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Volume 10
Issue 12
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Biomedicines, Volume 10, Issue 12 (December 2022) – 298 articles

Cover Story (view full-size image): The COVID-19 pandemic had a deep impact on patients with hematologic malignancies (HMs). HM patients show an impaired immune response due to both their disease and to the treatment they receive, which often causes profound immunosuppression or prolonged cytopenia. Most patients also suffered from delays in anticancer therapies during the pandemic and therefore reported high levels of anxiety and depression during COVID-19, because they represent a high-risk population and because they were concerned about their disease not being treated in an appropriate timeline. These conditions made HM patients more vulnerable to COVID-19 infection and to complications and to suffering from psychological distress. View this paper
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17 pages, 7588 KiB  
Article
Inactivation of Yes-Associated Protein Mediates Trophoblast Dysfunction: A New Mechanism of Pregnancy Loss Associated with Anti-Phospholipid Antibodies?
by Zengshu Huang, Zhijing Tang, Haiyun Guan, Wingting Leung, Lu Wang, Hexia Xia and Wei Zhang
Biomedicines 2022, 10(12), 3296; https://doi.org/10.3390/biomedicines10123296 - 19 Dec 2022
Cited by 3 | Viewed by 2241
Abstract
Pregnancy morbidity induced by anti-phospholipid antibodies (aPL+/PM+) is mainly thought to arise from placental abnormalities. We attempted to investigate the effect of aPL on the activity of Yes-associated protein (YAP) in the trophoblast and how YAP regulated human trophoblasts function. Thus, HTR-8 cells [...] Read more.
Pregnancy morbidity induced by anti-phospholipid antibodies (aPL+/PM+) is mainly thought to arise from placental abnormalities. We attempted to investigate the effect of aPL on the activity of Yes-associated protein (YAP) in the trophoblast and how YAP regulated human trophoblasts function. Thus, HTR-8 cells were treated with IgG purified from aPL+/PM+ women or normal controls. We found that aPL+/PM+ IgG impacted YAP activity via abrogating YAP expression. Further investigation of the anti-β2GPI-IgG/β2GPI complex showed an inhibition of nuclear YAP level and translocation in a dose-dependent manner, which might be rescued by progesterone in HTR-8 cells. YAP overexpression or knockdown HTR-8 cells were established for the evaluation of cell function and related gene expression in vitro. Loss of YAP arrested cell cycles in the G2/M phase, accelerated cell apoptosis by increasing the ratio of Bax/Bcl2, and disrupted MMP2/9-mediated cell migration and angiogenesis tube formation by VEGF. These findings support a new mechanism of PM associated with aPL through which YAP inactivation induced by aPL perturbs the trophoblast cell cycle, apoptosis, migration, and angiogenesis, finally developing into pregnancy failure. Full article
(This article belongs to the Special Issue 10th Anniversary of Biomedicines-Immunopathogenesis of Autoimmune Diseases)
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Figure 1

Figure 1
<p>aPL-positive IgG affects YAP expression and nuclear localization in HTR-8 cells. aPL+ IgG was extracted from serum aPL-positive women carriers with pregnancy morbidity history and aPL− IgG from normal women for comparison. (<b>A</b>) YAP and phospho-Ser 127 YAP in HTR-8 cells treated with aPL− IgG (100 μg/mL) and aPL+ IgG (100 μg/mL) for 24 h were immunoblotted. Subgroup analysis was performed based on pregnancy status (<b>B</b>) and single or more aPL+ IgG (<b>C</b>). Single aPL+ IgG indicated that only aCL IgG or anti-β2GPI IgG was positive. More than one aPL+ IgG indicated both aCL IgG and anti-β2GPI IgG positivity. The anti-β2GPI-IgG monoclonal antibody and human β2GPI (anti-β2GPI-IgG/β2GPI) complex was also prepared. (<b>D</b>) The anti-β2GPI-IgG/β2GPI complex suppressed YAP expression of HTR-8 cells in a dose-dependent manner, with concentrations ranging from 0.1/1 to 10/100 μg/mL with a binding ratio of 1:10. Quantitation of total YAP and p-YAP levels from blots (normalized to GAPDH) are shown as the mean± SEM (<b>B</b>–<b>D</b>). Mann–Whitney U test; ns indicates no significance, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. (<b>E</b>) Incubation with the anti-β2GPI-IgG/β2GPI complex for 6 h caused cytoplasmic retention of YAP in HTR-8 cells. The cellular distribution of YAP was shown with representative immunostaining images of HTR-8 cells after 6 h of exposure to different concentrations of anti-β2GPI-IgG/β2GPI. Scale bars, 50 μm. NC, control; IC, anti-β2GPI-IgG/β2GPI complex.</p> Full article ">Figure 2
<p>Progesterone regulates the expression of YAP in HTR-8 cells. (<b>A</b>) Progesterone enhances the expression of YAP and is partly reversed by RU486 in HTR-8 cells. HTR-8 cells were treated with control vehicle (DMSO) or progesterone (P<sub>4</sub>) (10<sup>−6</sup> M), alone or in combination with RU486 (10<sup>−5</sup> M), for 24 h or 48 h. The YAP protein expression in each group was analyzed by immunoblotting and GAPDH served as internal control. The results of quantification are presented as the mean ± SEM. One-way ANOVA test; ns indicates nonsignificant, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. (<b>B</b>) Pre-treatment of P<sub>4</sub> (10<sup>−6</sup> M) for 24 h promotes nuclear translocation of YAP in HTR-8 cells administrated by anti-β2GPI-IgG (1 μg/mL)/β2GPI (10 μg/mL). Scale bars, 100 μm. NC, control; IC, the anti-β2GPI-IgG/β2GPI complex; IC+P, the anti-β2GPI-IgG/β2GPI complex with progesterone pretreatment.</p> Full article ">Figure 3
<p>Downregulated YAP induces cell apoptosis and blocks cell cycle progression. (<b>A</b>) Confirmation of YAP overexpression and knockdown in HTR-8 cells after transfection with YAP-OE plasmid (YAP-OE) or YAP siRNA (si-YAP) by immunoblotting. Vector plasmid and control siRNA served as controls. (<b>B</b>) Analysis of cell cycle in HTR-8 cells transfected with YAP siRNA transfection was performed using flow cytometer. The si-NC group served as the control. The phases of G0/G1, S, and G2/M were calculated. (<b>C</b>) The percentage of apoptotic cells in si-YAP HTR-8 cells was determined by using flow cytometer. (<b>D</b>) Immunoblotting of Bax and Bcl2 protein levels in HTR-8 cells transfected with YAP-OE plasmid or YAP siRNA. The ratio of Bax and Bcl2 protein level was quantified. All results are shown as mean ± SEM. Student’s <span class="html-italic">t</span> test; * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3 Cont.
<p>Downregulated YAP induces cell apoptosis and blocks cell cycle progression. (<b>A</b>) Confirmation of YAP overexpression and knockdown in HTR-8 cells after transfection with YAP-OE plasmid (YAP-OE) or YAP siRNA (si-YAP) by immunoblotting. Vector plasmid and control siRNA served as controls. (<b>B</b>) Analysis of cell cycle in HTR-8 cells transfected with YAP siRNA transfection was performed using flow cytometer. The si-NC group served as the control. The phases of G0/G1, S, and G2/M were calculated. (<b>C</b>) The percentage of apoptotic cells in si-YAP HTR-8 cells was determined by using flow cytometer. (<b>D</b>) Immunoblotting of Bax and Bcl2 protein levels in HTR-8 cells transfected with YAP-OE plasmid or YAP siRNA. The ratio of Bax and Bcl2 protein level was quantified. All results are shown as mean ± SEM. Student’s <span class="html-italic">t</span> test; * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>YAP knockdown impairs HTR-8 cell migration ability. (<b>A</b>) The wound healing assay was conducted between si-NC HTR-8 and si-YAP HTR-8 cells groups and the representative images were taken at 0 h, 20 h, and 30 h. Scale bars are 100 µm. (<b>B</b>) The recovery rates of HTR-8 cells from the two groups are summarized. (<b>C</b>) The mRNA levels of MMP2 and MMP9 in HTR-8 cells transfected with control siRNA or YAP siRNA for 48 h were determined by RT-qPCR. Results are shown as mean ± SEM. Student’s <span class="html-italic">t</span> test; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 5
<p>YAP-mediated VEGF expression from HTR-8 cell is essential for tube forming capability of HUVECs. HUVECs were seeded onto Matrigel and incubated with culture supernatant from HTR-8 cells transfected with scrambled siRNA or YAP siRNA. (<b>A</b>) The tube formation assay was analyzed after 3 h. Representative images are shown. Scale bars are 100 µm. (<b>B</b>) The VEGF protein expression in HTR-8 cells 72 h after transfection with scrambled siRNA and YAP siRNA was measured by immunoblotting (normalized to GAPDH). Results are shown as mean ± SEM. Student’s <span class="html-italic">t</span> test; *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 5 Cont.
<p>YAP-mediated VEGF expression from HTR-8 cell is essential for tube forming capability of HUVECs. HUVECs were seeded onto Matrigel and incubated with culture supernatant from HTR-8 cells transfected with scrambled siRNA or YAP siRNA. (<b>A</b>) The tube formation assay was analyzed after 3 h. Representative images are shown. Scale bars are 100 µm. (<b>B</b>) The VEGF protein expression in HTR-8 cells 72 h after transfection with scrambled siRNA and YAP siRNA was measured by immunoblotting (normalized to GAPDH). Results are shown as mean ± SEM. Student’s <span class="html-italic">t</span> test; *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 6
<p>The phosphorylation of MAPK-ERK ascends after YAP inhibition in HTR-8 cells. YAP-OE plasmid (YAP-OE) or YAP siRNA (si-YAP) were transfected into HTR-8 cells which were harvested after 72 h. (<b>A</b>) Immunoblotting of the p-ERK, ERK, p-p38, and p38 proteins in YAP-OE or si-YAP HTR-8 cells. (<b>B</b>) p-ERK and p-p38 protein levels were normalized to ERK or p38 protein levels. GAPDH served as internal control. All values are presented as mean ± SEM. Student’s <span class="html-italic">t</span> test; * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 7
<p>Schematic diagram of the hypothesized molecular mechanism for YAP protein involvement in aPL-induced pregnancy complications. aPL was previously demonstrated to recognize β2GPI on the trophoblast surface and provoke intra-cellular response dependent upon Toll-like receptor (TLR) or LDL receptor family member apoE receptor 2 (ApoER2) [<a href="#B15-biomedicines-10-03296" class="html-bibr">15</a>,<a href="#B17-biomedicines-10-03296" class="html-bibr">17</a>]. We assume that aPL interacting with β2GPI-ApoER2 or β2GPI-TLR on the trophoblast induces aberrant inactivation of YAP protein and thereafter impacts the expression of cell apoptosis, migration, and angiogenesis genes, which can be rescued by progesterone.</p> Full article ">
15 pages, 2642 KiB  
Article
Re-Analysis of Single-Nucleus Transcriptomics Reveals Diverse Dorsal Root Ganglia Macrophage Responses Following Peripheral Nerve Injury
by Nea Korvenlaita and Lauri Louhivuori
Biomedicines 2022, 10(12), 3295; https://doi.org/10.3390/biomedicines10123295 - 19 Dec 2022
Cited by 2 | Viewed by 2103
Abstract
An increasing amount of evidence points to an important role of macrophages in peripheral nerve injury (PNI) and associated pain. Peripheral nerve macrophages facilitate the regeneration, while dorsal root ganglia (DRG) macrophages might propagate the injury after a PNI. These differences might be [...] Read more.
An increasing amount of evidence points to an important role of macrophages in peripheral nerve injury (PNI) and associated pain. Peripheral nerve macrophages facilitate the regeneration, while dorsal root ganglia (DRG) macrophages might propagate the injury after a PNI. These differences might be explained by various in vivo models of PNIs or non-uniform methodologies to phenotype the macrophages. Unbiased methods to phenotype macrophages using single whole cell or nucleus transcriptomics have been rarely applied on PNIs outside the nerves themselves. Here, we compare the effects of the transection or crush of the sciatic nerve and spinal nerve transection on the DRG macrophage phenotypes utilizing a publicly available single-nucleus transcriptomic DRG dataset. Our results demonstrate that unique and time-dependent DRG macrophage gene expression profiles were produced by the three PNI models with particular macrophage clusters being enriched that were dependent on the severity of the neuronal injury score. PNI associated DRG macrophages were not purely anti- or pro-inflammatory. These results suggest that various functions of DRG macrophage subtypes are carefully orchestrated upon a PNI. These findings open a new avenue for studying the DRG macrophage subtypes in PNIs and encourage further unbiased phenotyping efforts to better understand their relevance in neuropathic pain. Full article
(This article belongs to the Special Issue Macrophages in Health and Non-infectious Disease 3.0)
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Figure 1
<p>Single-nucleus transcriptomics show a proportional increase in the number of macrophages in the mouse DRG after PNI. (<b>A</b>) UMAP dimensional reduction plot showing the clustering of all cell types. Clusters 0 to 16 are annotated as neurons, immune cells, or other. (<b>B</b>) The expression of marker genes used for the annotation of neurons (<span class="html-italic">Rbfox3</span>), other cell types (<span class="html-italic">Sparc</span>) and immune cells (<span class="html-italic">Ptprc</span>). (<b>C</b>) Nuclei from the Immune_11 cluster were re-clustered to obtain clusters for different immune cell subtypes. (<b>D</b>) Macrophage marker genes (<span class="html-italic">Lyz2, Mrc1, Csf1r,</span> and <span class="html-italic">Ccr2</span>). (<b>E</b>) Neutrophil marker genes (<span class="html-italic">S100a8, S100a9,</span> and <span class="html-italic">Retnlg</span>). (<b>F</b>) NK/T cell marker genes (<span class="html-italic">Ms4a4b, Nkg7,</span> and <span class="html-italic">Cd3g</span>). (<b>G</b>) B cell marker genes (<span class="html-italic">Cd79a, Cd79b, Ighd,</span> and <span class="html-italic">Ighm</span>). (<b>H</b>) Percent of macrophage nuclei from the total nuclei at different time points after PNI. The scale of the x-axis is transformed to log2. The gradient color in (<b>B</b>) and (<b>D</b>–<b>G</b>) represents the level of the normalized gene expression in individual nuclei. Abbreviations: spinal nerve transection (SpNT); sciatic nerve crush (Crush); sciatic nerve transection (ScNT).</p> Full article ">Figure 2
<p>The time course of DRG macrophage responses to peripheral nerve injuries are dependent on the site and type of injury. (<b>A</b>) Mean enrichment score of stress and inflammation associated genes following different PNI models. (<b>B</b>) Mean enrichment score of DAM associated genes. All nuclei annotated as macrophages were included in the mean score in (<b>A</b>,<b>B</b>). (<b>C</b>) Mean enrichment score of neuronal injury associated genes. All nuclei annotated as neurons were included in the mean score calculation. (<b>D</b>) Co-expression of the classical M1 (y-axes) and M2 (x-axes) marker genes in individual macrophage nuclei after PNI models. Axes represent the normalized expression values. The scales of the x-axes in (<b>A</b>–<b>C</b>) are transformed to log2. Abbreviations: spinal nerve transection (SpNT); sciatic nerve crush (Crush); sciatic nerve transection (ScNT); disease-associated macrophage (DAM).</p> Full article ">Figure 3
<p>Spinal nerve transection modulates inflammatory responses of macrophages, while sciatic nerve crush affects translational capacity of macrophages. (<b>A</b>) The number of statistically significant DEGs in macrophages after different PNI models. (<b>B</b>) The number of overlapping and unique DEGs at 72 h and 168 h time points after the SpNT or Crush. (<b>C</b>) Scaled log2 fold change to naïve shown for the DEGs which were statistically significant only in the Crush model (time points 72 h and 168 h compared to the same time points of the SpNT as in (<b>B</b>)). (<b>D</b>) Scaled log2 fold change to naïve shown for the DEGs which were statistically significant only in the SpNT model. (<b>E</b>,<b>F</b>) Number of overlapping and unique DEGs at different time points in SpNT or Crush, respectively. (<b>G</b>) Fifteen most enriched pathways in the SpNT groups and their LogP values were determined using Metascape pathway analysis tool. (<b>H</b>) Fifteen most enriched pathways in the Crush groups. Abbreviations: spinal nerve transection (SpNT); sciatic nerve crush (Crush); sciatic nerve transection (ScNT); differentially expressed (DE); DE gene (DEG).</p> Full article ">Figure 4
<p>Macrophages can be divided into transcriptionally distinct clusters which correspond to varying phenotypes. (<b>A</b>) UMAP dimensional reduction plot showing the clustering of macrophages into nine distinct clusters. (<b>B</b>) Top 5 marker genes defining the macrophage clusters. (<b>C</b>) Relative proportion of each macrophage cluster shown for the different nerve injury models over time. The y-axis corresponds to the percentage of individual cluster from all macrophages and the x-axis scale is transformed to log2 for the visualization. (<b>D</b>) Summary of the most enriched pathways among the distinct macrophage clusters. The color gradient represents the LogP values calculated by the Metascape pathway analysis tool. Marker genes having adjusted <span class="html-italic">p</span>-value &lt; 0.05 were used, and if over 200 genes were detected, the first 200 of them were included in the analysis. Abbreviations: spinal nerve transection (SpNT); sciatic nerve crush (Crush); sciatic nerve transection (ScNT).</p> Full article ">Figure 5
<p>Macrophages from different tissues have distinct transcriptomic profiles and exhibit remarkable heterogeneity. (<b>A</b>) DRG macrophage expression of the phagocytosis-related engulfment receptors. Left panel shows the expression for each macrophage cluster and right panel shows the expression for each PNI model and time point. (<b>B</b>) DRG macrophage expression of the peripheral macrophage genes. (<b>C</b>) DRG macrophage expression of the inflammatory genes. (<b>D</b>) DRG macrophage expression of chemotaxis-inducing genes. (<b>E</b>) Percentage of <span class="html-italic">Gpnmb</span> positive DRG macrophages in each macrophage cluster. (<b>F</b>) Percentage of <span class="html-italic">Gpnmb</span> positive macrophages at different time points after the nerve injury models. (<b>G</b>) Co-expression of <span class="html-italic">Gpnmb</span> and <span class="html-italic">Spp1</span> following different nerve injury models. Abbreviations: spinal nerve transection (SpNT); sciatic nerve crush (Crush); sciatic nerve transection (ScNT); central nervous system (CNS); spinal cord injury (SCI).</p> Full article ">Figure 6
<p>Sciatic and spinal nerve injuries show distinct effects on DRG macrophages.</p> Full article ">
21 pages, 1470 KiB  
Review
New Insights into the Use of Empagliflozin—A Comprehensive Review
by Joanna Forycka, Joanna Hajdys, Julia Krzemińska, Piotr Wilczopolski, Magdalena Wronka, Ewelina Młynarska, Jacek Rysz and Beata Franczyk
Biomedicines 2022, 10(12), 3294; https://doi.org/10.3390/biomedicines10123294 - 19 Dec 2022
Cited by 18 | Viewed by 7175
Abstract
Empagliflozin is a relatively new drug that, as an inhibitor of the sodium–glucose cotransporter 2 (SGLT2), causes increased urinary glucose excretion and thus contributes to improved glycemic control, better glucose metabolism, reduced glucotoxicity and insulin resistance. Although its original use was to induce [...] Read more.
