Cells

20 pages, 17068 KiB

Open AccessArticle

Cognitive Functions, Neurotransmitter Alterations, and Hippocampal Microstructural Changes in Mice Caused by Feeding on Western Diet

by Raly James Perez Custodio, Zaynab Hobloss, Maiju Myllys, Reham Hassan, Daniela González, Jörg Reinders, Julia Bornhorst, Ann-Kathrin Weishaupt, Abdel-latif Seddek, Tahany Abbas, Adrian Friebel, Stefan Hoehme, Stephan Getzmann, Jan G. Hengstler, Christoph van Thriel and Ahmed Ghallab

Cells 2023, 12(18), 2331; https://doi.org/10.3390/cells12182331 - 21 Sep 2023

Cited by 3 | Viewed by 2622

Abstract

Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is the most common chronic liver disease in Western countries. It is becoming increasingly evident that peripheral organ-centered inflammatory diseases, including liver diseases, are linked with brain dysfunctions. Therefore, this study aims to unravel the effect [...] Read more.

Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is the most common chronic liver disease in Western countries. It is becoming increasingly evident that peripheral organ-centered inflammatory diseases, including liver diseases, are linked with brain dysfunctions. Therefore, this study aims to unravel the effect of MASLD on brain histology, cognitive functions, and neurotransmitters. For this purpose, mice fed for 48 weeks on standard (SD) or Western diet (WD) were evaluated by behavioral tests, followed by sacrifice and analysis of the liver-brain axis including histopathology, immunohistochemistry, and biochemical analyses. Histological analysis of the liver showed features of Metabolic Dysfunction-Associated Steatohepatitis (MASH) in the WD-fed mice including lipid droplet accumulation, inflammation, and fibrosis. This was accompanied by an elevation of transaminase and alkaline phosphatase activities, increase in inflammatory cytokine and bile acid concentrations, as well as altered amino acid concentrations in the blood. Interestingly, compromised blood capillary morphology coupled with astrogliosis and microgliosis were observed in brain hippocampus of the WD mice, indicating neuroinflammation or a disrupted neurovascular unit. Moreover, attention was impaired in WD-fed mice along with the observations of impaired motor activity and balance, enhanced anxiety, and stereotyped head-twitch response (HTR) behaviors. Analysis of neurotransmitters and modulators including dopamine, serotonin, GABA, glutamate, and acetylcholine showed region-specific dysregulation in the brain of the WD-fed mice. In conclusion, the induction of MASH in mice is accompanied by the alteration of cellular morphology and neurotransmitter expression in the brain, associated with compromised cognitive functions. Full article

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Induction of steatohepatitis by 48 weeks of Western diet (WD) and standard diet (SD). (A) Body weight and liver-to-body-weight ratio. (B) Liver enzyme activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in plasma. (C) Hematoxylin-eosin (HE) staining, immunostaining with antibodies directed against the pan-leukocyte marker CD45, and visualization of fibrosis by Sirius red. Data are presented as the mean ± SE of 12 mice per test. Mann-Whitney U; ***: p < 0.001. Full article ">Figure 2
Clinical chemistry and immunostaining of arginase1 and glutamine synthetase in mice after 48 weeks of Western diet (WD) and standard diet (SD). (A) TNF-α, IL-10, IL-12, and IL-6 in plasma. (B) Sum bile acids (BA) from plasma taken from the portal vein, hepatic vein, and left heart chamber. (C) Ammonia (plasma). (D) Urea (plasma). (E) Arginine (plasma). (F). Glutamine and glutamate (plasma). (G) Quantification of arginase1 and glutamine synthetase immunostainings from whole slide scans. (H). Representative immunostainings for arginase1 (periportal zonation) and glutamine synthetase (GS) (pericentral zonation). Data are presented as the mean ± SE of 12 mice per test. (A) Mann-Whitney U, (B–H) two-way ANOVA with Tukey’s multiple comparisons; *: p ≤ 0.05, **: p < 0.01; ***: p < 0.001. Full article ">Figure 3
Motor, anxiety, impulsivity, and stereotypical behaviors of mice following 48 weeks of Western diet (WD) and standard diet (SD). (A,B) Total distance (cm) and movement duration (s) during the Open-field test (OFT). (C,D) Latency of fall (s) and falling frequency (#) in the Rotarod test (RT). (E,F) The center and peripheral field explorations (s) in the OFT. (G,H) Entry and time spent (%) in the open arms during the Elevated-plus maze test (EPMT). (I,J) Latency of fall (s) and falling frequency (#) in the Cliff-avoidance test (CAT). (K) Total counts of Head-twitch responses (HTR). (L) Total counts of jumps. (M) Total counts of buried marbles during the Marble-burying test (MBT). Data are presented as the mean ± SE of 20 mice per test. (A–F) two-way ANOVA with Tukey’s multiple comparisons, (G–M) two-tailed unpaired t-test. *: p ≤ 0.05, **: p < 0.01; ***: p < 0.001. Representative tracks, heatmaps, and photographs (marble burying) are shown. Full article ">Figure 4
Cognitive behaviors of mice after 48 weeks of Western diet (WD) and standard diet (SD). (A,B) Investigation time (s) and discrimination index in the Novel-object recognition test (NORT). (C,D) Recognition and preference indices in the Object-based recognition test (OBAT). (E,F) Arms entry (#) and spontaneous alternations (%) in the Y-maze test (YMT). (G–I) Latency (s), errors, and repeat errors (#) during the acquisition phase of the Barnes maze test (BMT). (J) Visits per quadrant during the Probe trial fifth day of BMT. Data are presented as the mean ± SE of 20 mice per test. Compared to SD group, (A–F) two-tailed unpaired t-test, (D,E) two-way ANOVA with Tukey’s multiple comparisons. **: p < 0.01; ***: p < 0.001. Representative tracks and heatmaps per experiment are shown. Full article ">Figure 5
Neurotransmitter levels in specific brain regions of mice 48 weeks after Western diet (WD) and standard diet (SD). Data are presented as mean ± SE of 11 mice. Mann-Whitney U; *: p ≤ 0.05, **: p < 0.01. Full article ">Figure 6
Immunostaining for serotonin on tissue sections of the hippocampus and cortex of the brain of mice 48 weeks after Western diet (WD) and standard diet (SD). Full article ">Figure 7
Histological alterations of hippocampal tissue after 48 weeks of Western diet (WD). (A) Immunostaining for the endothelial marker CD31, the astrocyte marker GFAP, and the microglia marker lba1. DAPI visualizes nuclei. (B) 3D reconstructed individual structures: microvessel segments (from the area indicated in (A)), astrocyte, microglial cell, and the intertwined network of all three structures. (C–E) Quantifications of microvessels, astrocytes, and microglia. Data are presented as mean ± SE of 12 mice. Mann-Whitney U; *: p ≤ 0.05, **: p < 0.01. Full article ">

69 pages, 4604 KiB

Open AccessReview

Insight and Recommendations for Fragile X-Premutation-Associated Conditions from the Fifth International Conference on FMR1 Premutation

by Flora Tassone, Dragana Protic, Emily Graves Allen, Alison D. Archibald, Anna Baud, Ted W. Brown, Dejan B. Budimirovic, Jonathan Cohen, Brett Dufour, Rachel Eiges, Nicola Elvassore, Lidia V. Gabis, Samantha J. Grudzien, Deborah A. Hall, David Hessl, Abigail Hogan, Jessica Ezzell Hunter, Peng Jin, Poonnada Jiraanont, Jessica Klusek, R. Frank Kooy, Claudine M. Kraan, Cecilia Laterza, Andrea Lee, Karen Lipworth, Molly Losh, Danuta Loesch, Reymundo Lozano, Marsha R. Mailick, Apostolos Manolopoulos, Veronica Martinez-Cerdeno, Yingratana McLennan, Robert M. Miller, Federica Alice Maria Montanaro, Matthew W. Mosconi, Sarah Nelson Potter, Melissa Raspa, Susan M. Rivera, Katharine Shelly, Peter K. Todd, Katarzyna Tutak, Jun Yi Wang, Anne Wheeler, Tri Indah Winarni, Marwa Zafarullah and Randi J. Hagerman Show full author list Hide full author list

Cells 2023, 12(18), 2330; https://doi.org/10.3390/cells12182330 - 21 Sep 2023

Cited by 16 | Viewed by 6243

Abstract

The premutation of the fragile X messenger ribonucleoprotein 1 (FMR1) gene is characterized by an expansion of the CGG trinucleotide repeats (55 to 200 CGGs) in the 5’ untranslated region and increased levels of FMR1 mRNA. Molecular mechanisms leading to fragile [...] Read more.

The premutation of the fragile X messenger ribonucleoprotein 1 (FMR1) gene is characterized by an expansion of the CGG trinucleotide repeats (55 to 200 CGGs) in the 5’ untranslated region and increased levels of FMR1 mRNA. Molecular mechanisms leading to fragile X-premutation-associated conditions (FXPAC) include cotranscriptional R-loop formations, FMR1 mRNA toxicity through both RNA gelation into nuclear foci and sequestration of various CGG-repeat-binding proteins, and the repeat-associated non-AUG (RAN)-initiated translation of potentially toxic proteins. Such molecular mechanisms contribute to subsequent consequences, including mitochondrial dysfunction and neuronal death. Clinically, premutation carriers may exhibit a wide range of symptoms and phenotypes. Any of the problems associated with the premutation can appropriately be called FXPAC. Fragile X-associated tremor/ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), and fragile X-associated neuropsychiatric disorders (FXAND) can fall under FXPAC. Understanding the molecular and clinical aspects of the premutation of the FMR1 gene is crucial for the accurate diagnosis, genetic counseling, and appropriate management of affected individuals and families. This paper summarizes all the known problems associated with the premutation and documents the presentations and discussions that occurred at the International Premutation Conference, which took place in New Zealand in 2023. Full article

(This article belongs to the Special Issue The Molecular Basis of Fragile X Syndrome and Other FMR1-Associated Conditions)

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Figure 1
Molecular mechanisms leading to FMR1-PM-associated conditions. Three nonexclusive models are proposed for how CGG repeats contribute to the pathogenesis of PM conditions, including FXTAS. First, the cotranscriptional R-loop formation, which compromises genomic stability and triggers a DNA-damage response that can activate inflammatory cascades [<a href="#B116-cells-12-02330" class="html-bibr">116</a>,<a href="#B117-cells-12-02330" class="html-bibr">117</a>]. Second, CGG-repeat RNAs can elicit a gain-of-function toxicity through RNA gelation into nuclear foci and sequestration of various rCGG-repeat-binding proteins, leading to their functional depletion [<a href="#B25-cells-12-02330" class="html-bibr">25</a>,<a href="#B26-cells-12-02330" class="html-bibr">26</a>]. Third, repeat-associated non-AUG (RAN)-initiated translation generates potentially toxic proteins that accumulate within intranuclear neuronal inclusions in FXTAS patients. The relative contribution from each mechanism to downstream sequelae, such as mitochondrial dysfunction and neuronal death, and their potential synergies in disease pathogenesis, are areas of ongoing research in the field. Adapted from [<a href="#B118-cells-12-02330" class="html-bibr">118</a>]. Full article ">Figure 2
FXPAC involvement across the lifespan. Full article ">Figure 3
(a–c) H&E and ubiquitin staining (brown). Inclusions in astrocytes (a), neurons (b), and Purkinje cells (c); (d) H&E. White-matter disease in cerebellum; (e) Cortical atrophy and venrticulomegalia; (f) Perl’s staining. Iron deposition in capillaries; (g,h) Iba1 staining. Activated microglia; (i,k) GFAP staining. Activated astrocytes; (j) Iba1 staining. Senescent microglia; (l) H&E. Microbleeding; (m,n) H&E and ubiquitin staining (brown). Inclusions in endothelial cells. Arrows and asterisks are indicating the pathology of interest. Arrowhead in (c) points to nucleolus. Full article ">Figure 4
Lived experience perspective. Results from an anonymous Survey Monkey questionnaire distributed via email to the member based of Fragile X New Zealand (n = 38). (a) Binary data (blue = yes; gray = no); (b) Preferred terminology by survey respondents; (c,d). Example quotes in response to query about (c) what is helpful in research and (d) what is unhelpful in research. Full article ">Figure 5
Lived experience perspectives from member based of NFXF and FXAA. (a) Results from an anonymous Survey Monkey questionnaire distributed via email to the member based of NFXF and FXAA (n = 255); (b) Example quotes. Full article ">Figure 6
Attendees at the 5th International Conference on FMR1 Premutation. Full article ">

14 pages, 1088 KiB

Open AccessReview

Emerging Roles of Ubiquitination in Biomolecular Condensates

by Peigang Liang, Jiaqi Zhang and Bo Wang

Cells 2023, 12(18), 2329; https://doi.org/10.3390/cells12182329 - 21 Sep 2023

Cited by 1 | Viewed by 1824

Abstract

Biomolecular condensates are dynamic non-membrane-bound macromolecular high-order assemblies that participate in a growing list of cellular processes, such as transcription, the cell cycle, etc. Disturbed dynamics of biomolecular condensates are associated with many diseases, including cancer and neurodegeneration. Extensive efforts have been devoted [...] Read more.

Biomolecular condensates are dynamic non-membrane-bound macromolecular high-order assemblies that participate in a growing list of cellular processes, such as transcription, the cell cycle, etc. Disturbed dynamics of biomolecular condensates are associated with many diseases, including cancer and neurodegeneration. Extensive efforts have been devoted to uncovering the molecular and biochemical grammar governing the dynamics of biomolecular condensates and establishing the critical roles of protein posttranslational modifications (PTMs) in this process. Here, we summarize the regulatory roles of ubiquitination (a major form of cellular PTM) in the dynamics of biomolecular condensates. We propose that these regulatory mechanisms can be harnessed to combat many diseases. Full article

(This article belongs to the Special Issue Advances in Ubiquitination and Deubiquitination Research)

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13 pages, 2693 KiB

Open AccessArticle

Novel Variant in CEP250 Causes Protein Mislocalization and Leads to Nonsyndromic Autosomal Recessive Type of Progressive Hearing Loss

by Minjin Kang, Jung Ah Kim, Mee Hyun Song, Sun Young Joo, Se Jin Kim, Seung Hyun Jang, Ho Lee, Je Kyung Seong, Jae Young Choi, Heon Yung Gee and Jinsei Jung

Cells 2023, 12(18), 2328; https://doi.org/10.3390/cells12182328 - 21 Sep 2023

Cited by 2 | Viewed by 1283

Abstract

Genetic hearing loss is the most common hereditary sensorial disorder. Though more than 120 genes associated with deafness have been identified, unveiled causative genes and variants of diverse types of hearing loss remain. Herein, we identified a novel nonsense homozygous variant in CEP250 [...] Read more.

