Cells

24 pages, 7743 KiB

Open AccessArticle

HOTAIR Participation in Glycolysis and Glutaminolysis Through Lactate and Glutamate Production in Colorectal Cancer

by Laura Cecilia Flores-García, Verónica García-Castillo, Eduardo Pérez-Toledo, Samuel Trujano-Camacho, Oliver Millán-Catalán, Eloy Andrés Pérez-Yepez, Jossimar Coronel-Hernández, Mauricio Rodríguez-Dorantes, Nadia Jacobo-Herrera and Carlos Pérez-Plasencia

Cells 2025, 14(5), 388; https://doi.org/10.3390/cells14050388 - 6 Mar 2025

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Abstract

Metabolic reprogramming plays a crucial role in cancer biology and the mechanisms underlying its regulation represent a promising study area. In this regard, the discovery of non-coding RNAs opened a new regulatory landscape, which is in the early stages of investigation. Using a [...] Read more.

Metabolic reprogramming plays a crucial role in cancer biology and the mechanisms underlying its regulation represent a promising study area. In this regard, the discovery of non-coding RNAs opened a new regulatory landscape, which is in the early stages of investigation. Using a differential expression model of HOTAIR, we evaluated the expression level of metabolic enzymes, as well as the metabolites produced by glycolysis and glutaminolysis. Our results demonstrated the regulatory effect of HOTAIR on the expression of glycolysis and glutaminolysis enzymes in colorectal cancer cells. Specifically, through the overexpression and inhibition of HOTAIR, we determined its influence on the expression of the enzymes PFKFB4, PGK1, LDHA, SLC1A5, GLUD1, and GOT1, which had a direct impact on lactate and glutamate production. These findings indicate that HOTAIR plays a significant role in producing “oncometabolites” essential to maintaining the bioenergetics and biomass necessary for tumor cell survival by regulating glycolysis and glutaminolysis. Full article

(This article belongs to the Special Issue Non-Coding and Coding RNAs in Targeted Cancer Therapy)

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23 pages, 850 KiB

Open AccessReview

Insights into the Roles of GLP-1, DPP-4, and SGLT2 at the Crossroads of Cardiovascular, Renal, and Metabolic Pathophysiology

by Melania Gaggini, Laura Sabatino, Adrian Florentin Suman, Kyriazoula Chatzianagnostou and Cristina Vassalle

Cells 2025, 14(5), 387; https://doi.org/10.3390/cells14050387 - 6 Mar 2025

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Abstract

In recent years, new drugs for the treatment of type 2 diabetes (T2D) have been proposed, including glucagon-like peptide 1 (GLP-1) agonists or sodium–glucose cotransporter 2 (SGLT2) inhibitors and dipeptidyl peptidase-4 (DPP-4) inhibitors. Over time, some of these agents (in particular, GLP-1 agonists [...] Read more.

In recent years, new drugs for the treatment of type 2 diabetes (T2D) have been proposed, including glucagon-like peptide 1 (GLP-1) agonists or sodium–glucose cotransporter 2 (SGLT2) inhibitors and dipeptidyl peptidase-4 (DPP-4) inhibitors. Over time, some of these agents (in particular, GLP-1 agonists and SGLT2 inhibitors), which were initially developed for their glucose-lowering actions, have demonstrated significant beneficial pleiotropic effects, thus expanding their potential therapeutic applications. This review aims to discuss the mechanisms, pleiotropic effects, and therapeutic potential of GLP-1, DPP-4, and SGLT2, with a particular focus on their cardiorenal benefits beyond glycemic control. Full article

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13 pages, 3592 KiB

Open AccessArticle

The Beneficial Role of the Thyroid Hormone Receptor Beta 2 (thrb2) in Facilitating the First Feeding and Subsequent Growth in Medaka as Fish Larval Model

by Jiaqi Wu, Ke Lu, Ruipeng Xie, Chenyuan Zhu, Qiyao Luo and Xu-Fang Liang

Cells 2025, 14(5), 386; https://doi.org/10.3390/cells14050386 - 6 Mar 2025

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Abstract

During the early growth stages of fish larvae, there are significant challenges to their viability, so improving their visual environment is essential to promoting their growth and survival. Following the successful knockout of thyroid hormone receptor beta 2 (thrb2) using Clustered [...] Read more.

During the early growth stages of fish larvae, there are significant challenges to their viability, so improving their visual environment is essential to promoting their growth and survival. Following the successful knockout of thyroid hormone receptor beta 2 (thrb2) using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, there was an increase in the expression of UV opsin (short-wave-sensitive 1, sws1), while the expression of other cone opsins was significantly decreased. Further analysis of the retinal structure demonstrated that the thrb2 knockout resulted in an increased lens thickness and a decreased thickness of the ganglion cell layer (GCL), outer plexiform layer (OPL), and outer nuclear layer (ONL) in the retina. The slowing down of swimming speed under light conditions in thrb2^−/− may be related to the decreased expression of phototransduction-related genes such as G protein-coupled receptor kinase 7a (grk7a), G protein-coupled receptor kinase 7b (grk7b), and phosphodiesterase 6c (pde6c). Notably, thrb2^−/− larvae exhibited a significant increase in the amount and proportion of first feeding, and their growth rate significantly exceeded that of wild-type controls during the week after feeding. This observation suggests that although the development of the retina may be somewhat affected, thrb2^−/− larvae show positive changes in feeding behaviour and growth rate, which may be related to their enhanced ability to adapt to their environment. These results provide novel insights into the function of the thrb2 gene in the visual system and behaviour and may have implications in areas such as fish farming and genetic improvement. Full article

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24 pages, 7156 KiB

Open AccessArticle

Selective Azapeptide CD36 Ligand MPE-298 Regulates oxLDL-LOX-1-Mediated Inflammation and Mitochondrial Oxidative Stress in Macrophages

by Mukandila Mulumba, Catherine Le, Emmanuelle Schelsohn, Yoon Namkung, Stéphane A. Laporte, Maria Febbraio, Marc J. Servant, Sylvain Chemtob, William D. Lubell, Sylvie Marleau and Huy Ong

Cells 2025, 14(5), 385; https://doi.org/10.3390/cells14050385 - 6 Mar 2025

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Abstract

Macrophage mitochondrial dysfunction, caused by oxidative stress, has been proposed as an essential event in the progression of chronic inflammation diseases, such as atherosclerosis. The cluster of differentiation-36 (CD36) and lectin-like oxLDL receptor-1 (LOX-1) scavenger receptors mediate macrophage uptake of oxidized low-density lipoprotein [...] Read more.

Macrophage mitochondrial dysfunction, caused by oxidative stress, has been proposed as an essential event in the progression of chronic inflammation diseases, such as atherosclerosis. The cluster of differentiation-36 (CD36) and lectin-like oxLDL receptor-1 (LOX-1) scavenger receptors mediate macrophage uptake of oxidized low-density lipoprotein (oxLDL), which contributes to mitochondrial dysfunction by sustained production of mitochondrial reactive oxygen species (mtROS), as well as membrane depolarization. In the present study, the antioxidant mechanisms of action of the selective synthetic azapeptide CD36 ligand MPE-298 have been revealed. After binding to CD36, MPE-298 was rapidly internalized by and simultaneously induced CD36 endocytosis through activation of the Lyn and Syk (spleen) tyrosine kinases. Within this internalized complex, MPE-298 inhibited oxLDL/LOX-1-induced chemokine ligand 2 (CCL2) secretion, abolished the production of mtROS, and prevented mitochondrial membrane potential depolarization in macrophages. This occurred through the inhibition of the multiple-component enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) by oxLDL-activated LOX-1, which was further supported by the reduced recruitment of the p47phox subunit and small GTPase (Rac) 1/2/3 into the plasma membrane. A new mechanism for alleviating oxLDL-induced oxidative stress and inflammation in macrophages is highlighted using the CD36 ligand MPE-298. Full article

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Figure 1
Intracellular localization of MPE-298 following its internalization in macrophages. (A) Representative immunofluorescence images of fixed RAW264.7 and J774A.1 cells treated with fluorescent ATTO-465-MPE298. (B,C) Relative fluorescent units (RFU) of ATT-465-MPE-298 kinetics of internalization after PBS (total) or acid wash (internalized) of the cells in RAW264.7 and J774A.1 cells, respectively. (D,E) Intracellular localization of ATTO-465-MPE-298 with late-endosomal and lysosomal markers, respectively. (F) Uptake of Dil-oxLDL in RAW264.7-and J774A.1 cell lines. Scale bar size: 20 µm. Full article ">Figure 2
Intracellular localization of the CD36-MPE-298 complex following its internalization in macrophages. (A,B) mCD36-GFPspark-transfected RAW264.7 cells were stained with Rab7 (late endosome) and Lamp1 (lysosome) markers, respectively. Pearson’s correlation coefficients are presented as a function of time. Data are presented as the mean ± SEM. A one-way ANOVA test with Dunnett’s comparison post-test was performed. * p < 0.05, ** p < 0.01, and *** p < 0.005 vs. vehicle. (C) Kinetics of CD36 internalization in a BRET-based assay in J774A.1 cells transiently co-expressing mCD36-RlucII and rGFP-CAAX in the presence of MPE-298 (100 nM) or oxLDL (25 μg/mL) at different times. (D) Dose–response of MPE-298 in a BRET-based CD36 internalization assay in co-transfected J774A.1 macrophages. (E) Dose–response curves of MPE-298 analogs 3f, 7e, and 21 using a BRET-based CD36 internalization in co-transfected J774A.1 cells. Scale bar size: 50 µm. Full article ">Figure 3
CD36-mediated MPE-298 and oxLDL intracellular signaling pathways in macrophages cell lines. (A) Representative Western blots of kinetic studies of total and phosphorylated Src (Tyr 416), Lyn (Tyr 397), and Syk (Tyr 348) kinases in RAW264.7 macrophages treated with MPE-298 or oxLDL. Relative quantification values are expressed as the ratio of phosphorylated/total protein normalized to the control. Data are expressed as the mean ± SEM of the fold change over the control (CTL) (n = 3 different experiments). A one-way ANOVA test with Dunnett’s comparison post-test was performed. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. CTL. (B) Internalization of ATTO-465-MPE-298 (500 nM) in the presence of PP1, PP2 (Src inhibitors), or cytochalasin D (Cyto D, inhibitor of actin polymerization). Data are presented as the mean relative fluorescence units (RFUs) ± SEM (n = 3 experiments performed in triplicate). (C,D) BRET-based assay of oxLDL- or MPE-298-induced CD36 endocytosis in mCD36-RlucII/rGFP-CAAX co-transfected J774A.1 cells exposed to PP1, Cyto D, and piceatannol (Pice) (n = 3 independent experiments performed in triplicate). Data are expressed as the percentage of DMSO/vehicle. Data are presented as mean ± SEM. A two-way ANOVA test with Tukey’s multiple comparison post-test was performed. ** p < 0.01 and *** p < 0.001 vs. DMSO/vehicle. & p < 0.05 and && p < 0.01 vs. DMSO/oxLDL. Full article ">Figure 4
MPE-298 modulatory effects on mitochondrial oxidative stress elicited by oxLDL in murine RAW264.7 cell lines macrophages. (A) CCL2 secretion in the supernatants of cells exposed to different concentrations of MPE-298 or oxLDL for 24 h. Data are presented as the mean ± SEM (n = 3 experiments performed in triplicate). (B) Mitochondrial reactive oxygen species production (mtROS) and (C) mitochondrial membrane potential (ΔΨM) in RAW264.7 cells treated with different concentrations of MPE-298 or oxLDL for 4 h. (D) Inhibitory effect of MPE-298 (100 nM) on oxLDL-induced CCL2 secretion in cells stimulated with different concentrations of oxLDL for 24 h. (E,F) MPE-298 dose–response inhibition of oxLDL (25 μg/mL)-induced mtROS production and ΔΨM loss, respectively. (G) CD36-mediated inhibitory effect of MPE-298 on CCL2 secretion in the presence or absence of the CD36 inhibitor, SSO (100 μM). (H) BRET-based assay of oxLDL- and MPE-298-induced CD36 endocytosis in the absence or presence of SSO in mCD36-RlucII/rGFP-CAAX co-transfected J774A.1 cells. Data in (A,D,G) are presented as mean ± SEM; data in (B,C,E,F,H) are expressed as the percentage of vehicle and presented as mean ± SEM. n = 3 independent experiments, each conducted in triplicate. In (A–C,E,F), a one-way ANOVA test with Dunnett’s comparison post-test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. vehicle. # p < 0.05 and ## p < 0.01 vs. oxLDL/CTL. In (D,G,H) a two-way ANOVA test with Tukey’s multiple comparison post-test was performed. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (CTL)/vehicle or DMSO/vehicle; ## p < 0.01 and ### p < 0.001 vs. oxLDL/vehicle or oxLDL/DMSO. Full article ">Figure 5
MPE-298 attenuates mitochondrial oxidative stress induced by oxLDL by mitigating LOX-1 signaling pathways in murine RAW264.7 macrophage cells. The assessment of MPE-298’s inhibitory effects on oxLDL-induced mtROS production in the presence or absence of the pharmacological inhibitors of endocytosis (A) PP1 (3 μg/mL), cytochalasin D (Cyto D, 2 μg/mL), and (B) sulfo-N-succinimidyl oleate (SSO, 100 μM) or 2-bromopalmitate (2-BP, 100 μM). (C) Assessment of the BRET-based assay following oxLDL- and MPE-298-induced CD36 endocytosis in mCD36-RlucII/rGFP-CAAX co-transfected J774A.1 cells exposed to 2-BP. (D) Effect of MPE-298 inhibition on ox-LDL-induced ΔΨM or the presence of the pharmacological inhibitors PP1, Cyto D, SSO, or 2-BP. (E) Western blots of the kinetic studies of the total and phosphorylated JNK (Thr183/Tyr185) and p66Shc (Ser36) in RAW264.7 macrophages treated with MPE-298 (100 nM) and oxLDL (25 μg/mL). Representative blots of 3 experiments. (F,G) Inhibitory effect of MPE-298 on oxLDL-induced mtROS production and ΔΨM loss, respectively, in the presence or absence of the pharmacological inhibitor of LOX-1, BI-0115 (BI, 5 μM). (H) The BRET-based assay in mCD36-RlucII/rGFP-CAAX co-transfected J774A.1 cells exposed to oxLDL and MPE-298 in the absence or presence of BI. All data are expressed as the mean ± SEM of three independent experiments each conducted in triplicate. Data are expressed either as the percentage of vehicle (C,H) or as the fold change relative to the vehicle and presented as the mean ± SEM. (A,B,D,F,G) A one-way ANOVA test with Dunnett’s comparison post-test was performed. * p < 0.05 and ** p < 0.01 vs. vehicle (no-treatment); # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 vs. oxLDL-treated; & p < 0.05 and && p < 0.01 vs. MPE-298/oxLDL. (C,H) A two-way ANOVA test with Tukey’s multiple comparison post-test was performed. ** p < 0.01 and *** p < 0.001 vs. DMSO/vehicle; && p < 0.01 and &&& p < 0.001 2-BP vs. DMSO. Full article ">Figure 6
MPE-298 attenuates oxLDL-induced mitochondrial damage in a CD36-dependent manner in primary M1 bone-marrow-derived macrophages (BMDM). (A) Western blots of the total and phosphorylated Src (Tyr416), Lyn (Tyr397), and JNK (Thr183/Tyr185) kinases and p66Shc (Ser36) M1-phenotype BMDMs. Differentiated cells from wild-type (WT) and CD36-KO mice were preincubated with or without the LOX-1 inhibitor BI-0115 (5 μM), followed by treatment with MPE-298 (100 nM) or oxLDL (25 μg/mL), or with a combination of MPE-298 and oxLDL. Representative blots of two experiments. The effect of MPE-298 on oxLDL-induced (B) mtROS production and (C) ΔΨM loss in the absence or presence of BI-0115. The experiments are expressed as the mean ± SEM of two independent experiments each conducted in triplicate. Data were assessed as the fold change relative to vehicle and are presented as the mean ± SEM. Two-way ANOVA test with Tukey’s multiple comparison post-test was performed. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. vehicle; # p < 0.05 and ### p < 0.001 vs. oxLDL-treated cells. Full article ">Figure 7
MPE-298 inhibits LOX-1-mediated NOX2 activation by oxLDL in the plasma membrane. of RAW264.7 cells (A) Western blots of the cell lysates of MPE-298- or oxLDL-treated cells in the presence or absence of the LOX-1 inhibitor BI-0115 (5 μM) using antibodies against the total and phosphorylated JNK (Thr183/Tyr185) and p66Shc (Ser36). Representative blots of three experiments. Data are expressed as the fold change relative to the vehicle and presented as the mean ± SEM of the ratio of phosphorylated/total protein. (B) Western blots of the plasma membrane and cytosolic fractions from treated RAW264.7 cells using antibodies for p47phox and RAC1/2/3. Representative blots of three independent experiments. Data are expressed as the fold change over the vehicle and presented as the mean ± SEM. A one-way ANOVA test with Dunnett’s comparison post-test was performed. * p < 0.05; ** p < 0.01, and *** p < 0.001 vs. vehicle; # p < 0.05, ### p < 0.001 vs. oxLDL-treated. (C) Western blots of mitochondria and cytosolic membrane fractions from cells treated with MPE-298 (100 nM) or oxLDL (25 μg/mL). (n = 3 independent experiments). Full article ">Figure 8
Molecular mechanisms underlying CD36-mediated MPE-298 regulation of oxidative stress induced by oxLDL in macrophages. Binding of azapeptide MPE-298 induces macrophage CD36 endocytosis through the activation of the Src/Syk kinases pathway and the depalmitoylation of the receptor. The internalized MPE-298/CD36 complex prevents oxLDL/LOX-1-mediated recruitment of subunit p47phox and Rac1/2/3 GTPase, which are essential for the formation of the NADPH oxidase 2 (NOX2) complex, disrupting the oxLDL/LOX-1/NOX2-induced activation and translocation of JNK and p66Shc into the mitochondria. This prevents mitochondrial ROS production. ΔΨM: mitochondria membrane potential; EEs: early endosomes; JNK: c-Jun N-terminal protein kinase; LOX-1: lectin-like oxidized low-density lipoprotein receptor 1; NOX2: NADPH oxidase 2; oxLDL: oxidized low-density lipoprotein; p66Shc:Shc: protein 66 Src homology 2 domain (SH2) at C-terminal; ROS: reactive oxygen species; Syk: spleen tyrosine kinase. Created in BioRender. Mulumba, M. (2024) <a href="https://BioRender.com/k26k106" target="_blank">https://BioRender.com/k26k106</a>, (accessed on 28 February 2025). Full article ">

