unique to myeloid-derived suppressor cells (MDSCs). * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p>The correlation between ossification and mouse survival with metastatic synovial sarcoma; (<b>a</b>) Representative bar graph showing the overall survival of non-ossifying (<span class="html-italic">n</span> = 104) and ossifying (<span class="html-italic">n</span> = 20) mice, with statistical significance occurring for the survival of mice exhibiting ossification; (<b>b</b>) Kaplan Meier Survival curve of non-ossifying and ossifying mice. Shaded area represents 90% CI. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 6
<p>Histological sections of pulmonary metastases demonstrating a lack of ossification within tumors. Scale bars = 100 μm.</p> Full article ">
18 pages, 5095 KiB  
Article
Interpretation of the Epigenetic Signature of Facioscapulohumeral Muscular Dystrophy in Light of Genotype-Phenotype Studies
by Ana Nikolic, Takako I Jones, Monica Govi, Fabiano Mele, Louise Maranda, Francesco Sera, Giulia Ricci, Lucia Ruggiero, Liliana Vercelli, Simona Portaro, Luisa Villa, Chiara Fiorillo, Lorenzo Maggi, Lucio Santoro, Giovanni Antonini, Massimiliano Filosto, Maurizio Moggio, Corrado Angelini, Elena Pegoraro, Angela Berardinelli, Maria Antonetta Maioli, Grazia D’Angelo, Antonino Di Muzio, Gabriele Siciliano, Giuliano Tomelleri, Maurizio D’Esposito, Floriana Della Ragione, Arianna Brancaccio, Rachele Piras, Carmelo Rodolico, Tiziana Mongini, Frederique Magdinier, Valentina Salsi, Peter L. Jones and Rossella Tupleradd Show full author list remove Hide full author list
Int. J. Mol. Sci. 2020, 21(7), 2635; https://doi.org/10.3390/ijms21072635 - 10 Apr 2020
Cited by 16 | Viewed by 3804
Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the [...] Read more.
Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice. Full article
(This article belongs to the Special Issue Epigenetic Alterations in Neuromuscular Disorders)
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<p>Assessment of DNA methylation level in facioscapulohumeral muscular dystrophy (FSHD) index cases using MRSE1 (A) and MRSE2 (B) approaches: (<b>A</b>) The crude relationship between the two parameters is not significant (b = −0.028, <span class="html-italic">p</span> = 0.236; R<sup>2</sup> = 0.012). The strength of the relationship does not improve when adjusting for the age of patient, as we find no significant association between level of D4Z4 methylation and FSHD score (b = −0.035, <span class="html-italic">p</span> = 0.140; R<sup>2</sup> = 0.033). (<b>B</b>) The crude relationship is very weak but statistically significant (b = −0.047, <span class="html-italic">p</span> = 0.048; R<sup>2</sup> = 0.036). The strength of the relationship improves adjusting for the age of patient (b=−0.050, <span class="html-italic">p</span> = 0.036, R<sup>2</sup> = 0.057).</p> Full article ">Figure 2
<p>Comparison of D4Z4 DNA methylation level between FSHD index cases carrying 1–10 D4Z4 repeat units (RU) (FSHD1) or more than 10 D4Z4 RU (FSHD2): A box plot of D4Z4 methylation level assessed with MSRE1 (<b>A</b>) and MSRE2 (<b>B</b>) approaches in carriers of 1–10 or &gt;10 D4Z4 units. The circle indicates an outlier value.</p> Full article ">Figure 3
<p>Evaluation of D4Z4 methylation levels in subjects belonging to different clinical categories: A box plot of D4Z4 methylation levels assessed with MSRE1 assay (<b>A</b>) and MSRE2 (<b>B</b>) in probands and relatives stratified over clinical categories (A, B, C, and D).</p> Full article ">Figure 4
<p>Evaluation of the efficacy of D4Z4 methylation status as a marker for classifying diseased and non-diseased individuals. The receiver operating characteristic (ROC) curve was plotted to analyze the values of methylation at D4Z4 as a discriminator between affected and unaffected individuals.</p> Full article ">Figure 5