Empagliflozin is a relatively new drug that, as an inhibitor of the sodium–glucose cotransporter 2 (SGLT2), causes increased urinary glucose excretion and thus contributes to improved glycemic control, better glucose metabolism, reduced glucotoxicity and insulin resistance. Although its original use was to induce a hypoglycemic effect in patients with type 2 diabetes mellitus (T2DM), empagliflozin has also shown a number of other beneficial effects by demonstrating a nephroprotective effect, and it has proven to be a breakthrough in the treatment of heart failure (HF). Empagliflozin has been shown to reduce hospitalizations for HF and the number of deaths from cardiovascular causes. Empagliflozin treatment also reduces the incidence of renal events, including death from renal causes, as well as the risk of end-stage renal failure. Empagliflozin appears to be a fairly well-tolerated and safe drug. In patients with inadequate glycemic control, empagliflozin used in monotherapy or as an adjunct to therapy effectively lowers fasting blood glucose, postprandial blood glucose, average daily glucose levels, glycated hemoglobin A1C (HbA1C) and also leads to significant weight reduction in patients with T2DM. Unfortunately, there are some limitations, e.g., severe hypersensitivity reaction to the drug and a glomerular filtration rate (GFR) < 30 mL/min/1.73 m2. As with any drug, empagliflozin is also characterized by several side effects among which symptomatic hypotension, troublesome genital fungal infections, urinary tract infections and rare ketoacidosis are characteristic. Full article
(This article belongs to the Special Issue 10th Anniversary of Biomedicines—Advances in New Pharmacological Therapies)
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Figure 1

Figure 1
<p>Beneficial effects of empagliflozin administration [<a href="#B2-biomedicines-10-03294" class="html-bibr">2</a>,<a href="#B3-biomedicines-10-03294" class="html-bibr">3</a>,<a href="#B4-biomedicines-10-03294" class="html-bibr">4</a>,<a href="#B6-biomedicines-10-03294" class="html-bibr">6</a>,<a href="#B7-biomedicines-10-03294" class="html-bibr">7</a>]. HbA1c, glycated hemoglobin A1C.</p> Full article ">Figure 2
<p>(<b>A</b>) Properly functioning nephron. (<b>B</b>) The mechanism of nephron impairment in the course of long-term, inappropriately treated DM. (<b>C</b>) Effect of SGLT2 inhibitor empagliflozin on improving intraglomerular pressure and GFR in DM [<a href="#B42-biomedicines-10-03294" class="html-bibr">42</a>]. SGLT1, SGLT2—sodium-glucose transport proteins, JGA—juxtaglomerular apparatus.</p> Full article ">Figure 3
<p>Potential SGLT2 inhibitor benefits in heart failure [<a href="#B67-biomedicines-10-03294" class="html-bibr">67</a>].</p> Full article ">
13 pages, 4032 KiB  
Article
Arsenic Trioxide Triggers Apoptosis of Metastatic Oral Squamous Cells Carcinoma with Concomitant Downregulation of GLI1 in Hedgehog Signaling
by Raphael Luís Rocha Nogueira, Taís Bacelar Sacramento de Araújo, Ludmila Faro Valverde, Viviane Aline Oliveira Silva, Bruno Raphael Ribeiro Cavalcante, Erik Aranha Rossi, Kyan James Allahdadi, Mitermayer Galvão dos Reis, Thiago Almeida Pereira, Ricardo D. Coletta, Daniel Pereira Bezerra, Bruno Solano de Freitas Souza, Rosane Borges Dias and Clarissa A. Gurgel Rocha
Biomedicines 2022, 10(12), 3293; https://doi.org/10.3390/biomedicines10123293 - 19 Dec 2022
Cited by 4 | Viewed by 2560
Abstract
Given the lack of advances in Oral Squamous Cell Carcinoma (OSCC) therapy in recent years, pharmacological strategies to block OSCC-related signaling pathways have gained prominence. The present study aimed to evaluate the therapeutic potential of Arsenic Trioxide (ATO) concerning its antitumoral effects and [...] Read more.
Given the lack of advances in Oral Squamous Cell Carcinoma (OSCC) therapy in recent years, pharmacological strategies to block OSCC-related signaling pathways have gained prominence. The present study aimed to evaluate the therapeutic potential of Arsenic Trioxide (ATO) concerning its antitumoral effects and the inhibition of the Hedgehog (HH) pathway in OSCC. Initially, ATO cytotoxicity was assessed in a panel of cell lines. Cell viability, cell cycle, death patterns, and cell morphology were analyzed, as well as the effect of ATO on the expression of HH pathway components. After the cytotoxic assay, HSC3 cells were chosen for all in vitro assays. ATO increased apoptotic cell death and nuclear fragmentation in the sub-G1 cell cycle phase and promoted changes in cell morphology. In addition, the reduced expression of GLI1 indicated that ATO inhibits HH activity. The present study provides evidence of ATO as an effective cytotoxic drug for oral cancer treatment in vitro. Full article
(This article belongs to the Special Issue Malignant and Potentially Malignant Disorders of the Oral Cavity: Updates from Pathogenesis to Therapy 2.0)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The cytotoxic effect of ATO on membrane integrity of HSC3 cells. Numbers of cells with integrity of membrane correspond to mean ± SEM of three independent experiments carried out in duplicate. (*) <span class="html-italic">p</span> ≤ 0.05 compared to the negative control (CTL), and (#) when compared to the HSC3 NT group (non-treated cells) by ANOVA (analysis of variance) followed by Student Newman-Keuls test.</p> Full article ">Figure 2
<p>The effects of ATO on cell cycle phases (Sub-G1, G0-G1, S and G2-M) and internucleosomal DNA fragmentation in HSC3 cells at 24, 48 and 72 h of treatment. The vehicle was used (0.25% DMSO) as the negative control (CTL) and 5-FU (16.75 µM) as the positive control. The values correspond to means ± SEM from three independent experiments carried out in duplicate. Cellular debris was omitted from the analyses and 10,000 events were analyzed per sample. (*) <span class="html-italic">p</span> ≤ 0.05 when compared to the negative control (CTL), and (#) when compared to the HSC3 NT group (non-treated cells) by ANOVA (analysis of variance) followed by Student Newman-Keuls test.</p> Full article ">Figure 3
<p>The effects of ATO on the externalization of phosphatidylserine in HSC3 cells, determined by flow cytometry using Annexin V-FITC/Propidium iodide (Early apoptosis/Late apoptosis), after 48 (<b>A</b>) and 72 h (<b>B</b>) of treatment. Cellular debris was omitted from the analyses and 10,000 events were analyzed per sample. (*) <span class="html-italic">p</span> ≤ 0.05 when compared to the negative control (CTL) and (#) when compared to the HSC3 NT group (non-treated cells) by ANOVA (analysis of variance) followed by Student Newman-Keuls test.</p> Full article ">Figure 4
<p>Bright field and representative images of HSC3 cells under early apoptosis, late apoptosis and necrosis after 48 h (<b>A</b>) and 72 h (<b>B</b>) of treatment, using annexin V-FITC (Green)/Propidium Iodide (PI; Red) (Scale Bar: 100 µm).</p> Full article ">Figure 5
<p>The graphical representation of light scattering characteristics of HSC3 cells, as determined by flow cytometry after 48 h (<b>A</b>) and 72 h (<b>B</b>) of treatment. The percentage (%) of FSC (Forward Scatter) and SSC (Side Scatter) correspond to mean ± SEM of three independent experiments carried out in duplicate. (*) <span class="html-italic">p</span> ≤ 0.05 compared to the negative control (CTL), and (#) when compared to the HSC3 NT group (non-treated cells) by ANOVA (analysis of variance) followed by Student Newman-Keuls test.</p> Full article ">Figure 6
<p>The representative images of GLI1 protein staining (green) in HSC3 cells. Nuclei are in blue. (Scale Bar: 100 µm) (<b>A</b>). Graphic representation of Fluorescence Intensity (<b>B</b>). Gene expression of GLI1 (<b>C</b>). The vehicle was used (0.25% DMSO) as a negative control (CTL) and 5-FU (16.75 μM) was used as a positive control. The Cq value for each sample was normalized using GAPDH as a reference gene, then calibrated according to the Cq values obtained for the untreated HSC3 cell group (HSC3 NT). (*) <span class="html-italic">p</span> ≤ 0.05 when compared to the negative control (CTL), and (#) when compared to the HSC3 NT group (non-treated cells) by ANOVA (analysis of variance) followed by Student Newman-Keuls test.</p> Full article ">Figure 7
<p>The evaluation of GLI1 protein expression in HSC3 cells after 24 h of treatment with ATO. (<b>A</b>) Low GLI1 protein expression levels in HSC3 cells treated with ATO. The symbol (−) represents the absence and (+) represents the experimental group in each band in the following order: HSC3 NT (non-treated), DMSO, 5-FU, ATO 3.7 µM and ATO 7.4 µM. (<b>B</b>) Graphic representation of densitometry readings of three individual experiments. The value of each experimental condition was calibrated using GLI1/ß-actin intensity ratio and then DMSO (---) was used as the reference group. (*) <span class="html-italic">p</span> ≤ 0.05 when compared to HSC3 NT (non-treated cells) group and (#) when compared to 5-FU by ANOVA (analysis of variance) followed by Student Newman-Keuls test.</p> Full article ">
14 pages, 457 KiB  
Review
What Are the Roles of Proprotein Convertases in the Immune Escape of Tumors?
by Elham Mehranzadeh, Olatz Crende, Iker Badiola and Patricia Garcia-Gallastegi
Biomedicines 2022, 10(12), 3292; https://doi.org/10.3390/biomedicines10123292 - 19 Dec 2022
Cited by 6 | Viewed by 2547
Abstract
Protein convertases (PCs) play a significant role in post-translational procedures by transforming inactive precursor proteins into their active forms. The role of PCs is crucial for cellular homeostasis because they are involved in cell signaling. They have also been described in many diseases [...] Read more.
Protein convertases (PCs) play a significant role in post-translational procedures by transforming inactive precursor proteins into their active forms. The role of PCs is crucial for cellular homeostasis because they are involved in cell signaling. They have also been described in many diseases such as Alzheimer’s and cancer. Cancer cells are secretory cells that send signals to the tumor microenvironment (TME), remodeling the surrounding space for their own benefits. One of the most important components of the TME is the immune system of the tumor. In this review, we describe recent discoveries that link PCs to the immune escape of tumors. Among PCs, many findings have determined the role of Furin (PC3) as a paramount enzyme causing the TME to induce tumor immune evasion. The overexpression of various cytokines and proteins, for instance, IL10 and TGF-B, moves the TME towards the presence of Tregs and, consequently, immune tolerance. Furthermore, Furin is implicated in the regulation of macrophage activity that contributes to the increased impairment of DCs (dendritic cells) and T effector cells. Moreover, Furin interferes in the MHC Class_1 proteolytic cleavage in the trans-Golgi network. In tumors, the T cytotoxic lymphocytes (CTLs) response is impeded by the PD1 receptor (PD1-R) located on CTLs and its ligand, PDL1, located on cancer cells. The inhibition of Furin is a subtle means of enhancing the antitumor response by repressing PD-1 expression in tumors or macrophage cells. The impacts of other PCs in tumor immune escape have not yet been clarified to the extent that Furin has. Accordingly, the influence of other types of PCs in tumor immune escape is a promising topic for further consideration. Full article
(This article belongs to the Special Issue Tumor Microenvironment and Cancer Therapy)
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<p>Summary of pro- and anti-tumor factors [<a href="#B31-biomedicines-10-03292" class="html-bibr">31</a>].</p> Full article ">
12 pages, 504 KiB  
Article
Delphi-Based Consensus on Interstitial Lung Disease Screening in Patients with Connective Tissue Diseases (Croatian National-Based Study)
by Mislav Radić, Srđan Novak, Marko Barešić, Ana Hećimović, Dijana Perković, Jasna Tekavec-Trkanjec, Miroslav Mayer, Višnja Prus, Jadranka Morović-Vergles, Daniela Marasović Krstulović, Mislav Cerovec, Ljiljana Bulat Kardum, Miroslav Samaržija and Branimir Anić
Biomedicines 2022, 10(12), 3291; https://doi.org/10.3390/biomedicines10123291 - 19 Dec 2022
Cited by 10 | Viewed by 3375
Abstract
The aim of this study was to develop a Croatian Delphi-based expert consensus for screening interstitial lung disease (ILD) associated with connective tissue disease (CTD). A systematic literature review was conducted on risk factors for the development of ILD, prevalence and incidence of [...] Read more.
The aim of this study was to develop a Croatian Delphi-based expert consensus for screening interstitial lung disease (ILD) associated with connective tissue disease (CTD). A systematic literature review was conducted on risk factors for the development of ILD, prevalence and incidence of ILD, diagnostic and screening methods for ILD, and prognosis of ILD in idiopathic inflammatory myopathy (IIM), mixed connective tissue disease (MCTD), primary Sjögren’s syndrome (pSS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and systemic sclerosis (SSc) were performed. Based on the evidence found, experts developed questionnaires for screening and monitoring ILD in each CTD, which were provided via an online survey. Following the electronic survey, two screening algorithms were developed based on the consensus opinions. The detection strategy for ILD included high-resolution computed tomography (HRCT) in addition to pulmonary function testing for IIM, MCTD, and SSc. and pulmonary function testing for newly diagnosed pSS, RA and SLE. However, in patients with identified risk factors for ILD HRCT, these tests should also be performed. A screening strategy for early identification of patients with various CTD-ILD was first developed by a multidisciplinary team of rheumatologists, pulmonologists, and radiologists to identify early CTD patients at risk of ILD, a severe extra-articular manifestation of CTD. Full article
(This article belongs to the Special Issue Frontiers of Targeted Therapy and Predictors of Treatment Response in Systemic Sclerosis)
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<p>Screening for ILD in newly discovered MCTD, IIM, or SSc patients. Abbreviations: DLco, diffusion capacity for carbon monoxide; FVC, forced vital capacity; HRCT, high-resolution computed tomography; IIM, idiopathic inflammatory myopathy; ILD, interstitial lung disease; MCTD, mixed connective tissue disease; MDT; NSIP; SSc, systemic sclerosis. * Especially in patients with antisynthetase positive antibodies and anti-MDA5 in idiopathic inflammatory myopathy and anti-Scl-70/anti-topoisomerase I antibody in SSc. ** Clinical deterioration and/or decrease in FVC or DLco below 80% or by more than 10% compared to previous findings. <sup>†</sup> Most common symptoms include shortness of breath, exertional dyspnea, dry cough without phlegm, and weakness/extreme fatigue. Other symptoms include loss of appetite, unexplained weight loss, chest discomfort, dyspnea, tachypnea, and pulmonary hemorrhage.</p> Full article ">Figure 2
<p>Screening for ILD in newly discovered RA, pSS, or SLE patients. Abbreviations: DLco, diffusion capacity for carbon monoxide; FVC, forced vital capacity; ILD, interstitial lung disease; HRCT, high-resolution computed tomography; MDT; multidisciplinary team; pSS, primary Sjögren’s syndrome; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus. * In patients with identified risk factors include HRCT as screening methods. ** Clinical deterioration and/or decrease in FVC or DLco below 80% or by more than chest x compared to previous findings. <sup>†</sup> Most common symptoms include shortness of breath, exertional dyspnea, dry cough without phlegm, and weakness/extreme fatigue. Other symptoms include loss of appetite, unexplained weight loss, chest discomfort, dyspnea, tachypnea, and pulmonary hemorrhage.</p> Full article ">
24 pages, 2468 KiB  
Review
A Bio-Social Model during the First 1000 Days Optimizes Healthcare for Children with Developmental Disabilities
by Mark S. Scher
Biomedicines 2022, 10(12), 3290; https://doi.org/10.3390/biomedicines10123290 - 19 Dec 2022
Cited by 13 | Viewed by 3497
Abstract
Most children with developmental disabilities (DD) live in resource-limited countries (LMIC) or high-income country medical deserts (HICMD). A social contract between healthcare providers and families advocates for accurate diagnoses and effective interventions to treat diseases and toxic stressors. This bio-social model emphasizes reproductive [...] Read more.
Most children with developmental disabilities (DD) live in resource-limited countries (LMIC) or high-income country medical deserts (HICMD). A social contract between healthcare providers and families advocates for accurate diagnoses and effective interventions to treat diseases and toxic stressors. This bio-social model emphasizes reproductive health of women with trimester-specific maternal and pediatric healthcare interactions. Lifelong neuronal connectivity is more likely established across 80% of brain circuitries during the first 1000 days. Maladaptive gene-environment (G x E) interactions begin before conception later presenting as maternal-placental-fetal (MPF) triad, neonatal, or childhood neurologic disorders. Synergy between obstetrical and pediatric healthcare providers can reduce neurologic morbidities. Partnerships between healthcare providers and families should begin during the first 1000 days to address diseases more effectively to moderate maternal and childhood adverse effects. This bio-social model lowers the incidence and lessens the severity of sequalae such as DD. Access to genetic-metabolomic, neurophysiologic and neuroimaging evaluations enhances clinical decision-making for more effective interventions before full expression of neurologic dysfunction. Diagnostic accuracy facilitates developmental interventions for effective preschool planning. A description of a mother-child pair in a HIC emphasizes the time-sensitive importance for early interventions that influenced brain health throughout childhood. Partnership by her parents with healthcare providers and educators provided effective healthcare and lessened adverse effects. Effective educational interventions were later offered through her high school graduation. Healthcare disparities in LMIC and HICMD require that this bio-social model of care begin before the first 1000 days to effectively treat the most vulnerable women and children. Prioritizing family planning followed by prenatal, neonatal and child healthcare improves wellness and brain health. Familiarity with educational neuroscience for teachers applies neurologic diagnoses for effective individual educational plans. Integrating diversity and inclusion into medical and educational services cross socioeconomic, ethnic, racial, and cultural barriers with life-course benefits. Families require knowledge to recognize risks for their children and motivation to sustain relationships with providers and educators for optimal outcomes. The WHO sustainable development goals promote brain health before conception through the first 1000 days. Improved education, employment, and social engagement for all persons will have intergenerational and transgenerational benefits for communities and nations. Full article
(This article belongs to the Special Issue Neurodevelopmental Disabilities)
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<p>(<b>A</b>) The horizontal approach to the diagnostic process across maturing phenotypes over the first 1000 days. The WHO program diagram depicts the first 1000 days from conception through two years of age that illustrates changing clinical expressions. (<b>B</b>) The vertical approach to the diagnostic process depicted as the flow of information across biological systems through a hierarchy of networks. Each panel highlights a different set of networks at play in a biological system. Genomics networks represent interactions among DNA sequences that may give rise to longer-range as well as more local chromosomal structures that modulate gene activity, in addition to inducing synergistic effects on higher-order phenotypes. Genomics networks drive molecular networks composed of RNA, protein, metabolites, and other molecules in the system. Molecular networks are components of cellular networks in which the complex web of interactions among these networks gives rise to the complex phenotypes that define living systems. Tissue networks comprise cellular networks that are clearly influenced by the molecular and genomics networks, and organism networks comprise tissue networks that are clearly defined by the component cellular and molecular networks. Complex phenotypes like disease emerge from this complex web of interacting networks, given genetic and perturbations to the system [<a href="#B3-biomedicines-10-03290" class="html-bibr">3</a>].</p> Full article ">Figure 2
<p>(<b>A</b>) Proposed causal chain of events with maternal immune activation in humans, leading to a wide spectrum of neuronal dysfunctions and behavioral phenotypes observable in the juvenile, adult, or aged progeny. Abbreviations: AD, Alzheimer disease; ASD, autism spectrum disorder; MIA, maternal immune activation; PD, Parkinson disease [<a href="#B19-biomedicines-10-03290" class="html-bibr">19</a>]. (<b>B</b>) Pathways for neurodevelopmental resilience and susceptibility to maternal immune activation are depicted, induced by infectious or noninfectious stimuli with variable effects on the offspring. Whereas a substantial portion of offspring are resilient to maternal immune activation and do not acquire overt pathologies, neurodevelopmental sequelae occur in susceptible offspring. The neurodevelopmental consequences among the latter are heterogeneous and span a range of neurological and psychiatric disorders with varying temporal onsets (as indicated by the corresponding lengths of the arrows and the broken-lined circles). Note that the illustrated pathways among susceptible offspring represent risks but not deterministic relationships [<a href="#B16-biomedicines-10-03290" class="html-bibr">16</a>].</p> Full article ">Figure 2 Cont.