32 pages, 1292 KiB

Open AccessReview

Tryptophan and Its Metabolite Serotonin Impact Metabolic and Mental Disorders via the Brain–Gut–Microbiome Axis: A Focus on Sex Differences

by Mengyang Xu, Ethan Y. Zhou and Haifei Shi

Cells 2025, 14(5), 384; https://doi.org/10.3390/cells14050384 - 6 Mar 2025

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Abstract

The crisis of metabolic and mental disorders continues to escalate worldwide. A growing body of research highlights the influence of tryptophan and its metabolites, such as serotonin, beyond their traditional roles in neural signaling. Serotonin acts as a key neurotransmitter within the brain–gut–microbiome [...] Read more.

The crisis of metabolic and mental disorders continues to escalate worldwide. A growing body of research highlights the influence of tryptophan and its metabolites, such as serotonin, beyond their traditional roles in neural signaling. Serotonin acts as a key neurotransmitter within the brain–gut–microbiome axis, a critical bidirectional communication network affecting both metabolism and behavior. Emerging evidence suggests that the gut microbiome regulates brain function and behavior, particularly through microbial influences on tryptophan metabolism and the serotonergic system, both of which are essential for normal functioning. Additionally, sex differences exist in multiple aspects of serotonin-mediated modulation within the brain–gut–microbiome axis, affecting feeding and affective behaviors. This review summarizes the current knowledge from human and animal studies on the influence of tryptophan and its metabolite serotonin on metabolic and behavioral regulation involving the brain and gut microbiome, with a focus on sex differences and the role of sex hormones. We speculate that gut-derived tryptophan and serotonin play essential roles in the pathophysiology that modifies neural circuits, potentially contributing to eating and affective disorders. We propose the gut microbiome as an appealing therapeutic target for metabolic and affective disorders, emphasizing the importance of understanding sex differences in metabolic and behavioral regulation influenced by the brain–gut–microbiome axis. The therapeutic targeting of the gut microbiota and its metabolites may offer a viable strategy for treating serotonin-related disorders, such as eating and affective disorders, with potential differences in treatment efficacy between men and women. This review would promote research on sex differences in metabolic and behavioral regulation impacted by the brain–gut–microbiome axis. Full article

(This article belongs to the Special Issue Molecular and Cellular Advances in Gut-Brain Axis)

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18 pages, 1904 KiB

Open AccessArticle

The LSmAD Domain of Ataxin-2 Modulates the Structure and RNA Binding of Its Preceding LSm Domain

by Shengping Zhang, Yunlong Zhang, Ting Chen, Hong-Yu Hu and Changrui Lu

Cells 2025, 14(5), 383; https://doi.org/10.3390/cells14050383 - 6 Mar 2025

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Abstract

Ataxin-2 (Atx2), an RNA-binding protein, plays a pivotal role in the regulation of RNA, intracellular metabolism, and translation within the cellular environment. Although both the Sm-like (LSm) and LSm-associated (LSmAD) domains are considered to associated with RNA binding, there is still a lack [...] Read more.

Ataxin-2 (Atx2), an RNA-binding protein, plays a pivotal role in the regulation of RNA, intracellular metabolism, and translation within the cellular environment. Although both the Sm-like (LSm) and LSm-associated (LSmAD) domains are considered to associated with RNA binding, there is still a lack of experimental evidence supporting their functions. To address this, we designed and constructed several recombinants containing the RNA-binding domain (RBD) of Atx2. By employing biophysical and biochemical techniques, such as EMSA and SHAPE chemical detection, we identified that LSm is responsible for RNA binding, whereas LSmAD alone does not bind RNA. NMR and small-angle X-ray scattering (SAXS) analyses have revealed that the LSmAD domain exhibits limited structural integrity and poor folding capability. The EMSA data confirmed that both LSm and LSm-LSmAD bind RNA, whereas LSmAD alone cannot, suggesting that LSmAD may serve as an auxiliary role to the LSm domain. SHAPE chemical probing further demonstrates that LSm binds to the AU-rich, GU-rich, or CU-rich sequence, but not to the CA-rich sequence. These findings indicate that Atx2 can interact with the U-rich sequences in the 3′-UTR, implicating its role in poly(A) tailing and the regulation of mRNA translation and degradation. Full article

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Design, expression, and purification of the recombinant domains of Atx2 for biochemical analyses. (A) Domain architecture of Atx2. Atx2 contains a polyQ tract at its N-terminus, the LSm and LSmAD domains, and a PAM2 domain flanked by IDRs. The polyQ, LSm, LSmAD, and PAM2 domains of Atx2 are shown in khaki, blue, red, and green, respectively. GB1 and SUMO fusion tags are represented in purple and orange, respectively. The numbers are given for the start and end residues of structural regions. (B) Expression and purification of LSmAD: lane 1, molecular weight marker; lane 2, cell lysates (induced); lane 3, precipitate; lane 4, supernatant; lane 5, protein sample eluted with 20 mM imidazole; lane 6, protein sample eluted with 250 mM imidazole; lane 7, purified sample by SEC-FPLC. (C) Preparation of LSm and the M2 mutant of LSm-LSmAD: lane 1, protein marker; lane 2, LSm; lane 3, M2. The arrow represents the target protein of interest. Full article ">Figure 2
Structural and functional analysis of LSmAD. (A) CD spectrum of LSmAD. (B) HSQC spectrum of LSmAD. (C) Kratky plot of LSmAD. (D) EMSA for characterizing the interaction of LSmAD with AC-rich and AU-rich RNA. Left, EMSA for characterizing the interaction of LSmAD with AC-rich RNA; right, EMSA for characterizing the interaction of LSmAD with AU-rich RNA. The top black graph illustrates the gradual increase in protein dose. Lanes 1–7 represent the molar protein/RNA molar ratio of 0, 0.25, 0.5, 1, 2, 4, and 6, respectively. Full article ">Figure 3
(A) SEC-FPLC analysis of the interaction of LSm with AU-rich and AC-rich RNA sequences, respectively. The right graph shows the normalized curves. (B) EMSA analysis of M2 with the AU-rich RNA sequence. The numbers 1–4 represent the M2 to RNA molar ratios of 0, 5, 10, and 20. (C) Kratky plot of LSm (black) and M2 (red). Full article ">Figure 4
The LSm domain of Atx2 recognizes the U-rich sequences. The color bars represent reduced SHAPE reactivity. The residues are indicated on the X-axis, while the CA-rich, GU-rich, AU-rich, and CU-rich sequences of RNA are labeled in grey, blue, orange, and green, respectively. The height of the bar graph indicates signal strength. Colored upper bars of RNA only represent reduced SHAPE reactivity. Downward bars in the RNA + protein row indicate the degree of base protection after the addition of protein. (A) Control for SHAPE analysis. (B) SHAPE analysis was carried out for RE1: LSm binding to RE1 (row1) and M2 binding to RE1 (row2). (C) SHAPE analysis was executed for RE2: LSm binding to RE2 (row1) and M2 binding to RE2 (row2). (D) SHAPE analysis was performed for RE5: LSm binding to RE5 (row1) and M2 binding to RE5 (row2). Full article ">

58 pages, 5256 KiB

Open AccessReview

The Histomorphology to Molecular Transition: Exploring the Genomic Landscape of Poorly Differentiated Epithelial Endometrial Cancers

by Thulo Molefi, Lloyd Mabonga, Rodney Hull, Absalom Mwazha, Motshedisi Sebitloane and Zodwa Dlamini

Cells 2025, 14(5), 382; https://doi.org/10.3390/cells14050382 - 5 Mar 2025

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Abstract

The peremptory need to circumvent challenges associated with poorly differentiated epithelial endometrial cancers (PDEECs), also known as Type II endometrial cancers (ECs), has prompted therapeutic interrogation of the prototypically intractable and most prevalent gynecological malignancy. PDEECs account for most endometrial cancer-related mortalities due [...] Read more.

The peremptory need to circumvent challenges associated with poorly differentiated epithelial endometrial cancers (PDEECs), also known as Type II endometrial cancers (ECs), has prompted therapeutic interrogation of the prototypically intractable and most prevalent gynecological malignancy. PDEECs account for most endometrial cancer-related mortalities due to their aggressive nature, late-stage detection, and poor response to standard therapies. PDEECs are characterized by heterogeneous histopathological features and distinct molecular profiles, and they pose significant clinical challenges due to their propensity for rapid progression. Regardless of the complexities around PDEECs, they are still being administered inefficiently in the same manner as clinically indolent and readily curable type-I ECs. Currently, there are no targeted therapies for the treatment of PDEECs. The realization of the need for new treatment options has transformed our understanding of PDEECs by enabling more precise classification based on genomic profiling. The transition from a histopathological to a molecular classification has provided critical insights into the underlying genetic and epigenetic alterations in these malignancies. This review explores the genomic landscape of PDEECs, with a focus on identifying key molecular subtypes and associated genetic mutations that are prevalent in aggressive variants. Here, we discuss how molecular classification correlates with clinical outcomes and can refine diagnostic accuracy, predict patient prognosis, and inform therapeutic strategies. Deciphering the molecular underpinnings of PDEECs has led to advances in precision oncology and protracted therapeutic remissions for patients with these untamable malignancies. Full article

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Figure 1
Global incidence and mortality rates of endometrial cancer. (A) The graph depicts the global trends in ASIR for endometrial cancer over the last 30 years, from 1990 to 2019, in countries grouped by economic status. The figure shows an increase in the incidence of the disease, regardless of income. However, a trend is visible in that the higher the income of a country, the greater the incidence of endometrial cancer. The study of the global burden of disease classifies different regions of the world. (B) Map depicting high-, middle-, and low-income countries. (C) This figure shows the age-standardized incidence rate per 100,000 in 2019 for each of the adjacent GBD regions. High-income North America, Western Europe, and East Asia are the GBD regions with the highest incidence. GBD values can be used to calculate changes in the incidence of disease over time, represented as a percentage. (D) Shows the global increase in endometrial cancer incidence 1990–2019. This was calculated by dividing the ASIR in 2019 by the ASIR from 1990 and multiplying by 100. The map and heatmap indicate that regions associated with lower income have faster-growing rates of endometrial cancer incidence. The increase in the incidence in low-to middle-income regions is thought to be linked to changes in lifestyle and a higher prevalence of risk factors such as obesity and inactivity levels. ASIR: age-standardized incidence rate. SDI: socio-demographic index. GBD: Global Burden of Disease (Figure adapted from [<a href="#B3-cells-14-00382" class="html-bibr">3</a>]). Full article ">Figure 2
The histologic subtypes of poorly differentiated epithelial endometrial cancers (PDEECs). The histological subtypes of PDEECs are a heterogeneous group of aggressive endometrial malignancies characterized by high-grade cellular atypia, increased mitotic activity, and poor glandular differentiation. The subtypes depicted include serous carcinoma, high-grade endometrioid carcinoma, clear cell endometrial carcinoma, dedifferentiated endometrial carcinoma, differentiated endometrial carcinoma, and undifferentiated endometrial carcinoma. Each histological subtype is associated with distinct molecular alterations, prognostic implications, and treatment responses. Representative histological images stained with hematoxylin and eosin (H&E) highlight the morphological diversity among PDEECs. The immunohistochemical markers used for differential diagnosis include p53, Napsin A, and E-cadherin. Full article ">Figure 3
Molecular classification of poorly differentiated epithelial endometrial cancers (PDEECs). This classification provides insights into the biological diversity and clinical behavior of these aggressive tumors. Primarily defined through genomic and transcriptomic analyses, this classification has helped refine the diagnostic criteria, predict prognosis, and identify potential therapeutic targets. It divides PDEECs into four main subtypes, namely, POLE-ultramutated, microsatellite instability-high (MSI-H), copy number low (CNL), and copy number high (CNH), based on specific molecular markers. Full article ">Figure 4
POLE mutations within the exonuclease domain. POLE exonuclease domain mutations significantly influence PDEEC biology. They drive a hypermutated phenotype that promotes immune recognition, often leading to better patient outcomes despite high tumor grades. This unique profile positions POLE as a valuable prognostic and predictive marker in PDEECs, guiding therapeutic approaches that prioritize immune-based treatments (figure adapted from [<a href="#B105-cells-14-00382" class="html-bibr">105</a>]). Full article ">Figure 5
Overview of the PI3K/AKT/mTOR pathway. In healthy cells, this pathway is tightly regulated, with phosphatase and tensin homolog (PTEN) acting as a critical suppressor by dephosphorylating PIP3 back to PIP2, thereby reducing PI3K/AKT activity. Mutations or dysregulation of genes such as PIK3CA, PTEN, and AKT are common in PDEECs, where the PI3K/AKT/mTOR pathway contributes to uncontrolled cell growth, resistance to apoptosis, and enhanced survival. Consequently, this pathway is a significant focus for PDEEC research, with inhibitors targeting PI3K, AKT, and mTOR showing potential in treating various tumors, especially those that exhibit high pathway activation owing to genetic alterations (figure adapted from [<a href="#B142-cells-14-00382" class="html-bibr">142</a>]). Full article ">Figure 6
Overview of p53 pathway dysfunction. Dysfunction of the p53 pathway in PDEECs is a driving factor in tumor progression and therapy resistance. Understanding and targeting this dysfunction is central to improving treatment strategies for high-grade aggressive cancers. As research advances, the development of p53-targeted therapies, synthetic lethal approaches, and combination regimens holds promise for addressing the challenges associated with p53-mutant PDEECs (figure adapted from [<a href="#B159-cells-14-00382" class="html-bibr">159</a>]). Full article ">Figure 7
Mismatch repair (MMR) pathway and mechanism. MMR deficiency is a critical factor in the pathology and treatment of PDEECs. By contributing to genomic instability and high MSI, it not only promotes tumor progression but also presents an opportunity for targeted immunotherapy, which could enhance survival in affected patients. Identifying and understanding MMR deficiency allows clinicians to personalize treatment and may pave the way for novel therapeutic strategies for endometrial cancer (figure adapted from [<a href="#B163-cells-14-00382" class="html-bibr">163</a>]). Full article ">Figure 8
PARP inhibitors in homologous recombination deficiency (HRD) in PDEECs. PARP inhibitors have emerged as a promising therapeutic strategy for treating PDEECs with homologous recombination deficiency (HRD). HRD is a condition in which cells lose the ability to accurately repair double-strand DNA breaks via the homologous recombination (HR) pathway, often due to mutations or deficiencies in key genes, such as BRCA1, BRCA2, and PTEN. HRD makes cancer cells more reliant on alternative DNA repair mechanisms, such as those mediated by PARP. By inhibiting PARP, these drugs trap cancer cells through a cycle of DNA damage, ultimately leading to cell death. In PDEECs, PARP inhibitors show promise as a targeted treatment, particularly for subtypes that are resistant to conventional therapies (figure adapted from [<a href="#B60-cells-14-00382" class="html-bibr">60</a>]). Full article ">

