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Biomedicines
Volume 10
Issue 4
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Biomedicines, Volume 10, Issue 4 (April 2022) – 198 articles

Cover Story (view full-size image): Our research indicates a defect in proinsulin (PI) chaperone GRP94 leads to an accumulation of mishandled proinsulin in β-cells. This results in ER stress, inflammatory pathway activation, int-proteasome assembly, and GRP94-PI complex secretion. Simultaneously, increased MHC-I expression on β-cells enhances their visibility to immune cells. The secreted GRP94-PI complexes are taken up by antigen-presenting cells and cross-presented to CD8+ T-cells to trigger an immune attack against β-cells. View this paper
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16 pages, 6343 KiB  
Article
Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse Astrocytes
by Gary Stanley Fernandes, Rishabh Deo Singh and Kyeong Kyu Kim
Biomedicines 2022, 10(4), 928; https://doi.org/10.3390/biomedicines10040928 - 18 Apr 2022
Cited by 7 | Viewed by 3400
Abstract
Astrocyte-to-neuron reprogramming is a promising therapeutic approach for treatment of neurodegenerative diseases. The use of small molecules as an alternative to the virus-mediated ectopic expression of lineage-specific transcription factors negates the tumorigenic risk associated with viral genetic manipulation and uncontrolled differentiation of stem [...] Read more.
Astrocyte-to-neuron reprogramming is a promising therapeutic approach for treatment of neurodegenerative diseases. The use of small molecules as an alternative to the virus-mediated ectopic expression of lineage-specific transcription factors negates the tumorigenic risk associated with viral genetic manipulation and uncontrolled differentiation of stem cells. However, because previously developed methods for small-molecule reprogramming of astrocytes to neurons are multistep, complex, and lengthy, their applications in biomedicine, including clinical treatment, are limited. Therefore, our objective in this study was to develop a novel chemical-based approach to the cellular reprogramming of astrocytes into neurons with high efficiency and low complexity. To accomplish that, we used C8-D1a, a mouse astrocyte cell line, to assess the role of small molecules in reprogramming protocols that otherwise suffer from inconsistencies caused by variations in donor of the primary cell. We developed a new protocol by which a chemical mixture formulated with Y26732, DAPT, RepSox, CHIR99021, ruxolitinib, and SAG rapidly and efficiently induced the neural reprogramming of astrocytes in four days, with a conversion efficiency of 82 ± 6%. Upon exposure to the maturation medium, those reprogrammed cells acquired a glutaminergic phenotype over the next eleven days. We also demonstrated the neuronal functionality of the induced cells by confirming KCL-induced calcium flux. Full article
(This article belongs to the Section Biomedical Engineering and Materials)
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Figure 1

Figure 1
<p>(<b>a</b>) Schematic design of the protocol for converting mouse astrocytes into neuron-like cells. (<b>b</b>) Neuronal conversion efficiency determined on day 4 after differentiation for NLC4 normalized to control astrocytes. (<b>c</b>) Neuronal purity determined on day 4 after differentiation for NLC4 normalized to control astrocytes. (<b>d</b>) Bright-field images of astrocytes–converted neuron-like cells on days 0, 4, and 10. Scale bar, 100 μm. (<b>e</b>) Immunofluorescence labeling with Tuj1, Map2, NeuN, and DCX on astrocytes–converted neuron-like cells on days 0, 4, and 10. Scale bar, 50 μm. (<b>f</b>) mRNA transcript levels of key neuronal transcription factors were assessed by qRT–PCR on days 0, 4, and 10. (<b>g</b>) mRNA transcript levels of key neuronal markers were assessed by qRT–PCR on days 0, 4, and 10.</p> Full article ">Figure 2
<p>(<b>a</b>) Representative bright-field image of NLCs from mouse astrocytes. The role of the small molecules was investigated by individually removing them from the 6C cocktail. Scale bar, 100 μm. (<b>b</b>) Immunostaining with Tuj1 to investigate the role of the small molecules. Scale bar, 50 μm. (<b>c</b>) Neuronal conversion efficiency.</p> Full article ">Figure 3
<p>(<b>a</b>) mRNA transcript levels of the neurotransmitters vGLUT1, GABA, TH, and aCHAT were assessed by qRT–PCR on days 0, 4, and 10. (<b>b</b>) Immunostaining labeling of neuron-like cells with the marker Syn1 on day 12 Scale bar, 50 μm. (<b>c</b>) Quantification of neuronal subtypes. (<b>d</b>) Immunostaining labeling of neurons-like cells with the neuronal markers MAP2, vGLUT1, GABA, and TH on day 15. Scale bar, 20 μm.</p> Full article ">Figure 4
<p>(<b>a</b>) Schematic illustration of the experimental timeline for neuronal reprogramming and functional assessment. (<b>b</b>) Fluorescent image of a cell loaded with 2 μM Rhoda 2-AM over different time intervals. Calcium flux was stimulated using 10 mM KCL. The asterisk indicates the regions in which the fluorescence measurements were performed. Selected pseudo-color frames are baseline-subtracted images (ΔF) of the cell shown. (<b>c</b>) Increases in calcium, plotted as ΔF/F0, measured over the regions indicated in a are shown over time (30 frames/s). (<b>d</b>) Increases in calcium, plotted as ΔF/F<sub>0</sub>, measured using multiple ROIs shown over time (30 frames/s). (<b>e</b>) The average Δ<span class="html-italic">Fmax</span>/<span class="html-italic">F<sub>0</sub></span> from (<b>c</b>) was determined and compared with that from (<b>d</b>).</p> Full article ">
16 pages, 3430 KiB  
Article
Cell Type-Specific Anti-Adhesion Properties of Peritoneal Cell Treatment with Plasma-Activated Media (PAM)
by Myriam Holl, Marie-Lena Rasch, Lucas Becker, Anna-Lena Keller, Laura Schultze-Rhonhof, Felix Ruoff, Markus Templin, Silke Keller, Felix Neis, Franziska Keßler, Jürgen Andress, Cornelia Bachmann, Bernhard Krämer, Katja Schenke-Layland, Sara Y. Brucker, Julia Marzi and Martin Weiss
Biomedicines 2022, 10(4), 927; https://doi.org/10.3390/biomedicines10040927 - 18 Apr 2022
Cited by 9 | Viewed by 3015
Abstract
Postoperative abdominal adhesions are responsible for serious clinical disorders. Administration of plasma-activated media (PAM) to cell type-specific modulated proliferation and protein biosynthesis is a promising therapeutic strategy to prevent pathological cell responses in the context of wound healing disorders. We analyzed PAM as [...] Read more.
Postoperative abdominal adhesions are responsible for serious clinical disorders. Administration of plasma-activated media (PAM) to cell type-specific modulated proliferation and protein biosynthesis is a promising therapeutic strategy to prevent pathological cell responses in the context of wound healing disorders. We analyzed PAM as a therapeutic option based on cell type-specific anti-adhesive responses. Primary human peritoneal fibroblasts and mesothelial cells were isolated, characterized and exposed to different PAM dosages. Cell type-specific PAM effects on different cell components were identified by contact- and marker-independent Raman imaging, followed by thorough validation by specific molecular biological methods. The investigation revealed cell type-specific molecular responses after PAM treatment, including significant cell growth retardation in peritoneal fibroblasts due to transient DNA damage, cell cycle arrest and apoptosis. We identified a therapeutic dose window wherein specifically pro-adhesive peritoneal fibroblasts were targeted, whereas peritoneal mesothelial cells retained their anti-adhesive potential of epithelial wound closure. Finally, we demonstrate that PAM treatment of peritoneal fibroblasts reduced the expression and secretion of pro-adhesive cytokines and extracellular matrix proteins. Altogether, we provide insights into biochemical PAM mechanisms which lead to cell type-specific pro-therapeutic cell responses. This may open the door for the prevention of pro-adhesive clinical disorders. Full article
(This article belongs to the Section Cell Biology and Pathology)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Characterization of cell type-specific growth inhibition in primary human peritoneal cells. (<b>A</b>) Schematic of anatomical and histological features of the peritoneum. (<b>B</b>) Schematic of the experimental setup of PAM generation. (<b>C</b>) Representative brightfield microscopy of native primary fibroblasts and mesothelial cells. Scale bar represents 200 µm. (<b>D</b>) Representative IF microscopy of fibroblasts and mesothelial cells after PFA fixation and staining with specific antibodies against cytokeratin and fibronectin. Scale bar represents 200 µm. (<b>E</b>,<b>F</b>) Relative cell confluency of (<b>E</b>) fibroblasts and (<b>F</b>) mesothelial cells 72 h after incubation of different PAM dosages for 4 h (mean ± SD). (<b>G</b>) Representative brightfield microscopy of fibroblasts and mesothelial cells 72 h after 4 h incubation with indicated PAM dilutions for 4 h and control treatment. Scale bar represents 400 µm. (<b>H</b>) Relative cell confluency 72 h after incubation with indicated PAM dilutions for 4 h with and control treatment (mean ± SD; * <span class="html-italic">p</span> &lt; 0.05; paired <span class="html-italic">t</span>-test).</p> Full article ">Figure 2
<p>Characterization of cellular PAM effects using contact- and label-independent Raman imaging. Cells were incubated with 1:2 diluted PAM for 4 h and analyzed after indicated timepoints. (<b>A</b>) Schematic of the Raman microscope and an exemplary Raman spectrum representing a specific biomolecule. (<b>B</b>) Representative Raman spectra and characteristic bands that were used to identify the molecular components. (<b>C</b>) Raman intensity distribution heat maps assigned to nuclei (blue), lipids (yellow) and cytoplasmic proteins (green) of fibroblasts and mesothelial cells after 4 h of PAM incubation; scale bar represents 50 µm. (<b>D</b>,<b>E</b>) PCA demonstrated a separation in the PC-1 vs. PC-2 score plot for the nuclei component in fibroblasts (<b>D</b>) and mesothelial cells (<b>E</b>). (<b>F</b>,<b>G</b>) Corresponding PC-1 and PC-2 loading plot for the nuclei component indicating changes in DNA after PAM treatment of (<b>F</b>) fibroblasts and (<b>G</b>) mesothelial cells. (<b>H</b>,<b>I</b>) Statistical comparison (of the spectra for nuclei, lipids and cytoplasmatic proteins obtained in (<b>C</b>)) was performed by PCA and subsequent normalization of the PC score values to the mean values of controls. This enabled the assessment of molecular differences in nuclear, lipid and cytosolic protein composition; the data points represent average score values per donor (mean ± SD; * <span class="html-italic">p</span> &lt; 0.05; paired <span class="html-italic">t</span>-test).</p> Full article ">Figure 3
<p>PAM treatment induces anti-proliferative pathways in fibroblasts and initiates cell survival in mesothelial cells. Cells were incubated with 1:2 diluted PAM for 4 h and analyzed after indicated timepoints. (<b>A</b>) Relative cell viability after 4 h of PAM incubation relative to controls. (<b>B</b>) Relative γH2AX intensity in flow cytometry after PAM incubation relative to controls. (<b>C</b>,<b>D</b>) Relative flow cytometry fractions of cells in cell cycle phases S, G0/G1 and G2/M after PAM incubation in (<b>C</b>) fibroblasts and (<b>D</b>) mesothelial cells relative to controls. (<b>E</b>,<b>F</b>,<b>H</b>) DigiWest protein profiles of fibroblasts and mesothelial cells after PAM incubation relative to controls. (<b>E</b>) Heat map of log2 transformed DigiWest data. Data were median-centered, and hierarchical clustering was performed using complete linkage and Euclidean distance, utilizing the MultiExperiment Viewer (MeV version 4.9.0, [<a href="#B45-biomedicines-10-00927" class="html-bibr">45</a>]) software. Yellow indicates a high signal level; blue indicates a low signal level (compared to median). (<b>F</b>) Relative expression of the cell cycle-regulating factors CDK4, Cyclin D1, p21 and H3 (Ser10) in fibroblasts (black) and mesothelial cells (gray). (<b>G</b>) Relative caspase-3/7 activity after PAM incubation relative to controls. (<b>H</b>) Relative expression of the anti-proliferative and pro-apoptotic factors p-p53 and Rb and the cell survival factors AKT, p AKT, HSP27 and survivin in fibroblasts (black) and mesothelial cells (gray). Results are expressed as mean ± SD; * <span class="html-italic">p</span> &lt; 0.05 as determined by paired <span class="html-italic">t</span>-test.</p> Full article ">Figure 4
<p>PAM treatment attenuates protein biosynthesis and the secretion of pro-adhesive factors. Fibroblasts were incubated with 1:2 diluted PAM for 4 h and analyzed after indicated timepoints. (<b>A</b>) Representative IF microscopy after staining with anti-5mC antibodies and (<b>B</b>) relative genomic methylation level per nucleus (number of foci normalized to the control); the scale bar equals 10 µm. (<b>C</b>) DNMT activity level per cell. (<b>D</b>) DigiWest protein profiles of pro-adhesive factors collagen I, FGF receptor and FGF-10 and fibronectin relative to controls. (<b>E</b>) Cytokine multiplex assay of GM-CSF and IL-1b in fibroblast supernatants relative to control. (<b>F</b>,<b>G</b>) Representative IF microscopy of fibronectin (<b>F</b>). Scale bars represent 100 and 10 µm, respectively. (<b>G</b>,<b>H</b>) Representative Western blot of fibronectin (<b>G</b>), and relative of fibronectin expression (<b>H</b>) (analyzed from (<b>G</b>)). (<b>I</b>,<b>J</b>) Representative IF microscopy of collagen I (<b>I</b>). Scale bars represent 100 and 10 µm, respectively. (<b>J</b>,<b>K</b>) Representative Western blot of collagen I (<b>J</b>), and relative collagen I expression (<b>K</b>) (analyzed from (<b>J</b>)). (<b>L</b>) Relative extracellular hydroxyproline expression. (<b>M</b>) Relative MMP-2 expression. (<b>N</b>) Relative expression of TGF β. Results are expressed as mean ± SD; * <span class="html-italic">p</span> &lt; 0.05 as determined by paired <span class="html-italic">t</span>-test.</p> Full article ">Figure 5
<p>(<b>A</b>) Reactive species in PAM induce an intracellular increase in ROS and RNS, especially by cytoplasmic membrane impairment. This is followed by the induction of various intracellular response pathways such as altered genomic methylation patterns and signal transduction cascades involving attenuation of cell growth and protein biosynthesis by cell cycle arrest and p53-associated apoptosis. (<b>B</b>) Schematic model of pathological cell growth, cytokine secretion and secretion of pro-adhesive molecules such as collagen and fibronectin following peritoneal disruption of the superficial cell layer. (<b>C</b>) Hypothesized mode of action of PAM application including inhibition of fibroblast proliferation and attenuation of cytokine and ECM components secretion, and unhindered re-epithelialization by mesothelial cells.</p> Full article ">
19 pages, 1247 KiB  
Review
The Trinity: Interplay among Cancer Cells, Fibroblasts, and Immune Cells in Pancreatic Cancer and Implication of CD8+ T Cell-Orientated Therapy
by Yu-Hsuan Hung, Li-Tzong Chen and Wen-Chun Hung
Biomedicines 2022, 10(4), 926; https://doi.org/10.3390/biomedicines10040926 - 18 Apr 2022
Cited by 1 | Viewed by 2989
Abstract
The microenvironment in tumors is complicated and is constituted by different cell types and stromal proteins. Among the cell types, the abundance of cancer cells, fibroblasts, and immune cells is high and these cells work as the “Trinity” in promoting tumorigenesis. Although unidirectional [...] Read more.
The microenvironment in tumors is complicated and is constituted by different cell types and stromal proteins. Among the cell types, the abundance of cancer cells, fibroblasts, and immune cells is high and these cells work as the “Trinity” in promoting tumorigenesis. Although unidirectional or bidirectional crosstalk between two independent cell types has been well characterized, the multi-directional interplays between cancer cells, fibroblasts, and immune cells in vitro and in vivo are still unclear. We summarize recent studies in addressing the interaction of the “Trinity” members in the tumor microenvironment and propose a functional network for how these members communicate with each other. In addition, we discuss the underlying mechanisms mediating the interplay. Moreover, correlations of the alterations in the distribution and functionality of cancer cells, fibroblasts, and immune cells under different circumstances are reviewed. Finally, we point out the future application of CD8+ T cell-oriented therapy in the treatment of pancreatic cancer. Full article
(This article belongs to the Special Issue Pancreatic Cancer: From Mechanisms to Therapeutic Approaches)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Mechanisms underlying the interplay among pancreatic cancer cells, fibroblasts, and immune cells. (<b>A</b>) Collagen I around fibroblasts (brown) stimulates pancreatic cancer cells (red) and increases the Rho/SOX9 activation, which leads to CXCL5 expression to activate myeloid cells (purple). Myeloid cells, in turn, express arginase 1 to suppress CD8<sup>+</sup> T cell (green). (<b>B</b>) Fibroblasts (brown) modulate macrophage (purple) polarization and function via ROS, and M2 polarized macrophages assist the proliferation of pancreatic cancer cell (red) via macrophage colony-stimulating factor (M-CSF). (<b>C</b>) T<sub>reg</sub> (gray) express TGFβ to stimulate the expression of CCR1 ligands in fibroblasts (brown), and the CCR1 ligands recruit myeloid cells (purple) to promote the growth of pancreatic cancer cells (red). (<b>D</b>) Fibroblasts (brown) express CXCL12 to suppress CD8<sup>+</sup> T cells (green) and block immune surveillance against pancreatic cancer cells (red).</p> Full article ">Figure 2
<p>Treating pancreatic cancer with CD8<sup>+</sup> T cell-orientated approaches. Potential CD8<sup>+</sup> T cell-orientated treatments for pancreatic cancer were reviewed and blockade of IL6/TGFβ/CXCR4/STAT and activation of CD40/IL15/CD11b were proposed to be effective in pancreatic cancer therapy. Solid arrow represents direct effect, while dotted arrow represents indirect effect.</p> Full article ">Figure 3
<p>Scheme shows the potential effects of fibroblast on immune cell recruitment/function in pancreatic cancer. Based on the findings of recent studies, the potential effect of fibroblast on recruitment and function of CD8<sup>+</sup> T cell, Treg, macrophage, MDSC, and DC in pancreatic cancer is proposed.</p> Full article ">
18 pages, 1822 KiB  
Article
Tandem Repeat Diversity in Two Closely Related Hamster Species—The Chinese Hamster (Cricetulus griseus) and Striped Hamster (Cricetulus barabensis)
by Nadezhda G. Ivanova, Irina V. Kartavtseva, Vera N. Stefanova, Dmitrii I. Ostromyshenskii and Olga I. Podgornaya
Biomedicines 2022, 10(4), 925; https://doi.org/10.3390/biomedicines10040925 - 18 Apr 2022
Cited by 3 | Viewed by 3038
Abstract
The Chinese hamster (Cricetulus griseus) and striped hamster (Cricetulus barabensis) are very closely related species with similar karyotypes. The karyotypes differ from each other by one Robertsonian rearrangement and X-chromosome morphology. The level of the tandem repeat (TR) sequences’ [...] Read more.