<p>(<b>A</b>) Proposed causal chain of events with maternal immune activation in humans, leading to a wide spectrum of neuronal dysfunctions and behavioral phenotypes observable in the juvenile, adult, or aged progeny. Abbreviations: AD, Alzheimer disease; ASD, autism spectrum disorder; MIA, maternal immune activation; PD, Parkinson disease [<a href="#B19-biomedicines-10-03290" class="html-bibr">19</a>]. (<b>B</b>) Pathways for neurodevelopmental resilience and susceptibility to maternal immune activation are depicted, induced by infectious or noninfectious stimuli with variable effects on the offspring. Whereas a substantial portion of offspring are resilient to maternal immune activation and do not acquire overt pathologies, neurodevelopmental sequelae occur in susceptible offspring. The neurodevelopmental consequences among the latter are heterogeneous and span a range of neurological and psychiatric disorders with varying temporal onsets (as indicated by the corresponding lengths of the arrows and the broken-lined circles). Note that the illustrated pathways among susceptible offspring represent risks but not deterministic relationships [<a href="#B16-biomedicines-10-03290" class="html-bibr">16</a>].</p> Full article ">Figure 3
<p>(<b>A</b>) Microscopic appearance on H&amp; E staining of an infarcted zone of a uterus with placenta in situ in severe preeclampsia. The intravascular injection of Chinese ink via the uterine artery appears as patent radial and spiral arteries in the central part of the placental bed (blue arrows on the right and center) but absent arteries are associated with a large intervillous thrombus (blue arrow on the left). (<b>B</b>) Schematic diagram of placental vessels’ transformation showing normal (bottom line) and defective deep placentation. [<a href="#B21-biomedicines-10-03290" class="html-bibr">21</a>].</p> Full article ">Figure 4
<p>Two types of fetal inflammatory response have been described based on statistical analyses comparing the results of RNA sequencing from human cord blood samples of preterm neonates diagnosed with fetal inflammatory response syndromes type I and type II after birth associated with placental-cord histopathological findings. Placental examinations were performed by pathologists blinded to the clinical histories. FIRS type I was characterized by an upregulation of host immune responses, including neutrophil and monocyte functions, together with a proinflammatory cytokine storm and a downregulation of T cell processes. IL 6 was the representative cytokine that was excessively released. This process was correlated with acute and chronic lesions of chorioamnionitis and funisitis. In contrast, FIRS type II comprised a mild chronic inflammatory response involving perturbation of HLA transcripts, suggestive of fetal semi-allograft rejection. CXCL 10 was the representative chemokine that was excessively released. This process was depicted by findings of chronic chorioamnionitis, villitis or deciduitis. Abbreviations: IL 6, interleukin 6; CXCL 10, chemokine 10 [<a href="#B27-biomedicines-10-03290" class="html-bibr">27</a>].</p> Full article ">Figure 5
<p>The neuro-ontogenic process starts at gestational age (GA) weeks 2–3 with the constitution of the neural tube. At GA week 4, the rostral portion of the neural tube forms the prosencephalic, mesencephalic and romboencephalic vesicles. The prosencephalic vesicle then forms two vesicles that are destined to become the telencephalon and the diencephalon (thalamus, hypothalamus, and other structures). The schematic diagram represents the development of telencephalon. Initially, the telencephalic primordium is constituted by dividing neuroepithelial cells (often called neural stem cells), characterized by interkinetic nuclear migration, which form the ventricular zone (VZ). There, one type of cells, the radial glial cells, are particularly prominent and distinctive. At GA week 4, they undergo early exponential proliferation increasing the number of progenitor/intermediate progenitor cells and the thickness of VZ. Early born neurons are interneurons which move within the marginal zone (MZ) and intermediate zone (IZ). The MZ will eventually form cortical layer I and, for some authors, MZ could be identified also before the cortical plate (CP) formation as early MZ (eMZ). The IZ is a cell-sparse compartment and exists before the appearance of the CP. Radial glial cells and intermediate progenitor cells can be classified in two distinct subpopulations: apical, resident in VZ with bipolar fibers, and basal, which delaminate from VZ with unipolar basal fiber. Apical progenitor cells in the VZ and basal progenitor cells in subventricular zone (SVZ) are considered the main source of pyramidal neurons. Around GA week 5, the neurogenesis begins, and the neuronal precursors proliferate rapidly within the VZ. The neurons located in the first recognizable cortical layer, known as the preplate (PP), form the earliest synaptic connections. PP is a transient structure present before the appearance of the CP. Around week 7, the PP cells contribute to the subplate (SP) which remains below the CP after its formation and contains post-migratory pyramidal neurons and interneurons. Moreover, the accumulation of basal progenitor cells creates a distinct new compartment above the VZ, the SVZ. Here, they divide and are considered an additional source of intermediate progenitor cells. By GA week 8, radially migrating neurons from VZ and SVZ initiate the development of the layered CP forming from inside to outside. For example, pyramidal neurons eventually migrate outward along the radial glial cells [<a href="#B18-biomedicines-10-03290" class="html-bibr">18</a>].</p> Full article ">Figure 6
<p>Diagram depicts the theoretical translational framework for the use of educational neuroscience by the educator to develop effective individual educational plans. Results of brain structural and functional testing are offered by clinicians to educators regarding diagnosis and prognosis of the maturing child through the school years. Interpretation of these findings are based on knowledge of developmental neuroscience research applied to clinical decision making. Interventional strategies are developed by the interdisciplinary educational team of teachers, psychologists and therapists based on this information at multiple time points. Lessons plans are revised as a function of advancing school grades. These teaching strategies are generally applied across a community’s school system calibrated to resource-availability [<a href="#B66-biomedicines-10-03290" class="html-bibr">66</a>].</p> Full article ">
11 pages, 857 KiB  
Article
Cytokine Profiles Differentiate Symptomatic from Asymptomatic PTSD in Service Members and Veterans with Chronic Traumatic Brain Injury
by Ethan G. Smith, James Hentig, Carina Martin, Chelsea Wagner, Vivian A. Guedes, Katie A. Edwards, Christina Devoto, Kerri Dunbar, Michael J. Roy and Jessica M. Gill
Biomedicines 2022, 10(12), 3289; https://doi.org/10.3390/biomedicines10123289 - 19 Dec 2022
Cited by 2 | Viewed by 2090
Abstract
Traumatic brain injuries (TBI) and posttraumatic stress disorder (PTSD) are commonly observed comorbid occurrences among military service members and veterans (SMVs). In this cross-sectional study, SMVs with a history of TBI were stratified into symptomatic and asymptomatic PTSD groups based on posttraumatic stress [...] Read more.
Traumatic brain injuries (TBI) and posttraumatic stress disorder (PTSD) are commonly observed comorbid occurrences among military service members and veterans (SMVs). In this cross-sectional study, SMVs with a history of TBI were stratified into symptomatic and asymptomatic PTSD groups based on posttraumatic stress checklist-civilian (PCL-C) total scores. Blood-based biomarkers were assessed, and significant differential markers were associated with scores from multiple neurobehavioral self-report assessments. PCL-C cutoffs were total scores >50 (PTSD symptomatic) and <25 (asymptomatic). Cytokines IL6, IL8, TNFα, and IL10 were significantly elevated (p < 0.05–0.001) in the TBI+/PTSD symptomatic group compared to the TBI+/asymptomatic group. Cytokine levels of IL8, TNFα, and IL10 were strongly associated with PCL-C scores (0.356 < r > 0.624 for all, p < 0.01 for all), while TNFα and IL10 were additionally associated with NSI totals (r = 0.285 and r = 0.270, p < 0.05, respectively). This is the first study focused on PTSD symptom severity to report levels of circulating pro-inflammatory IL8, specifically in SMVs with TBI. These data suggest that within the military TBI population, there are unique cytokine profiles that relate to neurobehavioral outcomes associated with TBI and PTSD. Full article
(This article belongs to the Special Issue Molecular Research of Neurodegenerative and Psychiatric Diseases)
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<p><b>Group differences in plasma cytokine concentration.</b> The cytokine concentration distribution is shown for the PTSD symptomatic and asymptomatic control groups using boxplots indicating the median, IQR, and minimum and maximum values. Cytokines IL6 (<b>A</b>), IL8 (<b>B</b>), TNFα (<b>C</b>), and IL10 (<b>D</b>) were all elevated in the PTSD symptomatic group. The Mann-Whitney U test was used to assess group differences. <span class="html-italic">p</span> values are reported above each plot (* indicates statistical significance at the 0.05 level).</p> Full article ">Figure 2
<p><b>Scatter plots of significant correlations in the PTSD high group.</b> In the PTSD symptomatic group, significant correlations between cytokine concentrations and behavioral measures are represented via scatter plots. Spearman’s rho correlation coefficient (ρ) was calculated as the data were non-normal. The Spearman’s rho correlation coefficient is represented on each scatter plot with an asterisk indicating the significance level (<span class="html-italic">p</span> * &lt; 0.05, <span class="html-italic">p</span> ** &lt; 0.01).</p> Full article ">
14 pages, 1066 KiB  
Systematic Review
Prognostic and Predictive Biomarkers in Head and Neck Squamous Cell Carcinoma Treated with Radiotherapy—A Systematic Review
by Daniel H. Schanne, Alexander Koch, Olgun Elicin, Roland Giger, Michaela Medová, Yitzhak Zimmer and Daniel M. Aebersold
Biomedicines 2022, 10(12), 3288; https://doi.org/10.3390/biomedicines10123288 - 19 Dec 2022
Cited by 6 | Viewed by 2769
Abstract
Background: Radiotherapy is a mainstay in head and neck squamous cell carcinoma (HNSCC) treatment but is mostly applied without stratification by molecular diagnostics. Development of reliable biomarkers may have the potential to improve radiotherapy (RT) efficacy and reduce toxicity. We conducted a [...] Read more.
Background: Radiotherapy is a mainstay in head and neck squamous cell carcinoma (HNSCC) treatment but is mostly applied without stratification by molecular diagnostics. Development of reliable biomarkers may have the potential to improve radiotherapy (RT) efficacy and reduce toxicity. We conducted a systematic review to summarize the field of biomarkers in HNSCC treated by RT. Methods: Pubmed and EMBASE were searched independently by two researchers following pre-defined inclusion and exclusion criteria. Z curves were generated to investigate publication bias. OncoKB was used for identification of druggable targets. Results: 134 manuscripts remained for data extraction. 12% of tumors were AJCC/UICC stage I–II and 82% were stage III–IV. The most common biomarkers were proteins (39%), DNA (14%) and mRNA (9%). Limiting analysis to prospective data and statistically significant results, we found three potentially druggable targets: ERCC2, PTCH1 and EGFR. Regarding data quality, AJCC/UICC stage was missing in 32% of manuscripts. 73% of studies were retrospective and only 7% were based on prospective randomized trials. Z-curves indicated the presence of publication bias. Conclusion: An abundance of potential biomarkers in HNSCC is available but data quality is limited by retrospective collection, lack of validation and publication bias. Improved study design and reporting quality might accelerate successful development of personalized treatments in HNSCC. Full article
(This article belongs to the Special Issue Head and Neck Cancers: Diagnosis, Therapy, Molecular and Cellular Mechanisms)
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<p>Screening process.</p> Full article ">Figure 2
<p>Number of included publications over the pre-defined time period (<b>A</b>). Histogram of the reported patient numbers that were included per study (<b>B</b>).</p> Full article ">Figure 3
<p>Method of data collection for included studies (<b>A</b>). Reported anatomical site of primary tumor (<b>B</b>). Type of radiotherapy applied to included patients (<b>C</b>). Reported Outcomes and endpoints (<b>D</b>). AFR—Any failure rate, DFS—Disease-free survival, DMFS—Distant metastasis- free survival, DRR—Distant recurrence rate, DSS—Disease-specific survival, LRC—Locoregional recurrence, LRFS—Local relapse-free survival, LRR—Locoregional recurrence, OS—Overall survival, PFS—Progression-free survival.</p> Full article ">Figure 4
<p>Distribution of biomarkers among categories (<b>A</b>). Tissue of origin for biomarkers (<b>B</b>). Number of assessed biomarkers per included study (<b>C</b>). Number of independent publications per biomarker (<b>D</b>).</p> Full article ">
15 pages, 1801 KiB  
Article
Investigating the Antibacterial Properties of Prospective Scabicides
by Sara Taylor, Deonne Walther, Deepani D. Fernando, Pearl Swe-Kay and Katja Fischer
Biomedicines 2022, 10(12), 3287; https://doi.org/10.3390/biomedicines10123287 - 19 Dec 2022
Cited by 2 | Viewed by 3241
Abstract
Scabies is a dermatological disease found worldwide. Mainly in tropical regions, it is also the cause of significant morbidity and mortality due to its association with potentially severe secondary bacterial infections. Current treatment strategies for scabies do not consider the role of opportunistic [...] Read more.
Scabies is a dermatological disease found worldwide. Mainly in tropical regions, it is also the cause of significant morbidity and mortality due to its association with potentially severe secondary bacterial infections. Current treatment strategies for scabies do not consider the role of opportunistic bacteria, and here we investigate whether current and emerging scabicides can offer any anti-bacterial protection. Using the broth microdilution method, we examined antimicrobial potential of the current scabicide ivermectin and emerging scabies treatments: abametapir, mānuka oil, and its individual β-triketones. Our results demonstrate that the two novel scabicides abametapir and mānuka oil have antimicrobial properties against common scabies-associated bacteria, specifically Staphylococcus aureus, Streptococcus pyogenes, Streptococcus dysgalactiae subsp. equisimilis and Acinetobacter baumannii. The current scabicide ivermectin offers some antimicrobial activity and is capable of inhibiting the growth aforementioned bacteria. This research is important as it could help to inform future best treatment options of scabies, and scabies-related impetigo. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Host-Bacterial Pathogen Interactions: Novel Promising Drug Targets)
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<p>Structures of two commercial scabicides, ivermectin and permethrin, and five emerging scabicides, including a commercial head lice drug, abametapir, and four β-triketones commonly found in mānuka oil, flavesone, leptospermone, isoleptospermone, and grandiflorone.</p> Full article ">Figure 2
<p>(<b>a</b>) Minimum inhibitory concentrations (MIC) and (<b>b</b>) Minimum bactericidal concentrations (MBC) of mānuka oil (% <span class="html-italic">v</span>/<span class="html-italic">v</span>) against four scabies-associated bacteria.</p> Full article ">Figure 3
<p>(<b>a</b>) Minimum inhibitory concentrations (MIC ) and (<b>b</b>) Minimum bactericidal concentrations (MBC) of novel scabicides against four scabies-associated bacteria. For all Streptococcus strains, the pure Ivermectin formulation was used. For <span class="html-italic">S. aureus</span> and <span class="html-italic">A. baumannii</span>, the Ivomec Pour-on Solution was used. Ivermectin did not show bactericidal activity against all strains tested. In the above bar chart, the β-triketones are shown in a striped pattern, with Flavesone in red, isoleptospermone in blue, leptospermone in grey and grandiflorone in green stripes. Abemetapir is presented in solid grey and ivermectin in in solid black.</p> Full article ">
13 pages, 2626 KiB  
Article
Hypoxia-Induced Downregulation of miR-29 in Renal Tumor Cells Affects Collagen IV Subunit Expression through Multiple Sites
by Chuncheng Liu, Linan Liu, Jinlai Bo, Xian Lu, Donghui Qu, Gehui Liu, Zhiyan Jiang and Lu Cai
Biomedicines 2022, 10(12), 3286; https://doi.org/10.3390/biomedicines10123286 - 19 Dec 2022
Cited by 3 | Viewed by 1958
Abstract
Multiple tumor exacerbations and treatment procedures, such as extracellular matrix remodeling, metabolic reprogramming, immunological evasion, and resistance to chemotherapy and radiotherapy, are influenced by intratumoral hypoxia. It is becoming increasingly clear how hypoxia interacts with the extracellular matrix and how this affects the [...] Read more.
Multiple tumor exacerbations and treatment procedures, such as extracellular matrix remodeling, metabolic reprogramming, immunological evasion, and resistance to chemotherapy and radiotherapy, are influenced by intratumoral hypoxia. It is becoming increasingly clear how hypoxia interacts with the extracellular matrix and how this affects the growth of cancer. We analyzed the published sequencing results of hypoxia-stressed mouse kidney tumor cells and found that the expression of miR-29b was significantly downregulated. There are several sites that are complementary to the miR-29 seed sequence in the 3’ non-coding regions (3’UTRs) of various extracellular matrix-related genes, including collagen IV. We analyzed the sequences of the 3’UTRs of different subunits of collagen IV in different species and constructed the corresponding phylogenetic trees. We found that the 3’UTRs of Col4a1 and Col4a4 may have been subjected to particular evolutionary pressures. By cloning the 3’UTRs of collagen IV subunits into the psiCHECKTM-2 vector, we found that seven of the eight sites in the Col4a3–Col4a6 gene complementary to miR-29 were significantly repressed by miR-29a, b (except for the 7774–7781 of Col4a3 gene). The inhibitory efficiency of miR-29a, b on these seven sites was between 27% and 57%. The research on the regulation of miR-29 and extracellular matrix by hypoxia can provide a theoretical basis for tumor and fibrosis research and treatment. Full article
(This article belongs to the Section Cancer Biology and Oncology)
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<p>MiR-29 target genes analysis. (<b>A</b>) Significantly differentially expressed miRNAs after hypoxia treatment (<span class="html-italic">p</span> &lt; 0.05). (<b>B</b>) Top 20 significantly enriched GO functional categories. (<b>C</b>) Statistics on the number of binding sites in collagen IV subunits of different species. C: conserved site; P: poorly conserved site. (<b>D</b>) Venn diagram showing the overlapping miRNA among the miRNAs regulating <span class="html-italic">Col4a1</span> to <span class="html-italic">Col4a6</span> for mouse.</p> Full article ">Figure 2
<p>Analysis of <span class="html-italic">Col4a1</span> and <span class="html-italic">Col4a2</span> 3’UTRs and miR-29 binding sites. (<b>A</b>,<b>B</b>) Phylogenetic tree constructed from the 3’UTRs of <span class="html-italic">Col4a1</span> and <span class="html-italic">Col4a2</span> in multiple species. Analysis of miR-29 binding sites and flanking sequences in the 3’UTRs of <span class="html-italic">Col4a1</span> and <span class="html-italic">Col4a2</span> in multiple species.</p> Full article ">Figure 3
<p>MiR-29 directly regulates the 3’UTR of <span class="html-italic">Col4a3</span>. (<b>A</b>) Phylogenetic tree constructed from the 3’UTRs of <span class="html-italic">Col4a3</span> in multiple species. Analysis of miR-29 binding sites and flanking sequences in the 3’UTRs of <span class="html-italic">Col4a3</span> in multiple species. (<b>B</b>) The effect of miR-29a or miR-29b mimics on the relative luciferase activity of the psi-wt-4a3 vector (psiCHECK<sup>TM</sup>-2 containing wild-type 3’UTR of <span class="html-italic">Col4a3</span>), psi-m-c1-4a3 vector (psiCHECK<sup>TM</sup>-2 contained 3’UTR of <span class="html-italic">Col4a3</span> mutated at conserved site 1), psi-m-c2-4a3 vector, and the psi-m-c1&amp;c2-4a3 vector (psiCHECK<sup>TM</sup>-2 containing 3’UTR of <span class="html-italic">Col4a3</span> mutated at conserved sites 1 and 2) was examined. The data are shown as the mean ± SEM (<span class="html-italic">n</span> = 3). **, <span class="html-italic">p</span> &lt; 0.01. (<b>C</b>) Schematic diagram of complementary sequence sites and primer F1, R1, F2, and R2 sites. (<b>D</b>) The effect of miR-29a or miR-29b mimics on the relative luciferase activity of the psi-new1-wt-4a3 vector, psi-new1-m-c1-4a3 vector, psi-new1-m-p1-4a3 vector, and the psi- new1-m-c1&amp;p1-4a3 vector was examined. The data are shown as the mean ± SEM (<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. (<b>E</b>) The effect of miR-29a or miR-29b mimics on the relative luciferase activity of the psi-new2-wt-4a3 vector, psi-new2-m-c2-4a3 vector, psi-new2-m-p2-4a3 vector, and the psi-new2-m-c2&amp;p2-4a3 vector was examined. The data are shown as the mean ± SEM (<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 ">Figure 4
<p>MiR-29 directly regulated the 3’UTR of <span class="html-italic">Col4a4</span>. (<b>A</b>) Phylogenetic tree constructed from the 3’UTRs of <span class="html-italic">Col4a4</span> in multiple species. Analysis of miR-29 binding sites and flanking sequences in the 3’UTRs of <span class="html-italic">Col4a3</span> in multiple species. (<b>B</b>) Schematic diagram of complementary sequence site and primer sites. (<b>C</b>) The effect of miR-29a or miR-29b mimics on the relative luciferase activity of the psi-wt-4a4 vector and psi-m-c1-4a4 vector was examined. The data are shown as the mean ± SEM (<span class="html-italic">n</span> = 3). **, <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 5
<p>MiR-29 directly regulates the 3’UTR of <span class="html-italic">Col4a5</span>. (<b>A</b>) Phylogenetic tree constructed from the 3’UTRs of <span class="html-italic">Col4a5</span> in multiple species. Analysis of miR-29 binding sites and flanking sequences in the 3’UTRs of <span class="html-italic">Col4a5</span> in multiple species. (<b>B</b>) Schematic diagram of complementary sequence sites and primer sites. (<b>C</b>) The effect of miR-29a or miR-29b mimics on the relative luciferase activity of the psi-1-wt-4a5 vector, psi-1-m-c1-4a5 vector, psi-1-m-c2-4a5 vector, and the psi-1-m-c1&amp;c2-4a5 vector was examined. The data are shown as the mean ± SEM (<span class="html-italic">n</span> = 3). **, <span class="html-italic">p</span> &lt; 0.01. (<b>D</b>) The effect of miR-29a or miR-29b mimics on the relative luciferase activity of the psi-2-wt-4a5 vector and psi-2-m-p1-4a5 vector was examined. The data are shown as the mean ± SEM (<span class="html-italic">n</span> = 3). **, <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 6
<p>MiR-29 directly regulates the 3’UTR of <span class="html-italic">Col4a6</span>. (<b>A</b>) Phylogenetic tree constructed from the 3’UTRs of <span class="html-italic">Col4a6</span> in multiple species. Analysis of miR-29 binding site and flanking sequences in the 3’UTRs of <span class="html-italic">Col4a6</span> in multiple species. (<b>B</b>) Schematic diagram of complementary sequence site and primer sites. (<b>C</b>) The effect of miR-29a or miR-29b mimics on the relative luciferase activity of the psi-wt-4a6 vector and the psi-m-c1-4a6 vector was examined. The data are shown as the mean ± SEM (<span class="html-italic">n</span> = 3). **, <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
23 pages, 7651 KiB  
Article
Synbiotic Intervention Ameliorates Oxidative Stress and Gut Permeability in an In Vitro and In Vivo Model of Ethanol-Induced Intestinal Dysbiosis
by Dhara Patel, Chirayu Desai, Deepmala Singh, Virupakshi Soppina, Kirti Parwani, Farhin Patel and Palash Mandal
Biomedicines 2022, 10(12), 3285; https://doi.org/10.3390/biomedicines10123285 - 19 Dec 2022
Cited by 8 | Viewed by 2802
Abstract
Alcoholic liver disease (ALD) alters gut microbiota and tight junctions, causing bacterial components to enter the portal vein and induce oxidative stress-induced inflammation in the liver. Only corticosteroids and liver transplants are treatment options for severe alcoholic hepatitis. ALD’s pathophysiology is unknown. However, [...] Read more.
