International Journal of Molecular Sciences

18 pages, 465 KiB

Open AccessArticle

Optimising Aripiprazole Long-Acting Injectable: A Comparative Study of One- and Two-Injection Start Regimens in Schizophrenia with and Without Substance Use Disorders and Relationship to Early Serum Levels

by Giada Trovini, Ginevra Lombardozzi, Georgios D. Kotzalidis, Luana Lionetto, Felicia Russo, Angela Sabatino, Elio Serra, Simone Castorina, Giorgia Civita, Sara Frezza, Donatella De Bernardini, Giuseppe Costanzi, Marika Alborghetti, Maurizio Simmaco, Ferdinando Nicoletti and Sergio De Filippis

Int. J. Mol. Sci. 2025, 26(3), 1394; https://doi.org/10.3390/ijms26031394 - 6 Feb 2025

Viewed by 831

Abstract

Aripiprazole as a long-acting injectable (LAI) is initiated in oral aripiprazole-stabilised patients and needs, after first injection, 14 days supplementation of oral aripiprazole (one-injection start, OIS). Recently, an alternative two-injection start (TIS) was advanced, involving two 400 mg injections with a single 20 [...] Read more.

Aripiprazole as a long-acting injectable (LAI) is initiated in oral aripiprazole-stabilised patients and needs, after first injection, 14 days supplementation of oral aripiprazole (one-injection start, OIS). Recently, an alternative two-injection start (TIS) was advanced, involving two 400 mg injections with a single 20 mg oral supplementation of aripiprazole. We tested the two regimens in patients with schizophrenia (SCZ, n = 152, 90 men and 62 women) with (SUD⁺; n = 93) or without (SUD^–; n = 59) substance use disorders (SUDs), comparing OIS (n = 66) with TIS (n = 86) and SUD⁺ vs. SUD^–. For 26 patients, we measured weekly for one month, aripiprazole + dehydroaripiprazole (active moiety) levels. Patients were followed for three months after LAI with psychopathology and quality-of-life scales (BPRS, CGI-S, ACES, BIS-11, and WHOQOL). All groups improved in psychopathology with no differences between OSI and TIS and between SCZ–SUD⁺ and SCZ–SUD^–. The TIS group was associated with serum blood levels of the active moiety within the therapeutic window, while the OIS group showed peaks above the window, possibly exposing patients to toxicity. Treatments were well-tolerated. Here we showed no disadvantages for TIS vs. OIS and possibly increased safety. Shifting the initiation of aripiprazole LAIs to the TIS modality may be safe and pharmacokinetically advantageous. Full article

(This article belongs to the Special Issue Translating Molecular Psychiatry: From Biomarkers to Personalized Therapies)

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14 pages, 2834 KiB

Open AccessArticle

Complement Factor B Deficiency Is Dispensable for Female Fertility but Affects Microbiome Diversity and Complement Activity

by Manato Sunamoto, Kazunori Morohoshi, Ban Sato, Ryo Mihashi, Masafumi Inui, Mitsutoshi Yamada, Kenji Miyado and Natsuko Kawano

Int. J. Mol. Sci. 2025, 26(3), 1393; https://doi.org/10.3390/ijms26031393 - 6 Feb 2025

Viewed by 496

Abstract

Complement factor B (CFB) is a crucial component for the activation of the alternative pathway due to the formation of the C3 convertase with C3b, which further produces C3b to enhance the overall complement activity. Although Cfb is expressed not only in the [...] Read more.

Complement factor B (CFB) is a crucial component for the activation of the alternative pathway due to the formation of the C3 convertase with C3b, which further produces C3b to enhance the overall complement activity. Although Cfb is expressed not only in the immune tissues, but also in the reproductive tract, the physiological role of the alternative complement pathway in reproduction remains unclear. In this study, we addressed this issue by producing Cfb-knockout (KO) mice and analyzing their phenotypes. Sperm function, number of ovulated oocytes, and litter size were normal in KO mice. In contrast, the diversity of microbiomes in the gut and vaginal tract significantly increased in KO mice. Some serine protease activity in the serum from KO mice was lower than that of wild-type mice. Since the serum from KO mice showed significantly lower activity of the alternative complement pathway, CFB was found to be essential for this pathway. Our results indicate that although the alternative pathway is dispensable for normal fertility and development, it maintains the gut and vaginal microbiomes by suppressing their diversity and activating the alternative complement pathway. Full article