Genetic hearing loss is the most common hereditary sensorial disorder. Though more than 120 genes associated with deafness have been identified, unveiled causative genes and variants of diverse types of hearing loss remain. Herein, we identified a novel nonsense homozygous variant in CEP250 (c.3511C>T; p.Gln1171Ter) among the family members with progressive moderate sensorineural hearing loss in nonsyndromic autosomal recessive type but without retinal degeneration. CEP250 encodes C-Nap1 protein belonging to the CEP protein family, comprising 30 proteins that play roles in centrosome aggregation and cell cycle progression. The nonsense variant in CEP250 led to the early truncating protein of C-Nap1, which hindered centrosome localization; heterologous expression of CEP250 (c.3511C>T) in NIH3T3 cells within cilia expression condition revealed that the truncating C-Nap1 (p.Gln1171Ter) was not localized at the centrosome but was dispersed in the cytosol. In the murine adult cochlea, Cep250 was expressed in the inner and outer hair cells. Knockout mice of Cep250 showed significant hair cell degeneration and progressive hearing loss in auditory brainstem response. In conclusion, a nonsense variant in CEP250 results in a deficit of centrosome localization and hair cell degeneration in the cochlea, which is associated with the progression of hearing loss in humans and mice. Full article

(This article belongs to the Special Issue Cell Death in Health and Disease)

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Graphical abstract

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Discovery of new causative gene CEP250 mutations in patients with hearing loss through whole-exome sequencing methods. (A) Whole-exome sequencing analysis in the YUHL cohort was performed and CEP250 was found as a novel deafness gene in YUHL 251 family. (B) Pedigree and found mutations of YUHL251 (−21, 42-year-old female; −22, 40-year-old female; −23, 35-year-old female. (C) Hearing tests showed high-frequency sensorineural hearing loss in YUHL251-21. Red line (circle; air conduction, [ or <; bone conduction] refers to the right ear threshold and blue line (cross; air threshold,] or >; bone conduction) refers to the left ear threshold. (D) The autosomal recessive CEP250 c.3511C>T, p.Gln1171Ter gene mutation was discovered through whole-exome sequencing. (E) Evolutionary conservation of altered amino acid residues (Homo sapiens (human), Rattus norvegicus (rat), Mus musculus (mouse), Gallus gallus (chicken), Xenopus tropicalis (frog)). (F) Schematic diagram of pathogenic variants in CEP250. Abbreviations: YUHL, Yonsei University Hearing Loss. Asterisk (*) denotes a stop codon. Full article ">Figure 2
Expression of p.Gln1171Ter C-Nap1 in NIH3T3 cell line. (A,B) Immunoblotting analysis using Myc (A) and CEP250 (B) antibodies. CEP250 WT was expressed at 255 kDa (full length) and ~160 kDa (short form). CEP250 p.Gln1171Ter was detected at ~150 kDa, corresponding to the early truncated protein. (C) Immunocytochemistry was performed in NIH3T3 cells transfected with wild-type CEP250 and p.Gln1171Ter. It was stained using γ-tubulin (red), Dapi (blue), and C-Myc (green) antibodies in NIH3T3 cells. White arrows point to centrosome. Full article ">Figure 3
Cilia expression in NIH3T3 cells transfected with CEP250 and p.Gln1171Ter variant in immunostaining. Staining with Acetyl-⍺-tubulin (green), C-Myc (red, CEP250) antibody to confirm whether the expression of CEP250 p.Gln1171Ter mutation affects the production and growth of primary cilia. Both CEP250 WT and p.Gln1171Ter mutations confirmed the production of primary cilia. Green arrows point to primary cilia. Full article ">Figure 4
Expression and localization of Cep250 in the mouse cochlea. (A,B) Cep250 transcriptional expression in the organ of Corti at P1 (A) and P30 (B). Myo7a and Atoh1 are depicted as hair cell markers. HC, hair cell; SC, supporting cell. (C) Cep250 transcript expression in the spiral ganglion neuron at different ages. Calb2, Calb1, and Lypd1 are markers for type I, II, and III neurons, respectively. SGN, spiral ganglion neuron. (D,E) Immunohistochemistry was performed for wildtype and Cep250 knockout mice (P30). Cep250 (red) was expressed in the inner hair cells (IHCs), outer hair cells, and pillar cells in the organ of Corti of wildtype mice but not Cep250 knockout mice (D). Cep250 (red) is expressed in the spiral ganglion (dashed area) of wildtype mice but not in that of the Cep250 knockout mice (E). Dapi (blue) staining. WT, wildtype; Het, heterozygous Cep250 knockout mice; KO, homozygous Cep250 knockout mice. Full article ">Figure 5
Auditory brainstem response (ABR) test in the Cep250 knockout mice. ABR thresholds were measured at the stimuli of click and tone sounds. The number of Cep250 (+/+), (+/−), and (−/−) mice was 5, 11, and 5, respectively. ****, p < 0.001. Full article ">Figure 6
The cell survival rate in the Cep250 knockout mice at the age of 17 weeks. (A) Whole-mount immunostaining with phalloidin (green), anti-Cep250 antibody (red), and Dapi (blue) was performed through the whole cochlear turn. Tonotopic frequencies are noted. (B) The basal turn of the Cep250 knockout mouse showed a higher degeneration rate of the outer hair cell compared to that of WT mice. (C) Inner hair cell counts in apex, mid, and basal turns in CEP250 knockout mice. (D) Outer hair cell counts in apex, mid, and basal turns in Cep250 knockout mice. IHC, inner hair cells; OHC, outer hair cells; ns, not significant; ****, p < 0.001. Full article ">

15 pages, 1752 KiB

Open AccessReview

NLRP3 Inflammasome as a Potentially New Therapeutic Target of Mesenchymal Stem Cells and Their Exosomes in the Treatment of Inflammatory Eye Diseases

by Carl Randall Harrell, Valentin Djonov, Ana Antonijevic and Vladislav Volarevic

Cells 2023, 12(18), 2327; https://doi.org/10.3390/cells12182327 - 21 Sep 2023

Cited by 5 | Viewed by 1712

Abstract

Due to their potent immunoregulatory and angio-modulatory properties, mesenchymal stem cells (MSCs) and their exosomes (MSC-Exos) have emerged as potential game-changers in regenerative ophthalmology, particularly for the personalized treatment of inflammatory diseases. MSCs suppress detrimental immune responses in the eyes and alleviate ongoing [...] Read more.

Due to their potent immunoregulatory and angio-modulatory properties, mesenchymal stem cells (MSCs) and their exosomes (MSC-Exos) have emerged as potential game-changers in regenerative ophthalmology, particularly for the personalized treatment of inflammatory diseases. MSCs suppress detrimental immune responses in the eyes and alleviate ongoing inflammation in ocular tissues by modulating the phenotype and function of all immune cells that play pathogenic roles in the development and progression of inflammatory eye diseases. MSC-Exos, due to their nano-sized dimension and lipid envelope, easily bypass all barriers in the eyes and deliver MSC-sourced bioactive compounds directly to target cells. Although MSCs and their exosomes offer a novel approach to treating immune cell-driven eye diseases, further research is needed to optimize their therapeutic efficacy. A significant number of experimental studies is currently focused on the delineation of intracellular targets, which crucially contribute to the immunosuppressive and anti-inflammatory effects of MSCs and MSC-Exos. The activation of NLRP3 inflammasome induces programmed cell death of epithelial cells, induces the generation of inflammatory phenotypes in eye-infiltrated immune cells, and enhances the expression of adhesion molecules on ECs facilitating the recruitment of circulating leukocytes in injured and inflamed eyes. In this review article, we summarize current knowledge about signaling pathways that are responsible for NLRP3 inflammasome-driven intraocular inflammation and we emphasize molecular mechanisms that regulate MSC-based modulation of NLRP3-driven signaling in eye-infiltrated immune cells, providing evidence that NLRP3 inflammasome should be considered a potentially new therapeutic target for MSCs and MSC-Exo-based treatment of inflammatory eye diseases. Full article

(This article belongs to the Special Issue Updates on Mesenchymal Stem Cells-Derived Extracellular Vesicles)

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Molecular mechanisms and signaling pathways involved in the activation of NLRP3 inflammasome in eye-infiltrated immune cells. The activation of the NLRP3 inflammasome in immune cells is a tightly regulated, multi-step process. It requires 2 steps: priming and activation. The priming step of NLRP3 inflammasome activation is elicited by the activation of pattern recognition receptors (PRRs), including Toll-like receptors (TLR). These membrane-bound receptors are expressed on immune cells that recognize pathogen-associated molecular patterns (PAMPs) of microbial antigens or damage-associated molecular patterns (DAMPs) released from injured cells. Signals generated from activated TLR-2, TLR-4, TLR-5, and TLR-6 initiate phosphorylation and consequent activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), which, in turn, phosphorylate c-Jun, ATF-2, and CREB transcription factors that bind to the promoter regions of NLRP3 and pro-IL-1β, leading to their transcriptional upregulation. Full article ">Figure 2
Molecular mechanisms responsible for NLRP3-dependent generation of detrimental immune response in inflamed eyes. Activation of NLRP3 inflammasome results in increased production of IL-1β and IL-18 in tissue-infiltrated macrophages and neutrophils. IL-1β and IL-18 induce massive recruitment of circulating leukocytes in inflamed tissues, increased expansion of inflammatory Th1 and Th17 lymphocytes, enhanced production of inflammatory cytokines and chemokines in neutrophils and macrophages, attenuated proliferation of immunosuppressive T regulatory cells (Tregs), and increased antibody production in plasma cells. Full article ">Figure 3
The beneficial effects of MSCs and MSC-derived exosomes in the attenuation of NLRP3-driven eye injury and inflammation. MSCs secrete a wide array of immunosuppressive cytokines and immunoregulatory factors, such as IL-10, transforming growth factor-β (TGF-β), prostaglandin E2 (PGE2), and stanniocalcin (STC)-1, which efficiently suppress NLRP3 inflammasome activation and inhibited production of IL-1β and IL-18 in eye-infiltrated macrophages. MSC-derived PGE2 was found mainly responsible for the MSC-dependent generation of tolerogenic phenotype in DCs, which was followed by attenuated activation of naïve T cells. MSC-sourced IDO and Kynurenine were considered mainly responsible for the MSC-dependent regulation of T/NK/NKT cells’ phenotype and function. Full article ">

20 pages, 7512 KiB

Open AccessArticle

Human Preadipocytes Differentiated under Hypoxia following PCB126 Exposure during Proliferation: Effects on Differentiation, Glucose Uptake and Adipokine Profile

by Zeinab El Amine, Jean-François Mauger and Pascal Imbeault

Cells 2023, 12(18), 2326; https://doi.org/10.3390/cells12182326 - 21 Sep 2023

Cited by 1 | Viewed by 1187

Abstract

Persistent organic pollutants (POPs) accumulation and hypoxia are two factors proposed to adversely alter adipose tissue (AT) functions in the context of excess adiposity. Studies have shown that preadipocytes exposure to dioxin and dioxin-like POPs have the greatest deleterious impact on rodent and [...] Read more.

Persistent organic pollutants (POPs) accumulation and hypoxia are two factors proposed to adversely alter adipose tissue (AT) functions in the context of excess adiposity. Studies have shown that preadipocytes exposure to dioxin and dioxin-like POPs have the greatest deleterious impact on rodent and immortalized human preadipocyte differentiation, but evidence on human preadipocytes is lacking. Additionally, hypoxia is known to strongly interfere with the dioxin-response pathway. Therefore, we tested the effects of pre-differentiation polychlorinated biphenyl (PCB)126 exposure at 10 µM for 3 days and subsequent differentiation under hypoxia on human subcutaneous adipocytes (hSA) differentiation, glucose uptake and expression of selected metabolism- and inflammation-related genes. Pre-differentiation PCB126 exposure lowered the adenosine triphosphate (ATP) content, glucose uptake and leptin expression of mature adipocytes but had limited effects on differentiation under normoxia (21% O₂). Under hypoxia (3% O₂), preadipocytes ability to differentiate was significantly reduced as reflected by significant decreased lipid accumulation and downregulation of key adipocyte genes such as peroxisome proliferator-activated receptor gamma (PPARγ) and adiponectin. Hypoxia increased glucose uptake and glucose transporter 1 (GLUT1) expression but abolished the adipocytes insulin response and GLUT4 expression. The expression of pro-inflammatory adipokine interleukin-6 (IL-6) was slightly increased by both PCB126 and hypoxia, while IL-8 expression was significantly increased only following the PCB126-hypoxia sequence. These observations suggest that PCB126 does not affect human preadipocyte differentiation, but does affect the subsequent adipocytes population, as reflected by lower ATP levels and absolute glucose uptake. On the other hand, PCB126 and hypoxia exert additive effects on AT inflammation, an important player in the development of chronic diseases such as type 2 diabetes and cardiovascular diseases. Full article

(This article belongs to the Special Issue The Adipose Tissue: From “Cinderella” to “Lion King” Organ)

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15 pages, 2989 KiB

Open AccessArticle

Establishment and Characterization of Continuous Satellite Muscle Cells from Olive Flounder (Paralichthys olivaceus): Isolation, Culture Conditions, and Myogenic Protein Expression

by Sathish Krishnan, Selvakumari Ulagesan, Josel Cadangin, Ji-Hye Lee, Taek-Jeong Nam and Youn-Hee Choi

Cells 2023, 12(18), 2325; https://doi.org/10.3390/cells12182325 - 21 Sep 2023

Cited by 1 | Viewed by 1791

Abstract

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth [...] Read more.

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl₂ and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs. Full article

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Figure 1
(a) Paralichthys olivaceus (red color rectangle indicates the place where muscle tissue was collected). (b) Live and dead cells of olive flounder muscle cells stained with calcine AM and propidium iodide. Scale bar: 200 µm. Full article ">Figure 2
Phase contrast micrographs of olive flounder muscle satellite cells attachment and proliferation from days 0 to 6. Scale bar: 100 µm. Full article ">Figure 3
Explant primary cell isolation. Radial movement of cells from explant tissue. Scale bar—100 µm. Full article ">Figure 4
Optimal growth conditions for OFMCs. (a) FBS concentration, (b) temperature, (c) salt concentration, and (d) growth factors. ns—non-significant; *—significance (p < 0.01). Full article ">Figure 5
Four representative images of spontaneous immortalization of OFMC cells. (a) Large-scale apoptosis and senescence occurred in the OFMC cells. Green circle—sporadic cluster; blue circle—detaching and defragmented cell. (b) The remaining cells initiated to overcome the crisis. Red arrow—sporadic cells started proliferating into myocytes. (c) Cells began to actively divide again. (d) The formation of a confluent cell monolayer Scale bar: 100 µm. Full article ">Figure 6
Differentiation of OFMCs into myotubes. (a) Representative images of undifferentiated (myoblasts) and differentiated cells (myotubes) incubated with 2% horse serum. Magnification, 10×; Scale bar—100 μm. (b) MyoD, myogenin, and desmin protein expression levels in undifferentiated and differentiated cells. Results are means ± standard deviation of three independent experiments. ** p < 0.05 vs. corresponding control group (D-0). CON, control; D, day; UD, undifferentiation, D-0. (c) FITC-phalloidin and DAPI were used to visualize actin and nucleus staining, and the colocalization of phalloidin and DAPI staining is indicated in the merged images. Magnification 10×; scale bar—100 μm. green- cytoskeleton staining, blue-nucleus staining. Full article ">

21 pages, 1044 KiB

Open AccessReview

Regulatory Mechanisms That Guide the Fetal to Postnatal Transition of Cardiomyocytes

by Patrick G. Burgon, Jonathan J. Weldrick, Omar Mohamed Sayed Ahmed Talab, Muhammad Nadeer, Michail Nomikos and Lynn A. Megeney

Cells 2023, 12(18), 2324; https://doi.org/10.3390/cells12182324 - 21 Sep 2023

Cited by 1 | Viewed by 1673

Abstract

Heart disease remains a global leading cause of death and disability, necessitating a comprehensive understanding of the heart’s development, repair, and dysfunction. This review surveys recent discoveries that explore the developmental transition of proliferative fetal cardiomyocytes into hypertrophic postnatal cardiomyocytes, a process yet [...] Read more.