24 pages, 11432 KiB

Open AccessArticle

Podocyte A20/TNFAIP3 Controls Glomerulonephritis Severity via the Regulation of Inflammatory Responses and Effects on the Cytoskeleton

by Paulina Köhler, Andrea Ribeiro, Mohsen Honarpisheh, Ekaterina von Rauchhaupt, Georg Lorenz, Chenyu Li, Lucas Martin, Stefanie Steiger, Maja Lindenmeyer, Christoph Schmaderer, Hans-Joachim Anders, Dana Thomasova and Maciej Lech

Cells 2025, 14(5), 381; https://doi.org/10.3390/cells14050381 - 5 Mar 2025

Viewed by 323

Abstract

A20/Tnfaip3, an early NF-κB response gene and key negative regulator of NF-κB signaling, suppresses proinflammatory responses. Its ubiquitinase and deubiquitinase activities mediate proteasomal degradation within the NF-κB pathway. This study investigated the involvement of A20 signaling alterations in podocytes in the development of [...] Read more.

A20/Tnfaip3, an early NF-κB response gene and key negative regulator of NF-κB signaling, suppresses proinflammatory responses. Its ubiquitinase and deubiquitinase activities mediate proteasomal degradation within the NF-κB pathway. This study investigated the involvement of A20 signaling alterations in podocytes in the development of kidney injury. The phenotypes of A20Δpodocyte (podocyte-specific knockout of A20) mice were compared with those of control mice at 6 months of age to identify spontaneous changes in kidney function. A20Δpodocyte mice presented elevated serum urea nitrogen and creatinine levels, along with increased accumulation of inflammatory cells—neutrophils and macrophages—within the glomeruli. Additionally, A20Δpodocyte mice displayed significant podocyte loss. Ultrastructural analysis of A20 podocyte-knockout mouse glomeruli revealed hypocellularity of the glomerular tuft, expansion of the extracellular matrix, podocytopenia associated with foot process effacement, karyopyknosis, micronuclei, and podocyte detachment. In addition to podocyte death, we also observed damage to intracapillary endothelial cells with vacuolation of the cytoplasm and condensation of nuclear chromatin. A20 expression downregulation and CRISPR-Cas9 genome editing targeting A20 in a podocyte cell line confirmed these findings in vitro, highlighting the significant contribution of A20 activity in podocytes to glomerular injury pathogenesis. Finally, we analyzed TNFAIP3 transcription levels alongside genes involved in apoptosis, anoikis, NF-κB regulation, and cell attachment in glomerular and tubular compartments of kidney biopsies of patients with various renal diseases. Full article

(This article belongs to the Special Issue Innate Immunity in Health and Disease)

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12 pages, 1145 KiB

Open AccessArticle

Decreased Expression of Aquaporins as a Feature of Tubular Damage in Lupus Nephritis

by Melchior Maxime, Van Eycken Marie, Nicaise Charles, Duquesne Thomas, Longueville Léa, Collin Amandine, Decaestecker Christine, Salmon Isabelle, Delporte Christine and Soyfoo Muhammad

Cells 2025, 14(5), 380; https://doi.org/10.3390/cells14050380 - 5 Mar 2025

Viewed by 204

Abstract

Background: Tubulointerstitial hypoxia is a key factor for lupus nephritis progression to end-stage renal disease. Numerous aquaporins (AQPs) are expressed by renal tubules and are essential for their proper functioning. The aim of this study is to characterize the tubular expression of AQP1, [...] Read more.

Background: Tubulointerstitial hypoxia is a key factor for lupus nephritis progression to end-stage renal disease. Numerous aquaporins (AQPs) are expressed by renal tubules and are essential for their proper functioning. The aim of this study is to characterize the tubular expression of AQP1, AQP2 and AQP3, which could provide a better understanding of tubulointerstitial stress during lupus nephritis. Methods: This retrospective monocentric study was conducted at Erasme-HUB Hospital. We included 37 lupus nephritis samples and 9 healthy samples collected between 2000 and 2020, obtained from the pathology department. Immunohistochemistry was performed to target AQP1, AQP2 and AQP3 and followed by digital analysis. Results: No difference in AQP1, AQP2 and AQP3 staining location was found between healthy and lupus nephritis samples. However, we observed significant differences between these two groups, with a decrease in AQP1 expression in the renal cortex and in AQP3 expression in the cortex and medulla. In the subgroup of proliferative glomerulonephritis (class III/IV), this decrease in AQPs expression was more pronounced, particularly for AQP3. In addition, within this subgroup, we detected lower AQP2 expression in patients with higher interstitial inflammation score and lower AQP3 expression when higher interstitial fibrosis and tubular atrophy were present. Conclusions: We identified significant differences in the expression of aquaporins 1, 2, and 3 in patients with lupus nephritis. These findings strongly suggest that decreased AQP expression could serve as an indicator of tubular injury. Further research is warranted to evaluate AQP1, AQP2, and AQP3 as prognostic markers in both urinary and histological assessments of lupus nephritis. Full article

(This article belongs to the Special Issue Novel Autoantibodies in Systemic Autoimmune Diseases: Challenges and Perspectives)

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Representative staining of AQP1, AQP2 and AQP3 in lupus nephritis (LN) and healthy (CTRL) kidney tissue with a focus on glomerular (G), cortical (Cx), or medullary (Med) structures (field magnification 200×). Full article ">Figure 2
Semiquantitative analysis of AQP1, AQP2 and AQP3 staining in lupus nephritis (LN) and healthy (CTRL) kidney tissue. Results are displayed with box-and-whisker plots (min-max-median–Q1–Q3) and overlaying dots for individual data points (male patient in black). Data are expressed as percentage of labeled area within the region-of-interest (%ROI) (AQP1 Cortex: n = 8 for CTRL, n = 32 for LN; AQP1 Medulla: n = 8 for CTRL, n = 15 for LN; AQP2 cortex: n = 9 for CTRL, n = 31; AQP2 medulla: n = 9 for CTRL, n = 15; AQP3 cortex: n = 9 for CTRL, n = 33 for LN; AQP3 medulla: n = 9 for CTRL, n = 15 for LN; tested by Mann–Whitney). Full article ">Figure 3
Subgroup analysis of AQPs expression in lupus nephritis. (A) Proportion of staining in renal cortex (%ROI) according to proliferative LN (P-LN; Class III and IV considered as proliferative) or non-proliferative LN (NP-LN; Class I, II or V considered as non-proliferative) (B) Proportion of staining in renal cortex of proliferative LN (%ROI) according to absence (0) or presence (score of 1 or more) of tubular or interstitial damage according to the NIH LN activity and chronicity scoring system. Only significant results are shown. Results are displayed with box-and-whisker plots (min-max-median–Q1–Q3) and overlaying dots for individual data points (male patient in black). Data are expressed as percentage of labeled area within the region-of-interest (%ROI) ((A) AQP1 n = 8 for ctrl, n = 8 for NP-LN, n = 24 for P-LN, tested by Kruskal–Wallis with Dunn’s multiple comparison test; AQP3 n = 8 for ctrl, n = 8 for NP-LN, n = 24 for P-LN, tested by Kruskal–Wallis with Dunn’s multiple comparison test. (B) AQP2 interstitial leukocytes score n = 24, n = 10 for score 0, n = 15 for score 1 or more; AQP3 interstitial leukocytes score n = 24, n = 11 for score 0, n = 13 for score 1+; AQP3 tubular atrophy score n = 24, n = 11 for score 0, n = 13 for score 1; tested by Mann–Whitney). Full article ">

16 pages, 5754 KiB

Open AccessArticle

MiR-34b Regulates Muscle Growth and Development by Targeting SYISL

by Yuting Wu, Xiao Liu, Yonghui Fan, Hao Zuo, Xiaoyu Niu, Bo Zuo and Zaiyan Xu

Cells 2025, 14(5), 379; https://doi.org/10.3390/cells14050379 - 5 Mar 2025

Viewed by 143

Abstract

Non-coding genes, such as microRNA and lncRNA, which have been widely studied, play an important role in the regulatory network of skeletal muscle development. However, the functions and mechanisms of most non-coding RNAs in skeletal muscle regulatory networks are unclear. This study investigated [...] Read more.

Non-coding genes, such as microRNA and lncRNA, which have been widely studied, play an important role in the regulatory network of skeletal muscle development. However, the functions and mechanisms of most non-coding RNAs in skeletal muscle regulatory networks are unclear. This study investigated the function and mechanism of miR-34b in muscle growth and development. MiR-34b overexpression and interference tests were performed in C2C12 myoblasts and animal models. It was demonstrated that miR-34b significantly promoted mouse muscle growth and development in vivo, while miR-34b inhibited myoblast proliferation and promoted myoblast differentiation in vitro. Bioinformatics prediction using TargetScan for miRNA target identification and Bibiserv2 for potential miRNA–gene interaction analysis revealed a miR-34b binding site in the SYlSL sequence. The molecular mechanism of miR-34b regulating muscle growth and development was studied by co-transfection experiment, luciferase reporter gene detection, RNA immunoprecipitation, and RNA pull-down. MiR-34b can directly bind to SYISL and AGO2 proteins and regulate the expression of SYISL target genes p21 and MyoG by targeting SYISL, thereby regulating muscle growth and development. This study highlights that, as a novel regulator of myogenesis, miR-34b regulates muscle growth and development by targeting SYISL. Full article

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23 pages, 3082 KiB

Open AccessReview

Metaboloepigenetics: Role in the Regulation of Flow-Mediated Endothelial (Dys)Function and Atherosclerosis

by Francisco Santos, Hashum Sum, Denise Cheuk Lee Yan and Alison C. Brewer

Cells 2025, 14(5), 378; https://doi.org/10.3390/cells14050378 - 5 Mar 2025

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Abstract

Endothelial dysfunction is the main initiating factor in atherosclerosis. Through mechanotransduction, shear stress regulates endothelial cell function in both homeostatic and diseased states. Accumulating evidence reveals that epigenetic changes play critical roles in the etiology of cardiovascular diseases, including atherosclerosis. The metabolic regulation [...] Read more.

Endothelial dysfunction is the main initiating factor in atherosclerosis. Through mechanotransduction, shear stress regulates endothelial cell function in both homeostatic and diseased states. Accumulating evidence reveals that epigenetic changes play critical roles in the etiology of cardiovascular diseases, including atherosclerosis. The metabolic regulation of epigenetics has emerged as an important factor in the control of gene expression in diseased states, but to the best of our knowledge, this connection remains largely unexplored in endothelial dysfunction and atherosclerosis. In this review, we (1) summarize how shear stress (or flow) regulates endothelial (dys)function; (2) explore the epigenetic alterations that occur in the endothelium in response to disturbed flow; (3) review endothelial cell metabolism under different shear stress conditions; and (4) suggest mechanisms which may link this altered metabolism to the regulation of the endothelial epigenome by modulations in metabolite availability. We believe that metabolic regulation plays an important role in endothelial epigenetic reprogramming and could pave the way for novel metabolism-based therapeutic strategies. Full article

(This article belongs to the Collection Cardiovascular Disease: From Molecular and Cellular Mechanisms to Therapeutic Opportunities)