(This article belongs to the Special Issue Exploring the Molecular Mechanisms of the Microbiome in Reproductive Health and Reproductive Immunity)

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19 pages, 5241 KiB

Open AccessArticle

Quantitative Proteomic Analysis of Lysine Malonylation in Response to Salicylic Acid in the Roots of Platycodon grandiflorus

by Wanyue Ding, Yingying Duan, Yuqing Wang, Jizhou Fan, Weiyi Rao and Shihai Xing

Int. J. Mol. Sci. 2025, 26(3), 1392; https://doi.org/10.3390/ijms26031392 - 6 Feb 2025

Viewed by 478

Abstract

Salicylic acid, as a plant hormone, significantly affects the physiological and biochemical indexes of soluble sugar, malondialdehyde content, peroxidase, and superoxide dismutase enzyme activity in Platycodon grandiflorus. Lysine malonylation is a post-translational modification that involves various cellular functions in plants, though it [...] Read more.

Salicylic acid, as a plant hormone, significantly affects the physiological and biochemical indexes of soluble sugar, malondialdehyde content, peroxidase, and superoxide dismutase enzyme activity in Platycodon grandiflorus. Lysine malonylation is a post-translational modification that involves various cellular functions in plants, though it is rarely studied, especially in medicinal plants. In this study, the aim was to perform a comparative quantitative proteomic study of malonylation modification on P. grandiflorus root proteins after salicylic acid treatment using Western blot with specific antibodies, affinity enrichment and LC-MS/MS analysis methods. The analysis identified 1907 malonyl sites for 809 proteins, with 414 proteins and 798 modification sites quantified with high confidence. Post-treatment, 361 proteins were upregulated, and 310 were downregulated. Bioinformatics analysis revealed that malonylation in P. grandiflorus is primarily involved in photosynthesis and carbon metabolism. Physiological and biochemical analysis showed that salicylic acid treatment increased the malondialdehyde levels, soluble protein, superoxide dismutase, and peroxidase activity but did not significantly affect the total saponins content in P. grandiflorus. These findings provide an important basis for exploring the molecular mechanisms of P. grandiflorus following salicylic acid treatment and enhance understanding of the biological function of protein lysine malonylation in plants. Full article

(This article belongs to the Special Issue Advanced Plant Molecular Responses to Abiotic Stresses)

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20 pages, 4383 KiB

Open AccessArticle

Adsorption of Serum Fetuin onto Octacalcium Phosphate and Its Relation to Osteogenic Property

by Yuki Tsuboi, Ryo Hamai, Kyosuke Okuyama, Kaori Tsuchiya, Yukari Shiwaku, Kensuke Yamauchi and Osamu Suzuki

Int. J. Mol. Sci. 2025, 26(3), 1391; https://doi.org/10.3390/ijms26031391 - 6 Feb 2025

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Abstract

This study aimed to investigate how the chemical elements in relation to octacalcium phosphate (OCP) hydrolysis affect the osteoblastic differentiation in the presence of serum fetuin. The adsorption of fetuin onto OCP was examined in buffers having different degrees of supersaturation (DS) with [...] Read more.

This study aimed to investigate how the chemical elements in relation to octacalcium phosphate (OCP) hydrolysis affect the osteoblastic differentiation in the presence of serum fetuin. The adsorption of fetuin onto OCP was examined in buffers having different degrees of supersaturation (DS) with respect to OCP and hydroxyapatite (HA) at pH 7.4 and 37 °C. The osteoblastic differentiation of mesenchymal stem cells (MSCs) was evaluated in cultures with OCP and 0 to 0.8 mg/mL of fetuin. The amount of fetuin adsorbed increased with increasing DS in the buffer. In the MSC culture, the coexistence of OCP and 0.2–0.4 mg/mL of fetuin close to serum level increased alkaline phosphatase activity; however, the activity was suppressed by 0.2–0.8 mg/mL of fetuin. Transmission electron microscopy revealed de novo crystal formation on OCP in supersaturated buffer and culture media with respect to OCP and HA at lower fetuin concentrations. Infrared spectroscopy and DS estimation indicate that the hydrolysis of OCP with de novo apatite formation was promoted in the culture media at 0.2–0.4 mg/mL of fetuin. These results suggest that OCP may promote osteoblastic differentiation if the suitable conditions are attained regarding the chemical elements and fetuin adsorption around OCP. Full article

