International Journal of Molecular Sciences

13 pages, 11062 KiB

Open AccessArticle

Hyaluronic Acid and a Short Peptide Improve the Performance of a PCL Electrospun Fibrous Scaffold Designed for Bone Tissue Engineering Applications

by Dana Rachmiel, Inbar Anconina, Safra Rudnick-Glick, Michal Halperin-Sternfeld, Lihi Adler-Abramovich and Amit Sitt

Int. J. Mol. Sci. 2021, 22(5), 2425; https://doi.org/10.3390/ijms22052425 - 12 Mar 2021

Cited by 26 | Viewed by 5307

Abstract

Bone tissue engineering is a rapidly developing, minimally invasive technique for regenerating lost bone with the aid of biomaterial scaffolds that mimic the structure and function of the extracellular matrix (ECM). Recently, scaffolds made of electrospun fibers have aroused interest due to their [...] Read more.

Bone tissue engineering is a rapidly developing, minimally invasive technique for regenerating lost bone with the aid of biomaterial scaffolds that mimic the structure and function of the extracellular matrix (ECM). Recently, scaffolds made of electrospun fibers have aroused interest due to their similarity to the ECM, and high porosity. Hyaluronic acid (HA) is an abundant component of the ECM and an attractive material for use in regenerative medicine; however, its processability by electrospinning is poor, and it must be used in combination with another polymer. Here, we used electrospinning to fabricate a composite scaffold with a core/shell morphology composed of polycaprolactone (PCL) polymer and HA and incorporating a short self-assembling peptide. The peptide includes the arginine-glycine-aspartic acid (RGD) motif and supports cellular attachment based on molecular recognition. Electron microscopy imaging demonstrated that the fibrous network of the scaffold resembles the ECM structure. In vitro biocompatibility assays revealed that MC3T3-E1 preosteoblasts adhered well to the scaffold and proliferated, with significant osteogenic differentiation and calcium mineralization. Our work emphasizes the potential of this multi-component approach by which electrospinning, molecular self-assembly, and molecular recognition motifs are combined, to generate a leading candidate to serve as a scaffold for bone tissue engineering. Full article

(This article belongs to the Special Issue Molecular Recognition in Biological and Bioengineered Systems)

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27 pages, 2435 KiB

Open AccessReview

TRP Channels as Cellular Targets of Particulate Matter

by Alina Milici and Karel Talavera

Int. J. Mol. Sci. 2021, 22(5), 2783; https://doi.org/10.3390/ijms22052783 - 9 Mar 2021

Cited by 21 | Viewed by 5553

Abstract

Particulate matter (PM) is constituted by particles with sizes in the nanometer to micrometer scales. PM can be generated from natural sources such as sandstorms and wildfires, and from human activities, including combustion of fuels, manufacturing and construction or specially engineered for applications [...] Read more.

Particulate matter (PM) is constituted by particles with sizes in the nanometer to micrometer scales. PM can be generated from natural sources such as sandstorms and wildfires, and from human activities, including combustion of fuels, manufacturing and construction or specially engineered for applications in biotechnology, food industry, cosmetics, electronics, etc. Due to their small size PM can penetrate biological tissues, interact with cellular components and induce noxious effects such as disruptions of the cytoskeleton and membranes and the generation of reactive oxygen species. Here, we provide an overview on the actions of PM on transient receptor potential (TRP) proteins, a superfamily of cation-permeable channels with crucial roles in cell signaling. Their expression in epithelial cells and sensory innervation and their high sensitivity to chemical, thermal and mechanical stimuli makes TRP channels prime targets in the major entry routes of noxious PM, which may result in respiratory, metabolic and cardiovascular disorders. On the other hand, the interactions between TRP channel and engineered nanoparticles may be used for targeted drug delivery. We emphasize in that much further research is required to fully characterize the mechanisms underlying PM-TRP channel interactions and their relevance for PM toxicology and biomedical applications. Full article

(This article belongs to the Special Issue Nanoparticles and Their Interactions with Target Cells, Host Receptors and Soluble Factors)

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Figure 1
Schematic of the structure of (A) TRPA1, (B) TRPV1, (C) TRPM8 and (D) TRPV4 and the representative sites involved in the activation by PM. Modified from [<a href="#B51-ijms-22-02783" class="html-bibr">51</a>] (with permission). Full article ">Figure 2
Amorphous silica nanoparticles inhibit the chemical activation of TRPV4. Acute response elicited in cultured human airway epithelial (16HBE) cells by different concentration of 10 nm amorphous silica nanoparticles (SiNPs) and the corresponding TRPV4 inhibition ((a): 0 μg/mL, (b): 10 μg/mL, (c): 100 μg/mL, (d): 1000 μg/mL). (e) The influx in Ca2+ upon SiNPs challenging is concentration-dependent. (f) The inhibition of TRPV4 activation by agonist in the presence of SiNPs is concentration-dependent. Reproduced with permission from [<a href="#B24-ijms-22-02783" class="html-bibr">24</a>]. Full article ">Figure 3
Changes in interleukin 8 (IL-8) mRNA expression as an indicator of inflammation induced by DEP originating from a “black smoker” diesel truck, a diesel exhaust filter regeneration machine, an emission station and NIST SRM 2975. TRPA1 inhibitor HC-030031 reduces the inflammatory effect. The symbols * and # indicate significant induction relative to control and significant inhibition by HC-030031, respectively. Reproduced with permission from [<a href="#B98-ijms-22-02783" class="html-bibr">98</a>]. Full article ">Figure 4
Graphical summary of the activation of TRPA1, TRPC4, TRPM2, TRPM8 and TRPV3 channels by C60 fullerenes, cigarette smoke (CS), wood smoke (WS), diesel exhaust (DE), ultrafine ambient particles (UFP), coal fly ash (CFA), zinc NPs (ZnNP), lanthanide NPs (LnNP), silica NPs (SiNP) and DOX-coated gold nanorods (AuNP). Full article ">Figure 5
Graphical summary of the activation of TRPV1, TRPV2 and TRPV4 channels by titanium NPs (TiNP), carbon NPs (CNP), coal fly ash (CFA), silica NPs (SiNP), ultrafine ambient particles (UFP), cigarette smoke (CS), diesel exhaust (DE) and magnetic particles (mag NP). Full article ">

26 pages, 5689 KiB

Open AccessArticle

The Leukotriene Receptor Antagonist Montelukast Attenuates Neuroinflammation and Affects Cognition in Transgenic 5xFAD Mice

by Johanna Michael, Julia Zirknitzer, Michael Stefan Unger, Rodolphe Poupardin, Tanja Rieß, Nadine Paiement, Horst Zerbe, Birgit Hutter-Paier, Herbert Reitsamer and Ludwig Aigner

Int. J. Mol. Sci. 2021, 22(5), 2782; https://doi.org/10.3390/ijms22052782 - 9 Mar 2021

Cited by 25 | Viewed by 5648

Abstract

Alzheimer’s disease (AD) is the most common form of dementia. In particular, neuroinflammation, mediated by microglia cells but also through CD8+ T-cells, actively contributes to disease pathology. Leukotrienes are involved in neuroinflammation and in the pathological hallmarks of AD. In consequence, leukotriene signaling—more [...] Read more.

Alzheimer’s disease (AD) is the most common form of dementia. In particular, neuroinflammation, mediated by microglia cells but also through CD8+ T-cells, actively contributes to disease pathology. Leukotrienes are involved in neuroinflammation and in the pathological hallmarks of AD. In consequence, leukotriene signaling—more specifically, the leukotriene receptors—has been recognized as a potential drug target to ameliorate AD pathology. Here, we analyzed the effects of the leukotriene receptor antagonist montelukast (MTK) on hippocampal gene expression in 5xFAD mice, a commonly used transgenic AD mouse model. We identified glial activation and neuroinflammation as the main pathways modulated by MTK. The treatment increased the number of Tmem119+ microglia and downregulated genes related to AD-associated microglia and to lipid droplet-accumulating microglia, suggesting that the MTK treatment targets and modulates microglia phenotypes in the disease model compared to the vehicle. MTK treatment further reduced infiltration of CD8+T-cells into the brain parenchyma. Finally, MTK treatment resulted in improved cognitive functions. In summary, we provide a proof of concept for MTK to be a potential drug candidate for AD and provide novel modes of action via modulation of microglia and CD8+ T-cells. Of note, 5xFAD females showed a more severe pathology, and in consequence, MTK treatment had a more pronounced effect in the females compared to the males. The effects on neuroinflammation, i.e., microglia and CD8+ T-cells, as well as the effects on cognitive outcome, were dose-dependent, therefore arguing for the use of higher doses of MTK in AD clinical trials compared to the approved asthma dose. Full article

(This article belongs to the Special Issue Molecular, Cellular and Systemic Signature of Microglia)

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24 pages, 3128 KiB

Open AccessArticle

Vitamin D Compounds PRI-2191 and PRI-2205 Enhance Anastrozole Activity in Human Breast Cancer Models

by Beata Filip-Psurska, Mateusz Psurski, Artur Anisiewicz, Patrycja Libako, Ewa Zbrojewicz, Magdalena Maciejewska, Michał Chodyński, Andrzej Kutner and Joanna Wietrzyk

Int. J. Mol. Sci. 2021, 22(5), 2781; https://doi.org/10.3390/ijms22052781 - 9 Mar 2021

Cited by 10 | Viewed by 4208

Abstract

1,25-Dihydroxycholecalciferol, the hormonally active vitamin D₃ metabolite, is known to exhibit therapeutic effects against breast cancer, mainly by lowering the expression of estrogen receptors and aromatase activity. Previously, the safety of the vitamin D active metabolite (24R)-1,24-dihydroxycholecalciferol (PRI-2191) and 1,25(OH) [...] Read more.

1,25-Dihydroxycholecalciferol, the hormonally active vitamin D₃ metabolite, is known to exhibit therapeutic effects against breast cancer, mainly by lowering the expression of estrogen receptors and aromatase activity. Previously, the safety of the vitamin D active metabolite (24R)-1,24-dihydroxycholecalciferol (PRI-2191) and 1,25(OH)₂D₃ analog PRI-2205 was tested, and the in vitro activity of these analogs against different cancer cell lines was studied. We determined the effect of the two vitamin D compounds on anastrozole (An) activity against breast cancer based on antiproliferative activity, ELISA, flow cytometry, enzyme inhibition potency, PCR, and xenograft study. Both the vitamin D active metabolite and synthetic analog regulated the growth of not only estrogen receptor-positive cells (T47D and MCF-7, in vitro and in vivo), but also hormone-independent cancer cells such as SKBR-3 (HER-2-positive) and MDA-MB-231 (triple-negative), despite their relatively low VDR expression. Combined with An, PRI-2191 and PRI-2205 significantly inhibited the tumor growth of MCF-7 cells. Potentiation of the antitumor activity in combined treatment of MCF-7 tumor-bearing mice is related to the reduced activity of aromatase by both An (enzyme inhibition) and vitamin D compounds (switched off/decreased aromatase gene expression, decreased expression of other genes related to estrogen signaling) and by regulation of the expression of the estrogen receptor ERα and VDR. Full article

(This article belongs to the Special Issue Vitamin D and Other Molecules in Development and Biological Profiling of Cancer)

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Structures of vitamin D compounds. Full article ">Figure 2
The proliferation inhibition (A–D) of human breast cancer cell lines after treatment with analogs of 1,25(OH)2D3 alone or in combination with anastrozole. As indicated (A) MCF-7 cell line, (B) T47D cell line, (C) SKBR-3 cell line, and (D) MDA-MB-231 cell line after 120 h of treatment with calcitriol or PRI-2191 and PRI-2205 and anastrozole. Calcitriol and PRI-2191 and PRI-2005 were used at the dose of 100 nM (A,C,D) or 10 nM (B). Anastrozole was used at the dose of 0.1 mg/mL. The tests were performed 3 to 6 times, each in triplicate. The % (mean ± SD) of proliferation inhibition of cancer cells is shown on the graphs. The nonparametric Kruskal-Wallis ANOVA for multiple comparisons was performed. p < 0.05 was considered to be statistically significant; a—compared to anastrozole, b—compared to PRI-2205 alone. Full article ">Figure 3
Aromatase inhibition by calcitriol and vitamin D compounds PRI-2191 and PRI-2205 (Corning CYP19 inhibition test). The concentrations of the compounds were used according to the manufacturers’ protocol by referring to the concentrations of the positive control used (ketoconazole). Ethanol was used as a solvent for the compounds. The test was performed three times, each time in triplicates. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. p < 0.05 was considered to be statistically significant; * compared to calcitriol. Full article ">Figure 4
Expression of the estrogen receptors ERα and ERβ in the MCF-7 breast cancer cell line after 72 h of treatment with calcitriol, PRI-2191 and PRI-2205, and anastrozole as measured by flow cytometry. Calcitriol, PRI-2191, and PRI-2205 were used at the concentration of 100 nM and anastrozole at the concentration of 0.1 mg/mL. The test was performed three times, each time in duplicates. Full article ">Figure 5
Secretion of 17-β estradiol by MCF-7 cells after in vitro treatment with vitamin D analogs and anastrozole Calcitriol, PRI-2191, and PRI-2205 were tested at 100 nM, while anastrozole was tested at 0.1 mg/mL concentration. (A) After 48 h of treatment, the culture medium was replaced with fresh, colorless DMEM medium without the tested compounds. The culture media were collected after additional 24 h. Total time of cell culture = 72 h. The test was performed two times, each time in triplicates. (B) After 96 h of treatment, the medium was replaced with fresh, colorless DMEM medium without the tested compounds. The culture media were collected after additional 24 h. Total time of cell culture = 120 h. The test was performed three times, each time in triplicates. The nonparametric Kruskal-Wallis ANOVA was used for multiple comparisons. p < 0.05 was considered to be statistically significant; * compared to untreated control. Full article ">Figure 6
Changes in the mRNA profile of MCF-7 cells after treatment with calcitriol or vitamin D analogs with/without anastrozole. The figures presented above represent genes encoding molecules involved in estrogen metabolism. (A) BCAR3—breast cancer antiestrogen resistance protein 3; (B,C) ERα, ERβ—estrogen receptors α and β, respectively; (D) CYP19—aromatase; (E,F) ESRRA, ESRRG—Estrogen Related Receptor Alpha and Gamma, respectively; (G) GNRH1—Gonadotropin-Releasing Hormone 1; (H) SULT1E1—sulfotransferase family 1E, estrogen-preferring member 1; (I) HSD17B2—hydroxysteroid dehydrogenase 17 beta 2. PCR analysis was performed for two independent samples (each sample was tested in triplicate). Full article ">Figure 7
The antitumor effect of vitamin D analogs when used alone or in combination with anastrozole in MCF-7 tumor-bearing mice. Five days after tumor inoculation (tumor volume was measured), the mice received subcutaneous (s.c.) injections of PRI-2191 (1 μg/kg body weight (b.w.) in each injection) or PRI-2205 (10 μg/kg b.w. in each injection) and/or anastrozole (200 μg/mouse in each injection). PRI-2191 and PRI-2205 were administered three times a week, and the total dose was 17 μg/kg and 170 μg/kg respectively. Anastrozole was administered five times a week, at the total dose of 5.2 mg/mouse. (A,B) tumor growth kinetics after treatment with PRI-2191 (A) or PRI-2205 (B) alone or combined with anastrozole. (C,D) present the tumor growth inhibition (TGI) in % calculated according to the formula given in this article. (C) Red bars indicate TGI for combined PRI-2191 and anastrozole treatment. □—PRI-2191 alone, ▼—anastrozole alone treatment. (D) Green bars indicate TGI for combined PRI-2205 and anastrozole treatment, ○—PRI-2205 alone, ▼—anastrozole alone treatment. (E,F) body weight (b.w.) changes during the treatment in all the experimental groups. (G) The serum calcium level at the end of the experiment. The serum calcium levels were measured at the end of the experiment by using a Roche Diagnostic Kit. Data presented in mmol/L are expressed as mean with SD for each group, except for calcitriol, which was administered subcutaneously to a single NOD/SCID mouse at the dose of 1 µg/kg b.w. (sacrificed after 48 h). To demonstrated the safety of using both vitamin D analogs in comparison to calcitriol, the serum calcium level was evaluated in one mouse. (H) The 17β-estradiol serum levels after treatment. The 17-β estradiol levels were measured using the IBL ELISA kit, which is used for in vitro diagnostic quantitative determination of 17β-estradiol. Statistical analysis was performed using Kruskal–Wallis ANOVA, For calcium and 17β-estradiol level in serum * p < 0.05 compared to the control group. Full article ">Figure 8
The expression of selected proteins in MCF-7 tumor tissue. NOD/SCID mice were treated with PRI-2191, PRI-2205, and/or anastrozole. Primary antibodies were used in the following concentrations: anti-VDR (sc-1008) 1:400, anti-CYP27B1 (sc-67261) 1:500, and anti-ERα (sc-542) and anti-ERβ (sc-8974) 1:500 (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anti-β-actin (Sigma-Aldrich, Poznań, Poland) 1:500. ECF Western Blotting Reagent Pack (Amersham, GE Healthcare, Little Chalfont, Buckinghamshire, UK) was used for the analysis. Densitometric analysis was performed using ImageJ 1.43u (Wayne Rasband, National Institutes of Health, USA, <a href="https://imagej.nih.gov/ij/" target="_blank">https://imagej.nih.gov/ij/</a>, accessed on 8 March 2021). Full article ">