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Endothelial dysfunction develops at sites of disturbed flow. (A) Atheroprotective laminar shear stress (stable flow) acts on EC mechanosensors, which then activate signalling pathways. This leads to the active transcription of genes that encode transcription factors known to be important for EC homeostasis and identity, such as KLF2/4. The production of NO from L-arginine, by eNOS, is an important feature of a healthy endothelium as it acts on vascular smooth muscle cells to promote vasodilation. (B) On the contrary, oscillatory shear stress (disturbed flow) leads to the activation of alternative signalling pathways that end up upregulating DNMTs, repressing the expression of KLF2/4. Disturbed flow-induced endothelial dysfunction causes an upregulation of cell adhesion molecules, leading to an exacerbation in leukocyte adhesion and transcellular migration. Furthermore, oxidative stress also increases upon disturbed flow, contributing to the oxidation of LDL into oxidized LDL, which is taken up by macrophages, causing them to reprogram into foam cells, which are an important feature in the development of atherosclerosis. Abbreviations: Akt (or PKB), protein kinase B; cGMP, cyclic guanosine monophosphate; DNMT, DNA methyltransferase; eNOS, endothelial nitric oxide synthase; FOXO-1, forkhead box protein O1; GTP, guanosine triphosphate; ITPR3, gene that encodes inositol 1,4,5-trisphosphate receptor, type 3; KLF, Krüppel-like factor; LDL, low-density lipoprotein; NO, nitric oxide; NOS3, gene that encodes eNOS; O2−, superoxide; ONOO−, peroxynitrite; oxLDL, oxidized low-density lipoprotein; PECAM, platelet endothelial cell adhesion molecule; PI3K, phosphoinositide 3-kinase; ROS, reactive oxygen species; SIRT, sirtuin; TET, ten-eleven translocation; VE, vascular endothelial; VEGFR, vascular endothelial growth factor receptor. Figure created using BioRender. Full article ">Figure 2
The relationship between metabolism and the EC epigenome under laminar and oscillatory shear stress. (A) ECs localized in areas where stable (atheroprotective) flow is prevalent are characterized by an upregulation of KLF2, which acts to suppress glucose transport and glycolysis. Mitochondria in healthy cells are mostly tubular, which is suggestive of a relatively high engagement in mitochondrial metabolism. Fatty acid oxidation plays a major role in maintaining the intracellular pool of acetyl-CoA, which is possibly used in histone acetylation reactions to preserve EC identity. Laminar flow is also characterized by the expression of TET2, which possibly acts to demethylate atheroprotective genes, using TCA cycle-derived α-KG as a cofactor. (B) By contrast, in ECs localized in regions of disturbed flow (atheroprone), aerobic glycolysis predominates, which is consistent with the fragmentation of the mitochondrial network. The increase in glycolysis is likely to generate lactate, which can be used in histone lactylation reactions. Acetate- and de novo lipogenesis-derived acetyl-CoA may also be used in histone acetylation. The hypermethylation observed in dysfunctional ECs is possibly attributed to the altered methionine uptake from plasma and/or altered one-carbon metabolism. Methionine is converted into SAM by MAT. SAM subsequently donates its methyl group to be used in DNA and histone methylation reactions. Abbreviations: ACLY, ATP citrate lyase; ACSS, acetyl-CoA synthetase; α-KG, alpha-ketoglutarate; CoA, coenzyme A; DNMT, DNA methyltransferase; FAO, fatty acid oxidation; FASN, fatty acid synthase; HMT, histone methyltransferase; LDH, lactate dehydrogenase; MAT, S-adenosylmethionine synthetase; OXPHOS, oxidative phosphorylation; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; TCA, tricarboxylic acid; TET, ten-eleven translocation. Figure created using BioRender. Full article ">

14 pages, 1562 KiB

Open AccessArticle

A Cross-Sectional Exploratory Study of Rat Sarcoid (Ras) Activation in Women with and Without Polycystic Ovary Syndrome

by Sara Anjum Niinuma, Haniya Habib, Ashleigh Suzu-Nishio Takemoto, Priya Das, Thozhukat Sathyapalan, Stephen L. Atkin and Alexandra E. Butler

Cells 2025, 14(5), 377; https://doi.org/10.3390/cells14050377 - 5 Mar 2025

Viewed by 215

Abstract

Objective: Rat sarcoma (Ras) proteins, Kirsten, Harvey, and Neuroblastoma rat sarcoma viral oncogene homolog (KRAS, HRAS, and NRAS, respectively), are a family of GTPases, which are key regulators of cellular growth, differentiation, and apoptosis through signal transduction pathways modulated by growth factors [...] Read more.

Objective: Rat sarcoma (Ras) proteins, Kirsten, Harvey, and Neuroblastoma rat sarcoma viral oncogene homolog (KRAS, HRAS, and NRAS, respectively), are a family of GTPases, which are key regulators of cellular growth, differentiation, and apoptosis through signal transduction pathways modulated by growth factors that have been recognized to be dysregulated in PCOS. This study explores Ras signaling proteins and growth factor-related proteins in polycystic ovary syndrome (PCOS). Methods: In a well-validated PCOS database of 147 PCOS and 97 control women, plasma was batch analyzed using Somascan proteomic analysis for circulating KRas, Ras GTPase-activating protein-1 (RASA1), and 45 growth factor-related proteins. The cohort was subsequently stratified for BMI (body mass index), testosterone, and insulin resistance (HOMA-IR) for subset analysis. Results: Circulating KRas, and RASA1 did not differ between PCOS and control women (p > 0.05). EGF1, EGFR, and EGFRvIII were decreased in PCOS (p = 0.04, p = 0.04 and p < 0.001, respectively). FGF8, FGF9, and FGF17 were increased in PCOS (p = 0.02, p = 0.03 and p = 0.04, respectively), and FGFR1 was decreased in PCOS (p < 0.001). VEGF-D (p < 0.001), IGF1 (p < 0.001), IGF-1sR (p = 0.02), and PDGFRA (p < 0.001) were decreased in PCOS compared to controls. After stratifying for BMI ≤ 29.9 kg/m², EGFR FGF8, FGFR1 VEGF-D, IGF1, and IGF-1sR differed (p < 0.05) though EGF1, EGFRvIII, FGF8, FGFR1, and VEGF-D no longer differed; after subsequently stratifying for HOMA-IR, only FGFR1, VEGF-D, IGF1, and IGF-1sR differed between groups (p < 0.05). Conclusions: Several growth factors that activate Ras differ between women with and without PCOS, and when stratified for BMI and HOMA-IR, only FGFR1, VEGF-D, IGF1, and IGF-1sR differed; these appear to be inherent features of the pathophysiology of PCOS. Full article

(This article belongs to the Special Issue Ras Family of Genes and Proteins: Structure, Function and Regulation)

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Illustration of growth factors and their activation of Ras. The downward arrows indicate the lower plasma levels found in women with polycystic ovary syndrome (PCOS) independent of BMI, inflammation and insulin resistance (EGF: Epidermal Growth Factor, EFGR: Epidermal Growth Factor Receptor, FGF: Fibroblast Growth Factor, FGFR: Fibroblast Growth Factor Receptor, PDGF: Platelet-derived Growth Factor, PDGFR: Platelet-derived Growth Factor Receptor; VEGF: Vascular Endothelial Growth Factor, VEGFR: Vascular Endothelial Growth Factor Receptor, IGF: Insulin and Insulin-like Growth Factor; IGFR: Insulin and Insulin-like Growth Factor receptor, HGF: Hepatocyte Growth Factor, HGFR: Hepatocyte Growth Factor Receptor, C-MET: Mesenchymal-Epithelial Transition Factor, NGF: Nerve Growth Factor, NGFR: Nerve Growth Factor Receptor, TrkA: Tropomyosin receptor kinase A, GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor, GM-CSFR: Granulocyte-Macrophage Colony-Stimulating Factor Receptor, Ras: Rat Sarcoma Virus, RAF: Rapidly Accelerated Fibrosarcoma, MEK: Mitogen-activated Protein Kinase Kinase, ERK: Extracellular Signal-regulated Kinase. MAPK: Ras/mitogen-activated Protein Kinase, FRS2: Fibroblast Growth Factor Receptor Substrate, GRB2: Growth Factor Receptor-bound Protein-2, SOS: Son of Sevenless, SHC: Src Homology and Collagen, PIP2: Phosphatidylinositol 4,5-bisphosphate, PLCγ: Phospholipase C Gamma, DAG: Diacylglycerol, PKC: Protein Kinase C, PIK3: Phosphoinositide 3-Kinase, Akt: Protein Kinase B, mTOR: Mammalian Target of Rapamycin, SHP2: Src Homology 2 Domain Containing Phosphatase 2, JAK2: Janus Kinase 2, STAT: Signal Transducer and Activator of Transcription). Full article ">Figure 2
Volcano plot for differentially regulated genes in whole cohort (the black dots denote the non-significant genes, pink dots denote the genes with fold change of 1 and raw p-value > 0.05, and the red dots indicate the genes with fold change of 1 and raw p-value < 0.05. Full article ">Figure 3
Volcano plot for differentially regulated genes in on BMI matched (BMI ≤ 29.9 kg/m2) cohort (the black dots denote the non-significant genes, pink dots denote the genes with fold change of 1 and raw p-value > 0.05, and the red dots indicate the genes with fold change of 1 and raw p-value < 0.05. Full article ">Figure 4
Volcano plot for differentially regulated genes in on BMI (BMI ≤ 29.9 kg/m2) and IR (HOMA-IR ≤ 1.9) matched cohort (the black dots denote the non-significant genes, pink dots denote the genes with fold change of 1 and raw p-value > 0.05, and the red dots indicate the genes with fold change of 1 and raw p-value < 0.05. Full article ">Figure 5
Correlations of body mass index (BMI) with Ras-related proteins. BMI was associated positively with VEGF-sR3 (A), FGF5 (B), VEGF-sR2 (C), and VEGF-C (D) only in women with polycystic ovary syndrome (PCOS). Full article ">

15 pages, 746 KiB

Open AccessReview

Diabetic Retinopathy (DR): Mechanisms, Current Therapies, and Emerging Strategies

by Hyewon Seo, Sun-Ji Park and Minsoo Song

Cells 2025, 14(5), 376; https://doi.org/10.3390/cells14050376 - 4 Mar 2025

Viewed by 222

Abstract

Diabetic retinopathy (DR) is one of the most prevalent complications of diabetes, affecting nearly one-third of patients with diabetes mellitus and remaining a leading cause of blindness worldwide. Among the various diabetes-induced complications, DR is of particular importance due to its direct impact [...] Read more.

Diabetic retinopathy (DR) is one of the most prevalent complications of diabetes, affecting nearly one-third of patients with diabetes mellitus and remaining a leading cause of blindness worldwide. Among the various diabetes-induced complications, DR is of particular importance due to its direct impact on vision and the irreversible damage to the retina. DR is characterized by multiple pathological processes, primarily a hyperglycemia-induced inflammatory response and oxidative stress. Current gold standard therapies, such as anti-VEGF injections and photocoagulation, have shown efficacy in slowing disease progression. However, challenges such as drug resistance, partial therapeutic responses, and the reliance on direct eye injections—which often result in low patient compliance—remain unresolved. This review provides a comprehensive overview of the underlying molecular mechanisms in DR, the current therapies, and their unmet needs for DR treatment. Additionally, emerging therapeutic strategies for improving DR treatment outcomes are discussed. Full article

(This article belongs to the Special Issue Diabetes-Induced Organ Damage: Cellular Mechanisms and Therapeutic Opportunities)

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16 pages, 3369 KiB

Open AccessArticle

Few-Layer Graphene-Based Optical Nanobiosensors for the Early-Stage Detection of Ovarian Cancer Using Liquid Biopsy and an Active Learning Strategy

by Obdulia Covarrubias-Zambrano, Deepesh Agarwal, Joan Lewis-Wambi, Raul Neri, Andrea Jewell, Balasubramaniam Natarajan and Stefan H. Bossmann

Cells 2025, 14(5), 375; https://doi.org/10.3390/cells14050375 - 4 Mar 2025

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Abstract

Ovarian cancer survival depends strongly on the time of diagnosis. Detection at stage 1 must be the goal of liquid biopsies for ovarian cancer detection. We report the development and validation of graphene-based optical nanobiosensors (G-NBSs) that quantify the activities of a panel [...] Read more.

Ovarian cancer survival depends strongly on the time of diagnosis. Detection at stage 1 must be the goal of liquid biopsies for ovarian cancer detection. We report the development and validation of graphene-based optical nanobiosensors (G-NBSs) that quantify the activities of a panel of proteases, which were selected to provide a crowd response that is specific for ovarian cancer. These G-NBSs consist of few-layer explosion graphene featuring a hydrophilic coating, which is linked to fluorescently labeled highly selective consensus sequences for the proteases of interest, as well as a fluorescent dye. The panel of G-NBSs showed statistically significant differences in protease activities when comparing localized (early-stage) ovarian cancer with both metastatic (late-stage) and healthy control groups. A hierarchical framework integrated with active learning (AL) as a prediction and analysis tool for early-stage detection of ovarian cancer was implemented, which obtained an overall accuracy score of 94.5%, with both a sensitivity and specificity of 0.94. Full article

(This article belongs to the Special Issue Nanofluidics, Nanopores, and Nanomaterials for Understanding Biology)

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33 pages, 55731 KiB

Open AccessArticle

Extracellular Signaling Molecules from Adipose-Derived Stem Cells and Ovarian Cancer Cells Induce a Hybrid Epithelial-Mesenchymal Phenotype in a Bidirectional Interaction

by Vinícius Augusto Simão, Juliana Ferreira Floriano, Roberta Carvalho Cesário, Karolina da Silva Tonon, Larissa Ragozo Cardoso de Oliveira, Flávia Karina Delella, Fausto Almeida, Lucilene Delazari dos Santos, Fábio Rodrigues Ferreira Seiva, Débora Aparecida Pires de Campos Zuccari, João Tadeu Ribeiro-Paes, Russel J. Reiter and Luiz Gustavo de Almeida Chuffa

Cells 2025, 14(5), 374; https://doi.org/10.3390/cells14050374 - 4 Mar 2025

Viewed by 150

Abstract

Ovarian cancer (OC) is characterized by high mortality rates due to late diagnosis, recurrence, and metastasis. Here, we show that extracellular signaling molecules secreted by adipose-derived mesenchymal stem cells (ASCs) and OC cells—either in the conditioned medium (CM) or within small extracellular vesicles [...] Read more.

Ovarian cancer (OC) is characterized by high mortality rates due to late diagnosis, recurrence, and metastasis. Here, we show that extracellular signaling molecules secreted by adipose-derived mesenchymal stem cells (ASCs) and OC cells—either in the conditioned medium (CM) or within small extracellular vesicles (sEVs)—modulate cellular responses and drive OC progression. ASC-derived sEVs and CM secretome promoted OC cell colony formation, invasion, and migration while upregulating tumor-associated signaling pathways, including TGFβ/Smad, p38MAPK/ERK1/2, Wnt/β-catenin, and MMP-9. Additionally, OC-derived sEVs and CM induced a pro-tumorigenic phenotype in ASCs, enhancing their invasiveness and expression of tumor-associated factors. Notably, both ASCs and OC cells exhibited increased expression of E-cadherin and Snail/Slug proteins, key markers of epithelial/mesenchymal hybrid phenotype, enhancing cellular plasticity and metastatic potential. We also demonstrated that these cellular features are, at least in part, due to the presence of tumor-supportive molecules such as TNF-α, Tenascin-C, MMP-2, and SDF-1α in the CM secretome of ASCs and OC cells. In silico analyses linked these molecular changes to poor prognostic outcomes in OC patients. These findings highlight the critical role of sEVs and tumor/stem cell-derived secretome in OC progression through bidirectional interactions that impact cellular behavior and phenotypic transitions. We suggest that targeting EV-mediated communication could improve therapeutic strategies and patient outcomes. Full article

(This article belongs to the Topic The Role of Extracellular Vesicles as Modulators of the Tumor Microenvironment)

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19 pages, 1222 KiB

Open AccessReview

Research Progress on the Immune Function of Liver Sinusoidal Endothelial Cells in Sepsis

by Xinrui Wang, Zhe Guo, Yuxiang Xia, Xuesong Wang and Zhong Wang

Cells 2025, 14(5), 373; https://doi.org/10.3390/cells14050373 - 4 Mar 2025

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Abstract

Sepsis is a complex clinical syndrome closely associated with the occurrence of acute organ dysfunction and is often characterized by high mortality. Due to the rapid progression of sepsis, early diagnosis and intervention are crucial. Recent research has focused on exploring the pathological [...] Read more.