(This article belongs to the Special Issue Bioceramics: Challenges and Medical Applications of Calcium-Phosphate-Based Biocompatible Ceramics)

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17 pages, 3816 KiB

Open AccessArticle

SMURF1-Induced Ubiquitination of FTH1 Disrupts Iron Homeostasis and Suppresses Myogenesis

by Xia Xiong, Wen Li, Chunlin Yu, Mohan Qiu, Zengrong Zhang, Chenming Hu, Shiliang Zhu, Li Yang, Han Pen, Xiaoyan Song, Jialei Chen, Bo Xia, Shunshun Han and Chaowu Yang

Int. J. Mol. Sci. 2025, 26(3), 1390; https://doi.org/10.3390/ijms26031390 - 6 Feb 2025

Viewed by 413

Abstract

Ferritin heavy chain 1 (FTH1) is pivotal in the storage, release, and utilization of iron, plays a crucial role in the ferroptosis pathway, and exerts significant impacts on various diseases. Iron influences skeletal muscle development and health by promoting cell growth, ensuring energy [...] Read more.

Ferritin heavy chain 1 (FTH1) is pivotal in the storage, release, and utilization of iron, plays a crucial role in the ferroptosis pathway, and exerts significant impacts on various diseases. Iron influences skeletal muscle development and health by promoting cell growth, ensuring energy metabolism and ATP synthesis, maintaining oxygen supply, and facilitating protein synthesis. However, the precise molecular mechanisms underlying iron’s regulation of skeletal muscle growth and development remain elusive. In this study, we demonstrated that the conditional knockout (cKO) of FTH1 in skeletal muscle results in muscle atrophy and impaired exercise endurance. In vitro studies using FTH1 cKO myoblasts revealed notable decreases in GSH concentrations, elevated levels of lipid peroxidation, and the substantial accumulation of Fe²⁺, collectively implying the induction of ferroptosis. Mechanistically, E3 ubiquitin-protein ligase SMURF1 (SMURF1) acts as an E3 ubiquitin ligase for FTH1, thereby facilitating the ubiquitination and subsequent degradation of FTH1. Consequently, this activation of the ferroptosis pathway by SMURF1 impedes myoblast differentiation into myotubes. This study identifies FTH1 as a novel regulator of muscle cell differentiation and skeletal muscle development, implicating its potential significance in maintaining skeletal muscle health through the regulation of iron homeostasis. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Skeletal Muscle Metabolism)