Heart disease remains a global leading cause of death and disability, necessitating a comprehensive understanding of the heart’s development, repair, and dysfunction. This review surveys recent discoveries that explore the developmental transition of proliferative fetal cardiomyocytes into hypertrophic postnatal cardiomyocytes, a process yet to be well-defined. This transition is key to the heart’s growth and has promising therapeutic potential, particularly for congenital or acquired heart damage, such as myocardial infarctions. Although significant progress has been made, much work is needed to unravel the complex interplay of signaling pathways that regulate cardiomyocyte proliferation and hypertrophy. This review provides a detailed perspective for future research directions aimed at the potential therapeutic harnessing of the perinatal heart transitions. Full article

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16 pages, 2284 KiB

Open AccessReview

Neuroimmune Interactions in Fetal Alcohol Spectrum Disorders: Potential Therapeutic Targets and Intervention Strategies

by Sayani Mukherjee, Prashant Tarale and Dipak K. Sarkar

Cells 2023, 12(18), 2323; https://doi.org/10.3390/cells12182323 - 21 Sep 2023

Viewed by 1897

Abstract

Fetal alcohol spectrum disorders (FASD) are a set of abnormalities caused by prenatal exposure to ethanol and are characterized by developmental defects in the brain that lead to various overt and non-overt physiological abnormalities. Growing evidence suggests that in utero alcohol exposure induces [...] Read more.

Fetal alcohol spectrum disorders (FASD) are a set of abnormalities caused by prenatal exposure to ethanol and are characterized by developmental defects in the brain that lead to various overt and non-overt physiological abnormalities. Growing evidence suggests that in utero alcohol exposure induces functional and structural abnormalities in gliogenesis and neuron–glia interactions, suggesting a possible role of glial cell pathologies in the development of FASD. However, the molecular mechanisms of neuron–glia interactions that lead to the development of FASD are not clearly understood. In this review, we discuss glial cell pathologies with a particular emphasis on microglia, primary resident immune cells in the brain. Additionally, we examine the involvement of several neuroimmune molecules released by glial cells, their signaling pathways, and epigenetic mechanisms responsible for FASD-related alteration in brain functions. Growing evidence suggests that extracellular vesicles (EVs) play a crucial role in the communication between cells via transporting bioactive cargo from one cell to the other. This review emphasizes the role of EVs in the context of neuron–glia interactions during prenatal alcohol exposure. Finally, some potential applications involving nutritional, pharmacological, cell-based, and exosome-based therapies in the treatment of FASD are discussed. Full article

(This article belongs to the Special Issue Alcohol and Neuroimmunology)

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Figure 1
A schematic diagram showing ethanol-induced alterations in glial cell functions. Early ethanol exposure during development causes a shift from resting microglia and astrocytes to activated microglia and astrocyte phenotypes. These inflammatory glial phenotypes secrete extracellular vesicles containing inflammatory molecules, including cytokines, chemokines, and miRNAs, which are detrimental to neurons associated with regulation of stress-axis function. Ethanol exposure also alters oligodendrocyte functions, which has long-term effects on neuronal myelination, affecting synaptic plasticity. These events may result in FASD-associated cognitive, behavioral, and motor impairments. Full article ">Figure 2
A schematic diagram showing ethanol-induced changes in microglial cell functions. Early ethanol exposure during the developmental period causes phenotypic shift from resting microglia to activated microglia (M1). The most common signaling pathways that are upregulated during alcohol-induced microglial priming are TLR2/4, chemokine, cytokine, complement, MOR, NLRP3, NF-kB/TNF-α, and ROS signaling. The most common epigenetic alterations include decrease in DNMT1/3A, SIRT1, miR153, and MeCP2. All these altered signaling mechanisms trigger the release of neurotoxic factors from primed microglia and cause apoptotic death of neurons. Full article ">Figure 3
A schematic diagram showing the early intervention, therapeutic, and management strategies to improve the cognitive function in FASD. Extracellular vesicles may be considered as potential biomarkers as well as therapeutic vesicles for FASD. Full article ">

22 pages, 7521 KiB

Open AccessArticle

TRPV1 Channels Are New Players in the Reticulum–Mitochondria Ca²⁺ Coupling in a Rat Cardiomyoblast Cell Line

by Nolwenn Tessier, Mallory Ducrozet, Maya Dia, Sally Badawi, Christophe Chouabe, Claire Crola Da Silva, Michel Ovize, Gabriel Bidaux, Fabien Van Coppenolle and Sylvie Ducreux

Cells 2023, 12(18), 2322; https://doi.org/10.3390/cells12182322 - 20 Sep 2023

Cited by 5 | Viewed by 2060

Abstract

The Ca²⁺ release in microdomains formed by intercompartmental contacts, such as mitochondria-associated endoplasmic reticulum membranes (MAMs), encodes a signal that contributes to Ca²⁺ homeostasis and cell fate control. However, the composition and function of MAMs remain to be fully defined. Here, [...] Read more.

The Ca²⁺ release in microdomains formed by intercompartmental contacts, such as mitochondria-associated endoplasmic reticulum membranes (MAMs), encodes a signal that contributes to Ca²⁺ homeostasis and cell fate control. However, the composition and function of MAMs remain to be fully defined. Here, we focused on the transient receptor potential vanilloid 1 (TRPV1), a Ca²⁺-permeable ion channel and a polymodal nociceptor. We found TRPV1 channels in the reticular membrane, including some at MAMs, in a rat cardiomyoblast cell line (SV40-transformed H9c2) by Western blotting, immunostaining, cell fractionation, and proximity ligation assay. We used chemical and genetic probes to perform Ca²⁺ imaging in four cellular compartments: the endoplasmic reticulum (ER), cytoplasm, mitochondrial matrix, and mitochondrial surface. Our results showed that the ER Ca²⁺ released through TRPV1 channels is detected at the mitochondrial outer membrane and transferred to the mitochondria. Finally, we observed that prolonged TRPV1 modulation for 30 min alters the intracellular Ca²⁺ equilibrium and influences the MAM structure or the hypoxia/reoxygenation-induced cell death. Thus, our study provides the first evidence that TRPV1 channels contribute to MAM Ca²⁺ exchanges. Full article

(This article belongs to the Special Issue Intracellular Ca²⁺ Sensing: Roles from Ca²⁺ Stores to Cytosolic Microdomains)

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14 pages, 643 KiB

Open AccessReview

What Does the Future Hold for Biomarkers of Response to Extracorporeal Photopheresis for Mycosis Fungoides and Sézary Syndrome?

by Oleg E. Akilov

Cells 2023, 12(18), 2321; https://doi.org/10.3390/cells12182321 - 20 Sep 2023

Cited by 1 | Viewed by 1079

Abstract

Extracorporeal photopheresis (ECP) is an FDA-approved immunotherapy for cutaneous T-cell lymphoma, which can provide a complete response in some patients. However, it is still being determined who will respond well, and predictive biomarkers are urgently needed to target patients for timely treatment and [...] Read more.

Extracorporeal photopheresis (ECP) is an FDA-approved immunotherapy for cutaneous T-cell lymphoma, which can provide a complete response in some patients. However, it is still being determined who will respond well, and predictive biomarkers are urgently needed to target patients for timely treatment and to monitor their response over time. The aim of this review is to analyze the current state of the diagnostic, prognostic, and disease state-monitoring biomarkers of ECP, and outline the future direction of the ECP biomarker discovery. Specifically, we focus on biomarkers of response to ECP in mycosis fungoides and Sézary syndrome. The review summarizes the current knowledge of ECP biomarkers, including their limitations and potential applications, and identifies key challenges in ECP biomarker discovery. In addition, we discuss emerging technologies that could revolutionize ECP biomarker discovery and accelerate the translation of biomarker research into clinical practice. This review will interest researchers and clinicians seeking to optimize ECP therapy for cutaneous T-cell lymphoma. Full article

(This article belongs to the Collection Cutaneous Lymphoma Molecular Pathogenesis, Diagnosis and Management)

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21 pages, 3403 KiB

Open AccessArticle

Effect of Acute Enriched Environment Exposure on Brain Oscillations and Activation of the Translation Initiation Factor 4E-BPs at Synapses across Wakefulness and Sleep in Rats

by José Lucas Santos, Evlalia Petsidou, Pallavi Saraogi, Ullrich Bartsch, André P. Gerber and Julie Seibt

Cells 2023, 12(18), 2320; https://doi.org/10.3390/cells12182320 - 20 Sep 2023

Viewed by 1502

Abstract

Brain plasticity is induced by learning during wakefulness and is consolidated during sleep. But the molecular mechanisms involved are poorly understood and their relation to experience-dependent changes in brain activity remains to be clarified. Localised mRNA translation is important for the structural changes [...] Read more.

Brain plasticity is induced by learning during wakefulness and is consolidated during sleep. But the molecular mechanisms involved are poorly understood and their relation to experience-dependent changes in brain activity remains to be clarified. Localised mRNA translation is important for the structural changes at synapses supporting brain plasticity consolidation. The translation mTOR pathway, via phosphorylation of 4E-BPs, is known to be activate during sleep and contributes to brain plasticity, but whether this activation is specific to synapses is not known. We investigated this question using acute exposure of rats to an enriched environment (EE). We measured brain activity with EEGs and 4E-BP phosphorylation at cortical and cerebellar synapses with Western blot analyses. Sleep significantly increased the conversion of 4E-BPs to their hyperphosphorylated forms at synapses, especially after EE exposure. EE exposure increased oscillations in the alpha band during active exploration and in the theta-to-beta (4–30 Hz) range, as well as spindle density, during NREM sleep. Theta activity during exploration and NREM spindle frequency predicted changes in 4E-BP hyperphosphorylation at synapses. Hence, our results suggest a functional link between EEG and molecular markers of plasticity across wakefulness and sleep. Full article

(This article belongs to the Special Issue Synaptic Plasticity in Brain and Nerves: New Vistas in Health and Diseases)

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Graphical abstract

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Full article ">Figure 1
Experimental design. (A) Experimental groups. After a 24 h habituation period (with or without EEG baseline recording), rats were kept awake (yellow squares) for 3 h at the end of the next dark period either in their home cage (HC) or in the enriched-environment (EE) cage. Some animals were sacrificed for tissue harvest (black arrowhead) immediately after this awake period (EE and SD groups) or left undisturbed to sleep for 3 h in their HC (SDS and EES groups). In a final group, rats were maintained awake for 6 h (SDSD) in their HC and sacrificed at the same circadian time as that of the sleeping groups. EEGs were recorded only in the sleeping groups. (B) Representation of the areas of the cortex and cerebellum harvested. (C) Whole-cell (TOT) and synapse-specific (SN) protein extracts were obtained from each tissue sample and processed for Western blot analyses. (D) Representative fronto-parietal (FP) and fronto-cerebellar (FC) EEsG and activity traces for the 5 main brains states. Created with BioRender.com. Full article ">Figure 2
Effect of EE on wakefulness architecture and EEG. (A) Mean (±SEM) EEG power spectra (normalised to the mean across all frequencies (see <a href="#sec2-cells-12-02320" class="html-sec">Section 2</a>)) for AW and QW and both EEGs (FP and FC) separately for all groups (bsl and SD/EE periods). (B) Change in mean (±SEM) power density for AW (upper graph) and QW (lower graph) during the 3 h awake period in EE or HC expressed as % of corresponding baseline values (see <a href="#sec2-cells-12-02320" class="html-sec">Section 2</a>). EEG from both parietal (FP) and frontal (FC) EEGs for each group are represented for AW and QW. Time course (30 min bins) of sigma band in the parietal (FP) EEG for the EE and SD groups are represented as insets. For both (A,B), only the presence of significant differences with p > 0.001 and p > 0.0001 are reported underneath the traces for each group and EEG. Detailed statistics (Two-Way RM ANOVAs and level of significance for each comparison) can be found in the <a href="#app1-cells-12-02320" class="html-app">Data S1</a>. Full article ">Figure 3
Effect of EE on sleep architecture and EEG. (A) Vigilance states (5–95% confidence interval, CI) and bout duration (5–95% CI, N = 8/group) during the 3 h rest following the awake period in either the HC (SD) or EE cage (EE). Two-way RM ANOVA did not reveal any effect of group (SD vs. EE) or condition (bsl vs. post-SD/EE) on vigilance states. (B) Change in mean (±SEM) power density for NREM and REM over the 3 h period after SD in EE or HC expressed as % of corresponding baseline values (see <a href="#sec2-cells-12-02320" class="html-sec">Section 2</a>). The parietal and frontal EEGs are shown separately. For clarity, only the presence of significant differences from baseline values are shown underneath the traces. Detailed statistics (Two-Way RM ANOVAs and level of significance for each comparison) can be found in <a href="#app1-cells-12-02320" class="html-app">Data S1</a>. Post-EE vs. post-SD, *: p < 0.05, Sidak test. (C) Comparison of the time course of changes in NREM Delta, theta, sigma and beta frequency bands (mean ± SEM) in 30 min bins during the 3-h rest period after the awake period in an EE or HC (Two-way ANOVA Group effect, *: p < 0.05, **: p < 0.01, ***: p < 0.001, Sidak test). Comparison between frontal and parietal EEGs are shown in insets for each frequency band. There were also no differences between EEGs for the SD group (Two-way ANOVA, p > 0.05 for factors EEG and bins for each frequency band). SO and slow γ frequency bands (see <a href="#app1-cells-12-02320" class="html-app">Figure S3A</a>) did not show any significant differences between groups. (D) Changes in spindle characteristics (density, duration, frequency, amplitude) in rats from the EE group recorded from the parietal EEG (Paired t-test, **: p < 0.01). (E) Correlations between spindle density and changes (as % of baseline) in sigma and beta frequency bands during NREM post-SD/EE in the parietal EEG. (F) Correlations between changes in frequency band power during the 3 h of wakefulness in the HC and EE and changes in parietal EEG power during NREM sleep during the following 3 h rest period. Data points for the EE and SD groups were grouped but are shown separately in the scatter plot. (Right graphs) Scatter plots showing the correlation between power in the sigma band in AW and NREM beta power and spindle duration. Results for the SD (open circles) and EE (filled circles) groups are shown. *: p < 0.05, ***: p < 0.001, Pearson’s. Full article ">Figure 4
Sleep and experience-dependent changes in 4E-BP phosphorylation. (A) Representative Western Blot analysis of SN extracts obtained from the cerebellum of four different animals across the five groups indicated at the top. Phospo-4E-BPs (Thr37/46) are shown in green, 4E-BP2 protein in red. The individual channels are shown separately below. (B) Normalised mean (±SEM) phospo-4E-BPs (Thr37/46)/4E-BP2 (see <a href="#sec2-cells-12-02320" class="html-sec">Section 2</a>) for all brain regions pooled (N = 32) in SN (left graph) and TOT (right graph) extracts. Values are represented for all 4E-BP forms (α, β, γ) separately and grouped (α + β + γ; “ALL”). Awake groups are represented in grey and black, sleeping groups in light and dark blue and the circadian control group in orange. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, Sidak test. (C) Normalised mean (±SEM) signals from antibodies detecting P-4E-BPs/4E-BP2 across groups. Note the specific decrease in the hypophosphorylated 4E-BP (α) form in the EES group. (D) Average (±SEM) 4E-BP conversion index (i.e., γ/α ratio) across groups. (E) Distribution of changes of the γ 4E-BP form in SN in all 4 brain regions (N = 8/region). A two-way RM ANOVA revealed an interaction between Group × brain region (p = 0.017) with significantly lower and higher levels of γ 4E-BP form in the cerebellum (Cb) in the control sleeping group (SDS) and circadian group (SDSD), respectively, compared to more frontal areas (SDS and SDSD: Cb vs. SM: * p = 0.048; SDSD: Cb vs. FR: ** p = 0.008). Full article ">Figure 5
Relation between wakefulness and sleep EEG changes and synaptic 4E-BP measures. (A) Correlation matrix between changes in frequency band power in AW during the 3 h of wakefulness (in HC and EE) or sleep (NREM, IS and REM) during the rest period and changes in 4E-BP forms (α, β, γ) and conversion index (γ/α ratio). Results are shown for the SN fraction and separately for each EEG. Correlation coefficients were computed with datapoints from EE and SD groups combined. *: p < 0.05, **: p < 0.01, Pearson’s. (B) Scatter plots showing the correlation between theta power in AW and 4E-BP conversion index (γ/α ratio). Results for each EEG (FP = filled circles, FC = empty circle) and each group (EE = red, SD = black) are represented. Significance of correlations are shown for each EEG in the legend. (C) Scatter plots showing the correlation between Beta power in frontal EEG in REM sleep and 4E-BP conversion index (γ/α ratio) levels in SN. Results for each group (EE = red, SD = black) are represented. Significance of the correlation is shown next to the trendline. (D) Correlation matrix between changes in spindle characteristics (density, duration, frequency and amplitude) obtained from frontal (FC) and parietal (FP) EEGs and changes in 4E-BP forms (α, β, γ) and conversion index (γ/α ratio) at synapses. *: p < 0.05, Pearson’s. The scatter plot showing the correlation between spindle frequency obtained from the parietal EEG and 4E-BP conversion index (γ/α ratio) is shown on the right. Results for each group (EE = red, SD = black) and significance of correlations are shown. Full article ">

