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Biomedicines
Volume 10
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Biomedicines, Volume 10, Issue 1 (January 2022) – 197 articles

Cover Story (view full-size image): In space, astronauts are subjected to a prolonged state of microgravity that induces a myriad of physiological adaptations. Human spaceflight is associated with several cardiovascular risk factors that induce multiple structural and functional changes. Upon entering microgravity, cephalad fluid shift occurs and increases the stroke volume and cardiac output. Despite this increase, astronauts enter a state of hypovolemia. The absence of orthostatic pressure and a decrease in arterial pressures reduces the workload of the heart and is believed to be the underlying mechanism for the development of cardiac atrophy in space. In addition, parasympathetic overactivity and blood anemia are observed inflight, while orthostatic intolerance is a remarkable feature postflight. View this paper
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16 pages, 1161 KiB  
Review
Unveiling the Role of the Fatty Acid Binding Protein 4 in the Metabolic-Associated Fatty Liver Disease
by Juan Moreno-Vedia, Josefa Girona, Daiana Ibarretxe, Lluís Masana and Ricardo Rodríguez-Calvo
Biomedicines 2022, 10(1), 197; https://doi.org/10.3390/biomedicines10010197 - 17 Jan 2022
Cited by 20 | Viewed by 5489
Abstract
Metabolic-associated fatty liver disease (MAFLD), the main cause of chronic liver disease worldwide, is a progressive disease ranging from fatty liver to steatohepatitis (metabolic-associated steatohepatitis; MASH). Nevertheless, it remains underdiagnosed due to the lack of effective non-invasive methods for its diagnosis and staging. [...] Read more.
Metabolic-associated fatty liver disease (MAFLD), the main cause of chronic liver disease worldwide, is a progressive disease ranging from fatty liver to steatohepatitis (metabolic-associated steatohepatitis; MASH). Nevertheless, it remains underdiagnosed due to the lack of effective non-invasive methods for its diagnosis and staging. Although MAFLD has been found in lean individuals, it is closely associated with obesity-related conditions. Adipose tissue is the main source of liver triglycerides and adipocytes act as endocrine organs releasing a large number of adipokines and pro-inflammatory mediators involved in MAFLD progression into bloodstream. Among the adipocyte-derived molecules, fatty acid binding protein 4 (FABP4) has been recently associated with fatty liver and additional features of advanced stages of MAFLD. Additionally, emerging data from preclinical studies propose FABP4 as a causal actor involved in the disease progression, rather than a mere biomarker for the disease. Therefore, the FABP4 regulation could be considered as a potential therapeutic strategy to MAFLD. Here, we review the current knowledge of FABP4 in MAFLD, as well as its potential role as a therapeutic target for this disease. Full article
(This article belongs to the Special Issue NAFLD: From Mechanisms to Therapeutic Approaches)
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<p>Potential mechanisms of FABP4 involvement in MAFLD. FABP4 increases adipose tissue lipolysis through HSL activation, thereby releasing NEFAs to the bloodstream that are taken by the liver to synthesize triglycerides. Additionally, FABP4 is also released to the bloodstream through a non-conventional mechanism and its circulating levels are associated with a large number of metabolic diseases, including MAFLD and hepatocellular carcinoma. In the liver, FABP4 contributes to triglyceride synthesis and its accumulation as lipid droplets, potentially by regulating SCD-1. FABP4 contributes to liver endoplasmic reticulum stress, insulin resistance, and cellular death. Additionally, FABP4 may contribute to the oxidative stress induction of endoplasmic reticulum stress and inflammation in Kupffer cells, at least partially activating JNK and NF-κB and the UCP2-induced ROS production, altogether contributing to MASH progression, and increases proliferation and migration of hepatocellular carcinoma cells, potentially through activation of Akt/PI3K and ERK1/2. The liver <span class="html-italic">FABP4</span> mRNA levels have been found to be increased in hepatocellular carcinoma with liver steatosis, and contribute to liver cancer stem cells trans-differentiation. AC-PKA, adenylyl cyclase–protein kinase A; Akt, protein kinase B; β-ox, beta oxidation; ER, endoplasmic reticulum; ERK1/2, extracellular regulated kinase 1/2; FA, fatty acids; FABP4, fatty acid binding protein 4; GC-PKG, guanylyl cyclase–protein kinase G; HCC, hepatocellular carcinoma; HSL, hormone-sensitive lipase; JNK, Jun N-terminal kinase 1; LCSCs, liver cancer stem cells; TG, triglycerides; MAFL, metabolic-associated fatty liver; MAFLD, metabolic-associated fatty liver disease; MASH, metabolic-associated steatohepatitis; NEFAs, non-esterified fatty acids; NF-κB, nuclear factor kappa B; Ox, oxidative; PI3K, phosphatidylinositol 3 kinase; ROS, reactive oxygen species; SCD-1, stearoyl-CoA desaturase-1; UCP2, uncoupling protein 2.</p> Full article ">Figure 2
<p>Pharmacological approaches targeting FABP4 for MAFLD. Several therapeutic approaches potentially reduce MAFLD by reducing <span class="html-italic">FABP4</span> levels via <span class="html-italic">PPARγ</span> down-regulation. Dietary sphingomyelin, FLAVn-3, miR100, Theobromine, Tetrahydrocurcumin, and Hugan Qingzhi tablets reduce both liver steatosis and liver <span class="html-italic">FABP4</span> expression. Huang-Qi San also reduces plasma FABP4 levels. The liver <span class="html-italic">FABP4</span> mRNA levels are additionally reduced by other compounds improving MASH-related features. Liver inflammation is attenuated by Korean red ginseng, Verbenaceae, Saikosaponin-d, the combination of Silybin/Tangerin, and GFT505. Additionally, Dieckol reduces NLRP3 inflammasome and BMP9 attenuates macrophage infiltration. Oxidative stress is reduced by Saikosaponin-d and the combination of Silybin/Tangerin, endoplasmic reticulum stress by Saikosaponin-d and fibrosis by GFT505. The selective FABP4 inhibitor BMS309403 reduces fatty liver and inflammation through the inhibition of JNK and NF-κB, and reduced liver carcinogenesis by reducing thesphere-forming, proliferation, and clonality of liver cancer stem cells, potentially through Akt and ERK1/2 activation. HTS01037, another FABP4 inhibitor, reduces inflammation in Kupffer cells. Therefore, approaches aimed at FABP4 inhibition/reduction may be considered as promising therapeutic tools against MAFLD. BMP9, bone morphogenetic protein 9; ER, endoplasmic reticulum; HCC, hepatocellular carcinoma; FABP4, fatty acid binding protein 4; FLAVn-3, flavan-3-ols long-chain n-3 poly-unsaturated fatty acids (LC-PUFA); JNK, Jun N-terminal kinase 1; LCSCs, liver cancer stem cells; TG, triglycerides; MAFL, metabolic-associated fatty liver; MAFLD, metabolic-associated fatty liver disease; MASH, metabolic-associated steatohepatitis; NF-κB, nuclear factor kappa B; NLRP3, nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing; Ox, oxidative; PPARγ, peroxisome proliferator-activated receptor γ.</p> Full article ">
21 pages, 2342 KiB  
Review
Multitasking Na+/Taurocholate Cotransporting Polypeptide (NTCP) as a Drug Target for HBV Infection: From Protein Engineering to Drug Discovery
by Dariusz Zakrzewicz and Joachim Geyer
Biomedicines 2022, 10(1), 196; https://doi.org/10.3390/biomedicines10010196 - 17 Jan 2022
Cited by 15 | Viewed by 4379
Abstract
Hepatitis B virus (HBV) infections are among the major public health concerns worldwide with more than 250 million of chronically ill individuals. Many of them are additionally infected with the Hepatitis D virus, a satellite virus to HBV. Chronic infection frequently leads to [...] Read more.
Hepatitis B virus (HBV) infections are among the major public health concerns worldwide with more than 250 million of chronically ill individuals. Many of them are additionally infected with the Hepatitis D virus, a satellite virus to HBV. Chronic infection frequently leads to serious liver diseases including cirrhosis and hepatocellular carcinoma, the most common type of liver cancer. Although current antiviral therapies can control HBV replication and slow down disease progress, there is an unmet medical need to identify therapies to cure this chronic infectious disease. Lately, a noteworthy progress in fighting against HBV has been made by identification of the high-affinity hepatic host receptor for HBV and HDV, namely Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1). Next to its primary function as hepatic uptake transporter for bile acids, NTCP is essential for the cellular entry of HBV and HDV into hepatocytes. Due to this high-ranking discovery, NTCP has become a valuable target for drug development strategies for HBV/HDV-infected patients. In this review, we will focus on a newly predicted three-dimensional NTCP model that was generated using computational approaches and discuss its value in understanding the NTCP’s membrane topology, substrate and virus binding taking place in plasma membranes. We will review existing data on structural, functional, and biological consequences of amino acid residue changes and mutations that lead to loss of NTCP’s transport and virus receptor functions. Finally, we will discuss new directions for future investigations aiming at development of new NTCP-based HBV entry blockers that inhibit HBV tropism in human hepatocytes. Full article
(This article belongs to the Special Issue Non-canonical Functions of Membrane Proteins: Novel Opportunities for Drug Discovery)
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<p>Multiple sequence alignment of NTCP/Ntcps from different species. (<b>A</b>) Phylogenetic relationship between human NTCP (hNTCP; Uniprot: Q14973), chimpanzee Ntcp (chNtcp; H2Q8J0), rhesus monkey Ntcp (rhNtcp; F6YRK3), rat Ntcp (rNtcp, Uniprot: P26435) and mouse Ntcp (mNtcp; Uniprot: O08705). (<b>B</b>) Deduced amino acid sequences from above-mentioned species were aligned using EBI ClustalW algorithm. Positions of transmembrane domains (TMD) are indicated with the color code also used in <a href="#biomedicines-10-00196-f002" class="html-fig">Figure 2</a>. Identical amino acids among all species are marked with grey shading. The HBV/HDV preS1-peptide binding motifs of hNTCP <sub>84</sub>RLKN<sub>87</sub> and <sub>157</sub>KGIVISLVL<sub>165</sub> are marked with red boxes and amino acids regulating bile acid transport are labeled with black boxes. The highly conserved serine at position 267 that is relevant for bile acid binding and HBV/HDV infection is colored in red.</p> Full article ">Figure 2
<p>Three-dimensional model of human NTCP predicted using AlphaFold. (<b>A</b>) Schematic representation of nine transmembrane domains (TMDs I-IX) of human NTCP with indicated aa positions of the α-helices (Arabic numbers). Transmembrane domains are marked (Greek letters) and colored: I, IV and V (green, panel domain); II, III, IV (blue, core domain); and VII, VIII and IX (orange, core domain). N-terminal glycosylation of the N5 and N11 are demonstrated as “Y”. (<b>B</b>) Proposed membrane topology of human NTCP based on AlphaFold prediction (AF-Q14973-F1-model_v1). (<b>C</b>) Backbone structure of human NTCP protein, where α-helices are represented by coiled ribbons, and protein loops are shown as thin lines. Positions of N- and C-termini are labeled. Two identical structures are related by a 90-degree rotation. The model was visualized by the Protean 3D DNASTAR Software.</p> Full article ">Figure 3
<p>NTCP regions and amino acids essential for bile acid transport and HBV/HDV binding. The AlphaFold model of human NTCP (AF-Q14973-F1) was visualized with Protean 3D DNASTAR Software. (Top panel) To better visualize amino acids regulating bile acid transport, the NTCP “panel” domain (TMDs I, V and VI) was made transparent. Positions of core-localized amino acids, namely C44, G60, Q68, S105, N106, D115, S119, C170, I223, R252, E257, Q261, C266, S267, I279, F285, P286 and L287 are marked and colored in red. Two identical structures are related by a 90-degree rotation. (Lower panel) Transparent surface presentation of NTCP. Positions of amino acids involved in preS1-binding activity are labeled with colors (<sub>157</sub>KGIVISLVL<sub>165</sub> (green), 158G (black), <sub>84</sub>RLKN<sub>87</sub> (blue), 267S (red)) (top panel)<b>.</b> Two identical structures are related by a 180 and 90-degree rotation.</p> Full article ">
16 pages, 1987 KiB  
Article
Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease
by Lauren A. Newman, Zivile Useckaite, Jillian Johnson, Michael J. Sorich, Ashley M. Hopkins and Andrew Rowland
Biomedicines 2022, 10(1), 195; https://doi.org/10.3390/biomedicines10010195 - 17 Jan 2022
Cited by 37 | Viewed by 5531
Abstract
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools [...] Read more.
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. Full article
(This article belongs to the Special Issue NAFLD: From Mechanisms to Therapeutic Approaches)
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<p>Study workflow. Plasma samples from patients with non-alcoholic fatty liver disease and matched healthy controls were purchased from Discovery Life Sciences (DLS). Samples were aliquoted for miRNA quantitation directly from plasma and from EVs following their isolation by qEV size exclusion chromatography (SEC) and immunoprecipitation (IP). EVs isolated by qEV were characterised by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and protein expression. Figure was created using BioRender.com [<a href="https://app.biorender.com/" target="_blank">https://app.biorender.com/</a>; accessed 17 January 2022].</p> Full article ">Figure 2
<p>Characterisation of global circulating EV isolated from control, NAFL, and NASH subjects. (<b>a</b>) Particle concentration and size distribution by nanoparticle tracking analysis (NTA) (<span class="html-italic">n</span> = 5). Error bars denote SEM. (<b>b</b>) Representative TEM images of NASH patient and control global EVs. (<b>c</b>) Relative abundance of EV protein markers determined by mass spectrometry. Error bars denote SD. ** <span class="html-italic">p</span> ≤ 0.01.</p> Full article ">Figure 3
<p>Differential expression of miRNA biomarkers in NAFLD. Relative quantities of miR-122 (<b>a</b>), miR-192 (<b>b</b>), and miR-128-3p (<b>c</b>) normalised to cel-miR-54 in total circulating RNA, global EVs, and asialoglycoprotein receptor 1 (ASGR1) positive EVs isolated from patients with NAFL and NASH and from controls. Statistical analysis performed by the Kruskal-Wallis test with Dunn’s for multiple comparisons. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> ≤ 0.001. Error bars represent standard deviation.</p> Full article ">Figure 4
<p>Relative expression of miRNA in liver-specific EVs. Expressions of miR-122 (<b>a</b>), miR-192 (<b>b</b>), and miR-128-3p (<b>c</b>) in asialoglycoprotein receptor 1 positive (ASGR1+) EVs as a percentage of global EVs in NAFL and NASH patients compared with controls. Statistical analysis performed by the Kruskal–Wallis test with Dunn’s for multiple comparisons. * <span class="html-italic">p</span> ≤ 0.05. Error bars represent standard deviation.</p> Full article ">Figure 5
<p>Ordinal logistic regression models. Ordinal regression applied to distinguish control, NAFL and NASH subjects using ASGR1+ EV-derived miR-122 (<b>a</b>), miR-192 (<b>b</b>), and miR-128-3p (<b>c</b>).</p> Full article ">
33 pages, 2269 KiB  
Review
Detailed Molecular Mechanisms Involved in Drug-Induced Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis: An Update
by Laura Giuseppina Di Pasqua, Marta Cagna, Clarissa Berardo, Mariapia Vairetti and Andrea Ferrigno
Biomedicines 2022, 10(1), 194; https://doi.org/10.3390/biomedicines10010194 - 17 Jan 2022
Cited by 11 | Viewed by 7712
Abstract
Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are some of the biggest public health challenges due to their spread and increasing incidence around the world. NAFLD is characterized by intrahepatic lipid deposition, accompanied by dyslipidemia, hypertension, and insulin resistance, leading to [...] Read more.
Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are some of the biggest public health challenges due to their spread and increasing incidence around the world. NAFLD is characterized by intrahepatic lipid deposition, accompanied by dyslipidemia, hypertension, and insulin resistance, leading to more serious complications. Among the various causes, drug administration for the treatment of numerous kinds of diseases, such as antiarrhythmic and antihypertensive drugs, promotes the onset and progression of steatosis, causing drug-induced hepatic steatosis (DIHS). Here, we reviewed in detail the major classes of drugs that cause DIHS and the specific molecular mechanisms involved in these processes. Eight classes of drugs, among the most used for the treatment of common pathologies, were considered. The most diffused mechanism whereby drugs can induce NAFLD/NASH is interfering with mitochondrial activity, inhibiting fatty acid oxidation, but other pathways involved in lipid homeostasis are also affected. PubMed research was performed to obtain significant papers published up to November 2021. The key words included the class of drugs, or the specific compound, combined with steatosis, nonalcoholic steatohepatitis, fibrosis, fatty liver and hepatic lipid deposition. Additional information was found in the citations listed in other papers, when they were not displayed in the original search. Full article
(This article belongs to the Special Issue Drug-Induced Hepatotoxicity: From Pathology to Novel Therapeutic Approaches)
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<p>Schematic representation of the molecular mechanisms involved in NAFLD onset. 1. Fatty acid uptake: fatty acids are introduced in the cell by specific transporters such as CD36, FATP2/5 and caveolins, which are controlled by PPARγ transcriptional activity. 2. De novo lipogenesis: the liver synthesizes fatty acids starting from acetyl CoA. This mechanism is controlled by ChREBP and SREBP-1c activity. The new fatty acid can be stored as triglycerides or exported via VLDL formation. 3. Fatty acid export: VLDL particles are produced by lipidation of apoB100 in the ER and then they are transferred to the Golgi apparatus for a second lipidation that is necessary for maturation and export. 4. Fatty acid oxidation: Fatty acids introduced from the external environment or produced by de novo lipogenesis can be oxidized to form energy by mitochondrial and peroxisomal β-oxidation and by cytochrome ω-oxidation. All these processes produce ROS.</p> Full article ">Figure 2
<p>Schematic representation of one of the molecular mechanisms involved in amiodarone/perhexiline-induced NAFLD. The ATP depletion caused by amiodarone/perhexiline interference with OXPHOS leads to the reduced activity of the smooth endoplasmic reticulum Ca<sup>2+</sup> pump (SERCA). The reduction in ER Ca<sup>2+</sup> produces ER stress and the upregulation of CHOP activity with consequent increase in the activity of the lipid droplet proteins cell death activator (Cidea), cell death inducing DFFA like effector C (Cidec), and perilipin-2, which are involved in lipid accumulation.</p> Full article ">Figure 3
<p>Linezolid activity on mitochondrial ribosomes. Linezolid stops the protein synthesis in bacteria by binding their ribosomes. At the same time, this drug can bind human mitochondrial ribosomes inhibiting the bond of aminoacyl tRNAs, blocking the mtDNA translation and reducing the mitochondrial respiration chain complexes activity. This process, after several weeks, produces micro and microvacuolar steatosis.</p> Full article ">Figure 4
<p>Schematic representation of glucocorticoid receptor β (GRβ) contribution in hepatic lipid deposition. GRβ overexpression induces both the inhibition of the PPARα-FGF21 signal pathway, decreasing β-oxidation of fatty acid, and at the same time is involved in inflammatory process establishment by the secretion of TNF-α and upregulation of iNOS protein expression. This condition leads to further lipid deposition and macrophage hepatic infiltration.</p> Full article ">Figure 5
<p>Ibuprofen S<sup>+</sup> and R<sup>-</sup> structure. The majority of NSAIDs are commercialized as the racemate mixture.</p> Full article ">
13 pages, 759 KiB  
Article
Effects of Alirocumab on Triglyceride Metabolism: A Fat-Tolerance Test and Nuclear Magnetic Resonance Spectroscopy Study
by Thomas Metzner, Deborah R. Leitner, Karin Mellitzer, Andrea Beck, Harald Sourij, Tatjana Stojakovic, Gernot Reishofer, Winfried März, Ulf Landmesser, Hubert Scharnagl, Hermann Toplak and Günther Silbernagel
Biomedicines 2022, 10(1), 193; https://doi.org/10.3390/biomedicines10010193 - 17 Jan 2022
Cited by 4 | Viewed by 3404
Abstract
Background: PCSK9 antibodies strongly reduce LDL cholesterol. The effects of PCSK9 antibodies on triglyceride metabolism are less pronounced. The present study aimed to investigate in detail the effects of alirocumab on triglycerides, triglyceride-rich lipoproteins, and lipase regulators. Methods: A total of 24 patients [...] Read more.