21 pages, 4452 KiB

Open AccessArticle

Microglial Heterogeneity and Its Potential Role in Driving Phenotypic Diversity of Alzheimer’s Disease

by Stefano Sorrentino, Roberto Ascari, Emanuela Maderna, Marcella Catania, Bernardino Ghetti, Fabrizio Tagliavini, Giorgio Giaccone and Giuseppe Di Fede

Int. J. Mol. Sci. 2021, 22(5), 2780; https://doi.org/10.3390/ijms22052780 - 9 Mar 2021

Cited by 12 | Viewed by 4113

Abstract

Alzheimer’s disease (AD) is increasingly recognized as a highly heterogeneous disorder occurring under distinct clinical and neuropathological phenotypes. Despite the molecular determinants of such variability not being well defined yet, microglial cells may play a key role in this process by releasing distinct [...] Read more.

Alzheimer’s disease (AD) is increasingly recognized as a highly heterogeneous disorder occurring under distinct clinical and neuropathological phenotypes. Despite the molecular determinants of such variability not being well defined yet, microglial cells may play a key role in this process by releasing distinct pro- and/or anti-inflammatory cytokines, potentially affecting the expression of the disease. We carried out a neuropathological and biochemical analysis on a series of AD brain samples, gathering evidence about the heterogeneous involvement of microglia in AD. The neuropathological studies showed differences concerning morphology, density and distribution of microglial cells among AD brains. Biochemical investigations showed increased brain levels of IL-4, IL-6, IL-13, CCL17, MMP-7 and CXCL13 in AD in comparison with control subjects. The molecular profiling achieved by measuring the brain levels of 25 inflammatory factors known to be involved in neuroinflammation allowed a stratification of the AD patients in three distinct “neuroinflammatory clusters”. These findings strengthen the relevance of neuroinflammation in AD pathogenesis suggesting, in particular, that the differential involvement of neuroinflammatory molecules released by microglial cells during the development of the disease may contribute to modulate the characteristics and the severity of the neuropathological changes, driving—at least in part—the AD phenotypic diversity. Full article

(This article belongs to the Special Issue Microglia Heterogeneity and Its Relevance for Translational Research)

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Graphical abstract
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Microglial characterization in fAD cases. (a,d,g,j,m) APPA673V (fAD1); (b,e,h,k,n) APPA713V (fAD2); (c,f,i,l,o) PS1P117A (fAD3). Scale bar 1 mm (a–c) 200 μm (d–f); 50 μm (g–i). (a–i) Frontal cortex sections immunostained with the IBA1 antibody. (j–l) Morphological shape extracted and edited by imageJ software analysis. (m–o) Quantification of IBA1 immunoreactivity based on the intensity and number of pixels (data were normalized respect to the higher signal obtained among Alzheimer’s disease (AD) samples). Full article ">Figure 2
Microglial characterization in sAD cases. (a,d,g,j,m) sAD6; (b,e,h,k,n) sAD19; (c,f,i,l,o) sAD21. Scale bar 1 mm (a–c) 200 μm (d–f); 50 μm (g–i). (a–i) Frontal cortex sections immunostained with the IBA1 antibody. Arrows in (a) highlight the bilayer distribution of microglial cells across the frontal cortex. (j–l) Morphological shape extracted and edited by imageJ software analysis. (m–o) Quantification of IBA1 immunoreactivity based on the intensity and number of pixels (data were normalized respect to the higher signal obtained among AD samples). Full article ">Figure 3
Comparison between AD and control samples. Boxplots of the most significant analytes, IL-4, IL-6, IL-13, CCL17, MMP-7, CXCL13. * p < 0.05, ** p < 0.005, *** p < 0.001. For IL-6 the most extreme observations have been omitted for a better graphical representation. Full article ">Figure 4
Comparison between pro- and anti-inflammatory cytokines in AD and control samples. Boxplot of the pro-inflammatory (IFN-γ, IL-1ɑ, IL-2, IL-6, IL-12 p70, IL-18) and the anti-inflammatory (IL-1rn, IL-4, IL-13) cytokines are shown for AD (p-value < 0.0001) (a) and control (p-value 0.29) (b) group. * p < 0.05, *** p < 0.001. Dots indicate outlier values. Natural logarithm scale has been used for a better graphical representation. Full article ">Figure 5
Families of neuroinflammatory factors. (a) STRING network highlights the direct (physical) and indirect (functional) connection of elements belongs to each family. Known interactions: from curated databases (light blue), experimentally determined (purple); predicted interactions: gene neighborhood (green), gene fusions (red), gene co-occurrence (blue); other interactions: text mining (yellow), co-expression (black), protein homology (grey). (b) Pie graph indicates the relative distribution of cytokines (red), chemokines (green), MMPs (pink) and IIFs (blue) within the AD group. (c) The heat map shows correlations between the four neuroinflammatory classes and clinical, neuropathological and biological data. Aβ42 levels in the insoluble fraction of brain homogenates (Aβ42i); Aβ40 levels in the insoluble fraction of brain homogenates (Aβ40i); Aβ42 levels in the soluble fraction of brain homogenates (Aβ42s); Aβ40 levels in the soluble fraction of brain homogenates (Aβ40s). Full article ">Figure 6
(a) Hierarchical Cluster Analysis (HCA) shows the different subgroups of AD patients characterized by different expression of neuroinflammatory molecular factors. In abscissa, there are the AD patients and the ordinate corresponds to the complete linkage measured by Euclidean distance. The dashed red line represents the cut off for three clusters (CL): AD-CL1 in yellow; AD-CL2 in red; AD-CL3 in light blue. (b) Histogram concerning the relative abundance of each inflammatory family (cytokines in red, chemokines in green, matrix-metalloproteinases (MMPs) in cream and Innate immunity factors in blue) within each cluster. Full article ">

19 pages, 2328 KiB

Open AccessArticle

Plumbagin, a Biomolecule with (Anti)Osteoclastic Properties

by Sevinj Sultanli, Soni Ghumnani, Richa Ashma and Katharina F. Kubatzky

Int. J. Mol. Sci. 2021, 22(5), 2779; https://doi.org/10.3390/ijms22052779 - 9 Mar 2021

Cited by 14 | Viewed by 4217

Abstract

Plumbagin is a plant-derived naphthoquinone that is widely used in traditional Asian medicine due to its anti-inflammatory and anti-microbial properties. Additionally, plumbagin is cytotoxic for cancer cells due to its ability to trigger reactive oxygen species (ROS) formation and subsequent apoptosis. Since it [...] Read more.

Plumbagin is a plant-derived naphthoquinone that is widely used in traditional Asian medicine due to its anti-inflammatory and anti-microbial properties. Additionally, plumbagin is cytotoxic for cancer cells due to its ability to trigger reactive oxygen species (ROS) formation and subsequent apoptosis. Since it was reported that plumbagin may inhibit the differentiation of bone resorbing osteoclasts in cancer-related models, we wanted to elucidate whether plumbagin interferes with cytokine-induced osteoclastogenesis. Using C57BL/6 mice, we unexpectedly found that plumbagin treatment enhanced osteoclast formation and that this effect was most pronounced when cells were pre-treated for 24 h with plumbagin before subsequent M-CSF/RANKL stimulation. Plumbagin caused a fast induction of NFATc1 signalling and mTOR-dependent activation of p70S6 kinase which resulted in the initiation of protein translation. In line with this finding, we observed an increase in RANK surface expression after Plumbagin stimulation that enhanced the responsiveness for subsequent RANKL treatment. However, in Balb/c mice and Balb/c-derived RAW264.7 macrophages, these findings could not be corroborated and osteoclastogenesis was inhibited. Our results suggest that the effects of plumbagin depend on the model system used and can therefore either trigger or inhibit osteoclast formation. Full article

(This article belongs to the Special Issue Signal Transduction: From Molecular Pathways to Translational Research)

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Plumbagin does not change the phenotype of bone marrow-derived macrophages: (A) the influence of increasing concentrations of plumbagin in the presence of 25 ng/mL M-CSF was tested after 48 h by measuring cell viability (n = 3, with triplicates); (B) cell death triggered by increasing plumbagin concentrations was measured after the incubation of the cells with SYTOX after 48 h of treatment in the presence of M-CSF (n = 4); statistical analysis was performed comparing the results to the positive control (M-CSF) in (A) using Friedman, and in (B) using Mann–Whitney test; (C) the effect of 2 µM plumbagin was tested on the expression of the macrophage marker molecules F4/80, CD11b, CD80, CD86 and CD115, respectively. The graphs represent one typical example (n = 3). *: p ≤ 0.05; **: p ≤ 0.01; ****: p ≤ 0.0001. Full article ">Figure 2
Plumbagin pre-treatment of bone marrow-derived macrophages (BMDMs) facilitates osteoclastogenesis: (A,B) BMDMs were seeded in a 96-well plate and treated with M-CSF or 2 µM plumbagin on day 0. After 24 h, M-CSF/RANKL was added to the wells of M/R and PB-M/R samples. On day 3, the cells were stimulated with M-CSF, M-CSF/RANKL or plumbagin/M-CSF/RANKL, respectively, until the osteoclasts were fully differentiated; (A) shows representative pictures (magnification 100×) and (B) presents the quantification of TRAP stains (n = 6, five of them with duplicates); (C,D) BMDMs were stimulated as described in (A) on 24-well osteo assay surface plates before quantifying the resorbed area on day 10 (n = 3, with duplicates); statistical analysis was carried out comparing the results to the positive control (M-CSF/RANKL) using Friedman test in (B) using a Friedman test and a Mann–Whitney test in (D). *: p ≤ 0.05; ns: p > 0.05. Full article ">Figure 3
Plumbagin induces the rapid activation of cellular signalling in BMDMs: (A,B) Western blot analysis to test typical osteoclast signalling molecules was performed using antibodies against NFATc1 (A) and molecules of the NF-κB pathway (B). GAPDH and actin were used as loading controls. The blots are typical examples out of three independent experiments; (C) differentiating osteoclasts were analysed on day 3 by RT-PCR to determine the expression of the osteoclastic marker genes Acp5, Ctsk, Ocstamp, Dcstamp and Oscar, respectively (n = 4). Statistical analysis comparing the results to the positive control (M-CSF/RANKL) was performed using Mann–Whitney test; (D,E) BMDM were either pre-stimulated with plumbagin for 24 h before stimulation with 100 ng/mL LPS for 6 h or stimulated directly with LPS. (D) RT-PCR was performed to determine the expression of the osteoclastogenic, pro-inflammatory cytokine genes Il6, Il1 and Tnfa, respectively (n = 4); (E) the supernatants of the samples were subjected to ELISA to determine the levels of produced cytokines (n = 5). Statistical analysis comparing the results to the positive control (LPS) was carried out using a Mann–Whitney test in (D) and (E). *: p ≤ 0.05; **: p ≤ 0.01. Full article ">Figure 4
Plumbagin inhibits osteoclastogenesis in RAW264.7 macrophages. (A) RAW264.7 cells were treated with increasing concentrations of plumbagin for 24 h and cell viability was measured after incubation with SYTOX. Statistical analysis was performed comparing the results to the control in using a Mann–Whitney test (n = 4); (B,C) and (D) RAW264.7 cells were seeded in a 24 well plate and treated with RANKL or plumbagin (1 µM) /RANKL. On day 3, cells were restimulated with RANKL, plumbagin/RANKL, respectively, until the osteoclasts were fully differentiated; (B) shows representative pictures and (C) the quantification of TRAP stains (n = 3, with duplicates); (D) one day before the TRAP staining was performed, light microscopic pictures (magnification 100×) were taken to document cell viability and cell numbers. *: p ≤ 0.05; **: p ≤ 0.01. Full article ">Figure 5
Plumbagin impacts ROS production in the presence of M-CSF/RANKL in BMDMs: (A) cells were incubated for 48 h with plumbagin or treated with M-CSF/RANKL before ROS production was analysed in a (iso)luminol-based assay (n = 3); (B) cells were incubated for 24 h with M-CSF/RANKL, plumbagin or pre-treated with plumbagin before the addition of M-CSF/RANKL and ROS production was analysed in a (iso)luminol based assay on day 2 (n = 3); (C) BMDMs were incubated for 6 h, as indicated. Mitochondrial activity was measured using Mitotracker deep red stain for 30 min before FACS analysis (n = 4); (D) BMDMs were pre-treated for 24 h with plumbagin before the addition of M-CSF/RANKL, or treated with only M-CSF and M-CSF/RANKL, respectively. Mitochondrial activity was measured using Mitotracker deep red stain for 30 min before FACS analysis (n = 4); (E) BMDMs were differentiated as indicated and the mitochondrial copy number was determined by comparing mitochondrial and nuclear DNA levels (n = 5); (F) the expression of OxPhos components was analysed in the mitochondrial extracts from cells treated with M-CSF/RANKL or plumbagin pre-stimulated (PB-M/R) cells by Western blot analysis on day 3. The blot is one typical example out of 3 experiments. Statistical analysis was performed comparing the results to the untreated control for (C) and for (F) to the day 0 or positive control (M-CSF/RANKL) using a Mann–Whitney test. *: p ≤ 0.05; **: p ≤ 0.01. ns. p > 0.05. Full article ">Figure 6
Plumbagin pre-treatment of BMDM facilitates osteoclastogenesis: (A) the expression of the metabolic signalling molecules pAMPK, p4E-BP1 and p70S6K was analysed from whole cell lysates at the indicated time-points of plumbagin stimulation. The blots show one typical example out of 3 experiments; (B) translational activity was measured using a Click-IT assay and the subsequent measurement of the incorporated, Alexa488 click-labelled HPG-containing proteins. Cells were treated for 6 h as indicated before chasing for 30 min with HPG. Untreated unstained and untreated stained cells were used as negative controls for background fluorescence and background translational activity, respectively. Fixed cells and Alexa488 were then measured by FACS analysis (n = 4). Statistical analysis was performed by comparing results to the untreated control using a Friedman test; (C) RANK expression was measured by FACS analysis using a phycoerythrin (PE)-labelled antibody; (D) cells were pre-treated with plumbagin or 10 µM cycloheximide to block protein translation (n = 3). Statistical analysis was implemented by comparing the results to the positive control (M-CSF/RANKL) using Kruskal–Wallis test. * p > 0.05. Full article ">

23 pages, 1663 KiB

Open AccessReview

Prominent Role of Histone Modifications in the Regulation of Tumor Metastasis

by Mariam Markouli, Dimitrios Strepkos, Efthimia K. Basdra, Athanasios G. Papavassiliou and Christina Piperi

Int. J. Mol. Sci. 2021, 22(5), 2778; https://doi.org/10.3390/ijms22052778 - 9 Mar 2021

Cited by 21 | Viewed by 4962

Abstract

Tumor aggressiveness and progression is highly dependent on the process of metastasis, regulated by the coordinated interplay of genetic and epigenetic mechanisms. Metastasis involves several steps of epithelial to mesenchymal transition (EMT), anoikis resistance, intra- and extravasation, and new tissue colonization. EMT is [...] Read more.