Sepsis is a complex clinical syndrome closely associated with the occurrence of acute organ dysfunction and is often characterized by high mortality. Due to the rapid progression of sepsis, early diagnosis and intervention are crucial. Recent research has focused on exploring the pathological response involved in the process of sepsis. Liver sinusoidal endothelial cells (LSECs) are a special type of endothelial cell and an important component of liver non-parenchymal cells. Unlike general endothelial cells, which mainly provide a barrier function within the body, LSECs also have important functions in the clearance and regulation of the immune response. LSECs are not only vital antigen-presenting cells (APCs) in the immune system but also play a significant role in the development of infectious diseases and tumors through their specific immune regulatory pathways. However, in certain disease states, the functions of LSECs may be impaired, leading to immune imbalance and the development of organ failure. Investigating the immune pathways of LSECs in sepsis may provide new solutions for the prevention and treatment of sepsis and is crucial for maintaining microcirculation and improving patient outcomes. Full article

(This article belongs to the Special Issue Sepsis: Pathogenesis, Pathophysiology, Molecular and Cellular Mechanisms and Immunity)

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The scavenging function of liver sinusoidal endothelial cells plays a crucial role during infection. As blood flows through the liver sinusoids, substances such as antigen–antibody complexes and LPS can be cleared by the action of these cells. Specifically, the clearance of LPS relies on endocytosis receptors, such as stabilin, SR, and MR, while antigen–antibody complexes and viruses are removed via corresponding receptors during their passage through the liver sinusoids. Consequently, the content of LPS, SIC, and other related substances in the blood decreases significantly after flowing through the liver sinusoids. Full article ">Figure 2
(A) Schematic diagram of LPS clearance: LPS entering the systemic circulation binds to HDL in the blood to form an LPS–HDL complex. This complex circulates through the bloodstream and binds to endocytosis receptors on the surface of LSECs, primarily stabilized proteins. It is then transported intracellularly to lysosomes, where the LPS clearance effect is exerted. (B) Schematic diagram of the TLR4 signaling pathway: LPS entering the systemic circulation binds to LBP in the blood to form a complex. This complex interacts with TLR4 receptors on the surface of LSECs, activating the signaling pathway. When the MYD88 pathway is activated, it interacts with the downstream NF-KB pathway, promoting the release of TNF-α, IL-6, and IL-1β. When the TRIF pathway is activated, it stimulates the release of proteins such as selectins, ICAM-1, and VCAM-1, influencing the progression of inflammatory responses. Full article ">Figure 3
The increased expression of FABP4 in LSECs may represent a potential target for liver injury. Under certain conditions, the upregulated expression of FABP4 in LSECs activates the NF-κB/CXCL10 pathway, which leads to the recruitment of CXCR3+ macrophages and induces their polarization towards the M1 phenotype. This polarization leads to the release of a significant amount of proinflammatory mediators, such as TNF-α and IL-6, which continuously exacerbate the immune response. Conversely, the M2 phenotype of macrophages secretes anti-inflammatory factors, preventing excessive immune reactions. Full article ">

22 pages, 5457 KiB

Open AccessArticle

Development and Validation of AAV-Mediated Liver, Liver-VAT, and Liver-Brain SORT and Therapeutic Regulation of FASN in Hepatic De Novo Lipogenesis

by Ratulananda Bhadury, Mohammad Athar, Pooja Mishra, Chayanika Gogoi, Shubham Sharma and Devram S. Ghorpade

Cells 2025, 14(5), 372; https://doi.org/10.3390/cells14050372 - 4 Mar 2025

Viewed by 207

Abstract

Hepatic lipogenesis combined with elevated endoplasmic reticulum (ER) stress is central to non-alcoholic steatohepatitis (NASH). However, the therapeutic targeting of key molecules is considerably less accomplished. Adeno-associated virus (AAV)-mediated gene therapies offer a new solution for various human ailments. Comprehensive bio-functional validation studies [...] Read more.

Hepatic lipogenesis combined with elevated endoplasmic reticulum (ER) stress is central to non-alcoholic steatohepatitis (NASH). However, the therapeutic targeting of key molecules is considerably less accomplished. Adeno-associated virus (AAV)-mediated gene therapies offer a new solution for various human ailments. Comprehensive bio-functional validation studies are essential to assess the impact of AAVs in the target organ for developing both preclinical and clinical gene therapy programs. Here, we have established a robust and efficient protocol for high-titer AAV production to enable detailed Selective ORgan Targeting (SORT) of AAV1, 5, 7, and 8 in vivo. Our results for in vivo SORT showed single organ (liver) targeting by AAV8, no organ targeting by AAV1, and dual organ transduction (liver-brain and liver-VAT) by AAV5 and AAV7. Using a human dataset and preclinical murine models of NASH, we identified an inverse correlation between ER stress-triggered CRELD2 and the de novo lipogenesis driver FASN. Furthermore, liver-specific silencing of CRELD2 via AAV8-shCreld2 strongly supports the contribution of CRELD2 to de novo lipogenesis through FASN regulation. Thus, our study demonstrates a robust method for producing clinically translatable AAVs that could be readily adapted for liver and/or liver-VAT or liver-brain targeted gene therapy. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of Liver Diseases)

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15 pages, 826 KiB

Open AccessReview

Advances in Therapy of Adult Patients with Acute Lymphoblastic Leukemia

by Oscar Sucre, Saagar Pamulapati, Zeeshan Muzammil and Jacob Bitran

Cells 2025, 14(5), 371; https://doi.org/10.3390/cells14050371 - 4 Mar 2025

Viewed by 210

Abstract

The landscape of adult acute lymphoblastic leukemia (ALL) is dramatically changing. With very promising results seen with novel immunotherapeutics in the setting of relapsed and refractory disease, the prospect of using these agents in first-line therapy has prompted the development of multiple clinical [...] Read more.

The landscape of adult acute lymphoblastic leukemia (ALL) is dramatically changing. With very promising results seen with novel immunotherapeutics in the setting of relapsed and refractory disease, the prospect of using these agents in first-line therapy has prompted the development of multiple clinical trials addressing this question. This review seeks to outline and expand the current standard of care, as well as new advances, in the treatment of adult patients with ALL and address future areas of research. We expect the frontline integration of immuno-oncology agents such as bispecific T-cell engagers, antibody–drug conjugates, and chimeric antigen receptor (CAR) T cells may maintain or improve outcomes in adults while also minimizing toxicity. Treatment of ALL will continue to evolve as we focus on personalized, patient-centered approaches. Full article

(This article belongs to the Special Issue Cellular Therapy of Leukemia)

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24 pages, 2536 KiB

Open AccessFeature PaperArticle

THz Waves Improve Spatial Working Memory by Increasing the Activity of Glutamatergic Neurons in Mice

by Lequan Song, Zhiwei He, Ji Dong, Haoyu Wang, Jing Zhang, Binwei Yao, Xinping Xu, Hui Wang, Li Zhao and Ruiyun Peng

Cells 2025, 14(5), 370; https://doi.org/10.3390/cells14050370 - 3 Mar 2025

Viewed by 210

Abstract

Terahertz (THz) waves, a novel type of radiation with quantum and electronic properties, have attracted increasing attention for their effects on the nervous system. Spatial working memory, a critical component of higher cognitive function, is coordinated by brain regions such as the infralimbic [...] Read more.

Terahertz (THz) waves, a novel type of radiation with quantum and electronic properties, have attracted increasing attention for their effects on the nervous system. Spatial working memory, a critical component of higher cognitive function, is coordinated by brain regions such as the infralimbic cortex (IL) region of the medial prefrontal cortex and the ventral cornu ammonis 1 (vCA1) of hippocampus. However, the regulatory effects of THz waves on spatial working memory and the underlying mechanisms remain unclear. In this study, the effects of 0.152 THz waves on glutamatergic neuronal activity and spatial working memory and the related mechanisms were investigated in cell, brain slice, and mouse models. Cellular experiments revealed that THz waves exposure for 60 min significantly increased the intrinsic excitability of primary hippocampal neurons, enhanced glutamatergic neuron activity, and upregulated the expression of molecules involved in glutamate metabolism. In brain slice experiments, THz waves markedly elevated neuronal activity, promoted synaptic plasticity, and increased glutamatergic synaptic transmission within the IL and vCA1 regions. Molecular dynamics simulations found that THz waves could inhibit the ion transport function of glutamate receptors. Moreover, Y-maze tests demonstrated that mice exposed to THz waves exhibited significantly improved spatial working memory. Multiomics analyses indicated that THz waves could induce changes in chromatin accessibility and increase the proportion of excitatory neurons. These findings suggested that exposure to 0.152 THz waves increased glutamatergic neuronal activity, promoted synaptic plasticity, and improved spatial working memory, potentially through modifications in chromatin accessibility and excitatory neuron proportions. Full article

(This article belongs to the Section Cells of the Nervous System)

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Figure 1
THz waves increase glutamatergic neuron activity. (A–C) Action potentials of primary hippocampal neurons exposed to THz waves for 6, 30, and 60 min; (D) Fluorescence in situ hybridization of c-Fos, VGLUT2, and VGAT after THz waves exposure; (E–H) Western blot analyses of vesicle release-related molecules, glutamate receptor subunits, glutamate transporter metabolic enzymes, and postsynaptic molecules in primary hippocampal neurons. * Represents p < 0.05, ** Represents p < 0.01. Full article ">Figure 2
THz waves increase IL and vCA1 neuron activity and promote synaptic plasticity. (A) c-Fos immunofluorescence staining, scale bar = 1000 μm; (B) c-Fos+ cell number statistical analysis; (C) IL neuron firing raster plots and heatmaps; (D) IL neuron synchronous firing frequency and spike ratio statistical analysis; (E) IL neuron action potential; (F) IL neuron fEPSP representative trace; (G) IL neuron fEPSP statistical analysis; (H) vCA1 neuron firing raster plots and heatmaps; (I) vCA1 neuron synchronous firing frequency and spike ratio statistical analysis; (J) vCA1 neuron action potential; (K) vCA1 neuron fEPSP representative trace; (L) vCA1 neuron fEPSP statistical analysis. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001. Full article ">Figure 3
THz waves enhance IL and vCA1 neuron glutamatergic synaptic transmission. (A) sEPSC traces of IL neurons; (B) Quantitative analysis of frequency and amplitude of sEPSC of IL neurons; (C) sIPSC traces of IL neurons; (D) Quantitative analysis of frequency and amplitude of sIPSC of IL neurons; (E) Glutamate receptor currents in IL neurons; (F) Quantitative analysis of glutamate receptor currents; (G) Schematic diagram of viral injection for calcium imaging in the IL region; (H) IL neurons labeled with genetically encoded calcium indicators; (I) Statistical analysis of calcium activity in IL neurons; (J) sEPSC traces of vCA1 neurons; (K) Quantitative analysis of frequency and amplitude of sEPSC of vCA1 neurons; (L) sIPSC traces of vCA1 neurons; (M) Quantitative analysis of frequency and amplitude of sIPSC of vCA1 neurons; (N) Glutamate receptor currents in vCA1 neurons; (O) Quantitative analysis of glutamate receptor currents; (P) Schematic diagram of viral injection for calcium imaging in the vCA1 region; (Q) vCA1 neurons labeled with genetically encoded calcium indicators; (R) Statistical analysis of calcium activity in vCA1 neurons. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001. Full article ">Figure 4
Effects of THz waves on the working memory of mice. (A) Heatmap of the open field traces of the mice after THz waves exposure to the IL; (B) Statistical analysis of average speed; (C) Statistical analysis of central area time; (D) Heatmap of the traces of the mice in the novel object recognition experiment after THz waves exposure to the IL; (E) Statistical analysis of the discrimination index; (F) Schematic diagram of the novel arm experiment in the Y-maze after THz waves exposure to the IL; (G) Graph of the mouse movement trace in the novel arm experiment in the Y-maze; (H) Statistical analysis of the novel arm discrimination index; (I) Graph of the mouse trace in the spontaneous alternation experiment in the Y-maze after THz waves exposure to the IL; (J) Statistical analysis of the spontaneous alternation rate; (K) Schematic diagram of the delayed unpaired task in the T-maze after THz waves exposure to the IL; (L) Statistical analysis of task accuracy; (M) Graph of the mouse trace in the spontaneous alternation experiment in the Y-maze after THz waves exposure to the vCA1; (N) Statistical analysis of the spontaneous alternation rate; (O) Schematic diagram of the delayed unpaired task in the T-maze after THz waves exposure to the vCA1; (P) Statistical analysis of task accuracy. * represents p < 0.05, ** represents p < 0.01. Full article ">Figure 5
Effects of THz waves on neuronal chromatin accessibility and the genome. (A) Spearman correlation heatmap of read counts in the C and T groups. (B) Volcano plot of T-C contrast in ATAC-seq. (C) Bar plot of enriched GO terms from ATAC down peaks in the T group. (D) Bubble plot of enriched KEGG terms from ATAC down peaks in the T group. (E) ATAC-seq and peak density levels are shown at representative promoters. (F) snRNA-seq of brain tissue samples obtained from mice. Uniform manifold approximation and projection (UMAP) clustering of single-cell transcriptomes colored according to PC clusters (left) and significant cell-type clusters (right). (G) Percent bar plot of cell counts according to cell type clusters. (H) Volcano plot of the T-C contrast in the excitatory neuron cluster. (I) Expression levels of upregulated genes are shown for a representative gene (Epha4, Epha7, Hpca) in the excitatory neuron cluster. Full article ">Figure 6
Schematic diagram of the neuromodulatory effect of THz waves. Full article ">

22 pages, 4151 KiB

Open AccessArticle

Isolation and Functional Characterization of Endophytic Bacteria from Muscadine Grape Berries: A Microbial Treasure Trove

by Meenakshi Agarwal and Mehboob B. Sheikh

Cells 2025, 14(5), 369; https://doi.org/10.3390/cells14050369 - 3 Mar 2025

Viewed by 215

Abstract

Muscadine grapes are renowned for their unique traits, natural disease resistance, and rich bioactive compounds. Despite extensive research on their phytochemical properties, microbial communities, particularly endophytic bacteria, remain largely unexplored. These bacteria play crucial roles in plant health, stress tolerance, and ecological interactions. [...] Read more.