Background: PCSK9 antibodies strongly reduce LDL cholesterol. The effects of PCSK9 antibodies on triglyceride metabolism are less pronounced. The present study aimed to investigate in detail the effects of alirocumab on triglycerides, triglyceride-rich lipoproteins, and lipase regulators. Methods: A total of 24 patients with an indication for treatment with PCSK9 antibodies were recruited. There were two visits at the study site: the first before initiation of treatment with alirocumab and the second after 10 weeks of treatment. Fat-tolerance tests, nuclear magnetic resonance spectroscopy, and enzyme-linked immunosorbent assays were performed to analyze lipid metabolism. Results: A total of 21 participants underwent the first and second investigation. Among these, two participants only received alirocumab twice and 19 patients completed the trial per protocol. All of them had atherosclerotic vascular disease. There was no significant effect of alirocumab treatment on fasting triglycerides, post-prandial triglycerides, or lipoprotein-lipase regulating proteins. Total, large, and small LDL particle concentrations decreased, while the HDL particle concentration increased (all p < 0.001). Mean total circulating PCSK9 markedly increased in response to alirocumab treatment (p < 0.001). Whereas PCSK9 increased more than three-fold in all 19 compliant patients, it remained unchanged in those two patients with two injections only. Conclusion: Significant effects of alirocumab on triglyceride metabolism were not detectable in the ALIROCKS trial. The total circulating PCSK9 concentration might be a useful biomarker to differentiate non-adherence from non-response to PCSK9 antibodies. Full article
(This article belongs to the Special Issue Lipids and Lipoproteins in Atherosclerosis: A Commemorative Issue in Honor of Dr. Vladimir V Tertov)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Fat-tolerance test results with Lipotest<sup>®</sup> meal at baseline and after 10 weeks of treatment with alirocumab. Legend: Triglyceride plasma levels at baseline and after 10 weeks of alirocumab treatment, before Lipotest<sup>®</sup> meal consumption (t0), at 2 h (t2), and 4 h (t4). Among the trial completion population, 14 patients participated in the per-protocol optional fat-tolerance assessment (<span class="html-italic">n</span> = 14). The small circles at the upper part of the figure identify outlier measurements. Outliers are defined as values between 1.5× and 3× interquartile ranges from the end of a box. Paired-samples <span class="html-italic">t</span>-test with two-sided <span class="html-italic">p</span>-value was performed.</p> Full article ">Figure 2
<p>Plasma PCSK9 increase per patient in response to alirocumab treatment. Legend: The figure includes the 19 adherent patients and 2 non-adherent (NA) patients that did not receive alirocumab beyond week 2 (<span class="html-italic">n</span> = 21). The used enzyme-linked immunosorbent assay measured total plasma PCSK9 levels, and thus did not differentiate between bound and unbound PCSK9.</p> Full article ">
12 pages, 2341 KiB  
Article
Neuroprotective Effects of Novel Treatments on Acute Optic Neuritis—A Meta-Analysis
by Tsung-Hsien Tsai, Chao-Wen Lin, Li-Wei Chan, Teck-Boon Tew and Ta-Ching Chen
Biomedicines 2022, 10(1), 192; https://doi.org/10.3390/biomedicines10010192 - 17 Jan 2022
Viewed by 2217
Abstract
Optic neuritis, inflammation of the optic nerve, can cause visual impairment through retinal nerve fiber layer (RNFL) degeneration. Optical coherence tomography could serve as a sensitive noninvasive tool for measuring RNFL thickness and evaluating the neuroprotective effects of treatment. We conducted a meta-analysis [...] Read more.
Optic neuritis, inflammation of the optic nerve, can cause visual impairment through retinal nerve fiber layer (RNFL) degeneration. Optical coherence tomography could serve as a sensitive noninvasive tool for measuring RNFL thickness and evaluating the neuroprotective effects of treatment. We conducted a meta-analysis to compare RNFL loss between novel add-on treatments and corticosteroid therapy at least 3 months after acute optic neuritis. The outcome measures were mean differences (MDs) in (1) RNFL thickness compared with the baseline in the affected and unaffected eye and (2) LogMAR visual acuity (VA). Seven studies involving five novel agents (memantine, erythropoietin, interferon-beta, phenytoin, and clemastine) were analyzed. When compared with the baseline RNFL thickness of the affected eye, the neuroprotective effects of novel add-on treatments could not be demonstrated. The difference in visual outcomes was also not significant between the two treatment groups. One study revealed that phenytoin has the potential to alleviate RNFL loss when the baseline thickness of the unaffected eye is considered. Larger randomized controlled trials with suitable outcome measures are warranted to evaluate the neuroprotective effects of novel treatments. Further studies should also tailor therapies to specific patient populations and investigate a more targeted treatment for acute optic neuritis. Full article
(This article belongs to the Section Neurobiology and Clinical Neuroscience)
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<p>Flow diagram of the study selection process.</p> Full article ">Figure 2
<p>Methodological quality of included randomized controlled trials.</p> Full article ">Figure 3
<p>Mean differences in retinal nerve fiber layer thickness compared with baseline data of the affected eye. (CI, confidence interval; IV, inverse variance; SD, standard deviation; EPO, erythropoietin; INF, interferon).</p> Full article ">Figure 4
<p>Mean differences in retinal nerve fiber layer thickness compared with baseline data of the unaffected eye. (CI, confidence interval; IV, inverse variance; SD, standard deviation; INF, interferon).</p> Full article ">Figure 5
<p>Mean differences in LogMAR visual acuity. (CI, confidence interval; IV, inverse variance; SD, standard deviation; EPO, erythropoietin).</p> Full article ">
8 pages, 761 KiB  
Article
Pretherapeutic Serum Albumin as an Outcome Prognosticator in Head and Neck Adenoid-Cystic Carcinoma
by Marlene Friedl, Stefan Stoiber, Faris F. Brkic and Lorenz Kadletz-Wanke
Biomedicines 2022, 10(1), 191; https://doi.org/10.3390/biomedicines10010191 - 17 Jan 2022
Cited by 4 | Viewed by 2206
Abstract
Background: A head and neck adenoid-cystic carcinoma is a rare malignant tumor arising from the salivary gland tissues. The long-term survival outcome is poor due to a high risk of recurrences and distant metastasis. The identification of prognostic markers could contribute to a [...] Read more.
Background: A head and neck adenoid-cystic carcinoma is a rare malignant tumor arising from the salivary gland tissues. The long-term survival outcome is poor due to a high risk of recurrences and distant metastasis. The identification of prognostic markers could contribute to a better risk assessment of each patient. The aim of this study is to assess the potential prognostic value of serum albumin in patients with head and neck adenoid-cystic carcinomas. Patients and Methods: This retrospective cohort study included all patients treated for a head and neck adenoid-cystic carcinoma between 1993 and 1 June 2019 with available pretherapeutic albumin values and clinical follow-up data. The cohort was stratified into a high and low group according to the median albumin value. The log-rank test was used for comparing overall and disease-free survival. Results: A total of 37 patients with complete follow-up data and available pretreatment albumin values were available. The overall mortality and recurrence rates were 21.6% (n = 8) and 45.9% (n = 17), respectively. Survival was shorter in the low albumin group. In particular, the mean overall survival for the low and high albumin groups were 121.0 months and 142.8 months, respectively. However, the difference was not statistically significant (p = 0.155). A statistically significant difference was observed in context with disease-free survival (45.2 months, 95% confidence interval 31.7–58.8 months vs. 114.8 months, 95% confidence interval 79.3–150.4 months; p = 0.029). Conclusion: Our study suggests a potential prognostic value of serum albumin in patients with a head and neck ACC. A further, external validation of our results is warranted. Full article
(This article belongs to the Special Issue Liquid Biopsies in Cancer Diagnosis, Monitoring and Prognosis)
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<p>Kaplan–Meier survival curve for OS for patients stratified according to median pretherapeutic serum albumin value. The OS for the low albumin group (<span class="html-italic">n</span> = 19) was shorter than for the high albumin group (<span class="html-italic">n</span> = 18). A log-rank test revealed no significant difference (<span class="html-italic">p</span> = 0.155). OS: overall survival.</p> Full article ">Figure 2
<p>Kaplan–Meier survival curve for DFS for patients stratified according to median pretherapeutic serum albumin value. The DFS for the low albumin group (<span class="html-italic">n</span> = 19) was shorter than for the high albumin group (<span class="html-italic">n</span> = 18). A log-rank test revealed a statistically significant difference (<span class="html-italic">p</span> = 0.029). DFS: disease-free survival.</p> Full article ">
8 pages, 581 KiB  
Article
Elevated Plasma Apurinic/Apyrimidinic Endonuclease 1/Redox Effector Factor-1 Levels in Refractory Kawasaki Disease
by Yu-Ran Lee, Eun Young Bae, Hong Ryang Kil, Byeong-Hwa Jeon and Geena Kim
Biomedicines 2022, 10(1), 190; https://doi.org/10.3390/biomedicines10010190 - 17 Jan 2022
Viewed by 1731
Abstract
Kawasaki disease (KD) refers to systemic vasculitis of medium-sized vessels accompanied by fever. The multifunctional protein apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is a new biomarker for vascular inflammation. Here, we investigated the association between APE1/Ref-1 and KD. Three groups, including 32 patients with KD [...] Read more.
Kawasaki disease (KD) refers to systemic vasculitis of medium-sized vessels accompanied by fever. The multifunctional protein apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is a new biomarker for vascular inflammation. Here, we investigated the association between APE1/Ref-1 and KD. Three groups, including 32 patients with KD (KD group), 33 patients with fever (Fever group), and 19 healthy individuals (Healthy group), were prospectively analyzed. APE1/Ref-1 levels were measured, and the clinical characteristics of KD were evaluated. The mean age of all patients was 2.7 ± 1.8 years, but the Healthy group participants were older than the other participants. Fever duration was longer in the KD group than in the fever group. APE1/Ref-1 levels were significantly higher in the KD group (p = 0.004) than in the other two groups, but there was no difference between the healthy and fever groups. APE1/Ref-1 levels did not differ according to fever duration or coronary arterial lesion but were higher in refractory KD cases than in non-refractory cases. APE1/Ref-1 levels were significantly higher during the acute phase of KD. We propose that APE1/Ref-1 could be a beneficial biological marker for the diagnosis and prognosis of KD, especially in refractory KD. Full article
(This article belongs to the Special Issue Vascular Inflammation: From Target Molecules to Therapeutic Approaches)
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<p>APE1/Ref-1 levels in the three groups, i.e., KD, fever, and healthy control groups. (<b>A</b>) APE1/Ref-1 levels were higher in the KD group than in the fever group (** <span class="html-italic">p</span> = 0.019) and the healthy control group (* <span class="html-italic">p</span> = 0.007). (<b>B</b>) The ROC curve of APE1/Ref-1 predicting KD, the cutoff value of APE1/Ref-1 predicting KD compared with that in the fever group was 0.542 ng/mL (AUC = 0.682; sensitivity of 60.6%; specificity of 62.5%), and the cutoff value of APE1/Ref-1 predicting KD compared with that in the healthy group was 0.482 ng/mL (AUC = 0.734; sensitivity of 68.4%; specificity of 68.7%), KD, Kawasaki disease; HC, healthy control.</p> Full article ">Figure 2
<p>APE1/Ref-1 levels between the fever, non-refractory KD, and refractory KD groups. (<b>A</b>) APE1/Ref-1 levels between the fever and refractory KD groups showed a significant difference (** <span class="html-italic">p</span> = 0.011) and those between the non-refractory and refractory KD also showed a significant difference (* <span class="html-italic">p</span> = 0.046) (<b>B</b>) The ROC curve of APE1/Ref-1 predicting refractory KD, (Fever vs. refractory KD, AUC = 0.734, Refractory vs. non-refractory, AUC = 0.725).</p> Full article ">
17 pages, 2149 KiB  
Review
Activation of STAT and SMAD Signaling Induces Hepcidin Re-Expression as a Therapeutic Target for β-Thalassemia Patients
by Hanan Kamel M. Saad, Alawiyah Awang Abd Rahman, Azly Sumanty Ab Ghani, Wan Rohani Wan Taib, Imilia Ismail, Muhammad Farid Johan, Abdullah Saleh Al-Wajeeh and Hamid Ali Nagi Al-Jamal
Biomedicines 2022, 10(1), 189; https://doi.org/10.3390/biomedicines10010189 - 17 Jan 2022
Cited by 7 | Viewed by 5530
Abstract
Iron homeostasis is regulated by hepcidin, a hepatic hormone that controls dietary iron absorption and plasma iron concentration. Hepcidin binds to the only known iron export protein, ferroportin (FPN), which regulates its expression. The major factors that implicate hepcidin regulation include iron [...] Read more.
Iron homeostasis is regulated by hepcidin, a hepatic hormone that controls dietary iron absorption and plasma iron concentration. Hepcidin binds to the only known iron export protein, ferroportin (FPN), which regulates its expression. The major factors that implicate hepcidin regulation include iron stores, hypoxia, inflammation, and erythropoiesis. When erythropoietic activity is suppressed, hepcidin expression is hampered, leading to deficiency, thus causing an iron overload in iron-loading anemia, such as β-thalassemia. Iron overload is the principal cause of mortality and morbidity in β-thalassemia patients with or without blood transfusion dependence. In the case of thalassemia major, the primary cause of iron overload is blood transfusion. In contrast, iron overload is attributed to hepcidin deficiency and hyperabsorption of dietary iron in non-transfusion thalassemia. Beta-thalassemia patients showed marked hepcidin suppression, anemia, iron overload, and ineffective erythropoiesis (IE). Recent molecular research has prompted the discovery of new diagnostic markers and therapeutic targets for several diseases, including β-thalassemia. In this review, signal transducers and activators of transcription (STAT) and SMAD (structurally similar to the small mothers against decapentaplegic in Drosophila) pathways and their effects on hepcidin expression have been discussed as a therapeutic target for β-thalassemia patients. Therefore, re-expression of hepcidin could be a therapeutic target in the management of thalassemia patients. Data from 65 relevant published experimental articles on hepcidin and β-thalassemia between January 2016 and May 2021 were retrieved by using PubMed and Google Scholar search engines. Published articles in any language other than English, review articles, books, or book chapters were excluded. Full article
(This article belongs to the Section Cell Biology and Pathology)
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<p>Iron distribution in the human body. Iron is absorbed by the duodenum enterocytes and into the plasma, where transferrin delivers it to bone marrow for Hb synthesis by erythroid precursors and erythrocytes or to muscles for myoglobin synthesis. Excess iron in the blood circulation can be stored as ferritin molecules in the liver or macrophages. The regular daily iron loss (1–2 mg) occurs mainly through blood loss (hemorrhage or menstruation).</p> Full article ">Figure 2
<p>Hepcidin regulation on iron homeostasis: <span class="html-italic">hepcidin</span> synthesis is regulated at the transcriptional level by multiple stimuli. <span class="html-italic">Hepcidin</span> transcription increased with rising intra–extracellular iron concentrations and inflammation. In contrast, hepcidin production is suppressed in response to higher erythropoietic activity. Iron concentration in plasma is regulated by <span class="html-italic">hepcidin</span> through controlling <span class="html-italic">FPN</span> concentrations in iron exporting cells (duodenal, enterocytes, hepatocytes, and macrophages from liver and spleen). <b>➔</b>: resulting in or enhances expression and <b>⊥:</b> reduced expression.</p> Full article ">Figure 3
<p>Mechanism of iron dysregulation in β-thalassemia syndrome. Affected patients experience anemia because of IE and shortened red blood cell (RBCs) survival. This condition induces erythropoietin production, leading to enhanced erythropoiesis. The dramatic increase in erythroid expansion activates the erythroid factors, including GDF15, TWSG1, and ERFE secretion. Excessive erythroid factors suppress <span class="html-italic">hepcidin</span> expression in liver cells, resulting in iron overload due to increased iron absorption from duodenal enterocytes, an increase in iron from hepatocytes and the reticuloendothelial system. <b>➔</b><b>:</b> resulting in and <b>⊥</b><b>:</b> suppresses expression.</p> Full article ">Figure 4
<p>Several regulatory pathways, including JAK/STAT in <span class="html-italic">hepcidin</span> transcription. The activation of JAKs after ligand–receptor coupling stimulates phosphorylation of STATs, followed by STAT dimerization and nucleus translocation to activate <span class="html-italic">hepcidin</span> transcription. <b>➔:</b> Activation/Nucleus translocation.</p> Full article ">Figure 5
<p>Signal transduction of TGF-β and BMP. The binding of TGF-β to the TβRII dimer allows the ligand to bind to the TβRI dimer and stimulate TβRI kinase activity. In SMAD-mediated TGF-β signal transduction, TβRI phosphorylates cytoplasmic SMAD2 and SMAD3, which interact with SMAD4 after dissociating from TβRI. The two receptors activate the trimeric complex of SMAD2 and SMAD3, and a SMAD4 then enters the nucleus, where it interacts with the DNA-binding transcription factor (TF) and coregulators of the target gene. Similarly, the BMP signals run parallel to the TGF-β signals. In response to the binding of the BMP ligand to the BMPRII heteromeric receptor complex and BMPRI transmembrane kinase, receptor-activated SMAD1 and SMAD5 bind to SMAD4 and are transported to the nucleus to activate or inhibit transcription of <span class="html-italic">hepcidin</span>. <b>➔:</b> Activation and <b>⊥:</b> Inhibit.</p> Full article ">Figure 6
<p>Regulation of STAT and SMAD signaling pathway on hepcidin expression.</p> Full article ">
13 pages, 2191 KiB  
Article
Penetration of the SARS-CoV-2 Spike Protein across the Blood–Brain Barrier, as Revealed by a Combination of a Human Cell Culture Model System and Optical Biosensing
by Dániel Petrovszki, Fruzsina R. Walter, Judit P. Vigh, Anna Kocsis, Sándor Valkai, Mária A. Deli and András Dér
Biomedicines 2022, 10(1), 188; https://doi.org/10.3390/biomedicines10010188 - 17 Jan 2022
Cited by 26 | Viewed by 9976
Abstract
Since the outbreak of the global pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), several clinical aspects of the disease have come into attention. Besides its primary route of infection through the respiratory system, SARS-CoV-2 is known to have neuroinvasive capacity, causing [...] Read more.