<p>Analysis of the D4Z4 methylation status in Family C. (<b>A</b>) Pedigree of family C. The genetic profile (<b>A</b>) or the clinical and epigenetic features (<b>B</b>) of each individual are reported. Filled symbols represent affected people. (<b>C</b>) Genomic DNAs from the selected individuals were analyzed using the 4qA or 4qA-L sodium bisulfite sequencing (BSS) assay. The percent DNA methylation for the Q1 is indicated. Red boxes indicate methylated CpGs, blue boxes indicate unmethylated CpGs, and white boxes indicate no CpG at the expected site.</p> Full article ">Figure 6
<p>Analysis of the D4Z4 methylation status in family A. (<b>A</b>) Pedigree of family A. The genetic profile (<b>A</b>) or the clinical and epigenetic features (<b>B</b>) of each individual are reported. Filled symbols represent affected people (<b>C</b>) Genomic DNAs from the selected individuals were analyzed using the 4qA or 4qA-L BSS assay. The percentage DNA methylation for the Q1 is indicated. Red boxes indicate methylated CpGs, blue boxes indicate unmethylated CpGs, and white boxes indicate no CpG at the expected site.</p> Full article ">Figure 7
<p>Analysis of the D4Z4 methylation status B. (<b>A</b>) Pedigree of family B. The genetic profile (<b>A</b>) or the clinical and epigenetic features (<b>B</b>) of each individual are reported. Filled symbols represent affected people (<b>C</b>) Genomic DNAs from the selected individuals were analyzed using the 4qA BSS assay. The percentage of DNA methylation for the Q1 is indicated. Red boxes indicate methylated CpGs, blue boxes indicate unmethylated CpGs, and white boxes indicate no CpG at the expected site.</p> Full article ">Figure 8
<p>Patients recruitment and their clinical status.</p> Full article ">
17 pages, 1799 KiB  
Article
Identification and Characterization of the Lactating Mouse Mammary Gland Citrullinome
by Guangyuan Li, Coleman H. Young, Bryce Snow, Amanda O. Christensen, M. Kristen Demoruelle, Venkatesh V. Nemmara, Paul R. Thompson, Heather M. Rothfuss and Brian D. Cherrington
Int. J. Mol. Sci. 2020, 21(7), 2634; https://doi.org/10.3390/ijms21072634 - 10 Apr 2020
Cited by 6 | Viewed by 3635
Abstract
Citrullination is a post-translational modification (PTM) in which positively charged peptidyl-arginine is converted into neutral peptidyl-citrulline by peptidylarginine deiminase (PAD or PADI) enzymes. The full protein citrullinome in many tissues is unknown. Herein, we used mass spectrometry and identified 107 citrullinated proteins in [...] Read more.
Citrullination is a post-translational modification (PTM) in which positively charged peptidyl-arginine is converted into neutral peptidyl-citrulline by peptidylarginine deiminase (PAD or PADI) enzymes. The full protein citrullinome in many tissues is unknown. Herein, we used mass spectrometry and identified 107 citrullinated proteins in the lactation day 9 (L9) mouse mammary gland including histone H2A, α-tubulin, and β-casein. Given the importance of prolactin to lactation, we next tested if it stimulates PAD-catalyzed citrullination using mouse mammary epithelial CID-9 cells. Stimulation of CID-9 cells with 5 µg/mL prolactin for 10 min induced a 2-fold increase in histone H2A citrullination and a 4.5-fold increase in α-tubulin citrullination. We next investigated if prolactin-induced citrullination regulates the expression of lactation genes β-casein (Csn2) and butyrophilin (Btn1a1). Prolactin treatment for 12 h increased β-casein and butyrophilin mRNA expression; however, this increase was significantly inhibited by the pan-PAD inhibitor, BB-Cl-amidine (BB-ClA). We also examined the effect of tubulin citrullination on the overall polymerization rate of microtubules. Our results show that citrullinated tubulin had a higher maximum overall polymerization rate. Our work suggests that protein citrullination is an important PTM that regulates gene expression and microtubule dynamics in mammary epithelial cells. Full article
(This article belongs to the Special Issue Peptidylarginine Deiminases and Protein Deimination in Health and Disease)
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<p>One hundred seven citrullinated proteins were present in the lactation day 9 (L9) mouse mammary gland. (<b>A</b>) The Venn diagram shows the number of citrullinated proteins identified with a biotin-PG label (Cit) and without biotin-PG as the negative control (Control) in the L9 mouse mammary gland. The pie charts depict gene ontology (GO) analysis of citrullinated protein distribution at the biological process (<b>B</b>) and cellular compartment levels (<b>C</b>).</p> Full article ">Figure 2