Tumor aggressiveness and progression is highly dependent on the process of metastasis, regulated by the coordinated interplay of genetic and epigenetic mechanisms. Metastasis involves several steps of epithelial to mesenchymal transition (EMT), anoikis resistance, intra- and extravasation, and new tissue colonization. EMT is considered as the most critical process allowing cancer cells to switch their epithelial characteristics and acquire mesenchymal properties. Emerging evidence demonstrates that epigenetics mechanisms, DNA methylation, histone modifications, and non-coding RNAs participate in the widespread changes of gene expression that characterize the metastatic phenotype. At the chromatin level, active and repressive histone post-translational modifications (PTM) in association with pleiotropic transcription factors regulate pivotal genes involved in the initiation of the EMT process as well as in intravasation and anoikis resistance, playing a central role in the progression of tumors. Herein, we discuss the main epigenetic mechanisms associated with the different steps of metastatic process, focusing in particular on the prominent role of histone modifications and the modifying enzymes that mediate transcriptional regulation of genes associated with tumor progression. We further discuss the development of novel treatment strategies targeting the reversibility of histone modifications and highlight their importance in the future of cancer therapy. Full article

(This article belongs to the Special Issue Causalities and Regulations of Tumor Metastasis — in Memory of Professor Isaiah J. Fidler (1936–2020))

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Figure 1
Epigenetic mechanisms regulate critical checkpoints of metastasis. Metastasis checkpoints are regulated by highly coordinated epigenetic mechanisms. EMT is mainly characterized by loss of E-cadherin expression. This can be achieved through regulation of transcription factors ZEB1, 2, SNAIL, and TWIST1. The p300/CBP complex acetylates the promoter of ZEB1, which further activates BRG1, in order to suppress E-cadherin via histone methylation. Additionally, ZEB1/ZEB2 have the ability tο recruit the CtBP complex, which enables HDAC1 to suppress the E-cadherin gene. At the same time, TGF-β, which is activated by TGFBR2-mediated induction of SNAI1/2, can activate both JMJ3 demethylase and lncRNA-HIT. JMJ3 is part of a loop along with TGF-β and SNAI1, regulating each other’s induction. Histone methyltransferase G9 targets SNAI1 which recruits SIN3A to suppress E-cadherin via deacetylation. Additionally, SET8 monomethylates H4K20 and cooperates with TWIST1 to increase N-cadherin and suppress E-cadherin. The methyltransferase EZH2, part of the PRC2 complex and the MYC-activated miR-9 can also inhibit E-cadherin expression by histone methylation and targeting of the E-cadherin mRNA. During the second step of intravasation and anoikis resistance, cancer cells express anti-apoptotic factors, such as BCL2 as well as mesenchymal markers. FZD7 induces activation of TWIST1 which increases BCL2 and FLIP inhibits anoikis by suppressing caspase-8 production. Moreover, JMJD2B interacts with β-catenin to upregulate the mesenchymal marker vimentin and PHF8 is involved in elevation of integrins expression. In the final step of extravasation and colonization, the cell differentiation NDRG1 gene plays a crucial role in reversing EMT and promoting metastasis. Full article ">Figure 2
Histone acetylation regulates genes involved in cancer progression. Histone acetylation is an activating histone modification that favors gene transcription. H3K4Ac marks are present on the promoters of several genes, GLI1, SMO, FOXF1, Bmi1, and SIRT2, which are upregulated in cancer, favoring tumor progression. Upon SNAI1/2 activation, increased H3K9 acetylation has been observed on the promoter of TGFBR2, causing its upregulation. Critical acetyltransferases TIP60, GCN5, PCAF, and p300 catalyze histone acetylation of metastasis-promoting genes. hMOF, a H4K16 histone acetyltransferase normally maintains the expression of EMT-related tumor suppressor genes, such as TMS1, CDH1, and ESR1, but is often downregulated in cancer. TIP60 is also commonly downregulated in various tumors, where it prevents the activity of anti-tumor DDR and p53 pathways, indicating how reduction in histone acetylation promotes cancer progression. HDAC3 can also be detected on the promoters of activated mesenchymal genes, even though most HDACs are associated with gene repression. Upon Notch signaling activation, H3K4Ac is removed by HDAC3, allowing for Notch-mediated expression of EMT-related genes. Profilin-2 interacts with HDAC1 and inhibits its binding to the promoters of Smad2 and Smad3, causing Smad protein activation and subsequently enhancing TGF-β-induced EMT and angiogenesis. HDACs also catalyze H3K4/56 deacetylation at the CDH1 promoter, thus repressing E-cadherin. Similarly, HDAC1 and 2 are recruited by ZEB1 to CDH1 promoter, inducing its repression. In the same context, ZEB1 recruits SIRT1, a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, associated with a global H3K27 deacetylation and reduced H3K9Ac and H3K27Ac levels on the promoters of CDH1 and other epithelial genes, such as EPCAM, ST14, ESRP1, and RAB25, promoting EMT and metastasis. Finally, overexpression of FLIP leads to the upregulation of the anti-apoptotic Bcl-XL, inhibiting the anoikis apoptotic pathway. HDAC inhibition prevents this outcome by reducing FLIP levels and inducing apoptosis of cancer cells, suggesting that HDACs participate in anoikis resistance of cancer cells through yet unknown mechanisms. Full article ">Figure 3
Role of histone methylation in gene regulation during tumor progression. Trimethylation of H3K27 and H3K4 has been associated with suppression of epithelial genes or activation of mesenchymal ones, respectively. In this process, the PRMT5-MEP50 complex suppresses CDH1 and GAS1 by mediating H3K4 and H4R3 methylation. E-cadherin is suppressed by SNAI1-mediated PRC2 trimethylation of H3K27. PRC2 also suppresses KLF2 expression. JARID2 recruits both PRC2 and G9a to promote H3K27 and H3K9 methylation at the promoters of CDH1 and miR-200. G9a also silences the ep-CAM gene, promoting metastasis. The TRIM33/Smad2/3 complex inhibits the binding of HP1 on the DNA, leading to expression of GSC and MIXL1 mesenchymal genes. The PRMT5-MEP50 complex recruits WDR5 and causes H3R2 methylation, activating the EMT-promoting genes VIM and SNAI1/2. Over-expression of the histone demethylase, JARID1A leads to the activation of cyclin D1/E1 and ITGB1 expression, promoting tumor progression. The H3K27 demethylase, UTX, activates several pro-metastatic genes, such as MMP-9/11 and SIX1, after interacting with the MLL4 complex, which includes a H3K4 methyltransferase, thus causing enhancement of EMT and metastasis. The PHF8 demethylase upregulates the expression of Vimentin, Integrin, and Rho-associated protein kinase (ROCK) kinase by removing H3K9 methylation marks, thus favoring metastasis. JMJD2B, a H3K9 demethylase, induces the expression of Vimentin, after interacting with β-catenin and also demethylates the Integrin (ITGB2, ITGAM, ITGA9, ITGAB2) gene promoters, further promoting EMT and metastasis. JMJ3, a H3K27 histone demethylase is induced by TGF-β and activates SNAI1 expression to facilitate EMT. EZH2 establishes repressive H3K27me3 marks and downregulates FOXC, inducing metastasis. It also silences the CDH1, AXIN2, NKD1, PPP2R2B, PRICKLE1, SFRP5, RKIP, promoting EMT and cancer metastasis. SETDB1 methyltransferase amplification is responsible for cancer cell growth by methylating and stabilizing the oncogenic p53 mutants. In this context, PRMT6 methyltransferase reduces the expression of p21, promoting tumor cell migration. Full article ">

17 pages, 1757 KiB

Open AccessReview

Bcl-xL: A Focus on Melanoma Pathobiology

by Anna Maria Lucianò, Ana B. Pérez-Oliva, Victoriano Mulero and Donatella Del Bufalo

Int. J. Mol. Sci. 2021, 22(5), 2777; https://doi.org/10.3390/ijms22052777 - 9 Mar 2021

Cited by 21 | Viewed by 4485

Abstract

Apoptosis is the main mechanism by which multicellular organisms eliminate damaged or unwanted cells. To regulate this process, a balance between pro-survival and pro-apoptotic proteins is necessary in order to avoid impaired apoptosis, which is the cause of several pathologies, including cancer. Among [...] Read more.

Apoptosis is the main mechanism by which multicellular organisms eliminate damaged or unwanted cells. To regulate this process, a balance between pro-survival and pro-apoptotic proteins is necessary in order to avoid impaired apoptosis, which is the cause of several pathologies, including cancer. Among the anti-apoptotic proteins, Bcl-xL exhibits a high conformational flexibility, whose regulation is strictly controlled by alternative splicing and post-transcriptional regulation mediated by transcription factors or microRNAs. It shows relevant functions in different forms of cancer, including melanoma. In melanoma, Bcl-xL contributes to both canonical roles, such as pro-survival, protection from apoptosis and induction of drug resistance, and non-canonical functions, including promotion of cell migration and invasion, and angiogenesis. Growing evidence indicates that Bcl-xL inhibition can be helpful for cancer patients, but at present, effective and safe therapies targeting Bcl-xL are lacking due to toxicity to platelets. In this review, we summarized findings describing the mechanisms of Bcl-xL regulation, and the role that Bcl-xL plays in melanoma pathobiology and response to therapy. From these findings, it emerged that even if Bcl-xL plays a crucial role in melanoma pathobiology, we need further studies aimed at evaluating the involvement of Bcl-xL and other members of the Bcl-2 family in the progression of melanoma and at identifying new non-toxic Bcl-xL inhibitors. Full article

(This article belongs to the Special Issue Advances in Bcl-xL Research 2.0)

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18 pages, 1921 KiB

Open AccessReview

Interactions of Lipid Droplets with the Intracellular Transport Machinery

by Selma Yilmaz Dejgaard and John F. Presley

Int. J. Mol. Sci. 2021, 22(5), 2776; https://doi.org/10.3390/ijms22052776 - 9 Mar 2021

Cited by 16 | Viewed by 7533

Abstract

Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and [...] Read more.

Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and lipid droplets. There is a large recent body of work on these organelles, which have resulted in the introduction of new paradigms regarding the roles of membrane trafficking organelles. In this review, we discuss the roles of membrane trafficking in the life cycle of lipid droplets. This includes the complementary roles of lipid phase separation and proteins in the biogenesis of lipid droplets from endoplasmic reticulum (ER) membranes, and the attachment of mature lipid droplets to membranes by lipidic bridges and by more conventional protein tethers. We also discuss the catabolism of neutral lipids, which in part results from the interaction of lipid droplets with cytosolic molecules, but with important roles for both macroautophagy and microautophagy. Finally, we address their eventual demise, which involves interactions with the autophagocytotic machinery. We pay particular attention to the roles of small GTPases, particularly Rab18, in these processes. Full article

(This article belongs to the Special Issue Intracellular Membrane Transport: Models and Machines)

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Comparison of the formation of coatomer protein II (COPII) vesicles with biogenesis of LDs. (A) COPII polymerizes laterally to form coated buds, with cargo proteins being sorted into the vesicle through interactions of their cytoplasmic domains with the COPII coat. Coat assembly and sorting take place primarily through protein–protein interactions. (B) Lipid droplet (LD) formation begins with the synthesis of neutral lipids: triglycerides by diacylglycerol O-acetyltransferase 1 (DGAT1) or cholesterol by acetyl-CoA acetyltransferase (ACAT)1/2. Neutral lipids condense into lenses in ER membranes, which then bud to form LDs. DGAT2 and other LD-resident proteins with short hydrophobic loops that are stable in a lipid monolayer can move to LDs while still attached to ER. These processes are driven primarily by phase separation and biophysical processes of lipids, but with proteins playing important modulatory roles. Full article ">Figure 2
Interaction of LDs with other membranes. (A) Illustration of a direct connection. LDs can remain attached to the ER by direct membranous connections stabilized by seipin rings. These connections permit exchange of proteins such as lipid droplet assembly factor 1 (LDAF1) between LDs and ER membranes. (B) Examples of protein complexes which may tether LDs to ER in the absence of direct membranous connections, including SNX14 and the Rab18/NRZ complex. While Rab18 is required for recruitment of fatty acid synthase to LDs, the link representing binding between fatty acid synthetase (FAS) and Rab18 is speculative. (C) Examples of proteins and protein complexes tethering LDs to mitochondria. At least two perilipins (PLIN1 and PLIN5) are involved. The mitoguardin 2 (MIGA2) complex may be involved in forming three-way complexes involving mitochondria and LDs linked to ER via VAP-B. Full article ">Figure 3
Triglyceride catabolism by cytoplasmic lipases on the LD surface. ATGL catalyzes release of the first fatty acid, followed by HSL and MAG in sequence. While ATGL and MAG seem to have narrow specificity as shown here, HSL has some activity towards TAG. Full article ">Figure 4
Catabolism of LDs by autophagy. (A) Macroautophagy is a process by which the entire LD can be enveloped by a phagophore or isolation membrane which seals to become an autophagosome. The autophagosome then undergoes a maturation process and fuses with a lysosome, leading to digestion of the LD and release of free fatty acid, glycerol and cholesterol. (B) Microautophagy of LDs has also been reported. While this process is not currently well understood, it may involve the internalization of a small portion of the LD into an internal vesicle in the lysosome. This process may allow autophagy of lipid from LDs which are too large for macroautophagy. Full article ">

15 pages, 2842 KiB

Open AccessArticle

Deletion of Irf4 in T Cells Suppressed Autoimmune Uveitis and Dysregulated Transcriptional Programs Linked to CD4⁺ T Cell Differentiation and Metabolism

by Minkyung Kang, Hyun-Su Lee, Jin Kyeong Choi, Cheng-Rong Yu and Charles E. Egwuagu

Int. J. Mol. Sci. 2021, 22(5), 2775; https://doi.org/10.3390/ijms22052775 - 9 Mar 2021

Cited by 3 | Viewed by 3312

Abstract

Interferon regulatory factor-4 (IRF4) and IRF8 regulate differentiation, growth and functions of lymphoid and myeloid cells. Targeted deletion of irf8 in T cells (CD4-IRF8KO) has been shown to exacerbate colitis and experimental autoimmune uveitis (EAU), a mouse model of human uveitis. We therefore [...] Read more.