Since the outbreak of the global pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), several clinical aspects of the disease have come into attention. Besides its primary route of infection through the respiratory system, SARS-CoV-2 is known to have neuroinvasive capacity, causing multiple neurological symptoms with increased neuroinflammation and blood–brain barrier (BBB) damage. The viral spike protein disseminates via circulation during infection, and when reaching the brain could possibly cross the BBB, which was demonstrated in mice. Therefore, its medical relevance is of high importance. The aim of this study was to evaluate the barrier penetration of the S1 subunit of spike protein in model systems of human organs highly exposed to the infection. For this purpose, in vitro human BBB and intestinal barrier cell–culture systems were investigated by an optical biosensing method. We found that spike protein crossed the human brain endothelial cell barrier effectively. Additionally, spike protein passage was found in a lower amount for the intestinal barrier cell layer. These observations were corroborated with parallel specific ELISAs. The findings on the BBB model could provide a further basis for studies focusing on the mechanism and consequences of spike protein penetration across the BBB to the brain. Full article
(This article belongs to the Special Issue Biosensors at the Aid of Medicine)
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<p>The schematic representation of the SARS-CoV-2 and its surface spike protein structure with their structural descriptions and detailed mechanisms of the viral entry to cells during infection. Spike protein plays a crucial role in this process. While S1 subunit is responsible for anchoring the virion by binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) of the host cell, S2 subunit enhances the fusion of the viral and the host cell membranes. The fusion is mediated by the S2 subunit that is activated by the transmembrane protease serine 2 (TMPRSS2) cleaving the spike protein at the S1/S2 sites. Adapted from “An In-depth Look into the Structure of the SARS-CoV2 Spike Glycoprotein”, “Human Coronavirus Structure” and “Mechanism of SARS-CoV-2 Viral Entry” by <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 30 November 2021) [<a href="#B3-biomedicines-10-00188" class="html-bibr">3</a>].</p> Full article ">Figure 2
<p>Schematic representation of the biosensor device: the integrated optical Mach–Zehnder interferometer (MZI) for sensing the analyte (1), the microfluidic apparatus (syringe pump, tubes, PDMS microchannel) for fluid sample providing (2), the signal processing unit, namely a photomultiplier tubes (PMT) detector (3) with an oscilloscope (4), the microheater structure for bias point tuning (5). The working principle of the device is also presented: the evanescent field detection is based on the phase difference in the propagating light of the measuring arm (yellow waves) compared to the ones of the reference arm (red waves) (6). Phase difference can be induced by the binding of the target spike protein S1 subunit to the antibody-covered surface of the measuring arm. The figure was created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p> Full article ">Figure 3
<p>Results of the interferometric biosensing by the integrated optical MZI sensor. Both graphs show the signals detected after the introduction of the calibration samples (2 and 20 µg/mL). (<b>a</b>) Results obtained from samples after the passage of the spike protein S1 subunit across cell-free cell culture inserts and the BBB model. (<b>b</b>) Signals detected from buffer (0.1% BSA-RH) or samples after the permeability of the spike protein through the Caco-2 monolayer.</p> Full article ">Figure 4
<p>(<b>a</b>) Results of the transendothelial–transepithelial electrical resistance (TEER) measurement after SARS-CoV-2 spike protein (200 µg/mL) treatment in 0.1% bovine serum albumin (BSA)–Ringer–HEPES buffer or only after buffer treatment (0.1% BSA). (<b>b</b>) Detection of spike protein S1 subunit by ELISA in samples from the bottom compartment of cell culture inserts after spike protein treatment (200 µg/mL, 30 min). We also indicate results for inserts, which received only the carrier buffer (0.1% BSA) or the results of passage of spike protein across cell-free inserts. Caco-2: human intestinal epithelial cells. BBB: blood–brain barrier model.</p> Full article ">
12 pages, 2755 KiB  
Article
Effect of Ulinastatin on Syndecan-2-Mediated Vascular Damage in IDH2-Deficient Endothelial Cells
by Su-jeong Choi, Harsha Nagar, Jun Wan Lee, Seonhee Kim, Ikjun Lee, Shuyu Piao, Byeong Hwa Jeon and Cuk-Seong Kim
Biomedicines 2022, 10(1), 187; https://doi.org/10.3390/biomedicines10010187 - 17 Jan 2022
Cited by 4 | Viewed by 2213
Abstract
Syndecan-2 (SDC2), a cell-surface heparin sulfate proteoglycan of the glycocalyx, is mainly expressed in endothelial cells. Although oxidative stress and inflammatory mediators have been shown to mediate dysfunction of the glycocalyx, little is known about their role in vascular endothelial cells. In this [...] Read more.
Syndecan-2 (SDC2), a cell-surface heparin sulfate proteoglycan of the glycocalyx, is mainly expressed in endothelial cells. Although oxidative stress and inflammatory mediators have been shown to mediate dysfunction of the glycocalyx, little is known about their role in vascular endothelial cells. In this study, we aimed to identify the mechanism that regulates SDC2 expression in isocitrate dehydrogenase 2 (IDH2)-deficient endothelial cells, and to investigate the effect of ulinastatin (UTI) on this mechanism. We showed that knockdown of IDH2 induced SDC2 expression in human umbilical vein endothelial cells (HUVECs). Matrix metalloproteinase 7 (MMP7) influences SDC2 expression. When IDH2 was downregulated, MMP7 expression was increased, as was TGF-β signaling, which regulates MMP7. Inhibition of MMP7 activity using MMP inhibitor II significantly reduced SDC2, suggesting that IDH2 mediated SDC2 expression via MMP7. Moreover, expression of SDC2 and MMP7, as well as TGF-β signaling, increased in response to IDH2 deficiency, and treatment with UTI reversed this increase. Similarly, the increase in SDC2, MMP7, and TGF-β signaling in the aorta of IDH2 knockout mice was reversed by UTI treatment. These findings suggest that IDH2 deficiency induces SDC2 expression via TGF-β and MMP7 signaling in endothelial cells. Full article
(This article belongs to the Section Molecular and Translational Medicine)
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<p>Expression of Syndecans in IDH2 deficient HUVECs. HUVECs were transfected with dose dependent siIDH2 (10 pmol, 50 pmol) and siCON for 48h. mRNA expressions of (<b>a</b>) SDC1, (<b>b</b>) SDC2, (<b>c</b>) SDC3, (<b>d</b>) SDC4 were quantified by qPCR (<b>e</b>) protein level of SDC2 was detected by western blotting. β-actin was used as a loading control. Protein levels were quantified by densitometric analysis using Image-J software (Shown in the right panel). (<b>f</b>) Immunofluorescence analysis of HUVECs to compare the SDC2 expression in siCON and siIDH2 (50 pmol) transfected HUVECs (scale bar 100 μm). All data are presented as means ± SEM of three independent experiments, * <span class="html-italic">p</span> &lt; 0.05, compared with siCON.</p> Full article ">Figure 2
<p>Involvement of TGF-β and MMP7 signaling in relation to SDC2 shedding in IDH2 deficient HUVECs. HUVECs were transfected with 50 pmol of siIDH2 and siCON for 48 h. (<b>a</b>) mRNA expression of TGF-β was quantified by qPCR. Protein levels of (<b>b</b>) MMP7 (<b>c</b>) β-catenin and (<b>d</b>) TGF-β signaling-related proteins were detected by western blotting. β-actin was used as a loading control. Protein levels were quantified by densitometric analysis using Image-J software (Shown in the right panel). HUVECs were transfected with 50 pmol of siIDH2 and siCON, incubated for 24 h and treated with MMP inhibitor II (25 μM) for another 24 h. Protein levels of (<b>e</b>) MMP7 and SDC2 were detected by western blotting. β-actin was used as a loading control. Protein levels were quantified by densitometric analysis using Image-J software (Shown in the right panel). All data are presented as means ± SEM of three independent experiments, * <span class="html-italic">p</span> &lt; 0.05, compared with siCON, <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared with the 50 pmol siIDH2 treated cells.</p> Full article ">Figure 3
<p>Effect of UTI on SDC2, MMP7 and TGF-β signaling in IDH2 deficient HUVECs. HUVECs were transfected with 50 pmol of siIDH2 and siCON and 1000 U/mL of UTI. mRNA expressions of (<b>a</b>) SDC2 and (<b>c</b>) MMP7 were quantified by qPCR. Protein levels of (<b>b</b>) SDC2 and (<b>d</b>) MMP7 were detected by western blotting. (<b>e</b>) Protein level of TGF-β signaling was detected by western blotting. β-actin was used as a loading control. Protein levels were quantified by densitometric analysis using Image-J software (Shown in the right panel). All data are presented as means ± SEM of three independent experiments, * <span class="html-italic">p</span> &lt; 0.05, compared with siCON, <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared with the 50 pmol siIDH2 treated cells.</p> Full article ">Figure 4
<p>Effect of UTI on SDC2, MMP7 and TGF-β signaling in aorta from IDH2 KO mice. Aorta were isolated from WT and IDH2 KO mice and after peritoneal injection of UTI in WT and IDH2 KO mice. (<b>a</b>,<b>b</b>) Protein level of SDC2, (<b>c</b>,<b>d</b>) TGF-β and MMP7 were detected by western blotting. β-actin was used as a loading control. Protein levels were quantified by densitometric analysis using Image-J software (Shown in the right panel). All data are presented as means ± SEM, * <span class="html-italic">p</span> &lt; 0.05 compared with WT mice, <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 compared with UTI injected IDH2 KO mice (n = 10 mice/group).</p> Full article ">Figure 5
<p>Schematic representation of the proposed pathway of SDC2 activation in IDH2 deficiency condition.</p> Full article ">
4 pages, 214 KiB  
Editorial
Oral Microbiome, Oral Health and Systemic Health: A Multidirectional Link
by Elena Maria Varoni and Lia Rimondini
Biomedicines 2022, 10(1), 186; https://doi.org/10.3390/biomedicines10010186 - 17 Jan 2022
Cited by 14 | Viewed by 3157
Abstract
The oral cavity can be regarded as the mirror of systemic health, since many systemic diseases may have manifestations in the oral cavity, as in the case, among oral, potentially malignant disorders, of lupus erythematosus oral lichenoid lesions, and, vice-versa, oral diseases may [...] Read more.
The oral cavity can be regarded as the mirror of systemic health, since many systemic diseases may have manifestations in the oral cavity, as in the case, among oral, potentially malignant disorders, of lupus erythematosus oral lichenoid lesions, and, vice-versa, oral diseases may affect systemic health, impairing patient’s nutrition and wellbeing, reducing the quality of life and increasing stress and anxiety [...] Full article
(This article belongs to the Special Issue Malignant and Potentially Malignant Disorders of the Oral Cavity: Updates from Pathogenesis to Therapy)
15 pages, 4125 KiB  
Article
Ribosome Biogenesis Serves as a Therapeutic Target for Treating Endometriosis and the Associated Complications
by Cherry Yin-Yi Chang, An-Jen Chiang, Man-Ju Yan, Ming-Tsung Lai, Yun-Yi Su, Hsin-Yi Huang, Chan-Yu Chang, Ya-Hui Li, Pei-Fen Li, Chih-Mei Chen, Tritium Hwang, Chloe Hogg, Erin Greaves and Jim Jinn-Chyuan Sheu
Biomedicines 2022, 10(1), 185; https://doi.org/10.3390/biomedicines10010185 - 17 Jan 2022
Cited by 1 | Viewed by 4162
Abstract
Ribosome biogenesis is a cellular process critical for protein homeostasis during cell growth and multiplication. Our previous study confirmed up-regulation of ribosome biogenesis during endometriosis progression and malignant transition, thus anti-ribosome biogenesis may be effective for treating endometriosis and the associated complications. A [...] Read more.
Ribosome biogenesis is a cellular process critical for protein homeostasis during cell growth and multiplication. Our previous study confirmed up-regulation of ribosome biogenesis during endometriosis progression and malignant transition, thus anti-ribosome biogenesis may be effective for treating endometriosis and the associated complications. A mouse model with human endometriosis features was established and treated with three different drugs that can block ribosome biogenesis, including inhibitors against mTOR/PI3K (GSK2126458) and RNA polymerase I (CX5461 and BMH21). The average lesion numbers and disease frequencies were significantly reduced in treated mice as compared to controls treated with vehicle. Flow cytometry analyses confirmed the reduction of small peritoneal macrophage and neutrophil populations with increased large versus small macrophage ratios, suggesting inflammation suppression by drug treatments. Lesions in treated mice also showed lower nerve fiber density which can support the finding of pain-relief by behavioral studies. Our study therefore suggested ribosome biogenesis as a potential therapeutic target for treating endometriosis. Full article
(This article belongs to the Special Issue Advanced Research in Endometriosis 2.0)
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<p>Anti-ribosome biogenesis treatment in a mouse model of endometriosis induced in C57BL/6JNarl mice. (<b>a</b>) The schematic diagram indicates the timeline of procedures performed on donor and recipient mice. The recipient mice were further treated with GSK2126458 (GSK; <span class="html-italic">n</span> = 23), CX5461 (<span class="html-italic">n</span> = 25) and BMH-21 (<span class="html-italic">n</span> = 25) at the indicated drug dosages for three weeks. Vehicle-treated mice (<span class="html-italic">n</span> = 25) were utilized as controls. (<b>b</b>) Tissue section staining revealed the presence of stroma (anti-vimentin) or glands (anti-cytokeratin) in the collected lesions. (<b>c</b>) The disease frequencies and average lesion numbers in drug-treated mice were counted and compared with that in control mice. (<b>d</b>) Statistical differences between vehicle and drug-treated groups were compared by chi-squire test. The <span class="html-italic">p</span> values were presented as *: <span class="html-italic">p</span> value &lt; 0.05 and **: <span class="html-italic">p</span> value &lt; 0.01.</p> Full article ">Figure 2
<p>Regulation of inflammation in the peritoneal cavity of mice with induced endome triosis by anti-ribosome biogenesis agents. (<b>a</b>) Multicolor flow cytometry was used to assess myeloid cell populations in the peritoneal fluid of mice with induced endometriosis. Neutrophils (Ly6G<sup>+</sup>) were quantified by the sequential gating strategy. (<b>b</b>) The depicted cellular gating is representative of five individual experiments (<span class="html-italic">n</span> = 25 in each group). The naive group serves as the negative control. (<b>c</b>) The bar chart summarized the calculated numbers of neutrophils in each group. Statistical differences between vehicle and drug-treated groups were compared by using <span class="html-italic">t</span>-test. The <span class="html-italic">p</span> values were presented as **: <span class="html-italic">p</span> value &lt; 0.01, and ***: <span class="html-italic">p</span> value &lt; 0.001.</p> Full article ">Figure 3
<p>Regulation of macrophage populations in the peritoneal cavity of mice with induced endometriosis by anti-ribosome biogenesis agents. (<b>a</b>) Multicolor flow cytometry was applied to gate the myeloid cell populations in the peritoneal fluid of mice with induced endometriosis. Macrophage subsets from each mouse were further analyzed by detecting the expression of F4/80 for large peritoneal macrophages (LpM) or MHC-II for small peritoneal macrophages (SpM). (<b>b</b>) The depicted cellular gating is representative of five individual experiments (<span class="html-italic">n</span> = 25 in each group). Naïve mice serve as the negative controls. (<b>c</b>) The bar charts summarized the calculated numbers of LpM (left) or SpM (middle) and the ratios (SpM/LpM) of these two (right) in each group. Statistical differences between vehicle and drug-treated groups were compared by using <span class="html-italic">t</span>-test. The <span class="html-italic">p</span> values were presented as *: <span class="html-italic">p</span> value &lt; 0.05, and **: <span class="html-italic">p</span> value &lt; 0.01.</p> Full article ">Figure 4
<p>Regulation of nerve fiber growth in endometriotic lesions of mice with induced endometriosis. (<b>a</b>) The protein levels of NGF (<b>left</b>) and IGF-1 (<b>right</b>) in the peritoneal fluid were analyzed by sandwich ELISA. The data were normalized with the average levels in mice treated with vehicle (<span class="html-italic">n</span> = 15 for each group). (<b>b</b>) Immunofluorescence staining was performed to detect the presence of nerve fibers (PGP9.5<sup>+</sup>) in endometriotic lesions from different groups (<b>left</b>). The fluorescence intensity was averaged by using data from 10 independent tissue sections (<b>right</b>). Statistical differences between vehicle and drug-treated groups were compared by <span class="html-italic">t</span>-test. The <span class="html-italic">p</span> values were presented as *: <span class="html-italic">p</span> value &lt; 0.05, **: <span class="html-italic">p</span> value &lt; 0.01, and ***: <span class="html-italic">p</span> value &lt; 0.001.</p> Full article ">Figure 5
<p>Expression of inflammatory mediators in peritoneal tissue, spinal cord and brain. RNA was extracted from peritoneal tissue (blue), spinal cord (red) and posterior insula (green) in the brain of experimental mice and subjected to cDNA synthesis. qPCR was performed on cDNA samples (<span class="html-italic">n</span> = 6 for each group) to detect the expression levels of inflammatory mediators, including (<b>a</b>) COX-2, (<b>b</b>) IL-6, (<b>c</b>) IL-1β and (<b>d</b>) TNF-α in different tissues (<a href="#app1-biomedicines-10-00185" class="html-app">Table S2</a>). The data were normalized with the average levels in naïve mice. Statistical differences between vehicle and drug-treated groups were compared by using <span class="html-italic">t</span>-test. The <span class="html-italic">p</span> values were presented as *: <span class="html-italic">p</span> value &lt; 0.05, **: <span class="html-italic">p</span> value &lt; 0.01 and ***: <span class="html-italic">p</span> value &lt; 0.001.</p> Full article ">Figure 6
<p>Impacts of anti-ribosome biogenesis agents on pain relief in endometriosis mice. The behavior study was performed to monitor (<b>a</b>) tunnel-entering activity and (<b>b</b>) abdominal grooming of disease mice treated with different drugs within five minutes. Touch test (von Frey) was also performed on (<b>c</b>) abdomen and (<b>d</b>) hind paws of mice to quantify pain-sensitivity. The vehicle-treated mice were utilized as the untreated controls whereas naïve mice were utilized as healthy controls. The data were averaged from three independent experiments (<span class="html-italic">n</span> = 15 for each group). Statistical differences between vehicle and drug-treated groups were compared by using one-way ANOVA (Kruskal-Wallis) test. The <span class="html-italic">p</span> values were presented as *: <span class="html-italic">p</span> value &lt; 0.05, **: <span class="html-italic">p</span> value &lt; 0.01, and ***: <span class="html-italic">p</span> value &lt; 0.001.</p> Full article ">Figure 7
<p>Ribosome biogenesis serves as a therapeutic target for treating endometriosis. The ribosome biogenesis machinery in cells determines energy homeostasis, which is critical to maintain cell growth and proliferation. Endometriosis-induced inflammatory cytokines, e.g., IL-1β and TNF-α, can activate PI3K/AKT/mTOR signaling [<a href="#B34-biomedicines-10-00185" class="html-bibr">34</a>], which subsequently promotes RNA polymerase I-mediated rRNA transcription and editing/processing, resulting in continuous cell growth. Development of endometriotic lesions in peritoneal cavity triggers long-term inflammation and neuron fiber formation, leading to chronic pelvic pain. Up-regulation of ribosome biogenesis by endometriosis-related effectors, such as SNORD116, RPLP2 and RPL38 [<a href="#B12-biomedicines-10-00185" class="html-bibr">12</a>], may speed up the whole process and provide sufficient energy for further aggressive progression. Blockage of ribosome biogenesis by inhibitors against PI3K/mTOR or RNA polymerase-1 can cause genometoxic shock due to energy shortage and ribosomal protein imbalance, leading to cell cycle arrest and apoptosis via p53/p21 pathway.</p> Full article ">
12 pages, 1095 KiB  
Article
Plasma Proteomics in Healthy Subjects with Differences in Tissue Glucocorticoid Sensitivity Identifies A Novel Proteomic Signature
by Nicolas C. Nicolaides, Manousos Makridakis, Rafael Stroggilos, Vasiliki Lygirou, Eleni Koniari, Ifigeneia Papageorgiou, Amalia Sertedaki, Jerome Zoidakis and Evangelia Charmandari
Biomedicines 2022, 10(1), 184; https://doi.org/10.3390/biomedicines10010184 - 16 Jan 2022
Cited by 2 | Viewed by 2589
Abstract
Significant inter-individual variation in terms of susceptibility to several stress-related disorders, such as myocardial infarction and Alzheimer’s disease, and therapeutic response has been observed among healthy subjects. The molecular features responsible for this phenomenon have not been fully elucidated. Proteomics, in association with [...] Read more.