<p>Histone H2A, α-tubulin, and β-casein were citrullinated in the L9 mouse mammary gland. L9 mouse mammary glands were harvested in HEPES buffer, then equal concentrations of lysate were labeled with biotin-PG (Cit) or without the probe as the negative control (Ctrl). Citrullinated proteins were enriched with streptavidin-conjugated agarose beads. Enriched citrullinated proteins were then subjected to Western blot analysis with anti-histone H2A, α-tubulin, or β-casein antibodies. A 5% input sample was collected before enrichment and served as the loading control.</p> Full article ">Figure 3
<p>Prolactin induced histone H2A citrullination in mouse mammary epithelial CID-9 cells. CID-9 cells were treated with vehicle or 5 µg/mL prolactin for 10 and 30 min. Equal amounts of lysate from each time point were labeled with biotin-PG and enriched with streptavidin-conjugated agarose beads. A 5% input sample was removed before enrichment and served as the loading control. Citrullinated histones and 5% input samples were examined by Western blot analysis, and membranes were probed with the anti-histone H2A antibody. The top panel shows a representative Western blot, while the bottom panel represents the quantification of multiple Western blots using BioRad Image Lab 4.0. Data are presented as means +/− SEM and separated using ANOVA SNK (<span class="html-italic">n</span> = 3, * <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 4
<p>Inhibiting citrullination blocked prolactin induced expression of milk proteins β-casein and butyrophilin in mouse mammary epithelial CID-9 cells. (<b>A</b>) CID-9 cells were treated with vehicle or 5 µg/mL prolactin for 1, 6, 12, or 24 h. Total RNA was extracted, reverse transcribed, and subjected to qPCR performed with primers specific to Csn2 and Btn1a1 or Gapdh as the endogenous controls (<span class="html-italic">n</span> &lt; 4, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001). (<b>B</b>) CID-9 cells were pre-treated with vehicle or 2 µM of BB-ClA for 1 h, followed by 5 µg/mL prolactin treatment for 12 h. Total RNA was extracted, reverse transcribed, and subjected to qPCR performed with primers specific to Csn2 and Btn1a1 or Gapdh as the endogenous control (<span class="html-italic">n</span> = 3, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001). Data are presented as means +/− SEM and separated using ANOVA SNK.</p> Full article ">Figure 5
<p>Prolactin stimulated α-tubulin citrullination in mouse mammary epithelial CID-9 cells. CID-9 cells were treated with vehicle or 5 µg/mL prolactin for 10 and 30 min. Equal amounts of lysate from each time point were labeled with biotin-PG and enriched with streptavidin-conjugated agarose beads. A 5% input sample was removed before enrichment and served as the loading control. Citrullinated histones and 5% input samples were examined by Western blot analysis, and membranes were probed with the anti-α-tubulin antibody. The top panel shows a representative Western blot, while the bottom panel represents the quantification of multiple Western blots using BioRad Image Lab 4.0. Data are presented as means +/− SEM and separated using ANOVA SNK (<span class="html-italic">n</span> = 3, * <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>Citrullination altered tubulin polymerization rates. (<b>A</b>) Purified porcine brain tubulin was incubated with vehicle (native), purified PAD enzyme with calcium (active PAD), or purified PAD enzyme without calcium (inactive PAD). Native, active PAD, and inactive PAD tubulin samples were polymerized into microtubules and rates measured every 20 s through the entire polymerization range until steady-state equilibrium was reached. The tubulin polymerization Vmax was calculated as the maximum 5 min average slope of polymerization for all three samples. The active PAD and inactive PAD Vmax values are represented as fold change compared to native tubulin Vmax. All values are expressed as