14 pages, 17035 KiB

Open AccessArticle

Modeling of FAN1-Deficient Kidney Disease Using a Human Induced Pluripotent Stem Cell-Derived Kidney Organoid System

by Sun Woo Lim, Dohyun Na, Hanbi Lee, Xianying Fang, Sheng Cui, Yoo Jin Shin, Kang In Lee, Jae Young Lee, Chul Woo Yang and Byung Ha Chung

Cells 2023, 12(18), 2319; https://doi.org/10.3390/cells12182319 - 20 Sep 2023

Cited by 2 | Viewed by 1543

Abstract

Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this [...] Read more.

Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this study was to explore the possibility of using a human induced pluripotent stem cell (hiPSC)-derived kidney organoid system for modeling FAN1-deficient kidney disease, also known as KIN. We generated kidney organoids using WTC-11 (wild-type) hiPSCs and FAN1-mutant hiPSCs which include KIN patient-derived hiPSCs and FAN1-edited hiPSCs (WTC-11 ^FAN1+/−), created using the CRISPR/Cas9 system in WTC-11-hiPSCs. Kidney organoids from each group were treated with 20 nM of mitomycin C (MMC) for 24 or 48 h, and the expression levels of Ki67 and H2A histone family member X (H2A.X) were analyzed to detect DNA damage and assess the viability of cells within the kidney organoids. Both WTC-11-hiPSCs and FAN1-mutant hiPSCs were successfully differentiated into kidney organoids without structural deformities. MMC treatment for 48 h significantly increased the expression of DNA damage markers, while cell viability in both FAN1-mutant kidney organoids was decreased. However, these findings were observed in WTC-11-kidney organoids. These results suggest that FAN1-mutant kidney organoids can recapitulate the phenotype of FAN1-deficient kidney disease. Full article

(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)

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Figure 1
FAN1 gene mutation in a patient with karyomegalic interstitial nephritis (KIN). (A) Hematoxylin and eosin (H&E) staining of kidney tissues of a patient with karyomegalic interstitial nephritis. Scale bar = 100 μm. White arrows in A point to karyomegaly. (B) Patient PBMC with KIN showing FAN1 gene mutation on deletion c.1985-1944delTTGGGTGGAT on 15q13.3. (C) Pedigree of a family showing individuals affected by karyomegalic interstitial nephritis resulting in the appearance of p.Gly663llefs*54. Full article ">Figure 2
Establishment of CRISPR/Cas9 ribonucleoproteins (RNP)-mediated FAN1 gene editing in WTC-11 hiPSCs. (A) Target site for guide RNA (gRNA) targeting FAN1. Target sites are indicated by a green color in exon 2 of the FAN1 gene. The downward-pointing arrowhead indicates the position of the canonical cut site and predicted specificity based on the number and distribution of homoeologous SNPs at the corresponding target site/PAM. (B) PAM sequences (5′-NGG-3′) in target site are indicated by red letters. The table shows no mismatched number with gRNA. The asterisk indicates on-target gRNA. (C) Indel sequences after transfecting WTC-11 hiPSCs with gRNA using the CRISPR/Cas9 RNP method. Indel sequences are indicated by red letters (a green color indicates a target site). (D) Read number of In-del frequency. Indel of 1bp (A ins) in the target sequence read of about 50%. (E) Morphology of FAN1-gene-edited WTC-11 iPSC (WTC-11FAN1+/−). (F,G) Flow cytometry analysis and immunofluorescence image of cells expressing NANOG, SSEA-4, and TRA-1-81 in WTC-11FAN1+/− hiPSCs. Scale bar = 50 μm. (H) Immunofluorescence staining of three germ layer markers. Ectoderm, mesoderm, and entoderm differentiation were detected using PAX4, SM22a, and FOX2A, respectively. Scale bar = 50 μm. (I) Expression analysis via RT-PCR of FAN1 and GAPDH in WTC-11 and WTCFAN1+/− hiPSCs. All expression levels were normalized against GAPDH expression level. (J) Expression analysis via immunoblot of FAN1 and β-actin in WTC-11 and WTCFAN1+/− hiPSCs. All expression levels were normalized against the β-actin expression level. Data are presented as mean ± standard error. *, p < 0.05 vs. WTC-11 iPSC. Full article ">Figure 3
Differentiation of kidney organoids from WTC-11, WTC-11FAN1+/−, and KIN patient hiPSCs. (A) Schematic timeline of the hiPSC differentiation protocol. (B) Representative immunofluorescence images of podocalyxin (PODXL), lotus tetragonolobus lectin (LTL), and e-cadherin (ECAD) kidney organoids from WTC-11, WTC-11FAN1+/−, and KIN patient, respectively. (C) Quantification of PODXL, LTL or ECAD-positive cells per organoid in each group. Scale bar = 50 or 100 μm. Full article ">Figure 4
Effect of mitomycin C (MMC) treatment in kidney organoids from WTC-11, WTC-11FAN1+/−, and KIN patient hiPSCs. (A) Each kidney organoid was treated with 20 nM MMC. After 24 h or 48 h of incubation, each kidney organoid was stained with Ki67 antibody. Images with DAPI show nuclei positive for the Ki67 antibody. Scale bar = 50 or 100 μm. (B) Quantification of Ki67-positive cells in kidney organoids from WTC-11, WTC-11FAN1+/−, and KIN patient hiPSCs. (C) Flow cytometry gating strategy illustrating a viable cell population being subgated to the level of podocalyxin+ (PODXL), lotus tetragonolobus lectin+ (LTL), or e-cadherin+ (ECAD) in kidney organoid cells from WTC-11, WTC-11FAN1+/−, and the KIN patient hiPSCs, respectively, after treatment with MMC for 24 h or 48 h. (D–F) Quantification of percentage of viable cells in PODXL+, LTL+, or ECAD+ in cells from each kidney organoid. (G,H) Immunoblot analysis and its quantification of H2A.X in kidney organoids from WTC-11, WTC-11FAN1+/−, and KIN patient hiPSCs after treatment with MMC for 24 h or 48 h. Data were normalized against the β-actin expression level. All data are presented as mean ± standard error. *, p < 0.05 vs. Nil group or 24 h MMC group. Full article ">

20 pages, 3016 KiB

Open AccessReview

Biochemical and Molecular Pathways in Neurodegenerative Diseases: An Integrated View

by Nitesh Sanghai and Geoffrey K. Tranmer

Cells 2023, 12(18), 2318; https://doi.org/10.3390/cells12182318 - 20 Sep 2023

Cited by 6 | Viewed by 5445

Abstract

Neurodegenerative diseases (NDDs) like Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) are defined by a myriad of complex aetiologies. Understanding the common biochemical molecular pathologies among NDDs gives an opportunity to decipher the overlapping and numerous cross-talk mechanisms of [...] Read more.

Neurodegenerative diseases (NDDs) like Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) are defined by a myriad of complex aetiologies. Understanding the common biochemical molecular pathologies among NDDs gives an opportunity to decipher the overlapping and numerous cross-talk mechanisms of neurodegeneration. Numerous interrelated pathways lead to the progression of neurodegeneration. We present evidence from the past pieces of literature for the most usual global convergent hallmarks like ageing, oxidative stress, excitotoxicity-induced calcium butterfly effect, defective proteostasis including chaperones, autophagy, mitophagy, and proteosome networks, and neuroinflammation. Herein, we applied a holistic approach to identify and represent the shared mechanism across NDDs. Further, we believe that this approach could be helpful in identifying key modulators across NDDs, with a particular focus on AD, PD, and ALS. Moreover, these concepts could be applied to the development and diagnosis of novel strategies for diverse NDDs. Full article

(This article belongs to the Special Issue Neurodegenerative and Neurologic Disease: Genes, Mechanisms, and Therapies)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Schematic presentation of various biochemical cross-talks and their detrimental manifestations (A–I) in the brain provoked by oxidative stress and their implications in the progress of neurodegenerative diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). Brain is highly vulnerable to oxidative stress due to low regenerative capacity, enrichment of polyunsaturated fatty acids, high dependency on mitochondria for adenosine triphosphate (ATP) generation, elevated glucose demand, high concentration of metals like ferrous ion (Fe+2), cuprous ion (Cu+), zinc ion (Zn+2) and calcium ion (Ca+2), glutamate-induced excitotoxicity, high oxygen (O2) consumption, and relatively low antioxidant system. These multiple factors initiate various reaction pathways to create redox disbalance called oxidative and nitrosative stress in the brain, implicated in various NDDs. (A). The triplet unstable O2 undergoes reduction to produce the precursor of all radicals called superoxide anion radical (O2•−) via NAD(P)H oxidases (NOXs) pathway, i.e., one-electron trans-membrane transfer to (O2) [<a href="#B95-cells-12-02318" class="html-bibr">95</a>]. (B). Antioxidant superoxide dismutase (SOD1) undergoes dismutation to scavenge (O2•−) to produce hydrogen peroxide (H2O2). (C). The weakly liganded (Fe+2) and (Cu+) undergo reduction to produce nature’s most vulnerable oxidant hydroxyl radical (HO•) through Fenton’s reaction and Haber-Weiss reaction. (D). The final 4th electron reduction of H2O2 in the presence of antioxidants, like glutathione peroxidase (Gpx), catalase (cat), and peroxiredoxin system (Prx), forms water (H2O). (E). Overactivation of neuronal nitric oxide synthase (nNOS) produces nitric oxide (NO•) radicals from L-arginine, which create nitrosative stress by modification of thiol group (SH) containing proteins. (F). Excessive superoxide anion radicals lead to inactivation of nitric oxide production and switch the biology to production of highly potent oxidant peroxynitrite anion (ONOO−), which leads to the nitrosative stress by (SH) modification of free tyrosine (Tyr) residues to form 3-nitrotyrosine (3-NO2Tyr) (G), which act as a versatile biomarker of nitrosative stress and NDDs. (H). Highly reactive and mutagenic oxidant (HO•) damages the nucleic acid deoxyribonucleic acid/ribonucleic acid (DNA/RNA) to form oxidative products 8-hydroxy-2′-deoxyguanosine(8-OHdG) and (8-OxoG), and acts as a universal biomarker for oxidative stress and NDDs (important to note that guanine is the most oxidation prone nucleobase because of low reduction potential [<a href="#B96-cells-12-02318" class="html-bibr">96</a>]). Further, HO• radical causes lipid peroxidation of lipid-rich neuronal membranes, resulting in the death of neurons. Lipid peroxides (ROO.) act as a biomarker of oxidative stress and NDDs. Created with BioRender.com (accessed on 19 September 2023). Full article ">Figure 2
Schematic presentation of various biochemical cross-talks, involving calcium ion (Ca+2), ferrous ion (Fe+2), and Zinc ion (Zn+2) implicated in the progress of neurodegenerative diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). (A). Excitotoxicity (neuronal death) is triggered by the excessive release of excitatory neurotransmitter glutamate (neurotoxic) from the presynaptic neuron and leads to activation of various biochemical cascades leading to neurotoxicity and hence, neuronal death. This process is initiated by the activation of the N-methyl-D-aspartic acid receptors (NMDAR) by excessive glutamate at postsynaptic neurons and thereby the release and accumulation of toxic intraneuronal Ca2+. (B). Glutamate-mediated excitotoxicity is increased because of the astrocyte-mediated downregulation of excitatory amino acid transporters 2 (EAAT2), which slows down the uptake of glutamate from the synaptic cleft and incites the excitotoxicity cascade. (C). Ca2+ overload initiates most of the deleterious downstream mechanisms of the cascade, through increasing Ca2+ overload in mitochondria, induction of proteases (calpains and caspases), decreasing the proton gradient (ΔpH), mitochondrial membrane potential (ΔΨm) and adenosine triphosphate (ATP), activation of phospholipase A2 (PLA2) pathway initiating downstream activation of arachidonic acid and prostaglandin E2 (PGE2), aggravation of mitochondrial and endoplasmic reticulum stress leading to superoxide dismutase (SOD1) and TAR DNA-binding protein (TDP-43) aggregation. (D). Surge of reactive oxygen species (ROS) like hydrogen peroxide (H2O2) and hydroxyl radical (HO•) and reactive nitrogen species (RNS) like nitric oxide (NO•) radical, formation of peroxynitrite anion (ONOO−) increases the intraneuronal Zn2+ mobilization, which targets mitochondria and further exacerbates Ca2+ dysregulation and ROS production. (E). Ca+2 and Fe+2 dysregulation participates in the ferroptosis death of neurons. Iron dysregulation leads to Ca2+ dysregulation and vice versa. Excessive glutamate increases the Fe+2 intake inside the neurons, thereby leading to excitotoxicity and lipid peroxidation via Fenton’s reaction called Ferroptosis. Created with BioRender.com. Full article ">Figure 3
Schematic presentation of various common neuronal biochemical pathways perturbed or compromised in multiple neurodegenerative diseases, such as AD, PD, and ALS. The key points in the pathway and the selected disease-associated proteins are demonstrated in this picture. 1. Protein Quality Control (PQC) proteostasis network: molecular chaperones, including heat shock proteins (Hsp90, Hsp70, and Hsp40), regulate protein folding and maturation. Ubiquitin-proteosome system (UPS) is a crucial protein degradation pathway and is important for PQC and homeostasis. Any defect in the PQC leads to neurodegeneration (AD, PD, ALS). Decline of proteostasis is the hallmark of ageing and it decreases with age, leading to the accumulation of toxic and non-functional aggregates. 2. Autophagy-Lysosome Pathway (a,b,c,d,e.): Perturbations throughout the pathway, from initiation of autophagosome formation to degradation in the autolysosomes, have been suggested to be involved in neurodegenerative diseases like AD, PD, and ALS and further, could build an accumulation of pathogenic and toxic protein aggregates and defective mitochondria. a. Autophagy initiation defects due to decreased expression of protein Beclin1 in case of AD. b. Loss of sequestration into autophagosomes due to mutations in the gene-encoding p62/optineurin in the case of ALS, and mitophagy defects due to mutations in the gene-encoding protein PINK1/Parkin in the case of PD c. Defects in the maturation of autophagosome are due to decreasing expression of PICLAM protein in the case of AD, whereas mutation in SIGMAR1 gene in the case of ALS. c. Defects in vesicle trafficking (lysosome to membrane) are due to the mutations in the gene-encoding protein dynactin/profilin in case of ALS. 3. Dysregulation of mitochondrial quality control (MQC): including a (mitochondrial damage), b (mitochondrial fusion and fission dynamics), c (selective autophagy of mitochondria called mitophagy) results in decreased ATP production and dysfunctional proteostasis network. 4. Axonal transport defects in AD, PD, and ALS and underlying mechanisms: Defective axonal transport is due to perturbed anterograde and retrograde transport mechanisms involving mitochondrial kinesin and endosomal transport protein dynein. Further, disrupted neurofilament (NF) in forms of phosphorylated NF in the case of AD, PD, and ALS and microtubules (including α-Tubulin and β-Tubulin) are involved in the impairment of transport across neurons. 5. Protein Seeding and Propagation: Dysfunction of Intracellular propagation and seeding of toxic protein aggregates involved in the disease progression in case of AD, PD, and ALS. Created with BioRender.com. Full article ">