Alcoholic liver disease (ALD) alters gut microbiota and tight junctions, causing bacterial components to enter the portal vein and induce oxidative stress-induced inflammation in the liver. Only corticosteroids and liver transplants are treatment options for severe alcoholic hepatitis. ALD’s pathophysiology is unknown. However, acetaldehyde’s toxic effects cause oxidative stress and intestinal permeability. This study investigates the influence of a synbiotic (a combination of aged garlic extract (AGE) and Lactobacillus rhamnosus MTCC1423) on colonic oxidative stress and inflammation in ALD male Wistar rats and Caco2 cells. MDA measurement by HPLC in CaCo2 cells, blood serum, and colon tissue demonstrated that synbiotic treatment in the ALD model reduces oxidative stress. Further, fecal high-throughput 16S rRNA gene sequencing revealed the microbiome’s shift towards Firmicutes in the synbiotic group compared to ethanol. In addition, DCFDA labeling and H/E staining demonstrate that the synbiotic is beneficial in inhibiting the development of ALD. In the colon, the synbiotic reduces the activation of CYP2E1 and the inflammatory markers TNF-a and IL-6 while elevating the mRNA expression of ZO-1, occludin, and IL-10. Synbiotics colonize Lactobacillus to restore barrier function and microbiota and reduce colon oxidative stress. Thus, a synbiotic combination can be used in ALD treatment. Full article
(This article belongs to the Special Issue Gut Dysbiosis: Molecular Mechanisms and Therapies 2.0)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The appearance of Caco-2 cells is enhanced by synbiotic preventive therapy: Caco-2 cells were given the following dosage: (<b>A</b>) control, (<b>B</b>) ALD model: 100 mM ethanol, (<b>C</b>) synbiotic (100 mM + 10 g/mL AGE+ 10<sup>9</sup> CFU/mL cell-free supernatant) and 100 mM ethanol and restored the morphology of cells. All pictures were captured at an ×100.</p> Full article ">Figure 2
<p>The effects of AGE and synbiotic on Caco-2 cells treated with ethanol. Tight junctions (<b>A</b>) ZO-1 and (<b>B</b>) occludin mRNA expression, as well as presentation of markers of inflammation (<b>C</b>) TNF-a, (<b>D</b>) CYP2E1, (<b>E</b>) IL-6, and anti-inflammatory indicator (<b>F</b>) IL-10, are all reported. The statistical analysis was calculated as mentioned in <a href="#sec2dot11-biomedicines-10-03285" class="html-sec">Section 2.11</a>. Mean −/+ SD was determined for N = 6 replicates. For statistical analysis, GraphPad Prism 7 and one-way ANOVA in conjunction with the Tukey post hoc test were calculated. When associated with the ethanol group, significance (<span class="html-italic">p</span>-value) is defined as ** <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 2 Cont.
<p>The effects of AGE and synbiotic on Caco-2 cells treated with ethanol. Tight junctions (<b>A</b>) ZO-1 and (<b>B</b>) occludin mRNA expression, as well as presentation of markers of inflammation (<b>C</b>) TNF-a, (<b>D</b>) CYP2E1, (<b>E</b>) IL-6, and anti-inflammatory indicator (<b>F</b>) IL-10, are all reported. The statistical analysis was calculated as mentioned in <a href="#sec2dot11-biomedicines-10-03285" class="html-sec">Section 2.11</a>. Mean −/+ SD was determined for N = 6 replicates. For statistical analysis, GraphPad Prism 7 and one-way ANOVA in conjunction with the Tukey post hoc test were calculated. When associated with the ethanol group, significance (<span class="html-italic">p</span>-value) is defined as ** <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 3
<p>TEER assessment of Caco-2 integrity: TEER investigation of the impact of probiotics and AGE individually or as synbiotic on the functionality of the intestinal barrier produced by ethanol in monolayers of Caco-2 cells. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>. Mean −/+ SD was determined for N = 6 replicates. For statistical analysis, GraphPad Prism 7 and one-way ANOVA in conjunction with the Tukey post hoc test were calculated. When associated with the ethanol group, significance (<span class="html-italic">p</span>-value) is defined as *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>Synbiotic (AGE + <span class="html-italic">L. rhamnosus</span>) affects gut dysbiosis caused by ethanol dosage. (<b>A</b>) Body weight and (<b>B</b>) relative weight and length proportion of the colon are represented above. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>. Mean −/+ SD was determined for N = 6 replicates. For statistical analysis, GraphPad Prism 7 and one-way ANOVA in conjunction with the Tukey post hoc test were calculated. When associated with the ethanol group, significance (<span class="html-italic">p</span>-value) is defined as *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 5
<p>The influence of the synbiotic on the reduction of ethanol-mediated inflammation in the colonic epithelium as assessed by histopathological changes. The colon of a rat in its raw state (100×). These are the microscopic images: (<b>A</b>) control rats (<b>B</b>) rats fed 100 mM ethanol in increasing amounts (<b>C</b>) rats given ethanol and synbiotic (10<sup>9</sup> CFU/day <span class="html-italic">L. rhamnosus</span> and 200 mg/kg AGE).</p> Full article ">Figure 6
<p>Confocal images of lipogenesis staining by Oil red O as standard and C2 stain is the novel lipid stain (<b>A</b>) control rats (<b>B</b>) rats exposed to 100 ethanol in increasing amounts (<b>C</b>) rats administrated with ethanol and synbiotic (10<sup>9</sup> CFU/day <span class="html-italic">L. rhamnosus</span> MTCC1423 and 200 mg/kg AGE). The staining of lipid droplets (ethanol group), disruption of the colon epithelial layer in the presence of ethanol, and regaining of the epithelial barriers in the company of the two treatments (AGE and synbiotic) are represented by arrows.</p> Full article ">Figure 7
<p>Synbiotic (AGE + <span class="html-italic">L. rhamnosus</span> MTCC 1423) mRNA expression on ethanol-induced gut dysbiosis. Expression of (<b>A</b>) ZO-1 and (<b>B</b>) occludin, (<b>C</b>) anti-inflammatory biomarker HO-1 as well as inflammatory biomarkers (<b>D</b>) CYP2E1, (<b>E</b>) TNF-a, and (<b>F</b>) IL-6. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>. Mean −/+ SD was determined for N = 6 replicates. For statistical analysis, GraphPad Prism 7 and one-way ANOVA in conjunction with the Tukey post hoc test were calculated. When associated with the ethanol group, significance (<span class="html-italic">p</span>-value) is defined as * <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.</p> Full article ">Figure 7 Cont.
<p>Synbiotic (AGE + <span class="html-italic">L. rhamnosus</span> MTCC 1423) mRNA expression on ethanol-induced gut dysbiosis. Expression of (<b>A</b>) ZO-1 and (<b>B</b>) occludin, (<b>C</b>) anti-inflammatory biomarker HO-1 as well as inflammatory biomarkers (<b>D</b>) CYP2E1, (<b>E</b>) TNF-a, and (<b>F</b>) IL-6. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>. Mean −/+ SD was determined for N = 6 replicates. For statistical analysis, GraphPad Prism 7 and one-way ANOVA in conjunction with the Tukey post hoc test were calculated. When associated with the ethanol group, significance (<span class="html-italic">p</span>-value) is defined as * <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.</p> Full article ">Figure 8
<p>(<b>A</b>) Synbiotic (AGE + <span class="html-italic">Lactobacillus rhamnosus</span>) affects ethanol-induced intestinal dysbiosis when used alone or in combination. (<b>B</b>) Fluorescence staining and spectroscopy at 485/525 nm were conducted following carboxy-H2-DCFDA incubation. The scale of the images is 4.0 μm. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 8 Cont.
<p>(<b>A</b>) Synbiotic (AGE + <span class="html-italic">Lactobacillus rhamnosus</span>) affects ethanol-induced intestinal dysbiosis when used alone or in combination. (<b>B</b>) Fluorescence staining and spectroscopy at 485/525 nm were conducted following carboxy-H2-DCFDA incubation. The scale of the images is 4.0 μm. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 9
<p>On Caco-2 cells, a graphical depiction of the synbiotic (AGE + <span class="html-italic">L. rhamnosus</span>) analysis of MDA concentration using the HPLC method. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 10
<p>The HPLC technique determined the concentration of MDA in rat serum samples. The effect of the synbiotic (AGE + <span class="html-italic">Lactobacillus rhamnosus</span>) on gut dysbiosis is investigated in the presence of ethanol. Results reveal that synbiotic preventive therapy is more beneficial in treating ALD. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 11
<p>HPLC analysis of MDA concentration of rat colon tissue in synbiotic (AGE + <span class="html-italic">L. rhamnosus</span>) preventive treatment has a protective effect on rat colon tissue by lowering MDA concentration even in ethanol. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>. Mean −/+ SD was determined for N = 6 replicates. For statistical analysis, GraphPad Prism 7 and one-way ANOVA in conjunction with the Tukey post hoc test were calculated. When associated with the ethanol group, significance (<span class="html-italic">p</span>-value) is defined as ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 12
<p>The mRNA expression of (<b>A</b>) IL-10, (<b>B</b>) AMPK, and (<b>C</b>) SREBP-1c in Caco2 cells is illustrated here. The anti-inflammatory markers IL-10 and AMPK were considerably increased after synbiotic (AGE + <span class="html-italic">L. rhamnosus</span>) treatment, whereas the inflammatory marker SREBP-1c was decreased. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>. Mean −/+ SD was determined for N = 6 replicates. For statistical analysis, GraphPad Prism 7 and one-way ANOVA in conjunction with the Tukey post hoc test were calculated. When associated with the ethanol group, significance (<span class="html-italic">p</span>-value) is defined as ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 13
<p>The microbiome of the acute alcoholic male rat model using control, ethanol, and ethanol-and-synbiotic (ES). Taxonomic profiles. Comparative representation of (<b>A</b>) Alpha Diversity: Red: Control; Green: Ethanol-and-synbiotic; Purple: Ethanol and (<b>B</b>) Beta Diversity, (<b>C</b>) phylum level, (<b>D</b>) genus level of selected genera among the groups, normalized by the control group. Applying a two-way ANOVA multiple comparison test between the mean of N = 3 to each group, significance is defined as a <span class="html-italic">p</span>-value &lt; 0.05, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 13 Cont.
<p>The microbiome of the acute alcoholic male rat model using control, ethanol, and ethanol-and-synbiotic (ES). Taxonomic profiles. Comparative representation of (<b>A</b>) Alpha Diversity: Red: Control; Green: Ethanol-and-synbiotic; Purple: Ethanol and (<b>B</b>) Beta Diversity, (<b>C</b>) phylum level, (<b>D</b>) genus level of selected genera among the groups, normalized by the control group. Applying a two-way ANOVA multiple comparison test between the mean of N = 3 to each group, significance is defined as a <span class="html-italic">p</span>-value &lt; 0.05, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 13 Cont.
<p>The microbiome of the acute alcoholic male rat model using control, ethanol, and ethanol-and-synbiotic (ES). Taxonomic profiles. Comparative representation of (<b>A</b>) Alpha Diversity: Red: Control; Green: Ethanol-and-synbiotic; Purple: Ethanol and (<b>B</b>) Beta Diversity, (<b>C</b>) phylum level, (<b>D</b>) genus level of selected genera among the groups, normalized by the control group. Applying a two-way ANOVA multiple comparison test between the mean of N = 3 to each group, significance is defined as a <span class="html-italic">p</span>-value &lt; 0.05, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 14
<p>(<b>A</b>) Oil Red O-stained Caco-2 cells. Compared to the control group, the accumulation of lipids in Caco-2 cells increased in the ethanol group but was reduced significantly by synbiotic treatment at 100X. (<b>B</b>) Graphical representation of the Oil red O absorbance. The statistical analysis was calculated as mentioned in <a href="#sec2dot10-biomedicines-10-03285" class="html-sec">Section 2.10</a>, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">
18 pages, 2321 KiB  
Article
Ivermectin Affects Neutrophil-Induced Inflammation through Inhibition of Hydroxylysine but Stimulation of Cathepsin G and Phenylalanine Secretion
by Svetlana I. Galkina, Ekaterina A. Golenkina, Marina V. Serebryakova, Natalia V. Fedorova, Alexander L. Ksenofontov, Vladimir I. Stadnichuk and Galina F. Sud’ina
Biomedicines 2022, 10(12), 3284; https://doi.org/10.3390/biomedicines10123284 - 19 Dec 2022
Cited by 3 | Viewed by 4713
Abstract
The invasion and integrin-dependent adhesion of neutrophils to lung tissues and their secretion lead to the development of pneumonia in various pulmonary pathologies, including acute respiratory distress syndrome in coronavirus disease. We studied the effect of ivermectin, a possible therapeutic agent for inflammation [...] Read more.
The invasion and integrin-dependent adhesion of neutrophils to lung tissues and their secretion lead to the development of pneumonia in various pulmonary pathologies, including acute respiratory distress syndrome in coronavirus disease. We studied the effect of ivermectin, a possible therapeutic agent for inflammation and cancer, on integrin-dependent neutrophil adhesion to fibronectin and the concomitant secretion. Ivermectin did not affect the attachment of neutrophils to the substrate and the reactive oxygen species production but sharply inhibited the adhesion-induced release of hydroxylysine and stimulated the release of phenylalanine and cathepsin G. Hydroxylysine is a product of lysyl hydroxylase, which is overexpressed in tumor cells with an increased ability to invade and metastasize. The inhibition of hydroxylysine release by ivermectin, by analogy, may indicate the suppression of neutrophil invasion into tissue. The increase in the release of phenylalanine in our experiments coincided with the secretion of cathepsin G, which indicates the possible role of this enzyme in the cleavage of phenylalanine. What is the substrate in such a reaction is unknown. We demonstrated that exogenously added angiotensin II (1–8) can serve as a substrate for phenylalanine cleavage. Mass spectrometry revealed the formation of angiotensin II (1–7) in the secretion of neutrophils, which attached to fibronectin in the presence of ivermectin and exogenous angiotensin II (1–8), indicating a possible involvement of ivermectin in the inactivation of angiotensin II. Full article
(This article belongs to the Collection Feature Papers in Immunology and Immunotherapy)
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Figure 1

Figure 1
<p>Attachment of neutrophils to fibrinogen- or fibronectin-coated substrata ((<b>A</b>,<b>B</b>), respectively). Neutrophils (2 × 10<sup>6</sup> cells/mL) were incubated for 25 min at 37 °C, 5% CO<sub>2</sub> in control conditions or in the presence of ivermectin. Shown are the percentages (means ± SEM, <span class="html-italic">n</span> = 3) of firmly attached cells from the total number of cells in the sample.</p> Full article ">Figure 2
<p>Scanning electron images of human neutrophils that were adhered to fibronectin-coated substrates for 25 min under control conditions (<b>A</b>) or in the presence of 50 µM ivermectin (<b>B</b>); in the presence of 50 nM TNF alone (<b>C</b>) or in combination with 50 µM ivermectin (<b>D</b>); in the presence of 1 µg/mL LPS alone (<b>E</b>) or in combination with 50 µM ivermectin (<b>F</b>); in the presence 200 µM L-NAME alone (<b>G</b>) or in combination with 50 µM ivermectin (<b>H</b>). The figures show typical images observed in three independent experiments.</p> Full article ">Figure 3
<p>Area occupied by neutrophils that were adhered for 25 min to a fibronectin-coated substrate for 25 min under control conditions or in the presence of 50 µM ivermectin alone or in combination with 50 nM TNF, 1 µg/mL LPS, or 200 µM L-NAME. **** <span class="html-italic">p</span> &lt; 0.0001, as shown one-way ANOVA, Tukey’s multiple comparison test.</p> Full article ">Figure 4
<p>Effect of ivermectin on the formation of ROS during adhesion of neutrophils to fibronectin. H<sub>2</sub>DCF-DA-stained neutrophils were incubated in fibronectin-coated 96-well plates for 60 min at 37 °C in 5% CO<sub>2</sub> under control conditions and in the presence of additives. (<b>A</b>) Phase-contrast and fluorescence images of H<sub>2</sub>DCF-DA-stained neutrophils that were attached to fibronectin for 25 min under control conditions or in the presence of ivermectin or PMA. (<b>B</b>) Representative DCF fluorescence kinetic curves for control, ivermectin- and PMA-treated neutrophils. Stimuli were added at the moment marked with the “syringe” icon. (<b>C</b>) DCF fluorescence intensity measured after 30 min incubation under control conditions and in the presence of ivermectin or vehicle (DMSO, 5 μL/mL). Values are means ± SEM from three independent experiments.</p> Full article ">Figure 5
<p>Effect of ivermectin on the free amino acid secretion by neutrophils during adhesion to fibronectin. Neutrophils were attached to fibronectin-coated substrates for 25 min under control conditions or in the presence of 50 μM ivermectin. The number of amino acids is presented as a percentage of the total content of free amino acids found (mean value ± standard error). Amino acid profiles were obtained by summing up the results of three independent experiments. ****—Significant differences compared to the value for the same amino acid under control conditions (<span class="html-italic">p</span> &lt; 0.0001), as indicated by two-way ANOVA with Tukey’s multiple comparison test.</p> Full article ">Figure 6
<p>SDS-PAGE separation of proteins secreted by neutrophils upon adhesion to fibronectin. Human neutrophils were attached to fibronectin-coated substrates for 25 min incubation under control conditions or in the presence of 50 μM ivermectin. The extracellular medium was collected and the proteins were extracted. After concentration, the proteins were subjected to separation in 15% SDS-PAGE under non-reduced conditions. The gels were stained with Coomassie brilliant blue. The figures show typical protein profiles observed in three independent experiments.</p> Full article ">Figure 7
<p>MALDI-TOF MS analysis of peptides in the extracellular environment of neutrophils after adhesion. Neutrophils were attached to fibronectin for 90 min in the presence of 50 μM angiotensin II (1–8) alone ((<b>A</b>): control) or in combination with 50 μM ivermectin ((<b>B</b>): ivermectin) or 10 μg/mL cytochalasin D ((<b>C</b>): cytochalasin D). Samples of the extracellular medium were taken and after removal of non-attached neutrophils by centrifugation, they were incubated for 120 min at 3 °C. Aliquots of the extracellular medium were taken and subjected to mass spectrometry.</p> Full article ">
12 pages, 2430 KiB  
Article
EMT and Tumor Turning Point Analysis in 3D Spheroid Culture of HNSCC and Mesenchymal Stem Cells
by Sabine Brylka and Florian Böhrnsen
Biomedicines 2022, 10(12), 3283; https://doi.org/10.3390/biomedicines10123283 - 19 Dec 2022
Cited by 3 | Viewed by 2130
Abstract
The prognosis, metastasis, and behavior of head and neck squamous cancer cells are influenced by numerous factors concerning the tumor microenvironment, intercellular communication, and epithelial-to-mesenchymal transition (EMT). The aim of this study was to examine the codependent interaction of the mesenchymal stroma with [...] Read more.
