Cells

19 pages, 9482 KiB

Open AccessArticle

Fermented Soybean Paste Attenuates Biogenic Amine-Induced Liver Damage in Obese Mice

by Ju-Hwan Yang, Eun-Hye Byeon, Dawon Kang, Seong-Geun Hong, Jinsung Yang, Deok-Ryong Kim, Seung-Pil Yun, Sang-Won Park, Hyun-Joon Kim, Jae-Won Huh, So-Yong Kim, Young-Wan Kim and Dong-Kun Lee

Cells 2023, 12(5), 822; https://doi.org/10.3390/cells12050822 - 6 Mar 2023

Cited by 10 | Viewed by 2978

Abstract

Biogenic amines are cellular components produced by the decarboxylation of amino acids; however, excessive biogenic amine production causes adverse health problems. The relationship between hepatic damage and biogenic amine levels in nonalcoholic fatty liver disease (NAFLD) remains unclear. In this study, mice were [...] Read more.

Biogenic amines are cellular components produced by the decarboxylation of amino acids; however, excessive biogenic amine production causes adverse health problems. The relationship between hepatic damage and biogenic amine levels in nonalcoholic fatty liver disease (NAFLD) remains unclear. In this study, mice were fed a high-fat diet (HFD) for 10 weeks to induce obesity, presenting early-stage of NAFLD. We administered histamine (20 mg/kg) + tyramine (100 mg/kg) via oral gavage for 6 days to mice with HFD-induced early-stage NAFLD. The results showed that combined histamine and tyramine administration increased cleaved PARP-1 and IL-1β in the liver, as well as MAO-A, total MAO, CRP, and AST/ALT levels. In contrast, the survival rate decreased in HFD-induced NAFLD mice. Treatment with manufactured or traditional fermented soybean paste decreased biogenically elevated hepatic cleaved PARP-1 and IL-1β expression and blood plasma MAO-A, CRP, and AST/ALT levels in HFD-induced NAFLD mice. Additionally, the biogenic amine-induced reduction in survival rate was alleviated by fermented soybean paste in HFD-induced NAFLD mice. These results show that biogenic amine-induced liver damage can be exacerbated by obesity and may adversely affect life conservation. However, fermented soybean paste can reduce biogenic amine-induced liver damage in NAFLD mice. These results suggest a beneficial effect of fermented soybean paste on biogenic amine-induced liver damage and provide a new research perspective on the relationship between biogenic amines and obesity. Full article

(This article belongs to the Special Issue Liver Diseases: From Molecular Mechanism to Therapeutic Aspect)

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Figure 1
Combined effect of biogenic amines administered by repeated oral gavage in mice fed a normal chow diet (NCD). (A) Timeline of repeated oral gavage administrations of biogenic amines. Changes in body weight (B), food intake (C), and water intake (D) after repeated oral gavage administrations of biogenic amines. (E) Changes in survival rate after repeated oral gavage administrations of biogenic amines by concentration. (F) Changes in plasma C-reactive protein (CRP) levels after repeated oral gavage administrations of biogenic amines by concentration. (G) Changes in survival rate following repeated oral gavage administration of histamine and tyramine. (H) Changes in plasma CRP levels after repeated oral gavage administrations of either histamine or tyramine. * p < 0.05, ** p < 0.01 vs. carboxymethylcellulose (CMC). Data are shown as mean ± SEM. BA, biogenic amine; His, histamine; Tyr, tyramine. Full article ">Figure 2
Effect of administration of single biogenic amines or their mixture on liver damage markers in mice fed an NCD. (A) Changes in IL-1β expression levels after repeated oral gavage administrations of histamine, tyramine, and combined biogenic amines. (B) Osteopontin expression levels changed after oral gavage administrations of histamine, tyramine, and combined biogenic amines. * p < 0.05 vs. CMC. Data are shown as mean ± SEM. IL-1β, interleukin-1 beta. Full article ">Figure 3
Establishing an HFD-induced NAFLD mice model by evaluating leptin resistance. (A) Intraperitoneal glucose tolerance test to evaluate leptin resistance after 10 weeks of feeding mice either an NCD or HFD. (B) The area under the curve corresponding to <a href="#cells-12-00822-f003" class="html-fig">Figure 3</a>A. (C) Fasting plasma glucose levels. (D) Plasma leptin levels. *** p < 0.001, **** p < 0.0001 vs. CMC. Data are shown as mean ± SEM. Full article ">Figure 4
Effect of biogenic amines on liver damage in mice fed an HFD. (A) Changes in survival rate following single or repeated oral gavage administrations of biogenic amines after feeding an HFD. (B) Changes in IL-1β following single or repeated oral gavage administrations of biogenic amines after feeding an HFD. (C) Comparison of changes in survival rate between NCD- and HFD-fed groups after repeated oral gavage administrations of biogenic amines. (D) Comparison of changes in blood CRP levels between NCD-fed, HFD-fed, and HFD-fed + biogenic amines administration groups. Changes in liver MAO-A (E), MAO-B (F), total MAO (G) levels, and total bile acid levels in the blood (H). * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. NCD + CMC. Data are shown as mean ± SEM. MAO, monoamine oxidase. Full article ">Figure 5
Reduction in biogenic amine-induced toxic effects by fermented soybean paste in obesity. (A) Effect of TSBP on survival rate changes caused by biogenic amine administrations and HFD-induced obesity. (B) Effect of MSBP on survival rate changes caused by biogenic amine administrations and HFD-induced obesity. (C) Changes in blood AST and (D) ALT levels caused by biogenic amines and fermented soybean paste in HFD-induced obesity. * p < 0.05, *** p < 0.001 vs. HFD + CMC; # p < 0.05, ## p < 0.01 vs. HFD + histamine 20 mg/kg + tyramine 100 mg/kg. Data are shown as mean ± SEM. TSBP, traditionally made fermented soybean paste; MSBP, manufactured fermented soybean paste. Full article ">Figure 6
Reduction in biogenic amine-induced hepatic damage by fermented soybean paste in developmental NAFLD. (A) Changes in IL-1β expression levels by biogenic amines and fermented soybean paste in HFD-induced NAFLD liver tissue. (B) Changes in cleaved PARP-1 expression levels caused by biogenic amines and fermented soybean paste in HFD-induced NAFLD liver tissue. (C) Changes in blood CRP levels caused by biogenic amines and fermented soybean paste in HFD-induced NAFLD. Changes in MAO-A (D), MAO-B (E), and total MAO (F) levels caused by biogenic amines and fermented soybean paste in HFD-induced NAFLD liver tissue. * p < 0.05, **** p < 0.0001, vs. NCD + CMC; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001, vs. HFD + histamine 20 mg/kg + tyramine 100 mg/kg. Data are shown as mean ± SEM. Full article ">Figure 7
Putative role of fermented soybean paste extract in biogenic amine-induced hepatic damage in NAFLD. A large amount of combined biogenic amine ingestion may exacerbate hepatic function by increasing IL-1β expression, although activation of MAO degrades biogenic amines in NAFLD. In addition, biogenic amines enhance the cleavage of PARP-1, which may be upregulated by fatty liver disease. However, fermented soybean paste extracts are probably involved in the degradation of biogenic amines, reducing biogenic amine-induced hepatic damage in NAFLD. Full article ">Figure A1
The full-length and whole Western blot images corresponding to <a href="#cells-12-00822-f002" class="html-fig">Figure 2</a>. (A) IL-1β. (B) β-actin for IL-1β. (C) Osteopontin. (D) β-actin for osteopontin. The arrows indicate the appropriate molecular weight of each protein. Full article ">Figure A2
The full-length Western blot images corresponding to <a href="#cells-12-00822-f004" class="html-fig">Figure 4</a>B. (A) IL-1β. (B) β-actin for IL-1β. The arrows indicate the appropriate molecular weight of each protein. Full article ">Figure A3
The full-length Western blot images corresponding to <a href="#cells-12-00822-f006" class="html-fig">Figure 6</a>A. (A) IL-1β. (B) β-actin for IL-1β. The arrows indicate the appropriate molecular weight of each protein. Full article ">Figure A4
The full-length Western blot images corresponding to <a href="#cells-12-00822-f006" class="html-fig">Figure 6</a>B. (A) PARP-1. (B) β-actin for PARP-1. The arrows indicate the appropriate molecular weight of each protein. Full article ">

17 pages, 2019 KiB

Open AccessFeature PaperArticle

Electrophysiological Activity of Primary Cortical Neuron-Glia Mixed Cultures

by Noah Goshi, Hyehyun Kim, Gregory Girardi, Alexander Gardner and Erkin Seker

Cells 2023, 12(5), 821; https://doi.org/10.3390/cells12050821 - 6 Mar 2023

Cited by 7 | Viewed by 4502

Abstract

Neuroinflammation plays a central role in many neurological disorders, ranging from traumatic brain injuries to neurodegeneration. Electrophysiological activity is an essential measure of neuronal function, which is influenced by neuroinflammation. In order to study neuroinflammation and its electrophysiological fingerprints, there is a need [...] Read more.

Neuroinflammation plays a central role in many neurological disorders, ranging from traumatic brain injuries to neurodegeneration. Electrophysiological activity is an essential measure of neuronal function, which is influenced by neuroinflammation. In order to study neuroinflammation and its electrophysiological fingerprints, there is a need for in vitro models that accurately capture the in vivo phenomena. In this study, we employed a new tri-culture of primary rat neurons, astrocytes, and microglia in combination with extracellular electrophysiological recording techniques using multiple electrode arrays (MEAs) to determine the effect of microglia on neural function and the response to neuroinflammatory stimuli. Specifically, we established the tri-culture and its corresponding neuron-astrocyte co-culture (lacking microglia) counterpart on custom MEAs and monitored their electrophysiological activity for 21 days to assess culture maturation and network formation. As a complementary assessment, we quantified synaptic puncta and averaged spike waveforms to determine the difference in excitatory to inhibitory neuron ratio (E/I ratio) of the neurons. The results demonstrate that the microglia in the tri-culture do not disrupt neural network formation and stability and may be a better representation of the in vivo rat cortex due to its more similar E/I ratio as compared to more traditional isolated neuron and neuron-astrocyte co-cultures. In addition, only the tri-culture displayed a significant decrease in both the number of active channels and spike frequency following pro-inflammatory lipopolysaccharide exposure, highlighting the critical role of microglia in capturing electrophysiological manifestations of a representative neuroinflammatory insult. We expect the demonstrated technology to assist in studying various brain disease mechanisms. Full article

(This article belongs to the Topic Applications of Biomedical Technology and Molecular Biological Approach in Brain Diseases)

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(a) Bright-field and (b) epifluorescence images of the tri-culture at DIV 21 on the well MEA. The cultures were immunostained for the three cell types of interest: neurons—anti-βIII-tubulin (red), astrocytes—anti-GFAP (green), microglia—anti-Iba1 (orange), and the general nuclear stain DAPI (blue). (Scale bar = 100 µm). Representative extracellular recordings taken at DIV 7 and DIV 21 from (c) co-cultures and (d) tri-cultures. Comparisons of the (e) percentage of active channels, (f) spike frequency, and (g) synchrony between co-cultures (red) and tri-cultures (blue). The solid lines show the fitted linear mixed effects model (treating individual cultures as a random effect) with a b-spline basis. The shaded regions are the 95% confidence interval. An asterisk above an individual box indicates a significant difference of the estimated marginal means of the fitted curves between that timepoint and DIV 7 of the same culture type, while the bars indicate the significance between the co- and tri-culture at that timepoint (n = 8, from three independent dissections). * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no significant difference. Scale bar = 100 µm. Full article ">Figure 2
(a) Bright-field and (b) epifluorescence images of the tri-culture at DIV 21 in the platform MEA with microfluidic encapsulation. The cultures were immunostained for the three cell types of interest: neurons—anti-βIII-tubulin (red), astrocytes—anti-GFAP (green), microglia—anti-Iba1 (orange), and the general nuclear stain DAPI (blue). (Scale bar = 100 µm). Comparisons of the (c) percent active channels, (d) spike frequency, and (e) synchrony between co-cultures (red) and tri-cultures (blue) cultured in a two-chambered microfluidic device. The solid lines show the fitted linear mixed effects model (treating individual cultures as a random effect) with a b-spline basis. The shaded regions are the 95% confidence interval. An asterisk above an individual box indicates a significant difference of the estimated marginal means of the fitted curves between that timepoint and DIV 7 of the same culture type, while the bars indicate the significance between the co- and tri-culture at that timepoint (n = 5, from two independent dissections). * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no significant difference. Full article ">Figure 3
(a) Representative fluorescence images of co- and tri-cultures at DIV 21. The cultures are stained for the excitatory pre-synaptic marker VGlut1 (Green), post-synaptic marker PSD-95 (red). The above image also shows the co-localization with MAP-2 (white). (Scale bar = 10 µm). Comparison of the density of (b) PSD-95 puncta, (c) VGlut1 puncta, and (d) co-localized puncta. In all, three cases, a two-way ANOVA found no interaction between culture type and time in culture. Therefore, the asterisk indicates the significance of the main effect between timepoints (n = 4, from two independent dissections). * p < 0.05, ** p < 0.01, ns indicates no significant difference. Trendlines are visual guides only. Full article ">Figure 4
Comparison of the E/I ratio in tri- and co-cultures at DIV 21. (a) Representative waveforms of narrow-spiking and broad-spiking neurons recorded at DIV 21. The line represents the average spike waveform, while the shaded region is one standard deviation. (b) Percent excitatory neurons in the tri- and co-cultures at DIV 21 (n = 8 MEAs (249 total units) from three independent dissections). * p < 0.05. Full article ">Figure 5
Comparing the change in (a) percent active channels and (b) spike frequency following exposure to 5 µg/mL LPS between co-cultures (red) and tri-cultures (blue). The lines show the fitted liner mixed effects model (treating individual cultures as a random effect) with a b-spline basis. Asterisks indicate a significant difference in the estimated marginal means of the fitted curves between control and LPS treated tri-cultures at that timepoint (n = 4, from two independent dissections). * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">

14 pages, 2561 KiB

Open AccessArticle

Increased Radiation Sensitivity in Patients with Phelan-McDermid Syndrome

by Sarah Jesse, Lukas Kuhlmann, Laura S. Hildebrand, Henriette Magelssen, Martina Schmaus, Beate Timmermann, Stephanie Andres, Rainer Fietkau and Luitpold V. Distel

Cells 2023, 12(5), 820; https://doi.org/10.3390/cells12050820 - 6 Mar 2023

Viewed by 1953

Abstract

Phelan-McDermid syndrome is an inherited global developmental disorder commonly associated with autism spectrum disorder. Due to a significantly increased radiosensitivity, measured before the start of radiotherapy of a rhabdoid tumor in a child with Phelan-McDermid syndrome, the question arose whether other patients with [...] Read more.