Muscadine grapes are renowned for their unique traits, natural disease resistance, and rich bioactive compounds. Despite extensive research on their phytochemical properties, microbial communities, particularly endophytic bacteria, remain largely unexplored. These bacteria play crucial roles in plant health, stress tolerance, and ecological interactions. This study represents the first comprehensive effort to isolate, identify, and functionally characterize the bacterial endophytes inhabiting muscadine grape berries using a culture-dependent approach. We isolated diverse bacterial species spanning six genera—Bacillus, Staphylococcus, Paenibacillus, Calidifontibacillus, Curtobacterium, and Tatumella. Microscopic and physiological analysis revealed variations in bacterial morphology, with isolates demonstrating adaptability to varied temperatures. Cluster-based analysis indicated functional specialization among the isolates, with species from Pseudomonadota and Actinomycetota exhibiting superior plant growth-promoting abilities, whereas Bacillota species displayed potential biocontrol and probiotic properties. Among them, Tatumella ptyseos demonstrated exceptional plant growth-promoting traits, including indole-3-acetic acid production, nitrogen fixation, phosphate solubilization, and carbohydrate fermentation. Additionally, Bacillus spp. showed presumptive biocontrol potential, while Paenibacillus cineris emerged as a potential probiotic candidate. The identification of Calidifontibacillus erzurumensis as a novel endophytic species further expands the known biodiversity of grape-associated microbes. These findings provide insights into the metabolic diversity and functional roles of muscadine grape-associated endophytes, highlighting their potential for agricultural and biotechnological applications. Full article

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Morphological diversity of bacterial endophytes isolated from different muscadine berry genotypes. (A) Representative images of muscadine grape cultivars used in this study. (B) Plate images represent colony morphology and color and shape of bacterial endophytes grown on LB agar plates. Scale bar: 2.5 µm. (C) Microscopic images of individual bacterial isolates; both DIC and those stained with the membrane marker FM1-43FX shown. Scale bar: 2.5 µm. Full article ">Figure 2
Phylogenetic tree of bacterial endophytes isolated from muscadine berries. The tree illustrates the evolutionary relationships among the bacterial isolates. The tree was constructed using partial 16S rRNA gene sequences, analyzed by the neighbor-joining method. Bootstrap values (expressed as percentages) are shown at branch points, indicating the reliability of the clustering. Full article ">Figure 3
Growth profile of bacterial isolates in different media at varying temperatures. (A) Growth profiles of bacterial isolates cultured in LB broth. (B) Growth profiles of bacterial isolates cultured in MRS broth. Strains were incubated at three different temperatures: 25 °C, 30 °C, and 37 °C. Full article ">Figure 4
Biochemical characterization of individual bacterial isolates. (A) Phosphate solubilization assay, (B) screening for nitrogen fixation (C) IAA production assay, (D) sugar fermentation assay, (E) motility assay, (F) DNase assay, and (G) hemolysis assay. All assays were conducted at least three times, and representative images for each test are shown. Full article ">Figure 5
Cell survival assay under gastrointestinal condition. Individual bacterial isolates were incubated in test media for 4 h, and cell viability was determined using the CFU method. (A) AM-36, (B) AM-38, (C) AM-39, (D) AM-40, (E) AM-41, (F) AM-42, (G) AM-44, (H) AM-46, (I) AM-48. The percentage of cell survival was then calculated and shown for each condition. Full article ">Figure 6
Adaptive responses of bacterial strains for survival under stress conditions. (A) Phenomenon of spore formation by Bacillus sp. as a stress response. (B) Plate assay showing the tolerance of strains to gastric juice after 4 h of incubation along with untreated control. Dilutions were made at a 1:10 ratio. For gastric juice treatment, cells were diluted 100 times before plating the 1st dilution. Note: For strain AM-44, the 5th and 6th dilutions under the gastric juice condition show agar puncture due to the pipette tip, rather than colony growth. (C) Representative microscopic images of cells treated with gastric juice compared to untreated control cells. The red arrow indicates a cell bearing a spore, while the green arrow indicates released spores. Scale bar: 1.0 µm. (D) Scatter plot showing cell length measurements of strain AM-40, comparing treated and untreated cells. In total, 100 cells were analyzed. p values were calculated using one-way ANOVA, with significant differences observed (**** p < 0.0001). Full article ">Figure 7
Biochemical test results and cluster analysis of bacterial isolates. (A) Heatmap depicting the positive (green) and negative (red) outcomes of biochemical tests across bacterial isolates and functional traits. The x-axis lists individual isolates, while the y-axis corresponds to functional traits. (B) Hierarchical clustering dendrogram of bacterial isolates based on their biochemical test profiles, grouping isolates with similar functional traits. (C) Hierarchical clustering dendrogram of functional traits, grouping traits with similar patterns across isolates. Abbreviations: Spo—Sporulation; ND—Nutrient Depletion; Phos—Phosphate; G—Glucose; G, L/S—Glucose, Lactose, or Sucrose, N2—Nitrogen. Full article ">Figure 8
Potential function of muscadine grape berry bacterial endophytes. Based on the traits exhibited by the bacterial isolates, their potential functions for various applications were predicted. These functions include roles in nutrient cycling, stress tolerance, plant growth promotion, vinification, and biotechnological applications, highlighting their versatility in different environmental and industrial contexts. Full article ">

16 pages, 8619 KiB

Open AccessArticle

mir-276a Is Required for Muscle Development in Drosophila and Regulates the FGF Receptor Heartless During the Migration of Nascent Myotubes in the Testis

by Mathieu Preußner, Maik Bischoff and Susanne Filiz Önel

Cells 2025, 14(5), 368; https://doi.org/10.3390/cells14050368 - 3 Mar 2025

Viewed by 221

Abstract

MicroRNAs function as post-transcriptional regulators in gene expression and control a broad range of biological processes in metazoans. The formation of multinucleated muscles is essential for locomotion, growth, and muscle repair. microRNAs have also emerged as important regulators for muscle development and function. [...] Read more.

MicroRNAs function as post-transcriptional regulators in gene expression and control a broad range of biological processes in metazoans. The formation of multinucleated muscles is essential for locomotion, growth, and muscle repair. microRNAs have also emerged as important regulators for muscle development and function. In order to identify new microRNAs required for muscle formation, we have performed a large microRNA overexpression screen. We screened for defects during embryonic and adult muscle formation. Here, we describe the identification of mir-276a as a regulator for muscle migration during testis formation. The mir-276a overexpression phenotype in testis muscles resembles the loss-of-function phenotype of heartless. A GFP sensor assay reveals that the 3′UTR of heartless is a target of mir-276a. Furthermore, we found that mir-276a is essential for the proper development of indirect flight muscles and describe a method for determining the number of nuclei for each of the six longitudinal muscle fibers (DLMs), which are part of the indirect flight muscles. Full article

(This article belongs to the Special Issue Skeletal Muscle Differentiation and Epigenetics - Volume II)

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Graphical abstract

Graphical abstract
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The expression of UAS-mir276a causes defects during muscle formation: (A–C) lateral view of staged 16 embryos stained with anti-β3-Tubulin to visualize the muscle pattern. 20× Objective (A) Wild-type muscle pattern of a homozygous DMef2-GAL4 embryo. The asterisk marks the lateral transverse muscles. The arrowheads point to the dorsal muscles. (B) Embryo expressing UAS-mir276b with DMef2-GAL4. Lateral muscles appear shorter in comparison to the lateral muscles of DMef2-GAL4 embryos (asterisks). (C) Embryo expressing UAS-mir276a with DMef2-GAL4. Large gaps between the dorsal muscles are seen (arrowheads). (D,E) Testis of wild-type adults showing an elongated coiled-like testis. Scale bar D 20 μm. (E) Muscles of a wild-type testis covering the whole testis, including the tip of the testis, asterisk. Scale bar 50 μm (F) Testis of a male expressing UAS-mir276a with DMef2-GAL4. Scale bar 20 μm. (F’) Testis muscles of the boxed area in (F) are shown in a higher magnification. Holes in the musculature are marked with asterisks. Scale bar 20 μm. Full article ">Figure 2
Founder cells and fusion-competent myoblasts are specified: (A) Overview of a 24 h APF genital disc expressing UAS-mCD8-GFP with DMef2-GAL4 and stained with anti-Mef2 and DAPi. (A’) Myoblasts labeled with Mef2 (red, arrowhead). (B) Overview of a 24 h APF genital disc co-expressing UAS-mCD8-GFP and UAS-mir276a with DMef2-GAL4 and stained with anti-Mef2 and DAPi. (B’) Myoblasts labeled with Mef2 (red, arrowhead). (C) Overview of a 24 h APF genital disc expressing UAS-mCD8-GFP with DMef2-GAL4 and stained with anti-Lmd and DAPi. (C’) Myoblasts labeled with Lmd (red, arrowheads). (D) Overview of a 24 h APF genital disc co-expressing UAS-mCD8-GFP and UAS-mir276a with DMef2-GAL4 and stained with anti-Lmd and DAPi. (D’) Myoblasts labeled with Lmd (red, arrowheads). Arrows mark nascent myotubes. Scale bars 20 μm. vs = seminal vesicles. Full article ">Figure 3
GFP sensor assay to show that heartless is an in vivo target of mir-276a: (A–C) Analysis of adult testes dissected from Drosophila males. The framed areas show how far testis muscles colonize the testis. The asterisk marks the tip of the testis. (A) Wild type. (B) Dominant-negative UAS-htl expressed with DMef2-GAL4. (C) Expression of UAS-mir276a with D-Mef2-GAL4 and stained with Phalloidin-TRITC. (D–G) GFP sensor assay. (D,D’) Wild-type, third-instar larvae express no eGFP. (E,E’) Recombinant third-instar larvae carrying two copies of DMef2-GAL4 >> UAS-htl-3′UTR-eGFP with a strong eGFP expression. (F,F’) Recombinant third-instar larvae carrying one copy of DMef2-GAL4 >> UAS-htl-3′UTR-eGFP with detectable eGFP expression. (G,G’) Recombinant third-instar larva carrying one copy of DMef2-GAL4 >> UAS-htl-3′UTR-eGFP and one copy of UAS-mir276a express no eGFP. Full article ">Figure 4
The number of indirect flight muscles is reduced in UAS-mir-276a-expressing animals: (A) Longitudinal view. Scheme of the six indirect flight muscles in the Drosophila adult. (B) Confocal image of mir-276a overexpressing DLMs stained with Phalloidin and DAPi. Only three DLMs are seen. Scale bar 60 μm. (C) Longitudinal paraffin section of an adult wild-type fly stained with hematoxylin and eosin. Six DLM muscles are visible (asterisks). Scale bar 100 μm. (D) Longitudinal paraffin section of an adult fly expressing UAS-mir-276a in myoblasts with DMef2-GAL4. Three DLMs are detectable (asterisks). Scale bar 150 μm. (E–G) Transverse sections of indirect flight muscles of adult flies stained with hematoxylin. 10× Objective. (E) DMef2-GAL4 fly with a normal number of indirect flight muscles (asterisks) on each site of the thorax. (F) Adult fly expressing UAS-mir276a in myoblasts, showing an abnormal size and the number of indirect flight muscles (asterisks). (G) Adult fly expressing UAS-mir276a only in adult myoblasts with 1151-GAL4, showing an abnormal size and a number of indirect flight muscles (asterisks). On the right side only four DLMs are detectable. Full article ">Figure 5
Determination of nuclei for each DLM muscle: (A) Three-dimensional view of the six DLM muscles (asterisks) marked with UAS-mCD8-GFP, DAPi, and Phalloidin. Scale bar 30 μm. (B) Cross-section of the flight muscles that are highlighted by the yellow line and the asterisks in (A). Scale bar 20 μm. (C) Illustration of the individual work steps for quantifying the cell nuclei of the six DLMs marked by asterisks. Scale bar 30 μm. (D) Illustration of the accuracy of the Imaris 9.3 software in the counting of nuclei. Higher magnification of the area marked in (C). Scale bar 10 μm. (E) Quantification of the nuclei in the DLMS. Full article ">Figure 6
RNAi-mediated knockdown of heartless and stumps in DLMs: Three-dimensional view of (A) wild-type DLMs expressing UAS-mCD8-GFP with DMef2-GAL4, (B) UAS-htl-RNAi expressing DLMs with DMef2-GAL4, and (C) UAS-stumps-RNAi expressing DLMs with DMef2-GAL4. (D) Total number of nuclei in DLMs from Dmef2 >> UAS-mCD8-eGFP, Dmef2 >> UAS-htl-RNAi, and DMef2 >> UAS-stumps-RNAi flies. (E) Number of nuclei within each DLM muscle for the six DLM muscles of DMef2 >> UAS-mCD8-eGFP, DMef2 >> UAS-htl-RNAi, and DMef2 >> UAS-stumps-RNAi. (F,G) Transverse sections of DLMs. (F) Wild type. (G) DMef2-GAL4 >> UAS-htl-RNAi. Scale bars in A–C 30 μm, scale bars in F and G 100 μm. Full article ">

18 pages, 4966 KiB

Open AccessArticle

Exploring Metabolic Shifts in Kidney Cancer and Non-Cancer Cells Under Pro- and Anti-Apoptotic Treatments Using NMR Metabolomics

by Lucia Trisolini, Biagia Musio, Beatriz Teixeira, Maria Noemi Sgobba, Anna Lucia Francavilla, Mariateresa Volpicella, Lorenzo Guerra, Anna De Grassi, Vito Gallo, Iola F. Duarte and Ciro Leonardo Pierri

Cells 2025, 14(5), 367; https://doi.org/10.3390/cells14050367 - 2 Mar 2025

Viewed by 307

Abstract

This study investigates the metabolic responses of cancerous (RCC) and non-cancerous (HK2) kidney cells to treatment with Staurosporine (STAU), which has a pro-apoptotic effect, and Bongkrekic acid (BKA), which has an anti-apoptotic effect, individually and in combination, using ¹H NMR metabolomics to [...] Read more.

This study investigates the metabolic responses of cancerous (RCC) and non-cancerous (HK2) kidney cells to treatment with Staurosporine (STAU), which has a pro-apoptotic effect, and Bongkrekic acid (BKA), which has an anti-apoptotic effect, individually and in combination, using ¹H NMR metabolomics to identify metabolite markers linked to mitochondrial apoptotic pathways. BKA had minimal metabolic effects in RCC cells, suggesting its role in preserving mitochondrial function without significantly altering metabolic pathways. In contrast, STAU induced substantial metabolic reprogramming in RCC cells, disrupting energy production, redox balance, and biosynthesis, thereby triggering apoptotic pathways. The combined treatment of BKA and STAU primarily mirrored the effects of STAU alone, with BKA showing little capacity to counteract the pro-apoptotic effects. In non-cancerous HK2 cells, the metabolic alterations were far less pronounced, highlighting key differences in the metabolic responses of cancerous and non-cancerous cells. RCC cells displayed greater metabolic flexibility, while HK2 cells maintained a more regulated metabolic state. These findings emphasize the potential for targeting cancer-specific metabolic vulnerabilities while sparing non-cancerous cells, underscoring the value of metabolomics in understanding apoptotic and anti-apoptotic mechanisms. Future studies should validate these results in vivo and explore their potential for personalized treatment strategies. Full article

(This article belongs to the Collection Feature Papers in Mitochondria)

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12 pages, 1951 KiB

Open AccessBrief Report

Spheroids Composed of Reaggregated Neonatal Porcine Islets and Human Endothelial Cells Accelerate Development of Normoglycemia in Diabetic Mice

by Mohsen Honarpisheh, Yutian Lei, Antonia Follenzi, Alessia Cucci, Cristina Olgasi, Ekaterine Berishvili, Fanny Lebreton, Kevin Bellofatto, Lorenzo Piemonti, Antonio Citro, Francesco Campo, Cataldo Pignatelli, Olivier Thaunat, Elisabeth Kemter, Martin Kraetzl, Eckhard Wolf, Jochen Seissler, Lelia Wolf-van Buerck and VANGUARD Consortium

Cells 2025, 14(5), 366; https://doi.org/10.3390/cells14050366 - 2 Mar 2025

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Abstract

The engraftment of transplanted islets depends on the rapid establishment of a novel vascular network. The present study evaluated the effects of cord blood-derived blood outgrowth endothelial cells (BOECs) on the viability of neonatal porcine islets (NPIs) and the post-transplant outcome of grafted [...] Read more.