the mean ± SEM. Means were separated using Student’s <span class="html-italic">t</span>-test and * indicates significantly different means (<span class="html-italic">n</span> = 6, * <span class="html-italic">p</span> &lt; 0.05). (<b>B</b>) After completion of the polymerization assay, 4 µgs of native, active PAD, and inactive PAD tubulin samples were labelled with biotin-PG and examined by Western blot. The top panel shows a representative Western blot, while the bottom panel represents the quantification of multiple Western blots using ThermoFisher iBright Analysis software. Densitometry results for citrullinated tubulin for active PAD and inactive PAD samples were normalized to native tubulin to allow for comparison across different experiments. Data are presented as means +/− SEM and separated using the student Student’s <span class="html-italic">t</span>-test (<span class="html-italic">n</span> = 6, * <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 7
<p>β-casein was citrullinated in mouse and human milk. L9 mouse milk and three human breast milk samples were labelled with biotin-PG (Cit) or without biotin-PG (Ctrl), enriched with streptavidin beads, and then separated by Western blot. PVDF membranes containing enriched citrullinated proteins were then probed with anti-β-casein antibodies. A 5% input sample was collected before enrichment and served as the loading control.</p> Full article ">
13 pages, 429 KiB  
Review
Genes and Diet in the Prevention of Chronic Diseases in Future Generations
by Marica Franzago, Daniele Santurbano, Ester Vitacolonna and Liborio Stuppia
Int. J. Mol. Sci. 2020, 21(7), 2633; https://doi.org/10.3390/ijms21072633 - 10 Apr 2020
Cited by 73 | Viewed by 17269
Abstract
Nutrition is a modifiable key factor that is able to interact with both the genome and epigenome to influence human health and fertility. In particular, specific genetic variants can influence the response to dietary components and nutrient requirements, and conversely, the diet itself [...] Read more.
Nutrition is a modifiable key factor that is able to interact with both the genome and epigenome to influence human health and fertility. In particular, specific genetic variants can influence the response to dietary components and nutrient requirements, and conversely, the diet itself is able to modulate gene expression. In this context and the era of precision medicine, nutrigenetic and nutrigenomic studies offer significant opportunities to improve the prevention of metabolic disturbances, such as Type 2 diabetes, gestational diabetes, hypertension, and cardiovascular diseases, even with transgenerational effects. The present review takes into account the interactions between diet, genes and human health, and provides an overview of the role of nutrigenetics, nutrigenomics and epigenetics in the prevention of non-communicable diseases. Moreover, we focus our attention on the mechanism of intergenerational or transgenerational transmission of the susceptibility to metabolic disturbances, and underline that the reversibility of epigenetic modifications through dietary intervention could counteract perturbations induced by lifestyle and environmental factors. Full article
(This article belongs to the Special Issue Epigenetics of Diabetes and Related Complications)
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<p>Interactions among genes, diet and human health.</p> Full article ">
30 pages, 2485 KiB  
Review
Lipotoxicity and Diabetic Nephropathy: Novel Mechanistic Insights and Therapeutic Opportunities
by Lucas Opazo-Ríos, Sebastián Mas, Gema Marín-Royo, Sergio Mezzano, Carmen Gómez-Guerrero, Juan Antonio Moreno and Jesús Egido
Int. J. Mol. Sci. 2020, 21(7), 2632; https://doi.org/10.3390/ijms21072632 - 10 Apr 2020
Cited by 203 | Viewed by 15659
Abstract
Lipotoxicity is characterized by the ectopic accumulation of lipids in organs different from adipose tissue. Lipotoxicity is mainly associated with dysfunctional signaling and insulin resistance response in non-adipose tissue such as myocardium, pancreas, skeletal muscle, liver, and kidney. Serum lipid abnormalities and renal [...] Read more.