The prognosis, metastasis, and behavior of head and neck squamous cancer cells are influenced by numerous factors concerning the tumor microenvironment, intercellular communication, and epithelial-to-mesenchymal transition (EMT). The aim of this study was to examine the codependent interaction of the mesenchymal stroma with head and neck squamous cell carcinoma (HNSCC) in a 3D spheroid structure. To simulate stroma-rich and -poor 3D tumor microenvironments, cells of the established cell SCC-040 were cultured with human mesenchymal stromal cells (MSCs), forming 3D stroma-tumor spheroids (STSs). STSs were compared to uniform spheroids of SCC-040 and MSC, respectively. The expressions of CD24, β-catenin, SNAI2, and ZEB2 were analyzed via RT-qPCR. The immunohistochemical expressions of E-cadherin, connexin 43, vimentin, and emmprin were analyzed, and protein expression pathways as well as Akt signaling were assessed via protein analysis. A promotive effect on the expressions of EMT markers ZEB2 (p = 0.0099), SNAI2 (p = 0.0352), and β-catenin (p = 0.0031) was demonstrated in STSs, as was the expression of Akt pathway proteins mTOR (p = 0.007), Erk1/2 (p = 0.0045), and p70 S6 Kinase (p = 0.0016). Our study demonstrated a change in genetic expression patterns early on in tumor development, indicating a tumor turning point. Full article
(This article belongs to the Section Biomedical Engineering and Materials)
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<p>Immunohistochemical staining of vimentin in MSCs, Co1/1, Co3/1, Co5/1, and SCCs; magnification 40×. **** = <span class="html-italic">p</span> ≤ 0.0001, ** = <span class="html-italic">p</span> ≤ 0.01.</p> Full article ">Figure 2
<p>Immunohistochemical staining of connexin 43 in MSCs, Co1/1, Co3/1, Co5/1, and SCCs; magnification 40×. **** = <span class="html-italic">p</span> ≤ 0.0001, * = <span class="html-italic">p</span> ≤ 0.05.</p> Full article ">Figure 3
<p>Immunohistochemical staining of E-cadherin in MSCs, Co 1/1, Co3/1, Co5/1, and SCCs; magnification 40×. **** = <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">Figure 4
<p>Immunohistochemical staining of emmprin in MSCs, Co1/1, Co3/1, Co5/1, and SCCs; magnification 40×. ** = <span class="html-italic">p</span> ≤ 0.01.</p> Full article ">Figure 5
<p>RT-qPCR analysis of β-catenin, SNAI2, ZEB2, and CD24 in MSCs, Co1/1, Co3/1, Co5/1, and SCCs (*** = <span class="html-italic">p</span> ≤ 0.01, ** = <span class="html-italic">p</span> ≤ 0.01, * = <span class="html-italic">p</span> ≤ 0.05).</p> Full article ">Figure 6
<p>Protein level analysis of S6 ribosomal protein, mTOR, p70 S6 kinase, and Erk1/2 in MSCs, Co1/1, Co3/1, Co5/1, and SCCs (** = <span class="html-italic">p</span> ≤ 0.01, * = <span class="html-italic">p</span> ≤ 0.05).</p> Full article ">
14 pages, 2434 KiB  
Article
Bioengineered Ciprofloxacin-Loaded Chitosan Nanoparticles for the Treatment of Bovine Mastitis
by Preeti Yadav, Awadh Bihari Yadav, Preksha Gaur, Vartika Mishra, Zul-I Huma, Neelesh Sharma and Young-Ok Son
Biomedicines 2022, 10(12), 3282; https://doi.org/10.3390/biomedicines10123282 - 19 Dec 2022
Cited by 14 | Viewed by 2654
Abstract
Mastitis is the most devastating economic disease in dairy cattle. Mastitis in dairy cattle frequently occurs during the dry period or during early lactation. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus)are the main causative agents of mastitis [...] Read more.
Mastitis is the most devastating economic disease in dairy cattle. Mastitis in dairy cattle frequently occurs during the dry period or during early lactation. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus)are the main causative agents of mastitis in India. S. aureus can form microabscesses in the udder and develop a subclinical form of mastitis. This bacterial property hinders an effective cure during the lactation period. Antimicrobials used for treatments have a short half-life at the site of action because of frequent milking; thereforethey are unable to maintain the desired drug concentration for effective clearance of bacteria. We demonstrated the potential of ciprofloxacin-encapsulated nanocarriersthat can improve the availability of drugs and provide an effective means for mastitis treatment. These drug-loaded nanoparticles show low toxicity and slow clearance from the site of action. Antimicrobial activity against clinical strains of E. coli and S. aureus showed that the zone of inhibition depended on the dose (0.5 mg to 2 mg/mL nanoparticle solution from 11.6 to 14.5 mm and 15 to 18 mm). These nanoparticles showed good antimicrobial activity in broth culture and agar diffusion assay against bacteria. Full article
(This article belongs to the Topic Microfluidics Applied in Nanomedicine and Pharmaceutics)
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Graphical abstract

Graphical abstract
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<p>Surface morphology of CPX-loaded chitosan nanoparticles after lyophilization with trehalose cryoprotectant using scanning electron microscopy (SEM) image analysis.</p> Full article ">Figure 2
<p>In vitro drug release study of ciprofloxacin hydrochloride-loaded chitosan nanoparticles in (<b>A</b>) PBS buffer at pH 5.2 and (<b>B</b>) PBS buffer at pH 7.4.</p> Full article ">Figure 3
<p>Cellular uptake of FITC dye-loaded chitosan nanoparticles (<b>A</b>) histogram of cells and (<b>B</b>) percentage of cell uptake of dye-loaded nanoparticles using flow cytometry analysis (<span class="html-italic">n</span> = 3, ±SD).</p> Full article ">Figure 4
<p>Antimicrobial activity of CPX-loaded nanoparticles against clinical isolate <span class="html-italic">S. aureus</span> and <span class="html-italic">E. coli</span>. (<b>A</b>) agar diffusion assay, (<b>B</b>) activity of equal amount of CPX and CPX-loaded nanoparticles, and (<b>C</b>) broth culture method at different doses of nanoparticles (<span class="html-italic">n</span> = 3, ±SD).</p> Full article ">Figure 5
<p>Toxicity assay after cells were exposed to CPX-loaded nanoparticles. (<b>A</b>) The trypan blue dye exclusion assay in 3T3 cells exposed todifferent doses of CPX, CPX-NPs, or blank NPs (1, 10, 100, and 1000 μg/mL); (<b>B</b>) hemolysis assay to assess membrane damage by CPX-loaded NPs; (<b>C</b>) neutral red dye uptake assay, cells exposed tociprofloxacin (CPX), chitosan blank nanoparticles (BNPs), and CPX-loaded chitosan nanoparticles (CPX-CS NPs) at different concentrations. Triton X-100 0.1% used as a positive control and normal saline (NS) used as negative control.</p> Full article ">Figure 5 Cont.
<p>Toxicity assay after cells were exposed to CPX-loaded nanoparticles. (<b>A</b>) The trypan blue dye exclusion assay in 3T3 cells exposed todifferent doses of CPX, CPX-NPs, or blank NPs (1, 10, 100, and 1000 μg/mL); (<b>B</b>) hemolysis assay to assess membrane damage by CPX-loaded NPs; (<b>C</b>) neutral red dye uptake assay, cells exposed tociprofloxacin (CPX), chitosan blank nanoparticles (BNPs), and CPX-loaded chitosan nanoparticles (CPX-CS NPs) at different concentrations. Triton X-100 0.1% used as a positive control and normal saline (NS) used as negative control.</p> Full article ">Figure 6
<p>CPX-loaded chitosan nanoparticle exposure to the cells and membrane damage of cells assessed by lipid peroxidation assay (<span class="html-italic">n</span> = 3, ±SD).</p> Full article ">
16 pages, 1013 KiB  
Review
Seventy Years of Treating Delusional Disorder with Antipsychotics: A Historical Perspective
by Alexandre González-Rodríguez, José A. Monreal, Mentxu Natividad and Mary V. Seeman
Biomedicines 2022, 10(12), 3281; https://doi.org/10.3390/biomedicines10123281 - 18 Dec 2022
Cited by 10 | Viewed by 10189
Abstract
For many decades, delusional disorder (DD) has been considered a treatment-resistant disorder, with antipsychotics acknowledged as the best, though imperfect, treatment. It is possible that the discovery of the right drug could turn treatment resistance into treatment response. The goal of this narrative [...] Read more.
For many decades, delusional disorder (DD) has been considered a treatment-resistant disorder, with antipsychotics acknowledged as the best, though imperfect, treatment. It is possible that the discovery of the right drug could turn treatment resistance into treatment response. The goal of this narrative review is to provide a historical perspective of the treatment of DD since the introduction of antipsychotics 70 years ago. The following search terms were used to scan the literature: antipsychotics AND “delusional disorder”. Findings were that therapy for DD symptoms has changed over time. Initial reports suggested that the drug of choice was the antipsychotic pimozide, and that this drug was especially effective for the somatic subtype of DD. Subsequent studies demonstrated that other antipsychotics, for instance, risperidone and olanzapine, were also highly effective. Treatment response may vary according to the presence or absence of specific symptoms, such as cognitive defect and depression. Clozapine, partial D2 agonists, and long-acting injectable drugs may be more effective than other drugs, but the evidence is not yet in. Because of the absence of robust evidence, treatment guidelines for the optimal management of DD are not yet available. Full article
(This article belongs to the Special Issue Antipsychotics: 70 Years)
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<p>Flow diagram of included studies.</p> Full article ">Figure 2
<p>Molecular structure of the main antipsychotics used to treat patients with delusional disorders.</p> Full article ">
16 pages, 1089 KiB  
Article
Effectiveness and Safety Profiles of Biological Therapies in Inflammatory Bowel Disease: Real Life Data from an Active Pharmacovigilance Project
by Maria Antonietta Barbieri, Anna Viola, Giuseppe Cicala, Edoardo Spina and Walter Fries
Biomedicines 2022, 10(12), 3280; https://doi.org/10.3390/biomedicines10123280 - 18 Dec 2022
Cited by 7 | Viewed by 3209
Abstract
Post-marketing surveillance is essential to evaluate the risk/benefit profile of drugs; however, pharmacovigilance studies comparing persistence and safety of biologic therapies in patients with inflammatory bowel disease (IBD) are scant. The aim of this study was to prospectively investigate persistence together with safety [...] Read more.
Post-marketing surveillance is essential to evaluate the risk/benefit profile of drugs; however, pharmacovigilance studies comparing persistence and safety of biologic therapies in patients with inflammatory bowel disease (IBD) are scant. The aim of this study was to prospectively investigate persistence together with safety profiles of biologics in a cohort of patients diagnosed with Crohn’s Disease (CD) or ulcerative colitis (UC) followed by the IBD unit of Messina and treated with infliximab (IFX), adalimumab (ADA), golimumab (GOL), vedolizumab (VED), and ustekinumab (UST) from 2017 through 2021. Descriptive and treatment persistence analyses with predictors for discontinuation and occurrence of adverse drug reactions (ADRs) were performed. A total of 675 IBD patients were enrolled. A higher persistence rate was noted for UST and ADA in the first year (83.8% and 83.1%, respectively) and for IFX in the fifth year of treatment (58.1%). GOL, VED, and UST—all used as second/third-line therapies—seemed to have a higher risk of non-persistence than IFX (in order HR: 2.19; CI 95%: 1.33–3.61, 1.45; 1.04–2.04, 2.25; 1.25–4.07) as well as switchers and those who had at least one ADR (18.1; 13.22–24.68 and 1.55; 1.20–1.99, respectively). The reported ADRs, which were generally mild–moderate, were largely known. However, real-world data should be implemented to further study undetected safety concerns, including risk of malignancy. Full article
(This article belongs to the Special Issue Novel Therapeutic Approaches in Inflammatory Bowel Diseases 3.0)
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<p>Kaplan–Meier curve for the persistence of biologics in patients with IBD. Abbreviations: ADA = adalimumab; CI = confidence interval; GOL = golimumab; IFX = infliximab; VED = vedolizumab; UST = ustekinumab.</p> Full article ">Figure 2
<p>Predictors of non-persistence of biologics in patients with IBD. Abbreviations: 5-ASA = mesalazine; ADA = adalimumab; ADRs = adverse drug reactions; CD = Crohn’s disease; GOL = golimumab; IFX = infliximab; VED = vedolizumab; UC = ulcerative colitis; UST = ustekinumab; yrs = years.</p> Full article ">
17 pages, 2317 KiB  
Article
Transposons Acting as Competitive Endogenous RNAs: In-Silico Evidence from Datasets Characterised by L1 Overexpression
by Mauro Esposito, Nicolò Gualandi, Giovanni Spirito, Federico Ansaloni, Stefano Gustincich and Remo Sanges
Biomedicines 2022, 10(12), 3279; https://doi.org/10.3390/biomedicines10123279 - 17 Dec 2022
Cited by 3 | Viewed by 2199
Abstract
LINE L1 are transposable elements that can replicate within the genome by passing through RNA intermediates. The vast majority of these element copies in the human genome are inactive and just between 100 and 150 copies are still able to mobilize. During evolution, [...] Read more.
LINE L1 are transposable elements that can replicate within the genome by passing through RNA intermediates. The vast majority of these element copies in the human genome are inactive and just between 100 and 150 copies are still able to mobilize. During evolution, they could have been positively selected for beneficial cellular functions. Nonetheless, L1 deregulation can be detrimental to the cell, causing diseases such as cancer. The activity of miRNAs represents a fundamental mechanism for controlling transcript levels in somatic cells. These are a class of small non-coding RNAs that cause degradation or translational inhibition of their target transcripts. Beyond this, competitive endogenous RNAs (ceRNAs), mostly made by circular and non-coding RNAs, have been seen to compete for the binding of the same set of miRNAs targeting protein coding genes. In this study, we have investigated whether autonomously transcribed L1s may act as ceRNAs by analyzing public dataset in-silico. We observed that genes sharing miRNA target sites with L1 have a tendency to be upregulated when L1 are overexpressed, suggesting the possibility that L1 might act as ceRNAs. This finding will help in the interpretation of transcriptomic responses in contexts characterized by the specific activation of transposons. Full article
(This article belongs to the Special Issue microRNAs in Health and Disease)
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<p>The KO of <span class="html-italic">DNMT1</span> gene leads to autonomous L1 transcription. (<b>A</b>). Top-10 deregulated TE families in <span class="html-italic">DNMT1</span> model. L1 is the most upregulated TE family with 1660 elements. (<b>B</b>). Rationale of our method for detecting autonomous transcription of L1 elements. Autonomous transcription of L1s will produce a higher amount of <span class="html-italic">Inside</span> fragments with both reads mapped inside the L1 consensus resulting in a higher <span class="html-italic">Inside/Outside</span> ratio. (<b>C</b>). Analysis to detect autonomous transcription of L1 elements. Upon the KO of <span class="html-italic">DNMT,</span> the upregulation of L1s derives by autonomous transcription of elements. (<b>D</b>). Top-10 deregulated TE families in naive T <span class="html-italic">CD4<sup>+</sup></span> cells. L1 is the most upregulated TE family with 10,794 elements. (<b>E</b>). Rationale of our method for detecting non-autonomous transcription of L1 elements. Transcription of L1s embedded in other transcriptional units will produce a low amount of Inside fragments. (<b>F</b>). Analysis to detect autonomous transcription of L1 elements. In naive T CD4+ cells, the upregulation of L1s derives by a non-independent transcription of elements. Given that the two distributions are similar the overexpression of L1s is due to their transcription as part of other transcriptional units.</p> Full article ">Figure 2
<p>L1 transcripts might act as ceRNA. (<b>A</b>). Overlap analysis of L1s and protein-coding genes. Regions of upregulated genes are significantly enriched to contain upregulated L1 fragments. We found particularly interesting the strong enrichment in the 3′UTRs. (<b>B</b>). Analysis of miRNA target sites sharing between autonomously transcribed L1s and the 3′UTRs of protein-coding genes in <span class="html-italic">DNMT1</span> model. The upregulated genes share a significantly higher number of miRNA target sites with <span class="html-italic">active</span> L1s, adding support to a possible ceRNA activity of L1 transcripts. (<b>C</b>). Analysis to identify miRNAs sequestered by L1s. Each analyzed miRNA is represented by a point with the X-axis indicating the delta between the proportion of targeted upregulated genes and the total upregulated genes proportion. The Y-axis represents the -log10(FDR) of the proportional statistical test applied. The 117 miRNA in green represent the most probable pool of miRNAs that are undergoing the ceRNA activity from L1s. (<b>D</b>). In this wordcloud are shown the 117 miRNAs whose size font is proportional to the absolute number of experimentally validated target genes upregulated in <span class="html-italic">DNMT1</span> model. The let-7 family is among the top-10 miRNAs with the higher number of connections to these upregulated genes. (<b>E</b>). Correlation analysis between miR-128-1 (Y-axis) and L1 (X-axis) expression levels in Geuvadis dataset. Upon the L1 overexpression, the miR-128-1-5p levels concordantly increases probably as part of defense cellular mechanisms. (<b>F</b>). Correlation analysis between let-7a-1 (Y-axis) and L1 (X-axis) expression levels in Geuvadis dataset. No significant associations were found.</p> Full article ">Figure 3
<p>L1 could act as ceRNA when artificially overexpressed. (<b>A</b>). TE subfamilies significantly deregulated in the ORFeus-OE model. In this experiment, the L1 construct is the most upregulated TE with a log2 fold-change of 10.92. (<b>B</b>). Analysis to detect autonomous transcription of L1. In ORFeus-OE cells, the upregulation of L1 is deriving from an autonomous transcription of the ORFeus element. (<b>C</b>). Analysis of miRNA target sites sharing between artificial L1 construct and the 3′UTRs of protein-coding genes in ORFeus-OE model. The upregulated genes share a significantly high number of miRNA target sites with the L1 construct, reflecting a possible ceRNA activity of the artificial construct transcript.</p> Full article ">Figure 4
<p>L1 ceRNA activity potentially relies on autonomous L1 transcription and Ago2 levels. (<b>A</b>). Top-10 deregulated TE families in <span class="html-italic">ATRX</span> model. Both in iPSC cells and in neurons, L1 family is the most upregulated TE family. (<b>B</b>). Analysis to detect autonomous transcription of L1 elements in <span class="html-italic">ATRX</span> model. Upon the KO of ATRX, the upregulation of L1 elements seems not to derive from an autonomous transcription of elements in neurons. (<b>C</b>). Analysis of miRNA target sites sharing between <span class="html-italic">active</span> L1s and the 3′UTRs of protein-coding genes in <span class="html-italic">ATRX</span> model. Downregulated genes show a higher number of miRNA target sites in common with overexpressed L1s. (<b>D</b>). Comparative analysis of Ago2 expression levels in all analyzed datasets.</p> Full article ">
12 pages, 911 KiB  
Article
Epidemiological, Clinical, and Genomic Profile in Head and Neck Cancer Patients and Their Families
by Thiago Celestino Chulam, Fernanda Bernardi Bertonha, Rolando André Rios Villacis, João Gonçalves Filho, Luiz Paulo Kowalski and Silvia Regina Rogatto
Biomedicines 2022, 10(12), 3278; https://doi.org/10.3390/biomedicines10123278 - 17 Dec 2022
Cited by 2 | Viewed by 2177
Abstract
Inherited cancer predisposition genes are described as risk factors in head and neck cancer (HNC) families. To explore the clinical and epidemiological data and their association with a family history of cancer, we recruited 74 patients and 164 relatives affected by cancer. The [...] Read more.