Interferon regulatory factor-4 (IRF4) and IRF8 regulate differentiation, growth and functions of lymphoid and myeloid cells. Targeted deletion of irf8 in T cells (CD4-IRF8KO) has been shown to exacerbate colitis and experimental autoimmune uveitis (EAU), a mouse model of human uveitis. We therefore generated mice lacking irf4 in T cells (CD4-IRF4KO) and investigated whether expression of IRF4 by T cells is also required for regulating T cells that suppress autoimmune diseases. Surprisingly, we found that CD4-IRF4KO mice are resistant to EAU. Suppression of EAU derived in part from inhibiting pathogenic responses of Th17 cells while inducing expansion of regulatory lymphocytes that secrete IL-10 and/or IL-35 in the eye and peripheral lymphoid tissues. Furthermore, CD4-IRF4KO T cells exhibit alterations in cell metabolism and are defective in the expression of two Ikaros zinc-finger (IKZF) transcription factors (Ikaros, Aiolos) that are required for lymphocyte differentiation, metabolism and cell-fate decisions. Thus, synergistic effects of IRF4 and IkZFs might induce metabolic reprogramming of differentiating lymphocytes and thereby dynamically regulate relative abundance of T and B lymphocyte subsets that mediate immunopathogenic mechanisms during uveitis. Moreover, the diametrically opposite effects of IRF4 and IRF8 during EAU suggests that intrinsic function of IRF4 in T cells might be activating proinflammatory responses while IRF8 promotes expansion of immune-suppressive mechanisms. Full article

(This article belongs to the Section Molecular Immunology)

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Generation and characterization of mice with targeted deletion of irf4 in T cell. (A) Irf4fl/fl mice were crossed with CD4-Cre mice to generate mice deficient of IRF4 in CD4+ T cells (CD4-IRF4KO). The homozygote KO mice (CD4creIRF4fl/fl) were identified by PCR analysis of mouse tail genomic DNA. (B,C) Purified CD4+ T cells or CD19+ B cells from lymph nodes or spleen of wild-type (WT) or CD4-IRFK4O mice were analyzed for IRF4 expression by Western blot (B) or RT-PCR (C) analysis. Images of the full-length Western blot gels are provided (<a href="#app1-ijms-22-02775" class="html-app">Supplementary Figure S1</a>). (D) CD4+ T cells from LN and spleen of WT or CD4-IRF4KO mice were stimulated with anti-CD3/anti-CD28 for 3 days and analyzed by RT-PCR. (E) CD4+ T cells from LN/spleen and splenic CD19+ B cells of WT or CD4-IRF4KO mice were isolated 21 days after immunization with IRBP/CFA and analyzed by intracellular cytokine staining assay. Results represent more than 3 independent studies **** p < 0.0001. N.S.: Not significant Full article ">Figure 2
Inducible deletion of IRF4 in CD4+ T cells confers protection from experimental autoimmune uveitis (EAU). EAU was induced by immunization of C57BL/6J or CD4-IRF4KO mice with the uveitogenic peptide, IRBP651−670 in complete Freund’s adjuvant (CFA) (n = 12). Disease progression was monitored and assessed by fundoscopy, histology, optical coherence tomography (OCT) and electroretinography (ERG). (A) Fundus images of the retina at day 21 after EAU induction were taken using an otoendoscopic imaging system. Black arrow indicates inflammation with blurred optic disc margins and enlarge juxtapapillary areas; blue arrows indicate retinal vasculitis; white arrows indicate yellow-whitish retinal and choroidal infiltrates. Severe uveitis was observed in the WT compared to the CD4-IRF4KO mouse eyes as indicated by the clinical EAU scores. (B) Histologic sections are of eyes harvested 21 days after immunization with IRBP peptide and H&E staining reveal substantial numbers of inflammatory cells in the vitreous of WT compared to the CD4-IRF4KO eyes. EAU in the WT mice is characterized by the development massive retinal in-folding (*), a hallmark feature of severe uveitis. V, vitreous; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE retinal pigmented epithelial layer. Black arrows, lymphocytes; Asterisks, retinal folds. (C) Representative OCT images show marked increase of inflammatory cells (white arrows) in the vitreous and optic nerve (OPN). (D) ERG analysis of the retina on day 20 after EAU induction. The averages of light- or dark-adapted ERG a-wave or b-wave amplitudes are plotted as a function of flash luminance. Data presented as the mean ± SEM of four mice in each group of at least three independent experiments. (E) Cells from lymph nodes and spleen of IRBP651-670 immunized WT or CD4-IRF4KO mice were re-stimulated ex vivo and 1 × 107 cells were adoptively transferred to naïve WT mice. Disease scores determined by masked investigators indicate reduced EAU symptoms in mice that received CD4-IR4KO cells. Results represent 3 independent studies. * p < 0.05; ** p < 0.01, **** p < 0.0001. Full article ">Figure 3
Th1 and Th17 cells are reduced in CD4-IRF4KO mice during EAU. CD4+ T cells from the eye, lymph nodes or spleen of WT and CD4-IRF4KO mice immunized with IRBP/CFA were sorted and analyzed by the intracellular cytokine staining assay. Numbers in quadrants represent percentage CD4+ T cells expressing IL-17 and/or IFN-γ (A), ROR-γt (B) or ROR-γt and IL-17 (C). Data represent at least three independent experiments and were analyzed using Student’s t-test (two-tailed). * p < 0.05; ** p < 0.01; *** p < 0.001. ****: p < 0.0001. Full article ">Figure 4
Regulatory T and B cells that produce IL-10 or IL-35 are expanded during EAU in CD4-IRF4KO mice. (A) Lymphocytes in the eyes of WT or CD4-IRF4KO mice immunized with IRBP/CFA were stained with CD4 or B220 Abs and numbers in quadrants represent percent B cells and T cells. (B–D) CD4+ T cells in eyes or lymph nodes (B,C) or B220+ B cells in the spleen of EAU mice (D) were analyzed by intracellular cytokine staining assay. Numbers in quadrants represent percent CD4+ T cells expressing IL-10 (B) or IL-35 (C) or B cells secreting IL-35 (D). Data represent three independent experiments. Analysis was performed by Student’s t-test (two-tailed). * p < 0.05; ** p < 0.01; **** p < 0.0001. Full article ">Figure 5
Mice lacking IRF4 in CD4+ T cells exhibit altered expression of Ikaros Zinc Finger (IKZF) and Th17 signature genes. RNA was isolated from control (WT) and CD4-IRF4KO CD4+ T cells at day 21 post-immunization with IRBP/CFA, and subjected to RNA-Seq analyses. (A,B) Heatmap derived from global RNA-Seq analysis identified upregulated and downregulated genes in IRF4-deficient T cells. (C) KEGG pathway analysis shows effects of IRF4 on the transcription of genes that regulate Th1, Th2 or Th17 differentiation. (D,E) Heatmaps reveal differential expression of Th17 signature (D) and IKZF (E) genes by WT and CD4-IRF4KO T cells. (F) RNA was isolated from control (WT) and CD4-IRF4KO T cells at day 21 post immunization with IRBP/CFA and subjected to qPCR analyses. (G) A Gene Ontology enrichment analysis of the differentially expressed genes was performed to evaluate enriched biological processes. Data represent at least three independent experiments. ** p < 0.01; **** p < 0.0001. ns: Not significant Full article ">Figure 6
Loss of IRF4 in CD4+ T cells induced changes in cell metabolism during EAU. (A,B) CD4+ T cells derived from WT and CD4-IRF4KO mice during EAU were subjected to KEGG pathway analysis to determine whether loss of IRF4 expression in CD4+ T cells correlates with changes in the expression of metabolic gene. (B) Representative heatmap showing alterations in expression of metabolic pathway genes of WT and CD4-IRF4KO T cells. (C,D) Purified CD4+ T cells were isolated from WT or CD4-IRF4KO EAU mice (C) Images of the full-length Western blot gels are provided (<a href="#app1-ijms-22-02775" class="html-app">Supplementary Figure S2</a>). Glycolic assays were performed using the Seahorse Glycolytic Rate assay (D). Results are presented as time-dependent changes in oxygen consumption rate (OCR). Data represent three independent experiments. Full article ">

25 pages, 8426 KiB

Open AccessArticle

Synthesis and Pharmacological In Vitro Investigations of Novel Shikonin Derivatives with a Special Focus on Cyclopropane Bearing Derivatives

by Nadine Kretschmer, Antje Hufner, Christin Durchschein, Katrin Popodi, Beate Rinner, Birgit Lohberger and Rudolf Bauer

Int. J. Mol. Sci. 2021, 22(5), 2774; https://doi.org/10.3390/ijms22052774 - 9 Mar 2021

Cited by 9 | Viewed by 3301

Abstract

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of [...] Read more.

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of the roots of Onosma paniculata as well as a synthetic derivative of the most active constituent showed promising results in metastatic melanoma cell lines. In the current study, we address the question whether we can generate further derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells) using the XTT viability assay. We identified (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity. Furthermore, pharmacological investigations including the ApoToxGlo^TM Triplex assay, LDH assay, and cell cycle measurements revealed that this compound induced apoptosis and reduced cells in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be promising drug leads in the treatment of melanoma. Full article

(This article belongs to the Special Issue Skin Inflammation Aging and Diseases)

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20 pages, 4204 KiB

Open AccessArticle

Stress and Nasal Allergy: Corticotropin-Releasing Hormone Stimulates Mast Cell Degranulation and Proliferation in Human Nasal Mucosa

by Mika Yamanaka-Takaichi, Yukari Mizukami, Koji Sugawara, Kishiko Sunami, Yuichi Teranishi, Yukimi Kira, Ralf Paus and Daisuke Tsuruta

Int. J. Mol. Sci. 2021, 22(5), 2773; https://doi.org/10.3390/ijms22052773 - 9 Mar 2021

Cited by 9 | Viewed by 11452

Abstract

Psychological stress exacerbates mast cell (MC)-dependent inflammation, including nasal allergy, but the underlying mechanisms are not thoroughly understood. Because the key stress-mediating neurohormone, corticotropin-releasing hormone (CRH), induces human skin MC degranulation, we hypothesized that CRH may be a key player in stress-aggravated nasal [...] Read more.

Psychological stress exacerbates mast cell (MC)-dependent inflammation, including nasal allergy, but the underlying mechanisms are not thoroughly understood. Because the key stress-mediating neurohormone, corticotropin-releasing hormone (CRH), induces human skin MC degranulation, we hypothesized that CRH may be a key player in stress-aggravated nasal allergy. In the current study, we probed this hypothesis in human nasal mucosa MCs (hM-MCs) in situ using nasal polyp organ culture and tested whether CRH is required for murine M-MC activation by perceived stress in vivo. CRH stimulation significantly increased the number of hM-MCs, stimulated both their degranulation and proliferation ex vivo, and increased stem cell factor (SCF) expression in human nasal mucosa epithelium. CRH also sensitized hM-MCs to further CRH stimulation and promoted a pro-inflammatory hM-MC phenotype. The CRH-induced increase in hM-MCs was mitigated by co-administration of CRH receptor type 1 (CRH-R1)-specific antagonist antalarmin, CRH-R1 small interfering RNA (siRNA), or SCF-neutralizing antibody. In vivo, restraint stress significantly increased the number and degranulation of murine M-MCs compared with sham-stressed mice. This effect was mitigated by intranasal antalarmin. Our data suggest that CRH is a major activator of hM-MC in nasal mucosa, in part via promoting SCF production, and that CRH-R1 antagonists such as antalarmin are promising candidate therapeutics for nasal mucosa neuroinflammation induced by perceived stress. Full article

(This article belongs to the Section Molecular Biology)