The Chinese hamster (Cricetulus griseus) and striped hamster (Cricetulus barabensis) are very closely related species with similar karyotypes. The karyotypes differ from each other by one Robertsonian rearrangement and X-chromosome morphology. The level of the tandem repeat (TR) sequences’ evolutional variability is high. The aim of the current work was to trace the TR distribution on the chromosomes of two very closely related species. The striped hamster genome has not yet been sequenced. We classified the Chinese hamster TR in the assemblies available and then compared the mode of the TR distribution in closely related species. Chinese and striped hamsters are separate species due to the relative species specificity of Chinese hamster TR and prominent differences in the TR distribution in both species. The TR variation observed within homologous striped hamster chromosomes is caused by a lack of inbreeding in natural populations. The set of TR tested could be used to examine the CHO lines’ instability that has been observed in heterochromatic regions. Full article
(This article belongs to the Special Issue Advances in Molecular Cytogenetics)
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Figure 1

Figure 1
<p>Molecular phylogeny of the <span class="html-italic">Cricetinae</span> subfamily based on the mitochondrial cytochrome b and 12S rRNA genes and the nuclear vWF gene ([<a href="#B27-biomedicines-10-00925" class="html-bibr">27</a>] adapted).</p> Full article ">Figure 2
<p><span class="html-italic">C. griseus</span> metaphase plates after FISH with the TR (tandem repeat) probes. Order of the probes is according to <a href="#biomedicines-10-00925-t004" class="html-table">Table 4</a>. Those TR probes are shown that did not give any answer on <span class="html-italic">C. barabensis</span> plates. Bar 10 mcm.</p> Full article ">Figure 3
<p><span class="html-italic">C. griseus</span> (I) and <span class="html-italic">C. barabensis</span> (II) karyotypes after FISH with the TR probes indicated. Top row—chromosomes’ schemes and their numbers for both species. The number of chromosomes 6/7 of <span class="html-italic">C. griseus</span>, which corresponds to chromosome 4 of <span class="html-italic">C. barabensis</span>, is given in brackets. Order of the probes is arranged according to <a href="#biomedicines-10-00925-t002" class="html-table">Table 2</a>. Only the TR probes that gave the signal on <span class="html-italic">C. barabensis</span> methaphase plates are shown. Bar 10 mcm.</p> Full article ">Figure 4
<p>Signals’ variability on homologous chromosome 6 (upper row) and 5 (lower row) of <span class="html-italic">C. barabensis</span> is presented, and the probes that were used are indicated. Pairs of homologous chromosomes from different metaphase plates are shown. Bar 5 mcm.</p> Full article ">
16 pages, 4641 KiB  
Article
Extremely Low-Frequency Electromagnetic Fields Increase Cytokines in Human Hair Follicles through Wnt/β-Catenin Signaling
by Ju-Hye Choi, Yu-Mi Kim, Hee-Jung Park, Myeong-Hyun Nam and Young-Kwon Seo
Biomedicines 2022, 10(4), 924; https://doi.org/10.3390/biomedicines10040924 - 18 Apr 2022
Cited by 4 | Viewed by 4723
Abstract
Hair loss is a chronic disorder that affects many people; however, a complete treatment has not yet been developed. Therefore, new therapeutic agents for preventing hair loss must be developed, and electromagnetic field (EMF) therapy has been proven to be a promising medical [...] Read more.
Hair loss is a chronic disorder that affects many people; however, a complete treatment has not yet been developed. Therefore, new therapeutic agents for preventing hair loss must be developed, and electromagnetic field (EMF) therapy has been proven to be a promising medical treatment in various fields, including hair loss treatment. This study evaluated the effect of extremely low-frequency electromagnetic field (ELF-EMF) intensity and exposure time by analyzing the expression of cytokines and anagen-related molecules, which influence hair activation and growth, in hair bulb spheroid (HBS) and hair follicle (HF) organ cultures. ELF-EMFs did not induce toxicity in the HBSs, as verified via the lactate dehydrogenase (LDH) assay. Moreover, an ELF-EMF intensity of 5–20 G promoted the expression of ALP, versican, β-catenin, and several cytokines (VEGF, PDGF, FGF-10, and ET-1) in HBSs. Immunohistochemical staining showed that ELF-EMF at an intensity of 5–20 G upregulated ALP and β-catenin and decreased TUNEL staining in HBS. Moreover, HFs exposed to ELF-EMF for 60 min exhibited an increase in hair length and a 1.5-fold increase in IL-4, ICAM-1, ALP, and versican mRNA expression compared to the control. Immunohistochemical staining indicated that 60 min of ELF-EMF can increase the expression of ALP and β-catenin and decreases TUNEL staining in organ cultures. Collectively, our results demonstrated that ELF-EMF exposure at a 10 G intensity for 60 min promoted hair shaft growth in HFs due to the effect of cytokines and adhesion molecules via the Wnt/β-catenin pathway. Therefore, ELF-EMF is a promising treatment for hair loss. Full article
(This article belongs to the Special Issue Cellular Mechanisms of Physical Stimulation)
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Figure 1

Figure 1
<p>Photograph of the ELF-EMF device in a 5% CO<sub>2</sub> incubator at 37 °C. ELF-EMF was generated using a pair of Helmholtz coils. (<b>A</b>) Helmholtz coil; (<b>B</b>) function generator; (<b>C</b>) power supply.</p> Full article ">Figure 2
<p>(<b>A</b>) Isolation of DP cells from hair follicles. (<b>B</b>) Establishment of the hair bulb spheroid (HBS) in vitro model consisting of human DP and ORS cells. (<b>C</b>) Evaluation of the cytotoxic effects of various ELF-EMF intensities (2, 5, 10, 20, and 50 G) on HBSs. Cytotoxicity was measured using the LDH assay kit. (<b>D</b>) Effects of various ELF-EMF intensities (2, 5, 10, 20, and 50 G) on HBS apoptosis. Apoptosis was measured via qPCR analysis of caspase-3. Each bar represents the mean ± standard error of independent experiments performed in triplicate (<span class="html-italic">n</span> = 3). Significant differences were determined via one-way ANOVA with Tukey’s post hoc test; <sup>†</sup> <span class="html-italic">p</span> &gt; 0.5 compared to the control. Original magnification: 100×. Scale bar: 200 μm.</p> Full article ">Figure 3
<p>Immunohistochemical staining of ALP, β-catenin, and TUNEL assay in the hair bulb spheroid (HBS) in vitro model after simulation with ELF-EMF. (<b>A</b>) Scale bar: 100 μm. The brown color indicates positive staining. Original magnification: 200×; Scale bars: 100 μm and 50 μm. (<b>B</b>) Percentage of positive cells in (<b>A</b>). The values were calculated using QuPath software. Each bar represents the mean ± standard error of independent experiments performed in triplicate (<span class="html-italic">n</span> = 3). Significant differences were determined via the non-parametric Kruskal–Wallis test; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 4
<p>Gene and protein expression levels detected in hair bulb spheroids (HBSs) treated with ELF-EMF. Gene expression of VEGF, PDGF, FGF-10, ET-1, ICAM, and versican measured by qPCR using β-actin as an internal control. Each bar represents the mean ± standard error of independent experiments performed in triplicate (<span class="html-italic">n</span> = 3). Significant differences were determined via the non-parametric Kruskal–Wallis test; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 5
<p>Effects of various ELF-EMF exposure times (15, 60, and 180 min) on human hair follicles (HFs). (<b>A</b>) Photograph of increased hair length on days 7 and 14. Original magnification: 40×; scale bar: 1 mm; (<b>B</b>) Increased hair length of human HFs (<span class="html-italic">n</span> = 6, female patient samples). (<b>C</b>) Evaluation of the potential cytotoxic effect of different ELF-EMF exposure times on HFs. Cytotoxicity was measured using an LDH assay kit. (<b>D</b>) Effect of the exposure times of ELF-EMFs on HF apoptosis. Apoptosis was measured via qPCR analysis of caspase-3. Each bar represents the mean ± standard error of independent experiments performed in triplicate. Significant differences were determined via one-way ANOVA coupled with Tukey’s post hoc test (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01). <sup>†</sup> <span class="html-italic">p</span>, <sup>††</sup> <span class="html-italic">p</span> &gt; 0.5 compared to each control. The results are representative of at least six hair follicles per condition.</p> Full article ">Figure 6
<p>Gene and protein expression levels detected in human hair follicles (HFs) treated with ELF-EMF. (<b>A</b>) Gene expression of ALP, ICAM-1, IL-4, and versican measured by qPCR using β-actin as an internal control. (<b>B</b>) Protein expression of β-catenin, Wnt3α, ALP, and versican, using β-actin as an internal control. (<b>C</b>) Protein expression of (<b>B</b>). Relative expression intensities were calculated using ImageJ. Each bar represents the mean ± standard error of independent experiments performed in triplicate (<span class="html-italic">n</span> = 3). Significant differences were determined by one-way ANOVA with Tukey’s post hoc test (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 7
<p>Immunohistochemical staining of ALP, β-catenin, and TUNEL in the hair follicles (HFs) after treatment with ELF-EMF for 14 days. Scale bar: 100μm. The arrows indicate positive staining. The dotted lines indicate dermal papilla. Original magnification: 200×. Scale bar: 100 μm. (<b>B</b>) Percentage of positive cells (ALP) or intensity of expression (β-catenin and TUNEL) in (<b>A</b>). The values were calculated using QuPath software. Each bar represents the mean ± standard error of independent experiments performed in triplicate (<span class="html-italic">n</span> = 3). Significant differences were determined via the non-parametric Kruskal–Wallis test; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
12 pages, 1428 KiB  
Article
Effects of Reversal of Hypotension on Cerebral Microcirculation and Metabolism in Experimental Sepsis
by Fabio Silvio Taccone, Fuhong Su, Xinrong He, Lorenzo Peluso, Katia Donadello, Sabino Scolletta, Daniel De Backer and Jean-Louis Vincent
Biomedicines 2022, 10(4), 923; https://doi.org/10.3390/biomedicines10040923 - 18 Apr 2022
Cited by 4 | Viewed by 2139
Abstract
The effects of reversal of hypotension on the cerebral microcirculation, oxygenation, and metabolism in septic shock remain unclear. In 12 sheep, peritonitis was induced by injection of feces into the abdominal cavity. At the onset of septic shock (mean arterial pressure (MAP) < [...] Read more.
The effects of reversal of hypotension on the cerebral microcirculation, oxygenation, and metabolism in septic shock remain unclear. In 12 sheep, peritonitis was induced by injection of feces into the abdominal cavity. At the onset of septic shock (mean arterial pressure (MAP) < 65 mmHg, unresponsive to fluid challenge), a norepinephrine infusion was titrated in eight sheep to restore a MAP ≥ 75 mmHg; the other four sheep were kept hypotensive. The microcirculation of the cerebral cortex was evaluated using side-stream dark-field video-microscopy. Brain partial pressure of oxygen (PbtO2) was measured, and cerebral metabolism was assessed using microdialysis. All animals developed septic shock after a median of 15 (14–19) h. When MAP was raised using norepinephrine, the PbtO2 increased significantly (from 41 ± 4 to 55 ± 5 mmHg), and the cerebral lactate/pyruvate ratio decreased (from 47 ± 13 to 28 ± 4) compared with values at shock onset. Changes in the microcirculation were unchanged with restoration of MAP and the glutamate increased further (from 17 ± 11 to 23 ± 16 μM), as it did in the untreated animals. In septic shock, the correction of hypotension with vasopressors may improve cerebral oxygenation but does not reverse the alterations in brain microcirculation or cerebral metabolism. Full article
(This article belongs to the Special Issue Bacterial and Viral Infection and Sepsis: From Pathogenesis to Therapeutic Strategies)
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<p>Cardiac output, mean arterial pressure, the PaO<sub>2</sub>/FiO<sub>2</sub> ratio, and lactate levels in the two groups at different study time-points. NE, norepinephrine. Data are presented as mean ± SEM. * <span class="html-italic">p</span> &lt; 0.05 from Sidak post hoc analysis.</p> Full article ">Figure 2
<p>Time-course of the proportion of small perfused vessels (PPV), the functional capacity density (FCD), the mean flow index (MFI), and the heterogeneity index of PPV (PPV-HI) in the cerebral microcirculation in the two groups of animals. NE, norepinephrine. Data are presented as mean ± SEM.</p> Full article ">Figure 3
<p>Time-course of the brain oxygen pressure (PbtO<sub>2</sub>), the cerebral lactate/pyruvate ratio, cerebral glycerol, and cerebral glutamate in the two groups. NE, norepinephrine. Data are presented as mean ± SEM. * <span class="html-italic">p</span> &lt; 0.05 from Sidak post hoc analysis.</p> Full article ">
3 pages, 199 KiB  
Editorial
Molecular Mechanisms in Lysosomal Storage Diseases: From Pathogenesis to Therapeutic Strategies
by Valeria De Pasquale, Melania Scarcella and Luigi Michele Pavone
Biomedicines 2022, 10(4), 922; https://doi.org/10.3390/biomedicines10040922 - 17 Apr 2022
Cited by 3 | Viewed by 1980
Abstract
Lysosomal storage diseases (LSDs) are a group of metabolic diseases caused by inborn mutations of lysosomal enzymes, which lead to lysosome substrate accumulation in various cell types [...] Full article
(This article belongs to the Special Issue Molecular Mechanisms in Lysosomal Storage Diseases: From Pathogenesis to Therapeutic Strategies)
15 pages, 666 KiB  
Review
Oncology Drug Repurposing for Sepsis Treatment
by Izabela Rumienczyk, Maria Kulecka, Małgorzata Statkiewicz, Jerzy Ostrowski and Michal Mikula
Biomedicines 2022, 10(4), 921; https://doi.org/10.3390/biomedicines10040921 - 17 Apr 2022
Cited by 6 | Viewed by 2874
Abstract
Sepsis involves life-threatening organ dysfunction caused by a dysregulated host response to infection. Despite three decades of efforts and multiple clinical trials, no treatment, except antibiotics and supportive care, has been approved for this devastating syndrome. Simultaneously, numerous preclinical studies have shown the [...] Read more.
Sepsis involves life-threatening organ dysfunction caused by a dysregulated host response to infection. Despite three decades of efforts and multiple clinical trials, no treatment, except antibiotics and supportive care, has been approved for this devastating syndrome. Simultaneously, numerous preclinical studies have shown the effectiveness of oncology-indicated drugs in ameliorating sepsis. Here we focus on cataloging these efforts with both oncology-approved and under-development drugs that have been repositioned to treat bacterial-induced sepsis models. In this context, we also envision the exciting prospect for further standard and oncology drug combination testing that could ultimately improve clinical outcomes in sepsis. Full article
(This article belongs to the Special Issue 10th Anniversary of Biomedicines—Sepsis: Diagnostics and Therapeutics)
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<p>Schematic depiction of molecular pathways and processes involved in cellular responses upon pathogen infection and respective oncology drugs repurposed for experimental sepsis treatment. The names of oncology drugs discussed herein are in red font next to their molecular targets. Following toll-like receptor (TLR) activation, the myeloid differentiation primary response protein 88 (MYD88) together with MYD88 adaptor-like protein (MAL) are recruited to the TLRs initiating the cascade of molecular events activating mitogen-activated protein kinase (MAPK) components including MEK and extracellular signal-regulated kinase (ERK) that ultimately mobilize chromatin recruitment of interferon regulatory factors (IRFs), nuclear factor (NF)-κB, and activator protein 1 (AP-1) to gene loci initiating expression of the specific immune responses. ERK also indirectly influences translation by regulating MAPK-interacting kinase (MNK) that phosphorylates the eukaryotic translation initiation factor 4E (eIF4E) at Ser209 from a cap-binding complex leading to the translation of transcripts encoding pro-inflammatory cytokines, including tumor necrosis factor (TNF)α. Anaplastic lymphoma kinase (ALK) participates with the epidermal growth factor receptor (EGFR) to promote AKT stimulation, which then activates NF-kB and IRF3 factors to induce the expression of proinflammatory cytokines and interferon β (IFN β). In the nucleus, the poly (ADP-ribose) polymerase 1 (PARP1) acts as a transcriptional co-regulator of the NF-κB transcriptional factor while the topoisomerase 1 (TOP1) facilitates polymerase 2 RNA (RNAP2) recruitment to the genes encoding pro-inflammatory mediators. Checkpoint proteins including programmed cell death protein 1 (PD-1) and PD-1 ligand (PDL-1) play an essential role in transitioning from a hyper- to hypo-inflammatory response. Both PD-1 and PDL-1 are expressed on immune cells while PDL-1 is also expressed on non-immune cells.</p> Full article ">
14 pages, 727 KiB  
Review
The Role of the Adipokine Resistin in the Pathogenesis and Progression of Epithelial Ovarian Cancer
by Klaudia Parafiniuk, Wiktoria Skiba, Anna Pawłowska, Dorota Suszczyk, Aleksandra Maciejczyk and Iwona Wertel
Biomedicines 2022, 10(4), 920; https://doi.org/10.3390/biomedicines10040920 - 16 Apr 2022
Cited by 10 | Viewed by 4039
Abstract
Obesity is a civilization disease associated with an increased risk of developing cardiovascular diseases, diabetes, and some malignancies. The results concerning the relationship between obesity and epithelial ovarian cancer (EOC) are inconclusive. The higher incidence of neoplasms in obese subjects has led to [...] Read more.