Inherited cancer predisposition genes are described as risk factors in head and neck cancer (HNC) families. To explore the clinical and epidemiological data and their association with a family history of cancer, we recruited 74 patients and 164 relatives affected by cancer. The germline copy number alterations were evaluated in 18 patients using array comparative genomic hybridization. Two or more first-degree relatives with HNC, tobacco-associated tumor sites (lung, esophagus, and pancreas), or other related tumors (breast, colon, kidney, bladder, cervix, stomach carcinomas, and melanoma) were reported in 74 families. Ten index patients had no exposure to any known risk factors. Family members presented tumors of 19 topographies (30 head and neck, 26 breast, 21 colon). In first-degree relatives, siblings were frequently affected by cancer (n = 58, 13 had HNC). Breast cancer (n = 21), HNC (n = 19), and uterine carcinoma (n = 15) were commonly found in first-degree relatives and HNC in second-degree relatives (n = 11). Nineteen germline genomic imbalances were detected in 13 patients; three presented gains of WRD genes. The number of HNC patients, the degree of kinship, and the tumor types detected in each relative support the role of heredity in these families. Germline alterations may potentially contribute to cancer development. Full article
(This article belongs to the Special Issue Head and Neck Tumors 2.0)
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<p>(<b>A</b>). Pedigree of one family showing two generations of patients with head and neck cancer (II:4 and III:3) and other tumor types, in relatives of the index patient (III:1). (<b>B</b>). Graphical representation of the gains encompassing the genes of the WDR family.</p> Full article ">
26 pages, 1417 KiB  
Review
Markers of Bronchiolitis Obliterans Syndrome after Lung Transplant: Between Old Knowledge and Future Perspective
by Dalila Cavallaro, Marco Guerrieri, Stefano Cattelan, Gaia Fabbri, Sara Croce, Martina Armati, David Bennett, Antonella Fossi, Luca Voltolini, Luca Luzzi, Alberto Salvicchi, Piero Paladini, Adriano Peris, Miriana d’Alessandro, Paolo Cameli, Elena Bargagli, Tuscany Transplant Group and Laura Bergantini
Biomedicines 2022, 10(12), 3277; https://doi.org/10.3390/biomedicines10123277 - 17 Dec 2022
Cited by 6 | Viewed by 3495
Abstract
Bronchiolitis obliterans syndrome (BOS) is the most common form of CLAD and is characterized by airflow limitation and an obstructive spirometric pattern without high-resolution computed tomography (HRCT) evidence of parenchymal opacities. Computed tomography and microCT analysis show abundant small airway obstruction, starting from [...] Read more.
Bronchiolitis obliterans syndrome (BOS) is the most common form of CLAD and is characterized by airflow limitation and an obstructive spirometric pattern without high-resolution computed tomography (HRCT) evidence of parenchymal opacities. Computed tomography and microCT analysis show abundant small airway obstruction, starting from the fifth generation of airway branching and affecting up to 40–70% of airways. The pathogenesis of BOS remains unclear. It is a multifactorial syndrome that leads to pathological tissue changes and clinical manifestations. Because BOS is associated with the worst long-term survival in LTx patients, many studies are focused on the early identification of BOS. Markers may be useful for diagnosis and for understanding the molecular and immunological mechanisms involved in the onset of BOS. Diagnostic and predictive markers of BOS have also been investigated in various biological materials, such as blood, BAL, lung tissue and extracellular vesicles. The aim of this review was to evaluate the scientific literature on markers of BOS after lung transplant. We performed a systematic review to find all available data on potential prognostic and diagnostic markers of BOS. Full article
(This article belongs to the Section Molecular and Translational Medicine)
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<p>Flowchart of the study.</p> Full article ">Figure 2
<p>The most relevant circulating biomarkers and gene expression.</p> Full article ">Figure 3
<p>The radiological and tissue markers of BOS.</p> Full article ">
32 pages, 5394 KiB  
Review
Amyloid Disassembly: What Can We Learn from Chaperones?
by Zaida L. Almeida and Rui M. M. Brito
Biomedicines 2022, 10(12), 3276; https://doi.org/10.3390/biomedicines10123276 - 17 Dec 2022
Cited by 11 | Viewed by 3243
Abstract
Protein aggregation and subsequent accumulation of insoluble amyloid fibrils with cross-β structure is an intrinsic characteristic of amyloid diseases, i.e., amyloidoses. Amyloid formation involves a series of on-pathway and off-pathway protein aggregation events, leading to mature insoluble fibrils that eventually accumulate in multiple [...] Read more.
Protein aggregation and subsequent accumulation of insoluble amyloid fibrils with cross-β structure is an intrinsic characteristic of amyloid diseases, i.e., amyloidoses. Amyloid formation involves a series of on-pathway and off-pathway protein aggregation events, leading to mature insoluble fibrils that eventually accumulate in multiple tissues. In this cascade of events, soluble oligomeric species are formed, which are among the most cytotoxic molecular entities along the amyloid cascade. The direct or indirect action of these amyloid soluble oligomers and amyloid protofibrils and fibrils in several tissues and organs lead to cell death in some cases and organ disfunction in general. There are dozens of different proteins and peptides causing multiple amyloid pathologies, chief among them Alzheimer’s, Parkinson’s, Huntington’s, and several other neurodegenerative diseases. Amyloid fibril disassembly is among the disease-modifying therapeutic strategies being pursued to overcome amyloid pathologies. The clearance of preformed amyloids and consequently the arresting of the progression of organ deterioration may increase patient survival and quality of life. In this review, we compiled from the literature many examples of chemical and biochemical agents able to disaggregate preformed amyloids, which have been classified as molecular chaperones, chemical chaperones, and pharmacological chaperones. We focused on their mode of action, chemical structure, interactions with the fibrillar structures, morphology and toxicity of the disaggregation products, and the potential use of disaggregation agents as a treatment option in amyloidosis. Full article
(This article belongs to the Special Issue Protein Aggregation: Molecular Mechanisms, Determinants and Therapeutic Approaches)
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<p>Structural polymorphism of amyloid fibrils. (<b>A</b>) 3D morphology models of reconstructed amyloid fibrils with different number of protofilaments: one (black), two (green), three (orange), four (blue), and five (yellow) protofilaments. Adapted from [<a href="#B2-biomedicines-10-03276" class="html-bibr">2</a>]. (<b>B</b>) Cryo-electron microscopy structures of the core regions of amyloid protofilaments of amyloid-β 1-42 (Aβ42) (PDB code: 7Q4B) [<a href="#B3-biomedicines-10-03276" class="html-bibr">3</a>], α-synuclein (PDB code: 6XYO) [<a href="#B4-biomedicines-10-03276" class="html-bibr">4</a>], and transthyretin (TTR) (PDB code: 6SDZ) [<a href="#B5-biomedicines-10-03276" class="html-bibr">5</a>] from patients with sporadic Alzheimer’s disease, sporadic synucleinopathy, and hereditary Val30Met TTR amyloidosis, respectively. The (<b>B</b>) upper panel displays the backbone and side chains of one dimer pair, while the (<b>B</b>) lower panel illustrates the cross-β structure of stacked dimers along the fibril axis. Structures produced with UCSF Chimera [<a href="#B6-biomedicines-10-03276" class="html-bibr">6</a>] using the respective PDB codes.</p> Full article ">Figure 2
<p>Three-dimensional structure representation of L-PGDS. Image produced with UCSF Chimera [<a href="#B6-biomedicines-10-03276" class="html-bibr">6</a>] using the crystallographic structure of L-PGDS wild-type in its apo form (PDB code: 4IMN).</p> Full article ">Figure 3
<p>Three-dimensional structure representation of the native TTR homotetramer. The regions of TTR implicated in the interaction with Aβ are highlighted in blue. Image produced with UCSF Chimera [<a href="#B6-biomedicines-10-03276" class="html-bibr">6</a>] using the crystallographic structure of wild-type TTR in a complex with thyroxine (PDB code: 1ICT).</p> Full article ">Figure 4
<p>Three-dimensional structure representation of CyP40. The N-terminal domain of cyclophilin (residues 1–183) is shown in yellow, whereas the α-helical C-terminal TPR domain (residues 214–362) is shown in blue [<a href="#B66-biomedicines-10-03276" class="html-bibr">66</a>]. Image produced with UCSF Chimera [<a href="#B6-biomedicines-10-03276" class="html-bibr">6</a>] using the crystallographic structure of CyP40 (PDB code: 1IHG).</p> Full article ">Figure 5
<p>Chemical structures of chemical chaperones capable of disaggregating amyloid deposits.</p> Full article ">Figure 6
<p>Chemical structures of tryptophan derivatives with disruptive effects on preformed amyloid structures.</p> Full article ">Figure 7
<p>Chemical structures of anthraquinone derivatives with disruptive effects on preformed amyloid fibrils.</p> Full article ">Figure 8
<p>Chemical structures of benzophenone derivatives with disruptive action on preformed amyloid species.</p> Full article ">Figure 9
<p>Chemical structures of catechin derivatives reported to disassemble amyloid fibrillar structures.</p> Full article ">Figure 10
<p>Chemical structures of tetracycline and anthracycline derivatives able to disassemble preformed amyloid structures.</p> Full article ">Figure 11
<p>Chemical structures of NSAIDs (A) and paracetamol (B) with the ability to disaggregate amyloid fibrils in vitro.</p> Full article ">Figure 12
<p>Chemical structures of porphyrin and phthalocyanine derivatives known to disassemble preformed amyloid fibrils.</p> Full article ">Figure 13
<p>Chemical structures of surfactants capable of disrupting preformed amyloid fibrils. The CMC values shown were determined at 25 °C [<a href="#B200-biomedicines-10-03276" class="html-bibr">200</a>,<a href="#B204-biomedicines-10-03276" class="html-bibr">204</a>,<a href="#B205-biomedicines-10-03276" class="html-bibr">205</a>,<a href="#B206-biomedicines-10-03276" class="html-bibr">206</a>,<a href="#B207-biomedicines-10-03276" class="html-bibr">207</a>,<a href="#B208-biomedicines-10-03276" class="html-bibr">208</a>].</p> Full article ">
11 pages, 536 KiB  
Article
Oxaliplatin-Related Hypersensitivity Reactions: A Single Institution Series and Literature Review
by Francesca Barbin, Michele Ghidini, Alessandra Panichi, Gianluca Tomasello, Claudia Bareggi, Barbara Galassi, Nerina Denaro, Fiorella Ruatta, Carolina Cauchi, Maria Grazia Rossino and Ornella Garrone
Biomedicines 2022, 10(12), 3275; https://doi.org/10.3390/biomedicines10123275 - 17 Dec 2022
Cited by 4 | Viewed by 3728
Abstract
Oxaliplatin-based chemotherapy is extensively used for the treatment of gastrointestinal tumors and other malignancies. Oxaliplatin-related hypersensitivity reactions (HSRs) are common during antitumor treatment. Several studies have been conducted to identify predictive risk factors for oxaliplatin-related HSRs, but findings remain controversial. No definitive approach [...] Read more.
Oxaliplatin-based chemotherapy is extensively used for the treatment of gastrointestinal tumors and other malignancies. Oxaliplatin-related hypersensitivity reactions (HSRs) are common during antitumor treatment. Several studies have been conducted to identify predictive risk factors for oxaliplatin-related HSRs, but findings remain controversial. No definitive approach has been identified to reduce the risk of developing HSRs. The aim of this article is to provide an overview of oxaliplatin-related HSRs, and to report our institution’s experience. With our work, we reviewed available data from the literature and described our case series. A total of 153 patients were treated with oxaliplatin and 17 developed an HSR. On the whole, 70.6% of reactions were Grade 3, mostly with respiratory and cutaneous symptoms. Steroids and antihistamines were administered to reduce hypersensitivity symptoms and prevent further reactions. A stronger premedication and prolonged time of infusion resulted in milder reactions or absence of subsequent reactions. We did not find any clear predictive factor for the development of HSRs. Although it is not possible to cancel the risk of oxaliplatin-based HSRs, strategies to reduce the risk of occurrence could be stronger premedication and prolonged time of infusion. Full article
(This article belongs to the Section Molecular and Translational Medicine)
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<p>Symptoms of hypersensitivity to oxaliplatin.</p> Full article ">
16 pages, 3632 KiB  
Article
Development and Characterization of Phage-Display-Derived Novel Human Monoclonal Antibodies against the Receptor Binding Domain of SARS-CoV-2
by Ji Woong Kim, Sung Won Min, Jichul Lee, Ha Gyeong Shin, Hye Lim Choi, Ha Rim Yang, Ji Hyun Lee, Yea Bin Cho, Hyunbo Shim and Sukmook Lee
Biomedicines 2022, 10(12), 3274; https://doi.org/10.3390/biomedicines10123274 - 17 Dec 2022
Cited by 7 | Viewed by 5854
Abstract
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in an ongoing global pandemic crisis, caused by the life-threatening illness coronavirus disease 2019 (COVID-19). Thus, the rapid development of monoclonal antibodies (mAbs) to cope with COVID-19 is urgently necessary. In this study, we used [...] Read more.
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in an ongoing global pandemic crisis, caused by the life-threatening illness coronavirus disease 2019 (COVID-19). Thus, the rapid development of monoclonal antibodies (mAbs) to cope with COVID-19 is urgently necessary. In this study, we used phage display to develop four human mAbs specific to the receptor-binding domain (RBD) of SARS-CoV-2. Our intensive in vitro functional analyses demonstrated that K102.1, an anti-SARS-CoV-2 RBD-specific mAb, exerted potent neutralizing activity against pseudoviral and live viral infection and the interaction between SARS-CoV-2 RBD and human angiotensin-converting enzyme 2. Monotherapy with K102.1 also revealed the therapeutic potential against SARS-CoV-2 infection in vivo. Further, this study developed a sandwich enzyme-linked immunosorbent assay with a non-competing mAb pair, K102.1 and K102.2, that accurately detected the RBDs of SARS-CoV-2 wild-type and variants with high sensitivity in the picomolar range. These findings suggest that the phage-display-based mAb selection from an established antibody library may be an effective strategy for the rapid development of mAbs against the constantly evolving SARS-CoV-2. Full article
(This article belongs to the Special Issue Biomedicines: 10th Anniversary)
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<p>Isolation and characterization of SARS-CoV-2 RBD-specific mAbs. (<b>A</b>) Following biopanning using phage-display technology with wild-type SARS-CoV-2 RBD-coupled magnetic beads, four scFv clones specific to SARS-CoV-2 RBD were selected from the human synthetic antibody library and converted to generate IgG mAbs (K101.1–4). (<b>B</b>) Following overproduction and purification, the dissociation equilibrium constants of the selected mAbs were individually measured using surface plasmon resonance. The data are representative of two independent experiments.</p> Full article ">Figure 2
<p>Concentration-dependent neutralizing effect of selected mAbs against interactions between hACE2 and RBDs of SARS-CoV-2. Interactions between recombinant hACE2 and RBDs of wild-type (<b>A</b>), Alpha (<b>B</b>), or Delta (<b>C</b>) variant SARS-CoV-2 were determined in the presence or absence of increasing concentrations of K102.1 (red), K102.2 (blue), K102.3 (yellow), and K102.4 (green). All values represent the mean ± SD of duplicate measurements from one of two independent experiments. The IC<sub>50</sub> values were determined by log (inhibitor) response of nonlinear regression.</p> Full article ">Figure 3
<p>Concentration-dependent neutralizing effect of selected mAbs against pseudoviral SARS-CoV-2 infection. Neutralizing activity of K102.1 (red), K102.2 (blue), K102.3 (yellow), and K102.4 (green) against infection with pseudoviral wild-type (<b>A</b>), Alpha (<b>B</b>), and Delta (<b>C</b>) variant SARS-CoV-2. Values represent the mean ± SD of duplicate measurements from one of two independent experiments. The IC<sub>50</sub> values were determined by log (inhibitor) response of nonlinear regression.</p> Full article ">Figure 4
<p>Neutralizing effect of selected mAb against live SARS-CoV-2 infection. Wild-type SARS-CoV-2-induced CPE on Vero E6 cells was observed in the absence or presence of indicated concentrations of K102.1 at 120 h using bright-filed microscopy. Scale bars: 200 μm.</p> Full article ">Figure 5
<p>In vivo efficacy evaluation of K102.1 in hACE2 transgenic mice infected with wild-type SARS-CoV-2. (<b>A</b>) Schematic description of the experimental design for in vivo efficacy evaluation of K102.1 in wild-type SARS-CoV-2-infected K18-hACE2 transgenic mice (<span class="html-italic">n</span> = 5). The level of the viral E (<b>B</b>) or RdRp (<b>C</b>) gene expression in lung tissue was measured using RT-qPCR at 5 dpi. Data were statistically analyzed using two-tailed Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span>  &lt; 0.01). (<b>D</b>) Two representative histopathological images of the lung tissue in each group at 5 dpi. Scale bar (40×): 500 µm; scale bar (400×): 50 µm. All values represent the mean ± SD of five biological replicates.</p> Full article ">Figure 6
<p>Identification of SARS-CoV-2 RBD-specific monoclonal antibody pair for sandwich ELISA. (<b>A</b>) Sandwich ELISA was conducted with untagged forms of K102.1 or K102.2 as capture antibodies and HA-tagged K102.1 (K102.1-HA) or K102.2 (K102.2-HA) as detection antibodies. Values represent the mean ± SD of duplicate measurements from one of two independent experiments. Data were statistically analyzed using two-tailed Student’s t-test (*** <span class="html-italic">p</span> &lt; 0.001). (<b>B</b>) Additional binding of K102.2-HA (blue) to wild-type SARS-CoV-2 RBD after K102.1 (red) was saturated, as measured by surface plasmon resonance. The curves are representative of two independent experiments.</p> Full article ">Figure 7
<p>Generation and characterization of SARS-CoV-2 RBD sandwich ELISA. (<b>A</b>) Schematic representation of sandwich ELISA. (<b>B</b>) Optimizing the capture antibody (K102.1) concentration by sandwich ELISA. Microplate wells were coated with the indicated concentrations of K102.1, followed by incubation of the plates with 0.1 mg RBD of wild-type SARS-CoV-2 (antigen) and 2.5 μg/mL K102.2-HA (detection antibody). (<b>C</b>) Microplate wells were coated with 5 μg/mL K102.1, followed by incubation with 0.1 μg RBD of wild-type SARS-CoV-2. Titration of the indicated concentrations of K102.2-HA (detection) antibody was evaluated by sandwich ELISA. (<b>D</b>) The wild-type SARS-CoV-2 RBD calibration curve was constructed using 5 μg/mL capture antibody (K102.1) and 1 μg/mL detection antibody (K102.2-HA) to determine the limit of detection. All values represent the mean ± SD of triplicate measurements from six independent experiments.</p> Full article ">
18 pages, 1940 KiB  
Review
Management of Familial Hypercholesterolemia with Special Emphasis on Evinacumab
by Julia Krzemińska, Ewelina Młynarska, Ewa Radzioch, Magdalena Wronka, Jacek Rysz and Beata Franczyk
Biomedicines 2022, 10(12), 3273; https://doi.org/10.3390/biomedicines10123273 - 16 Dec 2022
Cited by 2 | Viewed by 2981
Abstract
Familial hypercholesterolemia (FH) is an underdiagnosed disease that contributes to a significant number of cardiovascular incidents through high serum Low-Density Lipoprotein Cholesterol (LDL-C) values. Its treatment primarily requires healthy lifestyle and therapy based on statins, ezetimibe and Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9); [...] Read more.