Phelan-McDermid syndrome is an inherited global developmental disorder commonly associated with autism spectrum disorder. Due to a significantly increased radiosensitivity, measured before the start of radiotherapy of a rhabdoid tumor in a child with Phelan-McDermid syndrome, the question arose whether other patients with this syndrome also have increased radiosensitivity. For this purpose, the radiation sensitivity of blood lymphocytes after irradiation with 2Gray was examined using the G0 three-color fluorescence in situ hybridization assay in a cohort of 20 patients with Phelan-McDermid syndrome from blood samples. The results were compared to healthy volunteers, breast cancer patients and rectal cancer patients. Independent of age and gender, all but two patients with Phelan-McDermid syndrome showed significantly increased radiosensitivity, with an average of 0.653 breaks per metaphase. These results correlated neither with the individual genetic findings nor with the individual clinical course, nor with the respective clinical severity of the disease. In our pilot study, we saw a significantly increased radiosensitivity in lymphocytes from patients with Phelan-McDermid syndrome, so pronounced that a dose reduction would be recommended if radiotherapy had to be performed. Ultimately, the question arises as to the interpretation of these data. There does not appear to be an increased risk of tumors in these patients, since tumors are rare overall. The question, therefore, arose as to whether our results could possibly be the basis for processes, such as aging/preaging, or, in this context, neurodegeneration. There are no data on this so far, but this issue should be pursued in further fundamentally based studies in order to better understand the pathophysiology of the syndrome. Full article

(This article belongs to the Special Issue Advances in Cancer Genomics)

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Graphical abstract

7 pages, 1695 KiB

Open AccessCommentary

Molecular Regulation of Autophagy and Asymmetric Cell Division by Cancer Stem Cell Marker CD133

by Hideki Izumi, Yasuhiko Kaneko and Akira Nakagawara

Cells 2023, 12(5), 819; https://doi.org/10.3390/cells12050819 - 6 Mar 2023

Cited by 3 | Viewed by 2511

Abstract

CD133, also called prominin-1, is widely known as a cancer stem cell marker, and its high expression correlates with a poor prognosis in many cancers. CD133 was originally discovered as a plasma membranous protein in stem/progenitor cells. It is now known that Src [...] Read more.

CD133, also called prominin-1, is widely known as a cancer stem cell marker, and its high expression correlates with a poor prognosis in many cancers. CD133 was originally discovered as a plasma membranous protein in stem/progenitor cells. It is now known that Src family kinases phosphorylate the C-terminal of CD133. However, when Src kinase activity is low, CD133 is not phosphorylated by Src and is preferentially downregulated into cells through endocytosis. Endosomal CD133 then associates with HDAC6, thereby recruiting it to the centrosome via dynein motors. Thus, CD133 protein is now known to localize to the centrosome as endosomes as well as to the plasma membrane. More recently, a mechanism to explain the involvement of CD133 endosomes in asymmetric cell division was reported. Here, we would like to introduce the relationship between autophagy regulation and asymmetric cell division mediated by CD133 endosomes. Full article

(This article belongs to the Special Issue Cell Biology: State of the Art and Perspectives in Japan)

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The signal transduction pathway of CD133. (A) CD133 is phosphorylated in its intracellular C-terminal domain by Src family tyrosine kinases, resulting in the binding and activation of the p85 subunit of phosphoinositide 3-kinase (PI-3K). Activated PI-3K then signals downstream targets such as Akt to promote cell proliferation. CD133 is also stabilized by binding to histone deacetylase 6 (HDAC6), which increases the transcriptional activity of β-catenin, promotes cell proliferation, and inhibits cell differentiation. (B) Under conditions in which Src family kinases are inactivated, unphosphorylated CD133 is transported from the plasma membrane to intracellular regions via endocytosis. After endocytosis, CD133 endosomes are transported along microtubules to centrosomes, assisted by HDAC6 and dynein motors. Endosomal CD133 localizes to the centrosome, which traps GABARAP, and inhibits the GABARAP-ULK1 interaction and initiation of autophagy. These figures were partly modified from the Journal of Japanese Biochemical Society 2022, 94, 415–418. doi:10.14952/SEIKAGAKU.2022.940415 [<a href="#B12-cells-12-00819" class="html-bibr">12</a>]. Full article ">Figure 2
Symmetry-breaking of autophagic activity during cytokinesis in CD133-positive human neuroblastoma cells. (A) Representative images of CD133 and β-catenin during cytokinesis in SK-N-DZ cells transfected with control or CD133 siRNA. CD133 is red, β-catenin is green, and DAPI (DNA) is blue. Phase contrast images are also shown. Arrows show the pericentrosomal localization of CD133. Arrowheads show the predominant nuclear localization of β-catenin. Scale bars: 10 μm. (B) Symmetry-breaking of autophagic activity during cytokinesis. During cytokinesis, CD133 endosomes asymmetrically localize to the centrosome and suppress autophagy. In autophagy-repressed daughter cells, the nuclear localization of β-catenin is enhanced and suppresses p62/SQSTM1 gene expression. On the other hand, in daughter cells in which CD133 endosomes do not localize to the centrosome, autophagy is fully activated, and β-catenin remains, localizing to the plasma membrane. In addition, the gene expression of p62/SQSTM1 is promoted. This figure (B) was partly modified from the Journal of Journal of Japanese Biochemical Society 2022, 94, 415–418. doi:10.14952/SEIKAGAKU.2022.940415 [<a href="#B12-cells-12-00819" class="html-bibr">12</a>]. Full article ">

18 pages, 2635 KiB

Open AccessArticle

Intermittent Lead Exposure Induces Behavioral and Cardiovascular Alterations Associated with Neuroinflammation

by Liana Shvachiy, Ângela Amaro-Leal, Tiago F. Outeiro, Isabel Rocha and Vera Geraldes

Cells 2023, 12(5), 818; https://doi.org/10.3390/cells12050818 - 6 Mar 2023

Cited by 10 | Viewed by 2939

Abstract

The nervous system is the primary target for lead exposure and the developing brain appears to be especially susceptible, namely the hippocampus. The mechanisms of lead neurotoxicity remain unclear, but microgliosis and astrogliosis are potential candidates, leading to an inflammatory cascade and interrupting [...] Read more.

The nervous system is the primary target for lead exposure and the developing brain appears to be especially susceptible, namely the hippocampus. The mechanisms of lead neurotoxicity remain unclear, but microgliosis and astrogliosis are potential candidates, leading to an inflammatory cascade and interrupting the pathways involved in hippocampal functions. Moreover, these molecular changes can be impactful as they may contribute to the pathophysiology of behavioral deficits and cardiovascular complications observed in chronic lead exposure. Nevertheless, the health effects and the underlying influence mechanism of intermittent lead exposure in the nervous and cardiovascular systems are still vague. Thus, we used a rat model of intermittent lead exposure to determine the systemic effects of lead and on microglial and astroglial activation in the hippocampal dentate gyrus throughout time. In this study, the intermittent group was exposed to lead from the fetal period until 12 weeks of age, no exposure (tap water) until 20 weeks, and a second exposure from 20 to 28 weeks of age. A control group (without lead exposure) matched in age and sex was used. At 12, 20 and 28 weeks of age, both groups were submitted to a physiological and behavioral evaluation. Behavioral tests were performed for the assessment of anxiety-like behavior and locomotor activity (open-field test), and memory (novel object recognition test). In the physiological evaluation, in an acute experiment, blood pressure, electrocardiogram, and heart and respiratory rates were recorded, and autonomic reflexes were evaluated. The expression of GFAP, Iba-1, NeuN and Synaptophysin in the hippocampal dentate gyrus was assessed. Intermittent lead exposure induced microgliosis and astrogliosis in the hippocampus of rats and changes in behavioral and cardiovascular function. We identified increases in GFAP and Iba1 markers together with presynaptic dysfunction in the hippocampus, concomitant with behavioral changes. This type of exposure produced significant long-term memory dysfunction. Regarding physiological changes, hypertension, tachypnea, baroreceptor reflex impairment and increased chemoreceptor reflex sensitivity were observed. In conclusion, the present study demonstrated the potential of lead intermittent exposure inducing reactive astrogliosis and microgliosis, along with a presynaptic loss that was accompanied by alterations of homeostatic mechanisms. This suggests that chronic neuroinflammation promoted by intermittent lead exposure since fetal period may increase the susceptibility to adverse events in individuals with pre-existing cardiovascular disease and/or in the elderly. Full article

(This article belongs to the Special Issue The Function of Glial Cells in the Neuroinflammatory and Neuroimmunological Responses Ⅱ)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Locomotor and exploratory behaviors and anxiety-like behavior assessed by the open-field test: (a) Total number of entries in the different regions. (b) Time spent in the central zone. (c) Total travelled distance in the apparatus. (d) Average velocity of the animals in the apparatus. Values are expressed as the mean ± SD for nIntPb = 15 and nCtrl = 18 for each evaluated time-point. Symbols denote statistically significant differences inter (Ctrl vs. IntPb—* p < 0.05; *** p < 0.001) and intra groups (Ctrl 12 weeks vs. Ctrl 20 weeks vs. 28 weeks—+ p < 0.05, ++++ p < 0.0001; IntPb 12 weeks vs. IntPb 20 weeks vs. 28 weeks—## p < 0.01,); two-way ANOVA, Tuckey’s multiple comparison test. Full article ">Figure 2
Episodic long-term memory assessed by novel object recognition test. (a) Percentage of exploration time of sample (S) and novel (N) objects by each group at the different time points. (b) Novelty recognition index is calculated by the equation presented in the <a href="#sec2-cells-12-00818" class="html-sec">Section 2</a>. Values are expressed as the mean ± SD for nIntPb = 15 and nCtrl = 18 for each evaluated time-point. Symbols denote statistically significant differences inter groups; * p < 0.05 ** p < 0.01, *** p < 0.001; paired Student’s t-test (a) and two-way ANOVA, Tukey’s multiple comparison test (b). Full article ">Figure 3
Cardiovascular and respiratory parameters evaluated along the three timepoints. (a) Systolic, diastolic, and mean blood pressure assessed from the femoral artery. (b) Heart rate values were calculated from the electrocardiogram. (c) Respiratory rate calculated from basal tracheal pressure. Values are expressed as the mean ± SD for nIntPb = 12 and nCtrl = 12 for each evaluated time-point. The symbols denote statistically significant differences inter (Ctrl vs. IntPb—* p < 0.05; **** p < 0.001) and intra groups (IntPb 12 weeks vs. IntPb 20 weeks vs. 28 weeks—# p < 0.05, ## p < 0.01, #### p < 0.0001); two-way ANOVA, Tuckey’s multiple comparison test. Full article ">Figure 4
Variation of baro- and chemoreflex expression along the three time points for all groups. (a) Baroreflex gain calculated from the variation in heart rate and blood pressure after phenylephrine stimulation. (b) Chemoreceptor reflex sensitivity calculated from the variation in respiratory rate upon lobeline stimulation. Values are expressed as the mean ± SD for nIntPb = 12 and nCtrl = 12 for each evaluated time-point. The symbols denote statistically significant differences inter groups (Ctrl vs. IntPb—* p < 0.05; ** p < 0.01; *** p < 0.001); two-way ANOVA, Tuckey’s multiple comparison test. Full article ">Figure 5
Neuroinflammation evaluated through astrocytic (GFAP) and microglial (Iba1) markers by immunohistochemistry. (a) Representative images of the GFAP (1:500)-stained astrocytes. (b) Quantification of GFAP-positive cells. (c) Representative images of the Iba1 (1:250)-stained microglia. (d) Quantification of Iba1-positive cells. Images were acquired using a confocal point scanning microscope (Zeiss LSM 880 with Airyscan) with a 20× objective. The scale bar is 50 µm or 20 µm for stained images. Values are expressed as the mean ± SD for nIntPb = 3–4 and nCtrl = 3–4 for each evaluated time-point. The symbols denote statistically significant differences inter groups (Ctrl vs. IntPb—*** p < 0.001, **** p < 0.0001); two-way ANOVA, Tuckey’s multiple comparison test. Full article ">Figure 6
Synaptic alterations (Syn) and neurodegeneration (NeuN) results evaluated by the immunohistochemistry technique. (a) Representative images of the Syn (1:500)-stained pre-synapses. (b) Histogram of the fluorescence intensity of Syn staining. (c) Representative images of the NeuN (1:500)-stained neurons. (d) Histogram of NeuN-positive cells’ quantification. Images were acquired using a confocal point scanning microscope (Zeiss LSM 880 with Airyscan) with 20× objective. Scale bar is 50 µm for stained images. Values are expressed as the mean ± SD for nIntPb = 3–4 and nCtrl = 3–4 for each evaluated time-point. The symbols denote statistically significant differences inter groups (Ctrl vs. IntPb—* p < 0.05, ** p < 0.01); two-way ANOVA, Tuckey’s multiple comparison test. Full article ">

19 pages, 2469 KiB

Open AccessArticle

Nucleolin Regulates Pulmonary Artery Smooth Muscle Cell Proliferation under Hypoxia by Modulating miRNA Expression

by Jihui Lee and Hara Kang

Cells 2023, 12(5), 817; https://doi.org/10.3390/cells12050817 - 6 Mar 2023

Cited by 5 | Viewed by 2122

Abstract

Hypoxia induces the abnormal proliferation of vascular smooth muscle cells (VSMCs), resulting in the pathogenesis of various vascular diseases. RNA-binding proteins (RBPs) are involved in a wide range of biological processes, including cell proliferation and responses to hypoxia. In this study, we observed [...] Read more.

Hypoxia induces the abnormal proliferation of vascular smooth muscle cells (VSMCs), resulting in the pathogenesis of various vascular diseases. RNA-binding proteins (RBPs) are involved in a wide range of biological processes, including cell proliferation and responses to hypoxia. In this study, we observed that the RBP nucleolin (NCL) was downregulated by histone deacetylation in response to hypoxia. We evaluated its regulatory effects on miRNA expression under hypoxic conditions in pulmonary artery smooth muscle cells (PASMCs). miRNAs associated with NCL were assessed using RNA immunoprecipitation in PASMCs and small RNA sequencing. The expression of a set of miRNAs was increased by NCL but reduced by hypoxia-induced downregulation of NCL. The downregulation of miR-24-3p and miR-409-3p promoted PASMC proliferation under hypoxic conditions. These results clearly demonstrate the significance of NCL–miRNA interactions in the regulation of hypoxia-induced PASMC proliferation and provide insight into the therapeutic value of RBPs for vascular diseases. Full article

(This article belongs to the Section Cell Proliferation and Division)

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24 pages, 2454 KiB

Open AccessReview

Pathogenesis Underlying Neurological Manifestations of Long COVID Syndrome and Potential Therapeutics

by Albert Leng, Manuj Shah, Syed Ameen Ahmad, Lavienraj Premraj, Karin Wildi, Gianluigi Li Bassi, Carlos A. Pardo, Alex Choi and Sung-Min Cho

Cells 2023, 12(5), 816; https://doi.org/10.3390/cells12050816 - 6 Mar 2023

Cited by 93 | Viewed by 14906

Abstract

The development of long-term symptoms of coronavirus disease 2019 (COVID-19) more than four weeks after primary infection, termed “long COVID” or post-acute sequela of COVID-19 (PASC), can implicate persistent neurological complications in up to one third of patients and present as fatigue, “brain [...] Read more.