Significant inter-individual variation in terms of susceptibility to several stress-related disorders, such as myocardial infarction and Alzheimer’s disease, and therapeutic response has been observed among healthy subjects. The molecular features responsible for this phenomenon have not been fully elucidated. Proteomics, in association with bioinformatics analysis, offer a comprehensive description of molecular phenotypes with clear links to human disease pathophysiology. The aim of this study was to conduct a comparative plasma proteomics analysis of glucocorticoid resistant and glucocorticoid sensitive healthy subjects and provide clues of the underlying physiological differences. For this purpose, 101 healthy volunteers were given a very low dose (0.25 mg) of dexamethasone at midnight, and were stratified into the 10% most glucocorticoid sensitive (S) (n = 11) and 10% most glucocorticoid resistant (R) (n = 11) according to the 08:00 h serum cortisol concentrations determined the following morning. One month following the very-low dose dexamethasone suppression test, DNA and plasma samples were collected from the 22 selected individuals. Sequencing analysis did not reveal any genetic defects in the human glucocorticoid receptor (NR3C1) gene. To investigate the proteomic profile of plasma samples, we used Liquid Chromatography–Mass Spectrometry (LC-MS/MS) and found 110 up-regulated and 66 down-regulated proteins in the S compared to the R group. The majority of the up-regulated proteins in the S group were implicated in platelet activation. To predict response to cortisol prior to administration, a random forest classifier was developed by using the proteomics data in order to distinguish S from R individuals. Apolipoprotein A4 (APOA4) and gelsolin (GSN) were the most important variables in the classification, and warrant further investigation. Our results indicate that a proteomics signature may differentiate the S from the R healthy subjects, and may be useful in clinical practice. In addition, it may provide clues of the underlying molecular mechanisms of the chronic stress-related diseases, including myocardial infarction and Alzheimer’s disease. Full article
(This article belongs to the Section Molecular and Translational Medicine)
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<p>Heatmap (<b>left</b>) and Volcano plot (<b>right</b>) of proteins quantified in patients with complete (hypersensitive) or not complete (resistant) response to cortisol. Heatmap shows the abundance of proteins passing the ±0.585 log2 fold change threshold, in the two groups. Volcano plot includes proteins with presence in at least 35% of the analyzed samples (in one of the two groups) and illustrates the log2 fold change (x axis) as a function of the Mann–Whitney p value (y axis). Red color marks proteins passing the 1.5 (or 0.67)-fold change (equivalent to ±0.585 in the logarithmic scale).</p> Full article ">Figure 2
<p>Variable importance for the 14 proteins used to train the random forest classifier so as to distinguish between responders (hypersensitive) and non-responders (resistant) to cortisol: (<b>A</b>) Multiway importance plots depicting the mean decrease in accuracy as a function of the mean minimal depth (left) and of the mean decrease in the Gini index (right); (<b>B</b>) Plot showing structure of the forest with respect to the distribution of the mean minimal depth across trees for each variable; (<b>C</b>) Predictive value of the top two important variables (APOA4 and GSN) in detecting non-responders, based on their normalized protein intensity areas across samples.</p> Full article ">
10 pages, 2019 KiB  
Article
Serpinin in the Skin
by Cristina Fraquelli, Jasmine Hauzinger, Christian Humpel, Maria Nolano, Vincenzo Provitera, Vinay Kumar Sharma, Peng Loh, Zenon Pidsudko, Georgios Blatsios and Josef Troger
Biomedicines 2022, 10(1), 183; https://doi.org/10.3390/biomedicines10010183 - 16 Jan 2022
Viewed by 2527
Abstract
The serpinins are relatively novel peptides generated by proteolytic processing of chromogranin A and they are comprised of free serpinin, serpinin-RRG and pGlu-serpinin. In this study, the presence and source of these peptides were studied in the skin. By Western blot analysis, a [...] Read more.
The serpinins are relatively novel peptides generated by proteolytic processing of chromogranin A and they are comprised of free serpinin, serpinin-RRG and pGlu-serpinin. In this study, the presence and source of these peptides were studied in the skin. By Western blot analysis, a 40 kDa and a 50 kDa protein containing the sequence of serpinin were detected in the trigeminal ganglion and dorsal root ganglia in rats but none in the skin. RP-HPLC followed by EIA revealed that the three serpinins are present in similar, moderate amounts in rat dorsal root ganglia, whereas in the rat skin, free serpinin represents the predominant molecular form. There were abundant serpinin-positive cells in rat dorsal root ganglia and colocalization with substance P was evident. However, much more widespread distribution of the serpinins was found in dorsal root ganglia when compared with substance P. In the skin, serpinin immunoreactivity was found in sensory nerves and showed colocalization with substance P; as well, some was present in autonomic nerves. Thus, although not exclusively, there is evidence that serpinin is a constituent of the sensory innervation of the skin. The serpinins are biologically highly active and might therefore be of functional significance in the skin. Full article
(This article belongs to the Special Issue Neuropeptides in Biomedicines)
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<p>Western blot analysis of serpinin immunoreactivities in rat TG, rat DRG and rat skin. Note the presence of two strong bands at 40 kDa and 50 kDa in both TG and DRG representing two larger molecular forms containing the sequence of serpinin, whereas no bands were observed in the skin. Actin represents an internal standard at 42 kDa as a control.</p> Full article ">Figure 2
<p>RP-HPLC and EIA for serpinin-like immunoreactivity from extracts of rat DRG and skin. In DRGs (brown line), there were two peaks present, in fraction 29 and 33 and a shoulder in fraction 34 representing serpinin-RRG (“RRG”), free serpinin (“Serp”) and pGlu-serpinin (“pGlu”), respectively. In the skin (blue line), the immunoreactivities in fraction 29 and 34 were approximately equal to that in DRGs, but the immunoreactive peak in fraction 33 was much higher than in DRGs. The elution positions of these three serpinins are indicated by arrows.</p> Full article ">Figure 3
<p>Serpinin-LI in rat thoracic DRG showing double-immunofluorescence of serpinin/SP. Many serpinin immuno-positive cells were observed in these ganglia (green), the cells featured a diameter of 20–30 µm and were arranged mainly in clusters (<b>A</b>–<b>C</b>). Furthermore, double-immunofluorescent studies of serpinin ((<b>D</b>), green) with SP-IR ((<b>E</b>), red) revealed colocalization (indicated by arrows in (<b>D</b>,<b>E</b>), arrows in the superimposed image (<b>F</b>), orange) but most serpinin positive cells were devoid of SP-IR. Scale bars represent 50 µm.</p> Full article ">Figure 4
<p>Confocal images demonstrating the presence of serpinin IR within nerve fibers in human skin from control subjects. On the left, the merged images (<b>A</b>–<b>D</b>) show colocalization of serpinin with neural markers. On the right, the unmerged images (<b>A1</b>,<b>A2</b>,<b>B1</b>,<b>B2</b>,<b>C1</b>,<b>C2</b>,<b>D1</b>,<b>D2</b>) allow to better appreciate the different stainings. (<b>A</b>): serpinin (green) colocalization with SP (red) in a sensory fiber within a dermal papilla. (<b>B</b>): serpinin (green) colocalization with the pan neuronal marker PGP 9.5 (red) in the fibers included in a nerve bundle in the reticular dermis. (<b>C</b>): serpinin (green) colocalization with PGP 9.5 (red) in pilomotor sympathetic nerves from an arrector pili muscle. (<b>D</b>): serpinin (green) colocalization with PGP 9.5 (red) in sudomotor sympathetic nerves around sweat tubules. Scale bar is 30 µm in (<b>A</b>) and 50 µm in (<b>B</b>–<b>D</b>). SP = substance P; PGP 9.5 = Pan neuronal marker protein gene product 9.5; ULEX = Ulex europaeus agglutinin 1.</p> Full article ">
21 pages, 754 KiB  
Review
Impact of Sarcopenia and Myosteatosis in Non-Cirrhotic Stages of Liver Diseases: Similarities and Differences across Aetiologies and Possible Therapeutic Strategies
by Annalisa Cespiati, Marica Meroni, Rosa Lombardi, Giovanna Oberti, Paola Dongiovanni and Anna Ludovica Fracanzani
Biomedicines 2022, 10(1), 182; https://doi.org/10.3390/biomedicines10010182 - 16 Jan 2022
Cited by 17 | Viewed by 3642
Abstract
Sarcopenia is defined as a loss of muscle strength, mass and function and it is a predictor of mortality. Sarcopenia is not only a geriatric disease, but it is related to several chronic conditions, including liver diseases in both its early and advanced [...] Read more.
Sarcopenia is defined as a loss of muscle strength, mass and function and it is a predictor of mortality. Sarcopenia is not only a geriatric disease, but it is related to several chronic conditions, including liver diseases in both its early and advanced stages. Despite the increasing number of studies exploring the role of sarcopenia in the early stages of chronic liver disease (CLD), its prevalence and the relationship between these two clinical entities are still controversial. Myosteatosis is characterized by fat accumulation in the muscles and it is related to advanced liver disease, although its role in the early stages is still under researched. Therefore, in this narrative review, we firstly aimed to evaluate the prevalence and the pathogenetic mechanisms underlying sarcopenia and myosteatosis in the early stage of CLD across different aetiologies (mainly non-alcoholic fatty liver disease, alcohol-related liver disease and viral hepatitis). Secondly, due to the increasing prevalence of sarcopenia worldwide, we aimed to revise the current and the future therapeutic approaches for the management of sarcopenia in CLD. Full article
(This article belongs to the Special Issue New Challenges in the Study of Liver Diseases: From Molecular Pathogenesis to Therapeutic Approaches)
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<p>Pathogenic mechanisms linking sarcopenia development in the context of non-alcoholic Fatty Liver Disease (NAFLD), Alcoholic Liver Disease (ALD) and Viral hepatitis. NAFLD and sarcopenia share common underlying pathogenic mechanisms, such as IR, chronic inflammation, mitochondrial dysfunction, nutritional deficiencies, and reduction in physical activity. In the context of NAFLD, the key pathogenic event is the presence of insulin resistance (IR), which is associated with compensatory hyperinsulinemia. Insulin plays a primary role in different tissues among which liver, adipose tissue and muscle. IR in adipose tissue triggers the activation of lipolysis, favouring in turn the release of free fatty acids (FFAs) into the bloodstream. At the hepatic level, the reduced insulin signalling hampers the phosphorylation of Forkhead box O1-phosphorylated (FOXO1), whereby forcing gluconeogenesis, while the enhanced influx of FFAs induces fat deposition, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and reactive oxygen species (ROS) production. Moreover, hyperinsulinemia stimulates de novo lipogenesis, through sterol regulatory element-binding protein-1c (SREBP1c). In muscle tissue, IR suppresses glycogen synthesis and muscle mass enlargement. In addition, both sarcopenia and NAFLD are shaped by a chronic inflammation, characterized by tumour necrosis factor alpha (TNFα) and interleukin 1 (IL1) over-secretion. Similarly, Vitamin D deficiency participates to sarcopenia onset. The gut microbiota and the somatotropic axis are involved both in sarcopenia and NAFLD, but their effect on sarcopenia development and progression in NAFLD is not well established (<b>left</b> panel). Likewise, in Alcoholic Liver disease (ALD), ethanol exerts a detrimental effect on both muscle and liver, through its conversion into acetaldehyde, which induces ROS production, mitochondrial dysfunction, promotes the activation of hepatic stellate cells (HSCs) producing extracellular matrix (ECM) deposition, and interferes with hepatic ureagenesis with consequent increase in ammonia levels. Hyperammonaemia per se blunted protein synthesis in skeletal muscle and stimulates myostatin, a member of transforming growth factor beta (TGFβ) (<b>middle</b> panel). Less is known regarding the mechanisms linking chronic viral hepatitis and sarcopenia. In this background, chronic inflammation promotes the protein catabolism in muscle cells, by ubiquitin-proteasome system (UPS) (<b>right</b> panel).</p> Full article ">
15 pages, 4111 KiB  
Article
Differential Expression and Localization of EHBP1L1 during the First Wave of Rat Spermatogenesis Suggest Its Involvement in Acrosome Biogenesis
by Massimo Venditti and Sergio Minucci
Biomedicines 2022, 10(1), 181; https://doi.org/10.3390/biomedicines10010181 - 16 Jan 2022
Cited by 8 | Viewed by 2426
Abstract
The identification and characterization of new proteins involved in spermatogenesis is fundamental, considering that good-quality gametes are basic in ensuring proper reproduction. Here, we further analyzed the temporal and spatial localization during the first spermatogenic wave of rat testis of EHBP1L1, which is [...] Read more.
The identification and characterization of new proteins involved in spermatogenesis is fundamental, considering that good-quality gametes are basic in ensuring proper reproduction. Here, we further analyzed the temporal and spatial localization during the first spermatogenic wave of rat testis of EHBP1L1, which is involved in vesicular trafficking due to the CH and bMERB domains, which bind to actin and Rab8/10, respectively. Western blot and immunofluorescence analyses showed that EHBP1L1 protein expression started at 21 days post-partum (dpp) concomitantly with the appearance of primary spermatocytes (I SPC). In subsequent stages, EHBP1L1 specifically localized together with actin in the perinuclear cytoplasm close to the acrosomal and Golgian regions of spermatids (SPT) during the different phases of acrosome biogenesis (AB). Moreover, it was completely absent in elongated SPT and in mature spermatozoa, suggesting that its role was completed in previous stages. The combined data, also supported by our previous report demonstrating that EHBP1L1 mRNA was expressed by primary (I) and secondary (II) SPC, lead us to hypothesize its specific role during AB. Although these results are suggestive, further studies are needed to better clarify the underlying molecular mechanisms of AB, with the aim to use EHBP1L1 as a potential new marker for spermatogenesis. Full article
(This article belongs to the Special Issue Biochemical and Cellular Mechanisms Regulating ER-to-Golgi Vesicular Trafficking in Health and Disease)
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<p>EHBP1L1 protein level during the first wave of rat spermatogenesis. (<b>A</b>): Western blot analysis showing the expression of EHBP1L1 (164 kDa, top section) and ACTIN (42 kDa, bottom section) during rat testis post-natal development at 7, 14, 21, 28, 35, 42, and 60 days postpartum (dpp). (<b>B</b>): Histogram showing the relative expression levels of EHBP1L1 in all the analyzed samples. Data were normalized with ACTIN and reported as EHBP1L1/ACT OD ratio. All data represent the mean ± SEM. a vs. b: <span class="html-italic">p</span> &lt; 0.01. Western blot experiment was performed in triplicate.</p> Full article ">Figure 2
<p>Histology and staging of the developing rat testis. Hematoxylin-eosin staining of tissue sections at 7 (<b>A</b>), 14 (<b>B</b>), 21 (<b>C</b>), 28 (<b>D</b>), 35 (<b>E</b>), 42 (<b>F</b>), 60 (<b>G</b>) dpp, in which the most representative cell types are highlighted. Scale bars represent 20 μm. Arrowhead: gonocytes; striped arrowhead: spermatogonia (SPG); arrow: spermatocyte (SPC); dotted arrow: round spermatid (rSPT); striped arrow: elongated spermatid (eSPT); asterisk: spermatozoa (SPZ) tails.</p> Full article ">Figure 3
<p>Localization of EHBP1L1 during the first wave of rat spermatogenesis (7–21 dpp). (<b>A</b>,<b>D</b>,<b>G</b>) DAPI-fluorescent nuclear staining (blue) and PNA lectin acrosome staining (red). (<b>B</b>,<b>E</b>,<b>H</b>) EHBP1L1 fluorescence (green). (<b>C</b>,<b>F</b>,<b>I</b>) Merged fluorescent channels (blue/red/green). (<b>A</b>–<b>C</b>) 7 dpp testis; (<b>D</b>–<b>F</b>) 14 dpp; (<b>G</b>–<b>I</b>) 21 dpp. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. Arrowhead: gonocytes; striped arrowhead: SPG; arrow: I SPC.</p> Full article ">Figure 4
<p>Localization of EHBP1L1 during the first wave of rat spermatogenesis (28–42 dpp). (<b>A</b>,<b>D</b>,<b>G</b>) DAPI-fluorescent nuclear staining (blue) and PNA lectin acrosome staining (red). (<b>B</b>,<b>E</b>,<b>H</b>) EHBP1L1 fluorescence (green). (<b>C</b>,<b>F</b>,<b>I</b>) Merged fluorescent channels (blue/red/green). (<b>A</b>–<b>C</b>) 28 dpp testis; (<b>D</b>–<b>F</b>) 35 dpp; (<b>G</b>–<b>I</b>) 42 dpp. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. Striped arrowhead: SPG; arrow: I SPC; dotted arrow: rSPT.</p> Full article ">Figure 5
<p>Localization of EHBP1L1 in adult rat testis (60 dpp). (<b>A</b>) EHBP1L1 localization in different stages of adult rat spermatogenesis. To better show the differences between them, the 14 stages comprising the whole spermatogenic cycle are arbitrarily sub-divided into four parts. Scale bars represent 20 μm. Arrow: I SPC; dotted arrow: rSPT; striped arrow: eSTP. (<b>B</b>) EHBP1L1 localization in SPT in comparison with a schematic representation of the different phases of acrosome biogenesis. In A and B merged images, the blue channel represents DAPI-fluorescent nuclear staining, the red channel represents PNA lectin acrosome staining, and the green channel represents EHBP1L1 fluorescent signal.</p> Full article ">Figure 6
<p>Co-localization of EHBP1L1 and ACTIN during the first wave of rat spermatogenesis (7–28 dpp). (<b>A</b>,<b>D</b>,<b>G</b>,<b>J</b>) DAPI-fluorescent nuclear staining (blue) and ACTIN staining (red). (<b>B</b>,<b>E</b>,<b>H</b>,<b>K</b>) EHBP1L1 fluorescence (green). (<b>C</b>,<b>F</b>,<b>I</b>,<b>L</b>) Merged fluorescent channels (blue/red/green). The intermediate yellow–orange tint indicates EHBP1L1 and ACTIN co-localization. (<b>A</b>–<b>C</b>) 7 dpp testis; (<b>D</b>–<b>F</b>) 14 dpp; (<b>G</b>–<b>I</b>) 21 dpp; (<b>J</b>–<b>L</b>) 28 dpp. (<b>M</b>–<b>P</b>) Negative controls for the same time points, obtained by omitting the primary antibodies. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. Arrowhead: gonocytes; striped arrowhead: SPG; arrow: I SPC; dotted arrow: rSPT.</p> Full article ">Figure 7
<p>Co-localization of EHBP1L1 and ACTIN during the first wave of rat spermatogenesis (35–60 dpp). (<b>A</b>,<b>D</b>,<b>G</b>,<b>J</b>) DAPI-fluorescent nuclear staining (blue) and ACTIN staining (red). (<b>B</b>,<b>E</b>,<b>H</b>,<b>K</b>) EHBP1L1 fluorescence (green). (<b>C</b>,<b>F</b>,<b>I</b>,<b>L</b>) Merged fluorescent channels (blue/red/green). The intermediate yellow–orange tint indicates EHBP1L1 and ACTIN co-localization. (<b>A</b>–<b>C</b>) 35 dpp testis; (<b>D</b>–<b>F</b>) 42 dpp; (<b>G</b>–<b>I</b>) 60 dpp. (<b>J</b>–<b>L</b>) Negative controls for the same time points, obtained by omitting the primary antibodies. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. Arrow: I SPC; dotted arrow: rSPT; striped arrow: eSPT.</p> Full article ">Figure 8
<p>Co-localization of EHBP1L1 and GOLGB1 at 42 and 60 dpp. (<b>A</b>,<b>D</b>) DAPI-fluorescent nuclear staining (blue) and GOLGB1 staining (red). (<b>B</b>,<b>E</b>) EHBP1L1 fluorescence (green). (<b>C</b>,<b>F</b>) Merged fluorescent channels (blue/red/green). (<b>A</b>–<b>C</b>) 42 dpp testis; (<b>D</b>–<b>F</b>) 60 dpp. GOLGB1 staining, initially green, was modified into red to obtain the merged channels. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. Arrow: I SPC; dotted arrow: rSPT; striped arrow: eSPT.</p> Full article ">Figure 9
<p>EHBP1L1 protein level in isolated germ cells. (<b>A</b>) Western blot analysis showing the expression of EHBP1L1 (164 kDa, top section) and ACTIN (42 kDa, bottom section), in I and II SPC and SPT enriched fractions. (<b>B</b>) Histogram showing the relative expression levels of EHBP1L1 in all the analyzed samples. Data were normalized with ACTIN and reported as EHBP1L1/ACT OD ratio. All data represent the mean ± SEM. a vs. b: <span class="html-italic">p</span> &lt; 0.01. Western blot experiment was performed in triplicate.</p> Full article ">Figure 10
<p>EHBP1L1 protein level and localization in rat and human SPZ. (<b>A</b>) Western blot analysis showing the expression of EHBP1L1 (164 kDa, top section) and ACTIN (42 kDa, bottom section) in rat (lane 1) and human (lane 2) SPZ. Each Western blot experiment was performed in triplicate. (<b>B</b>) Immunofluorescence analysis of EHBP1L1 in rat and human SPZ. Scale bars represent 10 μm.</p> Full article ">
12 pages, 1667 KiB  
Article
A Unique Immune-Related Gene Signature Represents Advanced Liver Fibrosis and Reveals Potential Therapeutic Targets
by Pil-Soo Sung, Chang-Min Kim, Jung-Hoon Cha, Jin-Young Park, Yun-Suk Yu, Hee-Jung Wang, Jin-Kyeoung Kim and Si-Hyun Bae
Biomedicines 2022, 10(1), 180; https://doi.org/10.3390/biomedicines10010180 - 16 Jan 2022
Cited by 6 | Viewed by 3461
Abstract
Innate and adaptive immune responses are critically associated with the progression of fibrosis in chronic liver diseases. In this study, we aim to identify a unique immune-related gene signature representing advanced liver fibrosis and to reveal potential therapeutic targets. Seventy-seven snap-frozen liver tissues [...] Read more.