Lipotoxicity is characterized by the ectopic accumulation of lipids in organs different from adipose tissue. Lipotoxicity is mainly associated with dysfunctional signaling and insulin resistance response in non-adipose tissue such as myocardium, pancreas, skeletal muscle, liver, and kidney. Serum lipid abnormalities and renal ectopic lipid accumulation have been associated with the development of kidney diseases, in particular diabetic nephropathy. Chronic hyperinsulinemia, often seen in type 2 diabetes, plays a crucial role in blood and liver lipid metabolism abnormalities, thus resulting in increased non-esterified fatty acids (NEFA). Excessive lipid accumulation alters cellular homeostasis and activates lipogenic and glycogenic cell-signaling pathways. Recent evidences indicate that both quantity and quality of lipids are involved in renal damage associated to lipotoxicity by activating inflammation, oxidative stress, mitochondrial dysfunction, and cell-death. The pathological effects of lipotoxicity have been observed in renal cells, thus promoting podocyte injury, tubular damage, mesangial proliferation, endothelial activation, and formation of macrophage-derived foam cells. Therefore, this review examines the recent preclinical and clinical research about the potentially harmful effects of lipids in the kidney, metabolic markers associated with these mechanisms, major signaling pathways affected, the causes of excessive lipid accumulation, and the types of lipids involved, as well as offers a comprehensive update of therapeutic strategies targeting lipotoxicity. Full article
(This article belongs to the Special Issue Molecular Mechanisms Involved in Diabetic Nephropathy)
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<p>Lipotoxicity origin. The positive energy balance (high fat and/or carbohydrates diet) is one of the main promoters associated with obesity development. Hypertrophy and hyperplasia of white adipose tissue is a process commonly observed in the progression of obesity. The abdominal subcutaneous deposit has been associated with a greater increase in plasma non-esterified fatty acids (NEFA), a characteristic finding of insulin-resistant patients. Although the onset of lipotoxicity is unknown, altered lipid signaling by white adipose tissue and dysregulation in adipokines production is a key factor in restricting the lipid storage capacity observed in adipocytes. This limitation in the lipid deposit activates a vicious circle that leads to specific adaptations in energy metabolism in certain tissues such as the skeletal muscle, heart, liver, pancreas, and kidney, thus activating signaling pathways associated with gluco(neo)genesis in the presence of active lipogenesis. <span class="html-italic">Created with BioRender.com.</span></p> Full article ">Figure 2
<p>Effect of lipotoxicity on kidney nephron (<b>left</b>) and main pathways of action and detoxification of non-esterified fatty acids (NEFA) in podocytes and tubular cells (<b>right</b>). In brackets are shown selected references on lipotoxicity-mediated mechanisms. Adapted from Wikimedia glomerule image (CC BY-SA 4.0 Author: M. Komorniczak).</p> Full article ">Figure 3
<p>Targeting lipotoxicity in DN. The intrarenal lipids reduction has been positively correlated with renoprotective effects observed in the progression of experimental diabetic nephropathy. On the left side, there are represented different strategies focused on enhancing signaling pathways that are considered beneficial to reduce lipid accumulation (adiponectin/PPAR signaling and cholesterol efflux) and prevent other derived damages such as inflammation and oxidative stress. On the right side are shown different approaches focused on blocking signaling pathways that have a harmful effect in the context of diabetic nephropathy (glucose excretion, cholesterol synthesis, lipid synthesis, and accumulation and FA uptake). ABCA1, ATP-Binding Cassette Transporter A1; ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; CBR1, Carbonyl reductase 1; CCR2, C-C chemokine receptor type 2; C5AR, complement component 5a receptor 1; FA, fatty acid; FATP3/4, fatty acid transport protein 3/4; GLP-1, glucagon-like peptide 1; LXR, live X receptor; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PPARα/γ, peroxisome proliferator-activated receptors α/γ; SGLT2, sodium-glucose co-transporter 2 inhibitors; SREBP, sterol regulatory element-binding proteins; TGFβ, transforming growth factor-beta; VEGF-B, vascular endothelial growth factor-beta; VEGFR1, vascular endothelial growth factor receptor. <span class="html-italic">Created with BioRender.com.</span></p> Full article ">
21 pages, 1207 KiB  
Review
Inducible Polarized Secretion of Exosomes in T and B Lymphocytes
by Victor Calvo and Manuel Izquierdo
Int. J. Mol. Sci. 2020, 21(7), 2631; https://doi.org/10.3390/ijms21072631 - 10 Apr 2020
Cited by 41 | Viewed by 6191
Abstract
Exosomes are extracellular vesicles (EV) of endosomal origin (multivesicular bodies, MVB) constitutively released by many different eukaryotic cells by fusion of MVB to the plasma membrane. However, inducible exosome secretion controlled by cell surface receptors is restricted to very few cell types and [...] Read more.