The development of long-term symptoms of coronavirus disease 2019 (COVID-19) more than four weeks after primary infection, termed “long COVID” or post-acute sequela of COVID-19 (PASC), can implicate persistent neurological complications in up to one third of patients and present as fatigue, “brain fog”, headaches, cognitive impairment, dysautonomia, neuropsychiatric symptoms, anosmia, hypogeusia, and peripheral neuropathy. Pathogenic mechanisms of these symptoms of long COVID remain largely unclear; however, several hypotheses implicate both nervous system and systemic pathogenic mechanisms such as SARS-CoV2 viral persistence and neuroinvasion, abnormal immunological response, autoimmunity, coagulopathies, and endotheliopathy. Outside of the CNS, SARS-CoV-2 can invade the support and stem cells of the olfactory epithelium leading to persistent alterations to olfactory function. SARS-CoV-2 infection may induce abnormalities in innate and adaptive immunity including monocyte expansion, T-cell exhaustion, and prolonged cytokine release, which may cause neuroinflammatory responses and microglia activation, white matter abnormalities, and microvascular changes. Additionally, microvascular clot formation can occlude capillaries and endotheliopathy, due to SARS-CoV-2 protease activity and complement activation, can contribute to hypoxic neuronal injury and blood–brain barrier dysfunction, respectively. Current therapeutics target pathological mechanisms by employing antivirals, decreasing inflammation, and promoting olfactory epithelium regeneration. Thus, from laboratory evidence and clinical trials in the literature, we sought to synthesize the pathophysiological pathways underlying neurological symptoms of long COVID and potential therapeutics. Full article

(This article belongs to the Special Issue Insights into Molecular and Cellular Mechanisms of NeuroCOVID)

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Neuroinvasion and persistent viral shedding. SARS-CoV-2 employs the ACE2 receptor to invade the stem cells, perivascular cells, sustentacular cells, and Bowman’s gland cells in the olfactory epithelium; this leads to chronic thinning of filia and loss of olfactory bulb volume. Additionally, there is an association between areas of hypometabolism in the cortex, cerebellum, and brainstem with the spatial distribution of ACE2 receptors, though there is little evidence for direct neuroinvasion in these areas. Rather, it is hypothesized that these regions experience elevated levels of microglial activation, cytotoxic T lymphocyte infiltration, oxidative stress, and neurodegeneration and demyelination secondary to neuroinvasion. These mechanisms likely persist due to the chronic presence of viral shedding specifically in the gastrointestinal tract where there exists ACE2 co-regulation of DDC and involvement of the dopamine metabolic pathway. Figure was created with the BioRender software. Full article ">Figure 2
Systemic and neurological immune response. The systemic immune and inflammatory response to SARS-CoV-2 infection can continue for months after the acute recovery phase, inducing a state of persistent systemic inflammation with upregulated cytokines, such as IFN-β, IFN-λ1, IFN-γ, IL-2, IL-6, IL-17, CXCL8, CXCL9, and CXCL10. This prolonged cytokine release has been linked to activation of specific immune cell populations, such as non-classical and intermediate monocytes, as well as other cell types, such as fibroblasts and myeloid cells. From an aberrant Th2 cytokine pool, production of CCL11 is induced and leads to neuroinflammation with activation of resting microglia, which can further release increased levels of CCL11. This microglial reactivity can in turn cause reduced hippocampal neurogenesis, loss of myelinating oligodendrocytes and oligodendrocyte precursors, and ensuing subcortical white matter demyelination. These systemic and neurological mechanisms have been strongly associated with a range of cognitive impairments and neuropsychiatric symptoms. Figure was created with the BioRender software. Full article ">Figure 3
Blood–brain barrier disruption and microclot formation. SARS-CoV-2 can cause increased microclot formation through spike protein interactions with fibrinogen and serum protein A that promote fibril formation and resist fibrinolysis. Antiphospholipid antibodies are also present in long COVID and can precipitate microclot formation through IL-6, IL-8, VEGF, nitric oxide synthase, and NET release. These microclots also contain α2AP which inhibit plasmin and thus prevent the degradation of fibrin, further contributing to their fibrinolysis-resistant nature. Additionally, SARS-CoV-2 can induce BBB disruption through Mpro cleavage of NEMO in endothelial cells leading to cell death and string vessel formation. Figure was created with the BioRender software. Full article ">

20 pages, 2358 KiB

Open AccessSystematic Review

The Role of Preservation Solutions upon Saphenous Vein Endothelial Integrity and Function: Systematic Review and UK Practice Survey

by Georgia R. Layton, Shameem S. Ladak, Riccardo Abbasciano, Liam W. McQueen, Sarah J. George, Gavin J. Murphy and Mustafa Zakkar

Cells 2023, 12(5), 815; https://doi.org/10.3390/cells12050815 - 6 Mar 2023

Cited by 5 | Viewed by 2467

Abstract

The long saphenous vein is the most used conduit in cardiac surgery, but its long-term patency is limited by vein graft disease (VGD). Endothelial dysfunction is a key driver of VGD; its aetiology is multi-factorial. However emerging evidence identifies vein conduit harvest technique [...] Read more.

The long saphenous vein is the most used conduit in cardiac surgery, but its long-term patency is limited by vein graft disease (VGD). Endothelial dysfunction is a key driver of VGD; its aetiology is multi-factorial. However emerging evidence identifies vein conduit harvest technique and preservation fluids as causal in their onset and propagation. This study aims to comprehensively review published data on the relationship between preservation solutions, endothelial cell integrity and function, and VGD in human saphenous veins harvested for CABG. The review was registered with PROSPERO (CRD42022358828). Electronic searches of Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE databases were undertaken from inception until August 2022. Papers were evaluated in line with registered inclusion and exclusion criteria. Searches identified 13 prospective, controlled studies for inclusion in the analysis. All studies used saline as a control solution. Intervention solutions included heparinised whole blood and saline, DuraGraft, TiProtec, EuroCollins, University of Wisconsin (UoW), buffered, cardioplegic and Pyruvate solutions. Most studies demonstrated that normal saline appears to have negative effects on venous endothelium and the most effective preservation solutions identified in this review were TiProtec and DuraGraft. The most used preservation solutions in the UK are heparinised saline or autologous whole blood. There is substantial heterogeneity both in practice and reporting of trials evaluating vein graft preservation solutions, and the quality of existing evidence is low. There is an unmet need for high quality trials evaluating the potential for these interventions to improve long-term patency in venous bypass grafts. Full article

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3 pages, 200 KiB

Open AccessEditorial

Large-Scale Multi-Omic Approaches and Atlas-Based Profiling for Cellular, Molecular, and Functional Specificity, Heterogeneity, and Diversity in the Endocannabinoid System

by Jun Aoki and Masako Isokawa

Cells 2023, 12(5), 814; https://doi.org/10.3390/cells12050814 - 6 Mar 2023

Viewed by 1250

Abstract

The endocannabinoid system (ECS) is a widely-recognized lipid messenger system involved in many aspects of our our lives in health and diseases [...] Full article

(This article belongs to the Special Issue Large-Scale Multi-Omic Approaches and Atlas-Based Profiling for Cellular, Molecular, and Functional Specificity, Heterogeneity, and Diversity in the Endocannabinoid System)

18 pages, 3260 KiB

Open AccessArticle

17⍺-Estradiol Protects against HIV-1 Tat-Induced Endolysosome Dysfunction and Dendritic Impairments in Neurons

by Gaurav Datta, Nicole M. Miller and Xuesong Chen

Cells 2023, 12(5), 813; https://doi.org/10.3390/cells12050813 - 6 Mar 2023

Cited by 1 | Viewed by 1997

Abstract

HIV-1 Tat continues to play an important role in the development of HIV-associated neurocognitive disorders (HAND), which persist in 15–55% of people living with HIV even with virological control. In the brain, Tat is present on neurons, where Tat exerts direct neuronal damaging [...] Read more.

HIV-1 Tat continues to play an important role in the development of HIV-associated neurocognitive disorders (HAND), which persist in 15–55% of people living with HIV even with virological control. In the brain, Tat is present on neurons, where Tat exerts direct neuronal damaging effects by, at least in part, disrupting endolysosome functions, a pathological feature present in HAND. In this study, we determined the protective effects of 17α-estradiol (17αE2), the predominant form of estrogen in the brain, against Tat-induced endolysosome dysfunction and dendritic impairment in primary cultured hippocampal neurons. We demonstrated that pre-treatment with 17αE2 protected against Tat-induced endolysosome dysfunction and reduction in dendritic spine density. Estrogen receptor alpha (ERα) knockdown impairs the ability of 17αE2 to protect against Tat-induced endolysosome dysfunction and reduction in dendritic spine density. Furthermore, over-expressing an ERα mutant that fails to localize on endolysosomes impairs 17αE2′s protective effects against Tat-induced endolysosome dysfunction and reduction in dendritic spine density. Our findings demonstrate that 17αE2 protects against Tat-induced neuronal injury via a novel ERα-mediated and endolysosome-dependent pathway, and such a finding might lead to the development of novel adjunct therapeutics against HAND. Full article

(This article belongs to the Topic Molecular and Cellular Mechanisms of Diseases: HIV)

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13 pages, 2739 KiB

Open AccessCommunication

Phosphorylation of LKB1 by PDK1 Inhibits Cell Proliferation and Organ Growth by Decreased Activation of AMPK

by Sarah Borkowsky, Maximilian Gass, Azadeh Alavizargar, Johannes Hanewinkel, Ina Hallstein, Pavel Nedvetsky, Andreas Heuer and Michael P. Krahn

Cells 2023, 12(5), 812; https://doi.org/10.3390/cells12050812 - 6 Mar 2023

Cited by 5 | Viewed by 3986

Abstract

The master kinase LKB1 is a key regulator of se veral cellular processes, including cell proliferation, cell polarity and cellular metabolism. It phosphorylates and activates several downstream kinases, including AMP-dependent kinase, AMPK. Activation of AMPK by low energy supply and phosphorylation of LKB1 [...] Read more.

The master kinase LKB1 is a key regulator of se veral cellular processes, including cell proliferation, cell polarity and cellular metabolism. It phosphorylates and activates several downstream kinases, including AMP-dependent kinase, AMPK. Activation of AMPK by low energy supply and phosphorylation of LKB1 results in an inhibition of mTOR, thus decreasing energy-consuming processes, in particular translation and, thus, cell growth. LKB1 itself is a constitutively active kinase, which is regulated by posttranslational modifications and direct binding to phospholipids of the plasma membrane. Here, we report that LKB1 binds to Phosphoinositide-dependent kinase (PDK1) by a conserved binding motif. Furthermore, a PDK1-consensus motif is located within the kinase domain of LKB1 and LKB1 gets phosphorylated by PDK1 in vitro. In Drosophila, knockin of phosphorylation-deficient LKB1 results in normal survival of the flies, but an increased activation of LKB1, whereas a phospho-mimetic LKB1 variant displays decreased AMPK activation. As a functional consequence, cell growth as well as organism size is decreased in phosphorylation-deficient LKB1. Molecular dynamics simulations of PDK1-mediated LKB1 phosphorylation revealed changes in the ATP binding pocket, suggesting a conformational change upon phosphorylation, which in turn can alter LKB1’s kinase activity. Thus, phosphorylation of LKB1 by PDK1 results in an inhibition of LKB1, decreased activation of AMPK and enhanced cell growth. Full article

(This article belongs to the Topic Cell Signaling Pathways)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
LKB1 displays conserved PDK1-binding and -phosphorylation motifs and is phosphorylated by PDK1 in vitro. (A) Scheme of Drosophila LKB1 and sequence alignment of PDK1-binding and consensus-motif. DmLKB1—Drosophila melanogaster LKB1, HsLKB1—Homo sapiens LKB1, DrLKb1—Danio rerio LKB1 (zebrafish), CeLKB1—C. elegans LKB1. (B,C) S2R+ cells were co-transfected with HA-PDK1 and either wild type GFP-LKB1 and GFP-LKB1 E253A (B) or wild type GFP-LKB1 and GFP-LKB1 T253A (C) and GFP alone. GFP-proteins were immunoprecipitated and GFP(-LKB1) and HA-PDK1 (B) or Myc-PDK1 (C) were detected by immunoblotting. (D) Recombinant wild type or mutant MBP-LKB1 produced in E. coli was used in a 32P radioactive kinase assay with recombinant PDK1. CCB is colloidal coomassie blue, which was used to visualize the LKB1 input proteins. Full article ">Figure 2
GFP-LKB1 variants localize normally and do not impair fly survival but affect body size (A–C) Immunostainings of embryonic epidermis epithelial cells expressing GFP-LKB1 variants from its endogenous promoter. Discs large (Dlg) was used as marker for the lateral membrane and Bazooka (Baz) marks the apical junctions. (D) Flies with CRISPR/Cas9-mediated knockin of wild type LKB1, LKB1 T353A or LKB1 T353D display comparable survival rates (n = 100, N = 3), but different body sizes (E), n ≥ 35. Scales bars are 5 µm in (A–C). Error bars are standard error of the means. Significance was determined by one way ANOVA with Bonferroni multiple comparison test: * p < 0.05, n.s. not significant. Full article ">Figure 3
Simulation of LKB1 phosphorylation reveals changes in the ATP-binding pocket. (A) The RMSF of the protein Cα atoms averaged over the two samples is shown for human LKB1 (Blue) and human phospho-LKB1 (pLKB1, red). (B) Overlay of the two average structures for human LKB1 (blue) and human pLKB1 (orange). The average structures were obtained from 1 to 3 μs of the simulations. (C) The difference between human LKB1 and human pLKB1 average structures are shown for the two samples. For each Cα atom a shift distance is determined. The atoms with the 50% largest distances are described by a shifted value of 1. The remaining distances are ordered and linearly mapped on shift values between 0 and 1. The shift values are translated into the respective color codes. This non-linear procedure avoids that the parts, which are dramatically changed, blur the other parts. On the right-hand side, the cartoon representation of the phosphorylated protein is shown, colored based on the right-hand side figure, showing the degree of change in different parts of the protein between the two systems. (D) The distance between S60 and A194 residues over the simulation time. The protein is drawn and these two residues, along with the pT230 (corresponding to T353 in Drosophila LKB1) residue, are shown as stick representations. (E) The free volume available inside the ATP-binding pocket, obtained from 0.9 to 1.9 μs (represented with a dashed rectangle) of the first sample simulations, was calculated using CASTp and is shown in red spheres. (F) The distance between K78 and E98 residues over the simulation time along with the pT230 residue are shown in stick representations. (G) The free volume available inside the protein for the average structures, obtained from 2.0 to 2.4 μs (represented with a dashed rectangle) of the second sample simulations. Full article ">Figure 4
LKB1 T353 phosphorylation regulates AMPK activation, cell size and proliferation in vivo. (A) Western blots of embryonic lysates of LKB1 wt, T353A and T353D knockin flies against the indicated proteins. (B) In vitro kinase assay with recombinant GST-AMPK (aa 108–280) and the indicated LKB1 variants plus its cofactor Stlk. CCB is colloidal coomassie blue, which was used to visualize the GST-AMPK input proteins. (C–F) GFP-marked MARCM (mosaic analysis with a repressible cell marker) clones of LKB1 knockin-variants in otherwise wild type tissue in wing imaginal discs stained with a proliferation marker (Histone 3 phospho-S10, pH3) and Dlg to label cell boundaries. The size of GFP-marked cells was quantified (n > 200). Scale bars are 20 µm. Error bars are standard error of the means. Significance was determined by one way ANOVA with Bonferroni multiple comparison test: *** p < 0.001, n.s. not significant. (G) S2R+ cells were co-transfected with Mo25-HA, Stlk-Myc and either wild type GFP-LKB1 or GFP-LKB1 T353A. GFP alone was used as control. GFP proteins were immunoprecipitated and GFP(-LKB1), Mo25-HA and Stlk-Myc were detected by immunoblotting. Full article ">

13 pages, 1976 KiB

Open AccessArticle

Functional Deficiency of Interneurons and Negative BOLD fMRI Response

by Daniil P. Aksenov, Limin Li, Natalya A. Serdyukova, David A. Gascoigne, Evan D. Doubovikov and Alexander Drobyshevsky

Cells 2023, 12(5), 811; https://doi.org/10.3390/cells12050811 - 6 Mar 2023

Cited by 1 | Viewed by 2163

Abstract

The functional deficiency of the inhibitory system typically appears during development and can progress to psychiatric disorders or epilepsy, depending on its severity, in later years. It is known that interneurons, the major source of GABAergic inhibition in the cerebral cortex, can make [...] Read more.