Obesity is a civilization disease associated with an increased risk of developing cardiovascular diseases, diabetes, and some malignancies. The results concerning the relationship between obesity and epithelial ovarian cancer (EOC) are inconclusive. The higher incidence of neoplasms in obese subjects has led to the development of the adipokine hypothesis. Omental adipocyte cells interact with cancer cells, promoting their migration and metastasis via the secretion of adipokines, growth factors, and hormones. One of the adipokines is resistin. It was shown in vitro that resistin stimulates the growth and differentiation of ovarian cancer cells. Moreover, it increases the level of angiogenesis factors, e.g., matrix metalloproteinase 2 (MMP-2) and vascular epithelial growth factor (VEGF). Additionally, resistin induces epithelial–mesenchymal transition (EMT) and stemness in EOC cell lines. A positive correlation has been shown between a higher level of resistin expression and the stage of histological differentiation of EOC or the occurrence of lymph node metastases. In addition, the overexpression of resistin has been found to act as an independent factor determining disease-free survival as well as overall survival in EOC patients. Growing evidence supports the finding that resistin plays an important role in some mechanisms leading to the progression of EOC, though this issue still requires further research. Full article
(This article belongs to the Special Issue The Role of Inflammatory Cytokines in Cancer Progression)
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<p>The complex role of resistin in cancer development [<a href="#B31-biomedicines-10-00920" class="html-bibr">31</a>,<a href="#B78-biomedicines-10-00920" class="html-bibr">78</a>,<a href="#B96-biomedicines-10-00920" class="html-bibr">96</a>,<a href="#B97-biomedicines-10-00920" class="html-bibr">97</a>,<a href="#B98-biomedicines-10-00920" class="html-bibr">98</a>,<a href="#B99-biomedicines-10-00920" class="html-bibr">99</a>,<a href="#B100-biomedicines-10-00920" class="html-bibr">100</a>]. Reactive oxygen species (ROS); nicotinamide adenine dinucleotide phosphate (NADPH); mammalian target of rapamycin (mTOR); phosphatidylinositol 3-kinase (PI3K); protein kinase B (Akt); specificity protein 1 (Sp1); extracellular signal-regulated kinase (ERK); c-Jun N-terminal kinase (JNK); toll-like receptor 4 (TLR4); mitogen activated protein kinase (MAPK); transcription factor nuclear factor-kappa B (NFκβ).</p> Full article ">
13 pages, 3335 KiB  
Article
miR-195-5p Regulates Tight Junctions Expression via Claudin-2 Downregulation in Ulcerative Colitis
by Viviana Scalavino, Emanuele Piccinno, Antonio Lacalamita, Angela Tafaro, Raffaele Armentano, Gianluigi Giannelli and Grazia Serino
Biomedicines 2022, 10(4), 919; https://doi.org/10.3390/biomedicines10040919 - 16 Apr 2022
Cited by 14 | Viewed by 2976
Abstract
Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation associated with an increased intestinal permeability. Several studies have shown that microRNAs (miRNAs) are involved in the IBD pathogenesis. Here, we aimed to functionally characterize the role of miRNAs in the regulation of [...] Read more.
Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation associated with an increased intestinal permeability. Several studies have shown that microRNAs (miRNAs) are involved in the IBD pathogenesis. Here, we aimed to functionally characterize the role of miRNAs in the regulation of intestinal permeability and barrier function. We identified 18 dysregulated miRNAs in intestinal epithelial cells (IECs) from the ulcerative colitis (UC) mice model and control mice. Among them, down-regulated miR-195-5p targeted claudin-2 (CLDN2) and was involved in impaired barrier function. CLDN2 expression levels were increased in UC mice models and negatively correlated with miR-195-5p expression. We demonstrated that gain-of-function of miR-195-5p in colonic epithelial cell lines decreased the CLDN2 levels. This modulation, in turn, downregulated claudin-1 (CLDN1) expression at protein level but not that of occludin. Our data support a previously unreported role of miR-195-5p in intestinal tight junctions’ regulation and suggest a potential pharmacological target for new therapeutic approaches in IBD. Full article
(This article belongs to the Special Issue Non-coding RNAs in Health and Disease)
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<p>miRNA expression profile of colonic epithelial cells from Winnie and wild-type mice. (<b>A</b>) Hierarchical clustering using the 18 differentially expressed miRNAs (FDR &lt; 0.05 and fold change threshold &gt;2) discriminating colonic epithelial cells from Winnie mice compared to wild-type mice. Two principal clusters were identified on the basis of differential miRNA expression. (<b>B</b>) PCA based on the expression of differentially expressed miRNA in all samples. PCA showed evident clustering and confirmed the separation of Winnie and wild-type mice.</p> Full article ">Figure 2
<p>Validation of differentially expressed miRNAs. Expression levels of miR-195a-5p, miR-497a-5p, miR-99a-5p, and miR-199a-3p on miRNAs isolated from colonic IECs. miRNAs expression levels were quantified using qRT-PCR and the relative expression was normalized to the expression of endogenous control miR-186-5p. All tested miRNAs showed a significant change in expression, confirming microarray data. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 3
<p>miR-195a-5p targets CLDN2. (<b>A</b>) Sequence alignment of the miR-195a-5p base-pairing sites in the 3’-UTR of Cldn2 mRNA showing that the regions complementary to miR-195a-5p are highly conserved among mouse, human, and chimp. The “seed” sequences of miR-195a-5p complementary to Cldn2 are shown in white. (<b>B</b>) Cldn2 mRNA expression levels evaluated in the same set of RNA samples used in the microarray validation. Cldn2 levels were significantly higher in Winnie mice compared to wild-type mice. Cldn2 expression levels were normalized on the housekeeping gene Gapdh. The histograms represent the mean ± SEM. * <span class="html-italic">p</span> = 0.03. (C) Linear correlation between the expression of Cldn2 and the expression of miR-195a-5p. Cldn2 mRNA levels inversely correlated with miR-195a-5p expression levels. Black points represent correlation between mRNA and miRNA expression for each sample (r = −0.64; <span class="html-italic">p</span> = 0.04).</p> Full article ">Figure 4
<p>miR-195-5p regulates Cldn2 mRNA expression. (A) Cldn2 mRNA expression was analyzed by Real-Time PCR after transfection with miR-195-5p mimic in HT-29, Caco2, and T84 human cell lines. The increased intracellular amount of miR-195-5p (at 30 nM and 50 nM concentrations) led to a significant decrease of Cldn2 in all cell lines. (B) Cldn1 mRNA expression was analyzed by Real-Time PCR after transfection with miR-195-5p mimic in HT-29, Caco2, and T84 human cell lines. The levels of Cldn1 remained similar to mock-control in all cell lines. Expression data were normalized to the housekeeping gene Gapdh. Data are representative of four independent experiments (mean ± SEM). * <span class="html-italic">p</span> &lt; 0.01, ** <span class="html-italic">p</span> &lt; 0.001, *** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 5
<p>Regulation of CLDN2 protein expression by miR-195-5p in colonic epithelial cell lines. (<b>A</b>) Western blot analysis of CLDN2 protein expression in HT-29, Caco2, and T84 cell lines after miR-195-5p mimic transfection. A significant reduction of CLDN2 expression was detected in all cell lines. Raw data of the independent experiments of Western blot were reported in <a href="#app2-biomedicines-10-00919" class="html-app">Appendix A</a>. (<b>B</b>) Immunofluorescence staining of CLDN2 in HT-29, Caco2 and T84 cell cultures after miR-195-5p mimic transfection. In accordance with Western blot results, after transfection, the expression of CLDN2 decreased compared with mock-control. Single channel images of DAPI and CLDN2 staining before merge were reported in <a href="#app3-biomedicines-10-00919" class="html-app">Appendix B</a>. Data of WB were obtained by dividing the normalized transfected sample values by the normalized control sample values. β-Tubulin was used as housekeeping protein to normalize the data. Data are representative of four independent experiments. The histograms correspond to mean ± SEM. Scale bar = 50 µm ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p>CLDN1 and Occludin protein expression after miR-195-5p mimic transfection. (<b>A</b>) Representative blots of CLDN1 and occludin protein expression in three IECs cell lines, HT-29, Caco2, and T84 after transfection. Western blot quantitative analysis demonstrated inhibition of CLDN1 (<b>B</b>) but not occludin (<b>C</b>) in all cell lines, after miR-195-5p mimic transfection at 30 and 50 nM. Mock represents cells which have undergone transfection without miRNA mimic. Raw data of the independent experiments of Western blot were reported in <a href="#app2-biomedicines-10-00919" class="html-app">Appendix A</a>. Data were obtained by dividing the normalized transfected sample values to normalized mock-control sample values. β-Tubulin was used as housekeeping protein to normalize the data. Data are representative of four independent experiments. The histograms correspond to mean ± SEM. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01, *** p &lt; 0.001.</p> Full article ">Figure 7
<p>miR-195-5p, Cldn2, Cldn1, and Occludin expression in UC patients. Analysis of colonic tissue from UC and controls downloaded from the GEO database (GSE133059, GSE133060, GSE128682, and GSE87466). Mean expression data of miR-195-5p (<b>A</b>), Cldn2 (<b>B</b>), Cldn1 (<b>C</b>), and Occludin (<b>D</b>) were expressed as normalized expression values. LogFC value and FDR were reported for each dataset and each signal.</p> Full article ">
13 pages, 1786 KiB  
Article
Expression of the Costimulatory Molecule B7-H4 in the Decidua and Placental Tissues in Patients with Placental Abruption
by Monika Bączkowska, Magdalena Maria Dutsch-Wicherek, Ewa Przytuła, Jan Faryna, Cezary Wojtyła, Mohamed Ali, Anna Knafel and Michał Ciebiera
Biomedicines 2022, 10(4), 918; https://doi.org/10.3390/biomedicines10040918 - 16 Apr 2022
Cited by 2 | Viewed by 2572
Abstract
B7 homolog 4 protein (B7-H4), a member of the B7 family, is a immunomodulatory membrane protein. The aim of the study was to evaluate the expression of this protein in the decidua and placental tissues in case of placental abruption (PA) compared to [...] Read more.
B7 homolog 4 protein (B7-H4), a member of the B7 family, is a immunomodulatory membrane protein. The aim of the study was to evaluate the expression of this protein in the decidua and placental tissues in case of placental abruption (PA) compared to cases of retained placental tissue (RPT) and controls. Tissue samples were obtained from 47 patients with PA, 60 patients with RPT, and 41 healthy controls. The samples were stained for B7-H4 expression, analyzed by an expert pathologist, and a semi-quantitative scale was applied. A statistical analysis revealed that the expression of B7-H4 was significantly higher in the decidua in PA samples compared to samples from patients with RPT (p-value < 0.001) and healthy controls (p-value < 0.001). The expression of B7-H4 in the placental chorionic villus was significantly higher in PA samples in relation to samples from healthy controls (p-value < 0.001) but not in relation to RPT samples (p-value = 0.0853). This finding suggests that B7-H4 might play an important role in mechanisms restoring reproductive tract homeostasis. Further research is necessary in regard to the role of B7-H4 in PA. Full article
(This article belongs to the Topic Pathogenesis of Pregnancy-Related Complications)
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<p>B7-H4 immunoreactivity; (<b>a</b>) decidua–stage 0–no B7-H4 immunoreactivity; (<b>b</b>) decidua–stage 1–low B7-H4 immunoreactivity; (<b>c</b>) decidua–stage 2–moderate B7-H4 immunoreactivity; (<b>d</b>) decidua–stage 3–high B7-H4 immunoreactivity; (<b>e</b>) placental chorionic villus–stage 0–no B7-H4 immunoreactivity; (<b>f</b>) placental chorionic villus–stage 1–low B7-H4 immunoreactivity; (<b>g</b>) placental chorionic villus–stage 2–moderate B7-H4 immunoreactivity; (<b>h</b>) placental chorionic villus–stage 3–high B7-H4 immunoreactivity. Objective magnification ×40. ↖—Points some of the cells expressing B7-H4.</p> Full article ">Figure 2
<p>B7-H4 immunoreactivity in the decidua in samples from patients with placental abruption, retained placental tissue, and healthy controls. <span class="html-italic">n—Number of samples</span>.</p> Full article ">Figure 3
<p>B7-H4 immunoreactivity in the placental chorionic villus in samples from patients with placental abruption, retained placental tissue, and healthy controls. <span class="html-italic">n—Number of samples</span>.</p> Full article ">Figure 4
<p>The two-signal hypothesis. The mechanism of action of the costimulatory molecule B7-H4. <span class="html-italic">MHC—major histocompatibility complex, TCR—T-cell receptor, APC—antigen-presenting cell</span>.</p> Full article ">
19 pages, 4731 KiB  
Article
Prolonged Cadmium Exposure Alters Migration Dynamics and Increases Heterogeneity of Human Uterine Fibroid Cells—Insights from Time Lapse Analysis
by Yitang Yan, Min Shi, Rick Fannin, Linda Yu, Jingli Liu, Lysandra Castro and Darlene Dixon
Biomedicines 2022, 10(4), 917; https://doi.org/10.3390/biomedicines10040917 - 16 Apr 2022
Cited by 1 | Viewed by 2552
Abstract
Cadmium (Cd) is one of the most prevalent environmental heavy metal contaminants and is considered an endocrine disruptor and carcinogen. In women with uterine fibroids, there is a correlation between blood Cd levels and fibroid tumor size. In this study, fibroid cells were [...] Read more.
Cadmium (Cd) is one of the most prevalent environmental heavy metal contaminants and is considered an endocrine disruptor and carcinogen. In women with uterine fibroids, there is a correlation between blood Cd levels and fibroid tumor size. In this study, fibroid cells were exposed to 10 µM CdCl2 for 6 months and a fast-growing Cd-Resistant Leiomyoma culture, termed CR-LM6, was recovered. To characterize the morphological and mechanodynamic features of uterine fibroid cells associated with prolonged Cd exposure, we conducted time lapse imaging using a Zeiss confocal microscope and analyzed data by Imaris and RStudio. Our experiments recorded more than 64,000 trackable nuclear surface objects, with each having multiple parameters such as nuclear size and shape, speed, location, orientation, track length, and track straightness. Quantitative analysis revealed that prolonged Cd exposure significantly altered cell migration behavior, such as increased track length and reduced track straightness. Cd exposure also significantly increased the heterogeneity in nuclear size. Additionally, Cd significantly increased the median and variance of instantaneous speed, indicating that Cd exposure results in higher speed and greater variation in motility. Profiling of mRNA by NanoString analysis and Ingenuity Pathway Analysis (IPA) strongly suggested that the direction of gene expression changes due to Cd exposure enhanced cell movement and invasion. The altered expression of extracellular matrix (ECM) genes such as collagens, matrix metallopeptidases (MMPs), secreted phosphoprotein 1 (SPP1), which are important for migration contact guidance, may be responsible for the greater heterogeneity. The significantly increased heterogeneity of nuclear size, speed, and altered migration patterns may be a prerequisite for fibroid cells to attain characteristics favorable for cancer progression, invasion, and metastasis. Full article
(This article belongs to the Special Issue Advanced Research in Cell Motility)
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<p>Full track display of nuclear surface objects to illustrate migration patterns. The images were captured at time frame number 50 (at 245 min). (<b>A</b>). Ht-UtLM6 tracks showed relatively parallel bundles in multiple regions. (<b>B</b>). CR-LM6 tracks showed relatively random and disorganized patterns. Scale bar = 100 μm.</p> Full article ">Figure 2
<p>Distribution of Nuclear Track Straightness. (<b>A</b>) Tracks with various straightness values. These individual tracks were extracted from the full track displays in <a href="#biomedicines-10-00917-f001" class="html-fig">Figure 1</a>. The Straightness is defined as the ratio between Track Displacement and Track Length. Scale bar = 100 μm. (<b>B</b>) Distribution of Track Straightness in ht-UtLM6. The data labels shown on top of the bars are the absolute counts. The Frequency in <span class="html-italic">Y</span>-axis denotes the number of counts within a specific bin. (<b>C</b>) Distribution of Track Straightness in CR-LM6. (<b>D</b>) Superimposed probability density function curves of Straightness values for both populations.</p> Full article ">Figure 3
<p>Track Length analysis. (<b>A</b>) The full track displays for the top 6 longest tracks. The Track Length values calculated by Imaris are displayed at the top-left. A. ht-UtLM6 tracks were relatively smooth and straight. (<b>B</b>) CR-LM6 tracks had sharp turns and sometimes loops. Scale bar = 100 μm. (<b>C</b>,<b>D</b>). The histograms of Track Length distribution for ht-UtLM6 and CR-LM6. (<b>E</b>) The probability density function curves of Track Length for the two populations.</p> Full article ">Figure 4
<p>Prolonged Cd exposure enhanced instantaneous speed and increased speed variance. The instantaneous speed values were plotted against time frame numbers 0, 20, 40, 60, 80 and 100, corresponding to imaging times (in minutes) of 0, 95, 195, 295, 395 and 495, respectively. The five colored curves correspond to median (red), 25% and 75% quantiles (gold) and 10% and 90% quantiles (green). The speed reads above the blue dashed lines are greater than 100 µm/hour. The highest speed reads are marked by blue arrows. (<b>A</b>) ht-UTLM6. (<b>B</b>) CR-LM6. (<b>C</b>) The medians of instantaneous speeds at each time point were plotted against frame numbers. (<b>D</b>) The variances of instantaneous speeds at each time point were plotted against frame numbers.</p> Full article ">Figure 5
<p>Cd exposure increased heterogeneity in nuclear size. The nuclear size, shape and migration tracks are shown. (<b>A</b>). ht-UTLM6. (<b>B</b>). CR-LM6. Scale bar = 20 μm. (<b>C</b>,<b>D</b>). Scatterplots to show the distribution of nuclear size over time. Note the presence of large nuclei on top of the plots. The five colored curves correspond with median (red), 25% and 75% quantiles (gold) and 10% and 90% quantiles (green). (<b>C</b>). ht-UtLM6. (<b>D</b>). CR-LM6. As shown by the blue dashed line, in ht-UtLM6 there was no nuclear objects with a size greater than 300 µm<sup>2</sup>, whereas in CR-LM6 there were 190 nuclear objects with a size greater than 400 µm<sup>2</sup>. (<b>E</b>). Medians of nuclear size at each time point were plotted against frame number. (<b>F</b>). Variances of nuclear size at each time point were plotted against frame number.</p> Full article ">Figure 6
<p>Cd exposure induced rounding of nuclei. (<b>A</b>) Shapes of selected nuclei with various C/B ratios from ht-UTLM6 and CR-LM6 populations. The C/B ratio is defined as ratio between the length of the longest principal axis of a nucleus and the length of the second longest principal axis. Scale bar = 15 μm. (<b>B</b>) The distribution of C/B ratio in ht-UTLM6. The data labels shown on top of the bars are the absolute counts. (<b>C</b>) The distribution of C/B ratio in CR-LM6. (<b>D</b>) The overlay of inset B and inset C by probability density function. The <span class="html-italic">x</span>-axis is the C/B ratio and <span class="html-italic">y</span>-axis is probability density.</p> Full article ">Figure 7
<p>Cd exposure altered orientation of nuclei along migration tracks. The superimposed images show the location, shape, and orientation of nuclei. (<b>A</b>) The longest principal axis of ht-UTLM6 nucleus aligned with the direction of migration track at frames 5, 15, 30, 58, 76, and 100. (<b>B</b>) The longest principal axis of CR-LM6 nucleus was misaligned and occasionally perpendicular to the direction of migration track at frames 15, 69, 72, and 86. For nuclear orientations at all the time points, refer to <a href="#app1-biomedicines-10-00917" class="html-app">supplementary videos (S1 and S2)</a>. Scale bar = 20 μm.</p> Full article ">Figure 8
<p>IPA predicted that Cd exposure stimulated migration and invasion in CRLM6 cells based on the directions of the gene expression changes. (<b>A</b>). IPA function analysis derived from the PanCancer Pathways panel dataset (<span class="html-italic">z</span>-score 2.30). (<b>B</b>). IPA functions analysis derived from the PanCancer Progression panel dataset (<span class="html-italic">z</span>-score 2.35). The dashed lines with an arrow indicate that upregulation of gene expression is predicted to promote cell movement. The dashed lines with a T-bar demonstrate that downregulation of gene expression is predicted to promote cell movement.</p> Full article ">
12 pages, 1791 KiB  
Article
Post-Bariatric Hypoglycemia Is Associated with Endothelial Dysfunction and Increased Oxidative Stress
by Roberta Lupoli, Ilenia Calcaterra, Giuseppe Annunziata, Giancarlo Tenore, Carmen Rainone, Luigi Schiavo, Brunella Capaldo and Matteo Nicola Dario Di Minno
Biomedicines 2022, 10(4), 916; https://doi.org/10.3390/biomedicines10040916 - 16 Apr 2022
Cited by 8 | Viewed by 2958
Abstract
Post-bariatric hypoglycemia (PBH) is a potentially serious complication that may occur after bariatric surgery. Recurrent hypoglycemia may exert detrimental effects on vascular function. The aim of the present study was to evaluate endothelial function and oxygen reactive compounds in patients who experience PBH [...] Read more.