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Morphological and functional alterations in FTH1 knockout mice: (A) Representative images of gastrocnemius (GAS), soleus (SOL), extensor digitorum longus (EDL), and tibialis anterior (TA) muscles from control and FTH1 conditional knockout (cKO) mice. (B) Histological examination of GAS muscle sections stained with hematoxylin and eosin. The mean cross-sectional area of muscle fibers is presented on the right. (C) Immunohistochemical staining of GAS muscle fibers from mice using an anti-dystrophin antibody. The relative mean area is displayed on the right. The green is the dystrophin protein, and the blue is the nucleus. (D) Size distribution and the average diameter of muscle fibers in the gastrocnemius muscle of FTH1 cKO mice compared to control mice. (E) Force measurements obtained during the tetanic contraction of GAS muscles from control and FTH1 cKO mice. (F) Assessment of muscle endurance through forced treadmill running to exhaustion. Data are reported as means ± standard error of the mean (s.e.m.). * indicates p < 0.05; ** indicates p < 0.01. Full article ">Figure 2
Impact of FTH1 on ferroptosis in mouse myoblast cells: (A) Gene Ontology (GO) enrichment analysis conducted to identify significantly differentially expressed genes between FTH1 cKO myoblasts and their control counterparts. (B) Pathway enrichment analysis performed to elucidate the pathways associated with the significantly differentially expressed genes observed between FTH1 cKO myoblasts and controls. (C) Western blot analysis employed to assess the expression levels of ferroptosis-related proteins ACSL4 and COX2 in FTH1 cKO myoblasts compared to controls. (D) qPCR utilized to measure the mRNA expression levels of ACSL4 and COX2 in FTH1 cKO myoblasts relative to control cells. (E–H) The concentrations of GSH, MDA, and Fe2+ quantified and compared between FTH1 cKO myoblasts and control myoblasts to determine their relative levels. Data are reported as means ± standard error of the mean (s.e.m.). * indicates p < 0.05; ** indicates p < 0.01. Full article ">Figure 3
FTH1 regulates myoblast differentiation into myotubes via the ferroptosis pathway: (A) qPCR analysis of MyoG and MyoD mRNA expression in FTH1 cKO myoblasts relative to control cells. (B) Western blot assessment of MyoD protein levels in FTH1 cKO myoblasts compared to controls. (C) MyHC immunofluorescence staining to determine the myotube fusion index in FTH1 cKO myoblasts versus controls. Red: MyHC, Green, nucleus. (D–G) Quantification of GSH, MDA, and Fe2+ concentrations in FTH1 cKO myoblasts and control myoblasts, with and without DFO treatment. (H) qPCR analysis of MyoG and MyoD mRNA expression in FTH1 cKO myoblasts relative to controls, with and without DFO treatment. (I) MyHC immunofluorescence staining to assess the myotube fusion index in FTH1 cKO myoblasts compared to controls, with and without DFO treatment. Red: MyHC, Green, nucleus. Data are reported as means ± standard error of the mean (s.e.m.). * indicates p < 0.05; ** indicates p < 0.01, ns: not significant. Full article ">Figure 4
SMURF1 identified as the E3 ligase for FTH1: (A,B) Prediction of SMURF1 as the specific E3 ligase for FTH1 using the UbiBrowser database. (C) Confocal microscopy images showing the colocalization of SMURF1 (red) and FTH1 (green). (D) Reciprocal co-immunoprecipitation analysis revealing the interaction between SMURF1 and FTH1 in primary myoblasts cultured for 4 days in a differentiation medium. Atrogin1 used as native control, GAPDH used as loading control. (E) Validation of the interaction by reciprocal co-immunoprecipitation between GFP-tagged SMURF1 and Flag-tagged FTH1 in HEK293T cells. Data are reported as means ± standard error of the mean (s.e.m.). Full article ">Figure 5
SMURF1 promotes the degradation of FTH1: (A,B) The wild type SMURF1 (A) decreases the FTH1 protein level, but the C276S mutant (C) cannot downregulate FTH1. Cells expressing Flag-SMURF1 or Flag-SMURF1C276S treated with cycloheximide (CHX, 200 μg/mL). (C) SMURF1 is critical to the stability of FTH1 before or after erastin treatment. Cells expressing indicated shRNA constructs were treated with cycloheximide (CHX, 200 μg/mL) and erastin (0–12 h, 5 μM). Data are reported as means ± standard error of the mean (s.e.m.). ** indicates p < 0.01. ns: not significant. Full article ">Figure 6
SMURF1 promotes FTH1 ubiquitination and degradation: (A) Representative Western blot analysis and quantification of FTH1 protein levels in cells treated with Fer-1 (5 µM) for 12 h. (B) qPCR analysis of FTH1 mRNA expression in cells treated with Fer-1 (5 µM) for 12 h. (C) The immunoprecipitation (IP) of lysates from HEK 293T cells transfected with either control (Ctrl) or SMURF1-shRNA and pretreated with MG132 prior to collection, followed by detection with the indicated antibodies. (D) The IP of lysates from HEK 293T cells transfected with either Vector or SMURF1 overexpression constructs and pretreated with MG132 prior to collection, followed by detection with the indicated antibodies. Data are reported as means ± standard error of the mean (s.e.m.). ** indicates p < 0.01. Full article ">Figure 7
SMURF1 targets FTH1 to facilitate ferroptosis and impede skeletal muscle development: (A) Overview of the experimental groups and treatments employed in this study phase. (B) Western blot analysis depicting the protein levels of ACSL4 and COX2 in the specified cell treatments. (C–F) Quantification of GSH, MDA, GSSG, and Fe2+ concentrations in the indicated treated cells. (G) Immunofluorescence staining for MyHC in the specified cell treatments. Red: MyHC, Green, nucleus. (H,I) Western blot analysis of MyoG and MyoD protein expression in the indicated treated cells. (I) qRT-PCR analysis of MyoG and MyoD mRNA expression in the indicated treated cells. * indicates p < 0.05; ** indicates p < 0.01. Full article ">