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Graphical abstract

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Full article ">Figure 1
Human nasal mucosa mast cells (hM-MCs) and the mucosal epithelium in nasal polyps (NPs) express corticotropin-releasing hormone receptor type 1 (CRH-R1). (a) CRH-R1 expression on c-Kit+ or (b) tryptase+ hM-MCs in situ. An arrow denotes double+ MCs. (c) Percentage of tryptase and CRH-R1 or corticotropin-releasing hormone receptor type 2 (CRH-R2) double+ MCs. n = 5. (d) CRH expression within the mucosal epithelium in NPs (an arrow). (e) CRH-R1 and CRH-R2 expression within the mucosal epithelium in NPs. Scale bar = 20 µm. Error bars indicate the standard error of the mean (SEM). ** p < 0.01. MC, mast cell; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; CRH-R1, CRH receptor type 1; CRH-R2, CRH receptor type 2; DAPI, diamidino-2-phenylindole. Full article ">Figure 2
CRH increased tryptase+ hM-MC numbers and stimulated their degranulation. (a) Toluidine blue histochemistry with 1 day organ-cultured human NPs treated with vehicle or CRH (10−7 M). An arrow denotes hM-MCs in the lamina propria. Top, non-degranulated MCs in vehicle-treated NPs. Bottom, degranulated MCs in CRH-treated NPs. n = 5; scale bar = 20 µm. (b) Tryptase immunohistochemistry with 1 day organ-cultured human NPs treated with vehicle or CRH (10−7 M). Scale bar = 50 µm. (c) Quantitative immunohistomorphometry of tryptase+ cells in the lamina propria of organ-cultured NPs with CRH, anti-SCF (stem cell factor neutralizing antibody; 1 μg/mL), and/or antalarmin (10−6 M); n = 6. (d) Tryptase immunohistochemistry. An arrow denotes MC degranulation induced by CRH. Scale bar = 10 µm. (e) Percentage of degranulated MCs treated with CRH, anti-SCF (1 μg/mL), and/or antalarmin (10−6 M) analyzed by tryptase immunohistochemistry; n = 6. (f) Electron microscope images of CRH-induced MC degranulation. Scale bar = 1 µm. (g) Diameter of tryptase+ hM-MC granules; n = 6; scale bar = 10 µm. Error bars indicate SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001. MC, mast cell; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; anti-SCF, stem cell factor neutralizing antibody. Full article ">Figure 3
CRH increased tryptase+ hM-MC proliferation but not the apoptosis of hM-MCs. CRH-R1 gene silencing showed that CRH-R1 small interfering RNA (siRNA) inhibited the increase in tryptase+ hM-MC number and degranulation by CRH in situ. (a) Tryptase/Ki-67 double immunofluorescence. The arrow denotes tryptase and Ki-67 double+ cells within human nasal polyps treated with CRH; n = 6; scale bar = 10 µm. (b) Quantitative immunohistomorphometry of tryptase/proliferating cell nuclear antigen (PCNA) double+ cells; n = 4. (c) Quantitative immunohistomorphometry of tryptase/terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) double+ cells; n = 4. (d) Quantitative immunohistomorphometry of tryptase+ cells in the lamina propria of organ-cultured NPs with CRH-R1 siRNA-treated human NPs; n = 4. (e) Percentage of degranulated hM-MCs in the lamina propria with CRH-R1 siRNA-treated human NPs; n = 4. Error bars indicate SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, N.S. = not significant. MC, mast cell; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; CRH-R1, CRH receptor type 1; CRH-R1 siRNA, CRH-R1 siRNA-treated NPs; SCR, scrambled siRNA-treated NPs; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Full article ">Figure 4
CRH increased stem cell factor (SCF) expression in the mucosal epithelium in NPs. (a) SCF immunofluorescence of organ-cultured human NPs treated with vehicle or CRH (10−7 M). CRH significantly increased SCF expression within the epithelium of 1 day organ-cultured human NPs. The arrow denotes SCF-positive immunoreactivity within the epithelium. n = 6; scale bar = 50 µm. (b) Double immunofluorescence for AE1/3/SCF and (c) tryptase/SCF of CRH-treated NPs. Most of the SCF+ cells were epithelial cells. Some tryptase+ hM-MCs within the epithelium expressed SCF. The arrow denotes SCF+ hM-MCs; n = 4 in (b) and 6 in (c). Scale bar = 10 µm in (b), 20 µm in (c). (d) SCF immunoreactivity in NP epithelium with CRH-R1 siRNA-treated human NPs. The arrow denotes SCF-positive immunoreactivity within the epithelium. n = 4; scale bar = 20 µm. Error bars indicate SEM. *p < 0.05, ** p < 0.01. MC, mast cell; SCF, stem cell factor; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; CRH-R1, CRH receptor type 1; CRH-R1 siRNA, CRH-R1 siRNA-treated NPs; SCR, scrambled siRNA-treated NPs; DAPI, diamidino-2-phenylindole. Full article ">Figure 5
CRH increased the expression of CRH-R1 on tryptase+ hM-MCs and upregulated tryptase immunoreactivity within the hM-MCs. Percentage of (a) tryptase+ and (b) c-Kit+ MCs with CRH-R1 in the lamina propria of organ-cultured human NPs, analyzed by double immunofluorescence of tryptase or c-Kit/CRH-R1. n = 7 in (a) and 4 in (b). (c) Tryptase immunoreactivity within the hM-MCs in NPs treated with vehicle or CRH. CRH treatment significantly increased tryptase immunoreactivity within hM-MCs. n = 5; scale bar = 10 µm. Error bars indicate SEM. * p < 0.05, N.S. = not significant. MC, mast cell; hM-MCs, human nasal mucosa MCs; NPs, nasal polyps; CRH, corticotropin-releasing hormone; CRH-R1, CRH receptor type 1. Full article ">Figure 6
Restraint stress increased mice nasal mucosa MC (mM-MC) numbers and degranulation. (a) Toluidine blue histochemistry of murine nasal mucosa. The arrow denotes mM-MCs in the lamina propria, and the arrowhead denotes a degranulated mM-MC. Scale bar = 20 µm. (b) Quantitative immunohistomorphometry of mM-MC. (c) The percentage of degranulated mM-MCs in the nasal mucosa after restraint stress and nasal application of antalarmin. Both acute and chronic restraint stress significantly increased mM-MC numbers in the nasal mucosa and stimulated their degranulation. The increased number of mM-MCs and their degranulation in stressed mouse nasal mucosa was significantly inhibited by nasal application of antalarmin. Control group, n = 4 (without stress); stress group, n = 4; stress + antalarmin (5 μg/g/day) group, n = 5; and antalarmin group, n = 4 (without stress). Error bars indicate SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. MC, mast cell; mM-MCs, mice nasal mucosa MCs. Full article ">

43 pages, 8002 KiB

Open AccessArticle

Design, Synthesis, and Evaluation of Novel 3-Carboranyl-1,8-Naphthalimide Derivatives as Potential Anticancer Agents

by Sebastian Rykowski, Dorota Gurda-Woźna, Marta Orlicka-Płocka, Agnieszka Fedoruk-Wyszomirska, Małgorzata Giel-Pietraszuk, Eliza Wyszko, Aleksandra Kowalczyk, Paweł Stączek, Andrzej Bak, Agnieszka Kiliszek, Wojciech Rypniewski and Agnieszka B. Olejniczak

Int. J. Mol. Sci. 2021, 22(5), 2772; https://doi.org/10.3390/ijms22052772 - 9 Mar 2021

Cited by 19 | Viewed by 4144

Abstract

We synthesized a series of novel 3-carboranyl-1,8-naphthalimide derivatives, mitonafide and pinafide analogs, using click chemistry, reductive amination and amidation reactions and investigated their in vitro effects on cytotoxicity, cell death, cell cycle, and the production of reactive oxygen species in a HepG2 cancer [...] Read more.

We synthesized a series of novel 3-carboranyl-1,8-naphthalimide derivatives, mitonafide and pinafide analogs, using click chemistry, reductive amination and amidation reactions and investigated their in vitro effects on cytotoxicity, cell death, cell cycle, and the production of reactive oxygen species in a HepG2 cancer cell line. The analyses showed that modified naphthalic anhydrides and naphthalimides bearing ortho- or meta-carboranes exhibited diversified activity. Naphthalimides were more cytotoxic than naphthalic anhydrides, with the highest IC₅₀ value determined for compound 9 (3.10 µM). These compounds were capable of inducing cell cycle arrest at G0/G1 or G2M phase and promoting apoptosis, autophagy or ferroptosis. The most promising conjugate 35 caused strong apoptosis and induced ROS production, which was proven by the increased level of 2′-deoxy-8-oxoguanosine in DNA. The tested conjugates were found to be weak topoisomerase II inhibitors and classical DNA intercalators. Compounds 33, 34, and 36 fluorescently stained lysosomes in HepG2 cells. Additionally, we performed a similarity-based assessment of the property profile of the conjugates using the principal component analysis. The creation of an inhibitory profile and descriptor-based plane allowed forming a structure–activity landscape. Finally, a ligand-based comparative molecular field analysis was carried out to specify the (un)favorable structural modifications (pharmacophoric pattern) that are potentially important for the quantitative structure–activity relationship modeling of the carborane–naphthalimide conjugates. Full article

(This article belongs to the Section Biochemistry)

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16 pages, 3351 KiB

Open AccessArticle

Combined Application of Pan-AKT Inhibitor MK-2206 and BCL-2 Antagonist Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia

by Anna Richter, Elisabeth Fischer, Clemens Holz, Julia Schulze, Sandra Lange, Anett Sekora, Gudrun Knuebel, Larissa Henze, Catrin Roolf, Hugo Murua Escobar and Christian Junghanss

Int. J. Mol. Sci. 2021, 22(5), 2771; https://doi.org/10.3390/ijms22052771 - 9 Mar 2021

Cited by 9 | Viewed by 3535

Abstract

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B-ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK-2206 promises meticulous pan-AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK-2206 [...] Read more.

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B-ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK-2206 promises meticulous pan-AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK-2206 on B-ALL cell lines and primary samples and investigate potential synergistic effects with BCL-2 inhibitor venetoclax to overcome limitations in apoptosis induction. MK-2206 incubation reduced AKT phosphorylation and influenced downstream signaling activity. Interestingly, after MK-2206 mono application tumor cell proliferation and metabolic activity were diminished significantly independently of basal AKT phosphorylation. Morphological changes but no induction of apoptosis was detected in the observed cell lines. In contrast, primary samples cultivated in a protective microenvironment showed a decrease in vital cells. Combined MK-2206 and venetoclax incubation resulted in partially synergistic anti-proliferative effects independently of application sequence in SEM and RS4;11 cell lines. Venetoclax-mediated apoptosis was not intensified by addition of MK-2206. Functional assessment of BCL-2 inhibition via Bax translocation assay revealed slightly increased pro-apoptotic signaling after combined MK-2206 and venetoclax incubation. In summary, we demonstrate that the pan-AKT inhibitor MK-2206 potently blocks B-ALL cell proliferation and for the first time characterize the synergistic effect of combined MK-2206 and venetoclax treatment in B-ALL. Full article

(This article belongs to the Special Issue Novel Agents and Mechanisms in Acute Leukemias)

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Figure 1

Figure 1
Expression of phosphorylated and total AKT protein determined by immunoblot and subsequent quantification. (a) SEM, RS4;11, REH and NALM-6 cells were incubated with the stated concentrations of MK-2206 for the indicated time periods. The displayed immunoblots are representative captions of at least two individual biological replicates. GAPDH was used as internal loading control. Blots were processed and cropped using Image Studio Lite 5.2 software and MS PowerPoint (2011) to improve clarity and conciseness. (b) Phospho (p)-AKT and total AKT band intensities of two independent biological replicates including the blots shown in <a href="#ijms-22-02771-f001" class="html-fig">Figure 1</a>a were determined using Image Studio Lite 5.2 software. AKT phosphorylation was calculated as the ratio of pAKT/total AKT and normalized to the DMSO control separately for every time point. One REH replicate was not quantified due to the very low basal pAKT expression and therefore low signal to noise ratio not acceptable for reliable quantification. Full article ">Figure 2
Effects of MK-2206 incubation on cell proliferation and metabolic activity. Dose-response curves of SEM, RS4;11, REH and NALM-6 cells incubated with increasing concentrations of MK-2206 for 48 h (a,c) and 72 h (b,d). Proliferation (a,b) was assessed by trypan blue staining and metabolic activity (c,d) was determined by WST-1 assay. Mean ± standard deviation of at least three individual biological replicates. Significance was determined by student’s t test after testing Gaussian distribution. To increase clarity, significance levels are not indicated within the graphs and instead listed in <a href="#app1-ijms-22-02771" class="html-app">Supplementary Tables S1–S4</a>. Full article ">Figure 3
Effects of MK-2206 on cell morphology and apoptosis induction. (a) Cells were incubated with increasing concentrations for 72 h. Cytospins were prepared and Pappenheim stained. Representative images of all cell lines treated with 0.5 µM MK-2206, 100-fold magnification. Images were acquired using the EVOS xl core microscope (Nikon). Red arrows: disintegrating cell membrane; light blue arrows: vacuolization; purple arrows: nuclear fragmentation; green arrows: cytoplasmic blebs; dark blue arrows: karyopyknosis. (b) SEM and RS4;11 cells were incubated with 0.25 µM MK-2206 for 72 h and subsequently stained with annexin V-FITC and PI for apoptosis analysis by flow cytometry. Cells positive for annexin V and negative for PI are indicated as early apoptotic whereas cells positive for both markers are considered late apoptotic/necrotic. (c) Exemplary dot plots of flow cytometric apoptosis determination in SEM cells. Full article ">Figure 4
Effects of MK-2206 on primary B-ALL blasts. (a,b) Protein expression of phosphorylated and total AKT was measured by immunofluorescence staining (a) and immunoblotting (b). Cells of patients #0122 and #0159 were orthotopically xenografted into NSG mice as described in [<a href="#B33-ijms-22-02771" class="html-bibr">33</a>,<a href="#B34-ijms-22-02771" class="html-bibr">34</a>] and spleen cells were subsequently harvested for further analysis. (a) Expression of pAKT was determined by immunofluorescence using the LSM780 confocal microscope (Zeiss) at 20-fold magnification (left panel). The right hand images are enlargements of the regions indicated in the left hand red boxes. (b) Cells were lysed and analyzed for phosphorylated and total AKT protein expression by immunoblot. GAPDH was used as internal loading control. Blots were processed and cropped using Image Studio Lite 5.2 software and MS PowerPoint (2011) to improve clarity and conciseness. (c) Genetic variants in both patients were detected using Cancer hotspot panel (Ion PGM System, Thermo Fisher Scientific) and previously described in [<a href="#B34-ijms-22-02771" class="html-bibr">34</a>]. (d,e) Xenograft-derived primary blasts were cultured on a murine stromal cell feeder layer system and cell viability and apoptosis induction were assessed after 72 h incubation with increasing concentrations of MK-2206. (d) Cell viability was determined by CellTiter Blue assay in technical triplicates. Mean ± standard deviation of three individual biological replicates. Significance was determined by student’s t test after testing Gaussian distribution. * P < 0.05. (e) Cells were stained with anti-human CD19, annexin V-FITC and PI for apoptosis analysis by flow cytometry. Cells positive for annexin V and negative for PI are indicated as early apoptotic whereas cells positive for both markers are considered late apoptotic/necrotic. Human primary blasts and murine feeder cells were discriminated by anti-human CD19 staining. Full article ">Figure 5
Combined application of MK-2206 (MK) and venetoclax (ven). (a,b) Cytotoxicity of MK-2206 (0.25 µM), venetoclax (10 nM) or the combination was determined by hemolysis assay and PBMC viability analysis. Blood of five healthy voluntary donors was used. Technical triplicates were performed for both assays. Mean ± standard deviation. (a) Hemolytic activity was assessed by hemoglobin release after 120 min incubation with the substances, 1% SDS (positive control) or PBS (negative control). (b) PBMCs were isolated by density gradient centrifugation and incubated with the respective inhibitors or 10 µM DMSO (control) for 24 h. Cell viability was determined by Calcein AM assay. (c,d) Assessment of cell proliferation and metabolic activity was performed by trypan blue staining and WST-1 assay, respectively and compared to DMSO-treated control cells. Cells were incubated with 0.25 µM MK-2206, 10 nM (SEM (c)) or 2.5 nM (RS4;11 (d)) venetoclax or both inhibitors for 72 h. (e) Effects of sequential application of MK-2206 and venetoclax on SEM and RS4;11 cells were determined by WST-1 assay. Substances were applied either simultaneously (MK + ven) or the second substance was added 24 h after the first (MK > ven or ven > MK). Metabolic activity was assessed after 72 h. Mean ± standard deviation of at least three individual biological replicates. Significance was determined by one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001. Asterisks demonstrate significance compared to DMSO-treated control cells or other treatments when connected by lines. (f) Synergy was calculated according to the Bliss independence model with values > 0 indicating synergistic combinations [<a href="#B35-ijms-22-02771" class="html-bibr">35</a>]. Bliss values were calculated based on the mean biological response of at least four biological replicates. Cells were incubated with MK-2206, venetoclax or both (simultaneously or sequentially) and metabolic activity was assessed by WST-1 assay after 72 h. Full article ">Figure 6
Analysis of apoptotic processes after single or combined MK-2206 (MK; 0.25 µM) and venetoclax (ven; 10 nM (SEM) or 2.5 nM (RS4;11)) incubation in SEM and RS4;11. (a) Cells were incubated for 72 h and stained with annexin V-FITC and PI. The amount of apoptotic cells was determined by flow cytometry with cells single positive for annexin V called early aposptotic and cells positive for both markers late apoptotic/necrotic. Mean ± standard deviation of at least three individual biological replicates. Significance was determined by one-way ANOVA. *** P < 0.001. (b,c) Downstream apoptotic signaling was investigated by Bax translocation assay. Cells were treated with one or both inhibitors for 48 h and spun onto microscopic slides. Mitochondria were stained using MitoSpy™ red and Bax protein was stained in green. Images were taken using the Eclipse TE200 microscope (Nikon) and at 40-fold magnification. Four biological replicates were performed for each treatment and two to four individual images per biological replicate were considered for colocalization analysis. (b) Color-merged images were split into separate images for both channels and regions of interest were marked using Fiji program. Colocalization analysis of Bax and mitochondria was performed using the Coloc2 plugin in Fiji and Pearson’s coefficient were used to quantify the amount of Bax/mitochondria overlap. (c) Representative images of SEM and RS4;11 cells treated with DMSO (control), MK-2206, venetoclax or both. Mitochondria are stained in red and Bax in green. Yellow signals are a product of red and green signal overlay and indicate colocalization of both structures. Full article ">