Post-bariatric hypoglycemia (PBH) is a potentially serious complication that may occur after bariatric surgery. Recurrent hypoglycemia may exert detrimental effects on vascular function. The aim of the present study was to evaluate endothelial function and oxygen reactive compounds in patients who experience PBH compared with controls. We performed a cross-sectional study on subjects with PBH (HYPO) and those without (NO-HYPO), detected by seven-day continuous glucose monitoring (CGM) performed at least twelve months after bariatric surgery. We enrolled 28 post-bariatric subjects (17.9% males, mean age 40.6 ± 10.7 years), with 18 in the HYPO group and 10 in the NO-HYPO group. In the two groups, we measured brachial artery flow-mediated dilation (FMD), oxidized low-density lipoproteins (oxLDL) and reactive oxygen metabolites (D-ROMs). The HYPO group had significantly lower FMD values than the NO-HYPO group (3.8% ± 3.0 vs. 10.5% ± 2.0, p < 0.001). A significant correlation was found between FMD and the time spent in hypoglycemia (rho = −0.648, p < 0.001), the number of hypoglycemic events (rho = −0.664, p < 0.001) and the mean glucose nadir (rho = 0.532, p = 0.004). The HYPO group showed significantly higher levels of D-ROMs (416.2 ± 88.7 UCARR vs. 305.5 ± 56.3 UCARR, p < 0.001) and oxLDLs (770.5 ± 49.7 µEq/L vs. 725.1 ± 51.6 µEq/L, p = 0.035) compared to the NO-HYPO group. In the multiple linear regression analysis, hypoglycemia independently predicted FMD values (β = −0.781, p < 0.001), D-ROMs (β = 0.548, p = 0.023) and oxLDL levels (β = 0.409, p = 0.031). PBH is associated with impaired endothelial function accompanied by increased oxidative stress. Full article
(This article belongs to the Special Issue Biomedicines: 10th Anniversary)
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<p>Flow-mediated dilation in HYPO and NO-HYPO groups.</p> Full article ">Figure 2
<p>Scatter plot of Spearman correlations between flow-mediated dilation values (FMD) and: (<b>a</b>) % time spent at interstitial glucose (IG) &lt; 54 mg/dL; (<b>b</b>) number of hypoglycemic events; (<b>c</b>) mean IG nadir; (<b>d</b>) coefficient of variation (CV).</p> Full article ">Figure 3
<p>Flow-mediated dilation values (FMD) according to tertiles of % time spent in hypoglycemia (IG &lt; 54 mg/dL). IG: interstitial glucose. Lowest tertile: time in hypoglycemia ≤ 0.2%; Middle tertile: time in hypoglycemia 0.3–2.8%; Highest tertile: time in hypoglycemia &gt; 2.8%. Comparisons were made with Mann−Whitney U-test.</p> Full article ">Figure 4
<p>Oxidative stress-related biomarkers in HYPO and NO-HYPO groups: (<b>a</b>) D-ROMs; (<b>b</b>) oxLDL.</p> Full article ">
27 pages, 2980 KiB  
Review
Elucidating miRNA Function in Cancer Biology via the Molecular Genetics’ Toolbox
by Adam Azlan, Yaashini Rajasegaran, Khor Kang Zi, Aliaa Arina Rosli, Mot Yee Yik, Narazah Mohd Yusoff, Olaf Heidenreich and Emmanuel Jairaj Moses
Biomedicines 2022, 10(4), 915; https://doi.org/10.3390/biomedicines10040915 - 15 Apr 2022
Cited by 4 | Viewed by 2913
Abstract
Micro-RNA (miRNAs) are short non-coding RNAs of about 18–20 nucleotides in length and are implicated in many cellular processes including proliferation, development, differentiation, apoptosis and cell signaling. Furthermore, it is well known that miRNA expression is frequently dysregulated in many cancers. Therefore, this [...] Read more.
Micro-RNA (miRNAs) are short non-coding RNAs of about 18–20 nucleotides in length and are implicated in many cellular processes including proliferation, development, differentiation, apoptosis and cell signaling. Furthermore, it is well known that miRNA expression is frequently dysregulated in many cancers. Therefore, this review will highlight the various mechanisms by which microRNAs are dysregulated in cancer. Further highlights include the abundance of molecular genetics tools that are currently available to study miRNA function as well as their advantages and disadvantages with a special focus on various CRISPR/Cas systems This review provides general workflows and some practical considerations when studying miRNA function thus enabling researchers to make informed decisions in regards to the appropriate molecular genetics tool to be utilized for their experiments. Full article
(This article belongs to the Special Issue MicroRNA in Solid Tumor and Hematological Diseases 2.0)
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<p>miRNA loci amplification resulted in repeating miRNA sequences thus increasing basal expression of miRNA.</p> Full article ">Figure 2
<p>Single base disposition at miRNA locus.</p> Full article ">Figure 3
<p>(<b>A</b>): CpG site enrichment on miRNA locus resulting in miRNA suppression. CpG methylation brought about by DNMTs methylates DNA sequence in proximity to miRNA locus or direct locus methylation leads to suppression of miRNA expression. H3C: methyl group; (<b>B</b>): Histone state on miRNA locus controls accessibility of TFs. Histone modification leads to the formation of open or closed chromatin structure which in turn affect TFs binding onto miRNA locus where this would result in either miRNA activation or suppression depending on the transcriptional activity of the bounded factors. H3C: methyl group.</p> Full article ">Figure 4
<p>Tools for regulating miRNA; miRNA sponges use overexpression plasmids to produce miRNA target sequence which sequesters miRNAs reducing binding on target mRNA thus increasing target mRNA expression whereas antagomir is a complementary sequence of short RNA that binds mRNA via base pairing, the use of antagomir prevents miRNA from inducing mRNA cleavage thus resulting in increase in target expression.</p> Full article ">Figure 5
<p>miRNA mimic uses short RNA sequence which mimics target miRNA bases sequence, this acts as a tool in inducing miRNA activity which would result in downregulation of target mRNA.</p> Full article ">Figure 6
<p>(<b>A</b>) Using dCas9 to block transcription elongation. TFs: transcription factors; Pol II: RNA polymerase 2; (<b>B</b>) Using the nickase strategy to cut amplified loci. HDR: homology-directed repair; (<b>C</b>) Relieving the epigenetics state of miRNA locus to increase gene transcription. Act: Acetyl group; p300: Histone modifier; (<b>D</b>) Modulation of the CpG methylation to induce miRNA expression. Modulation of the CpG methylation to induce miRNA expression. H3C: methyl group.</p> Full article ">Figure 6 Cont.
<p>(<b>A</b>) Using dCas9 to block transcription elongation. TFs: transcription factors; Pol II: RNA polymerase 2; (<b>B</b>) Using the nickase strategy to cut amplified loci. HDR: homology-directed repair; (<b>C</b>) Relieving the epigenetics state of miRNA locus to increase gene transcription. Act: Acetyl group; p300: Histone modifier; (<b>D</b>) Modulation of the CpG methylation to induce miRNA expression. Modulation of the CpG methylation to induce miRNA expression. H3C: methyl group.</p> Full article ">Figure 7
<p>General workflow for studying miRNA dysfunction in cancer. Depending on experimental design miRNA study could be assessed via knock-in or loss of function study. Several CRISPR-based methods are available as listed in Figure for the desired experiments.</p> Full article ">
21 pages, 3533 KiB  
Article
Phosphatidylethanolamine Deficiency and Triglyceride Overload in Perilesional Cortex Contribute to Non-Goal-Directed Hyperactivity after Traumatic Brain Injury in Mice
by Lisa Hahnefeld, Alexandra Vogel, Robert Gurke, Gerd Geisslinger, Michael K. E. Schäfer and Irmgard Tegeder
Biomedicines 2022, 10(4), 914; https://doi.org/10.3390/biomedicines10040914 - 15 Apr 2022
Cited by 9 | Viewed by 2530
Abstract
Traumatic brain injury (TBI) is often complicated by long-lasting disabilities, including headache, fatigue, insomnia, hyperactivity, and cognitive deficits. In a previous study in mice, we showed that persistent non-goal-directed hyperactivity is a characteristic post-TBI behavior that was associated with low levels of endocannabinoids [...] Read more.
Traumatic brain injury (TBI) is often complicated by long-lasting disabilities, including headache, fatigue, insomnia, hyperactivity, and cognitive deficits. In a previous study in mice, we showed that persistent non-goal-directed hyperactivity is a characteristic post-TBI behavior that was associated with low levels of endocannabinoids in the perilesional cortex. We now analyzed lipidome patterns in the brain and plasma in TBI versus sham mice in association with key behavioral parameters and endocannabinoids. Lipidome profiles in the plasma and subcortical ipsilateral and contralateral brain were astonishingly equal in sham and TBI mice, but the ipsilateral perilesional cortex revealed a strong increase in neutral lipids represented by 30 species of triacylglycerols (TGs) of different chain lengths and saturation. The accumulation of TG was localized predominantly to perilesional border cells as revealed by Oil Red O staining. In addition, hexosylceramides (HexCer) and phosphatidylethanolamines (PE and ether-linked PE-O) were reduced. They are precursors of gangliosides and endocannabinoids, respectively. High TG, low HexCer, and low PE/PE-O showed a linear association with non-goal-directed nighttime hyperactivity but not with the loss of avoidance memory. The analyses suggest that TG overload and HexCer and PE deficiencies contributed to behavioral dimensions of post-TBI psychopathology. Full article
(This article belongs to the Special Issue Molecular Mechanisms in Brain Injury)
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<p>Time schedule of the behavioral experiments and plasma and tissue collection (cohort-2 of [<a href="#B15-biomedicines-10-00914" class="html-bibr">15</a>]). Abbreviations: BW, body weight; g, grams; <span class="html-italic">w</span>/<span class="html-italic">o</span>, with and without.</p> Full article ">Figure 2
<p>Heatmap of lipid species in ipsilateral and contralateral cortical and subcortical brain tissue 5.6 months after TBI versus sham surgery. Lipids were submitted to hierarchical clustering using Euclidean squared distance metrics. Each horizontal line is a lipid species; each vertical line is a mouse at one site (<span class="html-italic">n</span> = 16 sham, <span class="html-italic">n</span> = 15 TBI mice). Mice are depicted in the sequence of their IDs. The left dendrogram shows the lipid clusters. The red rectangle highlights the deregulated group of TBI Ctx ipsilateral. The color scale ranges from −3 to +3 SD.</p> Full article ">Figure 3
<p>Volcano plots of lipids in the plasma, ipsi-, and contralateral cortex and subcortex. The x-axes show the log2 difference between groups consisting in <span class="html-italic">n</span> = 16 sham and <span class="html-italic">n</span> = 15 TBI mice. Lipids that were increased in TBI are positive; those that were reduced are negative. The <span class="html-italic">y</span>-axis shows the negative logarithm of the <span class="html-italic">t</span>-test <span class="html-italic">p</span>-value. The dashed line indicates a <span class="html-italic">p</span>-value of 0.01. (<b>A</b>) Plasma. (<b>B</b>) Ctx-ipsi, ipsilateral perilesional cortex. (<b>C</b>) SubCtx-ipsi, ipsilateral subcortical brain. (<b>D</b>) Ctx-contra, contralateral cortex. (<b>E</b>) SubCtx-contra, contralateral subcortical brain. Abbreviations: TG, triacylglycerol; PC, phosphatidylcholine; SM, sphingomyelin; Cer, ceramide; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine.</p> Full article ">Figure 4
<p>Principal component analysis and association of lipid classes in the ipsilateral perilesional cortex of sham (<span class="html-italic">n</span> = 16, black) and TBI (<span class="html-italic">n</span> = 15, red) mice. Lipid species of different chain lengths and saturation were summed per lipid class. Scatters represent individual mice. (<b>A</b>) PCA component PC1 versus PC2. Asterisks show the loading. Circles are 95% CI. (<b>B</b>) Triacylglycerols versus sphingomyelins. Bubble size LPC. (<b>C</b>) Linear regression fit with 95% CI of PG versus PC. Bubble size LPC. (<b>D</b>) Linear regression of HexCer versus PS, bubble size LPC. (<b>E</b>) HexCer versus PE-O, bubble size PE. Abbreviations: TG, triacylglycerols; PE-O, phosphatidylethanolamines with ether-linkage; Cer, ceramides; HexCer, hexosylceramides; SM, sphingomyelin; SE, sterol ester; PC, phosphatidylcholines; LPC, lysoPC. PS, phosphatidylserine; PG, phosphatidylglycerol; LPG, lysoPG. Lipid units are AUC/IS (i.e., the ratio of the area under the curve of the analyte and the AUC of the internal standard).</p> Full article ">Figure 5
<p>Heatmap of the triacylglycerols in the plasma and ipsilateral and contralateral cortical and subcortical brain 5.6 months after TBI or sham surgery. Each horizontal line is a TG species; each vertical color line is one mouse at one site, each from left to right in the sequence of the mouse IDs (<span class="html-italic">n</span> = 16 sham, <span class="html-italic">n</span> = 15 TBI). The group blocks are indicated by lines between headers. The red rectangle highlights the ipsilateral TBI cortex. The left dendrogram shows the hierarchic clustering of TG according to concentrations using Euclidean distance metrics. The color scale ranges from −3 to +3 SD. Abbreviations: Ctx, cortex; Sub, subcortical brain tissue; TBI, traumatic brain injury; TG, triacylglycerol.</p> Full article ">Figure 6
<p>Accumulation of triacylglycerols in the ipsilateral cortex after TBI. (<b>A</b>) Box/scatter plot of triacylglycerols of different chain lengths and saturation 5.6 months after the injury. The scatters represent individual mice, <span class="html-italic">n</span> = 16 sham and <span class="html-italic">n</span> = 15 TBI. The box shows the interquartile range; the whiskers range from minimum to maximum. The data were compared via two-way ANOVA for the TG X group (sham versus TBI) and subsequent post hoc analysis according to Šidák. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001. The unit AUC/IS is the ratio of the area under the curve of the analyte of the mass spectrogram and the AUC of the internal standard (IS is listed in <a href="#app1-biomedicines-10-00914" class="html-app">Supplementary Methods</a>). (<b>B</b>) Oil Red O staining of neutral lipids in cryosections of TBI mice 12 months after the injury. The images show examples of brain sections at two different Bregma levels (−0.34 and −1.94 mm; according to the Paxinos and Franklin’s mouse brain atlas) from two different animals in order to illustrate that Oil Red O stains cells are consistently distributed at the border across the brain lesion irrespective of the Bregma position. The differences in lesion size are due to the different Bregma levels. The upper lesion cavity appears to be larger owing to a fusion with the lateral ventricle at Bregma −0.34. The left panel shows the overview; the right panels show high magnification views of the areas indicated by rectangles. Scale bars: 1 mm, 100 µm.</p> Full article ">Figure 7
<p>Scatter and polar plots of phosphatidylethanolamines in the ipsilateral cortex 5.6 months after TBI. (<b>A</b>) Scatter plot of phosphatidylethanolamines (PE and PE-O species) of different chain lengths and saturation, <span class="html-italic">n</span> = 16 sham mice and <span class="html-italic">n</span> = 15 TBI mice. Each scatter is a mouse. The data were compared via two-way ANOVA for PE (within subject factor) by group (sham versus TBI, between subject factors) and subsequent post hoc analysis according to Šidák. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001. (<b>B</b>) Association of phosphatidylethanolamines (key regulated PE and PE-O species) in the perilesional ipsilateral cortex with the endocannabinoids, palmitoylethanolamide (PEA, <b>B<sub>1</sub></b>), and oleoylethanolamide (OEA, <b>B<sub>2</sub></b>). The line shows the linear regression line; the shaded area is the 90% confidence interval. The slopes differed significantly from zero. AUC/IS: ratio of the area under the curve of the analyte and the AUC of the internal standard. Bubble sizes reveal behavioral circadian parameters indicative of median activity (mesor) and day-to-night fluctuations (amplitude). (<b>C</b>) Polar plots show median percentages of LPE, PE, and PE-O species in the ipsilateral cortex of sham and TBI mice, normalized to the mean of sham mice.</p> Full article ">Figure 8
<p>Behavior of sham (<span class="html-italic">n</span> = 16, grey) versus TBI (<span class="html-italic">n</span> = 15, red) mice. Behavioral data were obtained in different tasks in IntelliCages (PAA (place avoidance acquisition), PAEx (place avoidance extinction), PPL (place preference learning), sidedness, circadian mesor, amplitude, acrophase), spatial learning on a Barnes maze, and sensory tests of thermal and mechanical perception and nociception (hotplate, Hargreaves, dynamic von Frey). (<b>A</b>) The polar plot shows multiple dimensions of behavior. To allow for a combined analysis of different readouts/units, raw data were transformed into percentages of the median of sham mice, and mean percentages were plotted on radial y-axes. Error bars are SEM of TBI mice. (<b>B</b>) Multiple line plots representing individual mice of four candidate behavioral parameters that contributed most to the discrimination between groups.</p> Full article ">Figure 9
<p>Associations of non-goal-directed hyperactivity (<span class="html-italic">x</span>-axis) with lipid classes (<span class="html-italic">y</span>-axis) in the ipsilateral perilesional cortex of sham (<span class="html-italic">n</span> = 16, black) versus TBI (<span class="html-italic">n</span> = 15, red) mice. Non-goal-directed hyperactivity is represented by IntelliCage nighttime visits with nosepokes but without licks per hour (NPVisits/h) (<span class="html-italic">X</span>-axis). NPVisits/h were recorded daily, and the mouse’s nighttime average was used. NPVisits/h were associated with lipid classes on the <span class="html-italic">y</span>-axis. The bubble size is given by the respective lyso-form or by a related lipid. AUC/IS (unit) is the ratio of the area under the curve of the analyte and the AUC of the internal standard. Individual lipid species of different chain lengths and saturation were summed per lipid class. Scatters represent individual mice. The ellipses show the 85% CI of the prediction (outliers excluded). <span class="html-italic">P</span>-Values show statistically significant slopes of regression lines of TBI mice. (<b>A</b>–<b>H</b>) NPVisits/h versus lipids as indicated (<span class="html-italic">y</span>-axis). Bubble sizes as indicated. Abbreviations: TG, triacylglycerol; SM, sphingomyelin; SE, sterol ester; PI, phosphatidylinositol; LPI, lyso-PI; PG, phosphatidylglycerol; LPG, lyso-PG; HexCer, hexosylceramides; Cer, ceramides; PE, phosphatidylethanolamines; LPE, lyso-PE; PE-O, phosphatidylethanolamines with ether linkage; PS, phosphatidylserine; PC, phosphatidylcholine; LPC, lyso-PC. (<b>I</b>) Profile plot of key regulated PE, HexCer, PS, and TG per mouse. Bubble sizes show behavioral parameters as indicated. PAA, place avoidance acquisition; PAEx, place avoidance extinction.</p> Full article ">
15 pages, 2061 KiB  
Review
Inorganic Polyphosphate—Regulator of Cellular Metabolism in Homeostasis and Disease
by Filip Kus, Ryszard T. Smolenski and Marta Tomczyk
Biomedicines 2022, 10(4), 913; https://doi.org/10.3390/biomedicines10040913 - 15 Apr 2022
Cited by 11 | Viewed by 3867
Abstract
Inorganic polyphosphate (polyP), a simple anionic polymer consisting of even hundreds of orthophosphate units, is a universal molecule present in both simple and complex organisms. PolyP controls homeostatic processes in animals, such as blood coagulation, tissue regeneration, and energy metabolism. Furthermore, this polymer [...] Read more.