The engraftment of transplanted islets depends on the rapid establishment of a novel vascular network. The present study evaluated the effects of cord blood-derived blood outgrowth endothelial cells (BOECs) on the viability of neonatal porcine islets (NPIs) and the post-transplant outcome of grafted NPIs. Dispersed NPIs and human BOECs were reaggregated on microwell cell culture plates and tested for their anti-apoptotic and pro-angiogenic capacity by qRT-PCR and immunohistochemistry. The in vivo functionality was analyzed after transplantation into diabetic NOD-SCID IL2rγ^−/− (NSG) mice. The spheroids, which contained reaggregated neonatal porcine islet cells (REPIs) and BOECs, exhibited enhanced viability and a significantly elevated gene expression of VEGFA, angiopoetin-1, heme oxygenase-1, and TNFAIP3 (A20) in vitro. The development of normoglycemia was significantly faster in animals transplanted with spheroids in comparison to the only REPI group (median 51.5 days versus 60 days) (p < 0.05). Furthermore, intragraft vascular density was substantially increased (p < 0.01). The co-transplantation of prevascularized REPI-BOEC spheroids resulted in superior angiogenesis and accelerated in vivo function. These findings may provide a novel tool to enhance the efficacy of porcine islet xenotransplantation. Full article

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Figure 1
Generation and phenotypic characterization of REPIs and REPI-BOEC spheroids. (A) Phase contrast images of composite spheroids and REPIs generated in Sphericalplate 5D 24-well plates and stained for hCD31 (brown). Scale bar = 100 µm. (B) In vitro beta cell function measured by static glucose-stimulated insulin secretion (n = 6–7). (C) Cell viability at day 2 after cluster formation as determined by TUNEL assay (n = 3). (D) Real-time PCR analysis of porcine heme oxygenase-1 (HMOX-1) and A20 (TNFAIP3 gene expression at day 1 and 3 after spheroid formation (n = 4–6). Data shown are mean ± SD and represent the results of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 REPI-BOEC spheroids vs. REPIs. Scale bar = 100 µm. Full article ">Figure 2
Transplantation with REPI-BOEC spheroids improved diabetes reversal. (A) Life table analysis of diabetes reversal in diabetic NSG mice transplanted with 1500 IEQ REPIs (open cycles n = 10) or 1500 IEQ REPI-BOEC spheroids (black cycles, n = 8). Spheroid transplantation reduced the time to develop normoglycemia (p < 0.02 according to the log-rank test). (B–D) Intraperitoneal glucose tolerance test (IPGTT) performed 8–14 days after the development of persistent normoglycemia was similar in both transplant groups as assessed by measurement of blood glucose profiles (B), glucose clearance (AUC glucose 0–120 min) (C), and insulin secretion at 0 and 10 min after glucose challenge (D). (E) Quantification of grafted cells (day 7 after transplantation) immunostaining for Ki67 (cell proliferation), Nkx6.1 (marker of endocrine progenitor cells and mature beta cells), or MAFA (mature beta cells) and pan-cytokeratin (staining of all transplanted porcine cells) revealed no difference in groups transplanted with REPIs alone or with REPI-BOEC (n = 3). Full article ">Figure 3
Spheroids display proangiogenic capacity in vitro and improve revascularization of transplanted grafts in vivo. (A) Detection of blood vessels in the grafted area at the end of the observation period (posttransplant week 16). Representative images show a higher intragraft vascular network in mice transplanted with REPI-BOEC spheroids compared to REPIs alone. Left: Immunostaining for mouse CD31 (brown). Right: Double immunostaining for mouse CD31 (green) and human CD31 (red) showing the integration of human ECs into the vascular network. Scale bar = 100 µm. (B) Quantification of the vessel density (number of mCD31 positive cells) within the graft area. (C) Gene expression of the human angiogenic factors VEGFA and ANG1 in REPIs and REPI-BOEC spheroids relative to human GAPDH detected by qRT-PCR. Data are presented as mean ± SD and are generated from at least three independent experiments. ** p < 0.01, *** p < 0.001. Full article ">

25 pages, 19182 KiB

Open AccessArticle

Modification of RNF183 via m6A Methylation Mediates Podocyte Dysfunction in Diabetic Nephropathy by Regulating PKM2 Ubiquitination and Degradation

by Dongwei Guo, Yingxue Pang, Wenjie Wang, Yueying Feng, Luxuan Wang, Yuanyuan Sun, Jun Hao, Fan Li and Song Zhao

Cells 2025, 14(5), 365; https://doi.org/10.3390/cells14050365 - 1 Mar 2025

Viewed by 431

Abstract

Diabetic kidney disease (DKD) is a prevalent complication associated with diabetes in which podocyte dysfunction significantly contributes to the development and progression of the condition. Ring finger protein 183 (RNF183) is an ER-localized, transmembrane ring finger protein with classical E3 ligase activity. However, [...] Read more.

Diabetic kidney disease (DKD) is a prevalent complication associated with diabetes in which podocyte dysfunction significantly contributes to the development and progression of the condition. Ring finger protein 183 (RNF183) is an ER-localized, transmembrane ring finger protein with classical E3 ligase activity. However, whether RNF183 is involved in glomerular podocyte dysfunction, which is the mechanism of action of DKD, is still poorly understood. In this study, we first demonstrated that RNF183 expression in glomerular podocytes of patients with DKD decreased as the disease progressed. Additionally, our transcriptome sequencing analysis of kidney tissues from diabetic mice revealed a significant reduction in RNF183 expression within the kidney cortex. Similarly, the expression of RNF183 was significantly reduced both in the kidneys of diabetic mice and in human podocytes exposed to high glucose conditions. The downregulation of RNF183 resulted in a suppression of autophagic activity, an increase in apoptotic cell death, and reduced expression of cellular markers in HPC cells. We found that RNF183 was modified via N6-methyladenosine (m6A) RNA methylation. Meanwhile, treatment with meclofenamic acid 2 (MA2), an m6A demethylase inhibitor, resulted in the upregulation of RNF183 expression in HPC cells cultured in high glucose conditions. Furthermore, high glucose treatment decreased the transcription and protein levels in both the m6A writer methyltransferaselike3 (METTL3) and the m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). IGF2BP2 assisted with METTL3, which is jointly involved in the transcription of RNF183. Furthermore, we confirmed that RNF183 directly ubiquitinates M2 pyruvate kinase (PKM2) through co-immunoprecipitation (Co-IP) and liquid chromatography–mass spectrometry (LC-MS) experiments. The level of PKM2 ubiquitination was increased following RNF183 overexpression, leading to enhanced PKM2 protein degradation and subsequently alleviating high glucose-induced podocyte damage. The results of this study indicated that RNF183 was regulated via m6A methylation modification and that RNF183 expression was reduced in HPC cells treated with high glucose, which resulted in decreased PKM2 ubiquitination levels and subsequently aggravated podocyte injury. The findings suggest that RNF183 may serve as a potential therapeutic target for diabetic kidney injury, offering new insights into its role in the progression of DKD. Full article

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

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17 pages, 6414 KiB

Open AccessArticle

miR-204-5p Protects Nephrin from Enzymatic Degradation in Cultured Mouse Podocytes Treated with Nephrotoxic Serum

by George Haddad and Judith Blaine

Cells 2025, 14(5), 364; https://doi.org/10.3390/cells14050364 - 1 Mar 2025

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Abstract

Nephrin is an essential constituent of the slit diaphragm of the kidney filtering unit. Loss of nephrin expression leads to protein leakage into the urine, one of the hallmarks of kidney damage. Autoantibodies against nephrin have been reported in patients with minimal change [...] Read more.

Nephrin is an essential constituent of the slit diaphragm of the kidney filtering unit. Loss of nephrin expression leads to protein leakage into the urine, one of the hallmarks of kidney damage. Autoantibodies against nephrin have been reported in patients with minimal change disease and recurrent focal segmental glomerulosclerosis. Understanding the mechanism of nephrin loss may help improve or lead to the development of novel treatment strategies. In this study, we demonstrated the important function of miR-204-5p expression on the protection of nephrin from anti-nephrin antibodies present in nephrotoxic serum (NTS). In addition, we identified that aspartyl protease cathepsin D is one enzyme that may be involved in nephrin enzymatic degradation and that cathepsin D is a direct target of miR-204-5p gene regulation. The regulation of miR-204-5p expression was determined to be regulated by the long noncoding RNA Josd1-ps. In an NTS in vivo animal model, treatment with the pan aspartic protease inhibitor Pepstatin A ameliorated renal damage. Finally, we showed that the expression of miR-204-5p had a nephrin-protecting function in vitro. Developing a method of delivery of miR-204-5p specifically to podocytes in vivo may provide a novel method of nephroprotection against nephrin autoantibodies. Full article

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Figure 1
NTS targets nephrin when miR-204-5p expression is reduced. Immortalized mouse podocytes were treated with the miRNA control or miR-204-5p mimic or inhibitory sequences or left untreated prior to the addition of 1:200 nephrotoxic serum (NTS). The cell lysates were analyzed by Western blot (A) and probed for the podocyte’s markers nephrin, neph1, and podocin. Actin was used as a loading control. The blots are quantified in (B–D). The miR-204-5p sequence expressions were analyzed by real-time PCR, and the data were normalized to the expression of miR-16-5p (E). The presence of anti-nephrin antibodies in NTS was analyzed by Western blot (F) under native and reduced and denatured conditions. The immunofluorescence images in (G) show mouse podocytes transfected with miR-204-5p mimic, inhibitor, or control sequences prior to NTS treatment. The images were obtained using a Stedycon Abberior (STED) (Göttingen, Germany) and Olympus 1X81(Tokyo, Japan) confocal microscope and 100X objective lens. Nephrin is stained in green, actin is stained in red and the nucleus is stained with DAPI (blue). The scale bar represents 10 μm. The experiments were repeated 4 times (n = 4), and p values < 0.05 were considered significant, **** p < 0.0001. Full article ">Figure 2
Nephrin degradation is mediated by an aspartyl enzyme. Immortalized mouse podocytes were transfected with miR-204-5p mimic, inhibitor, or control sequences and treated with protease inhibitor cocktail before treatment with NTS. As shown by the Western blot (A) and quantitation (B), protease inhibition prevented the NTS-induced degradation of nephrin, even when miR-204 was inhibited. Mouse podocytes treated with the miR-204-5p inhibitory sequence and various protease inhibitors before NTS treatment showed that the aspartic acid protease inhibitor Pepstatin A rescued nephrin from degradation (C), and this was evident through Western blot quantitation (D). The experiments were repeated 4 times (n = 4). p values < 0.05 were considered significant ** p < 0.003, **** p < 0.001. Full article ">Figure 3
LAMP1 and cathepsin D are targets of miR-204-5p. The overexpression of the miR-204-5p mimic sequence reduced the expression of LAMP1 (A,C) but had no effect on nephrin (A,B) or cathepsin E (A,D), as determined by Western blot analysis. Using cathepsin ELISA, the overexpression of miR-204-5p decreased cathepsin D expression (E). The effect of cathepsin D’s enzymatic activity on nephrin is shown in (F) using SDS-PAGE gel and silver staining. Cathepsin D treatment results in nephrin fragments (arrows). The experiments were repeated 4 times (n = 4). * p < 0.05, *** p = 0.0007. Full article ">Figure 4
LncJosd1-ps regulates the expression of miR-204-5p. LncJosd1-ps sequence was cloned into a lentiviral vector (A) to overexpress lncJosd1-ps and an antisense oligo (ASO) was designed (B) to reduce lncJosd1-ps expression. (C) shows that the overexpression of lncJosd1-ps reduced miR-204-5p expression, whereas lncJosd1-ps inhibition increased miR-204-5p expression (D). The subcellular location of lncJosd1-ps was determined, and it shows nuclear as well as cytoplasmic expressions (E). The experiments were repeated 4 times (n = 4). p values < 0.05 were considered significant ** p < 0.003, *** p = 0.0002, **** p < 0.0001. Full article ">Figure 5
Validation of miR-204-5p targets by luciferase assay. The miR-204-5p binding sites within LAMP1 and cathepsin D 3′ UTR and within the lncJosd1-ps nucleotide sequence are shown in (A). miR-204-5p interaction with LAMP1 3′ UTR is validated by a luciferase assay using intact LAMP1 or mutated binding sites (B), cathepsin D (C), and lncJosd1-ps (D). The red letters indicate the complimentary nucleotides between miR-204-5p and the target genes sequences. The experiments were repeated 3 times (n = 3). * p value < 0.05 was considered significant from NT group. Full article ">Figure 6
The effects of NTS on kidney function in vivo. Male C57Bl/6 mice were injected with 100 μL of NTS via the tail vein and euthanized after 1 week. Kidneys were excised, and total RNA was extracted and analyzed by real-time PCR for the expression levels of miR-204-5p (A), lncJosd1 (B), KIM1 (C), NGAL (D), nephrin (E) and podocin (F). Albuminuria was significantly increased in mice treated with NTS compared to the control mice (G). Figure (H) shows glomerular damage induced by NTS (PAS stain, scale bar 50 µm). Seven to eight animals were included per group. * p < 0.05, ** p < 0.003, *** p = 0.0002, **** p < 0.0001. Full article ">Figure 7
Pepstatin A reduced kidney injury in the NTS model of immune-mediated kidney disease. Mouse kidney total RNA was analyzed for miR-204-5p and lncJosd1-ps expression in animals treated with NTS followed by IP injection of Pepstatin A (20 mg/Kg) or carrier (100 µL ethanol) on days 1, 3, and 5 (A,B). The expression levels of the kidney injury markers KIM1 (C) and NGAL (D) were determined at the mRNA level along with the podocyte markers nephrin (E) and podocin (F). Kidney morphology was assessed using PAS stain (G). Glomerulosclerosis was determined using a 0–4 scoring system, where 0 = 0%, 1 = 25%, 2 = 50%, 3 = 75%, and 4 = 100% glomerulosclerosis. All available cortical glomeruli in a PAS-stained tissue section were analyzed (H). Albuminuria was determined using the albumin-to-creatinine ratio (I). At least 5–8 animals were used per group. * p < 0.05 different from control group, # different from NTS group p < 0.002, NS = not significant from control; scale bars: 50 µm. Full article ">

18 pages, 1457 KiB

Open AccessReview

Sex Disparities in P53 Regulation and Functions: Novel Insights for Personalized Cancer Therapies

by Miriana Cardano, Giacomo Buscemi and Laura Zannini

Cells 2025, 14(5), 363; https://doi.org/10.3390/cells14050363 - 28 Feb 2025

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Abstract

Epidemiological studies have revealed significant sex differences in the incidence of tumors unrelated to reproductive functions, with females demonstrating a lesser risk and a better response to therapy than males. However, the reasons for these disparities are still unknown and cancer therapies are [...] Read more.

Epidemiological studies have revealed significant sex differences in the incidence of tumors unrelated to reproductive functions, with females demonstrating a lesser risk and a better response to therapy than males. However, the reasons for these disparities are still unknown and cancer therapies are generally sex-unbiased. The tumor-suppressor protein p53 is a transcription factor that can activate the expression of multiple target genes mainly involved in the maintenance of genome stability and tumor prevention. It is encoded by TP53, which is the most-frequently mutated gene in human cancers and therefore constitutes an attractive target for therapy. Recently, evidence of sex differences has emerged in both p53 regulations and functions, possibly providing novel opportunities for personalized cancer medicine. Here, we will review and discuss current knowledge about sexual disparities in p53 pathways, their role in tumorigenesis and cancer progression, and their importance in the therapy choice process, finally highlighting the importance of considering sex contribution in both basic research and clinical practice. Full article

(This article belongs to the Section Cell Signaling)

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Full article ">Figure 1
The multiple cellular pathways in which p53 is involved. In response to stress signals, p53 exerts different functions by regulating different mechanisms, which include cell cycle arrest, apoptosis, DNA repair, senescence, cellular metabolism, tumor microenvironment, ROS response, epigenetic modifications, EMT and inflammation. Image was created using pictures from Servier Medical Art, by Servier (<a href="http://smart.servier.com" target="_blank">http://smart.servier.com</a>). Full article ">Figure 2
p53 domain structure. Human p53 protein has a transactivation domain (TAD), a proline rich domain (PRD), a DNA-binding domain, a tetramerization domain (TET) and a regulatory domain (REG). Full article ">Figure 3
Graphical scheme representing p53 protein level maintenance in unstressed conditions (a) and its accumulation and activation in response to genotoxic stress (b). Full article ">Figure 4
(a) Graphical representation of p53–hormones interplay. (b) Mutated X-linked mRNA expression in male and female cells. If a gene mutation occurs in the male X chromosome, the mutated gene will be expressed. In female cells, the gene mutations on Xi chromosome will remain unexpressed. Images were created using pictures from Servier Medical Art, by Servier (<a href="http://smart.servier.com" target="_blank">http://smart.servier.com</a>). Full article ">Figure 5
Graphical representation of sex disparities in p53 functions. Sex differences exist in p53’s involvement in development and aging, cancer, Li Fraumeni syndrome and cancer therapy. Image was created using pictures from Servier Medical Art, by Servier (<a href="http://smart.servier.com" target="_blank">http://smart.servier.com</a>). Full article ">

12 pages, 1067 KiB

Open AccessReview

The Dual Role of cGAS-STING Signaling in COVID-19: Implications for Therapy

by Daniele Castro di Flora, João Paulo Zanardini Lara, Aline Dionizio and Marília Afonso Rabelo Buzalaf

Cells 2025, 14(5), 362; https://doi.org/10.3390/cells14050362 - 28 Feb 2025

Viewed by 186

Abstract

The progression of COVID-19 involves a sophisticated and intricate interplay between the SARS-CoV-2 virus and the host’s immune response. The immune system employs both innate and adaptive mechanisms to combat infection. Innate immunity initiates the release of interferons (IFNs) and pro-inflammatory cytokines, while [...] Read more.