20 pages, 2920 KiB

Open AccessArticle

Preclinical Evaluation of ^99mTc-ZHER2:41071, a Second-Generation Affibody-Based HER2-Visualizing Imaging Probe with a Low Renal Uptake

by Maryam Oroujeni, Sara S. Rinne, Anzhelika Vorobyeva, Annika Loftenius, Joachim Feldwisch, Per Jonasson, Vladimir Chernov, Anna Orlova, Fredrik Y. Frejd and Vladimir Tolmachev

Int. J. Mol. Sci. 2021, 22(5), 2770; https://doi.org/10.3390/ijms22052770 - 9 Mar 2021

Cited by 20 | Viewed by 4192

Abstract

Radionuclide imaging of HER2 expression in tumours may enable stratification of patients with breast, ovarian, and gastroesophageal cancers for HER2-targeting therapies. A first-generation HER2-binding affibody molecule [^99mTc]Tc-ZHER2:V2 demonstrated favorable imaging properties in preclinical studies. Thereafter, the affibody scaffold has been extensively [...] Read more.

Radionuclide imaging of HER2 expression in tumours may enable stratification of patients with breast, ovarian, and gastroesophageal cancers for HER2-targeting therapies. A first-generation HER2-binding affibody molecule [^99mTc]Tc-ZHER2:V2 demonstrated favorable imaging properties in preclinical studies. Thereafter, the affibody scaffold has been extensively modified, which increased its melting point, improved storage stability, and increased hydrophilicity of the surface. In this study, a second-generation affibody molecule (designated ZHER2:41071) with a new improved scaffold has been prepared and characterized. HER2-binding, biodistribution, and tumour-targeting properties of [^99mTc]Tc-labelled ZHER2:41071 were investigated. These properties were compared with properties of the first-generation affibody molecules, [^99mTc]Tc-ZHER2:V2 and [^99mTc]Tc-ZHER2:2395. [^99mTc]Tc-ZHER2:41071 bound specifically to HER2 expressing cells with an affinity of 58 ± 2 pM. The renal uptake for [^99mTc]Tc-ZHER2:41071 and [^99mTc]Tc-ZHER2:V2 was 25–30 fold lower when compared with [^99mTc]Tc-ZHER2:2395. The uptake in tumour and kidney for [^99mTc]Tc-ZHER2:41071 and [^99mTc]Tc-ZHER2:V2 in SKOV-3 xenografts was similar. In conclusion, an extensive re-engineering of the scaffold did not compromise imaging properties of the affibody molecule labelled with ^99mTc using a GGGC chelator. The new probe, [^99mTc]Tc-ZHER2:41071 provided the best tumour-to-blood ratio compared to HER2-imaging probes for single photon emission computed tomography (SPECT) described in the literature so far. [^99mTc]Tc-ZHER2:41071 is a promising candidate for further clinical translation studies. Full article

(This article belongs to the Special Issue Cancer Molecular Imaging 2.0)

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48 pages, 22172 KiB

Open AccessReview

A Review of the Pharmacological Activities and Recent Synthetic Advances of γ-Butyrolactones

by Joonseong Hur, Jaebong Jang and Jaehoon Sim

Int. J. Mol. Sci. 2021, 22(5), 2769; https://doi.org/10.3390/ijms22052769 - 9 Mar 2021

Cited by 50 | Viewed by 9018

Abstract

γ-Butyrolactone, a five-membered lactone moiety, is one of the privileged structures of diverse natural products and biologically active small molecules. Because of their broad spectrum of biological and pharmacological activities, synthetic methods for γ-butyrolactones have received significant attention from synthetic and [...] Read more.

γ-Butyrolactone, a five-membered lactone moiety, is one of the privileged structures of diverse natural products and biologically active small molecules. Because of their broad spectrum of biological and pharmacological activities, synthetic methods for γ-butyrolactones have received significant attention from synthetic and medicinal chemists for decades. Recently, new developments and improvements in traditional methods have been reported by considering synthetic efficiency, feasibility, and green chemistry. In this review, the pharmacological activities of natural and synthetic γ-butyrolactones are described, including their structures and bioassay methods. Mainly, we summarize recent advances, occurring during the past decade, in the construction of γ-butyrolactone classified based on the bond formation in γ-butyrolactone between (i) C5-O1 bond, (ii) C4-C5 and C2-O1 bonds, (iii) C3-C4 and C2-O1 bonds, (iv) C3-C4 and C5-O1 bonds, (v) C2-C3 and C2-O1 bonds, (vi) C3-C4 bond, and (vii) C2-O1 bond. In addition, the application to the total synthesis of natural products bearing γ-butyrolactone scaffolds is described. Full article

(This article belongs to the Special Issue Natural and Synthetic Lactones: Isolation, Synthesis, Biotransformations and Biological Activity)

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16 pages, 4271 KiB

Open AccessArticle

Rational Design of Adenylate Kinase Thermostability through Coevolution and Sequence Divergence Analysis

by Jian Chang, Chengxin Zhang, Huaqiang Cheng and Yan-Wen Tan

Int. J. Mol. Sci. 2021, 22(5), 2768; https://doi.org/10.3390/ijms22052768 - 9 Mar 2021

Cited by 8 | Viewed by 3088

Abstract

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or [...] Read more.

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic Escherichia coli (E. coli) ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature. Full article

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

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Figure 1

Figure 1
Residue Correlation Analysis (RCA) of Protein Sectors in ADK Family. (A) Heat map representation of RCA matrix rij for MSA of ADK. X− and y−axis coordinates both correspond to residue indices of ADK from E. coli, which has 214 residues. (B) Three dimensional scatter plot of the 214 residues in the space formed by the three eigenvectors of the second, the third, and the fourth largest eigenvectors. Each data point represented one position. After eliminating randomized background residues (gray), the rest of the positions could be clustered into four sectors, colored green, blue, orange, and magenta. (C) The relative entropy angle θ of thermophilic and mesophilic sequence profile at each position. Bars were colored by sectors. Residues whose angles were larger than 0.5 in the magenta sector were selected for further mutation (marked by arrow heads). (D) Amino acid distribution of thermophilic sequences at the chosen mutation positions. The first series of single-point mutations were S41K, D76V, G100N, I101R, P139K, P140S, K141R, and R206F. Full article ">Figure 2
Thermal stability and enzymatic activity of single-point mutants characterized by circular dichroism (CD) spectroscopy and room temperature enzymatic activity assay. (A) CD differential signal at 222nm of wild type and mutants S41A, D76N, G100N, I101T, P139K, P140S, K141A, K141P, and R206F from 40 °C to 70 °C. All data were normalized to a scale of zero to one. To be distinguished from each other, each curve was offset by one along the y-axis. (B) Forward enzymatic activity of wild type and eight mutants at 25 °C. [ATP] = 1 mM, [magnesium acetate (MgOAc2)] = 2 mM. Full article ">Figure 3
Correlation between melting temperature and enzymatic activity of wt and selected mutants of ADK. (A) Forward reaction activities (at 0.2 mM AMP and 1 mM ATP) versus melting temperature of ADK wild type and single mutants. (B) Temperature dependence of enzyme activity for ADK from E. coli, single mutant R206F, double mutant R206F/S41A and ADK from Aquifex aeolicus in the direction of ADP formation. The absolute value of 100% maximal activity was calculated as <math display="inline"><semantics> <mrow> <mn>458</mn> <mo>±</mo> <mn>4</mn> <mtext> </mtext> <msup> <mi>s</mi> <mrow> <mo>−</mo> <mn>1</mn> </mrow> </msup> </mrow> </semantics></math>. (C) Four double mutants, R206F/S41A, R206F/G100N, R206F/P139K, R206F/P140S, all displayed a moderate Tm increment lower than the sum of respective Tm rise from two related individual mutants. The synergistic effect lowered as the ΔTm sum increased. The dash line indicated the theoretical ΔTm distribution of double mutants compared to R206F provided that the synergistic effect was linear. Full article ">Figure 4
Mutations sites chosen by the relative entropy angle θ. (A) Residues colored according to our protein sectors color scheme on the “closed” conformation of ADK (PDB id: 1ANK chain A). ATP analog and AMP (both colored black) were shown in sticks. The structure on the right was obtained by rotating the diagram on the left by 90 degrees clockwise around the vertical axis. (B) Residues directly participating in substrate binding in E. coli ADK. All sectors except the magenta one were involved in ADK’s function execution. (C) Eight selected mutation sites were marked as magenta spheres in the tertiary structure of ADK. Full article ">

14 pages, 2574 KiB

Open AccessArticle

Comparative Analysis of CREB3 and CREB3L2 Protein Expression in HEK293 Cells

by Kentaro Oh-hashi, Ayumi Yamamoto, Ryoichi Murase and Yoko Hirata

Int. J. Mol. Sci. 2021, 22(5), 2767; https://doi.org/10.3390/ijms22052767 - 9 Mar 2021

Cited by 6 | Viewed by 3373

Abstract

We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 [...] Read more.

We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 and CREB3L2; however, the level of the full-length CREB3 protein, but not CREB3L2 protein, was not noticeably reduced by the monensin treatment. On the other hand, treatment with tunicamycin (Tm) shifted the molecular weight of the full-length CREB3L2 protein downward but abolished CREB3 protein expression. Thapsigargin (Tg) significantly increased the expression of only full-length CREB3L2 protein concomitant with a slight increase in the level of its cleaved form. Treatment with cycloheximide and MG132 revealed that both endogenous CREB3 and CREB3L2 are proteasome substrates. In addition, kifunensine, an α-mannosidase inhibitor, significantly increased the levels of both full-length forms. Consistent with these findings, cells lacking SEL1L, a crucial ER-associated protein degradation (ERAD) component, showed increased expression of both full-length CREB3 and CREB3L2; however, cycloheximide treatment downregulated full-length CREB3L2 protein expression more rapidly in SEL1L-deficient cells than the full-length CREB3 protein. Finally, we investigated the induction of the expression of several CREB3 and CREB3L2 target genes by Tg and BFA treatments and SEL1L deficiency. In conclusion, this study suggests that both endogenous full-length CREB3 and CREB3L2 are substrates for ER-associated protein degradation but are partially regulated by distinct mechanisms, each of which contributes to unique cellular responses that are distinct from canonical ER signals. Full article

(This article belongs to the Special Issue Physio-Pathological Role of ERAD and Its Pharmacological Modulation)

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23 pages, 7888 KiB

Open AccessArticle

Gene Expression-Related Changes in Morphologies of Organelles and Cellular Component Organization in Mucopolysaccharidoses

by Lidia Gaffke, Karolina Pierzynowska, Estera Rintz, Zuzanna Cyske, Izabela Giecewicz and Grzegorz Węgrzyn

Int. J. Mol. Sci. 2021, 22(5), 2766; https://doi.org/10.3390/ijms22052766 - 9 Mar 2021

Cited by 21 | Viewed by 3253

Abstract

Mucopolysaccharidoses (MPS) are inherited metabolic diseases characterized by accumulation of incompletely degraded glycosaminoglycans (GAGs) in lysosomes. Although primary causes of these diseases are mutations in genes coding for enzymes involved in lysosomal GAG degradation, it was demonstrated that storage of these complex carbohydrates [...] Read more.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases characterized by accumulation of incompletely degraded glycosaminoglycans (GAGs) in lysosomes. Although primary causes of these diseases are mutations in genes coding for enzymes involved in lysosomal GAG degradation, it was demonstrated that storage of these complex carbohydrates provokes a cascade of secondary and tertiary changes affecting cellular functions. Potentially, this might lead to appearance of cellular disorders which could not be corrected even if the primary cause of the disease is removed. In this work, we studied changes in cellular organelles in MPS fibroblasts relative to control cells. All 11 types and subtypes of MPS were included into this study to obtain a complex picture of changes in organelles in this group of diseases. Two experimental approaches were employed, transcriptomic analyses and electron microscopic assessment of morphology of organelles. We analyzed levels of transcripts of genes grouped into two terms included into the QuickGO database, ‘Cellular component organization’ (GO:0016043) and ‘Cellular anatomical entity’ (GO:0110165), to find that number of transcripts with significantly changed levels in MPS fibroblasts vs. controls ranged from 109 to 322 (depending on MPS type) in GO:0016043, and from 70 to 208 in GO:0110165. This dysregulation of expression of genes crucial for proper structures and functions of various organelles was accompanied by severe changes in morphologies of lysosomes, nuclei, mitochondria, Golgi apparatus, and endoplasmic reticulum. Interestingly, some observed changes occurred in all/most MPS types while others were specific to particular disease types/subtypes. We suggest that severe changes in organelles in MPS cells might arise from dysregulation of expression of a battery of genes involved in organelles’ structures and functions. Intriguingly, normalization of GAG levels by using recombinant human enzymes specific to different MPS types corrected morphologies of some, but not all, organelles, while it failed to improve regulation of expression of selected genes. These results might suggest reasons for inability of enzyme replacement therapy to correct all MPS symptoms, particularly if initiated at advanced stages of the disease. Full article

(This article belongs to the Special Issue Genetic and Metabolic Molecular Research of Lysosomal Storage Disease)

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21 pages, 4376 KiB

Open AccessArticle

Butyrate Improves Skin/Lung Fibrosis and Intestinal Dysbiosis in Bleomycin-Induced Mouse Models

by Hee Jin Park, Ok-Yi Jeong, Sung Hak Chun, Yun Hong Cheon, Mingyo Kim, Suhee Kim and Sang-Il Lee

Int. J. Mol. Sci. 2021, 22(5), 2765; https://doi.org/10.3390/ijms22052765 - 9 Mar 2021

Cited by 34 | Viewed by 5861

Abstract

Systemic sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Despite several studies on SSc treatments, effective treatments for SSc are still lacking. Since evidence suggests an association between intestinal microbiota and SSc, we focused on butyrate, [...] Read more.