Inorganic polyphosphate (polyP), a simple anionic polymer consisting of even hundreds of orthophosphate units, is a universal molecule present in both simple and complex organisms. PolyP controls homeostatic processes in animals, such as blood coagulation, tissue regeneration, and energy metabolism. Furthermore, this polymer is a potent regulator of inflammation and influences host immune response in bacterial and viral infections. Disturbed polyP systems have been related to several pathological conditions, including neurodegeneration, cardiovascular disorders, and cancer, but we lack a full understanding of polyP biogenesis and mechanistic insights into the pathways through which polyP may act. This review summarizes recent studies that describe the role of polyP in cell homeostasis and show how disturbances in polyP levels may lead to disease. Based on the collected findings, we highlight the possible usage of this polymer as a promising therapeutic tool in multiple pathologies. Full article
(This article belongs to the Special Issue Inorganic Phosphate Homeostasis and Signaling in Eukaryotic Cells)
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<p>Polyphosphate (polyP) regulates a variety of processes in different tissues in the human organism. In bone tissue, polyP stimulates osteogenesis and chondrogenesis, and promotes the growth and differentiation of bone marrow-derived mesenchymal stem cells (MSCs). In the brainstem, it can be taken up by activated astrocytes and act as a mediator of signal transmission. In the cardiovascular system, polyP can be released within extracellular vehicles by activated platelets, where it activates the contact pathway of blood clotting, stabilizes fibrin clot structure, and mediates proinflammatory responses by activating endothelial cells (ECs).</p> Full article ">Figure 2
<p>PolyP plays several roles in mitochondria and cell bioenergetics. The polymer is synthesized in mitochondria by ATP synthase, where it maintains calcium homeostasis and protects against the formation of calcium phosphate precipitates. It also regulates the processes of mitochondrial permeability transition and formation of mitochondrial permeability pores (mPTPores), thus maintaining mitochondrial fitness. PolyP is also involved in the purinergic system and the generation of extracellular nucleotides.</p> Full article ">Figure 3
<p>PolyP impact on the immune system is chain length-dependent. Bacterial long-chain polyP has an anti-inflammatory effect on myeloid cells, mainly macrophages, by downregulating the expressions of genes associated with antigen processing and antigen presentation, inhibiting the production and release of proinflammatory cytokines (e.g., CXCL10), and stimulating macrophage polarization towards the anti-inflammatory M2 phenotype. On the other hand, platelet-like short-chain polyP has proinflammatory activity, enhancing barrier permeability, upregulating the expression of the receptors necessary for leukocyte recruitment, and stimulating the release of neutrophil extracellular traps (NETs).</p> Full article ">Figure 4
<p>PolyP plays a dual role in cancer. On the one hand, it can act pro-tumorigenically by stimulating mTOR signaling or being utilized as a direct source of energy. Moreover, prostasomes of prostate cancer expose polyP on the surface, leading to cancer-associated thrombosis (CAT). On the other hand, polyP has been shown to block RNA polymerase I activity, reduce metastatic spread, and inhibit angiogenesis. PolyP also induces death in cisplatin-treated cancer cells.</p> Full article ">
28 pages, 3641 KiB  
Article
Evaluating Established Roles, Future Perspectives and Methodological Heterogeneity for Wilms’ Tumor 1 (WT1) Antigen Detection in Adult Renal Cell Carcinoma, Using a Novel N-Terminus Targeted Antibody (Clone WT49)
by Dorin Novacescu, Talida Georgiana Cut, Alin Adrian Cumpanas, Silviu Constantin Latcu, Razvan Bardan, Ovidiu Ferician, Cosmin-Ciprian Secasan, Andrei Rusmir and Marius Raica
Biomedicines 2022, 10(4), 912; https://doi.org/10.3390/biomedicines10040912 - 15 Apr 2022
Cited by 8 | Viewed by 3673
Abstract
Renal cell carcinoma (RCC) is arguably the deadliest form of genitourinary malignancy and is nowadays viewed as a heterogeneous series of cancers, with the same origin but fundamentally different metabolisms and clinical behaviors. Immunohistochemistry (IHC) is increasingly necessary for RCC subtyping and definitive [...] Read more.
Renal cell carcinoma (RCC) is arguably the deadliest form of genitourinary malignancy and is nowadays viewed as a heterogeneous series of cancers, with the same origin but fundamentally different metabolisms and clinical behaviors. Immunohistochemistry (IHC) is increasingly necessary for RCC subtyping and definitive diagnosis. WT1 is a complex gene involved in carcinogenesis. To address reporting heterogeneity and WT1 IHC standardization, we used a recent N-terminus targeted monoclonal antibody (clone WT49) to evaluate WT1 protein expression in 56 adult RCC (aRCC) cases. This is the largest WT1 IHC investigation focusing exclusively on aRCCs and the first report on clone WT49 staining in aRCCs. We found seven (12.5%) positive cases, all clear cell RCCs, showing exclusively nuclear staining for WT1. We did not disregard cytoplasmic staining in any of the negative cases. Extratumoral fibroblasts, connecting tubules and intratumoral endothelial cells showed the same exclusively nuclear WT1 staining pattern. We reviewed WT1 expression patterns in aRCCs and the possible explanatory underlying metabolomics. For now, WT1 protein expression in aRCCs is insufficiently investigated, with significant discrepancies in the little data reported. Emerging WT1-targeted RCC immunotherapy will require adequate case selection and sustained efforts to standardize the quantification of tumor-associated antigens for aRCC and its many subtypes. Full article
(This article belongs to the Special Issue Renal Cell Carcinoma: From Diagnosis to Identification of Novel Therapeutic Targets and Minimally Invasive Treatments)
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<p><b>HE staining</b>: (<b>A</b>) 200×, conventional solid clear cell renal carcinoma; (<b>B</b>) 200×, papillary architecture of RCC; (<b>C</b>) 400×, chromophobe RCC—clear cytoplasm with chromophobic perinuclear halo; (<b>D</b>) 200×, sarcomatoid dedifferentiation.</p> Full article ">Figure 2
<p><b>WT1 IHC staining</b>: (<b>A</b>) 200×, control image of a quasi-normal positive glomerulus; (<b>B</b>) 400×, positive fusiform cells outside of the tumor tissue, with a morphology suggestive of fibroblasts/myofibroblasts; (<b>C</b>) 400×, blood vessel with a majority of endothelial cells manifesting positive nuclear staining; (<b>D</b>) 400×, large blood vessel close-up, with scarce positive endothelial cells (<b>**</b>) and a single positive adjacent tumor cell (<b>*</b>); (<b>E</b>) 400×, moderate density of positive tumor cells (+2), manifesting predominantly high intensity nuclear staining; (<b>F</b>) 400×, low density (+1) tumor tissue, manifesting only 2 distinct positive cells, with weak-intensity (<b>*</b>) and moderate-intensity (<b>**</b>) nuclear staining.</p> Full article ">Figure 3
<p><b>WT1 IHC staining</b>: (<b>a</b>) 400×, positive connecting tubule cells, low-to-moderate nuclear staining; (<b>b</b>) 200×, positive renal corpuscles with severe degenerative lesions, compressed by neighboring tumor tissue, but with relatively unaffected podocytes.</p> Full article ">Figure 4
<p><b>High-density clear cell nuclear WT1 IHC staining (+3), moderate intensity.</b> (<b>a</b>) Case 1: 200×. (<b>b</b>) Case 2: 200×.</p> Full article ">
15 pages, 1047 KiB  
Article
The Immunogenicity and Safety of Three Types of SARS-CoV-2 Vaccines in Adult Patients with Immune-Mediated Inflammatory Diseases: A Longitudinal Cohort Study
by Ni Tien, Yu-Chang Chang, Po-Ku Chen, Hui-Ju Lin, Shih-Hsin Chang, Joung-Liang Lan, Po-Ren Hsueh, Ching-Kun Chang and Der-Yuan Chen
Biomedicines 2022, 10(4), 911; https://doi.org/10.3390/biomedicines10040911 - 15 Apr 2022
Cited by 8 | Viewed by 6340
Abstract
Patients with immune-mediated inflammatory diseases (IMID) were seldom enrolled in the studies of SARS-CoV-2 vaccines, and real-world data regarding the immunogenicity of different types of vaccines is limited. We aimed to assess the immunogenicity and safety of three types of vaccines (AZD1222, mRNA-1273, [...] Read more.
Patients with immune-mediated inflammatory diseases (IMID) were seldom enrolled in the studies of SARS-CoV-2 vaccines, and real-world data regarding the immunogenicity of different types of vaccines is limited. We aimed to assess the immunogenicity and safety of three types of vaccines (AZD1222, mRNA-1273, and BNT162b2) in 253 patients with IMID and 30 healthcare workers (HCWs). Plasma levels of IgG-antibody against SARS-CoV-2 targeting the receptor-binding domain of spike protein (anti-S/RBD-IgG) were determined by chemiluminescent immunoassay 3–4 weeks after the first-dose and second-dose vaccination. The positive rate and titers of anti-S/RBD-IgG were significantly higher in mRNA-1273 or BNT162b2 than in the AZD1222 vaccine. Immunogenicity was augmented after the second dose of any vaccine type in all IMID patients, suggesting that these patients should complete the vaccination series. Anti-S/RBD-IgG titers after first-dose vaccination were significantly lower in RA patients than pSS patients, but there was no significant difference after second-dose vaccination among five groups of IMID patients. The positive rate and titers of anti-S/RBD-IgG were significantly lower in patients receiving abatacept/rituximab therapy than in those receiving other DMARDs. All three SARS-CoV-2 vaccines showed acceptable safety profiles, and the common AEs were injection site reactions. We identified SLE as a significant predictor of increased autoimmunity and would like to promote awareness of the possibility of autoimmunity following vaccination. Full article
(This article belongs to the Special Issue Hypersensitivity to Drugs and Vaccines: Molecular Basis and Translational Research)
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<p>Study design workflow. IMID: immune-mediated inflammatory diseases immune-mediated inflammatory diseases; SLE: systemic lupus erythematosus; pSS: primary Sjögren’s syndrome; RA: rheumatoid arthritis; PsA: psoriatic arthritis; AOSD: adult-onset Still’s disease; HCWs: health-care workers; Anti-S/RBD-IgG Ab: IgG antibody against SARS-CoV-2 IgG antibody against SARS-CoV-2 specific for the receptor-binding domain (RBD) of the spike-1 (S1) protein.</p> Full article ">Figure 2
<p>Comparisons of anti-S/RBD-IgG positive rates and titers among different groups. Comparisons of anti-S/RBD-IgG positive rates (<b>A</b>) and titers (<b>B</b>) among three different types of SARS-CoV-2 vaccines. (<b>C</b>) Comparisons of anti-S/RBD-IgG positive rates and titers among the different groups of participants. (<b>D</b>) Comparisons of the anti-S/RBD-IgG positive rates and titers among the immunosuppressants, csDMARDs, bDMARDs, and tsDMARDs. The horizontal line within each figure indicates the cut-off value for positive anti-S/RBD-IgG. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, vs. after first dose or at baseline, as determined by Wilcoxon matched-pairs signed-rank test. <sup>#</sup> <span class="html-italic">p</span> &lt;0.05, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001, vs. AZD1222 vaccine, as determined by Kruskal-Wallis test using a post hoc Dunn’s test in (<b>B</b>). <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, vs. pSS group, as determined by Kruskal-Wallis test using a post hoc Dunn’s test in (<b>C</b>). <sup>#</sup> <span class="html-italic">p</span> &lt; 0.01, vs. ABT/RTX therapy, as determined by Kruskal-Wallis test using a post hoc Dunn’s test in (<b>D</b>). SLE: systemic lupus erythematosus; pSS: primary Sjögren’s syndrome; RA: rheumatoid arthritis (RA); SpA: spondyloarthropathies; AOSD: adult-onset Still’s disease; csDMARDs: conventional synthetic disease-modifying antirheumatic drugs; bDMARDs: biological DMARDs; tsDMARDs: targeted synthetic DMARDs; TNFi: tumor necrosis factor inhibitors; TCZ: tocilizumab; ABT: abatacept; RTX: rituximab; JAKi: Janus kinase inhibitors; MTX: methotrexate; MMF: mycophenolate.</p> Full article ">
11 pages, 1204 KiB  
Article
Expression and Prognostic Implication of PD-L1 in Patients with Urothelial Carcinoma with Variant Histology (Squamous Differentiation or Micropapillary) Undergoing Radical Cystectomy
by Jae-Hoon Chung, Chung-Un Lee, Dong-Hyeon Lee and Wan Song
Biomedicines 2022, 10(4), 910; https://doi.org/10.3390/biomedicines10040910 - 15 Apr 2022
Cited by 2 | Viewed by 2284
Abstract
The expression and prognostic role of programmed death ligand-1 (PD-L1) on tumor-infiltrating immune cells (TICs) has not been determined in urothelial carcinoma (UC) with variant histology. We retrospectively reviewed 90 patients (44 with micropapillary variant of UC (MPUC) and 46 with UC with [...] Read more.
The expression and prognostic role of programmed death ligand-1 (PD-L1) on tumor-infiltrating immune cells (TICs) has not been determined in urothelial carcinoma (UC) with variant histology. We retrospectively reviewed 90 patients (44 with micropapillary variant of UC (MPUC) and 46 with UC with squamous differentiation (UCSD)) who underwent radical cystectomy between January 2013 and December 2019. The expression of PD-L1 in TICs was measured using the VENTANA (SP-142) immunohistochemistry assay and dichotomized using a 5% cutoff value (positive ≥ 5%). Kaplan–Meier survival analysis was used to estimate recurrence-free survival (RFS), and multivariable Cox proportional hazard models were used to identify factors predicting tumor recurrence. Overall, positive PD-L1 expression in TICs was confirmed in 50 of 90 (55.6%) patients (40.1% (18/44) of MPUC and 69.9% (32/46) of UCSD). RFS was significantly shorter in patients with positive PD-L1 expression in TICs than in those with negative PD-L1 expression both in MPUC (p = 0.005) and UCSD (p = 0.046). Positive PD-L1 expression in TICs was significantly associated with an increased risk of tumor recurrence in both MPUC (HR = 1.85; 95% CI: 1.323–2.672; p = 0.017) and UCSD (HR = 1.58; 95% CI: 1.162–2.780; p = 0.032). In conclusion, positive PD-L1 expression in TICs was significantly associated with poorer RFS in both MPUC and UCSD patients. Our results support the use of adjuvant immunotherapy in these patients if they test positive for PD-L1 in their TICs. Full article
(This article belongs to the Topic Cancer Biology and Therapy)
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<p>Flow chart of study inclusion.</p> Full article ">Figure 2
<p>Representative images of PD-L1 expression in TICs using the VENTANA (SP142) immunohistochemistry assay. (<b>A</b>) Negative expression of PD-L1 in MPUC, (<b>B</b>) Positive expression of PD-L1 in MPUC. (<b>C</b>) Negative expression of PD-L1 in UCSD, (<b>D</b>) Positive expression of PD-L1 in UCSD. All images are ×200 magnification. PD-L1, programmed death-ligand 1; TICs, tumor-infiltrating immune cells; MPUC, micropapillary variant of urothelial carcinoma; UCSD, urothelial carcinoma with squamous differentiation.</p> Full article ">Figure 3
<p>Kaplan–Meier survival curves for RFS according to PD-L1 expression in TICs in patients with (<b>A</b>) MPUC and (<b>B</b>) UCSD. For patients with MPUC, the two-year overall RFS rate was significantly (<span class="html-italic">p</span> = 0.005) shorter in patients with positive expression of PD-L1 in TICs than in those with negative expression of PD-L1 in TICs (12.0% and 63.4%, respectively). Similarly, for patients with UCSD, PD-L1 expression in TICs also significantly (<span class="html-italic">p</span> = 0.046) affected the two-year overall RFS rate, which was 78.6% in patients with negative PD-L1 expression in TICs and 42.6% in PD-L1-positive patients.</p> Full article ">
17 pages, 3277 KiB  
Article
Testicular “Inherited Metabolic Memory” of Ancestral High-Fat Diet Is Associated with Sperm sncRNA Content
by Luís Crisóstomo, Matthieu Bourgery, Luís Rato, João F. Raposo, Rachel L. Batterham, Noora Kotaja and Marco G. Alves
Biomedicines 2022, 10(4), 909; https://doi.org/10.3390/biomedicines10040909 - 15 Apr 2022
Cited by 12 | Viewed by 3287
Abstract
Excessive adiposity caused by high-fat diets (HFDs) is associated with testicular metabolic and functional abnormalities up to grand-offspring, but the mechanisms of this epigenetic inheritance are unclear. Here we describe an association of sperm small non-coding RNA (sncRNA) with testicular “inherited metabolic memory” [...] Read more.