27 pages, 4896 KiB

Open AccessArticle

Differential Effects of Regulatory T Cells in the Meninges and Spinal Cord of Male and Female Mice with Neuropathic Pain

by Nathan T. Fiore, Brooke A. Keating, Yuting Chen, Sarah I. Williams and Gila Moalem-Taylor

Cells 2023, 12(18), 2317; https://doi.org/10.3390/cells12182317 - 20 Sep 2023

Cited by 7 | Viewed by 1832

Abstract

Immune cells play a critical role in promoting neuroinflammation and the development of neuropathic pain. However, some subsets of immune cells are essential for pain resolution. Among them are regulatory T cells (Tregs), a specialised subpopulation of T cells that limit excessive immune [...] Read more.

Immune cells play a critical role in promoting neuroinflammation and the development of neuropathic pain. However, some subsets of immune cells are essential for pain resolution. Among them are regulatory T cells (Tregs), a specialised subpopulation of T cells that limit excessive immune responses and preserve immune homeostasis. In this study, we utilised intrathecal adoptive transfer of activated Tregs in male and female mice after peripheral nerve injury to investigate Treg migration and whether Treg-mediated suppression of pain behaviours is associated with changes in peripheral immune cell populations in lymphoid and meningeal tissues and spinal microglial and astrocyte reactivity and phenotypes. Treatment with Tregs suppressed mechanical pain hypersensitivity and improved changes in exploratory behaviours after chronic constriction injury (CCI) of the sciatic nerve in both male and female mice. The injected Treg cells were detected in the choroid plexus and the pia mater and in peripheral lymphoid organs in both male and female recipient mice. Nonetheless, Treg treatment resulted in differential changes in meningeal and lymph node immune cell profiles in male and female mice. Moreover, in male mice, adoptive transfer of Tregs ameliorated the CCI-induced increase in microglia reactivity and inflammatory phenotypic shift, increasing M2-like phenotypic markers and attenuating astrocyte reactivity and neurotoxic astrocytes. Contrastingly, in CCI female mice, Treg injection increased astrocyte reactivity and neuroprotective astrocytes. These findings show that the adoptive transfer of Tregs modulates meningeal and peripheral immunity, as well as spinal glial populations, and alleviates neuropathic pain, potentially through different mechanisms in males and females. Full article

(This article belongs to the Special Issue Role of Glial Cells in Neuropathic Pain)

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19 pages, 2940 KiB

Open AccessArticle

Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties

by Rita V. Chertkova, Ilya P. Oleynikov, Alexey A. Pakhomov, Roman V. Sudakov, Victor N. Orlov, Marina A. Semenova, Alexander M. Arutyunyan, Vasily V. Ptushenko, Mikhail P. Kirpichnikov, Dmitry A. Dolgikh and Tatiana V. Vygodina

Cells 2023, 12(18), 2316; https://doi.org/10.3390/cells12182316 - 19 Sep 2023

Cited by 2 | Viewed by 1400

Abstract

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use [...] Read more.

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems. Full article

(This article belongs to the Special Issue Cytochrome C—Alternative Functions, New Interactions and Technological Applications)

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Locations of mutated residues in the 3D structure of CytC. Qualitative electrostatic potential surface of horse CytC WT (a) and 8Mut (b) are shown from the side of the heme cavity and when rotated by 180° around the vertical axis. ε-amino groups of Lys residues and carboxyl groups of Glu residues subjected to mutagenesis are marked with blue and red balls/numbers, respectively. The positions of unaltered Lys13 and Lys79 residues near heme cavity are marked with grey numbers. The images are obtained using the PyMOL program (pdb code 1HRC). Full article ">Figure 2
Isoelectric surfaces of CytC WT (a) and 8Mut (b). Blue and red surfaces correspond to charge +0.1 mV and −0.1 mV, respectively. Orientation of the molecules is the same as in <a href="#cells-12-02316-f001" class="html-fig">Figure 1</a>. Full article ">Figure 3
Typical oxygen consumption kinetics of CcO oxidizing CytC in the presence of TMPD and ascorbate: WT (black line) and CytC mutants: 2Mut (red line), 5Mut (green line), and 8Mut (blue line). The final concentrations in the measurement medium were CcO, 24 nM (black line) and 120 nM (red, green, and blue lines); ascorbate (5 mM); TMPD (0.1 mM). The addition of cytochromes (1 µM each) is marked with arrows with the letter c. The inset shows the dependences of the oxygen consumption rate on the concentration of CytC 2Mut, obtained in the presence of TMPD (1, red dots) and without it (2, black dots). Full article ">Figure 4
Typical kinetics of superoxide anion radical reduction during hypoxanthine oxidation by xanthine oxidase, measured by the reduction of CytC WT (black line) and mutant forms of CytC 2Mut (red line), CytC 5Mut (green line), and CytC 8Mut (blue line). CytC reduction was recorded spectrally in the two-wavelength mode by the difference in absorption at 550 nm and 535 nm (comparison wave). The concentration of CytC in the measurement medium in all experiments was 4 µM, hypoxanthine, 50 µM, and xanthine oxidase, 0.015 U/mL. The measurements were carried out in 50 mM K-phosphate buffer, pH 7.5, 0.1 mM EDTA. Full article ">Figure 5
The redox titration curves of CytC, wild-type (WT—transparent circles), and mutated (8Mut—blue circles) forms. Full article ">Figure 6
The longwave region of the absorption spectrum of ferric CytC: CytC (S) (magenta line), horse heart CytC (Sigma, Burlington, MA, USA), recombinant CytC WT (black line), and CytC 8Mut (blue line). For comparison, the spectrum of ferrous CytC 8Mut is also shown (red line, dotted line). Absorbance is normalized to the concentration of cytochromes in measurement samples that have been pre-oxidized with substoichiometric amounts of ferricyanide. The measurements were carried out in basic buffer, pH 7.6. Full article ">Figure 7
CD spectra of CytC WT (black line) and mutant variants 2Mut (red line), 5Mut (green line), and 8Mut (blue line). The spectra were normalized to the concentration of CytC in the studied samples, which was about 0.6 mM (measured from the band at 550 nm). The inset shows what the same CD spectra look like normalized to protein concentration based on far UV molar extinction at 205 nm, 210 nm, and 215 nm (<a href="#cells-12-02316-t004" class="html-table">Table 4</a> in [<a href="#B38-cells-12-02316" class="html-bibr">38</a>]). Three values are averaged. Full article ">Figure 8
Particle size of CytC samples: CytC (S) (black squares), horse heart CytC from Sigma, and mutant forms 2Mut (red circles), 5Mut (green triangles), and 8Mut (blue triangles) measured by dynamic light scattering. Full article ">

21 pages, 4862 KiB

Open AccessArticle

Characterisation of Lipoma-Preferred Partner as a Novel Mechanotransducer in Vascular Smooth Muscle Cells

by Alexandra Sporkova, Taslima Nahar, Mingsi Cao, Subhajit Ghosh, Carla Sens-Albert, Prisca Amayi Patricia Friede, Anika Nagel, Jaafar Al-Hasani and Markus Hecker

Cells 2023, 12(18), 2315; https://doi.org/10.3390/cells12182315 - 19 Sep 2023

Viewed by 5113

Abstract

In arteries and arterioles, a chronic increase in blood pressure raises wall tension. This continuous biomechanical strain causes a change in gene expression in vascular smooth muscle cells (VSMCs) that may lead to pathological changes. Here we have characterised the functional properties of [...] Read more.

In arteries and arterioles, a chronic increase in blood pressure raises wall tension. This continuous biomechanical strain causes a change in gene expression in vascular smooth muscle cells (VSMCs) that may lead to pathological changes. Here we have characterised the functional properties of lipoma-preferred partner (LPP), a Lin11–Isl1–Mec3 (LIM)-domain protein, which is most closely related to the mechanotransducer zyxin but selectively expressed by smooth muscle cells, including VSMCs in adult mice. VSMCs isolated from the aorta of LPP knockout (LPP-KO) mice displayed a higher rate of proliferation than their wildtype (WT) counterparts, and when cultured as three-dimensional spheroids, they revealed a higher expression of the proliferation marker Ki 67 and showed greater invasion into a collagen gel. Accordingly, the gelatinase activity was increased in LPP-KO but not WT spheroids. The LPP-KO spheroids adhering to the collagen gel responded with decreased contraction to potassium chloride. The relaxation response to caffeine and norepinephrine was also smaller in the LPP-KO spheroids than in their WT counterparts. The overexpression of zyxin in LPP-KO VSMCs resulted in a reversal to a more quiescent differentiated phenotype. In native VSMCs, i.e., in isolated perfused segments of the mesenteric artery (MA), the contractile responses of LPP-KO segments to potassium chloride, phenylephrine or endothelin-1 did not vary from those in isolated perfused WT segments. In contrast, the myogenic response of LPP-KO MA segments was significantly attenuated while zyxin-deficient MA segments displayed a normal myogenic response. We propose that LPP, which we found to be expressed solely in the medial layer of different arteries from adult mice, may play an important role in controlling the quiescent contractile phenotype of VSMCs. Full article

(This article belongs to the Special Issue Role of Vascular Smooth Muscle Cells in Cardiovascular Disease)

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44 pages, 552 KiB

Open AccessReview

Inborn Errors of Metabolism with Ataxia: Current and Future Treatment Options

by Tatiana Bremova-Ertl, Jan Hofmann, Janine Stucki, Anja Vossenkaul and Matthias Gautschi

Cells 2023, 12(18), 2314; https://doi.org/10.3390/cells12182314 - 19 Sep 2023

Viewed by 2012

Abstract

A number of hereditary ataxias are caused by inborn errors of metabolism (IEM), most of which are highly heterogeneous in their clinical presentation. Prompt diagnosis is important because disease-specific therapies may be available. In this review, we offer a comprehensive overview of metabolic [...] Read more.

A number of hereditary ataxias are caused by inborn errors of metabolism (IEM), most of which are highly heterogeneous in their clinical presentation. Prompt diagnosis is important because disease-specific therapies may be available. In this review, we offer a comprehensive overview of metabolic ataxias summarized by disease, highlighting novel clinical trials and emerging therapies with a particular emphasis on first-in-human gene therapies. We present disease-specific treatments if they exist and review the current evidence for symptomatic treatments of these highly heterogeneous diseases (where cerebellar ataxia is part of their phenotype) that aim to improve the disease burden and enhance quality of life. In general, a multimodal and holistic approach to the treatment of cerebellar ataxia, irrespective of etiology, is necessary to offer the best medical care. Physical therapy and speech and occupational therapy are obligatory. Genetic counseling is essential for making informed decisions about family planning. Full article

(This article belongs to the Special Issue Emerging Therapies for Hereditary Ataxia)

26 pages, 4596 KiB

Open AccessArticle

Let-7g Upregulation Attenuated the KRAS–PI3K–Rac1–Akt Axis-Mediated Bioenergetic Functions

by Kuang-Chen Hung, Ni Tien, Da-Tian Bau, Chun-Hsu Yao, Chan-Hung Chen, Jiun-Long Yang, Meng-Liang Lin and Shih-Shun Chen

Cells 2023, 12(18), 2313; https://doi.org/10.3390/cells12182313 - 19 Sep 2023

Cited by 1 | Viewed by 1673

Abstract

The aberrant activation of signaling pathways contributes to cancer cells with metabolic reprogramming. Thus, targeting signaling modulators is considered a potential therapeutic strategy for cancer. Subcellular fractionation, coimmunoprecipitation, biochemical analysis, and gene manipulation experiments revealed that decreasing the interaction of kirsten rat sarcoma [...] Read more.