The progression of COVID-19 involves a sophisticated and intricate interplay between the SARS-CoV-2 virus and the host’s immune response. The immune system employs both innate and adaptive mechanisms to combat infection. Innate immunity initiates the release of interferons (IFNs) and pro-inflammatory cytokines, while the adaptive immune response involves CD4+ Th lymphocytes, B lymphocytes, and CD8+ Tc cells. Pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPs), activating the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) signaling pathway, a crucial component of the innate immune response to SARS-CoV-2. This pathway fulfills a dual function during infection. In the early phase of infection, the virus can suppress cGAS-STING signaling to avoid immune detection. However, in the late stages, the activation of this pathway may trigger excessive inflammation and tissue damage, exacerbating disease severity. Modulating the cGAS-STING pathway, whether through agonists like dimeric amidobenzimidazole (diABZI) or inhibitors targeting viral proteins, such as 3CLpro, for example, offers a promising approach for personalized therapy to control the immune response and mitigate severe inflammation, ultimately improving clinical outcomes in patients with severe COVID-19. Full article

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27 pages, 2937 KiB

Open AccessArticle

Inflammatory Stimuli and Fecal Microbiota Transplantation Accelerate Pancreatic Carcinogenesis in Transgenic Mice, Accompanied by Changes in the Microbiota Composition

by Agnieszka Świdnicka-Siergiejko, Jarosław Daniluk, Katarzyna Miniewska, Urszula Daniluk, Katarzyna Guzińska-Ustymowicz, Anna Pryczynicz, Milena Dąbrowska, Małgorzata Rusak, Michał Ciborowski and Andrzej Dąbrowski

Cells 2025, 14(5), 361; https://doi.org/10.3390/cells14050361 - 28 Feb 2025

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Abstract

An association between gut microbiota and the development of pancreatic ductal adenocarcinoma (PDAC) has been previously described. To better understand the bacterial microbiota changes accompanying PDAC promotion and progression stimulated by inflammation and fecal microbiota transplantation (FMT), we investigated stool and pancreatic microbiota [...] Read more.

An association between gut microbiota and the development of pancreatic ductal adenocarcinoma (PDAC) has been previously described. To better understand the bacterial microbiota changes accompanying PDAC promotion and progression stimulated by inflammation and fecal microbiota transplantation (FMT), we investigated stool and pancreatic microbiota by 16s RNA-based metagenomic analysis in mice with inducible acinar transgenic expressions of KrasG12D, and age- and sex-matched control mice that were exposed to inflammatory stimuli and fecal microbiota obtained from mice with PDAC. Time- and inflammatory-dependent stool and pancreatic bacterial composition alterations and stool alpha microbiota diversity reduction were observed only in mice with a Kras mutation that developed advanced pancreatic changes. Stool Actinobacteriota abundance and pancreatic Actinobacteriota and Bifidobacterium abundances increased. In contrast, stool abundance of Firmicutes, Verrucomicrobiota, Spirochaetota, Desulfobacterota, Butyricicoccus, Roseburia, Lachnospiraceae A2, Lachnospiraceae unclassified, and Oscillospiraceae unclassified decreased, and pancreatic detection of Alloprevotella and Oscillospiraceae uncultured was not observed. Furthermore, FMT accelerated tumorigenesis, gradually decreased the stool alpha diversity, and changed the pancreatic and stool microbial composition in mice with a Kras mutation. Specifically, the abundance of Actinobacteriota, Bifidobacterium and Faecalibaculum increased, while the abundance of genera such as Lachnospiraceace A2 and ASF356, Desulfovibrionaceace uncultured, and Roseburia has decreased. In conclusion, pancreatic carcinogenesis in the presence of an oncogenic Kras mutation stimulated by chronic inflammation and FMT dynamically changes the stool and pancreas microbiota. In particular, a decrease in stool microbiota diversity and abundance of bacteria known to be involved in short-fatty acids production were observed. PDAC mouse model can be used for further research on microbiota–PDAC interactions and towards more personalized and effective cancer therapies. Full article

(This article belongs to the Section Tissues and Organs)

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Abundance of bacterial phyla in inflammation-induced pancreatic carcinogenesis. Relative abundance (%) of most common phyla in pancreas and stool samples (a). The differences between Kras/Cre mice and Cre mice 30 days and 120 days after saline and cerulein injections in the abundance of Actinobacteriota in pancreas samples (b), Actinobacteriota in stool (c), Verrucomicrobiota in stool (d), Desulfobacterota in stool (e), Firmicutes in stool (f). The median abundance (%) is indicated by a black line. The box represents the interquartile range. The whiskers extend to the upper adjacent value and the lower adjacent value and dots represent outliers. Statistically significant differences (p value < 0.05) between mice are marked * (for details see <a href="#app1-cells-14-00361" class="html-app">Table S4</a>). Sal—saline, CER—cerulein, Kras/Cre—mice with Kras mutation, Cre—mice without Kras mutation. Full article ">Figure 2
Abundance of bacterial genera in inflammation-induced pancreatic carcinogenesis. Relative abundance of most common phyla in pancreas and stool samples (a). The differences between Kras/Cre mice and Cre mice 30 days and 120 days after saline and cerulein injections in abundance of Bifidobacterium in pancreas samples (b), Butyricicoccus in stool (c), Clostridia UCG-014 in stool (d), Lachnospiraceae unclassified in stool (e), Lachnospiraceae A2 in stool (f), Oscillospiraceae unclassified in stool (g), Roseburia in stool (h), Erysipelotrichaceae uncultured in stool (i), Lachnoclostridium in stool (j) and Lachnospiraceae UCG-006 in stool (k). The median abundance (%) is indicated by a black line. The box represents the interquartile range. The whiskers extend to the upper adjacent value and the lower adjacent value and dots represent outliers. Statistically significant differences (p value < 0.05) between mice are marked * (for details: see <a href="#app1-cells-14-00361" class="html-app">Table S7</a>). Sal—saline, CER—cerulein, Kras/Cre—mice with Kras mutation, Cre—mice without Kras mutation. Full article ">Figure 3
The microbiota diversity in pancreas and stool samples in inflammation-induced carcinogenesis. Shannon index—pancreas (a), Shannon index—stool (b), Principal coordinates analysis (PCoA)—stool phyla (c), Principal coordinates analysis (PCoA)—stool genera (d), Principal coordinates analysis (PCoA)—pancreas phyla (e), Principal coordinates analysis (PCoA)—pancreas genera (f). (a,b): The median alpha diversity (Shannon index) is indicated by a black line. The box represents the interquartile range. The whiskers extend to the upper adjacent value and the lower adjacent value and dots represent outliers. Statistically significant differences (p value < 0.05) between mice are marked * (for details see <a href="#app1-cells-14-00361" class="html-app">Table S11</a>). Sal—saline, CER—cerulein, Kras/Cre—mice with Kras mutation, Cre—mice without Kras mutation. Full article ">Figure 4
Abundance of bacterial phyla after fecal microbiota transplantation associated pancreatic carcinogenesis. Relative abundance of the most common phyla in pancreas and stool samples in Kras/Cre mice and Cre mice after FMT and sham treatments (a). The differences between tested mice in abundance of Actinobacteriota in pancreas sample (b), Actinobacteriota in stool (c), Cyanobacteria in stool (d), Deferribacterota in stool (e), Desulfobacterota in stool (f), Proteobacteria in stool (g), and Spirochaetota in stool (h). The median abundance (%) is indicated by a black line. The box represents the interquartile range. The whiskers extend to the upper adjacent value and the lower adjacent value and dots represent outliers. Statistically significant differences (p value < 0.05) between mice are marked * (for details see <a href="#app1-cells-14-00361" class="html-app">Table S14</a>). Kras/Cre—mice with Kras mutation, Cre—mice without Kras mutation, FMT—fecal microbiota transplantation. Full article ">Figure 5
Abundance of bacterial genera after fecal microbiota transplantation associated pancreatic carcinogenesis. Relative abundance of the most common genera in pancreas and stool samples in Kras/Cre mice and Cre mice after FMT and sham treatments (a). The differences between tested mice in abundance in pancreatic tissue of Bifidobacterium (b), Dubosiella (c), Faecalibaculum (d), Roseburia (e), Desulfovibrionaceae (f), Lachnospiraceae A2 (g), Lachnospiraceae ASF356 (h), and Lachnospiraceae NK4A136 group (i). The median abundance (%) is indicated by a black line. The box represents the interquartile range. The whiskers extend to the upper adjacent value and the lower adjacent value and dots represent outliers. Statistically significant differences (p value < 0.05) between mice are marked * (for details see <a href="#app1-cells-14-00361" class="html-app">Table S17</a>). Kras/Cre—mice with Kras mutation, Cre—mice without Kras mutation, FMT—fecal microbiota transplantation. Full article ">Figure 6
Differences between Kras/Cre mice and Cre mice after fecal transplantation and sham treatments in abundance in stool of bacterial genera. Relative abundance of Bifidobacterium (a), Faecalibaculum (b), Roseburia (c), Desulfovibrionaceae uncultured (d), Lachnospiraceae A2 (e), Lachnospiraceae ASF356 (f). The median abundance (%) is indicated by a black line. The box represents the interquartile range. The whiskers extend to the upper adjacent value and the lower adjacent value and dots represent outliers. Statistically significant differences (p value < 0.05) between mice are marked * (for details see <a href="#app1-cells-14-00361" class="html-app">Table S20</a>). Kras/Cre mice—mice with Kras mutation, Cre mice—mice without Kras mutation, FMT—fecal microbiota transplantation. Full article ">Figure 7
Bacterial microbiota diversity in fecal microbiota transplantation associated pancreatic carcinogenesis. Alpha diversity index (Shannon index) of the microbiota in pancreas (a) and stool (b) samples in Kras/Cre mice and Cre mice after fecal microbiota transplantation and sham treatments. Principal coordinate analysis (PCoA) plot results based on Bray–Curtis dissimilarity distance at the genera level in Kras/Cre mice and Cre mice after fecal microbiota transplantation and sham treatments in pancreas (c) and stool (d) samples. Bacterial stool alpha diversity changes (Shannon index) over time after fecal microbiota transplantation in Cre mice (e) and Kras/Cre mice (f). (a,b,e,f): The median is indicated by a black line. The box represents the interquartile range. The whiskers extend to the upper adjacent value and the lower adjacent value and dots represent outliers. Statistically significant differences (p value < 0.05) between mice are marked * (for detail see <a href="#app1-cells-14-00361" class="html-app">Table S22</a>). Kras/Cre—mice with Kras mutation, Cre—mice without Kras mutation, FMT—fecal microbiota transplantation. Full article ">

30 pages, 1583 KiB

Open AccessReview

Endometriosis and Cytoskeletal Remodeling: The Functional Role of Actin-Binding Proteins

by Wioletta Arendt, Konrad Kleszczyński, Maciej Gagat and Magdalena Izdebska

Cells 2025, 14(5), 360; https://doi.org/10.3390/cells14050360 - 28 Feb 2025

Viewed by 150

Abstract

Endometriosis is a chronic, estrogen-dependent gynecological disorder characterized by the presence of endometrial-like tissue outside the uterine cavity. Despite its prevalence and significant impact on women’s health, the underlying mechanisms driving the invasive and migratory behavior of endometriotic cells remain incompletely understood. Actin-binding [...] Read more.

Endometriosis is a chronic, estrogen-dependent gynecological disorder characterized by the presence of endometrial-like tissue outside the uterine cavity. Despite its prevalence and significant impact on women’s health, the underlying mechanisms driving the invasive and migratory behavior of endometriotic cells remain incompletely understood. Actin-binding proteins (ABPs) play a critical role in cytoskeletal dynamics, regulating processes such as cell migration, adhesion, and invasion, all of which are essential for the progression of endometriosis. This review aims to summarize current knowledge on the involvement of key ABPs in the development and pathophysiology of endometriosis. We discuss how these proteins influence cytoskeletal remodeling, focal adhesion formation, and interactions with the extracellular matrix, contributing to the unique mechanical properties of endometriotic cells. Furthermore, we explore the putative potential of targeting ABPs as a therapeutic strategy to mitigate the invasive phenotype of endometriotic lesions. By elucidating the role of ABPs in endometriosis, this review provides a foundation for future research and innovative treatment approaches. Full article

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20 pages, 27145 KiB

Open AccessArticle

The Evolutionary Young Actin Nucleator Cobl Is Important for Proper Amelogenesis

by Hannes Janitzek, Jule González Delgado, Natja Haag, Eric Seemann, Sandor Nietzsche, Bernd Sigusch, Britta Qualmann and Michael Manfred Kessels

Cells 2025, 14(5), 359; https://doi.org/10.3390/cells14050359 - 28 Feb 2025

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Abstract

The actin cytoskeleton plays an important role in morphological changes of ameloblasts during the formation of enamel, which is indispensable for teeth to withstand wear, fracture and caries progression. This study reveals that the actin nucleator Cobl is expressed in ameloblasts of mandibular [...] Read more.

The actin cytoskeleton plays an important role in morphological changes of ameloblasts during the formation of enamel, which is indispensable for teeth to withstand wear, fracture and caries progression. This study reveals that the actin nucleator Cobl is expressed in ameloblasts of mandibular molars during amelogenesis. Cobl expression was particularly pronounced during the secretory phase of the enamel-forming cells. Cobl colocalized with actin filaments at the cell cortex. Importantly, our analyses show an influence of Cobl on both ameloblast morphology and cytoskeletal organization as well as on enamel composition. At P0, Cobl knock-out causes an increased height of ameloblasts and an increased F-actin content at the apical membrane. During the maturation phase, the F-actin density at the apical membrane was instead significantly reduced when compared to WT mice. At the same time, Cobl-deficient mice showed an increased carbon content of the enamel and an increased enamel surface of mandibular molars. These findings demonstrate a decisive influence of the actin nucleator Cobl on the actin cytoskeleton and the morphology of ameloblasts during amelogenesis. Our work thus expands the understanding of the regulation of the actin cytoskeleton during amelogenesis and helps to further elucidate the complex processes of enamel formation during tooth development. Full article

(This article belongs to the Section Tissues and Organs)

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