Excessive adiposity caused by high-fat diets (HFDs) is associated with testicular metabolic and functional abnormalities up to grand-offspring, but the mechanisms of this epigenetic inheritance are unclear. Here we describe an association of sperm small non-coding RNA (sncRNA) with testicular “inherited metabolic memory” of ancestral HFD, using a transgenerational rodent model. Male founders were fed a standard chow for 200 days (CTRL), HFD for 200 days (HFD), or standard chow for 60 days followed by HFD for 140 days (HFDt). The male offspring and grand-offspring were fed standard chow for 200 days. The sncRNA sequencing from epidydimal spermatozoa revealed signatures associated with testicular metabolic plasticity in HFD-exposed mice and in the unexposed progeny. Sperm tRNA-derived RNA (tsRNA) and repeat-derived small RNA (repRNA) content were specially affected by HFDt and in the offspring of HFD and HFDt mice. The grand-offspring of HFD and HFDt mice showed lower sperm counts than CTRL descendants, whereas the sperm miRNA content was affected. Although the causality between sperm sncRNAs content and transgenerational epigenetic inheritance of HFD-related traits remains elusive, our results suggest that sperm sncRNA content is influenced by ancestral exposure to HFD, contributing to the sperm epigenome up to the grand-offspring. Full article
(This article belongs to the Special Issue Non-coding RNAs in Health and Disease)
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<p>Distribution of sncRNA reads between different RNA biotypes across groups and generations. Results are expressed as the mean of normalized counts ± standard deviation. Generation F0 and F2: <span class="html-italic">n</span> = 3 per group. Generation F1: <span class="html-italic">n</span> = 2 per group. (<b>A</b>) Most representative sperm sncRNA biotypes, and (<b>B</b>) mtRNA and Y RNA. Data were tested by Wald’s test, corrected by the Benjamini–Hochberg method, using DESeq2. Significance was considered when <span class="html-italic">p</span> &lt; 0.1. * vs. CTRL; # vs. HFD.</p> Full article ">Figure 2
<p>Differently expressed sperm sncRNAs in Generation F0. Sperm sncRNA levels were compared between mice (Generation F0, <span class="html-italic">n</span> = 3 per group) fed with standard diet (CTRL), high-fat diet (HFD), or HFD for 60 days that was then replaced by a standard diet (HFDt). Results are presented as MA plots (mean log2 FC vs. log2 mean expression). Differently expressed sequences are highlighted and annotated. Data were tested by Wald’s test, corrected by the Benjamini-Hochberg method, using DESeq2. Significance was considered when <span class="html-italic">p</span> &lt; 0.1. (<b>A</b>) Transcription initiation RNA (tiRNA), HFD vs. CTRL; (<b>B</b>) tiRNA, HFDt vs. CTRL; (<b>C</b>) repeat-derived small RNA (repRNA), HFD vs. CTRL; (<b>D</b>) repRNA, HFDt vs. CTRL.</p> Full article ">Figure 3
<p>Differently expressed sperm miRNAs, transcription initiation RNA (tiRNA), and tRFs (tRNA-derived fragments) in F1 generation. Sperm sncRNA levels were compared between the offspring (Generation F1, <span class="html-italic">n</span> = 2 per group) of mice fed with standard diet (CTRL), high-fat diet (HFD), or HFD for 60 days that was then replaced by a standard diet (HFDt). Results are presented as MA plots (mean log2 FC vs. log2 mean expression). Differently expressed sequences are highlighted and annotated. Data were tested by Wald’s test, corrected by the Benjamini–Hochberg method, using DESeq2. Significance was considered when <span class="html-italic">p</span> &lt; 0.1. (<b>A</b>) MiRNA, HFD vs. CTRL; (<b>B</b>) miRNA, HFDt vs. CTRL; (<b>C</b>) miRNA, HFDt vs. HFD; (<b>D</b>) tiRNA, HFD vs. CTRL; (<b>E</b>) tiRNA, HFDt vs. CTRL; (<b>F</b>) tiRNA, HFDt vs. HFD; (<b>G</b>) tRF, HFD vs. CTRL; (<b>H</b>) tRF, HFDt vs. CTRL; (<b>I</b>) tRF, HFDt vs. HFD.</p> Full article ">Figure 4
<p>Differently expressed sperm piwi-interacting RNA (piRNA) and repeat-derived RNA (repRNA) sequences between the offspring (Generation F1, <span class="html-italic">n</span> = 2 per group) of mice fed with standard diet (CTRL), high-fat diet (HFD), or HFD for 60 days that was then replaced by a standard diet (HFDt). Results are expressed as MA plots (mean log2 FC vs. log2 mean expression). Differently expressed sequences are highlighted and annotated. Data were tested by Wald’s test, corrected by the Benjamini–Hochberg method, using DESeq2. Significance was considered when <span class="html-italic">p</span> &lt; 0.1. (<b>A</b>) PiRNA, HFD vs. CTRL; (<b>B</b>) piRNA, HFDt vs. CTRL; (<b>C</b>) piRNA, HFDt vs. HFD; (<b>D</b>) repRNA, HFD vs. CTRL; (<b>E</b>) repRNA, HFDt vs. CTRL; (<b>F</b>) repRNA, HFDt vs. HFD.</p> Full article ">Figure 5
<p>Differently expressed sperm sncRNA sequences between the grand-offspring (Generation F2, <span class="html-italic">n</span> = 3 per group) of mice fed with standard diet (CTRL), high-fat diet (HFD), or HFD for 60 days that was then replaced by a standard diet (HFDt). Results are expressed as MA plots (mean log2 FC vs. log2 mean expression). Differently expressed sequences are highlighted and annotated. Data were tested by Wald’s test, corrected by the Benjamini–Hochberg method, using DESeq2. Significance was considered when <span class="html-italic">p</span> &lt; 0.1. (<b>A</b>) MiRNA, HFD vs. CTRL; (<b>B</b>) miRNA, HFDt vs. CTRL; (<b>C</b>) miRNA, HFDt vs. HFD.</p> Full article ">Figure 6
<p>Gene ontology of the targets of differently expressed sperm sncRNAs, according to biological process. Targets of sncRNAs were estimated recurring to the online suite sRNAtools (<a href="https://bioinformatics.caf.ac.cn/sRNAtools/" target="_blank">https://bioinformatics.caf.ac.cn/sRNAtools/</a>; accessed on 15 August 2020). The output was then used for functional annotation based on the Gene Ontology Resource (<a href="http://geneontology.org/" target="_blank">http://geneontology.org/</a>; accessed on 17 August 2020). The annotation was performed by the topGO package run in R 4.1.0. GO terms with less than 10 annotated genes, and single gene targets were excluded from the analysis. CTRL—mice fed with standard diet, and their descendants; HFD—mice fed with high-fat diet, and their descendants; HFDt—mice fed with high-fat diet for 60 days that was then replaced by a standard diet, and their descendants.</p> Full article ">Figure 7
<p>Number of differently expressed sperm sncRNA sequences varies significantly in each generation and in each lineage of ancestral exposure to HFD. (<b>A</b>) Euler diagram illustrating the scale of differently expressed sperm sncRNA sequences in every generation and considering all the comparisons: HFD vs. CTRL, HFDt vs. CTRL, and HFDt vs. HFD. The breakdown of these numbers according to sncRNA biotype is provided in <a href="#biomedicines-10-00909-t001" class="html-table">Table 1</a>. (<b>B</b>) Schematic representation of “inherited metabolic memory” of testicular cells via sperm sncRNA. The exposure of the founder mice to HFD is the stimulus driving epigenetic changes in somatic cells, including Sertoli cells and spermatocytes. These changes are reflected in the sperm of the founders, which presented alterations in the number of tiRNAs and repRNAs, compared to CTRL. This epigenetic fingerprint will be the stimulus for epigenomic change in the offspring, especially during embryo development. Thus, the soma of the offspring will have a different epigenetic configuration that will, in its turn, influence the epigenome and the sncRNA content of produced sperm, distinct from the ancestors. This sperm sncRNA fingerprint will be the stimulus for the next generation, influencing the embryo development and the epigenome of the grand-offspring of the exposed mice.</p> Full article ">
18 pages, 41562 KiB  
Article
Recognition of Tumor Nidogen-1 by Neutrophil C-Type Lectin Receptors
by Ronit Vogt Sionov, Chrystelle Lamagna and Zvi Granot
Biomedicines 2022, 10(4), 908; https://doi.org/10.3390/biomedicines10040908 - 15 Apr 2022
Cited by 3 | Viewed by 2831
Abstract
Neutrophil-mediated cytotoxicity toward tumor cells requires cell contact and is mediated by hydrogen peroxide. We have recently shown that Cathepsin G expressed on the neutrophil surface interacts with tumor RAGE, and this interaction facilitates neutrophil cytotoxicity. Interruption of the Cathepsin G–RAGE interaction led [...] Read more.
Neutrophil-mediated cytotoxicity toward tumor cells requires cell contact and is mediated by hydrogen peroxide. We have recently shown that Cathepsin G expressed on the neutrophil surface interacts with tumor RAGE, and this interaction facilitates neutrophil cytotoxicity. Interruption of the Cathepsin G–RAGE interaction led to 50–80% reduction in cytotoxicity, suggesting that additional interactions are also involved. Here we show that blocking antibodies to the C-type lectin receptors (CLRs) Clec4e and Dectin-1, but not those to NKG2D, attenuated murine neutrophil cytotoxicity towards murine tumor cells, suggesting a contributing role for these CLRs in neutrophil recognition of tumor cells. We further observed that the CLRs interact with tumor Nidogen-1 and Hspg2, two sulfated glycoproteins of the basement membrane. Both Nidogen-1 and Hspg2 were found to be expressed on the tumor cell surface. The knockdown of Nidogen-1, but not that of Hspg2, led to reduced susceptibility of the tumor cells to neutrophil cytotoxicity. Altogether, this study suggests a role for CLR–Nidogen-1 interaction in the recognition of tumor cells by neutrophils, and this interaction facilitates neutrophil-mediated killing of the tumor cells. Full article
(This article belongs to the Collection Advances in Leukocyte Biology)
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<p>(<b>A</b>) Surface staining of tumor-elicited high-density neutrophils for Clec4e, Dectin-1 and NKG2D. (<b>B</b>) RT-PCR of indicted genes. Neut. = Neutrophils; BM = Bone marrow. (<b>C</b>) WB of supernatants from AT3 overexpressing tet-inducible form of Flag-tagged soluble sClec4e, sDectin and sNKG2D in the absence (Control) or presence of 1 μg/mL doxycycline (Dox.). bg = background band. (<b>D</b>) The sensitivity of AT3 overexpressing tet-inducible form of soluble sClec4e-Flag, sDectin-Flag and sNKG2D-Flag to neutrophil cytotoxicity. (<b>E</b>) WB of supernatant from AT3 overexpressing tet-inducible form of soluble sClec4e-Fc, sDectin-1-Fc and sNKG2D-Fc in the absence (Control) or presence of 1 μg/mL doxycycline (Dox.). (<b>F</b>) The sensitivity of AT3 overexpressing tet-inducible form of soluble sClec4e-Fc, sDectin-Fc and sNKG2D-Fc to neutrophil cytotoxicity. ** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 2
<p>(<b>A</b>) Inhibition of neutrophil cytotoxicity using blocking antibodies to Clec4e. (<b>B</b>) Inhibition of neutrophil cytotoxicity using blocking antibodies to Dectin-1. (<b>C</b>) Blocking antibodies to NKG2D did not perturb neutrophil cytotoxicity. (<b>D</b>) The concomitant presence of both anti-Clec4e (1 μg/mL) and anti-RAGE (1 μg/mL) did not cause a stronger inhibition of neutrophil cytotoxicity towards AT3 cells than either antibody alone. (<b>E</b>–<b>G</b>) Immunoprecipitation of C-type lectin receptor Fc fusion proteins in the presence of sNKG2D-Flag (<b>E</b>) sClec4e-Flag (<b>F</b>) or sDectin-1-Flag (<b>G</b>) Pre-IP = Cell extract samples prior to immunoprecipitation. IP = Immunoprecipitated samples. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>(<b>A</b>) Cytotoxicity of neutrophils pretreated in vitro 30 min with 10 μM SYK inhibitor R406 towards 4T1 and AT3 cells. (<b>A</b>–<b>C</b>) Local tumor growth of 4T1 (<b>B</b>) or AT3 (<b>C</b>) in mice that have been given control or R788-containing diet. (<b>D</b>,<b>E</b>). Cytotoxicity of neutrophils isolated from 4T1 (<b>D</b>) or AT3 (<b>E</b>)-tumor bearing mice that have been given control or R788-containing diet. (<b>F</b>) Lungs from 4T1-bearing mice that have been given control or R788-containing diet for 23, 30 and 35 days. (<b>G</b>,<b>H</b>) Spleen from 4T1-bearing mice that have been given control or R788-containing diet for 23, 30 and 35 days. (<b>I</b>) Percentage neutrophils in blood samples from 4T1-bearing mice that have been given control or R788-containing diet for 23, 30 and 35 days. (<b>J</b>) The neutrophil count in 1 mL blood samples from 4T1-bearing mice that have been given control or R788-containing diet for 23, 30 and 35 days. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>(<b>A</b>) Binding of sDectin-Fc to the cell surface of AT3 cells. AT3 cells were incubated with supernatant containing sClec4e-Fc, sDectin-1-Fc or sNKG2D-Fc, followed by incubation with APC-anti-Fc antibodies (red histograms). Black histograms represent 2nd antibody alone. (<b>B</b>) Kaplan-Meier Plot of survival of Her-2 positive breast cancer patients with low (black) or high (red) expression of Nidogen-1. (<b>C</b>) Kaplan-Meier Plot of survival of Her-2 positive breast cancer patients with low (black) or high (red) expression of Hspg2. (<b>D</b>) RT-PCR analysis of Nidogen-1 and Hspg2 expression in various tumor cell lines and in neutrophils (Neut.). (<b>E</b>) Co-immunoprecipitation of endogenous Nidogen-1 and endogenous Hspg2 in AT3 cells overexpressing tet-inducible Fc fusion proteins as indicated. Induction of Fc fusion proteins was done by adding 1 μg/mL doxycycline (Dox.) to the culture. Pre-IP = Cell extract samples prior to immunoprecipitation. IP = Immunoprecipitated samples. (<b>F</b>) Immunoprecipitation of Nidogen-1-Flag using the indicated Fc fusion proteins as baits. The proteins were overexpressed in AT3 cells. (<b>G</b>) Immunoprecipitation study of Nidogen-1-Flag using Galectin-3-Fc fusion protein as a bait. The proteins were overexpressed in AT3 cells. Pre-IP = Cell extract samples prior to immunoprecipitation. IP = Immunoprecipitated samples.</p> Full article ">Figure 5
<p>(<b>A</b>) Western blot analysis of Nidogen-Fc expression in supernatants of AT3 cells used for cell binding studies in (<b>B</b>). (<b>B</b>) Nidogen-1-Fc binding to tumor cells. The cells were incubated with supernatant containing Nidogen-1-Fc followed by incubation with APC-anti-Fc antibodies (red histograms). Black histograms represent cells incubated with control supernatant and APC-anti-Fc antibodies. (<b>C</b>) Staining of tumor cells with anti-Nidogen-1 antibodies followed by 2nd FITC-anti-rat antibodies (red histograms). Black histograms represent cells incubated with 2nd antibody only. (<b>D</b>) Western blot analysis of Nidogen link region (aa 270–356) fused to Fc and Nidogen G2 region (aa 357–665) fused to Fc. (<b>E</b>) Binding of Nidogen-G2 region-Fc fusion protein to AT3 cells. AT3 cells were incubated with supernatant containing either the Nidogen link region (aa 270–356) fused to Fc or Nidogen G2 region (aa 357–665) fused to Fc followed by incubation with APC-anti-Fc antibodies (red histograms). Black histograms represent cells incubated with control supernatant and APC-anti-Fc antibodies. (<b>F</b>) Staining of AT3 cells with anti-Hspg2 antibodies followed by 2nd FITC-anti-rat antibodies (red histograms). Black histograms represent cells incubated with 2nd antibody only. (<b>G</b>) Co-immunoprecipitation of endogenous Hspg2 in AT3 cells overexpressing tet-inducible Nidogen-1-Fc fusion protein. Induction of the Fc fusion protein was done by adding 1 μg/mL doxycycline (Dox.) to the culture. Pre-IP = Cell extract samples prior to immunoprecipitation. IP = Immunoprecipitated samples. (<b>H</b>) Co-immunoprecipitation of endogenous Nidogen-1 and endogenous Hspg2 in LLC cells overexpressing tet-inducible sRAGE-Fc fusion protein. Induction of Fc fusion proteins was done by adding 1 μg/mL doxycycline (Dox.) to the culture. Pre-IP = Cell extract samples prior to immunoprecipitation. IP = Immunoprecipitated samples.</p> Full article ">Figure 6
<p>(<b>A</b>,<b>B</b>) Relative mRNA levels of Nidogen-1 in AT3 and LLC cells following transduction of two different shRNAs targeting Nidogen-1 (<b>A</b>), and the resulting susceptibility of these cells to neutrophil cytotoxicity (<b>B</b>). (<b>C</b>,<b>D</b>) Relative mRNA levels of Hspg2 in AT3 and LLC cells following transduction of two different shRNAs targeting Hspg2 (<b>C</b>), and the resulting susceptibility of these cells to neutrophil cytotoxicity (<b>D</b>). * <span class="html-italic">p &lt;</span> 0.05; ** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 7
<p>A proposed model of the neutrophil-tumor cell synapse. CLR – C-type Lectin Receptor; NID1 – Nidogen-1.</p> Full article ">
22 pages, 3821 KiB  
Article
Potent and Broad-Spectrum Bactericidal Activity of a Nanotechnologically Manipulated Novel Pyrazole
by Silvana Alfei, Debora Caviglia, Alessia Zorzoli, Danilo Marimpietri, Andrea Spallarossa, Matteo Lusardi, Guendalina Zuccari and Anna Maria Schito
Biomedicines 2022, 10(4), 907; https://doi.org/10.3390/biomedicines10040907 - 15 Apr 2022
Cited by 6 | Viewed by 2202
Abstract
The antimicrobial potency of the pyrazole nucleus is widely reported these days, and pyrazole derivatives represent excellent candidates for meeting the worldwide need for new antimicrobial compounds against multidrug-resistant (MDR) bacteria. Consequently, 3-(4-chlorophenyl)-5-(4-nitrophenylamino)-1H-pyrazole-4-carbonitrile (CR232), recently reported as a weak antiproliferative agent, was considered [...] Read more.