The aberrant activation of signaling pathways contributes to cancer cells with metabolic reprogramming. Thus, targeting signaling modulators is considered a potential therapeutic strategy for cancer. Subcellular fractionation, coimmunoprecipitation, biochemical analysis, and gene manipulation experiments revealed that decreasing the interaction of kirsten rat sarcoma viral oncogene homolog (KRAS) with p110α in lipid rafts with the use of naringenin (NGN), a citrus flavonoid, causes lipid raft-associated phosphatidylinositol 3-kinase (PI3K)−GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)−protein kinase B (Akt)-regulated metabolic dysfunction of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), leading to apoptosis in human nasopharyngeal carcinoma (NPC) cells. The use of lethal-7g (let-7g) mimic and let-7g inhibitor confirmed that elevated let-7g resulted in a decrease in KRAS expression, which attenuated the PI3K−Rac1−Akt−BCL-2/BCL-x_L-modulated mitochondrial energy metabolic functions. Increased let-7g depends on the suppression of the RNA-specificity of monocyte chemoattractant protein-induced protein-1 (MCPIP1) ribonuclease since NGN specifically blocks the degradation of pre-let-7g by NPC cell-derived immunoprecipitated MCPIP1. Converging lines of evidence indicate that the inhibition of MCPIP1 by NGN leads to let-7g upregulation, suppressing oncogenic KRAS-modulated PI3K–Rac1–Akt signaling and thereby impeding the metabolic activities of aerobic glycolysis and mitochondrial OXPHOS. Full article

(This article belongs to the Section Intracellular and Plasma Membranes)

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32 pages, 5221 KiB

Open AccessReview

The Chemical Inhibitors of Endocytosis: From Mechanisms to Potential Clinical Applications

by Olga Klaudia Szewczyk-Roszczenko, Piotr Roszczenko, Anna Shmakova, Nataliya Finiuk, Serhii Holota, Roman Lesyk, Anna Bielawska, Yegor Vassetzky and Krzysztof Bielawski

Cells 2023, 12(18), 2312; https://doi.org/10.3390/cells12182312 - 19 Sep 2023

Cited by 10 | Viewed by 5528

Abstract

Endocytosis is one of the major ways cells communicate with their environment. This process is frequently hijacked by pathogens. Endocytosis also participates in the oncogenic transformation. Here, we review the approaches to inhibit endocytosis, discuss chemical inhibitors of this process, and discuss potential [...] Read more.

Endocytosis is one of the major ways cells communicate with their environment. This process is frequently hijacked by pathogens. Endocytosis also participates in the oncogenic transformation. Here, we review the approaches to inhibit endocytosis, discuss chemical inhibitors of this process, and discuss potential clinical applications of the endocytosis inhibitors. Full article

(This article belongs to the Section Cellular Pathology)

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23 pages, 7211 KiB

Open AccessArticle

Unraveling the Role of Hepatic PGC1α in Breast Cancer Invasion: A New Target for Therapeutic Intervention?

by Kumar Ganesan, Cong Xu, Qingqing Liu, Yue Sui and Jianping Chen

Cells 2023, 12(18), 2311; https://doi.org/10.3390/cells12182311 - 19 Sep 2023

Cited by 3 | Viewed by 1159

Abstract

Breast cancer (BC) is the most common cancer among women worldwide and the main cause of cancer deaths in women. Metabolic components are key risk factors for the development of non-alcoholic fatty liver disease (NAFLD), which may promote BC. Studies have reported that [...] Read more.

Breast cancer (BC) is the most common cancer among women worldwide and the main cause of cancer deaths in women. Metabolic components are key risk factors for the development of non-alcoholic fatty liver disease (NAFLD), which may promote BC. Studies have reported that increasing PGC1α levels increases mitochondrial biogenesis, thereby increasing cell proliferation and metastasis. Moreover, the PGC1α/ERRα axis is a crucial regulator of cellular metabolism in various tissues, including BC. However, it remains unclear whether NAFLD is closely associated with the risk of BC. Therefore, the present study aimed to determine whether hepatic PGC1α promotes BC cell invasion via ERRα. Various assays, including ELISA, western blotting, and immunoprecipitation, have been employed to explore these mechanisms. According to the KM plot and TCGA data, elevated PGC1α expression was highly associated with a shorter overall survival time in patients with BC. High concentrations of palmitic acid (PA) promoted PGC1α expression, lipogenesis, and inflammatory processes in hepatocytes. Conditioned medium obtained from PA-treated hepatocytes significantly increased BC cell proliferation. Similarly, recombinant PGC1α in E0771 and MCF7 cells promoted cell proliferation, migration, and invasion in vitro. However, silencing PGC1α in both BC cell lines resulted in a decrease in this trend. As determined by immunoprecipitation assay, PCG1a interacted with ERRα, thereby facilitating the proliferation of BC cells. This outcome recognizes the importance of further investigations in exploring the full potential of hepatic PGC1α as a prognostic marker for BC development. Full article

(This article belongs to the Special Issue Cancer-Related Signaling Cascades: Current Knowledge and Potential Therapeutic Targets)

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26 pages, 8216 KiB

Open AccessArticle

Syringin Prevents 6-Hydroxydopamine Neurotoxicity by Mediating the MiR-34a/SIRT1/Beclin-1 Pathway and Activating Autophagy in SH-SY5Y Cells and the Caenorhabditis elegans Model

by Ru-Huei Fu, Syuan-Yu Hong and Hui-Jye Chen

Cells 2023, 12(18), 2310; https://doi.org/10.3390/cells12182310 - 19 Sep 2023

Cited by 1 | Viewed by 1625

Abstract

Defective autophagy is one of the cellular hallmarks of Parkinson’s disease (PD). Therefore, a therapeutic strategy could be a modest enhancement of autophagic activity in dopamine (DA) neurons to deal with the clearance of damaged mitochondria and abnormal protein aggregates. Syringin (SRG) is [...] Read more.

Defective autophagy is one of the cellular hallmarks of Parkinson’s disease (PD). Therefore, a therapeutic strategy could be a modest enhancement of autophagic activity in dopamine (DA) neurons to deal with the clearance of damaged mitochondria and abnormal protein aggregates. Syringin (SRG) is a phenolic glycoside derived from the root of Acanthopanax senticosus. It has antioxidant, anti-apoptotic, and anti-inflammatory properties. However, whether it has a preventive effect on PD remains unclear. The present study found that SRG reversed the increase in intracellular ROS-caused apoptosis in SH-SY5Y cells induced by neurotoxin 6-OHDA exposure. Likewise, in C. elegans, degeneration of DA neurons, DA-related food-sensitive behaviors, longevity, and accumulation of α-synuclein were also improved. Studies of neuroprotective mechanisms have shown that SRG can reverse the suppressed expression of SIRT1, Beclin-1, and other autophagy markers in 6-OHDA-exposed cells. Thus, these enhanced the formation of autophagic vacuoles and autophagy activity. This protective effect can be blocked by pretreatment with wortmannin (an autophagosome formation blocker) and bafilomycin A1 (an autophagosome–lysosome fusion blocker). In addition, 6-OHDA increases the acetylation of Beclin-1, leading to its inactivation. SRG can induce the expression of SIRT1 and promote the deacetylation of Beclin-1. Finally, we found that SRG reduced the 6-OHDA-induced expression of miR-34a targeting SIRT1. The overexpression of miR-34a mimic abolishes the neuroprotective ability of SRG. In conclusion, SRG induces autophagy via partially regulating the miR-34a/SIRT1/Beclin-1 axis to prevent 6-OHDA-induced apoptosis and α-synuclein accumulation. SRG has the opportunity to be established as a candidate agent for the prevention and cure of PD. Full article

(This article belongs to the Special Issue Autophagy in Parkinson's Disease)

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15 pages, 3303 KiB

Open AccessArticle

Lymphatic Defects in Zebrafish sox18 Mutants Are Exacerbated by Perturbed VEGFC Signaling, While Masked by Elevated sox7 Expression

by Silvia Moleri, Sara Mercurio, Alex Pezzotta, Donatella D’Angelo, Alessia Brix, Alice Plebani, Giulia Lini, Marialaura Di Fuorti and Monica Beltrame

Cells 2023, 12(18), 2309; https://doi.org/10.3390/cells12182309 - 19 Sep 2023

Cited by 2 | Viewed by 1453

Abstract

Mutations in the transcription factor-coding gene SOX18, the growth factor-coding gene VEGFC and its receptor-coding gene VEGFR3/FLT4 cause primary lymphedema in humans. In mammals, SOX18, together with COUP-TFII/NR2F2, activates the expression of Prox1, a master regulator in lymphatic identity and development. [...] Read more.

Mutations in the transcription factor-coding gene SOX18, the growth factor-coding gene VEGFC and its receptor-coding gene VEGFR3/FLT4 cause primary lymphedema in humans. In mammals, SOX18, together with COUP-TFII/NR2F2, activates the expression of Prox1, a master regulator in lymphatic identity and development. Knockdown studies have also suggested an involvement of Sox18, Coup-tfII/Nr2f2, and Prox1 in zebrafish lymphatic development. Mutants in the corresponding genes initially failed to recapitulate the lymphatic defects observed in morphants. In this paper, we describe a novel zebrafish sox18 mutant allele, sa12315, which behaves as a null. The formation of the lymphatic thoracic duct is affected in sox18 homozygous mutants, but defects are milder in both zygotic and maternal-zygotic sox18 mutants than in sox18 morphants. Remarkably, in sox18 mutants, the expression of the closely related sox7 gene is elevated where lymphatic precursors arise. Sox7 could thus mask the absence of a functional Sox18 protein and account for the mild lymphatic phenotype in sox18 mutants, as shown in mice. Partial knockdown of vegfc exacerbates lymphatic defects in sox18 mutants, making them visible in heterozygotes. Our data thus reinforce the genetic interaction between Sox18 and Vegfc in lymphatic development, previously suggested by knockdown studies, and highlight the ability of Sox7 to compensate for Sox18 lymphatic dysfunction. Full article

(This article belongs to the Special Issue Modeling Developmental Processes and Disorders in Zebrafish)

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The sox18sa12315 mutant behaves as expected for a null allele. (A) On the left is a schematic representation of the Sox18 protein, with the HMG-box domain in blue. The G > A transition and the premature stop codon introduced in the sa12315 mutant are indicated. Fragments of the electropherograms derived using Sanger sequencing of the region surrounding the mutation in wt (sox18+/+), heterozygous (sox18+/−) or homozygous mutants (sox18−/−) are reported on the right. The restriction site for the BstNI/MvaI enzymes (boxed sequence) is disrupted by the mutation. (B) Embryos derived from sa12315 heterozygote matings and injected with subcritical doses of sox7-MO were collected in several independent experiments and analyzed in vivo at 2 dpf and 3 dpf or fixed at around 30 hpf for ISH, as shown in (C). The histogram on the right shows the trunk–tail circulatory phenotypes observed at 3 dpf. In control embryos, i.e., uninjected or injected with a standard control MO (first and second bars, respectively), trunk–tail circulatory defects are present in a small percentage of embryos. On the contrary, the partial knockdown of sox7 causes a blockage in trunk–tail circulation in a dose-dependent manner (third and fourth bars). Circulatory defects are genotype-dependent (see <a href="#app1-cells-12-02309" class="html-app">Table S1</a>). (C) ISHs were performed on embryos derived from sa12315 heterozygote matings and injected with subcritical doses of sox7-MO, as shown in (B), fixed at around 30 hpf. Upper panels show control ISH performed with the endothelial marker cdh5, showing no gross alteration in embryos of the three different genotypes. Lower panels show ISHs performed with a probe for vsg1/plvapb, whose expression was particularly downregulated in double partial sox7/sox18 morphants [<a href="#B29-cells-12-02309" class="html-bibr">29</a>]. Higher magnification images of the trunk–tail regions of the embryos are also shown. Experiments were repeated twice; all plavpb stained embryos and a subset of cdh5 stained embryos were genotyped; numbers in each image refer to a single experiment. Lateral views, anterior to the left. Pictures were taken at 40× and 63× magnification, for lower and higher magnification images respectively. Full article ">Figure 2
Homozygous sox18 mutants show subtle but statistically significant defects in thoracic duct (TD) formation. (A) Confocal trunk images representing wt (+/+) and sox18sa12315 homozygous mutant larvae (−/−) in the Tg(lyve1b:DsRed) line at 5dpf. Large and small white arrowheads point to TD+ segments (of typical or thinner aspect, respectively) while asterisks indicate the absence of TD. (B) The graph reports the mean number of TD+ segments, counted along 10 consecutive trunk segments, together with the Standard Error of the Mean (SEM), in all analyzed embryos of the three genotypes (wt: sox18+/+, het: sox18+/−, hom: sox18−/−). Data were gathered in three independent experiments, and each symbol represents the number of TD+ segments of a single analyzed larva. n = number of larvae, ** = p < 0.01. TD = thoracic duct; DA = dorsal aorta; PCV = posterior cardinal vein. Lateral view, anterior to the left. Full article ">Figure 3
TD formation defects are exacerbated upon slight perturbation of Vegfc signaling. The progeny of sox18sa12315 heterozygote matings in the Tg(fli1a:EGFP)y1 line were injected with a subcritical dose of vegfc-MO or left uninjected; TD formation was analyzed at 5dpf. (A) Confocal trunk images of uninjected wt (+/+) and sox18 homozygous mutant (−/−) larvae. Arrowheads point to TD+ segments, while asterisks indicate the absence of TD; a smaller arrowhead marks a thinner TD+ segment. (B) The graph reports the mean number of TD+ segments, counted along 10 consecutive trunk segments, together with the SEM, for all analyzed larvae of each genotype (wt: sox18+/+, het: sox18+/−, hom: sox18−/−). Each symbol represents the number of TD+ segments of a single larva; data were gathered in several independent experiments. Uninjected larvae on the left are compared to larvae with partially reduced Vegfc on the right. n = number of larvae, * = p < 0.05; ** = p < 0.01; *** = p < 0.001. TD = thoracic duct; DA = dorsal aorta; PCV = posterior cardinal vein. Lateral view, anterior to the left. Full article ">Figure 4
The expression of sox7 in the PCV is upregulated in sox18 mutants, but not in sox18 morphants. (A) Representative images of sox7 ISH on embryos at around 26 hpf derived from matings of sox18sa12315 heterozygotes in the Tg(lyve1b:DsRed) line. Higher magnifications of the trunk region are shown below the full size images. Compared to wt embryos, the sox7 ISH signal in the PCV is elevated in the great majority of sox18−/− homozygotes and, to a lesser extent, in sox18+/− heterozygotes. Numbers in each image state the number of embryos with the reported phenotype over the total analyzed embryos in one representative experiment. Lateral views, anterior to the left. Pictures were taken at 40× and 63× magnification, for lower and higher magnification images respectively. (B) Left, representative ImageJ-modified images, used to perform the quantification of the sox7 ISH signal in the PCV and the DA (as described in Materials and Methods) on 26 hpf wt (+/+), sox18sa12315 heterozygotes (+/−) or homozygous mutants (−/−). The graph on the right shows the calculated PCV/DA ratio in each embryo; embryos are grouped based on their genotypes: mean values and SEM are indicated. (C) The same analysis was performed on sox7 ISH of sox18 morphants and control embryos, as shown in <a href="#app1-cells-12-02309" class="html-app">Figure S6</a>. The calculated PCV/DA ratio of each embryo is shown in the graph; mean values and SEM for std-MO injected embryos and sox18 morphants are indicated. n = number of embryos, ** = p < 0.01; DA = dorsal aorta; PCV = posterior cardinal vein. The analysis was also repeated on ISHs of sox18sa12315 mutants in the Tg(fli1a:EGFP)y1 reporter line with similar results. ISH experiments on sox18sa12315 mutants were repeated at least three times. Data shown in A and B were generated on different clutches of embryos. Full article ">

24 pages, 2343 KiB

Open AccessArticle

Altered Epigenetic Marks and Gene Expression in Fetal Brain, and Postnatal Behavioural Disorders, Following Prenatal Exposure of Ogg1 Knockout Mice to Saline or Ethanol

by Shama Bhatia, David Bodenstein, Ashley P. Cheng and Peter G. Wells

Cells 2023, 12(18), 2308; https://doi.org/10.3390/cells12182308 - 19 Sep 2023

Viewed by 1373

Abstract

Oxoguanine glycosylase 1 (OGG1) is widely known to repair the reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG), and more recently was shown to act as an epigenetic modifier. We have previously shown that saline-exposed Ogg1 −/− knockout progeny exhibited learning and memory [...] Read more.