The functional deficiency of the inhibitory system typically appears during development and can progress to psychiatric disorders or epilepsy, depending on its severity, in later years. It is known that interneurons, the major source of GABAergic inhibition in the cerebral cortex, can make direct connections with arterioles and participate in the regulation of vasomotion. The goal of this study was to mimic the functional deficiency of interneurons through the use of localized microinjections of the GABA antagonist, picrotoxin, in such a concentration that it did not elicit epileptiform neuronal activity. First, we recorded the dynamics of resting-state neuronal activity in response to picrotoxin injections in the somatosensory cortex of an awake rabbit; second, we assessed the altered neuronal and hemodynamic responses to whisker stimulation using BOLD fMRI and electrophysiology recordings; third, we evaluated brain tissue oxygen levels before and after picrotoxin injection. Our results showed that neuronal activity typically increased after picrotoxin administration, the BOLD responses to stimulation became negative, and the oxygen response was nearly abolished. Vasoconstriction during the resting baseline was not observed. These results indicate that picrotoxin provoked imbalanced hemodynamics either due to increased neuronal activity, decreased vascular response, or a combination of both. Full article

(This article belongs to the Special Issue Remodeling and Recovery in the Neurovascular Unit)

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The effect of picrotoxin (PTX) on resting state neuronal activity. An example of multi-unit neuronal activity is shown before the PTX injection (A),after the injection of a small concentration (50 μM) of PTX (B), which did not cause visible resting state neuronal hypersynchronization, and larger PTX concentrations: 166 μM—(C), 248 μM—(D), and 331 μM—(E), which caused resting state neuronal hypersynchronization. We used the smaller 50 μM PTX concentration and two thresholds to detect changes in neuronal activity (30% and 10% changes after injection relative to the baseline before injection). After the injection of 50 μM PTX, neuronal activity experienced both an increase and decrease for both thresholds (F,G) where increases were more common. For the control experiments, changes above 30% were not observed after the injection of artificial cerebrospinal fluid (H) but some changes above 10% were recorded (I). GABA-agonist muscimol (MSC), in contrast, decreased the activity of all neurons. (J,K). Examples of peri-event histograms are shown (L–O), where PTX decreased (L), did not change (M), or increased the activity of single neurons. MSC always decreased the activity of a single neuron (O). Arrows indicate the time of injection. Full article ">Figure 2
The effect of picrotoxin (PTX) on stimulus-evoked BOLD responses. Before the 50 μM PTX injection (A) a robust positive BOLD response was observed which extended through the depth of the cortex. After the injection of PTX, the BOLD response was negative (B). For control experiments with vehicle injection, the BOLD response was positive before (C) and after (D) injection. The averaged temporal profile from a region corresponding to the post-injection area exhibited a strong positive temporal profile before injection, which became negative after the injection of PTX (E,F). Both standard error bars (E) and 95% confidence intervals (F) are shown for better visibility. The average temporal profile from a region corresponding to the post-injection area exhibited a strong positive temporal profile before injection, which did not change after the control vehicle injection (G,H). Both standard error bars (G) and confidence intervals (H) are shown for the control vehicle injection. The blue bars indicate the timing of the stimulus presentation. The color bar indicates the correlation in each voxel on top of the support vector machine mask. Full article ">Figure 3
Responses of single neurons to stimulation. The typical effect of PTX was an increase in spiking activity in response to stimulation (A). Sometimes other behaviors were observed: a decrease in the response (B), or a consistent inhibitory response (C). Examples of a newly acquired response after the PTX injection (D) and abolishing the response (E) were associated with interneurons. The histogram (F) shows that the relative (to 100% baseline) magnitude of excitatory neuronal responses increased after the PTX injection but did not change after vehicle injection. The blue bars indicate the timing of the stimulus presentation. Asterisk indicates significance (p < 0.05). Full article ">Figure 4
Brain tissue oxygen (PO2) changes after GABA-antagonist picrotoxin (PTX) injection. The baseline of PO2 did not change after PTX (A) as well as PO2 response to whisker stimulation after control vehicle injection (B). However, PO2 response after PTX injection was nearly abolished (grey line), and sometimes transient below-baseline PO2 responses were observed (C). An example of this transient below-baseline response is shown by the dashed line (“after neg”). The statistics for PO2 responses are shown on (D): “after” includes trials with only above-baseline responses and “after + neg” includes trials with both above- and below-baseline responses. The data on (D) are normalized to the 100% baseline (before stimulus). The grey bar indicates the stimulus presentation. Asterisk indicates significance (p < 0.05), and two asterisks indicate p < 0.01. Full article ">

37 pages, 1061 KiB

Open AccessReview

Targeting Autophagy Using Long Non-Coding RNAs (LncRNAs): New Landscapes in the Arena of Cancer Therapeutics

by Aviral Kumar, Sosmitha Girisa, Mohammed S. Alqahtani, Mohamed Abbas, Mangala Hegde, Gautam Sethi and Ajaikumar B. Kunnumakkara

Cells 2023, 12(5), 810; https://doi.org/10.3390/cells12050810 - 6 Mar 2023

Cited by 18 | Viewed by 3665

Abstract

Cancer has become a global health hazard accounting for 10 million deaths in the year 2020. Although different treatment approaches have increased patient overall survival, treatment for advanced stages still suffers from poor clinical outcomes. The ever-increasing prevalence of cancer has led to [...] Read more.

Cancer has become a global health hazard accounting for 10 million deaths in the year 2020. Although different treatment approaches have increased patient overall survival, treatment for advanced stages still suffers from poor clinical outcomes. The ever-increasing prevalence of cancer has led to a reanalysis of cellular and molecular events in the hope to identify and develop a cure for this multigenic disease. Autophagy, an evolutionary conserved catabolic process, eliminates protein aggregates and damaged organelles to maintain cellular homeostasis. Accumulating evidence has implicated the deregulation of autophagic pathways to be associated with various hallmarks of cancer. Autophagy exhibits both tumor-promoting and suppressive effects based on the tumor stage and grades. Majorly, it maintains the cancer microenvironment homeostasis by promoting viability and nutrient recycling under hypoxic and nutrient-deprived conditions. Recent investigations have discovered long non-coding RNAs (lncRNAs) as master regulators of autophagic gene expression. lncRNAs, by sequestering autophagy-related microRNAs, have been known to modulate various hallmarks of cancer, such as survival, proliferation, EMT, migration, invasion, angiogenesis, and metastasis. This review delineates the mechanistic role of various lncRNAs involved in modulating autophagy and their related proteins in different cancers. Full article

(This article belongs to the Special Issue Autophagy and Inflammation in Chronic Disease)

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23 pages, 2301 KiB

Open AccessArticle

Large-Scale Polymorphism Analysis of Dog Leukocyte Antigen Class I and Class II Genes (DLA-88, DLA-12/88L and DLA-DRB1) and Comparison of the Haplotype Diversity between Breeds in Japan

by Jiro Miyamae, Masaharu Okano, Fumihiko Katakura, Jerzy K. Kulski, Tadaaki Moritomo and Takashi Shiina

Cells 2023, 12(5), 809; https://doi.org/10.3390/cells12050809 - 6 Mar 2023

Cited by 4 | Viewed by 2770

Abstract

Polymorphisms of canine leukocyte antigen (DLA) class I (DLA-88 and DLA-12/88L) and class II (DLA-DRB1) genes are important for disease susceptibility studies, but information on the genetic diversity among dog breeds is still lacking. To better elucidate the polymorphism [...] Read more.

Polymorphisms of canine leukocyte antigen (DLA) class I (DLA-88 and DLA-12/88L) and class II (DLA-DRB1) genes are important for disease susceptibility studies, but information on the genetic diversity among dog breeds is still lacking. To better elucidate the polymorphism and genetic diversity between breeds, we genotyped DLA-88, DLA-12/88L, and DLA-DRB1 loci using 829 dogs of 59 breeds in Japan. Genotyping by Sanger sequencing identified 89, 43, and 61 alleles in DLA-88, DLA-12/88L, and DLA-DRB1 loci, respectively, and a total of 131 DLA-88–DLA-12/88L–DLA-DRB1 haplotypes (88-12/88L-DRB1) were detected more than once. Of the 829 dogs, 198 were homozygotes for one of the 52 different 88-12/88L-DRB1 haplotypes (homozygosity rate: 23.8%). Statistical modeling suggests that 90% of the DLA homozygotes or heterozygotes with one or other of the 52 different 88-12/88L-DRB1 haplotypes within somatic stem cell lines would benefit graft outcome after 88-12/88L-DRB1-matched transplantation. As previously reported for DLA class II haplotypes, the diversity of 88-12/88L-DRB1 haplotypes varied remarkably between breeds but was relatively conserved within most breeds. Therefore, the genetic characteristics of high DLA homozygosity rate and poor DLA diversity within a breed are useful for transplantation therapy, but they may affect biological fitness as homozygosity progresses. Full article

(This article belongs to the Special Issue Major Histocompatibility Complex (MHC) in Health and Disease 2022)

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Figure 1
Schematic representations of haplotype structures and typing method for DLA-I genes. (A) Two different haplotype structures associated with DLA-12 and DLA-88L locus. Interlocus gene conversion events in cis or trans between DLA-88 and DLA-12 are likely to be responsible for generating the DLA-88L locus. (B) Flow chart for locus-specific DLA typing method for DLA-88, DLA-12, and DLA-88L genes with genomic DNA. White and black-filled boxes in the chart represent the schematic location of UTR and CDS regions of a DLA gene. Arrowheads indicate the location and direction of the primers with the primer names whose detailed information is described in <a href="#app1-cells-12-00809" class="html-app">Supplementary Table S1</a>. Full article ">Figure 2
Top 20, 21 or 22 most frequent alleles of DLA-88, DLA-12/88L and DLA-DRB1 loci detected for 829 dogs. The allele frequency and the proportion of dogs with each allele are described by a bar and line chart, respectively. The allele names are indicated below each graph. Full article ">Figure 3
Phylogenetic relationship among the DLA-88, DLA-12, and DLA-88L allele sequences. (A) Neighbor-joining phylogenetic tree with exon 2–intron 2–exon 3 sequences of 109 alleles of DLA-88, DLA-12, and DLA-88L. The DLA-12 and DLA-88L alleles are represented with orange and red characters, respectively. Alleles that were not detected from 829 dogs in our study sample are indicated with a black-filled circle. Nucleotide sequences of H-2D and H-2K are used as an outgroup of the tree. (B) Dot-match analysis between two alleles by GenomeMatcher. The sequence similarity between DLA-88*nov65 and DLA-88*501:01, DLA-88*017:01 or DLA-12*004:01 are displayed by lines. The lines are colored based on the similarity score calculated by the bl2seq program with parameter as follow: word size 10 and e-value threshold 0.1. When the similarity score was lower than 90, the line was not described at the region. Dashed lines indicate the boundary of exon and intron in each allele. Full article ">Figure 4
Comparisons of the proportion of dogs with each DLA-DRB1 allele in 10 breeds between the present Japanese and the previous UK study. Scatter plots based on the proportion of dogs with the DLA-DRB1 alleles in ten breeds are shown. The number of dogs analyzed and the number of observed alleles in the present Japanese and the previous UK study are described on the x- and y-axis in each plot, respectively. Dachshunds are summarized without classification by their size, such as miniature or kaninchen, and hair length, such as short or long, in both studies. An allele name is displayed alongside a marker when the proportion is above 5% in either the present or previous study. A red line in each plot represents a regression line. Also, a correlation coefficient (r) is indicated in each plot. Full article ">Figure 5
Percentage distribution of the 88-12/88L-DRB1 haplotypes in 24 breeds and mongrels. The cumulative bar chart is based on the frequency of all the 88-12/88L-DRB1 haplotypes detected from each breed listed in <a href="#cells-12-00809-t005" class="html-table">Table 5</a>. A numeral following the breed’s name indicates the number of different haplotypes detected in each breed. Parenthesis indicates the number of different 88-12/88L-DRB1 haplotypes with a frequency of 5% or higher. The breeds are sorted in ascending order by the percentage frequency of the most frequent haplotype in each breed. For example, the graph of Miniature Schnauzer at the bottom of the figure represents a total of nine sub-haplotypes of 88-12/88L-DRB1 haplotypes with a frequency of 5% or higher for three of them at >50%, 10–29%, and 5–9%, respectively. Full article ">Figure 6
Principal component analysis based on 88-12/88L-DRB1 haplotype frequencies among 24 breeds. The analysis based on the frequency of three-locus DLA haplotypes in each allele at field-1 level among (A) 24 breeds listed in <a href="#cells-12-00809-t005" class="html-table">Table 5</a> and (B) 22 breeds except for Shetland Sheepdog and Miniature Schnauzer from (A) are represented. The breeds that shared a three-loci haplotype with relatively high frequency (See <a href="#sec3dot6-cells-12-00809" class="html-sec">Section 3.6</a>. in detail) are grouped with a red circle. The contribution ratio of the first (PC1) and second component (PC2) are described on the x- and y-axis in parentheses, respectively. Full article ">Figure 7
The cumulative percentage of dogs matched for homozygous 88-12/88L-DRB1 haplotypes detected in this study. The horizontal and vertical axes indicate 52 types of 88-12/88L-DRB1 homozygous haplotype numbers, which are sorted in descending order of the haplotype frequency, and the number of dogs, respectively. The cumulative proportion of dogs was calculated using the 803 dogs with identified haplotypes. Full article ">

14 pages, 14481 KiB

Open AccessArticle

Estrogen Mediates the Sexual Dimorphism of GT1b-Induced Central Pain Sensitization

by Jaesung Lee, Seohyun Chung, Minkyu Hwang, Yeongkag Kwon, Seung Hyun Han and Sung Joong Lee

Cells 2023, 12(5), 808; https://doi.org/10.3390/cells12050808 - 6 Mar 2023

Cited by 5 | Viewed by 2503

Abstract

We have previously reported that the intrathecal (i.t.) administration of GT1b, a ganglioside, induces spinal cord microglia activation and central pain sensitization as an endogenous agonist of Toll-like receptor 2 on microglia. In this study, we investigated the sexual dimorphism of GT1b-induced central [...] Read more.