The antimicrobial potency of the pyrazole nucleus is widely reported these days, and pyrazole derivatives represent excellent candidates for meeting the worldwide need for new antimicrobial compounds against multidrug-resistant (MDR) bacteria. Consequently, 3-(4-chlorophenyl)-5-(4-nitrophenylamino)-1H-pyrazole-4-carbonitrile (CR232), recently reported as a weak antiproliferative agent, was considered to this end. To overcome the CR232 water solubility issue and allow for the determination of reliable minimum inhibitory concentration values (MICs), we initially prepared water-soluble and clinically applicable CR232-loaded nanoparticles (CR232-G5K NPs), as previously reported. Here, CR232-G5K NPs have been tested on several clinically isolates of Gram-positive and Gram-negative species, including MDR strains. While for CR232 MICs ≥ 128 µg/mL (376.8 µM) were obtained, very low MICs (0.36–2.89 µM) were observed for CR232-G5K NPs against all of the considered isolates, including colistin-resistant isolates of MDR Pseudomonas aeruginosa and Klebsiella pneumoniae carbapenemases (KPCs)-producing K. pneumoniae (0.72 µM). Additionally, in time–kill experiments, CR232-G5K NPs displayed a rapid bactericidal activity with no significant regrowth after 24 h on all isolates tested, regardless of their difficult-to-treat resistance. Conjecturing a clinical use of CR232-G5K NPs, cytotoxicity experiments on human keratinocytes were performed, determining very favorable selectivity indices. Collectively, due to its physicochemical and biological properties, CR232-G5K NPs could represent a new potent weapon to treat infections sustained by broad spectrum MDR bacteria. Full article
(This article belongs to the Special Issue 10th Anniversary of Biomedicines—Advances in New Pharmacological Therapies)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Chemical structure of CR232 and BBB4.</p> Full article ">Figure 2
<p>Time-killing curves performed with CR232-G5K NPs (at concentrations equal to 4 x MIC) on <span class="html-italic">P. aeruginosa</span> 265 and 259, <span class="html-italic">E. coli</span> 462, and <span class="html-italic">S. aureus</span> 187.</p> Full article ">Figure 3
<p>Regression models that better fit the dispersion graphs obtained when reporting the cell viability % vs. the concentration of samples at 24 h of exposure of CR232 and G5K in graph (<b>a</b>) of CR232-G5K NPs (<b>b</b>) and of the nano-formulated CR232 provided by the quantity of NPs administered (<b>c</b>).</p> Full article ">Figure 3 Cont.
<p>Regression models that better fit the dispersion graphs obtained when reporting the cell viability % vs. the concentration of samples at 24 h of exposure of CR232 and G5K in graph (<b>a</b>) of CR232-G5K NPs (<b>b</b>) and of the nano-formulated CR232 provided by the quantity of NPs administered (<b>c</b>).</p> Full article ">
17 pages, 3567 KiB  
Article
Investigating the Effects of Conditioned Media from Stem Cells of Human Exfoliated Deciduous Teeth on Dental Pulp Stem Cells
by Huong Thu Vu, Mi-Ran Han, Jun-Haeng Lee, Jong-Soo Kim, Ji-Sun Shin, Ji-Young Yoon, Jeong-Hui Park, Khandmaa Dashnyam, Jonathan Campbell Knowles, Hae-Hyoung Lee, Jong-Bin Kim and Jung-Hwan Lee
Biomedicines 2022, 10(4), 906; https://doi.org/10.3390/biomedicines10040906 - 15 Apr 2022
Cited by 10 | Viewed by 3666
Abstract
Pulp regeneration has recently attracted interest in modern dentistry. However, the success ratio of pulp regeneration is low due to the compromising potential of stem cells, such as their survival, migration, and odontoblastic differentiation. Stem cells from human exfoliated deciduous teeth (SHED) have [...] Read more.
Pulp regeneration has recently attracted interest in modern dentistry. However, the success ratio of pulp regeneration is low due to the compromising potential of stem cells, such as their survival, migration, and odontoblastic differentiation. Stem cells from human exfoliated deciduous teeth (SHED) have been considered a promising tool for regenerative therapy due to their ability to secrete multiple factors that are essential for tissue regeneration, which is achieved by minimally invasive procedures with fewer ethical or legal concerns than those of other procedures. The aim of this study is to investigate the potency of SHED-derived conditioned media (SHED CM) on dental pulp stem cells (DPSCs), a major type of mesenchymal stem cells for dental pulp regeneration. Our results show the promotive efficiency of SHED CM on the proliferation, survival rate, and migration of DPSCs in a dose-dependent manner. Upregulation of odontoblast/osteogenic-related marker genes, such as ALP, DSPP, DMP1, OCN, and RUNX2, and enhanced mineral deposition of impaired DPSCs are also observed in the presence of SHED CM. The analysis of SHED CM found that a variety of cytokines and growth factors have positive effects on cell proliferation, migration, anti-apoptosis, and odontoblast/osteogenic differentiation. These findings suggest that SHED CM could provide some benefits to DPSCs in pulp regeneration. Full article
(This article belongs to the Special Issue Dental Pulp Stem Cells (DPSCs) and Tissue Regeneration: Mechanisms Mediated by Direct, Paracrine or Autocrine Effects)
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<p>Graphic abstract. SHED were collected from the pulp tissue of primary teeth, and DPSC were collected from the pulp tissue of permanent teeth. The cytokines/growth factors contained in SHED-cultured conditioned media promoted DPSC proliferation, survival, migration, and cell differentiation in pulp regenerative therapy.</p> Full article ">Figure 2
<p>Effect of stem cells isolated from human pulp of exfoliated deciduous tooth-derived conditioned media (CM) on dental pulp stem cells (DPSCs) regarding proliferation and migration. (<b>a</b>) Presentative live and dead images of SHED 24 h after treatment with 100% CM versus 50% CM and SFM. Scale bar represents 200 µm. (<b>b</b>) The effect of SHED CM after treatment for 24 h on the proliferation of SHED was quantified by CCK-8 assay. <span class="html-italic">n</span> = 6. (<b>c</b>) Schematic of migration assays. A total of 4 × 10<sup>4</sup> cells in 100 µL were seeded in the upper compartment, while the lower compartment contained 350 µL 100% CM, 50% CM or SFM. (<b>d</b>) Representative images of migrated SHED in the lower compartment of membrane after 6 h of treatment. (<b>e</b>) Cells counted in the lower compartment in 6 random fields. All data are represented as the mean ± standard deviation (SD). The statistical significance was calculated using one-way analysis of variance (ANOVA) to compare groups. Represents ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001, scale bar = 200 µm.</p> Full article ">Figure 3
<p>Treatment with SHED CM improved the viability of DPSC after H<sub>2</sub>O<sub>2</sub> exposure. DPSCs were cultured with 200 µM H<sub>2</sub>O<sub>2</sub> for 12 h before shifting to 100% CM, 50% CM and SFM for another 12 h. (<b>a</b>) Representative live and dead images of DPSCs confirmed the antioxidative stress effect of SHED-CM. (<b>b</b>) Cell viability was determined by CCK-8 analysis. All data are represented as the mean ± SD. The statistical significance was calculated using one-way analysis of variance (ANOVA) to compare groups. Represents ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, ns = not significant, scale bar = 200 µm.</p> Full article ">Figure 4
<p>Cytokine profiling array. (<b>a</b>) The process of cytokine analysis is briefly described. SHED at the 5th passage achieved 70%–80% confluence and was replenished with serum-free αMEM. After 48 h, the conditioned medium (CM) was collected. Control conditioned media was collected from serum-free αMEM (SFM) under the same conditions as conditioned medium. All CM and SFM were stored at −80 °C to maintain the biological properties before cytokine antibody analysis. The Fullmoonbio Cytokine Profiling antibody array features 310 antibodies for profiling cytokines, and related biomarkers were used. The data were analyzed with the ExDEGA GraphicPlus and DAVID tools based on the Gene Ontology (GO) database. (<b>b</b>) A clustering heatmap based on the 50 top cytokines showed a significant difference between SFM and 100% CM. (<b>c</b>) Using the 50 top cytokine lists, the DAVID v6.8 analysis tool with the Gene Ontology Term Biological Process database and ExDEGA Graphic Plus were used to build a functional annotation chart. The graph shows that SHED CM containing growth factors and cytokines has a positive effect on cell proliferation, migration, survival, and osteogenic differentiation. Pathways/biological activities related to upregulation of proliferation, migration, odontoblast/osteogenic differentiation, and multiple biological functions (proliferation, migration, downregulation of cell apoptosis, differentiation including downregulation of cell apoptosis) are marked in different colored boxes (green, blue, orange, and red, respectively).</p> Full article ">Figure 4 Cont.
<p>Cytokine profiling array. (<b>a</b>) The process of cytokine analysis is briefly described. SHED at the 5th passage achieved 70%–80% confluence and was replenished with serum-free αMEM. After 48 h, the conditioned medium (CM) was collected. Control conditioned media was collected from serum-free αMEM (SFM) under the same conditions as conditioned medium. All CM and SFM were stored at −80 °C to maintain the biological properties before cytokine antibody analysis. The Fullmoonbio Cytokine Profiling antibody array features 310 antibodies for profiling cytokines, and related biomarkers were used. The data were analyzed with the ExDEGA GraphicPlus and DAVID tools based on the Gene Ontology (GO) database. (<b>b</b>) A clustering heatmap based on the 50 top cytokines showed a significant difference between SFM and 100% CM. (<b>c</b>) Using the 50 top cytokine lists, the DAVID v6.8 analysis tool with the Gene Ontology Term Biological Process database and ExDEGA Graphic Plus were used to build a functional annotation chart. The graph shows that SHED CM containing growth factors and cytokines has a positive effect on cell proliferation, migration, survival, and osteogenic differentiation. Pathways/biological activities related to upregulation of proliferation, migration, odontoblast/osteogenic differentiation, and multiple biological functions (proliferation, migration, downregulation of cell apoptosis, differentiation including downregulation of cell apoptosis) are marked in different colored boxes (green, blue, orange, and red, respectively).</p> Full article ">Figure 5
<p>SHED CM enhanced DPSC osteogenic differentiation after exposure to 25 μM H<sub>2</sub>O<sub>2</sub> for 24 h. (<b>a</b>) Relative gene expression, early marker at Day 3 and late marker at Day 7. (<b>b</b>) Alkaline phosphatase (ALP) and Alizarin red staining of DPSCs cultured in osteogenic differentiation media (OM) and 50% CM or SFM at Days 3, 7, 21 and 28. DPSCs cultured in growth media (αMEM, 10% FBS, 1% PS) were used as a negative control. At day 7, the upregulation of ALP activity expressed darker blue in SHED CM treatment. The red color of ARS showed stronger in SHED CM group due to the increase in the amount of mineral deposition, compared to SFM at day 21 and 28. All data are represented as the mean ± SD. The statistical significance was calculated using one-way analysis of variance (ANOVA) to compare groups. Represents * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001, ns = nonsignificant, scale bar = 200 µm.</p> Full article ">Figure 6
<p>Schematic diagram of the effect of SHED CM on DPSC culture and odontoblast differentiation. SHED CM increased cell proliferation and progenitor cell recruitment, protected cells from oxidative stress, and enhanced cell differentiation.</p> Full article ">
26 pages, 4257 KiB  
Review
Aloperine: A Potent Modulator of Crucial Biological Mechanisms in Multiple Diseases
by Muhammad Tahir, Sakhawat Ali, Wenting Zhang, Boqiang Lv, Wenge Qiu and Juan Wang
Biomedicines 2022, 10(4), 905; https://doi.org/10.3390/biomedicines10040905 - 15 Apr 2022
Cited by 7 | Viewed by 3007
Abstract
Aloperine is an alkaloid found in the seeds and leaves of the medicinal plant Sophora alopecuroides L. It has been used as herbal medicine in China for centuries due to its potent anti-inflammatory, antioxidant, antibacterial, and antiviral properties. Recently, aloperine has been widely [...] Read more.
Aloperine is an alkaloid found in the seeds and leaves of the medicinal plant Sophora alopecuroides L. It has been used as herbal medicine in China for centuries due to its potent anti-inflammatory, antioxidant, antibacterial, and antiviral properties. Recently, aloperine has been widely investigated for its therapeutic activities. Aloperine is proven to be an effective therapeutic agent against many human pathological conditions, including cancer, viral diseases, and cardiovascular and inflammatory disorders. Aloperine is reported to exert therapeutic effects through triggering various biological processes, including cell cycle arrest, apoptosis, autophagy, suppressing cell migration, and invasion. It has also been found to be associated with the modulation of various signaling pathways in different diseases. In this review, we summarize the most recent knowledge on the modulatory effects of aloperine on various critical biological processes and signaling mechanisms, including the PI3K, Akt, NF-κB, Ras, and Nrf2 pathways. These data demonstrate that aloperine is a promising therapeutic candidate. Being a potent modulator of signaling mechanisms, aloperine can be employed in clinical settings to treat various human disorders in the future. Full article
(This article belongs to the Section Molecular and Translational Medicine)
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<p>Modulatory effects of aloperine on apoptosis and cell cycle.</p> Full article ">Figure 2
<p>Modulatory effects of aloperine on autophagy and tumor cell invasion &amp; migration.</p> Full article ">Figure 3
<p>Modulatory effects of aloperine on PI3K/Akt/mTOR and Ras signaling.</p> Full article ">Figure 4
<p>Modulatory effects of aloperine on NF-κB and Nrf2 signaling.</p> Full article ">
20 pages, 1388 KiB  
Review
In Vitro Model of Human Trophoblast in Early Placentation
by Darina Bačenková, Marianna Trebuňová, Daša Čížková, Radovan Hudák, Erik Dosedla, Alena Findrik-Balogová and Jozef Živčák
Biomedicines 2022, 10(4), 904; https://doi.org/10.3390/biomedicines10040904 - 15 Apr 2022
Cited by 17 | Viewed by 7419
Abstract
The complex process of placental implantation and development affects trophoblast progenitors and uterine cells through the regulation of transcription factors, cytokines, adhesion receptors and their ligands. Differentiation of trophoblast precursors in the trophectoderm of early ontogenesis, caused by the transcription factors, such as [...] Read more.
The complex process of placental implantation and development affects trophoblast progenitors and uterine cells through the regulation of transcription factors, cytokines, adhesion receptors and their ligands. Differentiation of trophoblast precursors in the trophectoderm of early ontogenesis, caused by the transcription factors, such as CDX2, TEAD4, Eomes and GATA3, leads to the formation of cytotrophoblast and syncytiotrophoblast populations. The molecular mechanisms involved in placental formation inside the human body along with the specification and differentiation of trophoblast cell lines are, mostly due to the lack of suitable cell models, not sufficiently elucidated. This review is an evaluation of current technologies, which are used to study the behavior of human trophoblasts and other placental cells, as well as their ability to represent physiological conditions both in vivo and in vitro. An in vitro 3D model with a characteristic phenotype is of great benefit for the study of placental physiology. At the same time, it provides great support for future modeling of placental disease. Full article
(This article belongs to the Special Issue Gynecological Tumor and Placenta Development)
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<p>Trophoblast stem cells differentiate into more specialized trophoblast populations at an early stage of development. TSCs are further divided into mononuclear cytotrophoblasts and multinucleated primitive syncytium. Cytotrophoblasts have the ability to proliferate, differentiate and fuse with the syncytiotrophoblast, thereby promoting syncytial growth during ontogenesis. Cells of the cytotrophoblast divide and migrate externally. Through cell fusion, the cytotrophoblast forms a multinucleated syncytiotrophoblast. Thus, cytotrophoblasts are the basis of the syncytiotrophoblast and reside on the basement membrane that separates them from the villous stroma. Extravillous trophoblastic cells line the mother’s blood vessels and intersect with maternal cells in the decidua, as well as several types of maternal immune cells, including T cells, decidual natural killer cells (dNK) and stromal cells that provide structural support for the decidua.</p> Full article ">Figure 2
<p>Maternal spiral arteries deliver nutrients to the placenta. Structure of a placental anchoring villus and its different trophoblast subtypes. Precursors that reside in the villous cytotrophoblast (vCTB) layer either differentiate into multinuclear syncytiotrophoblasts (STBs) when surrounded by maternal blood or give rise to proliferative proximal cell column trophoblasts (pCCTs) upon attachment of villi to the maternal decidua. Following the differentiation into distal cell column trophoblasts (dCCTs), extravillous trophoblasts (EVTs) develop, breaking through the overlying STB layer. Differentiation of EVT subtype progenitors residing on the basement membrane of CCTs leads to interstitial cytotrophoblasts (iCTB) and endovascular cytotrophoblasts (eCTB) relocated to maternal helical arteries. iCTBs in the decidual stroma colonize blood vessels from the outside and communicate with uterine cells of various types, such as decimal stromal cell macrophages and uterine natural killer (NK) cells.</p> Full article ">Figure 3
<p>Pregnancy is characterized by elevated levels of specific cytokines at the fetal–maternal interface. Blastocysts contain pluripotent ESC stem cells that are derived from the inner cell mass (ICM). Complicated differentiation process of the trophoblast precursors in the trophectoderm (TE) of early ontogenesis is modulated by transcription factors, which act as transcriptional promoters, leading to the formation of the fetal part of the placenta. Placenta-specific transcription factors are involved in further trophoblast development via transcription of placenta-specific genes. At the beginning of pregnancy, there is a transcriptional enhancer factor 3 (TEAP4) binding Yes-associated protein (YAP) coactivator, which plays a key role in the proliferation and expression of trophoblast stem cells (TSCs) villous trophoblastic epithelium progenitors. Caudal-type homeobox 2 (CDX2) plays a crucial role in the development of fetus and its perinatal tissues. In the overexpression of the octamer-binding transcription factor 4 (Oct4) antagonist, transcription factor CDX2 is able to induce a trophoblast, its morphology and upregulation of trophoblast markers. Eomesodermin (Eomes), a factor behind CDX2, is overexpressed and conditions differentiation toward TE/TSC, making both CDX2 and Eomes strong candidates for key TE regulators. Decreased expression of OCT4 in ESCs results in the loss of pluripotency and the formation of a monolayer by trophoblast-like cells. CDX2 and Eomes have a key effect on the regulation of differentiation into the trophoblast line. Tead4 acts on CDX2 during preimplantation, leading to the initiation of TE formation.</p> Full article ">Figure 4
<p>Schematic development of a blastocyst. Mammalian blastocyst consists of two types of cell layers, the outer trophectoderm surrounded by pluripotent cells, forming the inner cell mass (ICM). Blastocysts give rise to three stem cell entities—the pluripotent embryonic stem cells (ESCs), which are derived from ICM developed from the epiblast, and two types of extraembryonic stem cells, primitive eXtraembryonic ENdoderm-derived (XEN) cells and trophoblast stem cells (TSCs) derived from extraembryonic ectoderm. Solid lines—description of blastocyst cell layers. Dotted lines—three types of blastocyst-derived stem cells.</p> Full article ">
11 pages, 775 KiB  
Review
Advanced Imaging in Cardiac Amyloidosis
by Dominik Waldmeier, Jan Herzberg, Frank-Peter Stephan, Marcus Seemann and Nisha Arenja
Biomedicines 2022, 10(4), 903; https://doi.org/10.3390/biomedicines10040903 - 15 Apr 2022
Cited by 5 | Viewed by 3932
Abstract
This review serves as a synopsis of multimodality imaging in cardiac amyloidosis (CA), which is a disease characterized by deposition of misfolded protein fragments in the heart. It emphasizes and summarizes the diagnostic possibilities and their prognostic values. In general, echocardiography is the [...] Read more.