Oxoguanine glycosylase 1 (OGG1) is widely known to repair the reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG), and more recently was shown to act as an epigenetic modifier. We have previously shown that saline-exposed Ogg1 −/− knockout progeny exhibited learning and memory deficits, which were enhanced by in utero exposure to a single low dose of ethanol (EtOH) in both Ogg1 +/+ and −/− progeny, but more so in Ogg1 −/− progeny. Herein, OGG1-deficient progeny exposed in utero to a single low dose of EtOH or its saline vehicle exhibited OGG1- and/or EtOH-dependent alterations in global histone methylation and acetylation, DNA methylation and gene expression (Tet1 (Tet Methylcytosine Dioxygenase 1), Nlgn3 (Neuroligin 3), Hdac2 (Histone Deacetylase 2), Reln (Reelin) and Esr1 (Estrogen Receptor 1)) in fetal brains, and behavioural changes in open field activity, social interaction and ultrasonic vocalization, but not prepulse inhibition. OGG1- and EtOH-dependent changes in Esr1 and Esr2 mRNA and protein levels were sex-dependent, as was the association of Esr1 gene expression with gene activation mark histone H3 lysine 4 trimethylation (H3K4me3) and gene repression mark histone H3 lysine 27 trimethylation (H3K27me3) measured via ChIP-qPCR. The OGG1-dependent changes in global epigenetic marks and gene/protein expression in fetal brains, and postnatal behavioural changes, observed in both saline- and EtOH-exposed progeny, suggest the involvement of epigenetic mechanisms in developmental disorders mediated by 8-oxoG and/or OGG1. Epigenetic effects of OGG1 may be involved in ESR1-mediated gene regulation, which may be altered by physiological and EtOH-enhanced levels of ROS formation, possibly contributing to sex-dependent developmental disorders observed in Ogg1 knockout mice. The OGG1- and EtOH-dependent associations provide a basis for more comprehensive mechanistic studies to determine the causal involvement of oxidative DNA damage and epigenetic changes in ROS-mediated neurodevelopmental disorders. Full article

(This article belongs to the Special Issue Epigenetic Control and Gene Regulatory Mechanisms in Development and Disease)

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Figure 1

Figure 1
Dosing and behavioural testing timeline. Ogg1 heterozygous mice were mated, and on gestational day (GD) 17, the dams received a single dose of EtOH (2 g/kg i.p.) or its saline vehicle. The pups were delivered spontaneously, weaned after 3 weeks and subjected to a series of behavioural tests including interaction-induced ultrasonic vocalization (USV) at 3–4 weeks of age, open field activity at 6 weeks of age, social interaction with a novel mouse at 7–8 weeks of age, female-induced USV at 4–5 months of age and prepulse inhibition at approximately 5 months of age. Full article ">Figure 2
Altered histone and DNA modifications in fetal brains of Ogg1 −/− mice exposed in utero to EtOH. GD 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg i.p.) or its saline vehicle were extracted 1, 6 and 24 h later from Ogg1 +/+ and −/− littermates and were assessed for histone and DNA modifications. Fetal brains from at least three litters were used to minimize potential litter effects, and the number of fetal brains for each group is shown in parentheses. (A). The following histone modifications were analyzed: H3K9ac (activation mark), H3K9me3 and H3K27me3 (repressive marks). See <a href="#app1-cells-12-02308" class="html-app">Supplementary Figure S1</a> for 1 h results for H3K9ac and H3K9me3 (no differences observed) and for 6 and 24 h results for H3K4me3 (activation mark, no differences observed). (B). Increase in 5-methylcytosine (5-mC) levels in saline-exposed Ogg1 −/− vs. +/+ fetal brains at 24 h, with a similar trend in EtOH-exposed fetal brains. The same DNA sample was used for both 5-mC and 5-hmC measurements. The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s test. Abbreviations: 5-mC: 5-methylcytosine; 5-hmC: 5-hydroxymethylcytosine; H3K4me3: histone 3 lysine 9 trimethylation; H3K9ac: histone 3 lysine 9 acetylation; H3K9me3: histone 3 lysine 9 trimethylation; H3K27me3: histone 3 lysine 27 trimethylation. Full article ">Figure 3
Altered gene expression in Ogg1 −/− fetal brains exposed in utero to EtOH. GD 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg i.p.) or its saline vehicle were extracted 6 and 24 h later from Ogg1 +/+ and −/− littermates. Fetal brains from at least three litters were used to minimize potential litter effects, and the number of fetal brains for each group is shown in parentheses. Fetal brains were homogenized, RNA was extracted using the TRIzol method and mRNA expression levels were measured via RT-qPCR. Gapdh was used as a control. Expression levels of various learning and memory candidate genes were measured (see <a href="#app1-cells-12-02308" class="html-app">Supplementary Figure S2</a>). Above are the results for OGG1- and EtOH-dependent differences in mRNA levels in fetal brains. The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s test. Abbreviations: Hdac2: histone deacetylase 2; Nlgn3: neuroligin 3; Reln: Reelin; Tet1: Ten-eleven translocation methylcytosine dioxygenase 1. Full article ">Figure 4
OGG1- and sex-dependent differences in Esr1 and Esr2 mRNA levels and their ratios in fetal brains exposed in utero to EtOH. GD 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg i.p.) or its saline vehicle were extracted 6 and 24 h later from Ogg1 +/+ and −/− littermates. Fetal brains from at least three litters were used to minimize potential litter effects, and the number of fetal brains for each group is shown in parentheses. Fetal brains were homogenized, RNA was extracted using the TRIzol method and mRNA expression levels were measured via RT-qPCR. Gapdh was used as a control. Above are the results for Esr1 and Esr2 mRNA levels as well as their ratios in fetal brains. The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s test. Abbreviations: Esr1: estrogen receptor 1; Esr2: estrogen receptor 2. Full article ">Figure 5
OGG1- and sex-dependent differences in ESR1 and ESR2 protein levels and their ratios in fetal brains exposed in utero to EtOH. GD 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg i.p.) or its saline vehicle were extracted 6 and 24 h later from Ogg1 +/+ and −/− littermates. Fetal brains from at least three litters were used to minimize potential litter effects, and the number of fetal brains for each group is shown in parentheses. Fetal brains were homogenized, and protein levels were quantified via western blot. GAPDH was used as a loading control. Above are the results for ESR1 and ESR2 proteins levels as well as their ratios in fetal brains. The significance of differences for each sex was determined by two-way ANOVA and a post hoc Tukey’s test. Abbreviations: ESR1: estrogen receptor 1; ESR2: estrogen receptor 2. Full article ">Figure 6
EtOH-mediated increased association of H3K27me3 with Esr1 gene expression in Ogg1 +/+ but not −/− fetal brains. GD 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg i.p.) or its saline vehicle were extracted 6 h later from Ogg1 +/+ and −/− littermates. Fetal brains from at least three litters were used to minimize potential litter effects, and the number of fetal brains for each group is shown in parentheses. (A). ChIP was performed using fetal brains, and extracted DNA was used to perform quantitative PCR using five different sets of primers directed against various regions of the Esr1 gene. The Esr1 gene has two promoter regions, marked with arrows, which can generate four transcript variants via gene splicing. The locations of the exons and introns on chromosome 10 are marked. The primer locations are relative to the first transcription start site (TSS). Regions 1–5 were chosen based on the Ensembl database reporting their association with activation or repressive epigenetic marks. Each of the regions was amplified after chromatin was immunoprecipitated using antibodies against H3K4me3 (active promoter) and H3K27me3 (inactive promoter) and histone H3 (control). This figure is not drawn to scale. (B). The association of the H3K27me3:H3 ratio was normalized to 1% input in various regions of Esr1 of fetal brains exposed in utero to saline or EtOH. (C). The association of the H3K4me3:H3 ratio was normalized to 1% input in various regions of Esr1 of fetal brains exposed in utero to saline or EtOH. The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s test. See <a href="#app1-cells-12-02308" class="html-app">Supplementary Figure S3</a> for controls and <a href="#app1-cells-12-02308" class="html-app">Figures S4 and S5</a> for sex-separated data. Full article ">Figure 7
Increased hyperactivity for saline- but not EtOH-exposed Ogg1 −/− vs. +/+ females, and decreased centre time for EtOH-exposed Ogg1 +/+ but not −/− female mice. For all behavioural studies, pregnant dams were treated with single dose of EtOH (2 g/kg i.p.) or its saline vehicle on GD 17, as shown in <a href="#cells-12-02308-f001" class="html-fig">Figure 1</a>, and progeny were delivered spontaneously. Fetal brains from at least three litters were used to minimize potential litter effects, and the number of mice tested for each group is shown in parentheses. Results show total distance travelled during the entire test (1 h), total distance travelled during the last 30 min of the test and total time spent in the centre zone (10 × 10 inches). The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s test. See <a href="#app1-cells-12-02308" class="html-app">Supplementary Figure S6a</a> for data for the nonsocial zone. Full article ">Figure 8
OGG1- and sex-dependent effect on social interaction and interaction-induced ultrasonic vocalizations. For all behavioural studies, pregnant dams were treated with a single dose of EtOH (2 g/kg i.p.) or its saline vehicle on GD 17, as described in <a href="#cells-12-02308-f001" class="html-fig">Figure 1</a>, and progeny were delivered spontaneously. Fetal brains from at least three litters were used to minimize potential litter effects, and the number of mice tested for each group is shown in parentheses. (Panel A). For social interaction, EtOH vs. saline decreased velocity and track length in Ogg1 +/− progeny, but not in +/+ or −/− littermates, although the latter exhibited a similar non-significant trend. (Panel B). As with social interaction, EtOH vs. saline increased interaction-induced ultrasonic vocalizations in Ogg1 +/− progeny, but not in +/+ or −/− littermates. The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s test. Full article ">Figure 9
Postulated epigenetic role of oxidative DNA damage initiated by reactive oxygen species (ROS) in neurodevelopmental disorders caused by physiological or ethanol-enhanced levels of ROS formation. ROS, which are naturally produced in the body and essential for normal development and life, can oxidatively damage DNA, resulting in multiple types of lesions. The most prevalent DNA lesion, 8-oxoguanine (8-oxoG), is developmentally pathogenic, and is repaired by oxoguanine glycosylase 1 (OGG1). Heterozygous (+/−) and particularly homozygous (−/−) Ogg1 knockout progeny have decreased DNA repair activity compared to their wild-type (+/+) littermates, and can accumulate oxidative DNA damage due to even physiological levels of ROS formation, leading to epigenetic changes in DNA methylation and modifications to histone proteins, exemplified by histone methylation and acetylation. These epigenetic changes can alter the expression of developmentally important genes, leading to neurodevelopmental disorders. Prenatal exposure to ROS-enhancing drugs like alcohol (ethanol, EtOH) can further enhance both the complexity and magnitude of epigenetic changes initiated by oxidative DNA damage, increasing the spectrum and severity of neurodevelopmental disorders. Full article ">

19 pages, 1623 KiB

Open AccessReview

Astrocytes Are a Key Target for Neurotropic Viral Infection

by Maja Potokar, Robert Zorec and Jernej Jorgačevski

Cells 2023, 12(18), 2307; https://doi.org/10.3390/cells12182307 - 19 Sep 2023

Cited by 2 | Viewed by 1560

Abstract

Astrocytes are increasingly recognized as important viral host cells in the central nervous system. These cells can produce relatively high quantities of new virions. In part, this can be attributed to the characteristics of astrocyte metabolism and its abundant and dynamic cytoskeleton network. [...] Read more.

Astrocytes are increasingly recognized as important viral host cells in the central nervous system. These cells can produce relatively high quantities of new virions. In part, this can be attributed to the characteristics of astrocyte metabolism and its abundant and dynamic cytoskeleton network. Astrocytes are anatomically localized adjacent to interfaces between blood capillaries and brain parenchyma and between blood capillaries and brain ventricles. Moreover, astrocytes exhibit a larger membrane interface with the extracellular space than neurons. These properties, together with the expression of various and numerous viral entry receptors, a relatively high rate of endocytosis, and morphological plasticity of intracellular organelles, render astrocytes important target cells in neurotropic infections. In this review, we describe factors that mediate the high susceptibility of astrocytes to viral infection and replication, including the anatomic localization of astrocytes, morphology, expression of viral entry receptors, and various forms of autophagy. Full article

(This article belongs to the Special Issue Astrocytes in CNS Disorders)

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Figure 1

Figure 1
Entry of viruses through the epithelial barrier, the lymphatic spread of viruses and neuroinvasion through the blood–brain barrier. (A) After entry through the epithelium, viruses may continue with subepithelial invasion by exploiting the network of lymphatics to enter lymph nodes. Viruses enter porous lymphatic capillaries and/or efferent lymphatic vessels to access the venous system. Circulating blood is the fastest and most effective way of viral dissemination to different tissues, including the central nervous system (CNS). (B) Entry into the delicate CNS parenchyma is restricted by an elaborate barrier network, most notably by the blood–brain barrier (BBB). Certain viruses can pass the BBB by the transcellular route by transferring through the lining of microvascular endothelial cells, where they may or may not replicate. In addition, viruses can pass the BBB by paracellular traversal between adjacent endothelial cells, with or without disruption of tight junctions. Viruses can also penetrate the BBB by transmigration within infected haematopoietic cells, known as the “Trojan horse” mechanism. Full article ">Figure 2
Viral entry receptors in astrocytes. Diverse plasma membrane receptors can mediate virus attachment to and entry into astrocytes. These include neuropilin-1 (NRP1), angiotensin-converting enzyme 2 (ACE2), type II transmembrane serine protease (TMPRSS2), TAM family receptors (TYRO-3 and AXL), dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), T-cell Ig and mucin domain 1 (TIM-1), heparan sulphate proteoglycans (HSPGs) comprising syndecans and glypicans, αvβ3 integrin, dipeptidylpeptidase 4 (DPP4), and cluster of differentiation 147 (CD147). Full article ">Figure 3
Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV), but not mosquito-only flavivirus (MOF), increase autophagy in human astrocytes. (a) Representative fluorescence micrographs documenting mock-infected human astrocytes (Control) and astrocytes after exposure to selected flaviviruses (TBEV, WNV, and MOF) for 48 h. Cells were expressing mRFP-EGFP-LC3, where LC3, a marker of autophagic compartments, is tandem fluorescent-tagged with mRFP (red fluorescence) and EGFP (green fluorescence). The pH of autophagosomes (AP) is close to neutral, which facilitates the fluorescence of both fluorophores, resulting in yellow objects. Fusion of AP with lysosomes yields autolysosomes (AL), i.e., organelles with acidic pH, where the EGFP fluorescence is quenched, resulting in red-only objects. Selected rectangular areas within the cells, enlarged at the corners (bottom, right), show superimposed images of mRFP and EGFP fluorescence. Arrows indicate AL. Adjacent, smaller panels display mRFP and EGFP fluorescence and co-localization masks (Col.mask) (co-localized objects correspond to AP) of the enlarged images. The white outlines in the large panels show the cell shape. (b) The total number of autophagic compartments (i) and the ratio of AL to AP (which is a measure of autophagic degradation activity) (ii) in mock-infected cells and after infection with TBEV, WNV, and MOF at 12, 24, and 48 h post-infection (hpi). Infection with TBEV increases the total number of autophagic compartments at all three time points tested, compared with mock-infected cells (i.e., controls at 48 hpi, which were confirmed to be comparable to controls at 12 and 24 hpi) (* p < 0.05, one-way ANOVA followed by Dunn’s test), but does not affect the AL:AP ratio (p > 0.05, one-way ANOVA). WNV infection induces an increase in the total number of autophagic compartments at 48 hpi (* p < 0.05, one-way ANOVA followed by Dunn’s test) and does not affect the ratio AL:AP (p > 0.05, one-way ANOVA). MOF infection does not affect the number of autophagic compartments or the AL: AP ratio compared with mock-infected cells at any time point tested (p > 0.05, one-way ANOVA). (c) Diameter of AP and AL in mock-infected cells and 48 hpi with selected flaviviruses. Infection with TBEV, WNV and MOF does not affect the size of the autophagic compartment compared with mock-infected cells (p > 0.05, one-way ANOVA). ALs are larger than APs in all experimental conditions (*** p < 0.001, Mann-Whitney U test). Full lines in the boxplots represent median values, and the dotted lines correspond to average values. The numbers below the boxplots are the number of cells (b) or compartments (c) analysed for each condition. Cells were infected with TBEV and MOF at an MOI of 0.1 and with WNV at an MOI of 1. Figure and figure legend reproduced from Tavcar Verdev et al. [<a href="#B46-cells-12-02307" class="html-bibr">46</a>] with permission, licensed under a Creative Commons Attribution 4.0 International License (<a href="http://creativecommons.org/licenses/by/4.0" target="_blank">http://creativecommons.org/licenses/by/4.0</a> (accessed on 1 August 2023)). Full article ">