Exosomes are extracellular vesicles (EV) of endosomal origin (multivesicular bodies, MVB) constitutively released by many different eukaryotic cells by fusion of MVB to the plasma membrane. However, inducible exosome secretion controlled by cell surface receptors is restricted to very few cell types and a limited number of cell surface receptors. Among these, exosome secretion is induced in T lymphocytes and B lymphocytes when stimulated at the immune synapse (IS) via T-cell receptors (TCR) and B-cell receptors (BCR), respectively. IS formation by T and B lymphocytes constitutes a crucial event involved in antigen-specific, cellular, and humoral immune responses. Upon IS formation by T and B lymphocytes with antigen-presenting cells (APC), the convergence of MVB towards the microtubule organization center (MTOC), and MTOC polarization to the IS, are involved in polarized exosome secretion at the synaptic cleft. This specialized mechanism provides the immune system with a finely-tuned strategy to increase the specificity and efficiency of crucial secretory effector functions of B and T lymphocytes. As inducible exosome secretion by antigen-receptors is a critical and unique feature of the immune system this review considers the study of the traffic events leading to polarized exosome secretion at the IS and some of their biological consequences. Full article
(This article belongs to the Special Issue Signaling and Organelle Polarization at the Immunological Synapse)
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<p>Exosome timeline and publications. A search was performed in PubMed on 2 April 2020 to find, for each year of publication, articles using the given term “exosomes” and the related term “small extracellular vesicles” as text word. Data are not normalized to the total number of biology and biomedicine research publications. Arrows on the graph indicate the year of publication of some milestone papers mentioned in the text.</p> Full article ">Figure 2
<p>T lymphocyte—antigen-presenting cells (APC) immune synapse (IS) and polarized secretion. Stages 0 and 1 are common for both Th and cytotoxic T lymphocytes (CTL) IS. After the initial scanning contact of TCR with pMHC on APC, Th effector T lymphocytes (upper panel) form mature IS with antigen-presenting B lymphocytes within several minutes. This IS lasts many hours during which de novo cytokine (i.e., IL-2, IFN-γ) production and secretion occur, which require continuous T-cell receptors (TCR) signaling. Primed effector CTL (lower panel) establish more transient, mature IS after scanning their target cells (i.e., a virus-infected cell), and deliver their lethal hits within a few minutes. Secretory lysosomes (lytic granules) are very rapidly transported (within very few minutes) towards the microtubule organization center (MTOC) (in the minus “–“ direction) and, almost simultaneously, the MTOC polarizes towards the central supramolecular activation complex (cSMAC) of the IS, an F-actin poor area that constitutes a secretory domain. MTOC translocation to the IS appears to be dependent on dynein anchored to the Adhesion and Degranulation Promoting Adapter Protein (ADAP) at the peripheral SMAC (pSMAC), which pulls MTOC in the minus direction. In both types of IS (lower zoom panel), the initial F-actin reorganization in the cell-to-cell contact area, followed by a decrease in F-actin at the cSMAC and an accumulation at the distal SMAC (dSMAC) appears to be involved in granule secretion. In stage 3, MVB fusion with the plasma membrane occurs in both types of IS and leads to TCR-containing exosome polarized secretion at the IS. The exosomes released in Th IS contain proapoptotic FasL and Apo2L and can induce target cell death or Th cell death (AICD). TCR–containing shedding microvesicles have been described in Th IS.</p> Full article ">Figure 3