18 pages, 3512 KiB

Open AccessReview

Cancer Stem Cells and the Renin–Angiotensin System in the Tumor Microenvironment of Melanoma: Implications on Current Therapies

by Ethan J. Kilmister and Swee T. Tan

Int. J. Mol. Sci. 2025, 26(3), 1389; https://doi.org/10.3390/ijms26031389 - 6 Feb 2025

Viewed by 527

Abstract

Multiple signaling pathways are dysregulated in melanoma, notably the Ras/RAF/MAPK/ERK and PI3K/AKT/mTOR pathways, which can be targeted therapeutically. The high immunogenicity of melanoma has been exploited using checkpoint inhibitors. Whilst targeted therapies and immune checkpoint inhibitors have improved the survival of patients with [...] Read more.

Multiple signaling pathways are dysregulated in melanoma, notably the Ras/RAF/MAPK/ERK and PI3K/AKT/mTOR pathways, which can be targeted therapeutically. The high immunogenicity of melanoma has been exploited using checkpoint inhibitors. Whilst targeted therapies and immune checkpoint inhibitors have improved the survival of patients with advanced melanoma, treatment resistance, their side effect profiles, and the prohibitive cost remain a challenge, and the survival outcomes remain suboptimal. Treatment resistance has been attributed to the presence of cancer stem cells (CSCs), a small subpopulation of pluripotent, highly tumorigenic cells proposed to drive cancer progression, recurrence, metastasis, and treatment resistance. CSCs reside within the tumor microenvironment (TME) regulated by the immune system, and the paracrine renin–angiotensin system, which is expressed in many cancer types, including melanoma. This narrative review discusses the role of CSCs and the paracrine renin–angiotensin system in the melanoma TME, and its implications on the current treatment of advanced melanoma with targeted therapy and immune checkpoint blockers. It also highlights the regulation of the Ras/RAF/MAPK/ERK and PI3K/AKT/mTOR pathways by the renin–angiotensin system via pro-renin receptors, and how this may relate to CSCs and treatment resistance, underscoring the potential for improving the efficacy of targeted therapy and immunotherapy by concurrently modulating the renin–angiotensin system. Full article

(This article belongs to the Special Issue Melanoma: Molecular Mechanisms and Therapy)

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10 pages, 1174 KiB

Open AccessArticle

Bisabolane Sesquiterpenes with Anti-Chlamydial Activity Isolated from Ligularia narynensis

by Na Gao, Yi-Lin He, Hui-Ming Qi, Hong-Ying Yang, Guo-Li Li, Zhao-Cai Li and Tong Shen

Int. J. Mol. Sci. 2025, 26(3), 1388; https://doi.org/10.3390/ijms26031388 - 6 Feb 2025

Viewed by 357

Abstract

Chlamydia are obligate intracellular bacterial pathogens affecting humans and animals, causing miscarriage, stillbirth, or weak fetuses in the late stages of pregnancy of goats and sheep. Because there is no commercial vaccine for chlamydia in animals, drug treatment has become the most effective [...] Read more.

Chlamydia are obligate intracellular bacterial pathogens affecting humans and animals, causing miscarriage, stillbirth, or weak fetuses in the late stages of pregnancy of goats and sheep. Because there is no commercial vaccine for chlamydia in animals, drug treatment has become the most effective curative method. Natural products, also known as secondary metabolites, are becoming one of the main sources used in new drug development because of their structural diversity and biodiversity. In natural products, plant sources play a major role in the development process of new drugs. In this study, five undescribed highly oxygenated bisabolane sesquiterpenes (Pararubin W, Pararubin X, Pararubin Y., Pararubin Z, and Pararubin AA) were isolated from whole plants of Ligularia narynensis. Their chemical structures were determined via analyses of HRESIMS, IR, 1D, and 2D NMR data, along with the assignment of their relative configurations. These compounds were tested for their anti-chlamydial activity. The results show that compounds 1 and 5 inhibited the growth of Chlamydia abortus in host cells in a dose-dependent manner. Full article

(This article belongs to the Special Issue Natural Products and Synthetic Compounds for Drug Development: 3rd Edition)