Systemic sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Despite several studies on SSc treatments, effective treatments for SSc are still lacking. Since evidence suggests an association between intestinal microbiota and SSc, we focused on butyrate, which has beneficial effects in autoimmune diseases as a bacterial metabolite. Here, we investigated the therapeutic potential of sodium butyrate (SB) using a bleomycin-induced fibrosis mouse model of SSc and human dermal fibroblasts (HDFs). SB attenuated bleomycin-induced dermal and lung fibrosis in mice. SB influenced fecal microbiota composition (phyla Actinobacteria and Bacteroidetes, genera Bifidobacterium and Ruminococcus_g2). SB controlled macrophage differentiation in mesenteric lymph nodes, spleen, and bronchoalveolar lavage cells of mice with bleomycin-induced skin fibrosis. Profibrotic and proinflammatory gene expression was suppressed by SB administration in skin. Furthermore, SB inhibited transforming growth factor β1-responsive proinflammatory expression with increased acetylation of histone 3 in HDFs. Subcutaneous SB application had antifibrogenic effects on the skin. Butyrate ameliorated skin and lung fibrosis by improving anti-inflammatory activity in a mouse model of SSc. Butyrate may exhibit indirect and direct anti-fibrogenic action on fibroblasts by regulating macrophage differentiation and inhibition of histone deacetylase 3. These findings suggest butyrate as an SSc treatment. Full article

(This article belongs to the Section Molecular Immunology)

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17 pages, 3399 KiB

Open AccessArticle

Reduction of Amyloid Burden by Proliferated Homeostatic Microglia in Toxoplasma gondii-Infected Alzheimer’s Disease Model Mice

by Ji-Hun Shin, Young Sang Hwang, Bong-Kwang Jung, Seung-Hwan Seo, Do-Won Ham and Eun-Hee Shin

Int. J. Mol. Sci. 2021, 22(5), 2764; https://doi.org/10.3390/ijms22052764 - 9 Mar 2021

Cited by 8 | Viewed by 4239

Abstract

In this study, we confirmed that the number of resident homeostatic microglia increases during chronic Toxoplasma gondii infection. Given that the progression of Alzheimer’s disease (AD) worsens with the accumulation of amyloid β (Aβ) plaques, which are eliminated through microglial phagocytosis, we hypothesized [...] Read more.

In this study, we confirmed that the number of resident homeostatic microglia increases during chronic Toxoplasma gondii infection. Given that the progression of Alzheimer’s disease (AD) worsens with the accumulation of amyloid β (Aβ) plaques, which are eliminated through microglial phagocytosis, we hypothesized that T. gondii-induced microglial proliferation would reduce AD progression. Therefore, we investigated the association between microglial proliferation and Aβ plaque burden using brain tissues isolated from 5XFAD AD mice (AD group) and T. gondii-infected AD mice (AD + Toxo group). In the AD + Toxo group, amyloid plaque burden significantly decreased compared with the AD group; conversely, homeostatic microglial proliferation, and number of plaque-associated microglia significantly increased. As most plaque-associated microglia shifted to the disease-associated microglia (DAM) phenotype in both AD and AD + Toxo groups and underwent apoptosis after the lysosomal degradation of phagocytosed Aβ plaques, this indicates that a sustained supply of homeostatic microglia is required for alleviating Aβ plaque burden. Thus, chronic T. gondii infection can induce microglial proliferation in the brains of mice with progressed AD; a sustained supply of homeostatic microglia is a promising prospect for AD treatment. Full article

(This article belongs to the Section Molecular Neurobiology)

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17 pages, 972 KiB

Open AccessReview

Dysbiosis in the Development of Type I Diabetes and Associated Complications: From Mechanisms to Targeted Gut Microbes Manipulation Therapies

by Gratiela Gradisteanu Pircalabioru, Nicolae Corcionivoschi, Ozan Gundogdu, Mariana-Carmen Chifiriuc, Luminita Gabriela Marutescu, Bogdan Ispas and Octavian Savu

Int. J. Mol. Sci. 2021, 22(5), 2763; https://doi.org/10.3390/ijms22052763 - 9 Mar 2021

Cited by 18 | Viewed by 5343

Abstract

Globally, we are facing a worrying increase in type 1 diabetes mellitus (T1DM) incidence, with onset at younger age shedding light on the need to better understand the mechanisms of disease and step-up prevention. Given its implication in immune system development and regulation [...] Read more.

Globally, we are facing a worrying increase in type 1 diabetes mellitus (T1DM) incidence, with onset at younger age shedding light on the need to better understand the mechanisms of disease and step-up prevention. Given its implication in immune system development and regulation of metabolism, there is no surprise that the gut microbiota is a possible culprit behind T1DM pathogenesis. Additionally, microbiota manipulation by probiotics, prebiotics, dietary factors and microbiota transplantation can all modulate early host–microbiota interactions by enabling beneficial microbes with protective potential for individuals with T1DM or at high risk of developing T1DM. In this review, we discuss the challenges and perspectives of translating microbiome data into clinical practice. Nevertheless, this progress will only be possible if we focus our interest on developing numerous longitudinal, multicenter, interventional and double-blind randomized clinical trials to confirm their efficacy and safety of these therapeutic approaches. Full article

(This article belongs to the Special Issue Gut Microbiota and Immunity)

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12 pages, 1136 KiB

Open AccessReview

Bone Marrow Aspirate Matrix: A Convenient Ally in Regenerative Medicine

by José Fábio Lana, Lucas Furtado da Fonseca, Gabriel Azzini, Gabriel Santos, Marcelo Braga, Alvaro Motta Cardoso Junior, William D. Murrell, Alberto Gobbi, Joseph Purita and Marco Antonio Percope de Andrade

Int. J. Mol. Sci. 2021, 22(5), 2762; https://doi.org/10.3390/ijms22052762 - 9 Mar 2021

Cited by 14 | Viewed by 4073

Abstract

The rise in musculoskeletal disorders has prompted medical experts to devise novel effective alternatives to treat complicated orthopedic conditions. The ever-expanding field of regenerative medicine has allowed researchers to appreciate the therapeutic value of bone marrow-derived biological products, such as the bone marrow [...] Read more.

The rise in musculoskeletal disorders has prompted medical experts to devise novel effective alternatives to treat complicated orthopedic conditions. The ever-expanding field of regenerative medicine has allowed researchers to appreciate the therapeutic value of bone marrow-derived biological products, such as the bone marrow aspirate (BMA) clot, a potent orthobiologic which has often been dismissed and regarded as a technical complication. Numerous in vitro and in vivo studies have contributed to the expansion of medical knowledge, revealing optimistic results concerning the application of autologous bone marrow towards various impactful disorders. The bone marrow accommodates a diverse family of cell populations and a rich secretome; therefore, autologous BMA-derived products such as the “BMA Matrix”, may represent a safe and viable approach, able to reduce the costs and some drawbacks linked to the expansion of bone marrow. BMA provides —it eliminates many hurdles associated with its preparation, especially in regards to regulatory compliance. The BMA Matrix represents a suitable alternative, indicated for the enhancement of tissue repair mechanisms by modulating inflammation and acting as a natural biological scaffold as well as a reservoir of cytokines and growth factors that support cell activity. Although promising, more clinical studies are warranted in order to further clarify the efficacy of this strategy. Full article

(This article belongs to the Section Molecular Biology)

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12 pages, 265 KiB

Open AccessReview

Machine Learning and Novel Biomarkers for the Diagnosis of Alzheimer’s Disease

by Chun-Hung Chang, Chieh-Hsin Lin and Hsien-Yuan Lane

Int. J. Mol. Sci. 2021, 22(5), 2761; https://doi.org/10.3390/ijms22052761 - 9 Mar 2021

Cited by 121 | Viewed by 13728

Abstract

Background: Alzheimer’s disease (AD) is a complex and severe neurodegenerative disease that still lacks effective methods of diagnosis. The current diagnostic methods of AD rely on cognitive tests, imaging techniques and cerebrospinal fluid (CSF) levels of amyloid-β1-42 (Aβ42), total tau protein and hyperphosphorylated [...] Read more.

Background: Alzheimer’s disease (AD) is a complex and severe neurodegenerative disease that still lacks effective methods of diagnosis. The current diagnostic methods of AD rely on cognitive tests, imaging techniques and cerebrospinal fluid (CSF) levels of amyloid-β1-42 (Aβ42), total tau protein and hyperphosphorylated tau (p-tau). However, the available methods are expensive and relatively invasive. Artificial intelligence techniques like machine learning tools have being increasingly used in precision diagnosis. Methods: We conducted a meta-analysis to investigate the machine learning and novel biomarkers for the diagnosis of AD. Methods: We searched PubMed, the Cochrane Central Register of Controlled Trials, and the Cochrane Database of Systematic Reviews for reviews and trials that investigated the machine learning and novel biomarkers in diagnosis of AD. Results: In additional to Aβ and tau-related biomarkers, biomarkers according to other mechanisms of AD pathology have been investigated. Neuronal injury biomarker includes neurofiliament light (NFL). Biomarkers about synaptic dysfunction and/or loss includes neurogranin, BACE1, synaptotagmin, SNAP-25, GAP-43, synaptophysin. Biomarkers about neuroinflammation includes sTREM2, and YKL-40. Besides, d-glutamate is one of coagonists at the NMDARs. Several machine learning algorithms including support vector machine, logistic regression, random forest, and naïve Bayes) to build an optimal predictive model to distinguish patients with AD from healthy controls. Conclusions: Our results revealed machine learning with novel biomarkers and multiple variables may increase the sensitivity and specificity in diagnosis of AD. Rapid and cost-effective HPLC for biomarkers and machine learning algorithms may assist physicians in diagnosing AD in outpatient clinics. Full article

(This article belongs to the Special Issue Biomarkers and Molecular Methodologies in Neurodegenerative Diseases Diagnostic)

20 pages, 2299 KiB

Open AccessArticle

Anti-Apoptotic Effect of Apelin in Human Placenta: Studies on BeWo Cells and Villous Explants from Third-Trimester Human Pregnancy

by Ewa Mlyczyńska, Małgorzata Myszka, Patrycja Kurowska, Monika Dawid, Tomasz Milewicz, Marta Bałajewicz-Nowak, Paweł Kowalczyk and Agnieszka Rak

Int. J. Mol. Sci. 2021, 22(5), 2760; https://doi.org/10.3390/ijms22052760 - 9 Mar 2021

Cited by 19 | Viewed by 3123

Abstract

Previously, we demonstrated the expression of apelin and G-protein-coupled receptor APJ in human placenta cell lines as well as its direct action on placenta cell proliferation and endocrinology. The objective of this study was to examine the effect of apelin on placenta apoptosis [...] Read more.

Previously, we demonstrated the expression of apelin and G-protein-coupled receptor APJ in human placenta cell lines as well as its direct action on placenta cell proliferation and endocrinology. The objective of this study was to examine the effect of apelin on placenta apoptosis in BeWo cells and villous explants from the human third trimester of pregnancy. The BeWo cells and villous explants were incubated with apelin (2 and 20 ng/mL) alone or with staurosporine for 24 to 72 h. First, we analysed the dose- and time-dependent effect of apelin on the expression of apoptotic factors on the mRNA level by real-time PCR and on the protein level using Western blot. Next, we checked caspase 3 and 7 activity by Caspase-Glo 3/7, DNA fragmentation by the Cell Death Detection ELISA kit and oxygen consumption by the MitoXpress-Xtra Oxygen Consumption assay. We found that apelin increased the expression of pro-survival and decreased proapoptotic factors on mRNA and protein levels in both BeWo cells and villous explants. Additionally, apelin inhibited caspase 3 and 7 activity and DNA fragmentation in staurosporine-induced apoptosis as also attenuated oxidative stress by increasing extracellular oxygen consumption. The antiapoptotic effect of apelin in BeWo cells was mediated by the APJ receptor and mitogen-activated protein kinase (ERK1/2/MAP3/1) and protein kinase B (AKT). The obtained results showed the antiapoptotic effect of apelin on trophoblast cells, suggesting its participation in the development of the placenta. Full article

(This article belongs to the Special Issue Adipokines in Health and Diseases)

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16 pages, 3740 KiB

Open AccessArticle

Increased Presence of Complement Factors and Mast Cells in Alveolar Bone and Tooth Resorption

by Kathrin Luntzer, Ina Lackner, Birte Weber, Yvonne Mödinger, Anita Ignatius, Florian Gebhard, Susann-Yvonne Mihaljevic, Melanie Haffner-Luntzer and Miriam Kalbitz

Int. J. Mol. Sci. 2021, 22(5), 2759; https://doi.org/10.3390/ijms22052759 - 9 Mar 2021

Cited by 3 | Viewed by 2782

Abstract

Periodontitis is the inflammatory destruction of the tooth-surrounding and -supporting tissue, resulting at worst in tooth loss. Another locally aggressive disease of the oral cavity is tooth resorption (TR). This is associated with the destruction of the dental mineralized tissue. However, the underlying [...] Read more.