<p>B lymphocytes form two classes of IS: antigen-capture/processing (upper panel) and antigen presentation to Th lymphocytes (lower panel). Upper panel, antigen recognition on APC is mediated by B-cell receptors (BCR). BCR triggering induces actin reorganization at the IS and MTOC and secretory lysosomes recruitment towards the IS and protease secretion at the extracellular synaptic cleft, facilitating antigen processing and extraction. Subsequently, antigen-BCR complexes are extracted and endocytosed in a clathrin-dependent process to early endosomes (stage 1) and, then to late endosomal, MHC-II<sup>+</sup> compartments (stage 2) where antigen and MHC-II molecules trafficking from Golgi (stages 3 and 4) converge. Coordinately with antigen endocytosis, antigen-BCR interaction promotes the biogenesis of this compartment to facilitate antigen processing. In this compartment, the antigen is additionally processed by proteases to form MHC-II/antigenic peptide complexes (pMHC-II) (stage 5) that, subsequently, are exported and distributed to the cell surface (stages 6 and 7). Lower panel, pMHC-II complexes are recognized by Th lymphocyte TCR forming a second secretory IS. This leads to a polarized phenotype leading to MTOC and MHC-II<sup>+</sup> compartment (MVB) polarization. Local secretion of exosomes containing pMHC-II complexes at the B lymphocyte IS side is represented (see text for further details).</p> Full article ">
18 pages, 1762 KiB  
Article
The Effect of Nanosystems on ATP-Binding Cassette Transporters: Understanding the Influence of Nanosystems on Multidrug Resistance Protein-1 and P-glycoprotein
by Francisco V.C. Mello, Gabriela N. de Moraes, Raquel C. Maia, Jennifer Kyeremateng, Surtaj Hussain Iram and Ralph Santos-Oliveira
Int. J. Mol. Sci. 2020, 21(7), 2630; https://doi.org/10.3390/ijms21072630 - 10 Apr 2020
Cited by 9 | Viewed by 3547
Abstract
The cancer multidrug resistance is involved in the failure of several treatments during cancer treatment. It is a phenomenon that has been receiving great attention in the last years due to the sheer amount of mechanisms discovered and involved in the process of [...] Read more.
The cancer multidrug resistance is involved in the failure of several treatments during cancer treatment. It is a phenomenon that has been receiving great attention in the last years due to the sheer amount of mechanisms discovered and involved in the process of resistance which hinders the effectiveness of many anti-cancer drugs. Among the mechanisms involved in the multidrug resistance, the participation of ATP-binding cassette (ABC) transporters is the main one. The ABC transporters are a group of plasma membrane and intracellular organelle proteins involved in the process of externalization of substrates from cells, which are expressed in cancer. They are involved in the clearance of intracellular metabolites as ions, hormones, lipids and other small molecules from the cell, affecting directly and indirectly drug absorption, distribution, metabolism and excretion. Other mechanisms responsible for resistance are the signaling pathways and the anti- and pro-apoptotic proteins involved in cell death by apoptosis. In this study we evaluated the influence of three nanosystem (Graphene Quantum Dots (GQDs), mesoporous silica (MSN) and poly-lactic nanoparticles (PLA)) in the main mechanism related to the cancer multidrug resistance such as the Multidrug Resistance Protein-1 and P-glycoprotein. We also evaluated this influence in a group of proteins involved in the apoptosis-related resistance including cIAP-1, XIAP, Bcl-2, BAK and Survivin proteins. Last, colonogenic and MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assays have also been performed. The results showed, regardless of the concentration used, GQDs, MSN and PLA were not cytotoxic to MDA-MB-231 cells and showed no impairment in the colony formation capacity. In addition, it has been observed that P-gp membrane expression was not significantly altered by any of the three nanomaterials. The results suggest that GQDs nanoparticles would be suitable for the delivery of other multidrug resistance protein 1 (MRP1) substrate drugs that bind to the transporter at the same binding pocket, while MSN can strongly inhibit doxorubicin efflux by MRP1. On the other hand, PLA showed moderate inhibition of doxorubicin efflux by MRP1 suggesting that this nanomaterial can also be useful to treat MDR (Multidrug resistance) due to MRP1 overexpression. Full article