Innate and adaptive immune responses are critically associated with the progression of fibrosis in chronic liver diseases. In this study, we aim to identify a unique immune-related gene signature representing advanced liver fibrosis and to reveal potential therapeutic targets. Seventy-seven snap-frozen liver tissues with various chronic liver diseases at different fibrosis stages (1: n = 12, 2: n = 12, 3: n = 25, 4: n = 28) were subjected to expression analyses. Gene expression analysis was performed using the nCounter PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA, USA). Biological meta-analysis was performed using the CBS Probe PINGSTM (CbsBioscience, Daejeon, Korea). Using non-tumor tissues from surgically resected specimens, we identified the immune-related, five-gene signature (CHIT1_FCER1G_OSM_VEGFA_ZAP70) that reliably differentiated patients with low- (F1 and F2) and high-grade fibrosis (F3 and F4; accuracy = 94.8%, specificity = 91.7%, sensitivity = 96.23%). The signature was independent of all pathological and clinical features and was independently associated with high-grade fibrosis using multivariate analysis. Among these genes, the expression of inflammation-associated FCER1G, OSM, VEGFA, and ZAP70 was lower in high-grade fibrosis than in low-grade fibrosis, whereas CHIT1 expression, which is associated with fibrogenic activity of macrophages, was higher in high-grade fibrosis. Meta-analysis revealed that STAT3, a potential druggable target, highly interacts with the five-gene signature. Overall, we identified an immune gene signature that reliably predicts advanced fibrosis in chronic liver disease. This signature revealed potential immune therapeutic targets to ameliorate liver fibrosis. Full article
(This article belongs to the Special Issue Liver Fibrosis: Molecular Mechanisms, Potential Therapeutic Targets and Clinical Biomarkers)
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<p>Flow chart of gene signature development and meta-analysis.</p> Full article ">Figure 2
<p>Clinical performance evaluation of the selected 5-gene signature. The clinical performance of the 5-gene signature was evaluated using receiver operating characteristic (ROC) analysis, cross-validation, and logistic regression analysis. (<b>A</b>) ROC analysis of 5-gene signature to the advanced fibrosis stage. (<b>B</b>) Clinical performance of the 5-gene signature in logistic regression analysis, cross-validation, and ROC analysis.</p> Full article ">Figure 3
<p>Expression of each gene comprising the gene signature in early stage versus late-stage liver fibrosis. Relative expressions of 5 genes in 24 patients with early-stage fibrosis and in 53 patients with late-stage fibrosis. ** <span class="html-italic">p</span> &lt; 0.01. (<b>A</b>) CHIT1. (<b>B</b>) FCER1G. (<b>C</b>) OSM. (<b>D</b>) VEGFA. (<b>E</b>) ZAP70.</p> Full article ">Figure 4
<p>Expression of each gene comprising the gene signature in each stage of liver fibrosis in the HBV ((<b>A</b>), <span class="html-italic">n</span> = 62) and non-HBV ((<b>B</b>), <span class="html-italic">n</span> = 15) subgroups.</p> Full article ">
11 pages, 1223 KiB  
Article
Predictive Factors for Skip Lymph Node Metastasis and Their Implication on Recurrence in Papillary Thyroid Carcinoma
by Young-Jae Ryu, Seong-Young Kwon, Soo-Young Lim, Yong-Min Na and Min-Ho Park
Biomedicines 2022, 10(1), 179; https://doi.org/10.3390/biomedicines10010179 - 16 Jan 2022
Cited by 8 | Viewed by 2444
Abstract
Skip lymph node (LN) metastases in papillary thyroid carcinoma (PTC) belong to N1b classification in the absence of central neck LN involvement. This study aimed to evaluate the predictive factors of skip metastases and their impact on recurrence in PTC patients with pN1b. [...] Read more.
Skip lymph node (LN) metastases in papillary thyroid carcinoma (PTC) belong to N1b classification in the absence of central neck LN involvement. This study aimed to evaluate the predictive factors of skip metastases and their impact on recurrence in PTC patients with pN1b. A total of 334 PTC patients who underwent total thyroidectomy with LN dissection (central and lateral neck compartment) followed by radioactive iodine ablation were included. Patients with skip metastases tended to have a small primary tumor (≤1 cm) and single lateral neck level involvement. Tumor size ≤ 1 cm was an important predictive factor for skip metastases. Univariate analysis for recurrence showed that patients with a central LN ratio > 0.68, lateral LN ratio > 0.21, and stimulated thyroglobulin (Tg) levels > 7.3 ng/mL had shorter RFS (recurrence-free survival). The stimulated Tg level was associated with shorter RFS on multivariate analysis (>7.3 vs. ≤7.3 ng/mL; hazard ratio, 4.226; 95% confidence interval, 2.226−8.022; p < 0.001). Although patients with skip metastases tended to have a small primary tumor and lower burden of lateral neck LN involvement, there was no association between skip metastases and RFS in PTC with pN1b. Stimulated Tg level was a strong predictor of recurrence. Full article
(This article belongs to the Special Issue Recent Advances in Thyroid Cancer: From Diagnosis to Treatment)
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<p>Flowchart of study population.</p> Full article ">Figure 2
<p>Kaplan-Meier curve according to the presence and absence of skip metastases. Skip metastases (<b>A</b>), Non-skip metastases (<b>B</b>).</p> Full article ">Figure 3
<p>Kaplan-Meier curve according to the level of stimulated thyroglobulin (Tg). Tg ≤ 7.3 ng/mL (<b>A</b>), Tg &gt; 7.3 ng/mL (<b>B</b>).</p> Full article ">Figure 4
<p>Receiver operating characteristic curves. Area under the curve of CLN ratio, LLN ratio, and stimulated Tg was 0.626, 0.633, and 0.763, respectively. CLN, central lymph node; LLN, lateral lymph node; Tg, thyroglobulin.</p> Full article ">
26 pages, 1480 KiB  
Review
Novel Insight into the Mechanisms of the Bidirectional Relationship between Diabetes and Periodontitis
by Federica Barutta, Stefania Bellini, Marilena Durazzo and Gabriella Gruden
Biomedicines 2022, 10(1), 178; https://doi.org/10.3390/biomedicines10010178 - 16 Jan 2022
Cited by 36 | Viewed by 8968
Abstract
Periodontitis and diabetes are two major global health problems despite their prevalence being significantly underreported and underestimated. Both epidemiological and intervention studies show a bidirectional relationship between periodontitis and diabetes. The hypothesis of a potential causal link between the two diseases is corroborated [...] Read more.
Periodontitis and diabetes are two major global health problems despite their prevalence being significantly underreported and underestimated. Both epidemiological and intervention studies show a bidirectional relationship between periodontitis and diabetes. The hypothesis of a potential causal link between the two diseases is corroborated by recent studies in experimental animals that identified mechanisms whereby periodontitis and diabetes can adversely affect each other. Herein, we will review clinical data on the existence of a two-way relationship between periodontitis and diabetes and discuss possible mechanistic interactions in both directions, focusing in particular on new data highlighting the importance of the host response. Moreover, we will address the hypothesis that trained immunity may represent the unifying mechanism explaining the intertwined association between diabetes and periodontitis. Achieving a better mechanistic insight on clustering of infectious, inflammatory, and metabolic diseases may provide new therapeutic options to reduce the risk of diabetes and diabetes-associated comorbidities. Full article
(This article belongs to the Special Issue Bacterial and Viral Infection and Sepsis: From Pathogenesis to Therapeutic Strategies)
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<p>Bidirectional relationship between periodontitis and diabetes. (<b>A</b>) Periodontitis diabetes direction. Periodontitis favors development/worsening of type 2 diabetes by three major mechanisms: (1) Dissemination of periodontal bacteria/bacterial products into the bloodstream. Bacteria/bacterial products can induce insulin resistance (a) by inhibiting hepatic glycogen synthesis, increasing hepatic gluconeogenesis, and (b) blocking the insulin receptor substrate via production of branched-chain amino acids (BCAA). (c) Dipeptidyl peptidase-4 (DPP4) produced by P. gingivalis (Pg-DPP4) can reduce glucose-induced insulin production by enhancing glucagon-like peptide 1 (GLP-1) degradation (d) P. gingivalis may alter insulin production by inducing β cell dedifferentiation. (2) Induction/magnification of systemic inflammation, favoring both (e) hepatic and (f) adipose tissue insulin resistance. (3) Gut dysbiosis induced by swallowed periodontal bacteria, favoring both (g) endotoxemia and (h) changes in the blood metabolome. (<b>B</b>) Diabetes periodontitis direction. Pathogenesis of periodontitis is depicted on the right hand side of the figure. Dysbiosis, inflammation, and destruction of the periodontium (green boxes) are characteristic features of periodontitis. Dysbiotic bacteria reduce the efficacy of the host immune response, while fuelling inflammation (open green arrow). In turn, inflammation-induced tissue breakdown favors dysbiosis (closed green arrow) closing the vicious cycle. Mechanisms linking diabetes to periodontitis are shown on the left hand side of the figure. Diabetes favors development/worsening of periodontitis by three major mechanisms. (1) Increasing periodontal dysbiosis and bacterial pathogenicity via IL-17. (2) Enhancing the host response to the bacterial challenge. Diabetes (a) alters complement and neutrophil function (which also affects susceptibility to infection a’), (b) increases myelopoiesis, enhances (c) the M1/M2 macrophage ratio, (d) the Th17/Treg lymphocyte ratio, thus raising inflammatory cytokines levels (dotted lines) and fueling inflammation. (3) Increasing periodontal destruction. Diabetes reduces new bone formation by enhancing apoptosis of bone-forming cells and by lowering periodontal ligament stem cells (PLSCs) proliferation and differentiation in osteoblasts (pink boxes). Diabetes enhances osteoclastogenesis by increasing RANKL release by osteocytes/osteoblasts, leading to osteoclast precursor (OCP) differentiation in osteoclasts (grey boxes). Diabetes augments gingiva tissue degradation by increasing release of metalloproteinases (MMP) and reactive oxygen species (ROS) by neutrophils and fibroblasts (violet boxes).</p> Full article ">Figure 2
<p>The hypothesis of trained innate immunity as the underlying mechanism of the bidirectional relationship between diabetes and periodontitis. (<b>A</b>) Periodontitis-induced release of bacterial products and inflammatory cytokines as well as (<b>B</b>) diabetes-induced hyperglycemia may induce metabolic/epigenetic rewiring of both peripheral myeloid cells (peripheral trained immunity) (<b>C</b>,<b>D</b>) and bone-marrow precursors (central trained immunity) (<b>E</b>). This generates hyper-active myeloid cells that can respond more effectively to a second unrelated challenge. (<b>F</b>) The graph shows that myeloid cells epigenetically trained by an earlier exposure to periodontitis-related bacterial products may display an enhanced response to hyperglycemia and thus exacerbate diabetes-related inflammation. (<b>G</b>) The graph shows that myeloid cells epigenetically trained by an earlier exposure to hyperglycemia may display an enhanced response to bacterial products and thus exacerbate periodontitis-related inflammation. (<b>H</b>) Regardless of whether hyperactive myeloid cells are first affected by either periodontitis or diabetes, trained immunity can have a deleterious effect on both conditions and may provide a rationale for their bidirectional relationship. HSC (hematopoietic stem cells), MMP (multipotent progenitors), GMP (granulocyte/macrophage progenitors).</p> Full article ">
13 pages, 2354 KiB  
Article
Detection Rate and Clinical Impact of PET/CT with 18F-FACBC in Patients with Biochemical Recurrence of Prostate Cancer: A Retrospective Bicentric Study
by Luca Filippi, Oreste Bagni, Carmelo Crisafulli, Ivan Cerio, Gabriele Brunotti, Agostino Chiaravalloti, Orazio Schillaci and Franca Dore
Biomedicines 2022, 10(1), 177; https://doi.org/10.3390/biomedicines10010177 - 15 Jan 2022
Cited by 9 | Viewed by 4295
Abstract
Our aim was to assess the detection rate (DR) of positron emission computed tomography (PET/CT) with anti-1-amino-3-[18F]-flurocyclobutane-1-carboxylic acid (18F-FACBC) in patients with biochemical recurrence (BCR) from prostate cancer (PC). As a secondary endpoint, we evaluated 18F-FACBC PET/CT’s impact [...] Read more.
Our aim was to assess the detection rate (DR) of positron emission computed tomography (PET/CT) with anti-1-amino-3-[18F]-flurocyclobutane-1-carboxylic acid (18F-FACBC) in patients with biochemical recurrence (BCR) from prostate cancer (PC). As a secondary endpoint, we evaluated 18F-FACBC PET/CT’s impact on patients management. Clinical records of 81 patients submitted to 18F-FACBC PET/CT due to PC BCR in two Italian Nuclear Medicine Units were retrospectively assessed. DR was gauged in the whole cohort and stratifying patients by discrete intervals of PSA levels. PET/CT’s impact on clinical management was scored as (1) major if it entailed an intermodality change (e.g., from systemic to loco-regional therapy); (2) minor if it led to an intramodality change (e.g., modified radiotherapy field). PET/CT’s DR resulted in 76.9% in the whole cohort, with a positive predictive value of 96.7%. Stratified by PSA quartile intervals, PET/CT’s DR was 66.7%, 71.4%, 78.9% and 90% for PSA 0.2–0.57 ng/mL, 0.58–0.99 ng/mL, 1–1.5 ng/mL and >1.5 ng/mL without significant difference among groups (p = 0.81). The most common sites of relapse were prostate bed and pelvic lymph nodes (59.3%). PET/CT impacted on clinical management in 33/81 cases (40.7%), leading to a major change in 30 subjects (90.9%). 18F-FACBC PET/CT localized recurrence in patients with BCR, with meaningful DR also at low PSA levels and significantly impacted on clinical management. Full article
(This article belongs to the Special Issue Prostate Cancer: from Molecular Imaging to Immunological and Target Therapies II)
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<p>Diagnostic flow-chart for patient selection.</p> Full article ">Figure 2
<p>Graphic illustration of PET/CT detection rate in enrolled patients, stratified into 4 groups according to discrete PSA intervals.</p> Full article ">Figure 3
<p>ROC curve analysis shows the accuracy of the PSA level (<b>A</b>) and PSA doubling time (<b>B</b>), measured prior to <sup>18</sup>F-FACBC PET/CT scan, for predicting positive results.</p> Full article ">Figure 4
<p>Box plot graph showing the PSA level distribution in patients with positive (<b>A</b>) and negative (<b>B</b>) <sup>18</sup>F-FABC PET/CT scans, 2-tailed <span class="html-italic">t</span>-test results were significant at <span class="html-italic">p</span> = 0.005. The 2 hot dots in panel A represent 2 subjects with PSA value strongly out of range (i.e., 5 and 7.8 ng/mL, respectively).</p> Full article ">Figure 5
<p>Graph illustration of the therapeutic changes determined by <sup>18</sup>F-FACBC PET/CT in patients classified according to modified PROMISE reading approach.</p> Full article ">Figure 6
<p>A 72-year-old patient 6 years post prostatectomy for pT3b pN0 ISUP Grade Group 4 prostate adenocarcinoma, presenting biochemical recurrence with PSA level prior to PET/CT of 0.38 ng/mL. (<b>A</b>) MIP image showing an area of increased tracer incorporation in the right pelvis (arrow). Fused corresponding PET/CT axial (<b>B</b>, arrow) and coronal (<b>C</b>, arrow) slices depicting a round-shaped right obturator lymph node, characterized by <sup>18</sup>F-FACBC pathological uptake, with a maximum diameter of 12 mm and a maximum standardized uptake value (SUVmax) of 7.7, superior to bone marrow SUVmean (3.1). Final diagnosis was T0N1M0 according to PROMISE. Intended therapy prior to PET/CT was androgen deprivation therapy, after the collegial discussion of <sup>18</sup>F-FACBC PET/CT’s results, the patient was submitted to stereotactic radiotherapy with PSA response.</p> Full article ">Figure 7
<p>A 74-year-old patient 5 years post radiotherapy for cT2b ISUP Grade Group 4 prostate adenocarcinoma, presenting biochemical recurrence with PSA level prior to PET/CT of 1.25 ng/mL. (<b>A</b>) MIP image showing an area of increased tracer incorporation in the left umbilical region (arrow). Fused corresponding PET/CT axial (<b>B</b>, arrow) and coronal (<b>C</b>, arrow) slices depicting a round-shaped para-aortic lymph node, located below the left kidney artery, characterized by <sup>18</sup>F-FACBC pathological uptake, with a maximum diameter of 14 mm and a maximum standardized uptake value (SUV<sub>max</sub>) of 10.1, superior to bone marrow SUV<sub>mean</sub> (3.3). Final diagnosis was T0N0M1a according to PROMISE. Intended therapy prior to PET/CT was androgen deprivation therapy, after the collegial discussion of <sup>18</sup>F-FACBC PET/CT’s results, the patient was submitted to stereotactic radiotherapy on the abdominal node with PSA response.</p> Full article ">
16 pages, 3892 KiB  
Article
Hypoxia Preconditioned Serum (HPS)-Hydrogel Can Accelerate Dermal Wound Healing in Mice—An In Vivo Pilot Study
by Jun Jiang, Ursula Kraneburg, Ulf Dornseifer, Arndt F. Schilling, Ektoras Hadjipanayi, Hans-Günther Machens and Philipp Moog
Biomedicines 2022, 10(1), 176; https://doi.org/10.3390/biomedicines10010176 - 14 Jan 2022
Cited by 8 | Viewed by 2942
Abstract
The ability to use the body’s resources to promote wound repair is increasingly becoming an interesting area of regenerative medicine research. Here, we tested the effect of topical application of blood-derived hypoxia preconditioned serum (HPS) on wound healing in a murine wound model. [...] Read more.