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Structures of compounds 1–5. Full article ">Figure 2
1H−1H COSY (blue bold) and key HMBC (red arrows) correlations of compounds 1–5. Full article ">Figure 3
NOESY (red double arrow) correlations of compound 1. Full article ">Figure 4
Anti-chlamydial effects of compounds 1–5. Various concentrations of the compounds were applied to the C. abortus strain GN6 cultured in McCoy cells. Anti-chlamydial activity was represented by the inclusion formation ratio, based on immunofluorescent staining of C. abortus inclusions. (A) Observation of C. abortus inclusions in cell cultures under treatment with tetracycline (5 μM) as a positive control or the tested compounds at the concentration of 100 μg/mL. (B) C. abortus inclusion formation ratio in cell cultures treated with the tested compounds at 100 μg/mL final concentrations. (C) Treatment with compound 1 reduces the inclusion formation ratio of C. abortus in cell cultures. (D) Treatment with compound 5 reduces the inclusion formation ratio of C. abortus in the cell cultures. These data are the mean ± SD and representative of three independent experiments. Full article ">

17 pages, 9073 KiB

Open AccessArticle

Genetic Diversity and Environmental Adaptation Signatures of the Great Seahorse (Hippocampus kelloggi) in the Coastal Regions of the Indo-Pacific as Revealed by Whole-Genome Re-Sequencing

by Wen-Xin Hao, Ying-Yi Zhang, Xin Wang, Meng Qu, Shi-Ming Wan and Qiang Lin

Int. J. Mol. Sci. 2025, 26(3), 1387; https://doi.org/10.3390/ijms26031387 - 6 Feb 2025

Viewed by 403

Abstract

The great seahorse (Hippocampus kelloggi) is one of the larger species within the seahorse group and is widely distributed in coastal areas of the Indo-Pacific. However, the natural resources of this species continue to decrease, rendering it a vulnerable species that [...] Read more.

The great seahorse (Hippocampus kelloggi) is one of the larger species within the seahorse group and is widely distributed in coastal areas of the Indo-Pacific. However, the natural resources of this species continue to decrease, rendering it a vulnerable species that faces a high risk of extinction. Therefore, there is an urgent need to conduct research on the genetic diversity of this species to protect its genetic resources. In this study, we conducted whole-genome re-sequencing (WGRS) on three H. kelloggi populations from the Red Sea (RS, n = 30), the Andaman Sea (AS, n = 13), and the South China Sea (SCS, n = 13), and a total of 1,398,936 high-quality single-nucleotide polymorphisms (SNPs) were identified. The results indicate that the average observed heterozygosity (Ho) and the average expected heterozygosity (He) for the RS, AS, and SCS populations are 0.2031 and 0.1987, 0.1914 and 0.1822, and 0.2083 and 0.2001, respectively. The three geographic populations exhibit a high degree of genetic differentiation with only a minimal gene flow between them. Consistently, in a population structure analysis, the three groups are also clearly distinguished, which is consistent with the results of the population differentiation coefficient. Demographic analyses revealed that the effective population size (Ne) of the SCS population underwent a dramatic bottleneck during the Last Glacial Maximum (LGM), followed by a substantial recovery, whereas the RS and AS populations maintained stable Ne values throughout this period. To investigate adaptive responses to climate change in the SCS population, we employed selective elimination analysis, which identified 21 candidate genes potentially involved in environmental adaptation. Of particular significance were myo5a, hps4, znf385a, msh3, and pfkfb4, which likely play crucial roles in the adaptive mechanisms of H. kelloggi. This comprehensive study not only illuminates the genetic diversity patterns of H. kelloggi but also provides a valuable foundation for future investigations into the species’ evolutionary adaptations. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