(This article belongs to the Special Issue Nanomedicine, Nanopharmacy and Nanobiomaterials)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Profile of cell viability of breast cancer cells exposed to a range of nanopolymers concentrations. MDA-MB-231 breast cancer cells were treated with (<b>A</b>) graphene quantum dots (GQDs), (<b>B</b>) mesoporous silica nanoparticles (MSN) or (<b>C</b>) polymeric lactic acid (PLA) for 24, 48 and 72 h. The MTT assay was performed and optical density was obtained at 570 nm. The graphs represent the mean ± standard deviation from three independent experiments. UT: Untreated cells. Statistical significance was analyzed by the 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 2
<p>Clonogenicity capacity following treatment of breast cancer cells with a range of (<b>A</b>) graphene quantum dots (GQDs), (<b>B</b>) mesoporous silica nanoparticles (MSN) or (<b>C</b>) polymeric lactic acid (PLA) concentrations. MDA-MB-231 breast cancer cells were treated with nanopolymers for 24 h, after which fresh media was replaced in plates. After colony formation, cells were stained with crystal violet. Colonies were dissolved and optical density was measured at 595 nm. The graphs represent the mean ± standard deviation from three independent experiments. UT: Untreated cells. Statistical significance was analyzed by the Student´s <span class="html-italic">t</span> test (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 3
<p>P-glycoprotein (Pgp) expression profile in nanopolymer-treated breast cancer cells. MDA-MB-231 cells were treated with 50 µM graphene quantum dots (GQDs), mesoporous silica nanoparticles (MSN) or polymeric lactic acid (PLA) for 72 h and had P-gp expression compared with untreated cells (<b>A</b>,<b>B</b>). Cells were stained with PE-conjugated Pgp monoclonal antibody (UIC2, Coulter, USA) and evaluated by flow cytometry. For better visualization of differences between untreated and nanopolymer-treated cells, histograms were merged (<b>C</b>). The results were expressed by Relative Fluorescence Intensity (RFI), calculated by the ratio between the fluorescence intensity in cells treated with UIC2 and the fluorescence intensity in cells.</p> Full article ">Figure 4
<p>Expression pattern of proteins related to drug resistance in nanopolymer-treated MDA-MB-231 cells. MDA-MB-231 cells were treated with graphene quantum dots (GQDs), mesoporous silica nanoparticles (MSN), polymeric lactic acid (PLA) during 24 h and expression of c-IAP1, XIAP, Bcl-2, BAK and Survivin proteins were analyzed by Western blotting. Hsc70 or β-actin was used as an internal control. The blots are representative of three independent experiments.</p> Full article ">Figure 5
<p>Effect of nanopolymer treatments on MRP1 efflux activity. Doxorubicin accumulation assay was used to measure MRP1 efflux activity. HEK293T cells transiently transfected with MRP1-GFP (green) were pre-treated with 0.1%, 0.25%, 0.5%, 1% Graphene (quantum dots) (<b>A</b>), (mesoporous) Silica (<b>B</b>), PLA nanoparticles (<b>C</b>) or 50 μM of MK571 (known MRP1 inhibitor), before incubation with doxorubicin (red) at 37 °C for 1 h. Images were acquired using confocal microscopy. GFP and doxorubicin were excited at 488 nm, and emission detected at 475/42 and 605/64 nm, respectively.</p> Full article ">
2 pages, 161 KiB  
Correction
Correction: Shoombuatong, W., et al. iQSP: A Sequence-Based Tool for the Prediction and Analysis of Quorum Sensing Peptides via Chou’s 5-Steps Rule and Informative Physicochemical Properties. Int. J. Mol. Sci. 2020, 21, 75
by Phasit Charoenkwan, Nalini Schaduangrat, Chanin Nantasenamat, Theeraphon Piacham and Watshara Shoombuatong
Int. J. Mol. Sci. 2020, 21(7), 2629; https://doi.org/10.3390/ijms21072629 - 10 Apr 2020
Cited by 4 | Viewed by 2188
Abstract
The authors wish to make the following corrections to this paper: [...] Full article
(This article belongs to the Section Molecular Informatics)
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