The ability to use the body’s resources to promote wound repair is increasingly becoming an interesting area of regenerative medicine research. Here, we tested the effect of topical application of blood-derived hypoxia preconditioned serum (HPS) on wound healing in a murine wound model. Alginate hydrogels loaded with two different HPS concentrations (10 and 40%) were applied topically on full-thickness wounds created on the back of immunocompromised mice. We achieved a significant dose-dependent wound area reduction after 5 days in HPS-treated groups compared with no treatment (NT). On average, both HPS-10% and HPS-40% -treated wounds healed 1.4 days faster than NT. Healed tissue samples were investigated on post-operative day 15 (POD 15) by immunohistology and showed an increase in lymphatic vessels (LYVE-1) up to 45% with HPS-40% application, while at this stage, vascularization (CD31) was comparable in the HPS-treated and NT groups. Furthermore, the expression of proliferation marker Ki67 was greater on POD 15 in the NT-group compared to HPS-treated groups, in accordance with the earlier completion of wound healing observed in the latter. Collagen deposition was similar in all groups, indicating lack of scar tissue hypertrophy as a result of HPS-hydrogel treatment. These findings show that topical HPS application is safe and can accelerate dermal wound healing in mice. Full article
(This article belongs to the Topic Animal Model in Biomedical Research)
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<p>Accelerated wound healing in HPS-10% and 40% treated wounds. (<b>A</b>) Representative macroscopic photographs of full-thickness wounds in the excisional murine skin model on post-operative day (POD) 0, 3, 5, 7, 9, 11, 13 and 15. Two treatment groups were tested, HPS-10% (<span class="html-italic">n</span> = 14) and HPS-40% (<span class="html-italic">n</span> = 14) vs. no treatment (NT) (<span class="html-italic">n</span> = 10). (<b>B</b>) Plot showing wound surface area (50 mm<sup>2</sup> wound area on POD 0) change from POD 0 to POD 15 in the HPS-10%/-40% and NT groups. HPS-40% group has smaller wound area compared to NT already at POD 5 and POD 7 (<span class="html-italic">p</span> = 0.04 and <span class="html-italic">p</span> = 0.002 respectively), HPS 10% group has smaller wound area compared to NT at POD 7 (<span class="html-italic">p</span> = 0.03). Wound healing kinetics of HPS-10% and -40% treatment groups showed to be significantly better than NT (<span class="html-italic">p</span> = 0.02 and <span class="html-italic">p</span> = 0.0002, respectively). Two-way repeated measures ANOVA with Tukey’s multiple comparisons test. Data points represent means ± SEM. * = <span class="html-italic">p</span> &lt; 0.05, ** = <span class="html-italic">p</span> &lt; 0.01, ns = non-significant.</p> Full article ">Figure 2
<p>CD31 DAB Immunostaining. (<b>A</b>) Quantitative measurement of CD31 Diaminobenzidine (DAB) staining in HPS-10% (<span class="html-italic">n</span> = 14) and 40% (<span class="html-italic">n</span> = 14) treated murine wounds vs. no treatment (NT) (<span class="html-italic">n</span> = 10) on POD 15 by color segmentation of DAB-positive cells calculated as pixels. Vascularization did not appear different in either HPS-treated group compared to the NT group. Data are means ± SEM. Ns = non-significant. One-way ANOVA with Tukey’s post-hoc test. (<b>B</b>–<b>D</b>) Representative high-power fields of CD31 DAB Immunostaining of HPS-10%, HPS-40% -treated wounds and NT wounds. Scale bar = 200 μm.</p> Full article ">Figure 3
<p>LYVE-1 DAB Immunostaining. (<b>A</b>) Quantitative measurement of lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) Diaminobenzidine (DAB) staining in HPS-10% (<span class="html-italic">n</span> = 14) and 40% (<span class="html-italic">n</span> = 14) treated murine wounds vs. no treatment (NT) (<span class="html-italic">n</span> = 10) on POD 15 by color segmentation of DAB-positive cells calculated as pixels. HPS-40% showed higher LYVE-1 expression compared to the NT group (<span class="html-italic">p</span> = 0.01). Data are means ± SEM. * = <span class="html-italic">p</span> &lt; 0.05, ns = non-significant. One-way ANOVA with Tukey’s post-hoc test. (<b>B</b>–<b>D</b>) Representative high-power fields of LYVE-1 DAB Immunostaining of HPS-10%, HPS-40% -treated wounds and NT wounds. Scale bar = 200 μm.</p> Full article ">Figure 4
<p>Ki67 DAB Immunostaining. (<b>A</b>) Quantitative measurement of Ki67 Diaminobenzidine (DAB) staining in HPS-10% (<span class="html-italic">n</span> = 14) and 40% (<span class="html-italic">n</span> = 14) treated murine wounds vs. no treatment (NT) (<span class="html-italic">n</span> = 10) on POD 15 by color segmentation of DAB-positive cells calculated as pixels. HPS-10% group showed the least Ki67 expression compared to HPS-40% (<span class="html-italic">p</span> = 0.03) and NT group (<span class="html-italic">p</span> = 0.02). Data are means ± SEM. * = <span class="html-italic">p</span> &lt; 0.05, ns = non-significant. One-way ANOVA with Tukey’s post-hoc test. (<b>B</b>–<b>D</b>) Representative high-power fields of Ki67 DAB Immunostaining of HPS-10%, HPS-40% treated wounds and NT wounds. Ki67 positive cells were mainly located on POD 15 in the dermal papillary cells as well as in the basal and suprabasal epithelial layers. Scale bar = 200 μm.</p> Full article ">Figure 5
<p>Masson-Goldner-Trichrom staining. (<b>A</b>) Quantitative measurement of collagen fiber deposits in the subdermal layers of HPS-10% (<span class="html-italic">n</span> = 14) and 40% (<span class="html-italic">n</span> = 14) treated murine wounds vs. no treatment (NT) (<span class="html-italic">n</span> = 10) on POD 15 by color segmentation of collagen-staining, calculated as pixels. There was no overproduction of collagen in HPS-treated groups compared to NT. Data are means ± SEM. Ns = non-significant. One-way ANOVA with Tukey’s post-hoc test. (<b>B</b>–<b>D</b>) Representative high-power fields of Masson-Goldner-Trichrom staining of HPS-10%, HPS-40%-treated wounds and NT wounds. Scale bar = 200 μm.</p> Full article ">
15 pages, 4563 KiB  
Article
Puerarin Attenuates Obesity-Induced Inflammation and Dyslipidemia by Regulating Macrophages and TNF-Alpha in Obese Mice
by Ji-Won Noh, Hee-Kwon Yang, Min-Soo Jun and Byung-Cheol Lee
Biomedicines 2022, 10(1), 175; https://doi.org/10.3390/biomedicines10010175 - 14 Jan 2022
Cited by 29 | Viewed by 3858
Abstract
Obesity causes low-grade inflammation that results in dyslipidemia and insulin resistance. We evaluated the effect of puerarin on obesity and metabolic complications both in silico and in vivo and investigated the underlying immunological mechanisms. Twenty C57BL/6 mice were divided into four groups: normal [...] Read more.
Obesity causes low-grade inflammation that results in dyslipidemia and insulin resistance. We evaluated the effect of puerarin on obesity and metabolic complications both in silico and in vivo and investigated the underlying immunological mechanisms. Twenty C57BL/6 mice were divided into four groups: normal chow, control (HFD), HFD + puerarin (PUE) 200 mg/kg, and HFD + atorvastatin (ATO) 10 mg/kg groups. We examined bodyweight, oral glucose tolerance test, serum insulin, oral fat tolerance test, serum lipids, and adipocyte size. We also analyzed the percentage of total, M1, and M2 adipose tissue macrophages (ATMs) and the expression of F4/80, tumor necrosis factor-α (TNF-α), C-C motif chemokine ligand 2 (CCL2), CCL4, CCL5, and C-X-C motif chemokine receptor 4. In silico, we identified the treatment-targeted genes of puerarin and simulated molecular docking with puerarin and TNF, M1, and M2 macrophages based on functionally enriched pathways. Puerarin did not significantly change bodyweight but significantly improved fat pad weight, adipocyte size, fat area in the liver, free fatty acids, triglycerides, total cholesterol, and HDL-cholesterol in vivo. In addition, puerarin significantly decreased the ATM population and TNF-α expression. Therefore, puerarin is a potential anti-obesity treatment based on its anti-inflammatory effects in adipose tissue. Full article
(This article belongs to the Special Issue Macrophages in Health and Non-infectious Disease 3.0)
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<p>PPI network between hub genes of puerarin and obesity and functional pathway analysis. (<b>a</b>) Sixty-nine overlapping genes. (<b>b</b>) Ligand-target protein network. (<b>c</b>) Top 10 genes at high levels of connectivity. (<b>d</b>) GO enrichment analysis. (<b>e</b>) KEGG pathway analysis. PPI: Protein-protein interaction, GO: Gene ontology, KEGG: Kyoto Encyclopedia of Genes and Genomes.</p> Full article ">Figure 2
<p>Molecular docking. (<b>a</b>) Puerarin and TNF. (<b>b</b>) Puerarin and M1 macrophage. (<b>c</b>) Puerarin and M2 macrophage. TNF (PDB ID: 2AZ5), M1 macrophage (PDB ID: 1GD0), and M2 macrophage (PDB ID: 1JIZ). The binding energy is written at the top right.</p> Full article ">Figure 3
<p>Weight- and safety-related outcomes. (<b>a</b>) Bodyweight. (<b>b</b>) Liver weight. (<b>c</b>) Epididymal fat pads weight and levels of (<b>d</b>) AST, (<b>e</b>) ALT, and (<b>f</b>) creatinine. Data represent mean ± standard error of the mean (SEM). # <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 versus the NC group, and * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 versus the control group. AST: aspartate aminotransferase, ALT: alanine aminotransferase, NC: normal chow, HFD: high fat diet, PUE: puerarin, ATO: atorvastatin.</p> Full article ">Figure 3 Cont.
<p>Weight- and safety-related outcomes. (<b>a</b>) Bodyweight. (<b>b</b>) Liver weight. (<b>c</b>) Epididymal fat pads weight and levels of (<b>d</b>) AST, (<b>e</b>) ALT, and (<b>f</b>) creatinine. Data represent mean ± standard error of the mean (SEM). # <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 versus the NC group, and * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 versus the control group. AST: aspartate aminotransferase, ALT: alanine aminotransferase, NC: normal chow, HFD: high fat diet, PUE: puerarin, ATO: atorvastatin.</p> Full article ">Figure 4
<p>Lipid and glucose metabolism. (<b>a</b>) NEFA, (<b>b</b>) total, (<b>c</b>) LDL cholesterol, (<b>d</b>) HDL cholesterol, (<b>e</b>) TG, (f) OFTT, (<b>g</b>) AUC of OFTT, (<b>h</b>) OGTT, (<b>i</b>) AUC of OGTT, (<b>j</b>) fasting blood glucose, (<b>k</b>) fasting insulin, and (<b>l</b>) HOMA-IR. Data represent mean ± standard error of the mean (SEM). # <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 versus the NC group, and * <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 versus the control group. NEFA: non-esterified fatty acid, LDL: low-density lipoprotein, HDL: high-density lipoprotein, TG: triglyceride, AUC: area under the curve, HOMA-IR: homeostatic model assessment for insulin resistance, NC: normal chow, HFD: high fat diet, PUE: puerarin, ATO: atorvastatin.</p> Full article ">Figure 5
<p>ATM populations. (<b>a</b>) CD45 + ATMs. (<b>b</b>) Flow cytometry result of CD45 + ATMs. (<b>c</b>) CD11c + ATMs, and (<b>d</b>) CD206 + ATMs. Data represent mean ± standard error of the mean (SEM). ### <span class="html-italic">p</span> &lt; 0.001 versus the NC group, and ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 versus the control group. ATMs: adipose tissue macrophages, NC: normal chow, HFD: high rat diet, PUE: puerarin, ATO: atorvastatin.</p> Full article ">Figure 6
<p>Pro-inflammatory gene expression. The expression of (<b>a</b>) F4/80, (<b>b</b>) TNF- α, (<b>c</b>) CCL2, (<b>d</b>) CCL4, (<b>e</b>) CCL5, and (<b>f</b>) CXCR4. Data represent mean ± standard error of the mean (SEM). # <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 versus the NC group, and * <span class="html-italic">p</span> &lt; 0.05 versus the control group. ATMs: adipose tissue macrophages, NC: normal chow, HFD: high rat diet, PUE: puerarin, ATO: atorvastatin.</p> Full article ">Figure 6 Cont.
<p>Pro-inflammatory gene expression. The expression of (<b>a</b>) F4/80, (<b>b</b>) TNF- α, (<b>c</b>) CCL2, (<b>d</b>) CCL4, (<b>e</b>) CCL5, and (<b>f</b>) CXCR4. Data represent mean ± standard error of the mean (SEM). # <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 versus the NC group, and * <span class="html-italic">p</span> &lt; 0.05 versus the control group. ATMs: adipose tissue macrophages, NC: normal chow, HFD: high rat diet, PUE: puerarin, ATO: atorvastatin.</p> Full article ">Figure 7
<p>Analysis of tissue microscopic results. (<b>a</b>) Histological images of liver and epididymal fat. (<b>b</b>) Fat area in the liver and (<b>c</b>) adipocyte size. Representative histological images were stained by hematoxylin and eosin (H &amp; E) and the scale bar indicates 5 μm in liver and 100 μm in epi fat. Data represent mean ± standard error of the mean (SEM). ### <span class="html-italic">p</span> &lt; 0.001 versus the NC group and *** <span class="html-italic">p</span> &lt; 0.001 versus the control group. NC: normal chow, HFD: high fat diet, PUE: puerarin, ATO: atorvastatin.</p> Full article ">
21 pages, 3173 KiB  
Article
A More Diverse Cervical Microbiome Associates with Better Clinical Outcomes in Patients with Endometriosis: A Pilot Study
by Cherry Yin-Yi Chang, An-Jen Chiang, Ming-Tsung Lai, Man-Ju Yan, Chung-Chen Tseng, Lun-Chien Lo, Lei Wan, Chia-Jung Li, Kuan-Hao Tsui, Chih-Mei Chen, Tritium Hwang, Fuu-Jen Tsai and Jim Jinn-Chyuan Sheu
Biomedicines 2022, 10(1), 174; https://doi.org/10.3390/biomedicines10010174 - 14 Jan 2022
Cited by 26 | Viewed by 7025
Abstract
Infection-induced chronic inflammation is common in patients with endometriosis. Although microbial communities in the reproductive tracts of patients have been reported, little was known about their dynamic profiles during disease progression and complication development. Microbial communities in cervical mucus were collected by cervical [...] Read more.
Infection-induced chronic inflammation is common in patients with endometriosis. Although microbial communities in the reproductive tracts of patients have been reported, little was known about their dynamic profiles during disease progression and complication development. Microbial communities in cervical mucus were collected by cervical swabs from 10 healthy women and 23 patients, and analyzed by 16S rRNA amplicon sequencing. The abundance, ecological relationships and functional networks of microbiota were characterized according to their prevalence, clinical stages, and clinical features including deeply infiltrating endometriosis (DIE), CA125, pain score and infertility. Cervical microbiome can be altered during endometriosis development and progression with a tendency of increased Firmicutes and decreased Actinobacteria and Bacteroidetes. Distinct from vaginal microbiome, upregulation of Lactobacillus, in combination with increased Streptococcus and decreased Dialister, was frequently associated with advanced endometriosis stages, DIE, higher CA125 levels, severe pain, and infertility. Significantly, reduced richness and diversity of cervical microbiome were detected in patients with more severe clinical symptoms. Clinical treatments against infertility can partially reverse the ecological balance of microbes through remodeling nutrition metabolism and transport and cell-cell/cell-matrix interaction. This study provides a new understanding on endometriosis development and a more diverse cervical microbiome may be beneficial for patients to have better clinical outcomes. Full article
(This article belongs to the Special Issue Microbial Ecology in Health and Disease 2.0)
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<p>Abundance and composition of cervical microbiomes among normal women and endometriosis patients at different stages. (<b>a</b>) The top-5 major phyla of bacteria in cervical microbiome of Taiwanese women. (<b>b</b>) Tree graphs of species annotation for cervical microbiomes of normal women and endometriosis patients were constructed and compared by GraPhlAn method. (<b>c</b>) Relative abundance of the top-10 major phyla of cervical microbiomes in different groups are shown in distribution histogram. (<b>d</b>) Heatmap based on unweighted unifrac distance was plotted to measure the dissimilarity coefficient between pairwise group samples. The three groups include cervical microbiomes from normal women, patients at stage I-II and patients at stage III–IV.</p> Full article ">Figure 2
<p>Microbial communities associated with endometriosis patients at different stages. (<b>a</b>) LEfSe (linear discriminant analysis (LDA) Effect Size) was performed to define microbial biomarkers for individual groups (normal women, patients at stage I-II, and stage III-IV) with statistical differences. The representative microbiotas for each group are shown with the LDA scores (left panel). The phylogenetic trees of dominant microorganisms are revealed in cladogram (right). (<b>b</b>) Ternary plots in Genus (<b>left</b>) and Species (<b>right</b>) taxonomic ranks were drawn based on relative abundance of top-10 OTUs among different groups. Circles represent dominant species and the size of those circles represent the relative abundance. (<b>c</b>) One-way ANOVA was performed to define the major microbes down-regulated (upper) or up-regulated (lower) during endometriosis progression. Statistical significance: ***, <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>Microbial flora in cervical mucus that associates with deeply infiltrating endometriosis (DIE). Major (<b>a</b>) phyla, (<b>b</b>) genera and (<b>c</b>) species in the cervix that show significant differences in abundance between patients with DIE and patients without were presented. Statistical significance was estimated by t-test and shown as *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 4
<p>Abundance and composition of cervical microbiomes in endometriosis patients with different CA125 levels and pain scores. (<b>a</b>) Relative abundance of the top-10 major phyla of cervical microbiomes in patients with different CA125 levels (<b>left</b>) and pain scores (<b>right</b>). (<b>b</b>) The rarefaction curves indicate the differences in biodiversity (accumulated OTU numbers) of group samples according to CA125 levels (<b>left</b>) or pain scores (<b>right</b>). Statistics were performed with two-way ANOVA method. One-way ANOVA was performed to define the major down-regulated or up-regulated microbes in cervical mucus of patients that associated with (<b>c</b>) higher CA125 levels or (<b>d</b>) higher pain scores. Statistical significance: *, <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>Community diversity of cervical microbiome in endometriosis patients correlate with the severity of clinical outcomes. (<b>a</b>) The community diversity in each patient group is revealed by a heat tree map, showing the taxonomic context from higher ranks in the center to lower ranks in peripheries (Kingdom to Species). The abundance of microbiotas in each community can be quantified and visualized by color and size of the nodes and edges. (<b>b</b>) The box-plot was generated to illustrate alpha diversity indices (Shannon diversity) in cervical microbiomes of patient groups with different levels of severities. The statistic for the linear trend is estimated by one-way ANOVA. (<b>c</b>) The t-SNE analysis diagram was plotted to calculate the actual and embedded distance among the patient groups with different levels of severities. (<b>d</b>) The heatmap indicates the average counts of genera that show consistently up-regulated or down-regulated among normal women, double-negative and double-positive patients. Genera with bold font show statistically significant differences in OTU abundance among the groups by one-way ANOVA (<a href="#app1-biomedicines-10-00174" class="html-app">Figure S2</a>). (<b>e</b>) The Venn diagram indicates annotated OTUs in each group. Values in the overlapping part represent common OTUs. The others are specific OTUs in each group. (<b>f</b>) The KEGG/L2 pathways that show significant fold changes between double-positive and double-negative patients are presented. Statistical significance: *, <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 6
<p>Abundance and composition of cervical microbiomes in endometriosis patients with different reproductive ability. (<b>a</b>) Relative abundance of the top-10 major phyla of cervical microbiomes in patients with different reproductive ability are shown in distribution histogram. (<b>b</b>) Firmicutes/Bacteroidetes (F/B) ratios in cervical microbiomes of patients between two groups are compared by t-test. (<b>c</b>) The rarefaction curves indicate the differences in biodiversity (accumulated OTU numbers) of patients with different reproductive ability. Statistics were performed with two-way ANOVA method. (<b>d</b>) One-way ANOVA indicates the trend of alterations in alpha-diversity according to the reproductive ability of patients by using Shannon (<b>left</b>) and Chao1 (<b>right</b>) methods. (<b>e</b>) The t-SNE analysis diagram was plotted to calculate the actual and embedded distance among different patient groups. (<b>f</b>) Phylogenetic heat trees were constructed to reveal the abundance and diversity of microbiotas in different patient groups. Statistical significance: *, <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 7
<p>Ecological relationship of bacterial community in cervical mucus in endometriosis patients with different reproductive ability. (<b>a</b>) The heatmap reveals relative abundance of genera that show statistical differences between fertile and infertile patients by t-test (<span class="html-italic">p</span> &lt; 0.05). Yellow boxes indicate the genera that show consistent correlation between microbial abundance and reproductive ability. (<b>b</b>) Spearman correlation matrices reveal inter-genus long-term drifts of cervical microbiomes from patients with different reproductive ability.</p> Full article ">Figure 8
<p>Functional impacts on KEGG pathways governed by altered cervical microbiomes in endometriosis patients with different reproductive ability. (<b>a</b>) As compared to fertile patients, the significant altered KEGG/L3 functions (in blue) in infertile patients were plotted by t-test. (<b>b</b>) Among the significantly altered KEGG/L3 functions in (<b>a</b>), the dot plot indicates the key functions (in green) that can be re-gained or re-suppressed in cured patients by anti-infertility treatments. (<b>c</b>) The pie chart indicates the proportions of 8 major KEGG/L2 functions which the key altered functions in (<b>b</b>) can be categorized into. The data in (<b>a</b>) to (<b>c</b>) were generated by using average scores of functional annotations among patient groups. (<b>d</b>) Among the key altered functions in (<b>b</b>), the box plots indicate nine KEGG/L3 functions which show significant linear trend among patient groups with different reproductive ability by one-way ANOVA. Statistical significance: *, <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 ">
16 pages, 4008 KiB  
Article
Systemic Injection of Oncolytic Vaccinia Virus Suppresses Primary Tumor Growth and Lung Metastasis in Metastatic Renal Cell Carcinoma by Remodeling Tumor Microenvironment
by Jee Soo Park, Myung Eun Lee, Won Sik Jang, Jongchan Kim, Se Mi Park, Keunhee Oh, Namhee Lee and Won Sik Ham
Biomedicines 2022, 10(1), 173; https://doi.org/10.3390/biomedicines10010173 - 14 Jan 2022
Cited by 9 | Viewed by 3284
Abstract
Immune checkpoint inhibitors and tyrosine kinase inhibitors are the first-line treatment for metastatic renal cell carcinoma (mRCC), but their benefits are limited to specific patient subsets. Here, we aimed to evaluate the therapeutic efficacy of JX-594 (pexastimogene devacirepvec, Pexa-vec) monotherapy by systemic injection [...] Read more.