This review serves as a synopsis of multimodality imaging in cardiac amyloidosis (CA), which is a disease characterized by deposition of misfolded protein fragments in the heart. It emphasizes and summarizes the diagnostic possibilities and their prognostic values. In general, echocardiography is the first diagnostic tool in patients with an identified systemic disease or unclear left ventricular hypertrophy. Several echocardiographic parameters will raise suspicion and lead to further testing. Cardiac magnetic resonance and scintigraphy with bone avid radiotracers are crucial for diagnosis of CA and even enable a distinction between different subtypes. The subject is illuminated with established guidelines and innovative recent publications to further improve early diagnosis of cardiac amyloidosis in light of current treatment options. Full article
(This article belongs to the Special Issue Novel Diagnostic and Therapeutic Approaches in Cardiac Amyloidosis)
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<p>Typical findings in transthoracic echocardiography: (<b>a</b>) Apical four-chamber view with left ventricular hypertrophy. (<b>b</b>) Echocardiographic strain imaging with ‘cherry-on-top’ sign.</p> Full article ">Figure 2
<p>Typical findings in cardiovascular magnetic resonance: (<b>a</b>) Cine image of a four-chamber view with left ventricular hypertrophy. (<b>b</b>) Late gadolinium enhancement of the left and right ventricular myocardium.</p> Full article ">Figure 3
<p>Typical findings in scintigraphy and single-photon emission computed tomography: (<b>a</b>) Anterior view of 99Tc-hydroxymethylene diphosphonate scintigraphy with myocardial uptake Perugini Grade 3. (<b>b</b>) Posterior view of the same study. (<b>c</b>) Left and right ventricular uptake of 99mTc-hydroxymethylene diphosphonate in single-photon emission computed tomography.</p> Full article ">
14 pages, 303 KiB  
Review
Translational Applications of Extracorporeal Shock Waves in Dental Medicine: A Literature Review
by Abdulmonem Alshihri
Biomedicines 2022, 10(4), 902; https://doi.org/10.3390/biomedicines10040902 - 14 Apr 2022
Cited by 6 | Viewed by 2590
Abstract
Extracorporeal shock wave therapy (ESWT) has been studied and applied extensively in medical practice for various applications including musculoskeletal, dermal, vascular, and cardiac indications. These indications have emerged from primary ESWT use in treating urolithiasis and cholelithiasis. Likewise, dental medicine has had its [...] Read more.
Extracorporeal shock wave therapy (ESWT) has been studied and applied extensively in medical practice for various applications including musculoskeletal, dermal, vascular, and cardiac indications. These indications have emerged from primary ESWT use in treating urolithiasis and cholelithiasis. Likewise, dental medicine has had its share of utilizing ESWT in various investigations. This review aimed to provide an up-to-date summary of ESWT use in preclinical and clinical dental medicine. There is growing interest in ESWT use stemming from its non-invasiveness, low cost, and safe qualities in addition to its proven regenerative biostimulating aspects. Targeted tissue and parameters of ESWT delivery continue to be an integral part of successful ESWT treatment to attain the clinical value of the anticipated dose’s effect. Full article
(This article belongs to the Special Issue Translational Research in Shock Wave Medicine)
14 pages, 519 KiB  
Review
Potential Therapeutic Targets and Promising Agents for Combating NAFLD
by Atsushi Umemura, Seita Kataoka, Keiichiro Okuda, Yuya Seko, Kanji Yamaguchi, Michihisa Moriguchi, Takeshi Okanoue and Yoshito Itoh
Biomedicines 2022, 10(4), 901; https://doi.org/10.3390/biomedicines10040901 - 14 Apr 2022
Cited by 10 | Viewed by 3528
Abstract
Nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), is a growing cause of liver cirrhosis and liver cancer worldwide because of the global increases in obesity, dyslipidemia, hypertension, and type 2 diabetes mellitus. Contrary to the advancements in therapies for viral hepatitis, [...] Read more.
Nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), is a growing cause of liver cirrhosis and liver cancer worldwide because of the global increases in obesity, dyslipidemia, hypertension, and type 2 diabetes mellitus. Contrary to the advancements in therapies for viral hepatitis, effective treatments remain unestablished for patients with NAFLD. NAFLD, including NASH, is characterized by steatosis, inflammation, hepatic necrosis, and fibrosis. Despite our understanding of its pathophysiology, there are currently no effective treatments for NAFLD. In this review, we provide an update on the known pathophysiological mechanisms involved in the development of NAFLD and the role of hepatic stellate cells, and summarize the potential therapeutic agents, including natural products, for NAFLD. Full article
(This article belongs to the Special Issue NAFLD: From Mechanisms to Therapeutic Approaches)
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<p>The actions of honokiol.</p> Full article ">
16 pages, 11453 KiB  
Article
Liver-Targeted Nanoparticles Facilitate the Bioavailability and Anti-HBV Efficacy of Baicalin In Vitro and In Vivo
by Weiming Xu, Yijun Niu, Xin Ai, Chengjie Xia, Ping Geng, Haiyan Zhu, Wei Zhou, Hai Huang and Xunlong Shi
Biomedicines 2022, 10(4), 900; https://doi.org/10.3390/biomedicines10040900 - 14 Apr 2022
Cited by 11 | Viewed by 2532
Abstract
The anti-hepatitis B virus (HBV) efficacy of baicalin (BA) is mediated by HBV-related hepatocyte nuclear factors (HNFs). However, this efficacy is severely limited by the low bioavailability of BA. Therefore, a novel liver-targeted BA liposome was constructed to promote the bioavailability and antiviral [...] Read more.
The anti-hepatitis B virus (HBV) efficacy of baicalin (BA) is mediated by HBV-related hepatocyte nuclear factors (HNFs). However, this efficacy is severely limited by the low bioavailability of BA. Therefore, a novel liver-targeted BA liposome was constructed to promote the bioavailability and antiviral ability of BA. The results showed that apolipoprotein A1 (ApoA1)–modified liposomes (BAA1) significantly enhanced BA’s cellular uptake and specific distribution in the liver. Furthermore, the substantial inhibitory effects of BAA1 on HBsAg, HBeAg, HBV RNA, and HBV DNA were assessed in HB-infected cells and mice. Western blotting, co-immunoprecipitation, and transcriptomics analysis further revealed that the enhanced anti-HBV efficacy of BAA1 was attributed to the interaction between hepatocyte nuclear factors (HNFs) and estrogen receptors (ERs). Based on the findings, we propose that the ApoA1-modified liposomes aid BA in inhibiting HBV transcription and replication by augmenting its bioavailability and the HNFs–ERs axis. Full article
(This article belongs to the Section Nanomedicine and Nanobiology)
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<p>Characteristics of the liver-targeted baicalin liposomes (BAA1). (<b>A</b>) The particle size was measured by using the Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., Malvern, UK). (<b>B</b>) Zeta potential was measured by using the Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., Malvern, UK).</p> Full article ">Figure 2
<p>Cumulative percentage drug release and cellular uptake from BA and BAA1. (<b>A</b>) Cumulative percentage drug release from free BA and BAA1. (<b>B</b>) The cellular uptake of BA and BAA1 in HepG2 cells. Data are mean ± SD, <span class="html-italic">n</span> = 3; ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>Liver-targeted baicalin liposomes (BAA1) and baicalin (BA)-inhibited HBV replication in HepG2 cells. (<b>A</b>) pHBV1.2-transfected HepG2 cells were treated with ETV (10 μM), BA (25–100 μM), and BAA1 (25–100 μM) for 2 days. Cell viability was detected using the CCK-8 kit. (<b>B</b>,<b>C</b>) HBsAg and HBeAg in the cell supernatants were detected by using ELISA assay kits. (<b>D</b>) HBV-DNA in the culture supernatants was detected by qPCR. (<b>E</b>,<b>F</b>) HBV total RNAs and pgRNA were determined by qRT-PCR. Data are presented as mean ± SD, <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 vs. control. ns, not significant.</p> Full article ">Figure 4
<p>Liver-targeted baicalin liposome (BAA1) and baicalin (BA)-inhibited HBV replication in AAVDJ-transfected HepG2 cells. (<b>A</b>,<b>B</b>) AAVDJ-transfected HepG2 cells were treated with ETV (10 μM), BA (25–100 μM), and BAA1 (25–100 μM). HBsAg and HBeAg in the culture supernatants were detected by using ELISA kits. (<b>C</b>) HBV-DNA (HBV virion) in the culture supernatants was detected by qPCR. (<b>D</b>,<b>E</b>) HBV total RNAs and pgRNA were determined by qRT-PCR. Data are presented as mean ± SD, <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 vs. control. ns, not significant.</p> Full article ">Figure 5
<p>ApoA1 modification enhanced the anti-HBV effects of BA liposomes. pHBV1.2-transfected HepG2 cells were treated with empty liposomes (LP), BA liposomes (BALP, 50 μM), BA (50 μM), and BAA1 (50 μM) for 2 days. (<b>A</b>,<b>B</b>) HBsAg and HBeAg in the culture supernatants were detected by using ELISA kits. (<b>C</b>) HBV-DNA (HBV virion) in the culture supernatants was detected by qPCR. (<b>D</b>,<b>E</b>) HBV total RNAs and pgRNA were determined by qRT-PCR. Data are presented as the mean ± SD, <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 vs. control. ns, not significant.</p> Full article ">Figure 6
<p>Liver-enriched transcription factors were influenced by BAA1 treatment. pHBV1.2-HepG2 cells were treated with BA (50 μM) or BAA1 (50 μM) for 4 days. (<b>A</b>,<b>C</b>) HNF1α, HNF4α, FOXA2, Erα, p-ERα, and p-p38 MAPK expression in pHBV1.2-transfected HepG2 cells (Western blotting). (<b>B</b>,<b>D</b>,<b>E</b>) the relative mRNA levels of HNF1α, HNF4α, FOXA2, hB1F, and PS2 (qRT-PCR). Data are presented as mean ± SD, <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 vs. control. ns, not significant. (<b>F</b>) The combination of ERα and HNF4α in pHBV1.2-transfected HepG2 cells was detected by Western blotting.</p> Full article ">Figure 7
<p>Transcriptomics analysis of BA- and BAA1-treated cells. pHBV1.2-transfected HepG2 cells were treated with 50 μM of BA or BAA1 for 4 days and subjected to transcriptomics analysis. (<b>A</b>) PCA analysis, (<b>B</b>) Venn analysis, and (<b>C</b>,<b>D</b>) GO and KEGG enrichment analyses between V and BAA1. (<b>E</b>,<b>F</b>) GO and KEGG enrichment analyses between BA and BAA1. The transcriptomics data were analyzed on the free online platform (<a href="http://www.majorbio.com" target="_blank">www.majorbio.com</a>, accessed on 24 January 2022).</p> Full article ">Figure 8
<p>Anti-HBV efficacy of different baicalin preparations in vivo. (<b>A</b>–<b>C</b>) HBV transient mice were hydrodynamically injected with pHBV1.2 plasmids. The infected mice received oral administration of BA (1, 5, 10 mg/kg); ETV (5 mg/kg); intraperitoneal injection of BALP (1, 5, 10 mg/kg); or BAA1 (1, 5, 10 mg/kg) once a day for 7 days. The mice were sacrificed for their serum HBsAg and HBV DNA, with detection by ELISA and qPCR, respectively. Data are presented as mean ± SD, <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 vs. control. ns, not significant. (<b>D</b>,<b>E</b>) The infected mice received oral administration of BA (5 mg/kg); ETV (5 mg/kg); intraperitoneal injection of BA (5 mg/kg); BALP (5 mg/kg); or BAA1 (5 mg/kg) once a day for 1 week. The mice were sacrificed for their serum HBsAg and HBV DNA. with measurements by ELISA and qPCR, respectively. Data are presented as mean ± SD, <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 vs. control. ns, not significant. (<b>F</b>) HE staining of the liver tissues (200× magnification). Area of necrosis (black dotted line). Scale bar = 100 μm.</p> Full article ">Figure 9
<p>Pharmacokinetics and tissue distribution of BA, BALP, and BAA1 at the dose of 100 mg/kg. (<b>A</b>) The plasma BA concentration-time profiles in the mouse plasma were detected by HPLC. Data are presented as mean ± SD, <span class="html-italic">n</span> = 3; **** <span class="html-italic">p</span> &lt; 0.0001. (<b>B</b>) Drug distribution in each tissue 2 h after administration, detected by HPLC. Data are presented as mean ± SD, <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
14 pages, 2345 KiB  
Article
Potential Wound Healing Effect of Gel Based on Chicha Gum, Chitosan, and Mauritia flexuosa Oil
by Maria Onaira Gonçalves Ferreira, Alessandra Braga Ribeiro, Marcia S. Rizzo, Antonia Carla de Jesus Oliveira, Josy Anteveli Osajima, Leticia M. Estevinho and Edson C. Silva-Filho
Biomedicines 2022, 10(4), 899; https://doi.org/10.3390/biomedicines10040899 - 14 Apr 2022
Cited by 9 | Viewed by 2755
Abstract
Wounds are considered a clinically critical issue, and effective treatment will decrease complications, prevent chronic wound formation, and allow rapid healing. The development of products based on naturally occurring materials is an efficient approach to wound healing. Natural polysaccharides can mimic the extracellular [...] Read more.
Wounds are considered a clinically critical issue, and effective treatment will decrease complications, prevent chronic wound formation, and allow rapid healing. The development of products based on naturally occurring materials is an efficient approach to wound healing. Natural polysaccharides can mimic the extracellular matrix and promote cell growth, thus making them attractive for wound healing. In this context, the aim of this work was to produce a gel based on chicha gum, chitosan, and Mauritia flexuosa oil (CGCHO) for wound treatment. TG and DTG analyzed the thermal behavior of the materials, and SEM investigated the surface roughness. The percentages of total phenolic compounds, flavonoids, and antioxidants were determined, presenting a value of 81.811 ± 7.257 µmol gallic acid/g Mauritia flexuosa oil, 57.915 ± 0.305 µmol quercetin/g Mauritia flexuosa oil, and 0.379 mg/mL, respectively. The anti-inflammatory was determined, presenting a value of 10.35 ± 1.46% chicha gum, 16.86 ± 1.00% Mauritia flexuosa oil, 10.17 ± 1.05% CGCHO, and 15.53 ± 0.65% chitosan, respectively. The materials were tested against Gram-negative (Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus) bacteria and a fungus (Candida albicans). The CGCHO formulation showed better antimicrobial activity against Gram-positive bacteria. In addition, an in vivo wound healing study was also performed. After 21 days of treatment, the epidermal re-epithelialization process was observed. CGCHO showed good thermal stability and roughness that can help in cell growth and promote the tissue healing process. In addition to the good results observed for the antimicrobial, antioxidant, anti-inflammatory activities and providing wound healing, they provided the necessary support for the healing process, thus representing a new approach to the wound healing process. Full article
(This article belongs to the Special Issue Structural and Biomechanical Properties of Supramolecular Nanofibers-Based Hydrogels in Biomedicine)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>FTIR spectra for (A) chicha gum (CG), (B) <span class="html-italic">Mauritia flexuosa</span> oil, (C) chicha gum/chitosan/<span class="html-italic">Mauritia flexuosa</span> oil gel (CGCHO), and (D) chitosan-based gel (CHB).</p> Full article ">Figure 2
<p>Scanning electron microscope (SEM) images of chicha gum/chitosan/<span class="html-italic">Mauritia flexuosa</span> oil gel (CGCHO) at the magnitudes of (<b>a</b>) 500 µm, (<b>b</b>) 300 µm, (<b>c</b>) 50 µm and (<b>d</b>) 40 µm.</p> Full article ">Figure 3
<p>Thermogravimetric curves (TG) of (A) chicha gum (CG), (B) <span class="html-italic">Mauritia flexuosa</span> oil, (C) chicha gum/chitosan/<span class="html-italic">Mauritia flexuosa</span> oil gel (CGCHO), and (D) chitosan-based gel (CHB). Derivative thermogravimetric curves (DTG) of CG (E), <span class="html-italic">Mauritia flexuosa</span> oil (F), CGCHO (G), and CHG (H).</p> Full article ">Figure 4
<p>Inhibition of hyaluronidase activity by chicha gum (CG), <span class="html-italic">Mauritia flexuosa</span> oil, chicha gum/chitosan/<span class="html-italic">Mauritia flexuosa</span> oil gel (CGCHO), and chitosan-based gel (CHB).</p> Full article ">Figure 5
<p>Photomicrographs of skin wounds on the (<b>A</b>) 3rd, (<b>B</b>) 7th, (<b>C</b>) 14th, and (<b>D</b>) 21st day of treatment for the group treated with chicha gum/chitosan/<span class="html-italic">Mauritia flexuosa</span> oil gel (CGCHO). H.E. stain. (Scale bar: 2 µm, 4 µm, and 10 µm). (<b>A</b>) Skin ulcer (*) with the epidermis on the right edge (arrows), fibrin clot (arrowheads), and inflammatory infiltrate of polymorphonuclear cells in the dermis (detail). (<b>B</b>) Continuing reepithelialization of the epidermis (arrowhead), presence of newly formed vessels in granulation tissue in the dermis (circle), and macrophages and new fibroblasts (square). (<b>C</b>) Epidermal reepithelialization (arrowheads) and extensive granulation tissue formation (circle), with neoformed vessels (arrows), and collagen fibers(square). (<b>D</b>) Presence of hairs follicle (bulbus-arrows) and sebaceous glands (arrowheads).</p> Full article ">
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