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The genetic information of Hippocampus kelloggi. The identification of high-quality SNPs and their distribution across 21 chromosomes of H. kelloggi. The gradient colors from green to red indicate an increase in SNP density within 0.1 Mb interval. Full article ">Figure 2
Population structure analyses of H. kelloggi. (A) A phylogenetic tree of the three analyzed populations based on genotype data. The blue, red and yellow backgrounds represent individuals in the SCS, AS, RS population, respectively. (B) A population structure map for K = 2~5. (C) Population structure revealed by PCA. The blue, red and yellow dots represent individuals in the SCS, AS and RS populations, respectively. (D) The decay of linkage disequilibrium in the three experimental populations. The X-axis represents physical location. The Y-axis represents the LD value (r2). The blue, red and yellow line represent the SCS, AS and RS populations, respectively. (E) The error rate of the cross validation (CV) for K = 1~10 (K value represents the number of subgroups of the population). Full article ">Figure 3
Demographic history of the three H. kelloggi populations in this study. Pink, green and blue dots represent individuals in the SCS, AS, RS populationd, respectively. The orange background represents the period of the Last Glacial Maximum (LGM). Full article ">Figure 4
Candidate gene exploration and enrichment analysis of H. kelloggi from the SCS population (SCS vs. RS). (A) A plot of the moving average FST (SCS vs. RS) values of the SNPs per chromosome. The blue line indicates the significant threshold for identifying putative selection regions (top 5 FST = 0.479, p-value < 0.05). (B) Distribution of the π-ratio (SCS/RS) on 21 chromosomes. The blue line indicates the significant threshold for identifying putative selection regions (top 5 log2(π-ratio SCS/RS) = 2.265, p-value < 0.05). (C) The distribution of the log2 (π-ratio) and FST. The RS population is the control group and the SCS population is the selection group. (D,E) Results of the GO and KEGG enrichment analysis of selected genes in the SCS population. Full article ">Figure 5
Candidate gene exploration and enrichment analysis of H. kelloggi from the SCS population (SCS vs. AS). (A) A plot of the moving average FST (SCS vs. AS) values of SNPs per chromosome. The blue line indicates the significant threshold for identifying putative selection regions (top 5 FST = 0.525, p-value < 0.05). (B) Distribution of the π-ratio (SCS/AS) on 21 chromosomes. The blue line indicates the significant threshold for identifying putative selection regions (top 5 log2(π-ratio SCS/AS) = 1.937, p-value < 0.05). (C) Distribution of the log2 (π-ratio) and FST. The AS population is the control group and the SCS population is the selection group. (D,E) Results of the GO and KEGG enrichment analysis of selected genes in the SCS population. Full article ">Figure 6
Information on the natural distribution of H. kelloggi and sampling sites. The H. kelloggi pattern map indicates their natural distribution density, and the blue, red, and yellow triangles represent the population sampling points for the SCS, AS, RS. Full article ">

14 pages, 3027 KiB

Open AccessArticle

Antibiofilm Activities of Halogenated Pyrimidines Against Enterohemorrhagic Escherichia coli O157:H7

by Hyejin Jeon, Yong-Guy Kim, Jin-Hyung Lee and Jintae Lee

Int. J. Mol. Sci. 2025, 26(3), 1386; https://doi.org/10.3390/ijms26031386 - 6 Feb 2025

Viewed by 313

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a significant public health concern due to its ability to form biofilms, enhancing its resistance to antimicrobials and contributing to its persistence in food processing environments. Traditional antibiotics often fail to target these biofilms effectively, leading to increased [...] Read more.

Enterohemorrhagic Escherichia coli (EHEC) is a significant public health concern due to its ability to form biofilms, enhancing its resistance to antimicrobials and contributing to its persistence in food processing environments. Traditional antibiotics often fail to target these biofilms effectively, leading to increased bacterial resistance. This study aims to explore the efficacy of novel antibiofilm agents, specifically halogenated pyrimidine derivatives, against EHEC. We screened pyrimidine and 31 halogenated pyrimidine derivatives for their antimicrobial and antibiofilm activities against EHEC using biofilm quantification assays, SEM analysis, motility, and curli production assessments. Our findings reveal that certain halogenated pyrimidine derivatives, notably 2-amino-5-bromopyrimidine (2A5BP), 2-amino-4-chloropyrrolo[2,3-d]pyrimidine (2A4CPP), and 2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (2,4DC5IPP) at 50 µg/mL, exhibited significant inhibitory effects on EHEC biofilm formation without affecting bacterial growth, suggesting a targeted antibiofilm action. These compounds effectively reduced curli production and EHEC motility, essential factors for biofilm integrity and development. qRT-PCR analysis revealed that two active compounds downregulated the expression of key curli genes (csgA and csgB), leading to reduced bacterial adhesion and biofilm formation. Additionally, in silico ADME–Tox profiles indicated that these compounds exhibit favorable drug-like properties and lower toxicity compared with traditional pyrimidine. This study highlights the potential of halogenated pyrimidine derivatives as effective antibiofilm agents against EHEC, offering a promising strategy for enhancing food safety and controlling EHEC infections. The distinct mechanisms of action of these compounds, particularly in inhibiting biofilm formation and virulence factors without promoting bacterial resistance, underscore their therapeutic potential. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition)