Immune checkpoint inhibitors and tyrosine kinase inhibitors are the first-line treatment for metastatic renal cell carcinoma (mRCC), but their benefits are limited to specific patient subsets. Here, we aimed to evaluate the therapeutic efficacy of JX-594 (pexastimogene devacirepvec, Pexa-vec) monotherapy by systemic injection in comparison with sunitinib monotherapy in metastatic orthotopic RCC murine models. Two highly metastatic orthotopic RCC models were developed to compare the treatment efficacy in the International Metastatic RCC Database Consortium favorable-risk and intermediate- or poor-risk groups. JX-594 was systemically injected through the peritoneum, whereas sunitinib was orally administered. Post-treatment, tumor microenvironment (TME) remodeling was determined using immunofluorescence analysis. Systemic JX-594 monotherapy injection demonstrated therapeutic benefit in both early- and advanced-stage mRCC models. Sunitinib monotherapy significantly reduced the primary tumor burden and number of lung metastases in the early-stage, but not in the advanced-stage mRCC model. Systemic JX-594 delivery remodeled the primary TME and lung metastatic sites by increasing tumor-infiltrating CD4/8+ T cells and dendritic cells. Systemic JX-594 monotherapy demonstrated significantly better therapeutic outcomes compared with sunitinib monotherapy in both early- and advanced-stage mRCCs by converting cold tumors into hot tumors. Sunitinib monotherapy effectively suppressed primary tumor growth and lung metastasis in early-stage mRCC. Full article
(This article belongs to the Special Issue Oncolytic Viruses as a Novel Form of Immunotherapy for Cancer III)
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<p>Two pulmonary metastatic orthotopic renal cell carcinoma murine models. (<b>A</b>) An early-stage metastatic renal cell carcinoma (mRCC) model was used to compare groups A (mJX-594 monotherapy) and B (sunitinib monotherapy). The advanced-stage mRCC model was used to compare groups C (mJX-594 monotherapy) and D (sunitinib monotherapy). (<b>B</b>) In vivo selected tumor cells from pulmonary metastases for reimplanted (1 × 10<sup>5</sup> cells/100 μL) and treatments started on the first day of implantation for groups A and B. For groups C and D, treatments started on the 11th day of implantation. mJX-594 was intraperitoneally injected every 3 days for three times, whereas sunitinib was treated by gavage daily for 7 days. After the 21st day of injection, analysis was performed.</p> Full article ">Figure 2
<p>JX-594 has a direct oncolytic effect against human and murine renal cell carcinoma (RCC). (<b>A</b>) Cell viability of human (786-O, A498, ACHN, Caki-1) and murine (Renca) RCC cell lines treated with escalating doses of JX-594 or mJX-594. (<b>B</b>) Cell cycle analyses of mJX-594-treated cells. Renca cells treated with mJX-594 (1 MOI) were analyzed by flow cytometry for DNA content at 24 h post-treatment. Representative cell cycle histograms (left) and quantification of cell cycle distribution (right). (<b>C</b>) Apoptosis of Renca cells treated with mJX-594 or control (PBS) was analyzed using the TUNEL assay; TUNEL-positive cells (pseudocolored red), nuclei (pseudocolored blue), scale bar: 200 μm. (<b>D</b>) Migration of Renca cells was evaluated and compared after the treatment with mJX-594 or control (PBS), scale bar: 200 μm. (<b>E</b>) Invasion of Renca cells was evaluated and compared after the treatment with mJX-594 or control (PBS), scale bar: 200 μm. Values are mean ± SD. PBS, phosphate-buffered saline. All experiments were conducted at least three times.</p> Full article ">Figure 3
<p>Representative images and comparisons of primary tumor burden and number of lung nodules in mJX-594-treated, sunitinib-treated, and control (PBS-treated) mice. (<b>A</b>) Comparison of primary tumor burden after the treatments in early-stage metastatic renal cell carcinoma (mRCC) model. (<b>B</b>) Comparison of lung metastasis after the treatments in early-stage mRCC model. (<b>C</b>) Comparison of primary tumor burden after the treatments in advanced-stage mRCC model. (<b>D</b>) Comparison of lung metastasis after the treatments in advanced-stage mRCC model. Therapeutic efficacy of mJX-594 and sunitinib treatment in early- and advanced-stage mRCC models. (<b>E</b>) Comparison of primary tumor burden after the treatments with mJX-594, sunitinib, or control (PBS) in early-stage mRCC model. (<b>F</b>) Comparison of lung metastasis after the treatments with mJX-594, sunitinib, or control (PBS) in early-stage mRCC model. (<b>G</b>) Comparison of primary tumor burden after the treatments with mJX-594, sunitinib, or control (PBS) in advanced-stage mRCC model. (<b>H</b>) Comparison of lung metastasis after the treatments with mJX-594, sunitinib, or control (PBS) in advanced-stage mRCC model. Values are mean ± SD. PBS, phosphate-buffered saline; ns, not significant; #, number.</p> Full article ">Figure 4
<p>mJX-594 systemic treatment activates anti-cancer immunity by converting immunosuppressive noninflamed tumors into inflamed tumors at primary tumor. (<b>A</b>) Representative images of primary tumors treated with mJX-594 systemic treatment. Tumor sections were stained for JX-594, CD31, CD8, CD4, CD11c, Foxp3, and PD-L1. Scale bars, 50 μm. (<b>B</b>) Quantifications of the JX-594+ area, CD31+ blood vessels, CD8+ T cells, CD4+ T cells, CD11c+ dendritic cells, Foxp3+ regulatory T cells, and PD-L1+ cells. Values are mean ± SD. * <span class="html-italic">p</span> &lt; 0.05 vs. control. 6 animals per group were used.</p> Full article ">Figure 5
<p>mJX-594 systemic treatment activates anti-cancer immunity by converting immunosuppressive noninflamed tumors into inflamed tumors at lung metastatic sites. (<b>A</b>) Representative images of lung metastatic sites treated with mJX-594 systemic treatment. Tumor sections were stained for JX-594, CD31, CD8, CD4, CD11c, Foxp3, and PD-L1. Scale bars, 50 μm. (<b>B</b>) Quantifications of the JX-594+ area, CD31+ blood vessels, CD8+ T cells, CD4+ T cells, CD11c+ dendritic cells, Foxp3+ regulatory T cells, and PD-L1+ cells. Values are mean ± SD. * <span class="html-italic">p</span> &lt; 0.05 vs. control. 6 animals per group were used.</p> Full article ">
13 pages, 1557 KiB  
Systematic Review
The Role of Neuropilin-2 in the Epithelial to Mesenchymal Transition of Colorectal Cancer: A Systematic Review
by Cristina Lungulescu, Valentina Ghimpau, Dan Ionut Gheonea, Daniel Sur and Cristian Virgil Lungulescu
Biomedicines 2022, 10(1), 172; https://doi.org/10.3390/biomedicines10010172 - 14 Jan 2022
Cited by 7 | Viewed by 2599
Abstract
Neuropilin-2 (NRP-2) expression has been found in various investigations on the expression and function of NRP-2 in colorectal cancer. The link between NRP-2 and colorectal cancer, as well as the mechanism that regulates it, is still mostly unclear. This systematic review was carried [...] Read more.
Neuropilin-2 (NRP-2) expression has been found in various investigations on the expression and function of NRP-2 in colorectal cancer. The link between NRP-2 and colorectal cancer, as well as the mechanism that regulates it, is still mostly unclear. This systematic review was carried out according to the Cochrane guidelines for systematic reviews. We searched PubMed, Embase®, MEDLINE, Allied & Complementary MedicineTM, Medical Toxicology & Environmental Health, DH-DATA: Health Administration for articles published before 1 October 2021. The following search terms were used: “neuropilin-2” “neuropilin 2”, “NRP2” and “NRP-2”, “colorectal cancer”, “colon cancer”. Ten articles researching either tumor tissue samples, cell lines, or mice models were included in this review. The majority of human primary and metastatic colon cancer cell lines expressed NRP-2 compared to the normal colonic mucosa. NRPs have been discovered in human cancers as well as neovasculature. The presence of NRP-2 appears to be connected to the epithelial–mesenchymal transition’s function in cancer dissemination and metastatic evolution. The studies were heterogeneous, but the data assessed indicates NRP-2 might have an impact on the metastatic potential of colorectal cancer cells. Nevertheless, further research is needed. Full article
(This article belongs to the Special Issue Epithelial to Mesenchymal Transition and Cellular Plasticity in Colon Cancer Progression: Future Perspectives for Diagnosis and Target Therapy)
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<p>Epithelial to Mesenchymal Transition.</p> Full article ">Figure 2
<p>Neuropilin 2 regulatory mechanism. NRP2 promotes TGFβ signaling and induces EMT.</p> Full article ">Figure 3
<p>PRISMA flow diagram of the study selection process.</p> Full article ">Figure 4
<p>Risk of bias summary for each included study. The Cochrane risk-of-bias assessment for the 10 articles included in this review. In terms of random sequence generation, 6 studies had a low risk of bias while 4 had high risk of bias. Concerning the risk of allocation concealment, 5 studies had a low risk of bias and 5 has a high risk of bias. In the selective reporting domain, 4 studies had a low risk of bias, 3 unclear, and 3 had high.</p> Full article ">
14 pages, 5440 KiB  
Article
Humoral Immune Response in IBD Patients Three and Six Months after Vaccination with the SARS-CoV-2 mRNA Vaccines mRNA-1273 and BNT162b2
by Richard Vollenberg, Phil-Robin Tepasse, Joachim Ewald Kühn, Marc Hennies, Markus Strauss, Florian Rennebaum, Tina Schomacher, Göran Boeckel, Eva Lorentzen, Arne Bokemeyer and Tobias Max Nowacki
Biomedicines 2022, 10(1), 171; https://doi.org/10.3390/biomedicines10010171 - 13 Jan 2022
Cited by 21 | Viewed by 4522
Abstract
Severe acute respiratory syndrome coronovirus-2 (SARS-CoV-2) is the cause of the coronavirus disease 2019 (COVID-19) pandemic. Vaccination is considered the core approach to containing the pandemic. There is currently insufficient evidence on the efficacy of these vaccines in immunosuppressed inflammatory bowel disease (IBD) [...] Read more.
Severe acute respiratory syndrome coronovirus-2 (SARS-CoV-2) is the cause of the coronavirus disease 2019 (COVID-19) pandemic. Vaccination is considered the core approach to containing the pandemic. There is currently insufficient evidence on the efficacy of these vaccines in immunosuppressed inflammatory bowel disease (IBD) patients. The aim of this study was to investigate the humoral response in immunosuppressed IBD patients after COVID-19 mRNA vaccination. In this prospective study, IgG antibody levels (AB) against the SARS-CoV-2 receptor-binding domain (spike-protein) were quantitatively determined. For assessing the potential neutralizing capacity, a SARS-CoV-2 surrogate neutralization test (sVNT) was employed in IBD patients (n = 95) and healthy controls (n = 38). Sera were examined prior to the first/second vaccination and 3/6 months after second vaccination. Patients showed lower sVNT (%) and IgG-S (AU/mL) AB both before the second vaccination (sVNT p < 0.001; AB p < 0.001) and 3 (sVNT p = 0.002; AB p = 0.001) and 6 months (sVNT p = 0.062; AB p = 0.061) after the second vaccination. Although seroconversion rates (sVNT, IgG-S) did not differ between the two groups 3 months after second vaccination, a significant difference was seen 6 months after second vaccination (sVNT p = 0.045). Before and three months after the second vaccination, patients treated with anti-tumor necrosis factor (TNF) agents showed significantly lower AB than healthy subjects. In conclusion, an early booster shot vaccination should be discussed for IBD patients on anti-TNF therapy. Full article
(This article belongs to the Special Issue Next-Generation Vaccines and Antivirals against SARS-CoV-2)
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<p>Study flow chart. Inclusion of IBD patients in the period from 01/2021 to 11/2021 on immunosuppressive medication and healthy controls. Blood collection within 48 h before the first and second vaccination, 3 and 6 months following the 2nd vaccination (+/− 7 days). Exclusion of patients and controls with suspected or confirmed SARS-CoV-2 infection, and ChAdOx1/Ad26.CoV2.S vaccination. IBD, inflammatory bowel disease; SARS-CoV-2, severe acute respiratory syndrome coronavirus type 2; vacc., vaccination.</p> Full article ">Figure 2
<p>SARS-CoV-2-S IgG and sVTN inhibition levels in IBD patient subgroups before the second vaccination (<b>a</b>) and 3 months after the second vaccination (<b>b</b>). Comparison of antibody levels between IBD patients on immunosuppressive therapy (anti-TNF, vedolizumab) and healthy controls before the second vaccination (<b>c</b>) and 3 months after the second vaccination (<b>d</b>), antibody levels of all IBD patients in relation to existing immunosuppressive therapy compared with healthy controls (<b>e</b>). Tukey boxplots, * <span class="html-italic">p</span> &gt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 2 Cont.
<p>SARS-CoV-2-S IgG and sVTN inhibition levels in IBD patient subgroups before the second vaccination (<b>a</b>) and 3 months after the second vaccination (<b>b</b>). Comparison of antibody levels between IBD patients on immunosuppressive therapy (anti-TNF, vedolizumab) and healthy controls before the second vaccination (<b>c</b>) and 3 months after the second vaccination (<b>d</b>), antibody levels of all IBD patients in relation to existing immunosuppressive therapy compared with healthy controls (<b>e</b>). Tukey boxplots, * <span class="html-italic">p</span> &gt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 3
<p>Humoral response of IBD patients and healthy controls before second vaccination, as well as 3 and 6 months after second vaccination. (<b>a</b>) Percentage seroconversion rates (sVTN &gt; 30%); (<b>b</b>) percentage seroconversion rates (SARS-CoV-2 S-IgG ≥ 50 AU/mL); (<b>c</b>) quantitative detection of SARS-CoV-2 S-IgG; (<b>d</b>) percentage sVTN inhibition, representation of the cutoff values (30% inhibition) as a dashed line. IBD: inflammatory bowel disease; vacc: vaccination. Tukey boxplots, * <span class="html-italic">p &lt;</span> 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
33 pages, 4421 KiB  
Review
Epigenetic Mechanisms as Emerging Therapeutic Targets and Microfluidic Chips Application in Pulmonary Arterial Hypertension
by Linh Ho, Nazir Hossen, Trieu Nguyen, Au Vo and Fakhrul Ahsan
Biomedicines 2022, 10(1), 170; https://doi.org/10.3390/biomedicines10010170 - 13 Jan 2022
Cited by 12 | Viewed by 6262
Abstract
Pulmonary arterial hypertension (PAH) is a disease that progress over time and is defined as an increase in pulmonary arterial pressure and pulmonary vascular resistance that frequently leads to right-ventricular (RV) failure and death. Epigenetic modifications comprising DNA methylation, histone remodeling, and noncoding [...] Read more.