We have previously reported that the intrathecal (i.t.) administration of GT1b, a ganglioside, induces spinal cord microglia activation and central pain sensitization as an endogenous agonist of Toll-like receptor 2 on microglia. In this study, we investigated the sexual dimorphism of GT1b-induced central pain sensitization and the underlying mechanisms. GT1b administration induced central pain sensitization only in male but not in female mice. Spinal tissue transcriptomic comparison between male and female mice after GT1b injection suggested the putative involvement of estrogen (E2)-mediated signaling in the sexual dimorphism of GT1b-induced pain sensitization. Upon ovariectomy-reducing systemic E2, female mice became susceptible to GT1b-induced central pain sensitization, which was completely reversed by systemic E2 supplementation. Meanwhile, orchiectomy of male mice did not affect pain sensitization. As an underlying mechanism, we present evidence that E2 inhibits GT1b-induced inflammasome activation and subsequent IL-1β production. Our findings demonstrate that E2 is responsible for sexual dimorphism in GT1b-induced central pain sensitization. Full article

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

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24 pages, 7673 KiB

Open AccessArticle

Perfusion Air Culture of Precision-Cut Tumor Slices: An Ex Vivo System to Evaluate Individual Drug Response under Controlled Culture Conditions

by Meng Dong, Kathrin Böpple, Julia Thiel, Bernd Winkler, Chunguang Liang, Julia Schueler, Emma J. Davies, Simon T. Barry, Tauno Metsalu, Thomas E. Mürdter, Georg Sauer, German Ott, Matthias Schwab and Walter E. Aulitzky

Cells 2023, 12(5), 807; https://doi.org/10.3390/cells12050807 - 4 Mar 2023

Cited by 4 | Viewed by 3772

Abstract

Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air–liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we [...] Read more.

Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air–liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses. Full article

(This article belongs to the Collection Advances in 3D Cell Culture)

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15 pages, 2686 KiB

Open AccessArticle

Changes in Liver Lipidomic Profile in G2019S-LRRK2 Mouse Model of Parkinson’s Disease

by Yaiza Corral Nieto, Sokhna M. S. Yakhine-Diop, Paula Moreno-Cruz, Laura Manrique García, Amanda Gabrielly Pereira, José A. Morales-García, Mireia Niso-Santano, Rosa A. González-Polo, Elisabet Uribe-Carretero, Sylvère Durand, Maria Chiara Maiuri, Marta Paredes-Barquero, Eva Alegre-Cortés, Saray Canales-Cortés, Adolfo López de Munain, Jordi Pérez-Tur, Ana Pérez-Castillo, Guido Kroemer, José M. Fuentes and José M. Bravo-San Pedro

Cells 2023, 12(5), 806; https://doi.org/10.3390/cells12050806 - 4 Mar 2023

Cited by 2 | Viewed by 3636

Abstract

The identification of Parkinson’s disease (PD) biomarkers has become a main goal for the diagnosis of this neurodegenerative disorder. PD has not only been intrinsically related to neurological problems, but also to a series of alterations in peripheral metabolism. The purpose of this [...] Read more.

The identification of Parkinson’s disease (PD) biomarkers has become a main goal for the diagnosis of this neurodegenerative disorder. PD has not only been intrinsically related to neurological problems, but also to a series of alterations in peripheral metabolism. The purpose of this study was to identify metabolic changes in the liver in mouse models of PD with the scope of finding new peripheral biomarkers for PD diagnosis. To achieve this goal, we used mass spectrometry technology to determine the complete metabolomic profile of liver and striatal tissue samples from WT mice, 6-hydroxydopamine-treated mice (idiopathic model) and mice affected by the G2019S-LRRK2 mutation in LRRK2/PARK8 gene (genetic model). This analysis revealed that the metabolism of carbohydrates, nucleotides and nucleosides was similarly altered in the liver from the two PD mouse models. However, long-chain fatty acids, phosphatidylcholine and other related lipid metabolites were only altered in hepatocytes from G2019S-LRRK2 mice. In summary, these results reveal specific differences, mainly in lipid metabolism, between idiopathic and genetic PD models in peripheral tissues and open up new possibilities to better understand the etiology of this neurological disorder. Full article

(This article belongs to the Special Issue Cell Biology: State-of-the-Art and Perspectives in Spain II)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Results of metabolic changes observed in striatum tissues for the control (Co) and parkinsonian genetic (GS-PD) or due to acute intoxication (ai-PD) groups (n = 5). (A) Heatmap with the average of the log2 area (± standard error of the mean (SEM)) showed by metabolite groups (amino acids, nitrogen bases, carbohydrates, lipids, nucleosides, nucleotides, and organic compounds). (B,C) Volcano plot graphs are shown. The log2 FC shows changes observed on GS-PD model (B) or ai-PD model (C) by comparison to the control mice for each metabolite (represented by each dot). The −log10 p value represents non-significant (grey color) or significant (red represents significantly down-regulated metabolites, whereas green represents significantly up-regulated metabolites. (D) Correlation analysis between changes observed in striatal neurons of GS-PD compared to controls and ai-PD compared to controls. Statistical analysis was performed by obtaining p value (° (p < 0.1), * (p < 0.05), ** (p < 0.01), **** (p < 0.0001)), and Pearson’s correlation coefficient (R) between the noted changes. Full article ">Figure 2
Results of metabolic changes observed in hepatic tissues for the control (Co) and parkinsonian genetic (GS-PD) or due to acute intoxication (ai-PD) groups (n = 4–5). (A) Heatmap with the average of the log2 area (± standard error of the mean (SEM)) showed by metabolite groups (amino acids, nitrogenous bases, carbohydrates, lipids, nucleosides, nucleotides and organic compounds). (B,C) Volcano plot graphs are shown. The log2 FC indicates the changes observed on GS-PD model (B) or ai-PD model (C) in comparison to control mice for each metabolite (represented by each dot). The −log10 p value represents non-significant (grey color) or significant (red represents significantly down-regulated metabolites, whereas green represents significantly up-regulated metabolites. (D) Correlation analysis between changes observed in striatal neurons of GS-PD or ai-PD compared to controls. Statistical analysis was performed by obtaining p value (° (p < 0.1), * (p < 0.05), ** (p < 0.01), *** p < 0.001), and Pearson’s correlation coefficients (R) between the observed changes). Full article ">Figure 3
Histograms showing the average (± standard error of the mean (SEM)) of log2-fold change (Log2 FC) concentrations of different metabolites significantly decreased (A,B) or increased (C) in the liver of genetic (carrying the p.G2019S mutation in LRRK2; GS-PD) and acute intoxication PD (ai-PD) mouse models (n = 4–5). For statistical analyses, p-values were calculated by one-way ANOVA (analyzing the metabolites individually) and differences were evaluated as statistically significant when p-values were: ° (p < 0.1), * (p < 0.05) and ** (p < 0.01). Full article ">Figure 4
Heatmaps showing the TTEST (p value) on the square above, and log 2-fold change (Log2 FC) on the square below for the different concentration of fatty acids (FA) (A), phospholipids in cell membranes (B) lipid-related metabolites (C) and bile acid metabolites (D) in the liver of WT group and genetic (carrying the p.G2019S mutation in LRRK2; GS-PD) and acute intoxication PD (ai-PD) mouse models (n = 4–5). For statistical data, p-values were estimated by one-way ANOVA (analyzing the metabolites separately) and differences were considered significant when p-values were: ° (p < 0.1), * (p < 0.05), ** (p < 0.01) and *** (p < 0.001). G, glycine; LC, long-chain; MC, medium-chain; PC, phosphatidylcholine; PE, phosphatidylethanolamine; T, taurine; VLC, very long-chain. Full article ">

40 pages, 4215 KiB

Open AccessReview

LIM Kinases, LIMK1 and LIMK2, Are Crucial Node Actors of the Cell Fate: Molecular to Pathological Features

by Elodie Villalonga, Christine Mosrin, Thierry Normand, Caroline Girardin, Amandine Serrano, Bojan Žunar, Michel Doudeau, Fabienne Godin, Hélène Bénédetti and Béatrice Vallée

Cells 2023, 12(5), 805; https://doi.org/10.3390/cells12050805 - 4 Mar 2023

Cited by 21 | Viewed by 5815

Abstract

LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the only two members of the LIM kinase family. They play a crucial role in the regulation of cytoskeleton dynamics by controlling actin filaments and microtubule turnover, especially [...] Read more.

LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the only two members of the LIM kinase family. They play a crucial role in the regulation of cytoskeleton dynamics by controlling actin filaments and microtubule turnover, especially through the phosphorylation of cofilin, an actin depolymerising factor. Thus, they are involved in many biological processes, such as cell cycle, cell migration, and neuronal differentiation. Consequently, they are also part of numerous pathological mechanisms, especially in cancer, where their involvement has been reported for a few years and has led to the development of a wide range of inhibitors. LIMK1 and LIMK2 are known to be part of the Rho family GTPase signal transduction pathways, but many more partners have been discovered over the decades, and both LIMKs are suspected to be part of an extended and various range of regulation pathways. In this review, we propose to consider the different molecular mechanisms involving LIM kinases and their associated signalling pathways, and to offer a better understanding of their variety of actions within the physiology and physiopathology of the cell. Full article

(This article belongs to the Special Issue LIM Kinases: From Molecular to Pathological Features)

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12 pages, 1114 KiB

Open AccessReview

Polyunsaturated Fatty Acids Drive Lipid Peroxidation during Ferroptosis

by Michael S. Mortensen, Jimena Ruiz and Jennifer L. Watts

Cells 2023, 12(5), 804; https://doi.org/10.3390/cells12050804 - 4 Mar 2023

Cited by 81 | Viewed by 9420

Abstract

Ferroptosis is a form of regulated cell death that is intricately linked to cellular metabolism. In the forefront of research on ferroptosis, the peroxidation of polyunsaturated fatty acids has emerged as a key driver of oxidative damage to cellular membranes leading to cell [...] Read more.

Ferroptosis is a form of regulated cell death that is intricately linked to cellular metabolism. In the forefront of research on ferroptosis, the peroxidation of polyunsaturated fatty acids has emerged as a key driver of oxidative damage to cellular membranes leading to cell death. Here, we review the involvement of polyunsaturated fatty acids (PUFAs), monounsaturated fatty acids (MUFAs), lipid remodeling enzymes and lipid peroxidation in ferroptosis, highlighting studies revealing how using the multicellular model organism Caenorhabditis elegans contributes to the understanding of the roles of specific lipids and lipid mediators in ferroptosis. Full article

(This article belongs to the Special Issue Cellular and Molecular Control of Lipid Metabolism)

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Non-enzymatic lipid peroxidation. Peroxidation is initiated by cellular ROS, where hydroxyl, alkoxyl or peroxyl radicals abstract a hydrogen from a PUFA acyl group (radical electrons denoted as red circle). A PUFA peroxide is formed by reacting with molecular oxygen and abstraction of a hydrogen from an adjacent membrane PUFA. Fenton chemistry contributes to further lipid radical formation, contributing to the chain reaction of lipid radicals attacking acyl groups on nearby unsaturated phospholipid molecules. Lipid peroxidation is terminated by actions of radical-trapping antioxidants or by reduction by catalyzed by glutathione peroxidase activity. Figure created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>, accessed on 1 February 2023. Full article ">Figure 2
Structures of common fatty acids and oxygenated derivatives. (A) Stearic acid (18:0) is a saturated fatty acid. (B) Oleic acid (18:1n-9) is a monounsaturated fatty acid. The single double bond is in the cis position, creating a kink in the fatty acid that prevents tight packing of fatty acids and contributes to membrane fluidity. (C) Dihommo-γ linolenic acid (DGLA, 20:3n-6) is a polyunsaturated fatty acid. Oxygenated derivatives are produced by cytochrome P450 (CYP) enzymes, forming an epoxide. The double bond that is converted to an epoxide depends on the position-specific isoform of CYP enzymes. The epoxides can be converted into diols by epoxide hydrolase (EH) enzymes. The EH enzymes are inhibited by AUDA. (D) Arachidonic acid (AA, 20:4n-6) is a polyunsaturated fatty acid. Shown are examples of oxygenated derivatives produced by lipoxygenase (LOX) enzymes and peroxidase activity. The location of the hydroperoxide is dependent on the position-specific isoform of LOX. The hydroperoxide can be further reduced by peroxidase activity, leading to a bioactive hydroxyl derivative. Full article ">Figure 3
Dietary DGLA causes ferroptosis of germ cells and sterility in C. elegans. (A) Schematic of the C. elegans fatty acid supplementation assay. Synchronized L1 larvae are plated onto agar plates containing DGLA and dietary E. coli, and incubated at 20 degrees until they reach adulthood, when they are scored as fertile or sterile. Sterile worms lack gametes due to ferroptosis of germ cells during development. (B) Mutant strains that are more sensitive to DGLA are known as enhancers, while mutant strains that are less sensitive to DGLA are known as suppressors. Often, enhancer strains contain mutations in protective genes, such as genes encoding GPX enzymes or genes required for MUFA production. Suppressor genes include genes needed to produce membrane PUFAs, or mutants that confer increased stress responses. Full article ">

16 pages, 331 KiB

Open AccessArticle

Relationship between Oxidative Stress and Left Ventricle Markers in Patients with Chronic Heart Failure

by Aušra Mongirdienė, Agnė Liuizė, Dovilė Karčiauskaitė, Eglė Mazgelytė, Arūnas Liekis and Ilona Sadauskienė

Cells 2023, 12(5), 803; https://doi.org/10.3390/cells12050803 - 4 Mar 2023

Cited by 9 | Viewed by 2223

Abstract

Oxidative stress is proposed in the literature as an important player in the development of CHF and correlates with left ventricle (LV) dysfunction and hypertrophy in the failing heart. In this study, we aimed to verify if the serum oxidative stress markers differ [...] Read more.