Familial hypercholesterolemia (FH) is an underdiagnosed disease that contributes to a significant number of cardiovascular incidents through high serum Low-Density Lipoprotein Cholesterol (LDL-C) values. Its treatment primarily requires healthy lifestyle and therapy based on statins, ezetimibe and Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9); however, there are also new treatment options that can be used in patients who do not respond to therapy, among which we highlight evinacumab. Elevated LDL-C values, together with clinical manifestations associated with cholesterol deposition (e.g., tendon xanthomas, xanthelasma and arcus cornealis) and family history are the main elements in the diagnosis of FH. Pathognomonic signs of FH include extensor tendon xanthomas; however, their absence does not exclude the diagnosis. Elevated LDL-C levels lead to premature Atherosclerotic Cardiovascular Disease (ASCVD), which is why early diagnosis and treatment of FH is essential. Evinacumab, a novelty in pharmacological practice, having a complex mechanism of action, causes desirable changes in lipid parameters in patients with homozygous form of familial hypercholesterolemia (HoFH). This review collects and summarizes the most important aspects of the new drug, especially being a discovery in the treatment of HoFH, giving these patients hope for a longer and more comfortable life. Full article
(This article belongs to the Special Issue 10th Anniversary of Biomedicines—Genetics of Cholesterol in Cardiovascular Disease: From Basics to Clinics)
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<p>Biochemical Structure of ANGPTL3 [<a href="#B21-biomedicines-10-03273" class="html-bibr">21</a>,<a href="#B22-biomedicines-10-03273" class="html-bibr">22</a>]. ANGPTL3, angiopoietin-like protein 3.</p> Full article ">Figure 2
<p>Symptoms Associated with Cholesterol Deposition in the Course of FH [<a href="#B2-biomedicines-10-03273" class="html-bibr">2</a>,<a href="#B3-biomedicines-10-03273" class="html-bibr">3</a>,<a href="#B5-biomedicines-10-03273" class="html-bibr">5</a>,<a href="#B6-biomedicines-10-03273" class="html-bibr">6</a>,<a href="#B7-biomedicines-10-03273" class="html-bibr">7</a>,<a href="#B8-biomedicines-10-03273" class="html-bibr">8</a>,<a href="#B9-biomedicines-10-03273" class="html-bibr">9</a>,<a href="#B26-biomedicines-10-03273" class="html-bibr">26</a>].</p> Full article ">Figure 3
<p>Basic Monitoring Data for Hypolipidemic Treatment [<a href="#B49-biomedicines-10-03273" class="html-bibr">49</a>,<a href="#B50-biomedicines-10-03273" class="html-bibr">50</a>,<a href="#B51-biomedicines-10-03273" class="html-bibr">51</a>,<a href="#B52-biomedicines-10-03273" class="html-bibr">52</a>].</p> Full article ">Figure 4
<p>Changes in Plasma Components Affected by Evinacumab after 24 Weeks [<a href="#B16-biomedicines-10-03273" class="html-bibr">16</a>]. ApoC-III, Apolipoprotein C-III; Lp(a), Lipoprotein(a).</p> Full article ">
14 pages, 9520 KiB  
Article
Ruminococcaceae_UCG-013 Promotes Obesity Resistance in Mice
by Jinlian Feng, Hongliang Ma, Yiting Huang, Jiangchao Li and Weidong Li
Biomedicines 2022, 10(12), 3272; https://doi.org/10.3390/biomedicines10123272 - 16 Dec 2022
Cited by 25 | Viewed by 2587
Abstract
Alterations in the gut microbiome have been linked to obesity and type 2 diabetes, in epidemiologic studies and studies of fecal transfer effects in germ-free mice. Here, we aimed to identify the effects of specific gut microbes on the phenotype of mice fed [...] Read more.
Alterations in the gut microbiome have been linked to obesity and type 2 diabetes, in epidemiologic studies and studies of fecal transfer effects in germ-free mice. Here, we aimed to identify the effects of specific gut microbes on the phenotype of mice fed a high-fat diet (HFD). After eight weeks of HFD feeding, male C57BL/6J mice in the HFD group ranking in the upper and lower quartiles for body weight gain were considered obese prone and obese resistant, respectively. 16S rRNA gene sequencing was used to determine the composition of the intestinal microbiota, and fecal transplantation (FMT) was conducted to determine whether the microbiota plays a causal role in phenotypic variation. Ruminococcaceae_UCG-013 was more abundant in the gut microbes of mice with a lean phenotype than in those with an obese phenotype. Ruminococcaceae_UCG-013 was identified as the most significant biomarker for alleviating obesity by random forest analysis. In a correlation analysis of serum parameters and body weight, Ruminococcaceae_UCG-013 was positively associated with serum HDL-C levels and negatively associated with serum TC, TG, and LDL-C levels. To conclude, Ruminococcaceae_UCG-013 was identified as a novel microbiome biomarker for obesity resistance, which may serve as a basis for understanding the critical gut microbes responsible for obesity resistance. Ruminococcaceae_UCG-013 may serve as a target for microbiome-based diagnoses and treatments in the future. Full article
(This article belongs to the Special Issue The Interplay of Gut Dysbiosis with Metabolic Syndrome)
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<p>The physiological changes in the Normal diet (ND), obesity-prone (OP), and obesity-resistant (OR) groups. (<b>A</b>) Mice representative images. (<b>B</b>) Body weight (<span class="html-italic">n</span> = 12 per group). ** <span class="html-italic">p</span> &lt; 0.01 and **** <span class="html-italic">p</span> &lt; 0.0001 vs. the OR group. (<b>C</b>) Energy intake. (<b>D</b>) Fat index. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 vs. the ND group; <span class="html-italic">##</span> Fat index was significantly altered in OP relative to OR (<span class="html-italic">p</span> &lt; 0.01). (<b>E</b>) Blood glucose levels were measured by an oral glucose tolerance test (OGTT) performed during the 16 weeks of the experiment. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001 and **** <span class="html-italic">p</span> &lt; 0.0001 vs. the OR group (<b>F</b>) Serum biochemical parameters. A sample is represented by each black dot. (<b>G</b>) Representative images of H&amp;E staining of liver sections (<span class="html-italic">n</span> = 6 per group). The scale bar for H&amp;E staining represents 100 µm. Data are expressed as the mean ± SD. **** <span class="html-italic">p</span> &lt; 0.0001, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01 and * <span class="html-italic">p</span> &lt; 0.05 were considered significant. <span class="html-italic">ns</span>, not significant.</p> Full article ">Figure 2
<p>Significantly different microbiota structures were observed between OR and OP groups. (<b>A</b>) Comparison of alpha diversity indices in the OR versus OP groups. (<b>B</b>) Partial least-square discriminant analysis (PLS-DA) scores plot of OTU level showing the groupings of ND (red) group, OP (green), and HF-OR (blue). (<b>C</b>) Extended error bar plot between the OP and OR groups at the genus level. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and **** <span class="html-italic">p</span> &lt; 0.0001 were considered significant. <span class="html-italic">ns</span>, not significant.</p> Full article ">Figure 3
<p>The specific gut microbiota linked to the development of obesity according to the machine learning algorithm. (<b>A</b>) Linear discriminant analysis (LDA) effect size (LEfSe) analyses identified bacterial biomarkers of OP and OR mice (LDA &gt; 2, <span class="html-italic">p</span> &lt; 0.05). (<b>B</b>) Ranking of species importance produced by random forest analyses. (<b>C</b>) The relative abundance of Ruminococcaceae_UCG-013 in different groups at week 8. (<b>D</b>) The relative abundance of Ruminococcaceae_UCG-013 in different groups at week 16. (<b>E</b>) Heatmap of Spearman’s correlation between the faecal microbiota with significant differences and serum biochemical parameters and body weight. An indicator of the degree of association is the intensity of the colour. (<b>F</b>) Co-occurrence network. Note: <span class="html-italic">p</span> &lt; 0.05, confidence interval = 95%; green nodes are gut microbial genera; orange nodes are serum biochemical indicators; orange and black lines represent positive and negative correlations, respectively. In addition, the width of the lines indicates the strength of the correlation. * <span class="html-italic">p</span> &lt; 0.05. ** <span class="html-italic">p</span> &lt; 0.01. <span class="html-italic">ns</span>, not significant.</p> Full article ">Figure 4
<p>PICRUSt2 analysis predicted metabolic pathways by PICRUSt2 using 16s rRNA gene sequences (Only the first 20 will be displayed). The rightmost is the <span class="html-italic">p</span>-value, and <span class="html-italic">p</span> &lt; 0.05 is statistically significant.</p> Full article ">Figure 5
<p>The physiological change and relative abundance of Ruminococcaceae_UCG-013 change in the Mice with obesity-prone fecal flora transplanted (FMT-OP)and Mice with obesity-resistant fecal flora transplanted (FMT-OR) groups. (<b>A</b>) Images of mice representative of the species. (<b>B</b>) Body weight. * <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. the FMT-OR group. (<b>C</b>) Energy intake. (<b>D</b>) Fat index. (<b>E</b>) Blood glucose levels were measured by an oral glucose tolerance test (OGTT) performed in the eight weeks of FMT. (<b>F</b>) Serum biochemical parameters. A sample is represented by each black dot. (<b>G</b>) The relative abundance of Ruminococcaceae_UCG-013 in different groups at week 8. (<b>H</b>) Representative images of H&amp;E staining of liver sections. The scale bar for H&amp;E staining represents 100 µm. Data are expressed as the mean ± SD (<span class="html-italic">n</span> = 8 per group). **** <span class="html-italic">p</span> &lt; 0.0001, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01 and * <span class="html-italic">p</span> &lt; 0.05 were considered significant. <span class="html-italic">ns</span>, not significant.</p> Full article ">
18 pages, 3321 KiB  
Article
Differential Regulation of MMPs, Apoptosis and Cell Proliferation by the Cannabinoid Receptors CB1 and CB2 in Vascular Smooth Muscle Cells and Cardiac Myocytes
by Bettina Greiner, Manuela Sommerfeld, Ulrich Kintscher, Thomas Unger, Kai Kappert and Elena Kaschina
Biomedicines 2022, 10(12), 3271; https://doi.org/10.3390/biomedicines10123271 - 16 Dec 2022
Cited by 5 | Viewed by 2252
Abstract
Cannabinoids (CB) are implicated in cardiovascular diseases via the two main receptor subtypes CB1R and CB2R. This study investigated whether cannabinoids regulate the activity of matrix metalloproteases (MMP-2, MMP-9) in vascular smooth muscle cells (VSMCs) and in cells of [...] Read more.
Cannabinoids (CB) are implicated in cardiovascular diseases via the two main receptor subtypes CB1R and CB2R. This study investigated whether cannabinoids regulate the activity of matrix metalloproteases (MMP-2, MMP-9) in vascular smooth muscle cells (VSMCs) and in cells of cardiac origin (H9c2 cell line). The influence of CB1- and CB2 receptor stimulation or inhibition on cell proliferation, apoptosis and glucose uptake was also evaluated. We used four compounds that activate or block CB receptors: arachidonyl-2-chloroethylamide (ACEA)—CB1R agonist, rimonabant—CB1R antagonist, John W. Huffman (JWH133)—CB2R agonist and CB2R antagonist—6-Iodopravadoline (AM630). Treatment of cells with the CB2R agonist JWH133 decreased cytokine activated secretion of proMMP-2, MMP-2 and MMP-9, reduced Fas ligand and caspase-3-mediated apoptosis, normalized the expression of TGF-beta1 and prevented cytokine-induced increase in glucose uptake into the cell. CB1R inhibition with rimonabant showed similar protective properties as the CB2R agonist JWH133, but to a lesser extent. In conclusion, CB1R and CB2R exert opposite effects on cell glucose uptake, proteolysis and apoptosis in both VSMCs and H9c2 cells. The CB2R agonist JWH133 demonstrated the highest protective properties. These findings may pave the way to a new treatment of cardiovascular diseases, especially those associated with extracellular matrix degradation. Full article
(This article belongs to the Special Issue Therapeutic Potential for Cannabis and Cannabinoids)
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<p>Effect of rimonabant (Rimona), AM630, ACEA and JWH-133 on IL-1α-induced secretion of MMP-9 (<b>a</b>), proMMP-2 (<b>b</b>) and MMP-2 (<b>c</b>) in VSMCs, 48 h after treatment. The <span class="html-italic">graphs</span> represent the <span class="html-italic">densitometric</span> analysis (mean ± SD; <span class="html-italic">n</span> = 11). Statistical analysis performed with <span class="html-italic">t</span>-test with Welch’s correction, * <span class="html-italic">p</span> &lt; 0.05; **** <span class="html-italic">p</span> &lt; 0.0001. (<b>d</b>) MMP-9 activity, gelatin zymography, VSMCs, representative zymogram. (<b>e</b>) ProMMP-2, MMP-2 activity VSMCs, representative zymogram.</p> Full article ">Figure 2
<p>(<b>a</b>) Representative Western blots of MMP-9 in VSMCs, 48 h after stimulation with IL-1α. (<b>b</b>) Densiometric analysis of proMMP-9. (<b>c</b>) Densiometric analysis of MMP-9. (<b>d</b>) Densiometric analysis of MMP (45–60 kDa).</p> Full article ">Figure 3
<p>(<b>A</b>). Effect of rimonabant, AM630, ACEA and JWH-133 on IL-1α-induced apoptosis in VSMCs. Magnification ×10. More condensed small cell nuclei with irregular form (blebbing) as well as more disintegrating cells were detected after IL-1α stimulation (<b>B</b>). (<b>a</b>) The ratio of normal cell nuclei to apoptotic cell nuclei in VSMC. The higher the bar, the more normal VSMC could be found in the treatment group. (<b>b</b>) The relation of apoptotic ratio in the treatment group to the ratio of the IL-1α-stimulated group. The resulting number expresses the factor of increased normal cell nuclei in comparison to the IL-1α group.</p> Full article ">Figure 4
<p>(<b>A</b>) (<b>a</b>) Caspase-3 expression signal in VSMCs, 48 h after treatment with rimonabant, AM 630, ACEA and JWH-133; fluorescent images obtained with Biorevo BZ 900 Microscope using 10× magnification. Exposure time was equal in all observed groups. (<b>b</b>) Intensity of caspase-3 expression signal. The results were attained by measuring three areas of interest of the fluorescent image and subtracting the background signal (<span class="html-italic">n</span> = 3). (<b>c</b>) Representative Western blots of caspase-3 in VSMCs, 48 h after stimulation with IL-1α. (<b>B</b>) (<b>a</b>) FasL expression signal in VSMCs, 48 h after treatment with rimonabant, AM 630, ACEA and JWH-133; fluorescent images obtained with Biorevo BZ 900 Microscope using 10× magnification. Exposure time was equal in all observed groups. (<b>b</b>) Intensity of FasL expression signal. The results were attained by measuring three areas of interest of the fluorescent image and subtracting the background signal (<span class="html-italic">n</span> = 3). (<b>c</b>) Intensity of TGF-beta1 expression signal in VSMCs, 48 h after treatment with rimonabant, AM 630, ACEA, and JWH-133, attained by measuring three areas of interest of the fluorescent image and subtracting the background signal. The measurements were repeated three times per image. Statistical testing was performed using unpaired <span class="html-italic">t</span>-tests. Significance was expressed when <span class="html-italic">p</span> &lt; 0.05; (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 4 Cont.
<p>(<b>A</b>) (<b>a</b>) Caspase-3 expression signal in VSMCs, 48 h after treatment with rimonabant, AM 630, ACEA and JWH-133; fluorescent images obtained with Biorevo BZ 900 Microscope using 10× magnification. Exposure time was equal in all observed groups. (<b>b</b>) Intensity of caspase-3 expression signal. The results were attained by measuring three areas of interest of the fluorescent image and subtracting the background signal (<span class="html-italic">n</span> = 3). (<b>c</b>) Representative Western blots of caspase-3 in VSMCs, 48 h after stimulation with IL-1α. (<b>B</b>) (<b>a</b>) FasL expression signal in VSMCs, 48 h after treatment with rimonabant, AM 630, ACEA and JWH-133; fluorescent images obtained with Biorevo BZ 900 Microscope using 10× magnification. Exposure time was equal in all observed groups. (<b>b</b>) Intensity of FasL expression signal. The results were attained by measuring three areas of interest of the fluorescent image and subtracting the background signal (<span class="html-italic">n</span> = 3). (<b>c</b>) Intensity of TGF-beta1 expression signal in VSMCs, 48 h after treatment with rimonabant, AM 630, ACEA, and JWH-133, attained by measuring three areas of interest of the fluorescent image and subtracting the background signal. The measurements were repeated three times per image. Statistical testing was performed using unpaired <span class="html-italic">t</span>-tests. Significance was expressed when <span class="html-italic">p</span> &lt; 0.05; (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 5
<p>(<b>A</b>) (<b>a</b>) VSMC, cell confluence difference (cell growth) at 24 h (confluence at 24 h-confluence at 0 h) and the confluence at 48 h (confluence at 48 h-confluence at 0 h) in the treatment groups. The experiment was performed using 10% FBS medium with and without IL-1α stimulation in all treatment groups. (<b>b</b>) VSMC, cell growth in dynamic. Representative graph obtained from IncuCyte Live-Cell Analysis System. Cell proliferation was monitored by analyzing the occupied area (% confluence) of cell images over 100 h. Analysis of the IncuCyte images was performed with Incucyte<sup>®</sup> Analysis Software. The experiment was performed using 10% FBS medium with and without IL-1α stimulation in all treatment groups. (<b>B</b>) The growth rate (confluence difference) of VSMCs estimated by IncuCyte live-cell analysis after treatment with compounds after IL-1α stimulation in 24 h (<b>a</b>) and 48 h (<b>b</b>). Statistical testing was performed using unpaired <span class="html-italic">t</span>-tests. Significance was expressed when <span class="html-italic">p</span> &lt; 0.05; <span class="html-italic">n</span> = 6–32 (* <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 5 Cont.
<p>(<b>A</b>) (<b>a</b>) VSMC, cell confluence difference (cell growth) at 24 h (confluence at 24 h-confluence at 0 h) and the confluence at 48 h (confluence at 48 h-confluence at 0 h) in the treatment groups. The experiment was performed using 10% FBS medium with and without IL-1α stimulation in all treatment groups. (<b>b</b>) VSMC, cell growth in dynamic. Representative graph obtained from IncuCyte Live-Cell Analysis System. Cell proliferation was monitored by analyzing the occupied area (% confluence) of cell images over 100 h. Analysis of the IncuCyte images was performed with Incucyte<sup>®</sup> Analysis Software. The experiment was performed using 10% FBS medium with and without IL-1α stimulation in all treatment groups. (<b>B</b>) The growth rate (confluence difference) of VSMCs estimated by IncuCyte live-cell analysis after treatment with compounds after IL-1α stimulation in 24 h (<b>a</b>) and 48 h (<b>b</b>). Statistical testing was performed using unpaired <span class="html-italic">t</span>-tests. Significance was expressed when <span class="html-italic">p</span> &lt; 0.05; <span class="html-italic">n</span> = 6–32 (* <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 6
<p>Concentration of glucose (<b>a</b>) and lactate (<b>b</b>) in the supernatant of VSMC after 48 h of treatment with compounds and IL-1α stimulation. Concentration of glucose (<b>c</b>) and lactate (<b>d</b>) in the supernatant of H9c2 cells after 48 h of treatment with compounds and IL-1α stimulation. The values are expressed as mean ± SD (VSMC, <span class="html-italic">n</span> = 3–9; H9C2 cells, <span class="html-italic">n</span> = 3). Statistical testing was performed using unpaired <span class="html-italic">t</span>-tests. (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 7
<p>The concentrations of potassium (<b>a</b>), sodium (<b>b</b>) and chloride (<b>c</b>), measured in cell supernatant of VSMCs 48 h after treatments with compounds and IL-1α stimulation.</p> Full article ">
21 pages, 5564 KiB  
Article
A Cyclopentanone Compound Attenuates the Over-Accumulation of Extracellular Matrix and Fibrosis in Diabetic Nephropathy via Downregulating the TGF-β/p38MAPK Axis
by Chunyin Tang, Meng Wang, Jieting Liu, Chunlei Zhang, Luxin Li, Yan Wu, Yanhui Chu, Dan Wu, Haifeng Liu and Xiaohuan Yuan
Biomedicines 2022, 10(12), 3270; https://doi.org/10.3390/biomedicines10123270 - 16 Dec 2022
Cited by 8 | Viewed by 1847
Abstract
Excessive accumulation of the extracellular matrix (ECM) is a crucial pathological process in chronic kidney diseases, such as diabetic nephropathy, etc. The underlying mechanisms of how to decrease ECM deposition to improve diabetic nephropathy remain elusive. The present study investigated whether cyclopentanone compound [...] Read more.