Periodontitis is the inflammatory destruction of the tooth-surrounding and -supporting tissue, resulting at worst in tooth loss. Another locally aggressive disease of the oral cavity is tooth resorption (TR). This is associated with the destruction of the dental mineralized tissue. However, the underlying pathomechanisms remain unknown. The complement system, as well as mast cells (MCs), are known to be involved in osteoclastogenesis and bone loss. The complement factors C3 and C5 were previously identified as key players in periodontal disease. Therefore, we hypothesize that complement factors and MCs might play a role in alveolar bone and tooth resorption. To investigate this, we used the cat as a model because of the naturally occurring high prevalence of both these disorders in this species. Teeth, gingiva samples and serum were collected from domestic cats, which had an appointment for dental treatment under anesthesia, as well as from healthy cats. Histological analyses, immunohistochemical staining and the CH-50 and AH-50 assays revealed increased numbers of osteoclasts and MCs, as well as complement activity in cats with TR. Calcifications score in the gingiva was highest in animals that suffer from TR. This indicates that MCs and the complement system are involved in the destruction of the mineralized tissue in this condition. Full article

(This article belongs to the Special Issue Osteoclastogenesis and Osteogenesis)

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(A) Von Kossa staining of the gingiva in control cats (C) (n = 4) and cats affected by gingivitis (G) (n = 6), periodontitis (PD) (n = 8) or tooth resorption (TR) (n = 11). Images were obtained at 100× magnification. In control and gingivitis sections, no crystals were observed (calcification score 0). In periodontitis samples, rare, tiny crystals, marked by black arrows, were present (calcification score 1). In this TR section many large crystals could be seen (calcification score 3). (B) There was a significant increase in the occurrence of crystals in the tooth resorption (TR) (n = 11) group compared with the control (C) (n = 4) and gingivitis (G) (n = 6) groups (both p ≤ 0.05). There was no significant difference between the periodontitis group (PD) (n = 8) and the other groups. Full article ">Figure 2
(A) Micro-Computed Tomography (μCT) image of a canine affected by tooth resorption (TR). The affected area is marked with a white rectangle. (B) Tartrate-resistant acid phosphatase staining of this tooth revealed many osteoclasts in this area, directly adjacent to the dentine of the tooth. The image was obtained at 400× magnification. (C) The evaluation showed significantly more osteoclasts in TR (n = 11) compared to periodontitis (PD) (n = 23) in both of the mineralized tissues, bone (p ≤ 0.01) and tooth (p ≤ 0.05). There was also a significant difference in the percentage of the eroded tooth surface between these groups (p ≤ 0.01). Full article ">Figure 3
(A) Image of the immunohistochemical histamine staining in a TR section. The image was taken at 200× magnification. MCs could be identified. (B) Immunohistochemical staining for histamine showed a significant increase of MCs in the TR group (n = 11) compared with the control (n = 7) and gingivitis groups (n = 10) (both p ≤ 0.01). There was no difference between the TR and periodontitis groups (n = 8). Full article ">Figure 4
(A) Immunohistochemical staining of the complement C5a receptor (C5aR). Images were taken at 400× magnification. The endothelial layer of the vessels in the gingiva of the controls (C) (n = 4) and cats affected by gingivitis (G) (n = 6), periodontitis (PD) (n = 7) or tooth resorption (TR) (n = 12) is marked by black arrows. In the controls and gingivitis animals, only a few endothelial layers were stained. The staining in general was less marked than in in the other two groups (PD, TR). (B) No significant differences were observed between the groups. However, there was a trend for a difference in periodontitis and TR compared to control and gingivitis regarding the endothelial layer. In addition, there was a trend for a difference in positively stained cells of the lamina propria between the control group and the other groups. Full article ">Figure 5
(A) Immunohistochemical staining of C5a in the gingiva of the controls (C) (n = 4) and of cats affected by gingivitis (G) (n = 6), periodontitis (PD) (n = 9) or tooth resorption (TR) (n = 12). Images were obtained at 400× magnification. No positive cells were observed in the control section. In the gingivitis section, a single positively stained cell was seen, marked by a black arrow. In the other two groups, notably more cells could be seen. (B) There was a trend for an increase in positively stained cells in the PD and TR groups compared with the other two groups. (C) Double immunofluorescence staining in the gingiva of a tooth affected by TR. Scale bar 50 μm. Mast cell granules (Avidin) were stained red, while C5a was stained green. Mast cells positive for C5a were observed, marked by white arrows. Full article ">Figure 6
(A) Immunohistochemical staining of complement factor C3 in the gingiva of the controls (C) (n = 4) and cats affected by gingivitis (G) (n = 6), periodontitis (PD) (n = 9) or tooth resorption (TR) (n = 12). Images were obtained at 400× magnification. In the control and gingivitis groups, no positively stained cells were observed. Two C3-positive cells were seen in periodontitis, marked by black arrows. The greatest number of positively stained cells could be observed in TR. (B) There was a statistically significant increase in C3-positive cells in the TR group compared with the other three groups (p ≤ 0.05). (C) Double immunofluorescence staining in the gingiva of a tooth affected by TR. Scale bar 50 μm. Mast cell granules (Avidin) were stained red, while C5 was stained green. In this section, one mast cell was positive for C3, marked by a white arrow. Full article ">Figure 7
Hemolytic assay for the detection of complement classical pathway function. The optical density of the serum samples of both the control (C) and tooth resorption (TR) groups were plotted against the dilution factor. The curves show the control (4 cats) and TR groups (12 cats) with different levels of complement function. In TR, 50% lysis occurred between serum dilutions 1:80 and 1:160, in the control group between the undiluted serum and dilution 1:10. There was a significant difference in the serum complement concentration between the two groups, being significantly higher in the TR group than in the control group (p ≤ 0.05). Full article ">Figure 8
Hemolytic assay for the detection of complement alternative pathway function. The optical density of the serum samples of both the control (C) and tooth resorption (TR) groups were plotted against the dilution factor. The curves show the control (4 cats) and TR groups (10 cats) with different levels of complement function. In both groups, 50% lysis occurred between serum dilutions 1:8 and 1:16. Nevertheless, there was a significant difference in the serum complement concentration between the two groups, being significantly higher in the TR group than in the control group (p ≤ 0.05). Full article ">

17 pages, 1125 KiB

Open AccessReview

Assessing Plasmin Generation in Health and Disease

by Adam Miszta, Dana Huskens, Demy Donkervoort, Molly J. M. Roberts, Alisa S. Wolberg and Bas de Laat

Int. J. Mol. Sci. 2021, 22(5), 2758; https://doi.org/10.3390/ijms22052758 - 9 Mar 2021

Cited by 28 | Viewed by 6953

Abstract

Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The kinetics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood [...] Read more.

Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The kinetics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID-19, or diet-induced obesity. Moreover, this review introduces the PG assay as a promising clinical and research method to monitor antifibrinolytic medications and screen for genetic or acquired fibrinolytic disorders. Full article

(This article belongs to the Special Issue Fibrinogen/Fibrin, Factor XIII and Fibrinolysis in Diseases)

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15 pages, 23641 KiB

Open AccessReview

Diabetes, Obesity, and Inflammation: Impact on Clinical and Radiographic Features of Breast Cancer

by Braden Miller, Hunter Chalfant, Alexandra Thomas, Elizabeth Wellberg, Christina Henson, Molly W. McNally, William E. Grizzle, Ajay Jain and Lacey R. McNally

Int. J. Mol. Sci. 2021, 22(5), 2757; https://doi.org/10.3390/ijms22052757 - 9 Mar 2021

Cited by 19 | Viewed by 5219

Abstract

Obesity, diabetes, and inflammation increase the risk of breast cancer, the most common malignancy in women. One of the mainstays of breast cancer treatment and improving outcomes is early detection through imaging-based screening. There may be a role for individualized imaging strategies for [...] Read more.

Obesity, diabetes, and inflammation increase the risk of breast cancer, the most common malignancy in women. One of the mainstays of breast cancer treatment and improving outcomes is early detection through imaging-based screening. There may be a role for individualized imaging strategies for patients with certain co-morbidities. Herein, we review the literature regarding the accuracy of conventional imaging modalities in obese and diabetic women, the potential role of anti-inflammatory agents to improve detection, and the novel molecular imaging techniques that may have a role for breast cancer screening in these patients. We demonstrate that with conventional imaging modalities, increased sensitivity often comes with a loss of specificity, resulting in unnecessary biopsies and overtreatment. Obese women have body size limitations that impair image quality, and diabetes increases the risk for dense breast tis-sue. Increased density is known to obscure the diagnosis of cancer on routine screening mammography. Novel molecu-lar imaging agents with targets such as estrogen receptor, human epidermal growth factor receptor 2 (HER2), pyrimi-dine analogues, and ligand-targeted receptor probes, among others, have potential to reduce false positive results. They can also improve detection rates with increased resolution and inform therapeutic decision making. These emerg-ing imaging techniques promise to improve breast cancer diagnosis in obese patients with diabetes who have dense breasts, but more work is needed to validate their clinical application. Full article

(This article belongs to the Special Issue Molecular Imaging in Diabetes, Obesity, and Infections 2.0)

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Imaging appearance of different breasts of increasing density using three types of imaging modalities. Digital mammography depicts how breasts appear more opaque as breast density increases. T1-weighted non-fat-suppressed MR images show increasing amount of fibroglandular tissue (FGT). T1-weighted fat-suppressed contrast-enhanced subtraction MR images show increasing amounts of background parenchymal enhancement (BPE), which reflects the vascularity of the fibroglandular tissue. (Adapted from [<a href="#B23-ijms-22-02757" class="html-bibr">23</a>]). Full article ">Figure 2
Image of ultrasound placement for ultrasound-guided biopsy. (A) Image of ultrasound-guided biopsy for medial breast lesions. (B) In the prefire position, needle tip (white arrowhead) should be placed at the margin of most lesions (black arrow), with needle parallel or near parallel to chest wall. (C) Postfire images should be documented and should definitively outline the needle traversing the lesion (black arrow). The tip of the needle may lie beyond the lesion margin in some cases, depending on the size of the target (white arrowhead). Full article ">Figure 3
Mammography images depicting different appearances of calcifications. (A) Classic appearance of breast arterial calcifications (BAC) (white arrow) that are easy to discriminate and associated with diabetes. (B) Groups of microcalcifications (yellow circles) in a woman with ductal carcinoma in situ (DCIS) is shown in its most typical appearance on digital breast tomosynthesis (DBT). (C) A patient with DCIS with mammographic findings showing segmental fine pleomorphic and fine-linear branching calcifications. (D) A skin calcification resembles microcalcifications (white arrow) and often requires alternative views to localize to the skin. Any or all of these findings can be seen in a single mammogram and can create a convoluted picture for radiologists. (Adapted from [<a href="#B65-ijms-22-02757" class="html-bibr">65</a>]). Full article ">Figure 4
Patterns of HER2–PET/CT compared with FDG–PET/CT, Maximum intensity projection. Lesion uptake was considered concerning for malignant disease when visually higher than blood pool. (A) Entire tumor burden showed concerning tracer uptake; (B) Major portion of the tumor burden showed concerning tracer uptake; (C) A very small subset of the tumor burden showed concerning tracer uptake; (D) The entire visualized tumor burden lacked concerning tracer uptake. Adapted from [<a href="#B98-ijms-22-02757" class="html-bibr">98</a>]. Full article ">Figure 5
Osteopontin-probe identifies triple-negative breast cancer using multispectral optoacoustic tomography in a murine model. Serial slices are shown with * indicating positive osteopontin-probe uptake within the orthotopic triple negative breast tumor. Adapted from [<a href="#B103-ijms-22-02757" class="html-bibr">103</a>]. Full article ">

33 pages, 727 KiB

Open AccessReview

Omics Approaches in Adipose Tissue and Skeletal Muscle Addressing the Role of Extracellular Matrix in Obesity and Metabolic Dysfunction

by Augusto Anguita-Ruiz, Mireia Bustos-Aibar, Julio Plaza-Díaz, Andrea Mendez-Gutierrez, Jesús Alcalá-Fdez, Concepción María Aguilera and Francisco Javier Ruiz-Ojeda

Int. J. Mol. Sci. 2021, 22(5), 2756; https://doi.org/10.3390/ijms22052756 - 9 Mar 2021

Cited by 17 | Viewed by 6133

Abstract

Extracellular matrix (ECM) remodeling plays important roles in both white adipose tissue (WAT) and the skeletal muscle (SM) metabolism. Excessive adipocyte hypertrophy causes fibrosis, inflammation, and metabolic dysfunction in adipose tissue, as well as impaired adipogenesis. Similarly, disturbed ECM remodeling in SM has [...] Read more.

Extracellular matrix (ECM) remodeling plays important roles in both white adipose tissue (WAT) and the skeletal muscle (SM) metabolism. Excessive adipocyte hypertrophy causes fibrosis, inflammation, and metabolic dysfunction in adipose tissue, as well as impaired adipogenesis. Similarly, disturbed ECM remodeling in SM has metabolic consequences such as decreased insulin sensitivity. Most of described ECM molecular alterations have been associated with DNA sequence variation, alterations in gene expression patterns, and epigenetic modifications. Among others, the most important epigenetic mechanism by which cells are able to modulate their gene expression is DNA methylation. Epigenome-Wide Association Studies (EWAS) have become a powerful approach to identify DNA methylation variation associated with biological traits in humans. Likewise, Genome-Wide Association Studies (GWAS) and gene expression microarrays have allowed the study of whole-genome genetics and transcriptomics patterns in obesity and metabolic diseases. The aim of this review is to explore the molecular basis of ECM in WAT and SM remodeling in obesity and the consequences of metabolic complications. For that purpose, we reviewed scientific literature including all omics approaches reporting genetic, epigenetic, and transcriptomic (GWAS, EWAS, and RNA-seq or cDNA arrays) ECM-related alterations in WAT and SM as associated with metabolic dysfunction and obesity. Full article

(This article belongs to the Special Issue Proteolysis of Extracellular Matrix in Human Disease)

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16 pages, 2647 KiB

Open AccessArticle

Epigenetic Changes Governing Scn5a Expression in Denervated Skeletal Muscle

by David Carreras, Rebecca Martinez-Moreno, Mel·lina Pinsach-Abuin, Manel M. Santafe, Pol Gomà, Ramon Brugada, Fabiana S. Scornik, Guillermo J. Pérez and Sara Pagans

Int. J. Mol. Sci. 2021, 22(5), 2755; https://doi.org/10.3390/ijms22052755 - 9 Mar 2021

Cited by 6 | Viewed by 3191

Abstract

The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel (Na_V1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also [...] Read more.

The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel (Na_V1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the SCN5A gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing SCN5A expression are not yet well understood. To gain insights into the molecular basis of SCN5A gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate Scn5a expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac Scn5a isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with Scn5a amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the Scn5a promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate Scn5a in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of Scn5a in denervated muscle and cardiac tissue, suggesting a conserved pathway for SCN5A expression among striated muscles. Full article

(This article belongs to the Special Issue Epigenetic Mechanisms of Cardiac Disease)

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Figure 1

Figure 1
Comparison of exon 6 sequence between reference cardiac adult isoform (top sequence) and the sequence obtained from the denervated skeletal muscle (bottom sequence). The electropherogram corresponds to the product obtained for the denervated muscle. Full article ">Figure 2
Differential RNA-seq analysis of denervated skeletal muscle compared to control, innervated muscle. (A) Pie Chart shows the percentage of genes with no significant expression changes (grey), significantly upregulated (orange) and significantly downregulated (blue). (B) Heatmap showing the 50 top upregulated and downregulated genes upon denervation. Each row represents the expression level of a particular gene in control (n = 4) and denervated samples (n = 4). Full article ">Figure 3
Gene Ontology (GO) enrichment annotation analysis of differentially expressed (DE) genes. GO categorization of DE genes in molecular functions (MF), cellular components (CC), biological processes (BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway (shown as -log10 p-value). Only the top ten significant upregulated and downregulated annotations are shown (p < 0.05). Full article ">Figure 4
Scn5a is the only gene upregulated within the topological associated domain (TAD). Limits of the TAD in which Scn5a is included, and changes in the expression of genes located within the TAD are shown as log2FC. Full article ">Figure 5
Enrichment of H3K27ac and H3K4me3 and binding of Gata4 near the Scn5a promoter in the denervated muscle. (A) Schematic view of the localization of the four primer pairs used by qPCR. Numbers indicate outermost left base pairs of amplicons. (B) Chromatin immunoprecipitation (ChIP) assays from control (white) and denervated (blue) samples using antibodies against H3K27ac, H3K4me3 and Gata4 and followed by qPCR. Results are shown as percentage of input (mean ± SE). H3K27ac (n = 6), H3K4me3 (n = 6) and Gata4 (n = 3). Significance was analyzed with t-test (* p < 0.05). Full article ">Figure 6
Denervation induces the activation of a super enhancer downstream of Scn5a. Integrative Genome Viewer (IGV) view of the H3K27ac distribution pattern in the Scn5a TAD and the reads of RNA-seq data in control and denervated gastrocnemius muscle samples. Expanded view of the super enhancer region located between Exog and Scn5a gene (bottom). Full article ">

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Int. J. Mol. Sci., Volume 22, Issue 5 (March-1 2021) – 555 articles

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