Oxidative stress is proposed in the literature as an important player in the development of CHF and correlates with left ventricle (LV) dysfunction and hypertrophy in the failing heart. In this study, we aimed to verify if the serum oxidative stress markers differ in chronic heart failure (CHF) patients’ groups depending on the LV geometry and function. Patients were stratified into two groups according to left ventricular ejection fraction (LVEF) values: HFrEF (<40% (n = 27)) and HFpEF (≥40% (n = 33)). Additionally, patients were stratified into four groups according to LV geometry: NG–normal left ventricle geometry (n = 7), CR–concentric remodeling (n = 14), cLVH–concentric LV hypertrophy (n = 16), and eLVF–eccentric LV hypertrophy (n = 23). We measured protein (protein carbonyl (PC), nitrotyrosine (NT-Tyr), dityrosine), lipid (malondialdehyde (MDA), oxidizes (HDL) oxidation and antioxidant (catalase activity, total plasma antioxidant capacity (TAC) markers in serum. Transthoracic echocardiogram analysis and lipidogram were also performed. We found that oxidative (NT-Tyr, dityrosine, PC, MDA, oxHDL) and antioxidative (TAC, catalase) stress marker levels did not differ between the groups according to LVEF or LV geometry. NT-Tyr correlated with PC (r_s = 0.482, p = 0.000098), and oxHDL (r_s = 0.278, p = 0.0314). MDA correlated with total (r_s = 0.337, p = 0.008), LDL (r_s = 0.295, p = 0.022) and non-HDL (r_s = 0.301, p = 0.019) cholesterol. NT-Tyr negatively correlated with HDL cholesterol (r_s = -0.285, p = 0.027). LV parameters did not correlate with oxidative/antioxidative stress markers. Significant negative correlations were found between the end-diastolic volume of the LV and the end-systolic volume of the LV and HDL-cholesterol (r_s = –0.935, p < 0.0001; r_s = –0.906, p < 0.0001, respectively). Significant positive correlations between both the thickness of the interventricular septum and the thickness of the LV wall and the levels of triacylglycerol in serum (r_s = 0.346, p = 0.007; r_s = 0.329, p = 0.010, respectively) were found. In conclusions, we did not find a difference in serum concentrations of both oxidant (NT-Tyr, PC, MDA) and antioxidant (TAC and catalase) concentrations in CHF patients’ groups according to LV function and geometry was found. The geometry of the LV could be related to lipid metabolism in CHF patients, and no correlation between oxidative/antioxidant and LV markers in CHF patients was found. Full article

(This article belongs to the Special Issue The Role of Oxidative Stress in Cardiovascular Diseases)

15 pages, 1567 KiB

Open AccessReview

Cancer-Associated Fibroblast: Role in Prostate Cancer Progression to Metastatic Disease and Therapeutic Resistance

by Martina Bedeschi, Noemi Marino, Elena Cavassi, Filippo Piccinini and Anna Tesei

Cells 2023, 12(5), 802; https://doi.org/10.3390/cells12050802 - 4 Mar 2023

Cited by 30 | Viewed by 3768

Abstract

Prostate cancer (PCa) is one of the most common cancers in European males. Although therapeutic approaches have changed in recent years, and several new drugs have been approved by the Food and Drug Administration (FDA), androgen deprivation therapy (ADT) remains the standard of [...] Read more.

Prostate cancer (PCa) is one of the most common cancers in European males. Although therapeutic approaches have changed in recent years, and several new drugs have been approved by the Food and Drug Administration (FDA), androgen deprivation therapy (ADT) remains the standard of care. Currently, PCa represents a clinical and economic burden due to the development of resistance to ADT, paving the way to cancer progression, metastasis, and to long-term side effects induced by ADT and radio-chemotherapeutic regimens. In light of this, a growing number of studies are focusing on the tumor microenvironment (TME) because of its role in supporting tumor growth. Cancer-associated fibroblasts (CAFs) have a central function in the TME because they communicate with prostate cancer cells, altering their metabolism and sensitivity to drugs; hence, targeted therapy against the TME, and, in particular, CAFs, could represent an alternative therapeutic approach to defeat therapy resistance in PCa. In this review, we focus on different CAF origins, subsets, and functions to highlight their potential in future therapeutic strategies for prostate cancer. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Cancers: Prostate Cancer)

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Therapeutic strategies targeting prostatic stromal microenvironment. (a) Search for articles appearing in PUBMED over the past 10 years (2011–2021) using the mesh terms “prostatic neoplasm/prostate CAFs/drug therapy” (red); “prostatic neoplasm/ICIs” (yellow); and “prostatic neoplasm/ADT” (blue). In the last 10 years, publications regarding therapies targeting the major components of TME have developed. (b) Search for articles appearing in PUBMED using the mesh terms “prostatic neoplasm” AND “extracellular matrix” (6%), “prostatic neoplasm” AND “cancer-associated fibroblast” (5%), and “prostatic neoplasm/immunology” (88%). Among PCa therapies against the major components of TME, immune therapies represent the most frequently searched, indicating a lack of research on CAFs in PCa. Full article ">Figure 2
Features and functions of CAFs in TME. The major roles played by CAFs are represented in the figure: CAFs promote angiogenesis, induce smooth muscle contraction, stimulate ECM remodeling and collagen deposition, and boost cancer growth and metastasis, chronic inflammation, and immune inhibition. Full article ">Figure 3
Potential sources of CAFs. In PCa, TME interacts with proximal and distal cells, such as bone-marrow-derived mesenchymal stem cells, healthy fibroblasts, adipocytes, pericytes, and endothelial cells, inducing the switch into CAFs. CAFs build paracrine communication with cancer cells by releasing factors, such as IL-6, IL-8, TGFβ, FGFs, VEGF, and GDF15, stimulating tumor growth, angiogenesis, and progression. Full article ">Figure 4
TME produces several stimuli able to induce fibroblast activation. CAFs can be divided into three substantial subpopulations: myofibroblastic, immune-regulatory, and antigen-presenting (apCAFs) CAFs. The first ones are responsible for the reorganization of the ECM, producing hyaluronate, fibronectin, and collagens and inducing morphological alteration and increased stiffness. Immune-regulatory CAFs and apCAFs are involved in cancer inflammation and the modulation of immune responses in the tumor microenvironment. Full article ">

12 pages, 2125 KiB

Open AccessCommunication

Tubule-Derived Follistatin Is Increased in the Urine of Rats with Renal Ischemia and Reflects the Severity of Acute Tubular Damage

by Izumi Nagayama, Kaori Takayanagi, Hajime Hasegawa and Akito Maeshima

Cells 2023, 12(5), 801; https://doi.org/10.3390/cells12050801 - 4 Mar 2023

Cited by 2 | Viewed by 2321

Abstract

Activin A, a member of the TGF-beta superfamily, is a negative regulator of tubular regeneration after renal ischemia. Activin action is controlled by an endogenous antagonist, follistatin. However, the role of follistatin in the kidney is not fully understood. In the present study, [...] Read more.

Activin A, a member of the TGF-beta superfamily, is a negative regulator of tubular regeneration after renal ischemia. Activin action is controlled by an endogenous antagonist, follistatin. However, the role of follistatin in the kidney is not fully understood. In the present study, we examined the expression and localization of follistatin in normal and ischemic rat kidneys and measured urinary follistatin in rats with renal ischemia to assess whether urinary follistatin could serve as a biomarker for acute kidney injury. Using vascular clamps, renal ischemia was induced for 45 min in 8-week-old male Wistar rats. In normal kidneys, follistatin was localized in distal tubules of the cortex. In contrast, in ischemic kidneys, follistatin was localized in distal tubules of both the cortex and outer medulla. Follistatin mRNA was mainly present in the descending limb of Henle of the outer medulla in normal kidneys but was upregulated in the descending limb of Henle of both the outer and inner medulla after renal ischemia. Urinary follistatin, which was undetectable in normal rats, was significantly increased in ischemic rats and peaked 24 h after reperfusion. There was no correlation between urinary follistatin and serum follistatin. Urinary follistatin levels were increased according to ischemic duration and were significantly correlated with the follistatin-positive area as well as the acute tubular damage area. These results suggest that follistatin normally produced by renal tubules increases and becomes detectable in urine after renal ischemia. Urinary follistatin might be useful to assess the severity of acute tubular damage. Full article

(This article belongs to the Special Issue Recent Advances in Development and Progression of Kidney Diseases)

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Figure 1
Localization of the follistatin protein in normal rat kidneys. (a) Localization of follistatin (brown) in normal rat kidneys was examined by immunostaining. Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 0.2 mm. (b–f) Identification of follistatin-positive tubules in rat normal kidneys using serial sections. Aquaporin 1, AQP1; Na-Cl co-transporter, NCC; aquaporin 2, AQP2. Asterisks indicate identical tubules. Bar = 50 μm. Full article ">Figure 2
Localization of the follistatin protein in kidneys after renal ischemia. (a) Localization of follistatin (brown) in kidneys after renal ischemia was examined by immunostaining (left panel). Bar = 2 mm. Quantitative analysis of follistatin-positive area (right panel). Five randomly selected fields of the kidney were assessed at ×100 magnification. The follistatin-positive area was measured by ImageJ. Values are the means ± S.E. (n = 6–8). * p < 0.05, ** p < 0.01 vs. 0 h (b,c) Localization of the follistatin protein in normal and ischemic kidneys. The kidneys were collected at 0 h and 24 h after reperfusion. Ischemia reperfusion, IR. Bar = 0.2 mm in (b), 50 μm in (c). Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 50 μm. (d) Localization of follistatin protein and megalin, aquaporin 1 (AQP1), uromodulin (UMOD), or aquaporin 2 (AQP2) in ischemic kidneys was evaluated using serial sections. Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 50 μm. (e) Localization of follistatin, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) in ischemic kidneys was evaluated using serial sections. Bar = 0.2 mm (upper panels), 50 μm (lower panels). (f) Localization of follistatin and activin A in ischemic kidneys was evaluated using serial sections. Bar = 50 μm. Asterisks indicate identical tubules. Full article ">Figure 3
Expression and localization of follistatin mRNA in normal and ischemic rat kidneys. (a) Localization of follistatin mRNA in normal kidneys was examined by in situ hybridization. Hybridization signals are shown as blue color. Bar = 0.2 mm (left panel), 50 μm (right panels). (b) Follistatin mRNA (blue) and uromodulin (brown) in normal kidneys was evaluated by double staining. Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 0.2 mm (left panel), 50 μm (right panels). (c) Localization of follistatin mRNA and aquaporin 1 (AQP1) in normal rat kidneys was evaluated using serial sections. Asterisks indicate identical tubules. (d) Follistatin mRNA (blue) and uromodulin (brown) in normal and ischemic kidneys at 24 after reperfusion was evaluated by double staining. Cortex, CO; outer medulla, OM. (e) Follistatin mRNA (blue) and aquaporin 2 (AQP2, brown) in normal and ischemic kidneys was evaluated by double staining. Inner medulla, IM. Bar = 50 μm in (c–e). (f) Quantitative analysis of follistatin mRNA expression by real-time PCR. Values (relative expression ratio to GAPDH) are means ± S.E. (n = 5–8). Full article ">Figure 4
Urinary follistatin levels significantly increased after renal ischemia. (a) Time course changes in urinary follistatin and serum creatinine levels after reperfusion (n = 8). (b) Urinary and serum follistatin levels in normal and renal ischemia (45 min) rats were measured by ELISA. Values are means ± S.E. (n = 8). *** p < 0.001; N.S., not significant. (c) Time course changes in urinary follistatin, urinary NGAL, KIM-1, and L-FABP after reperfusion. (d,e) Correlations between urinary follistatin and urinary NGAL (d) or urinary KIM-1 (e) at 24 h after reperfusion. Renal ischemia (45 min) was performed, and urine was collected 24 h after reperfusion. Full article ">Figure 5
Correlation between urinary follistatin and severity of kidney damage. (a,b) Serum creatinine and BUN in rats with renal ischemia at 24 h after reperfusion. N.S., not significant, * p < 0.05, *** p < 0.001 vs. 0 h. (c–e) Urinary follistatin, urinary NGAL, and urinary KIM-1 in rats with renal ischemia for the indicated periods at 24 h after reperfusion. N.S., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 h. (f) Localization of follistatin (brown) in kidneys after renal ischemia for 15, 30, and 45 min was examined by immunostaining. Bar = 2 mm. (g) Quantitative analysis of follistatin-positive area. Five randomly selected fields of the kidney were assessed at ×100 magnification. The follistatin-positive area was measured by ImageJ 1.53a. Values are the means ± S.E. (n = 5–6). N.S., not significant, * p < 0.05 vs. 0 h. (h) Semiquantitative analysis of the histological changes induced by renal ischemia. The ATN area was calculated as described in <a href="#sec2-cells-12-00801" class="html-sec">Section 2</a>. N.S., not significant, * p < 0.05, ** p < 0.01 vs. 0 h. (i,j). Correlation between urinary follistatin and follistatin-positive area (i) or ATN area (j) at 24 h after reperfusion. Full article ">Figure 6
Possible mechanism of follistatin excretion into urine after renal ischemia. Aquaporin 1, AQP1; aquaporin 2, AQP2; Na-Cl co-transporter, NCC. Full article ">

23 pages, 5352 KiB

Open AccessArticle

Study of the Bcl-2 Interactome by BiFC Reveals Differences in the Activation Mechanism of Bax and Bak

by Óscar Gonzalo, Andrea Benedi, Laura Vela, Alberto Anel, Javier Naval and Isabel Marzo

Cells 2023, 12(5), 800; https://doi.org/10.3390/cells12050800 - 3 Mar 2023

Cited by 2 | Viewed by 2196

Abstract

Evasion of apoptosis is one of the hallmarks of cancer cells. Proteins of the Bcl-2 family are key regulators of the intrinsic pathway of apoptosis, and alterations in some of these proteins are frequently found in cancer cells. Permeabilization of the outer mitochondrial [...] Read more.