Excessive accumulation of the extracellular matrix (ECM) is a crucial pathological process in chronic kidney diseases, such as diabetic nephropathy, etc. The underlying mechanisms of how to decrease ECM deposition to improve diabetic nephropathy remain elusive. The present study investigated whether cyclopentanone compound H8 alleviated ECM over-deposition and fibrosis to prevent and treat diabetic nephropathy. HK-2 cell viability after treatment with H8 was measured by an MTT assay. ECM alterations and renal fibrosis were identified in vitro and in vivo. A pharmacological antagonist was used to detect associations between H8 and the p38 mitogen-activated protein kinase (p38MAPK) signaling pathway. H8 binding was identified through computer simulation methods. Studies conducted on high glucose and transforming growth factor β1 (TGF-β1)-stimulated HK-2 cells revealed that the p38MAPK inhibitor SB 202190 and H8 had similar pharmacological effects. In addition, excessive ECM accumulation and fibrosis in diabetic nephropathy were remarkably improved after H8 administration in vivo and in vitro. Finally, the two molecular docking models further proved that H8 is a specific p38MAPK inhibitor that forms a hydrogen bond with the LYS-53 residue of p38MAPK. The cyclopentanone compound H8 alleviated the over-deposition of ECM and the development of fibrosis in diabetic nephropathy by suppressing the TGF-β/p38MAPK axis. Full article
(This article belongs to the Special Issue Pathological Mechanisms of Diabetic Nephropathy)
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Figure 1

Figure 1
<p>H8 mitigated ECM over-accumulation in HG-stimulated HK-2 cells. (<b>A</b>) The H8 chemical structure: (2E, 5E)-2,5-double [(2-fluoro-6-three fluoromethyl) benzyl methylene]-cyclopentone (C<sub>21</sub>H<sub>12</sub>F<sub>8</sub>O; relative molecular mass: 432.31). The effects of different concentrations of H8 on the proliferation of HK-2 cells at the various time points of (<b>B</b>) 24 h and (<b>C</b>) 48 h. HK-2 cells were co-treated with H8 (5, 10, and 15 μM) and HG (35 mM) for 48 h. (<b>D</b>) The mRNA expression levels were examined by qPCR, and the values were normalized to rps16 (α-SMA, TGF-β1, FN, Col IV, and E-Ca). (<b>E</b>) Western blot band. (<b>F</b>) The protein relative expression levels (α-SMA, TGF-β1, FN, Col IV, E-Ca, and the ratio of P-p38MAPK/p38MAPK). (<b>G</b>) The IF was performed using FN antibody, and DAPI was used for staining cell nuclei (scale bar = 50 μm). Data are shown as mean ± S.D. (<span class="html-italic">n</span> = 5). <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to Con group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to HG group; n.s. represents no significant difference in statistics compared to HG group.</p> Full article ">Figure 1 Cont.
<p>H8 mitigated ECM over-accumulation in HG-stimulated HK-2 cells. (<b>A</b>) The H8 chemical structure: (2E, 5E)-2,5-double [(2-fluoro-6-three fluoromethyl) benzyl methylene]-cyclopentone (C<sub>21</sub>H<sub>12</sub>F<sub>8</sub>O; relative molecular mass: 432.31). The effects of different concentrations of H8 on the proliferation of HK-2 cells at the various time points of (<b>B</b>) 24 h and (<b>C</b>) 48 h. HK-2 cells were co-treated with H8 (5, 10, and 15 μM) and HG (35 mM) for 48 h. (<b>D</b>) The mRNA expression levels were examined by qPCR, and the values were normalized to rps16 (α-SMA, TGF-β1, FN, Col IV, and E-Ca). (<b>E</b>) Western blot band. (<b>F</b>) The protein relative expression levels (α-SMA, TGF-β1, FN, Col IV, E-Ca, and the ratio of P-p38MAPK/p38MAPK). (<b>G</b>) The IF was performed using FN antibody, and DAPI was used for staining cell nuclei (scale bar = 50 μm). Data are shown as mean ± S.D. (<span class="html-italic">n</span> = 5). <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to Con group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to HG group; n.s. represents no significant difference in statistics compared to HG group.</p> Full article ">Figure 2
<p>H8 reduced the excessive expressions of ECM in HG-stimulated HK-2 cells, which was related to p38MAPK. (<b>A</b>) The mRNA expression levels were examined by qPCR, and the values were normalized to rps16 (α-SMA, TGF-β1, FN, Col IV, and E-Ca). (<b>B</b>) Western blot band. (<b>C</b>) The protein relative expression levels (α-SMA, TGF-β1, FN, Col IV, E-Ca, and the ratio of P-p38MAPK/p38MAPK). Data are shown as mean ± S.D. (<span class="html-italic">n</span> = 5). <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to Con group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to HG group; n.s. represents no significant difference in statistics.</p> Full article ">Figure 3
<p>H8 attenuated the over-deposition of ECM and inhibited p38MAPK in the TGF-β1-induced HK-2 cells. (<b>A</b>) The mRNA expression levels were examined by qPCR, and the values were normalized to rps16 (α-SMA, TGF-β1, FN, Col IV, and E-Ca). (<b>B</b>) Western blot band. (<b>C</b>) The protein relative expression levels (a-SMA, TGF-β1, FN, Col IV, E-Ca, and the ratio of P-p38MAPK/p38MAPK). (<b>D</b>) The IF was performed using FN antibody, and DAPI was used for staining cell nuclei (scale bar = 50 μm). Data are shown as mean ± S.D. (<span class="html-italic">n</span> = 5). <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to Con group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to TGF-β1 group; n.s. represents no significant difference in statistics.</p> Full article ">Figure 3 Cont.
<p>H8 attenuated the over-deposition of ECM and inhibited p38MAPK in the TGF-β1-induced HK-2 cells. (<b>A</b>) The mRNA expression levels were examined by qPCR, and the values were normalized to rps16 (α-SMA, TGF-β1, FN, Col IV, and E-Ca). (<b>B</b>) Western blot band. (<b>C</b>) The protein relative expression levels (a-SMA, TGF-β1, FN, Col IV, E-Ca, and the ratio of P-p38MAPK/p38MAPK). (<b>D</b>) The IF was performed using FN antibody, and DAPI was used for staining cell nuclei (scale bar = 50 μm). Data are shown as mean ± S.D. (<span class="html-italic">n</span> = 5). <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to Con group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to TGF-β1 group; n.s. represents no significant difference in statistics.</p> Full article ">Figure 4
<p>Molecular docking of H8 on the 1BL7 protein. (<b>A</b>) Modeling of H8 docking to 1BL7 was simulated with Pymol, and positions of H8 are shown in pink within the active site of 1BL7. (<b>B</b>) The green dashed line indicates a possible hydrogen bond between the connected residues and the ligand. (<b>C</b>) Modeling of H8 docking to 1BL7 was simulated with Discovery studio software (3D and 2D, San Diego, CA, USA; <a href="https://discover.3ds.com/discovery-studio-visualizer-download" target="_blank">https://discover.3ds.com/discovery-studio-visualizer-download</a>, accessed on 22 August 2022). All the data showed that H8 could interact with the 1BL7 protein.</p> Full article ">Figure 5
<p>H8 improved the glomerular and tubular pathological changes and renal function in <span class="html-italic">db/db</span> mice. (<b>A</b>) HE staining, Masson staining, and Sirius Red staining of kidney sections in mice (original magnification 400×; scale bar = 50 µm). Orange arrow represents the glomerular mesangial expansion; green arrows represent accumulation of fibrosis in renal tubule; and black arrow represents formations of collagen in renal tubule. (<b>B</b>) The experimental design for the DN treatment in mice. (<b>C</b>) BUN (mmol/L). (<b>D</b>) Scr (µmol/L). (<b>E</b>) UA (µmol/L). (<b>F</b>) GLU (mmol/L). (<b>G</b>) 24 h urinary protein (mg). (<b>H</b>) NAG (U/L). (<b>I</b>) Ucr (µmol/L). (<b>J</b>) Urinary albumin/creatinine ratio (mg/mmol). Data are shown as mean ± S.D. (<span class="html-italic">db/m n</span> = 8, <span class="html-italic">db/db n</span> = 6, and <span class="html-italic">db/db</span> + H8 <span class="html-italic">n</span> = 8). <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to Con group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 compared to <span class="html-italic">db/db</span> group; n.s. represents no significant difference in statistics.</p> Full article ">Figure 6
<p>H8 reduced the fibrosis in renal tubule and inhibited p38MPAK signaling pathway in <span class="html-italic">db/db</span> mice. (<b>A</b>) The mRNA expression levels were examined by qPCR, and the values were normalized to rps16 (α-SMA, TGF-β1, FN, Col IV, and E-Ca). (<b>B</b>) Western blot band. (<b>C</b>) The protein relative expression levels (α-SMA, TGF-β1, FN, Col IV, E-Ca, and the ratio of P-p38MAPK/p38MAPK). (<b>D</b>) IF was performed using α-SMA and FN antibodies, and DAPI was used for staining cell nuclei (scale bar = 50 μm); white arrows represent the protein expressions of α-SMA and FN in renal tubule. Data are shown as mean ± S.D. (<span class="html-italic">db/m n</span> = 8, <span class="html-italic">db/db n</span> = 6, and <span class="html-italic">db/db</span> + H8 <span class="html-italic">n</span> = 8). <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to Con group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to <span class="html-italic">db/db</span> group.</p> Full article ">Figure 7
<p>H8 ameliorated renal pathological changes and function in DN rats. (<b>A</b>) HE staining, Masson staining, and Sirius Red staining of kidney sections in rats (original magnification 400×; scale bar = 50 µm). Orange arrow represents the glomerular mesangial expansion; green arrows represent accumulation of fibrosis in renal tubule; black arrow represents collagen formations in renal tubule. (<b>B</b>) The experimental design for the DN treatment in rats. (<b>C</b>) BUN (mmol/L). (<b>D</b>) Scr (µmol/L). (<b>E</b>) UA (µmol/L). (<b>F</b>) GLU (mmol/L). (<b>G</b>) 24 h urinary protein (mg). (<b>H</b>) NAG (U/L). (<b>I</b>) Ucr (µmol/L). (<b>J</b>) Urinary albumin/creatinine ratio (mg/mmol). Data are shown as mean ± S.D. (for each group of rats, <span class="html-italic">n</span> = 8). <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to Con group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 compared to DN group; n.s. represents no significant difference in statistics.</p> Full article ">Figure 8
<p>H8 repressed renal fibrosis and p38MPAK signaling pathway in DN rats. (<b>A</b>) The mRNA expression levels were examined by qPCR, and the values were normalized to rps16 (α-SMA, TGF-β1, FN, Col IV, and E-Ca). (<b>B</b>) Western blot band. (<b>C</b>) The protein relative expression levels (α-SMA, TGF-β1, FN, Col IV, E-Ca, and the ratio of P-p38MAPK/p38MAPK). Data are shown as mean ± S.D. (<span class="html-italic">n</span> = 8/group). <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to Con group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to DN group.</p> Full article ">Figure 9
<p>The cyclopentanone compound H8 improved DN via suppressing TGF-β/p38MAPK axis in vitro and in vivo.</p> Full article ">
12 pages, 1217 KiB  
Review
Current Application of iPS Cells in the Dental Tissue Regeneration
by Sayuri Hamano, Risa Sugiura, Daiki Yamashita, Atsushi Tomokiyo, Daigaku Hasegawa and Hidefumi Maeda
Biomedicines 2022, 10(12), 3269; https://doi.org/10.3390/biomedicines10123269 - 16 Dec 2022
Cited by 4 | Viewed by 3139
Abstract
When teeth and periodontal tissues are severely damaged by severe caries, trauma, and periodontal disease, such cases may be subject to tooth extraction. As tooth loss leads to the deterioration of quality of life, the development of regenerative medicine for tooth and periodontal [...] Read more.
When teeth and periodontal tissues are severely damaged by severe caries, trauma, and periodontal disease, such cases may be subject to tooth extraction. As tooth loss leads to the deterioration of quality of life, the development of regenerative medicine for tooth and periodontal tissue is desired. Induced pluripotent stem cells (iPS cells) are promising cell resources for dental tissue regeneration because they offer high self-renewal and pluripotency, along with fewer ethical issues than embryonic stem cells. As iPS cells retain the epigenetic memory of donor cells, they have been established from various dental tissues for dental tissue regeneration. This review describes the regeneration of dental tissue using iPS cells. It is important to mimic the process of tooth development in dental tissue regeneration using iPS cells. Although iPS cells had safety issues in clinical applications, they have been overcome in recent years. Dental tissue regeneration using iPS cells has not yet been established, but it is expected in the future. Full article
(This article belongs to the Special Issue Cell Biology in Dentistry)
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Figure 1
<p>Schematic illustration of iPS cell production.</p> Full article ">Figure 2
<p>Characterization of iPDLSCs. (<b>A</b>) The pictures of iPS cells, iPS-NCs, and iPDLSCs. (<b>B</b>) iPDLSCs were cultured in osteogenic, adipogenic, and chondrogenic medium. Mineralization was investigated by Alizarin Red S staining. Fatty lipids were investigated by Oil Red O staining. Cartilage matrix was investigated with Alcian Blue staining. Scale bar = 200 μm. Arrows indicate positive cells. (<b>C</b>) The expression of MSC markers (CD29, CD90, and CD105) in iPDLSCs was examined by flow cytometric analysis. (<b>D</b>) The expression of PDL-related markers, osteoprotegerin and periostin (red), in iPDLSCs was examined by immunofluorescence staining. Nuclei were stained with DAPI (blue). Scale bars = 200 μm.</p> Full article ">Figure 3
<p>Schematic illustration of iPS cells’ application in dental tissue regeneration.</p> Full article ">
12 pages, 730 KiB  
Article
Ten Years of KPC-Kp Bloodstream Infections Experience: Impact of Early Appropriate Empirical Therapy on Mortality
by Silvia Corcione, Ilaria De Benedetto, Nour Shbaklo, Fabio Ranzani, Simone Mornese Pinna, Anna Castiglione, Silvia Scabini, Gabriele Bianco, Rossana Cavallo, Stefano Mirabella, Renato Romagnoli and Francesco Giuseppe De Rosa
Biomedicines 2022, 10(12), 3268; https://doi.org/10.3390/biomedicines10123268 - 16 Dec 2022
Cited by 8 | Viewed by 1918
Abstract
Background. In K. pneumoniae KPC (KPC-Kp) bloodstream infections (BSI), INCREMENT CPE score >7, Charlson Comorbidity Index (CCI) ≥3 and septic shock are recognized predictors of mortality, with a possible beneficial effect of combination therapy in seriously ill patients. Materials and Methods. We conducted [...] Read more.
Background. In K. pneumoniae KPC (KPC-Kp) bloodstream infections (BSI), INCREMENT CPE score >7, Charlson Comorbidity Index (CCI) ≥3 and septic shock are recognized predictors of mortality, with a possible beneficial effect of combination therapy in seriously ill patients. Materials and Methods. We conducted a ten-year retrospective study including all KPC-Kp BSI in patients ≥18 years of age with the aim to evaluate the characteristics and impact of appropriate empirical therapy, either monotherapy or combination therapy, and targeted therapy on mortality. Appropriate therapy was defined as at least one active antimicrobial agent with in vitro activity against KPC-kp demonstrated by susceptibility testing, administered within 48 h from blood culture collection. Results. The median age of the 435 analyzed patients was 66.09 years (IQR 54.87–73.98). The median CCI was 4. KPC-Kp colonization was present in 324 patients (74.48%). The probable origin of the KPC-Kp BSI was not identified in 136 patients (31.26%), whereas in 120 (27.59%) patients, it was CVC-related, and in 118 (27.13%), it was respiratory. Source control was achieved in 87 patients (72.5%) with CVC-related KPC-Kp BSI. The twenty-eight-day survival was 70.45% for empirical monotherapy, 63.88% for empirical combination therapy and 57.05% for targeted therapy (p = 0.0399). A probable source of KPC-Kp BSI other than urinary, CVC or abdominal [aHR 1.64 (IC 1.15–2.34) p = 0.006] and deferred targeted therapy [HR 1.67 (IC 1.12–2.51), p= 0.013] emerged as predictors of mortality, whereas source control [HR 0.62 (IC 0.44–0.86), p = 0.005] and ceftazidime/avibactam administration in empirical therapy [aHR 0.37 (IC 0.20–0.68) p = 0.002] appeared as protective factors. Discussion. These data underline the importance of source control together with timing appropriateness in the early start of empirical therapy over the choice of monotherapy or combination therapy and the use of ceftazidime/avibactam against KPC-Kp BSI. Full article
(This article belongs to the Section Microbiology in Human Health and Disease)
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<p>Kaplan–Meier estimate of patient survival for patients receiving appropriate empirical monotherapy and appropriate empirical combination therapy (<span class="html-italic">n</span> = 201).</p> Full article ">Figure 2
<p>Kaplan–Meier estimate of survival for patients administered empirical monotherapy, empirical combination therapy and targeted therapy (<span class="html-italic">n</span> = 435).</p> Full article ">Figure 3
<p>Kaplan–Meier estimate of survival from nephrotoxicity in colistin/aminoglycoside (AG)-treated patients (<span class="html-italic">n</span> = 194).</p> Full article ">
11 pages, 1124 KiB  
Article
Clinical Significance of Serum Elafin in Children with Inflammatory Bowel Disease
by Paulina Krawiec and Elżbieta Pac-Kożuchowska
Biomedicines 2022, 10(12), 3267; https://doi.org/10.3390/biomedicines10123267 - 16 Dec 2022
Cited by 4 | Viewed by 1944
Abstract
Background: The role of elafin in the pathophysiology of inflammatory bowel disease (IBD) has not been not elucidated. We aimed to evaluate serum elafin in children with IBD and assess its relationship with disease activity. Methods: We enrolled children with IBD in the [...] Read more.
Background: The role of elafin in the pathophysiology of inflammatory bowel disease (IBD) has not been not elucidated. We aimed to evaluate serum elafin in children with IBD and assess its relationship with disease activity. Methods: We enrolled children with IBD in the study group and children with functional abdominal pain in the control group. We evaluated serum elafin using enzyme-linked immunosorbent assay kits. Results: In children with IBD, serum elafin (mean ± SD: 4.192 ± 1.424 ng/mL) was significantly elevated compared with controls (mean ± SD: 3.029 ± 1.366 ng/mL) (p = 0.0005). Elafin was significantly increased in children in the active phase of IBD (mean ± SD: 4.424 ± 1.449 ng/mL) compared with the control group (p = 0.0003). In IBD remission, only children with ulcerative colitis (mean ± SD: 4.054 ± 1.536 ng/mL) had elevated elafin compared with controls (p = 0.004). ROC analysis revealed that the area under the curve (AUC) of serum elafin was 0.809 while discriminating patients with ulcerative colitis from the control group, and the AUC was 0.664 while differentiating patients with Crohn’s disease from the control group. Conclusions: Serum elafin was found to be elevated in our cohort of children with IBD, depending on disease activity. Serum elafin was increased in the active phases of both ulcerative colitis and Crohn’s disease, but only in the remission of ulcerative colitis. Elafin appears to be a potential candidate for a biomarker of ulcerative colitis. Full article
(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy 2.0)
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<p>Comparison of serum elafin between children with IBD and children from control group.</p> Full article ">Figure 2
<p>Serum elafin in patients with Crohn’s disease and ulcerative colitis in comparison with control group.</p> Full article ">Figure 3
<p>(<b>A</b>) ROC curve and area under ROC for elafin in recognition of ulcerative colitis; (<b>B</b>) ROC curve and area under ROC for elafin in recognition of Crohn’s disease.</p> Full article ">
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