Pulmonary arterial hypertension (PAH) is a disease that progress over time and is defined as an increase in pulmonary arterial pressure and pulmonary vascular resistance that frequently leads to right-ventricular (RV) failure and death. Epigenetic modifications comprising DNA methylation, histone remodeling, and noncoding RNAs (ncRNAs) have been established to govern chromatin structure and transcriptional responses in various cell types during disease development. However, dysregulation of these epigenetic mechanisms has not yet been explored in detail in the pathology of pulmonary arterial hypertension and its progression with vascular remodeling and right-heart failure (RHF). Targeting epigenetic regulators including histone methylation, acetylation, or miRNAs offers many possible candidates for drug discovery and will no doubt be a tempting area to explore for PAH therapies. This review focuses on studies in epigenetic mechanisms including the writers, the readers, and the erasers of epigenetic marks and targeting epigenetic regulators or modifiers for treatment of PAH and its complications described as RHF. Data analyses from experimental cell models and animal induced PAH models have demonstrated that significant changes in the expression levels of multiple epigenetics modifiers such as HDMs, HDACs, sirtuins (Sirt1 and Sirt3), and BRD4 correlate strongly with proliferation, apoptosis, inflammation, and fibrosis linked to the pathological vascular remodeling during PAH development. The reversible characteristics of protein methylation and acetylation can be applied for exploring small-molecule modulators such as valproic acid (HDAC inhibitor) or resveratrol (Sirt1 activator) in different preclinical models for treatment of diseases including PAH and RHF. This review also presents to the readers the application of microfluidic devices to study sex differences in PAH pathophysiology, as well as for epigenetic analysis. Full article
(This article belongs to the Special Issue Druggability of Proteins/Enzymes)
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<p>Major pathophysiological mechanisms that lead to vascular remodeling increased pulmonary artery and, thus, right-ventricle remodeling. The sequential pathological events of vascular remodeling in PAH include increased proliferation of smooth muscle cells, subsequent muscularization of peripheral pulmonary arteries, and medial hypertrophy in pulmonary muscular arteries. Further intimal fibrosis occurs by infiltration of inflammatory cells and progressive migration of smooth muscle cells. As a consequence, plexiform lesions and vessel occlusion cause reduced blood flow, resulting in PAH as progression of PAH.</p> Full article ">Figure 2
<p>Current therapeutic target pathways for PAH. The endothelin-1, prostacyclin, and nitric oxide pathways have been exploited in clinical therapeutics. These three pathways are critical pathways approved for PAH treatment. Abbreviations: NO, nitric oxide; NOS, nitric oxide synthase; eNOS, enthothelial nitric oxide synthase; GC, guanylyl cyclase; PDE5, phosphodiesterase type 5; GMP, guanosine monophosphate; cGMP, cyclic guanosine monophosphate; GTP, guanosine triphosphate; ET-1, endothelin-1; ETA, endothelin A; ETB, endothelin type A; ECE, endothelin converting enzyme; PIP, phosphatidylinositol phosphate; IP3, inositol 1,4,5-trisphosphate; COX, cyclooxygenase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate.</p> Full article ">Figure 3
<p>Potential epigenetic therapeutic targets for PAH. Targeting epigenetic modifications that involve PAH pathogenesis and right-heart failure to develop drugs for the treatment of PAH. Epigenetic mechanisms including demethylation of typical methylation motifs histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 9 trimethylation (H3K9me3), DNA methylation, methylation of histone 3 lysine 4 (H3K4), and acetylation and deacetylation of histone lysine residues, as well as miRNAs and lncRNAs, in PAH affect chromatin packaging, accessibility, and gene expression and permit transcriptional elements and transcription apparatus to modulate gene transcription. Activation of enzymes, such as methyltransferases, demethylases, acetyltransferases, and deacetylases, is required for transitional states of chromatin for regulating gene expression. Sirtuins regulate apoptosis, glycolysis shift, and mitochondrial biogenesis in PAH. These processes lead to hyperproliferation, apoptosis resistance, and inflammation of PASMCs and PAECs, resulting in PAH pathology and ultimately right-heart failure. Epigenetic modifications that have been targeted for drug treatment of PAH are shown in green. Epigenetic modulators that were experimentally tested and potential modulators are listed in red. Me3, trimethylation; Me. methylation; Ac, acetylation.</p> Full article ">Figure 4
<p>Targeting miRNAs can revert the phenotype of PAH to normal by inhibiting cell proliferation. During PAH pathogenesis, the expression of several miRNAs (miR-1281, miR-124, miR-140, miR34a, and miR-223) in smooth muscle cells of pulmonary artery is downregulated, while that of others (miR-424 and 503 and miR-17/92) is down and upregulated in endothelial cells. However, miRNA expression in the fibroblast still remains to be clarified. Under up- and downregulated conditions, the proliferation of cells, including smooth muscle, endothelial, and fibroblast cells within the pulmonary artery, occurs and, in turn, narrows the artery and decreases blood blow, while increasing the pressure, leading to PAH pathogenesis. Delivery of miRNA mimics to the downregulated miRNAs or that of antagomiRs to the upregulated miRNAs in these cells could be a possible way to return the situation to normal.</p> Full article ">
15 pages, 1953 KiB  
Article
Diagnostic Blood Biomarkers in Alzheimer’s Disease
by Jung Eun Park, Tamil Iniyan Gunasekaran, Yeong Hee Cho, Seong-Min Choi, Min-Kyung Song, Soo Hyun Cho, Jahae Kim, Ho-Chun Song, Kyu Yeong Choi, Jang Jae Lee, Zee-Yong Park, Woo Keun Song, Han-Seong Jeong, Kun Ho Lee, Jung Sup Lee and Byeong C. Kim
Biomedicines 2022, 10(1), 169; https://doi.org/10.3390/biomedicines10010169 - 13 Jan 2022
Cited by 24 | Viewed by 4774
Abstract
Potential biomarkers for Alzheimer’s disease (AD) include amyloid β1–42 (Aβ1–42), t-Tau, p-Tau181, neurofilament light chain (NFL), and neuroimaging biomarkers. Their combined use is useful for diagnosing and monitoring the progress of AD. Therefore, further development of a combination [...] Read more.
Potential biomarkers for Alzheimer’s disease (AD) include amyloid β1–42 (Aβ1–42), t-Tau, p-Tau181, neurofilament light chain (NFL), and neuroimaging biomarkers. Their combined use is useful for diagnosing and monitoring the progress of AD. Therefore, further development of a combination of these biomarkers is essential. We investigated whether plasma NFL/Aβ1–42 can serve as a plasma-based primary screening biomarker reflecting brain neurodegeneration and amyloid pathology in AD for monitoring disease progression and early diagnosis. We measured the NFL and Aβ1–42 concentrations in the CSF and plasma samples and performed correlation analysis to evaluate the utility of these biomarkers in the early diagnosis and monitoring of AD spectrum disease progression. Pearson’s correlation analysis was used to analyse the associations between the fluid biomarkers and neuroimaging data. The study included 136 participants, classified into five groups: 28 cognitively normal individuals, 23 patients with preclinical AD, 22 amyloid-negative patients with amnestic mild cognitive impairment, 32 patients with prodromal AD, and 31 patients with AD dementia. With disease progression, the NFL concentrations increased and Aβ1–42 concentrations decreased. The plasma and CSF NFL/Aβ1–42 were strongly correlated (r = 0.558). Plasma NFL/Aβ1–42 was strongly correlated with hippocampal volume/intracranial volume (r = 0.409). In early AD, plasma NFL/Aβ1–42 was associated with higher diagnostic accuracy than the individual biomarkers. Moreover, in preclinical AD, plasma NFL/Aβ1–42 changed more rapidly than the CSF t-Tau or p-Tau181 concentrations. Our findings highlight the utility of plasma NFL/Aβ1–42 as a non-invasive plasma-based biomarker for early diagnosis and monitoring of AD spectrum disease progression. Full article
(This article belongs to the Special Issue Molecular Mechanisms and Treatments on Neurodegenerative Diseases)
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<p>Biomarker concentrations in the CSF, plasma, and neuroimaging data. Data are presented as mean values of ATN (amyloid, tau, and neurodegeneration) biomarker concentrations in the CSF (<b>a</b>–<b>d</b>), plasma neurofilament light chain (NFL) concentrations (<b>e</b>), plasma Aβ<sub>1–42</sub> concentrations (<b>f</b>), CSF NFL/Aβ<sub>1–42</sub> (<b>g</b>), plasma NFL/Aβ<sub>1–42</sub> (<b>h</b>), standard uptake value ratio (SUVR) scores (<b>i</b>), and value of hippocampal volume/intracranial volume (ICV) (<b>j</b>). Statistical analysis was performed using SPSS version 25. ** <span class="html-italic">p</span> &lt; 0.001, statically significant group effect by ANOVA [groups: cognitively normal (CN) (n = 51), amnestic mild cognitive impairment (aMCI) (n = 54), and Alzheimer’s disease (AD) dementia (n = 31)]. * <span class="html-italic">p</span> &lt; 0.005, <sup>†</sup> <span class="html-italic">p</span> &lt; 0.05, significant difference between two indicated groups using ANCOVA adjusted for age and sex. (<b>k</b>) Brain cortical atrophy patterns as t-value maps in the preclinical AD, prodromal AD, and AD dementia groups. Preclinical AD (CN Aβ+) (n = 23), prodromal AD (aMCI Aβ+) (n = 32), and AD dementia (AD Aβ+) (n = 30) groups were compared with the CN Aβ− (n = 28) group to observe differences in point-wise cortical thickness using a general linear model with adjustments for age, sex, and field strength as covariates. Greater cortical atrophy was observed in the AD dementia group.</p> Full article ">Figure 2
<p>Correlation analysis, ROC curves, and biomarker dynamics. Pearson’s correlation analysis was used to analyse the correlations among CSF neurofilament light chain (NFL) and plasma NFL concentrations (<b>a</b>), CSF Aβ<sub>1–42</sub> and plasma Aβ<sub>1–42</sub> concentrations (<b>b</b>), CSF NFL/Aβ<sub>1–42</sub> and plasma NFL/Aβ<sub>1–42</sub> (<b>c</b>), and plasma NFL/Aβ<sub>1–42</sub> and hippocampal volume/intracranial volume (ICV) (<b>d</b>). Representative ROC curves and AUC values are shown for indicated diagnostic groups (<b>e</b>–<b>l</b>). CSF and plasma biomarkers and neuroimaging dynamics as the standard uptake value ratio (SUVR) scores. Symbols: sky blue circle, CN(Aβ−); orange circle, Pre-AD; light green circle, aMCI(Aβ−); red circle, Pro-AD; dark red circle, AD.</p> Full article ">Figure 3
<p>Dynamics of measurement. To compare biomarkers and neuroimaging data with different dynamic ranges, measurements were converted to z-scores (mean values of normalized biomarker levels of each group) based on the distribution in this study cohort. The plot indicates the mean z-scores for a given biomarker connected across progressively more affected diagnostic groups by a smoothing spin line using SigmaPlot 10.0 (<b>a</b>). The ∆z-score was calculated to compare the z-score differences between the cognitively normal (CN Aβ− and preclinical AD (CN Aβ+) groups (<b>b</b>).</p> Full article ">
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Article
The Effect of Coenzyme Q10 on Liver Injury Induced by Valproic Acid and Its Antiepileptic Activity in Rats
by Fahad Alqarni, Hala S. Eweis, Ahmed Ali, Aziza Alrafiah, Mohammed Alsieni, Shahid Karim and Mosleh Ayed Alkathyri
Biomedicines 2022, 10(1), 168; https://doi.org/10.3390/biomedicines10010168 - 13 Jan 2022
Cited by 11 | Viewed by 2824
Abstract
Valproic acid (VPA) has toxic metabolites that can elevate oxidative stress markers, and the hepatotoxicity of VPA has been reported. Coenzyme Q10 (CoQ10) is one of the most widely used antioxidants. The effect of CoQ10 on epileptogenesis and VPA hepatotoxicity were examined. Rats [...] Read more.
Valproic acid (VPA) has toxic metabolites that can elevate oxidative stress markers, and the hepatotoxicity of VPA has been reported. Coenzyme Q10 (CoQ10) is one of the most widely used antioxidants. The effect of CoQ10 on epileptogenesis and VPA hepatotoxicity were examined. Rats were randomly divided into five groups: the control group received 0.5% methylcellulose by oral gavages daily and saline by intraperitoneal injection three times weekly. The PTZ group received 1% methylcellulose by gavages daily and 30 mg/kg PTZ by intraperitoneal injection three times weekly. The valproic acid group received 500 mg/kg valproic acid by gavage and 30 mg/kg PTZ, as above. The CoQ10 group received 200 mg/kg CoQ10 by gavages daily and 30 mg/kg PTZ, as above. The Valproic acid + CoQ10 group received valproic acid and CoQ10, as above. Results: CoQ10 exhibited anticonvulsant activity and potentiated the anticonvulsant effect of VPA. CoQ10 combined with VPA induced a more significant reduction in oxidative stress and improved the histopathological changes in the brain and liver compared to VPA treatment. In addition, CoQ10 reduced the level of toxic VPA metabolites. These findings suggest that the co-administration of CoQ10 with VPA in epilepsy might have therapeutic potential by increasing antiepileptic activity and reducing the hepatotoxicity of VPA. Full article
(This article belongs to the Special Issue Neuroinflammation and Neuroinflammation-Induced Symptoms)
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<p>Effects of CoQ10 on the kindling course of Pentylenetetrazole. The data are expressed as the mean ± the standard error of the mean (<span class="html-italic">N</span> = 6), one-way ANOVA, and a post hoc Tukey’s test. * <span class="html-italic">p</span> &lt; 0.05 compared to PTZ alone group.</p> Full article ">Figure 2
<p>Effects of COQ10 on the kindling development, latency and duration in rats subjected to Pentylenetetrazole. The data are expressed as the mean ± the standard error of the mean (<span class="html-italic">N</span> = 6), one-way ANOVA, and post hoc Turkey’s test. * <span class="html-italic">p</span> &lt; 0.05 compared to the PTZ alone group; # <span class="html-italic">p</span> &lt; 0.05 for the PTZ/VPA + COQ10 group compared to the PTZ/VPA group.</p> Full article ">Figure 3
<p>Effects of COQ10 on a Pentylenetetrazole rat’s behavior according to the rota rod performance test. The data are expressed as the mean ± the standard error of the mean (<span class="html-italic">N</span> = 6), one-way ANOVA, and post hoc Tukey’s test. <span>$</span> <span class="html-italic">p</span> &lt; 0.05 compared to group I (control); * <span class="html-italic">p</span> &lt; 0.05, compared to PTZ alone group; # <span class="html-italic">p</span> &lt; 0.05 for the PTZ/VPA + COQ10 group compared to group PTZ/VPA.</p> Full article ">Figure 4
<p>Effect of the test drugs on the glutamate level in the hippocampus and cortex of rats subjected to PTZ kindling. The data are expressed as the mean ± the standard error of the mean (<span class="html-italic">N</span> = 6), one-way ANOVA, and post hoc Tukey’s test. COQ10: Coenzyme Q10; PTZ: Pentylenetetrazole; VPA: Valproic acid. <span>$</span> <span class="html-italic">p</span> &lt; 0.05, compared to the control group; * <span class="html-italic">p</span> &lt; 0.05, compared to the PTZ group alone.</p> Full article ">Figure 5
<p>Effect of CoQ10 on liver ALT, AST, and TNF. Pictured are the uric acid and urea of the PTZ/VPA-treated rats. The data are expressed as the mean ± the standard error of the mean, one-way ANOVA, and a post hoc Tukey’s test. ALT: Alanine aminotransferase AST; Aspartate aminotransferase; CoQ10: Coenzyme Q10; PTZ: Pentylenetetrazole; VPA: Valproic acid; TNF.a: tumor necrosis factor. <span>$</span> <span class="html-italic">p</span> &lt; 0.05, compared to group I (control); * <span class="html-italic">p</span> &lt; 0.05, compared to the PTZ-alone group; # <span class="html-italic">p</span> &lt; 0.05 for the PTZ/VPA + CoQ10 group compared to the PTZ/VPA group.</p> Full article ">Figure 6
<p>A photomicrograph of a parasagittal section of the hippocampus of all of the rat groups. (<b>A</b>) The negative control showing the normal architecture of the hippocampus. (<b>B</b>) The PTZ group showing the predominance of the degenerated, deeply stained, shrunken neurons with per neuronal spaces within the pyramidal layer (↑). Notice the pale, vacuolated areas in the molecular layer (M) suggesting the concurrent swelling/degeneration of the pyramidal cell axons (yellow ↑). (<b>C</b>) The PTZ/VPA group showing the presence of occasional, deeply darkly stained cells (↑). (<b>D</b>) The PTZ/COQ10 group showing most of the pyramidal cells, which have large rounded vesicular nuclei (red ↑). Few deeply stained cells are present with per neuronal spaces (black ↑). (<b>E</b>) The PTZ/VPA/COQ10 group showing the pyramidal layer which formed of pyramidal cells (↑) with vesicular nuclei and basophilic cytoplasm. The outer polymorphic layer, PM; the middle pyramidal layer, P; the inner molecular layer, M. Notice the presence of the lateral ventricle (lV). H&amp;E/scale bar 50.</p> Full article ">Figure 7
<p>Photomicrographs of the Toluidine blue-stained section of the hippocampus of all of the rat groups. (<b>A</b>) Control group showing that the cytoplasm of the pyramidal cells appears heavily studded with Nissl granules (↑). (<b>B</b>) PTZ group showing an apparent decrease in the Nissl granule content (red ↑) of the pyramidal cells of the pyramidal layer, compared to that of group I (control, non-epileptic rats). The pyramidal layer with few of the pyramidal cells has large vesicular nuclei (black ↑). (<b>C</b>) PTZ/VPA group showing an apparent increase in the Nissl granule content (↑). (<b>D</b>) PTZ/COQ10 group showing an apparent moderate increase in the Nissl granule content (↑) of the pyramidal cells of the pyramidal layer. (<b>E</b>) PTZ/VPA/COQ10 group showing an apparent increase in the Nissl granule content (↑). Toluidine blue; scale bar 50 µm.</p> Full article ">Figure 8
<p>A photomicrograph of the CA3 region of the hippocampus of all of the rat group. (A) Control group showing the positive reaction for GFAP immuno-staining in the cytoplasm of astrocytes (↑). (<b>B</b>) PTZ showing an apparent increase of the positive brownish reaction for GFAP immuno-staining in the cytoplasm of astrocytes, compared to that of group I (the negative control, non-epileptic rats). (<b>C</b>) PTZ/VPA showing an apparent decrease of the positive brownish reaction for GFAP immuno-staining in the cytoplasm of the astrocytes. Notice that the astrocytes present shorter processes (↑) compared to those of group II (the epileptic model group). (<b>D</b>) PTZ/COQ10 showing the apparent moderate decrease of the positive brownish reaction for GFAP immuno-staining in the cytoplasm of the astrocytes. (<b>E</b>) PTZ/VPA/COQ10 group showing the apparent decrease of the positive brownish reaction for GFAP immuno-staining in the cytoplasm of the astrocytes.</p> Full article ">Figure 9
<p>Photomicrographs of the rat liver section at the peri-central vein area of all of the rat groups. (<b>A</b>) Control group showing the polyhedral hepatocytes arranged radially in cords around the central vein (CV), with normal vesicular basophilic nuclei (white arrow). Notice the thin flat endothelial lining of the blood sinusoids (red arrows) and Von kupffer cells (black arrow). (<b>B</b>) PTZ group showing the hepatocytes with normal vesicular nuclei (arrow); some of them have a deep acidophilic cytoplasm. The binucleated hepatocytes are shown with white arrows. Notice the focal aggregation of mononuclear inflammatory cells between the hepatocytes with deep acidophilic cytoplasm and small, shrunken, deeply stained nuclei (Head arrow), and the sparse presence of the hypertrophied intra-sinusoidal cells (Von Kupffer cells) (red arrow) in-between the hepatocytes. (<b>C</b>) PTZ/VPA group showing the hypertrophied Von Kupffer cells (→) in-between the hepatocytes, which are surrounded by flat and low cubical cells (curved arrow). Notice the micro-vacuolation of the cytoplasm of some of the hepatocytes with small pyknotic nuclei (Head arrow), and the mononuclear cellular infiltration in-between the hepatocytes (red arrow). (<b>D</b>) PT Z/COQ10 showing the hepatocytes with normal architecture near to the control. (<b>E</b>) PTZ/VPA/COQ10 showing the polyhedral hepatocytes with normal vesicular basophilic nuclei and granular cytoplasm (white arrow). Notice that a few hepatocytes appeared with slightly vacuolated cytoplasm, especially in the perinuclear region (black arrows), the thin endothelial lining of the blood sinusoids (red arrows), and (<b>E</b>). H&amp;E stain-group/scale bar ((<b>A</b>–<b>I</b>) 50 µm).</p> Full article ">Figure 10
<p>Photomicrographs of the rat liver sections at the portal area of all of the rats groups. Control group showing the portal vein (PV), Bile ducts (<b>b</b>) and hepatic artery (<b>a</b>). Some are binucleated (black arrow) (<b>A</b>). PTZ group showing the dilated portal vein (PV). Notice the heavy cellular infiltration (*) (<b>B</b>). (<b>C</b>) PTZ/VPA group showing a few normally arranged hepatocytes (red arrow); some of them show the loss and fading of nuclei (karyolysis) (Black arrow) and megalocytes (yellow arrow). (<b>D</b>) PT Z/COQ10 showing the hepatocytes with normal architecture near to the control. (<b>E</b>) PTZ/VPA/COQ10 showing the proliferating bile duct (black arrow) and the presence of newly formed hepatocytic cells with relatively rounded to elongated, large, dark nuclei (red arrow) (<b>E</b>). H&amp;E stain/scale bar ((<b>A</b>–<b>I</b>) 50 µm).</p> Full article ">Figure 11
<p>Photomicrographs of the stained liver sections (portal vein) of all of the rat groups. (<b>A</b>) Control group showing fine, blue-stained fibers between the radially arranged hepatocytes around the central vein and around the portal area (P). (<b>B</b>) PTZ group showing condensed, green-stained fibers between the radially arranged hepatocytes around the central vein (CV) and around the portal area (P). (<b>C</b>) PTZ/VPA group showing the apparent moderate increase in the green collagenous fibers. (<b>D</b>) PTZ/COQ10 showing the apparent decrease in the green, collagenous fibers around the central vein (CV) and portal areas (PV). (<b>E</b>) PTZ/VPA/COQ10 group showing fine, green, collagenous connective tissue between the cords of the hepatocytes, around the central vein and the walls of large vessels. (→). Masson trichrome stain/scale bar ((<b>A</b>–<b>E</b>) 100 µm).</p> Full article ">
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