Evasion of apoptosis is one of the hallmarks of cancer cells. Proteins of the Bcl-2 family are key regulators of the intrinsic pathway of apoptosis, and alterations in some of these proteins are frequently found in cancer cells. Permeabilization of the outer mitochondrial membrane, regulated by pro- and antiapoptotic members of the Bcl-2 family of proteins, is essential for the release of apoptogenic factors leading to caspase activation, cell dismantlement, and death. Mitochondrial permeabilization depends on the formation of oligomers of the effector proteins Bax and Bak after an activation event mediated by BH3-only proteins and regulated by antiapoptotic members of the Bcl-2 family. In the present work, we have studied interactions between different members of the Bcl-2 family in living cells via the BiFC technique. Despite the limitations of this technique, present data suggest that native proteins of the Bcl-2 family acting inside living cells establish a complex network of interactions, which would fit nicely into “mixed” models recently proposed by others. Furthermore, our results point to differences in the regulation of Bax and Bak activation by proteins of the antiapoptotic and BH3-only subfamilies. We have also applied the BiFC technique to explore the different molecular models proposed for Bax and Bak oligomerization. Bax and Bak’s mutants lacking the BH3 domain were still able to associate and give BiFC signals, suggesting the existence of alternative surfaces of interaction between two Bax or Bak molecules. These results agree with the widely accepted symmetric model for the dimerization of these proteins and also suggest that other regions, different from the α6 helix, could be involved in the oligomerization of BH3-in groove dimers. Full article

(This article belongs to the Special Issue Regulation of Apoptosis in Health and Disease)

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16 pages, 3467 KiB

Open AccessArticle

Melatonin Inhibits VEGF-Induced Endothelial Progenitor Cell Angiogenesis in Neovascular Age-Related Macular Degeneration

by Liang-Wei Lin, Shih-Wei Wang, Wei-Chien Huang, Thanh Kieu Huynh, Chao-Yang Lai, Chih-Yuan Ko, Yi-Chin Fong, Jie-Jen Lee, Shun-Fa Yang and Chih-Hsin Tang

Cells 2023, 12(5), 799; https://doi.org/10.3390/cells12050799 - 3 Mar 2023

Cited by 16 | Viewed by 3300

Abstract

Neovascular age-related macular degeneration (AMD) is described as abnormal angiogenesis in the retina and the leaking of fluid and blood that generates a huge, dark, blind spot in the center of the visual field, causing severe vision loss in over 90% of patients. [...] Read more.

Neovascular age-related macular degeneration (AMD) is described as abnormal angiogenesis in the retina and the leaking of fluid and blood that generates a huge, dark, blind spot in the center of the visual field, causing severe vision loss in over 90% of patients. Bone marrow-derived endothelial progenitor cells (EPCs) contribute to pathologic angiogenesis. Gene expression profiles downloaded from the eyeIntegration v1.0 database for healthy retinas and retinas from patients with neovascular AMD identified significantly higher levels of EPC-specific markers (CD34, CD133) and blood vessel markers (CD31, VEGF) in the neovascular AMD retinas compared with healthy retinas. Melatonin is a hormone that is mainly secreted by the pineal gland, and is also produced in the retina. Whether melatonin affects vascular endothelial growth factor (VEGF)-induced EPC angiogenesis in neovascular AMD is unknown. Our study revealed that melatonin inhibits VEGF-induced stimulation of EPC migration and tube formation. By directly binding with the VEGFR2 extracellular domain, melatonin significantly and dose-dependently inhibited VEGF-induced PDGF-BB expression and angiogenesis in EPCs via c-Src and FAK, NF-κB and AP-1 signaling. The corneal alkali burn model demonstrated that melatonin markedly inhibited EPC angiogenesis and neovascular AMD. Melatonin appears promising for reducing EPC angiogenesis in neovascular AMD. Full article

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22 pages, 1062 KiB

Open AccessReview

Transcriptional Response to Hypoxia: The Role of HIF-1-Associated Co-Regulators

by Angelos Yfantis, Ilias Mylonis, Georgia Chachami, Marios Nikolaidis, Grigorios D. Amoutzias, Efrosyni Paraskeva and George Simos

Cells 2023, 12(5), 798; https://doi.org/10.3390/cells12050798 - 3 Mar 2023

Cited by 46 | Viewed by 6271

Abstract

The Hypoxia Inducible Factor 1 (HIF-1) plays a major role in the cellular response to hypoxia by regulating the expression of many genes involved in adaptive processes that allow cell survival under low oxygen conditions. Adaptation to the hypoxic tumor micro-environment is also [...] Read more.

The Hypoxia Inducible Factor 1 (HIF-1) plays a major role in the cellular response to hypoxia by regulating the expression of many genes involved in adaptive processes that allow cell survival under low oxygen conditions. Adaptation to the hypoxic tumor micro-environment is also critical for cancer cell proliferation and therefore HIF-1 is also considered a valid therapeutical target. Despite the huge progress in understanding regulation of HIF-1 expression and activity by oxygen levels or oncogenic pathways, the way HIF-1 interacts with chromatin and the transcriptional machinery in order to activate its target genes is still a matter of intense investigation. Recent studies have identified several different HIF-1- and chromatin-associated co-regulators that play important roles in the general transcriptional activity of HIF-1, independent of its expression levels, as well as in the selection of binding sites, promoters and target genes, which, however, often depends on cellular context. We review here these co-regulators and examine their effect on the expression of a compilation of well-characterized HIF-1 direct target genes in order to assess the range of their involvement in the transcriptional response to hypoxia. Delineating the mode and the significance of the interaction between HIF-1 and its associated co-regulators may offer new attractive and specific targets for anticancer therapy. Full article

(This article belongs to the Special Issue Gene Regulation by HIFs during Hypoxia 2022)

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Figure 1
Schematic representation of HIF-1α and its interacting co-regulators. Positive (+) and negative (−) effectors of the p300/CBP-HIF-1α interaction are also shown. Brackets indicate the interacting region of HIF-1α, in cases that this has been experimentally defined. Residues, modification of which is known to affect an interaction, are also indicated. Genes directly regulated by HIF-1 are shown in bold. See <a href="#cells-12-00798-t001" class="html-table">Table 1</a> and text for details and relevant references. Full article ">Figure 2
Heatmap of KEGG pathway analysis of common genes between JMJD1A, CDK8, TRIM28, ZMYND8 or NPM1 (as indicated) and LIST A (left panel) or LIST B (right panel). The -log10 FDR values for the various pathways were plotted for each of the overlapping gene sets. An FDR cut-off of 0.05 was used for statistical significance. Clustering was performed using the Nearest Point algorithm with Euclidean distance from SciPy [<a href="#B117-cells-12-00798" class="html-bibr">117</a>]. The heatmaps were plotted using the python Plotly package (Plotly Technologies Inc. Collaborative data science, Montréal, QC, Canada, 2015. <a href="https://plot.ly" target="_blank">https://plot.ly</a> accessed on 31 January 2023). Full article ">Figure 3
(A,B): Venn diagrams depicting the overlap between List A (A) or List B (B) genes and the five co-activator-dependent gene sets as indicated. (C,D) Dot plots of KEGG pathway analysis of List A (C) or List B (D) genes that do not overlap with any coregulator-dependent gene sets. Full article ">

16 pages, 2941 KiB

Open AccessArticle

Maternal and Intrauterine Influences on Feto-Placental Growth Are Accompanied by Sexually Dimorphic Changes in Placental Mitochondrial Respiration, and Metabolic Signalling Pathways

by Esteban Salazar-Petres, Daniela Pereira-Carvalho, Jorge Lopez-Tello and Amanda N. Sferruzzi-Perri

Cells 2023, 12(5), 797; https://doi.org/10.3390/cells12050797 - 3 Mar 2023

Cited by 2 | Viewed by 2753

Abstract

Adverse maternal environments such as small size, malnutrition, and metabolic conditions are known to influence fetal growth outcomes. Similarly, fetal growth and metabolic alterations may alter the intrauterine environment and affect all fetuses in multiple gestation/litter-bearing species. The placenta is the site of [...] Read more.

Adverse maternal environments such as small size, malnutrition, and metabolic conditions are known to influence fetal growth outcomes. Similarly, fetal growth and metabolic alterations may alter the intrauterine environment and affect all fetuses in multiple gestation/litter-bearing species. The placenta is the site of convergence between signals derived from the mother and the developing fetus/es. Its functions are fuelled by energy generated by mitochondrial oxidative phosphorylation (OXPHOS). The aim of this study was to delineate the role of an altered maternal and/or fetal/intrauterine environment in feto-placental growth and placental mitochondrial energetic capacity. To address this, in mice, we used disruptions of the gene encoding phosphoinositol 3-kinase (PI3K) p110α, a growth and metabolic regulator to perturb the maternal and/or fetal/intrauterine environment and study the impact on wildtype conceptuses. We found that feto-placental growth was modified by a perturbed maternal and intrauterine environment, and effects were most evident for wildtype males compared to females. However, placental mitochondrial complex I+II OXPHOS and total electron transport system (ETS) capacity were similarly reduced for both fetal sexes, yet reserve capacity was additionally decreased in males in response to the maternal and intrauterine perturbations. These were also sex-dependent differences in the placental abundance of mitochondrial-related proteins (e.g., citrate synthase and ETS complexes), and activity of growth/metabolic signalling pathways (AKT and MAPK) with maternal and intrauterine alterations. Our findings thus identify that the mother and the intrauterine environment provided by littermates modulate feto-placental growth, placental bioenergetics, and metabolic signalling in a manner dependent on fetal sex. This may have relevance for understanding the pathways leading to reduced fetal growth, particularly in the context of suboptimal maternal environments and multiple gestation/litter-bearing species. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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Illustrative figure representing the crosses used in the study. WT = wild-type; α/+ = Heterozygous Pik3ca-D933A mice; F = female fetus; M = male fetus. Full article ">Figure 2
Fetal and placental growth of WT conceptuses in response to littermate and/or maternal p110α deficiency. Fetal weight (A), placenta weight (B), LZ weight (C), and fetal weight/LZ weight (D) in females and males on day 18 of pregnancy. Data are from WT fetuses generated by WT x WT, WT x α/+, and α/+ x WT parental crosses (n = 1–2 fetuses/sex/dam with 5–12 dams/group) and are displayed as individual data points with mean ± S.E.M. Data were analysed by one-way ANOVA with Tukey post hoc pairwise comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, pairwise comparison). LZ: labyrinthine zone. Full article ">Figure 3
Placental mitochondrial bioenergetics of WT conceptuses in response to littermate and/or maternal p110α deficiency. Oxygen consumption in the placental LZ associated with CILEAK (A), CIOXPHOS (B), CI + CIIOXPHOS (C), CII (D), total ETS (E), reserve capacity (F), CILEAK/total ETS (G), and CIOXPHOS/total ETS (H) for females and males on day 18 of pregnancy. Data are from WT fetuses generated by WT x WT, WT x α/+, and α/+ x WT parental crosses (n = 1–2 fetuses/sex/dam with 5–12 dams/group) and are displayed as individual data points with mean ± S.E.M. Data were analysed by one-way ANOVA with Tukey post hoc pairwise comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, pairwise comparison). Full article ">Figure 4
Protein abundance of mitochondrial complexes and key mitochondrial regulatory proteins in placental labyrinth of WT conceptuses in response to littermate and/or maternal p110α deficiency. Relative protein abundance of mitochondrial complexes in females (A) and males (B), as well as citrate synthase (C), PGC1α (D), PPARγ (E), and UCP2 (F) in females and males. Representative images from each antibody and Ponceau staining are included. Data are from 1 WT fetus per dam generated by WT x WT (n = 4), WT x α/+ (n = 5), and α/+ x WT (n = 5) parental crosses and are displayed as individual data points with mean ± S.E.M. Data were analysed by one-way ANOVA and Tukey post hoc pairwise comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, pairwise comparison). Full article ">Figure 5
Abundance of growth and metabolic signalling proteins in placental labyrinth of WT conceptuses in response to littermate and/or maternal p110α deficiency. Female (A) and male (B) representative images from each antibody immunodetection and Ponceau staining for phosphorylated and total AKT, AMPKα, MAPK 44/42, and P38 MAPK. Total AKT, AMPKα, MAPK 44/42, and P38 MAPK protein levels in females and males (C), and AKT, AMPKα, MAPK 44/42, and P38 MAPK phosphorylation levels as a ratio to total protein in females and males (D). Data are from 1 WT fetus per dam generated by WT x WT (n = 4), WT x α/+ (n = 5), and α/+ x WT (n = 5) parental crosses and are displayed as individual data points with mean ± S.E.M. Data were analysed by one-way ANOVA with Tukey post hoc pairwise comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, pairwise comparison). p = phosphorylated, t = total. Full article ">

16 pages, 901 KiB

Open AccessReview

No Time to Die—How Islets Meet Their Demise in Transplantation

by Atharva Kale and Natasha M. Rogers

Cells 2023, 12(5), 796; https://doi.org/10.3390/cells12050796 - 3 Mar 2023

Cited by 18 | Viewed by 3382

Abstract

Islet transplantation represents an effective treatment for patients with type 1 diabetes mellitus (T1DM) and severe hypoglycaemia unawareness, capable of circumventing impaired counterregulatory pathways that no longer provide protection against low blood glucose levels. The additional beneficial effect of normalizing metabolic glycaemic control [...] Read more.

Islet transplantation represents an effective treatment for patients with type 1 diabetes mellitus (T1DM) and severe hypoglycaemia unawareness, capable of circumventing impaired counterregulatory pathways that no longer provide protection against low blood glucose levels. The additional beneficial effect of normalizing metabolic glycaemic control is the minimisation of further complications related to T1DM and insulin administration. However, patients require allogeneic islets from up to three donors, and the long-term insulin independence is inferior to that achieved with solid organ (whole pancreas) transplantation. This is likely due to the fragility of islets caused by the isolation process, innate immune responses following portal infusion, auto- and allo-immune-mediated destruction and β-cell exhaustion following transplantation. This review covers the specific challenges related to islet vulnerability and dysfunction that affect long-term cell survival following transplantation. Full article

(This article belongs to the Special Issue Islet Function and Dysfunction in Diabetes Mellitus and Transplantation)

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13 pages, 2073 KiB

Open AccessArticle

Methylglyoxal-Modified Albumin Effects on Endothelial Arginase Enzyme and Vascular Function

by Ebaa M. Alzayadneh, Alia Shatanawi, R. William Caldwell and Ruth B. Caldwell

Cells 2023, 12(5), 795; https://doi.org/10.3390/cells12050795 - 3 Mar 2023

Cited by 3 | Viewed by 2345

Abstract

Advanced glycation end products (AGEs) contribute significantly to vascular dysfunction (VD) in diabetes. Decreased nitric oxide (NO) is a hallmark in VD. In endothelial cells, NO is produced by endothelial NO synthase (eNOS) from L-arginine. Arginase competes with NOS for L-arginine to produce [...] Read more.