23 pages, 2332 KiB

Open AccessArticle

Impact of Elevated Brain IL-6 in Transgenic Mice on the Behavioral and Neurochemical Consequences of Chronic Alcohol Exposure

by Donna L. Gruol, Delilah Calderon, Salvador Huitron-Resendiz, Chelsea Cates-Gatto and Amanda J. Roberts

Cells 2023, 12(18), 2306; https://doi.org/10.3390/cells12182306 - 19 Sep 2023

Cited by 1 | Viewed by 1117

Abstract

Alcohol consumption activates the neuroimmune system of the brain, a system in which brain astrocytes and microglia play dominant roles. These glial cells normally produce low levels of neuroimmune factors, which are important signaling factors and regulators of brain function. Alcohol activation of [...] Read more.

Alcohol consumption activates the neuroimmune system of the brain, a system in which brain astrocytes and microglia play dominant roles. These glial cells normally produce low levels of neuroimmune factors, which are important signaling factors and regulators of brain function. Alcohol activation of the neuroimmune system is known to dysregulate the production of neuroimmune factors, such as the cytokine IL-6, thereby changing the neuroimmune status of the brain, which could impact the actions of alcohol. The consequences of neuroimmune–alcohol interactions are not fully known. In the current studies we investigated this issue in transgenic (TG) mice with altered neuroimmune status relative to IL-6. The TG mice express elevated levels of astrocyte-produced IL-6, a condition known to occur with alcohol exposure. Standard behavioral tests of alcohol drinking and negative affect/emotionality were carried out in homozygous and heterozygous TG mice and control mice to assess the impact of neuroimmune status on the actions of chronic intermittent alcohol (ethanol) (CIE) exposure on these behaviors. The expressions of signal transduction and synaptic proteins were also assessed by Western blot to identify the impact of alcohol–neuroimmune interactions on brain neurochemistry. The results from these studies show that neuroimmune status with respect to IL-6 significantly impacts the effects of alcohol on multiple levels. Full article

(This article belongs to the Special Issue Alcohol and Neuroimmunology)

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Figure 1
Diagram of alcohol exposure protocol. Full article ">Figure 2
Effects of CIE on 2BC alcohol drinking. (A,B). Graphs showing effect of cycle on average weekly alcohol intake (mean ± SEM) in male (A) and female (B) WT, +/−TG, and +/+TG mice in 2BC and 2BC-CIE groups during baseline and four cycles of exposure to air (air 1–4) or CIE (CIE 1–4). * = significant increase in drinking relative to baseline drinking (blue, WT; green, +/−TG; red, +/+TG; data from +/+TG mice were not included in the statistical analyses but are shown for reference). Numbers in parentheses are number of animals studied. In this and all other figures, a statistically significant difference is defined as p < 0.05. Full article ">Figure 3
Graphs showing effects of 2BC and 2BC-CIE on behavioral tests for negative affect/emotionality during the abstinence period. CIE actions are reflected in differences between effects of 2BC and 2BC-CIE. Behavioral tests include light/dark transfer (measures of time in light chamber) (A1), and total number of transitions between dark and light chamber (A2), open field test (distance traveled) (B) and immobility time (forced swim test) (C). In this and other figures, bar graphs show mean ± SEM values and individual data points. Numbers within bars or under bars are number of animals tested. Bars underneath symbols for significance indicate 2BC plus 2BC-CIE data are combined (i.e., no treatment effect). # = significantly different from WT, & = significantly different from +/−TG, @ = overall 2BC-CIE significantly lower than 2BC. Full article ">Figure 4
Effects of 2BC and 2BC-CIE on neuroimmune-related proteins. (A,B). Mean (± SEM) values for levels of IL-6 (A) and TNF-alpha (B) measured by ELISA in hippocampi from 2BC- and 2BC-CIE-treated WT, +/−TG, and +/+TG mice. (C,D). Mean (± SEM) levels of IL-6 signal transduction partners STAT3 (C1), pSTAT3 (C2), p42MAPK (D1), and pp42MAPK (D2), measured by Western blot analysis in hippocampi from 2BC- and 2BC-CIE-treated WT, +/−TG, and +/+TG mice. Representative Western blots are inserted above the corresponding data bars. Β = beta-actin (42 kD). The source Western blots are included in the <a href="#app1-cells-12-02306" class="html-app">Supplemental section</a> (<a href="#app1-cells-12-02306" class="html-app">Figure S1</a>). # = significantly different from WT. * = significant difference between 2BC and 2BC-CIE of the same genotype. Note, on these and other graphs showing data points, some data points may be masked by overlapping values. Numbers within or above the bars show the number of animals in the sample. Full article ">Figure 5
Effects of 2BC and 2BC-CIE on synaptic proteins. (A–G). Mean (± SEM) values for levels of GABAAR alpha-1 (A1) and alpha-5 (A2), VGAT (B), GAD65 (C1) and GAD 67 (C2), gephyrin (D), GluR1 naïve, NR1 (F), and PSD-95 (G) determined by Western blot in WT, +/−TG, and +/+TG mice exposed to 2BC/abstinence or 2BC-CIE-abstinence protocols. The source Western blots are included in the <a href="#app1-cells-12-02306" class="html-app">Supplemental section</a> (<a href="#app1-cells-12-02306" class="html-app">Figure S2</a>). # = significantly different from WT, & = significantly different from +/−TG, @ = overall 2BC-CIE significantly lower than 2BC. Full article ">

21 pages, 21543 KiB

Open AccessArticle

scRNA-seq Reveals the Mechanism of Fatty Acid Desaturase 2 Mutation to Repress Leaf Growth in Peanut (Arachis hypogaea L.)

by Puxuan Du, Quanqing Deng, Wenyi Wang, Vanika Garg, Qing Lu, Lu Huang, Runfeng Wang, Haifen Li, Dongxin Huai, Xiaoping Chen, Rajeev K. Varshney, Yanbin Hong and Hao Liu

Cells 2023, 12(18), 2305; https://doi.org/10.3390/cells12182305 - 19 Sep 2023

Cited by 1 | Viewed by 1713

Abstract

Fatty Acid Desaturase 2 (FAD2) controls the conversion of oleic acids into linoleic acids. Mutations in FAD2 not only increase the high-oleic content, but also repress the leaf growth. However, the mechanism by which FAD2 regulates the growth pathway has not [...] Read more.

Fatty Acid Desaturase 2 (FAD2) controls the conversion of oleic acids into linoleic acids. Mutations in FAD2 not only increase the high-oleic content, but also repress the leaf growth. However, the mechanism by which FAD2 regulates the growth pathway has not been elucidated in peanut leaves with single-cell resolution. In this study, we isolated fad2 mutant leaf protoplast cells to perform single-cell RNA sequencing. Approximately 24,988 individual cells with 10,249 expressed genes were classified into five major cell types. A comparative analysis of 3495 differentially expressed genes (DEGs) in distinct cell types demonstrated that fad2 inhibited the expression of the cytokinin synthesis gene LOG in vascular cells, thereby repressing leaf growth. Further, pseudo-time trajectory analysis indicated that fad2 repressed leaf cell differentiation, and cell-cycle evidence displayed that fad2 perturbed the normal cell cycle to induce the majority of cells to drop into the S phase. Additionally, important transcription factors were filtered from the DEG profiles that connected the network involved in high-oleic acid accumulation (WRKY6), activated the hormone pathway (WRKY23, ERF109), and potentially regulated leaf growth (ERF6, MYB102, WRKY30). Collectively, our study describes different gene atlases in high-oleic and normal peanut seedling leaves, providing novel biological insights to elucidate the molecular mechanism of the high-oleic peanut-associated agronomic trait at the single-cell level. Full article

(This article belongs to the Collection Feature Papers in Plant, Algae and Fungi Cell Biology)

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14 pages, 1015 KiB

Open AccessReview

Riboflavin and Its Derivates as Potential Photosensitizers in the Photodynamic Treatment of Skin Cancers

by Małgorzata Insińska-Rak, Marek Sikorski and Agnieszka Wolnicka-Glubisz

Cells 2023, 12(18), 2304; https://doi.org/10.3390/cells12182304 - 19 Sep 2023

Cited by 4 | Viewed by 2340

Abstract

Riboflavin, a water-soluble vitamin B2, possesses unique biological and physicochemical properties. Its photosensitizing properties make it suitable for various biological applications, such as pathogen inactivation and photodynamic therapy. However, the effectiveness of riboflavin as a photosensitizer is hindered by its degradation upon exposure [...] Read more.

Riboflavin, a water-soluble vitamin B2, possesses unique biological and physicochemical properties. Its photosensitizing properties make it suitable for various biological applications, such as pathogen inactivation and photodynamic therapy. However, the effectiveness of riboflavin as a photosensitizer is hindered by its degradation upon exposure to light. The review aims to highlight the significance of riboflavin and its derivatives as potential photosensitizers for use in photodynamic therapy. Additionally, a concise overview of photodynamic therapy and utilization of blue light in dermatology is provided, as well as the photochemistry and photobiophysics of riboflavin and its derivatives. Particular emphasis is given to the latest findings on the use of acetylated 3-methyltetraacetyl-riboflavin derivative (3MeTARF) in photodynamic therapy. Full article

(This article belongs to the Special Issue Skin Cancer: From Cellular and Molecular Mechanisms to Therapeutic Opportunities)

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19 pages, 1217 KiB

Open AccessReview

Multiple Roles of the RUNX Gene Family in Hepatocellular Carcinoma and Their Potential Clinical Implications

by Milena Krajnović, Bojana Kožik, Ana Božović and Snežana Jovanović-Ćupić

Cells 2023, 12(18), 2303; https://doi.org/10.3390/cells12182303 - 19 Sep 2023

Cited by 1 | Viewed by 1291

Abstract

Hepatocellular carcinoma (HCC) is one of the most frequent cancers in humans, characterised by a high resistance to conventional chemotherapy, late diagnosis, and a high mortality rate. It is necessary to elucidate the molecular mechanisms involved in hepatocarcinogenesis to improve diagnosis and treatment [...] Read more.

Hepatocellular carcinoma (HCC) is one of the most frequent cancers in humans, characterised by a high resistance to conventional chemotherapy, late diagnosis, and a high mortality rate. It is necessary to elucidate the molecular mechanisms involved in hepatocarcinogenesis to improve diagnosis and treatment outcomes. The Runt-related (RUNX) family of transcription factors (RUNX1, RUNX2, and RUNX3) participates in cardinal biological processes and plays paramount roles in the pathogenesis of numerous human malignancies. Their role is often controversial as they can act as oncogenes or tumour suppressors and depends on cellular context. Evidence shows that deregulated RUNX genes may be involved in hepatocarcinogenesis from the earliest to the latest stages. In this review, we summarise the topical evidence on the roles of RUNX gene family members in HCC. We discuss their possible application as non-invasive molecular markers for early diagnosis, prognosis, and development of novel treatment strategies in HCC patients. Full article

(This article belongs to the Special Issue Emerging Therapeutic Approaches for Chronic Liver Diseases)

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19 pages, 13972 KiB

Open AccessArticle

Pancreatic Islet Viability Assessment Using Hyperspectral Imaging of Autofluorescence

by Jared M. Campbell, Stacey N. Walters, Abbas Habibalahi, Saabah B. Mahbub, Ayad G. Anwer, Shannon Handley, Shane T. Grey and Ewa M. Goldys

Cells 2023, 12(18), 2302; https://doi.org/10.3390/cells12182302 - 19 Sep 2023

Cited by 2 | Viewed by 1492

Abstract

Islets prepared for transplantation into type 1 diabetes patients are exposed to compromising intrinsic and extrinsic factors that contribute to early graft failure, necessitating repeated islet infusions for clinical insulin independence. A lack of reliable pre-transplant measures to determine islet viability severely limits [...] Read more.

Islets prepared for transplantation into type 1 diabetes patients are exposed to compromising intrinsic and extrinsic factors that contribute to early graft failure, necessitating repeated islet infusions for clinical insulin independence. A lack of reliable pre-transplant measures to determine islet viability severely limits the success of islet transplantation and will limit future beta cell replacement strategies. We applied hyperspectral fluorescent microscopy to determine whether we could non-invasively detect islet damage induced by oxidative stress, hypoxia, cytokine injury, and warm ischaemia, and so predict transplant outcomes in a mouse model. In assessing islet spectral signals for NAD(P)H, flavins, collagen-I, and cytochrome-C in intact islets, we distinguished islets compromised by oxidative stress (ROS) (AUC = 1.00), hypoxia (AUC = 0.69), cytokine exposure (AUC = 0.94), and warm ischaemia (AUC = 0.94) compared to islets harvested from pristine anaesthetised heart-beating mouse donors. Significantly, with unsupervised assessment we defined an autofluorescent score for ischaemic islets that accurately predicted the restoration of glucose control in diabetic recipients following transplantation. Similar results were obtained for islet single cell suspensions, suggesting translational utility in the context of emerging beta cell replacement strategies. These data show that the pre-transplant hyperspectral imaging of islet autofluorescence has promise for predicting islet viability and transplant success. Full article

(This article belongs to the Special Issue Islet Transplantation)

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Cells, Volume 12, Issue 18 (September-2 2023) – 126 articles

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