Advanced glycation end products (AGEs) contribute significantly to vascular dysfunction (VD) in diabetes. Decreased nitric oxide (NO) is a hallmark in VD. In endothelial cells, NO is produced by endothelial NO synthase (eNOS) from L-arginine. Arginase competes with NOS for L-arginine to produce urea and ornithine, limiting NO production. Arginase upregulation was reported in hyperglycemia; however, AGEs’ role in arginase regulation is unknown. Here, we investigated the effects of methylglyoxal-modified albumin (MGA) on arginase activity and protein expression in mouse aortic endothelial cells (MAEC) and on vascular function in mice aortas. Exposure of MAEC to MGA increased arginase activity, which was abrogated by MEK/ERK1/2 inhibitor, p38 MAPK inhibitor, and ABH (arginase inhibitor). Immunodetection of arginase revealed MGA-induced protein expression for arginase I. In aortic rings, MGA pretreatment impaired acetylcholine (ACh)-induced vasorelaxation, which was reversed by ABH. Intracellular NO detection by DAF-2DA revealed blunted ACh-induced NO production with MGA treatment that was reversed by ABH. In conclusion, AGEs increase arginase activity probably through the ERK1/2/p38 MAPK pathway due to increased arginase I expression. Furthermore, AGEs impair vascular function that can be reversed by arginase inhibition. Therefore, AGEs may be pivotal in arginase deleterious effects in diabetic VD, providing a novel therapeutic target. Full article

(This article belongs to the Special Issue Diabetes and Its Complications: Cellular and Molecular Mechanism and New Therapeutic Opportunities)

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Figure 1
Elevation of arginase activity by exposure of MAEC to MGA (100 µM, 24 h) that was abrogated by pretreatment of cells with SB (10 µM), PD (10 µM), and ABH (1 mM). * p < 0.01 control vs. MGA, # p ≤ 0.0001 MGA vs. MGA + SB or PD, € p < 0.001 MGA vs. MGA + ABH. Values are expressed as means ± SE from 5 independent experiments carried out in triplicates. Full article ">Figure 2
Effect of MGA on arginase expression. (A) Immunoblotting analysis of arginase I showing increased expression due to MGA treatment (100 μM, 24 h) as compared to BSA (100 μM, 24 h). (B) Immunoblotting analysis of arginase II showing no change in expression after MGA treatment (100 μM, 24 h) as compared to BSA (100 μM, 24 h). Values are expressed as means ± SE from 5 independent experiments carried out in triplicates. * p < 0.05 vs. MGA. Full article ">Figure 3
Fluorescent microscopy images of fixed MAECs in 2% formaldehyde after incubation with DAF-2DA (5 μM, 40 min). All cells were pretreated with either BSA (100 µM, 24 h) in (A–D), or MGA (100 µM, 24 h) in (E–H). Panels of different cells treatments are as follows: (A) BSA without ACh induction, (B) BSA with ACh induction, (C) BSA with ACh induction and pretreated with L-NAME, (D) BSA with ACh induction and pretreated with ABH, (E) MGA without ACh induction, (F) MGA with ACh induction, (G) MGA with ACh induction and pretreated with L-NAME, (H) MGA with ACh induction and pretreated with ABH. Bar: 20 µm. Fluorescence reflects NO production, which was more intense in cells induced with acetylcholine than in cells without acetylcholine. MGA-treated cells had lower fluorescence, indicating lower NO production even with acetylcholine induction (F); however, when pretreated with ABH (H), fluorescence induced by ACh was intensified and NO was restored to a level higher than ACh-induced, MG-treated cells (F). L-NAME inhibitor abolished ACh-induced fluorescence, reflecting inhibition of eNOS activity and NO production. A quantification of DAF fluorescence intensity in the different treatment conditions is demonstrated in (I). Values are expressed as percentage of BSA (control); analyzed images were obtained from 3 independent experiments. * p < 0.05. ACh, acetylcholine (1 µM); L-NAME, N (G)-nitro-L-arginine methyl ester (1 µM); ABH, boronic acids 2(S)-amino-6-boronohexanoic acid (1 mM). Full article ">Figure 4
Dose-response relaxation curves for (A) endothelium-dependent vasorelaxant acetylcholine (ACh) in phenylephrine (1 μM)-preconstricted aortas from mice; (B) endothelium-independent vasorelaxant sodium nitroprusside (SNP) in phenylephrine (1 μM)-preconstricted aortas from mice. Dashed black line indicates responses in control conditions (BSA, 100 μM, 24 h); solid red line indicates responses in MGA-pretreated aortas (100 μM, 24 h); solid blue line indicates responses in MGA-treated aortas pretreated with ABH (1 mM, 24 h). n = 3 in each group; * p < 0.05 MGA vs. control or MGA + ABH. Full article ">Figure 5
A schematic diagram of AGE/RAGE interaction with arginase enzyme and its effects on endothelial cells and vascular function. Acetylcholine (ACh) stimulates eNOS to produce NO, which is released from endothelial cells to smooth muscle cells, inducing vasorelaxation. Circulating AGE binding to RAGE activates NADPH oxidase, producing ROS, and stimulates ERK1/2 and P38 MAPK, which induce activity/expression of arginase I enzyme. Upregulation of arginase I limits both arginine and NO production by eNOS. Limited arginine leads to uncoupling of eNOS, which further limits NO production and produces superoxide (O2.) that reacts with NO, generating peroxinitrite (ONOO-) and further reducing NO. Arginase activation produces urea and L-ornithine that is used to produce L-proline and polyamines involved in collagen formation and proliferation, respectively. Arginase II is expressed in mitochondria and may be regulated by AGE by a different mechanism not involving its expression. Abbreviations: AGE: advanced glycation end products, RAGE: receptor for advanced glycation end products, ROS: reactive oxygen species, ABH: arginase inhibitor, L-NAME: eNOS inhibitor. Some components of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (<a href="https://creativecommons.org/licenses/by/3.0/" target="_blank">https://creativecommons.org/licenses/by/3.0/</a>) (accessed on 3 November 2022). Full article ">

14 pages, 20243 KiB

Open AccessArticle

An Innovative Drug Repurposing Approach to Restrain Endometrial Cancer Metastatization

by Federica Torricelli, Elisabetta Sauta, Veronica Manicardi, Vincenzo Dario Mandato, Andrea Palicelli, Alessia Ciarrocchi and Gloria Manzotti

Cells 2023, 12(5), 794; https://doi.org/10.3390/cells12050794 - 3 Mar 2023

Cited by 4 | Viewed by 2364

Abstract

Background: Endometrial cancer (EC) is the most common gynecologic tumor and the world’s fourth most common cancer in women. Most patients respond to first-line treatments and have a low risk of recurrence, but refractory patients, and those with metastatic cancer at diagnosis, remain [...] Read more.

Background: Endometrial cancer (EC) is the most common gynecologic tumor and the world’s fourth most common cancer in women. Most patients respond to first-line treatments and have a low risk of recurrence, but refractory patients, and those with metastatic cancer at diagnosis, remain with no treatment options. Drug repurposing aims to discover new clinical indications for existing drugs with known safety profiles. It provides ready-to-use new therapeutic options for highly aggressive tumors for which standard protocols are ineffective, such as high-risk EC. Methods: Here, we aimed at defining new therapeutic opportunities for high-risk EC using an innovative and integrated computational drug repurposing approach. Results: We compared gene-expression profiles, from publicly available databases, of metastatic and non-metastatic EC patients being metastatization the most severe feature of EC aggressiveness. A comprehensive analysis of transcriptomic data through a two-arm approach was applied to obtain a robust prediction of drug candidates. Conclusions: Some of the identified therapeutic agents are already successfully used in clinical practice to treat other types of tumors. This highlights the potential to repurpose them for EC and, therefore, the reliability of the proposed approach. Full article

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Comparison of metastatic and non-metastatic EC transcriptomic profiles: (A) Computational framework overview of the integrative drug repurposing pipeline. (B) Table summarizing metastasis-based re-classification of Type 1 EC samples. (C) Volcano plot and a heat map showing significantly UP and DOWN-regulated genes constituting dGES (FDR < 0.05). (D,E) Functional enrichment analysis of dGES, Gene Ontology-Biological Processes (GO_BO), and Gene Ontology-Molecular Functions, respectively. Full article ">Figure 2
Pathway-based analysis of EC: (A,B) Heatmaps representing the distribution of the PDS values from GO_BP and GO_MF, respectively. (C) The distribution of PDS values identifies the PDSM value. (D,E) Graphical representation of macro-categories encompassing the pathways found significantly altered by Pathifier, relative to GO_BP and GO_MF respectively. (F–I) An example of how the cloud of points representing NM and M samples, and the principal curve describing its variation are projected onto the three leading principal components. Four of the most representative pathways found altered are shown. Full article ">Figure 3
Drug repurposing: (A–C) Drugs identified through cMap querying, classified by CS, PCL, and FDA status. (D) Example of the dataset containing the list and all information relative to compounds identified through DGIdb querying. (E) Representation of the number of specific drugs found for each DEG in the DGIdb. Full article ">Figure 4
Validation of the efficacy of the identified drugs: (A) Chord plot summarizing the classification of the drug candidates obtained from the signature-matching and pathway-based repurposing approaches. Edge width is proportional to the number of drugs that belong to a certain drug class, highlighted by different colors and cited in the legend. (B) Venn diagram showing the molecules identified by both approaches. (C) Histograms showing IC50 values for each candidate drug found in the GDSC database, for EC cell lines. Next to each bar is the reported specific value of the IC50 for the corresponding cell line. Full article ">

26 pages, 3795 KiB

Open AccessReview

Gut-Microbiota-Derived Metabolites Maintain Gut and Systemic Immune Homeostasis

by Juanjuan Wang, Ningning Zhu, Xiaomin Su, Yunhuan Gao and Rongcun Yang

Cells 2023, 12(5), 793; https://doi.org/10.3390/cells12050793 - 2 Mar 2023

Cited by 158 | Viewed by 11127

Abstract

The gut microbiota, including bacteria, archaea, fungi, viruses and phages, inhabits the gastrointestinal tract. This commensal microbiota can contribute to the regulation of host immune response and homeostasis. Alterations of the gut microbiota have been found in many immune-related diseases. The metabolites generated [...] Read more.

The gut microbiota, including bacteria, archaea, fungi, viruses and phages, inhabits the gastrointestinal tract. This commensal microbiota can contribute to the regulation of host immune response and homeostasis. Alterations of the gut microbiota have been found in many immune-related diseases. The metabolites generated by specific microorganisms in the gut microbiota, such as short-chain fatty acids (SCFAs), tryptophan (Trp) and bile acid (BA) metabolites, not only affect genetic and epigenetic regulation but also impact metabolism in the immune cells, including immunosuppressive and inflammatory cells. The immunosuppressive cells (such as tolerogenic macrophages (tMacs), tolerogenic dendritic cells (tDCs), myeloid-derived suppressive cells (MDSCs), regulatory T cells (Tregs), regulatory B cells (Breg) and innate lymphocytes (ILCs)) and inflammatory cells (such as inflammatory Macs (iMacs), DCs, CD4 T helper (Th)1, CD4Th2, Th17, natural killer (NK) T cells, NK cells and neutrophils) can express different receptors for SCFAs, Trp and BA metabolites from different microorganisms. Activation of these receptors not only promotes the differentiation and function of immunosuppressive cells but also inhibits inflammatory cells, causing the reprogramming of the local and systemic immune system to maintain the homeostasis of the individuals. We here will summarize the recent advances in understanding the metabolism of SCFAs, Trp and BA in the gut microbiota and the effects of SCFAs, Trp and BA metabolites on gut and systemic immune homeostasis, especially on the differentiation and functions of the immune cells. Full article

(This article belongs to the Collection Gut Microbiota-Derived Metabolites and Host Gut-Brain Communication)

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The gut microbiota maintains the homeostasis of the gut and systemic immune system through the metabolites. Metabolites from the gut microbiota such as short-chain fatty acids (SCFAs), tryptophan metabolites (Trps), and bile acid metabolites (BAs) promote the differentiation and function of immune-suppressive cells and inhibit the inflammatory cells. DC, dendritic cell; iMac, inflammatory macrophage; Th1, T helper 1; Th2, T helper 2; Th17, T helper 17; NK, natural killer cell; NKT, natural killer T cell; MDSC, myeloid-derived suppressor cell; tMac, tolerogenic macrophage; tDC, tolerogenic dendritic cell; Treg, regulatory T cells; Tr1, type 1 regulatory T cells; Breg, regulatory B cell; ILC3, innate lymphoid cell 3; CD8αα, CD4+CD8αα+ intestinal intraepithelial lymphocyte. Full article ">Figure 2
Regulation of gut-microbiota-derived metabolites in different immune cells. Gut-microbiota-derived metabolites such as SCFAs, Trp and BA metabolites can promote differentiation and function of immune-suppressive cells (such as tolerogenic macrophages (tMacs), tolerogenic dendritic cells (tDCs), myeloid-derived suppressor cells (MDSCs), T regulatory Foxp3+ cells (Treg), type 1 regulatory T cells (Tr1), B regulatory cells (Breg), innate lymphoid cells (ILCs) and CD4+CD8+αα cells), and inhibit inflammatory cells (such as CD4+T helper (Th1), CD4+Th2, CD4+Th17, CD8, B cells, natural killer (NK) cells, NKT cells and neutrophils). Full article ">Figure 3
Gut-microbiota-derived metabolites promote the differentiation and function of tolerogenic macrophages through the receptors expressed in the macrophages such as short-chain fatty acids (SCFAs) through membrane receptors such as GPR43, tryptophan metabolites (Trps) through the AhR nuclear receptor and bile acid metabolites (BAs) through the TGR5 membrane receptor and/or FXR nuclear receptor. iMac, inflammatory macrophages; tMacs, tolerogenic macrophages; TGR5, Takeda G protein-coupled receptor 5; FXR, farnesoid X receptor; PXR, pregnane X receptor; NCOR1, nuclear receptor corepressor 1; cAMP, adenosine monophosphate; PKA, protein kinase A; SOCS3, suppressor of cytokine signaling 3; CYP450, cytochrome P450; FGF19, fibroblast growth factor 19; NLRP3, NOD-like receptor thermal protein domain associated protein 3; GPR43, G-protein coupled receptor 43; HDAC, histone deacetylase; NF-κB, nuclear factor-kappa B; PI3K, phosphatidylinositol 3 kinase; Akt, protein kinase B; mTOR, mammalian target of rapamycin; TLR4, Toll-like receptor 4; 3-HAA, 3-hydroxyanthranilic acid; SP1, specificity protein 1; His, histamine; AhR, aryl hydrocarbon receptor; Rac1, ras-related C3 botulinum toxin substrate 1; mIL-1β, mature interleukin -1β; TNFα, tumor necrosis factor α. Full article ">Figure 4
Gut-microbiota-derived metabolites promote differentiation of Treg, Tr1 and RORγt+ Treg cells. SCFAs, short-chain fatty acids; Trps, tryptophan metabolites; BAs, bile acid metabolites; GPR43, G-protein coupled receptor 43; HDAC, histone deacetylase; Foxp3, forkhead box protein p3; IDO, indoleamine 2,3-dioxygenase 1; Aldh1A2, aldehyde dehydrogenase 1A2; AhR, aryl hydrocarbon receptor; 3-HAA, 3-hydroxyanthranilic acid; Kyn, kynurenine; mitoROS, mitochondrial reactive oxygen species; NR4A1, nuclear receptor subfamily 4, group A, member 1; RORγt, retinoid-related orphan receptor-γt; VDR, vitamin D receptor; FXR, farnesoid X receptor; LCA, lithocholic acid; DCA, deoxycholic acid; DC, dendritic cells; Tr1, type 1 regulatory T cells. Full article ">

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Cells, Volume 12, Issue 5 (March-1 2023) – 147 articles

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