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Antibiofilm screening of pyrimidine and 31 halogenated pyrimidine derivatives and pyrimidine against EHEC. Biofilm formation (indicated as bars) was quantified after 24 h culture in 96-well plates without shaking in the presence of each compound at 0, 20, or 100 μg/mL. Planktonic cell growth was also measured and indicated as dots. The red-fonted numbers 5, 23, and 31 in the x-axis of the upper panel represent the most active compounds. * p < 0.05 versus non-treated controls. “N” indicates “none”, referring to the absence of any treatment. Full chemical names and structures are shown in <a href="#app1-ijms-26-01386" class="html-app">Table S1</a>. Full article ">Figure 2
Effects of halogenated pyrimidines on biofilm formation and cell growth: EHEC biofilm formation was quantified with or without the presence of 2A5BP (A), 2A4CPP (B), or (2,4DC5IPP) (C) after 24 h culture in 96-well polystyrene plates. Planktonic cell growths in the presence of 2A5BP (D) and 2A4CPP (E). Bacteriostatic activity of 2A4CPP (F). * p < 0.05 versus non-treated controls. Full article ">Figure 3
Effects of halogenated pyrimidines on curli production and cell motility: Colony morphology in the presence of 2A5BP and 2A4CPP (A). Curli formation in the presence of 2A5BP and 2A4CPP with Congo red staining (B). Swimming motility in the presence of 2A5BP and 2A4CPP (C,D). Swarming motility (E,F). * p < 0.05 versus non-treated controls. Yellow scale bars represent 0.5 cm (A). Full article ">Figure 4
SEM analysis of EHEC biofilms and qRT-PCR analysis. SEM images of cells treated with 2A5BP (A) or 2A4CPP (B) and gene expression by 2A5BP and 2A4CPP (C). Red and yellow scale bars represent 5 µm and 2 µm, respectively. * p < 0.05 versus non-treated controls. Full article ">Figure 5
Toxicity of halogenated pyrimidines in the plant and nematode models: Radish seed germination rate (A), total length of radish seedlings (B), and representative plant images (C). cultured with or without different concentrations of 2A5BP, 2A4CPP, and PP at 25 °C. C. elegans survival was assessed in the presence or absence of 2A5BP (D), 2A4CPP (E), and PP (F) for 10 days. The red scale bar in (C) represents 2 cm. Full article ">

14 pages, 11016 KiB

Open AccessArticle

Rubiadin Mediates the Upregulation of Hepatic Hepcidin and Alleviates Iron Overload via BMP6/SMAD1/5/9-Signaling Pathway

by Xueting Xie, Linyue Chang, Xinyue Zhu, Fengbei Gong, Linlin Che, Rujun Zhang, Lixin Wang, Chenyuan Gong, Cheng Fang, Chao Yao, Dan Hu, Weimin Zhao, Yufu Zhou and Shiguo Zhu

Int. J. Mol. Sci. 2025, 26(3), 1385; https://doi.org/10.3390/ijms26031385 - 6 Feb 2025

Viewed by 395

Abstract

Iron overload disease is characterized by the excessive accumulation of iron in the body. To better alleviate iron overload, there is an urgent need for safe and effective small molecule compounds. Rubiadin, the active ingredient derived from the Chinese herb Prismatomeris tetrandra, possesses [...] Read more.

Iron overload disease is characterized by the excessive accumulation of iron in the body. To better alleviate iron overload, there is an urgent need for safe and effective small molecule compounds. Rubiadin, the active ingredient derived from the Chinese herb Prismatomeris tetrandra, possesses notable anti-inflammatory and hepatoprotective properties. Nevertheless, its impact on iron metabolism remains largely unexplored. To determine the role of rubiadin on iron metabolism, Western blot analysis, real-time PCR analysis, and the measurement of serum iron were performed. Herein, we discovered that rubiadin significantly downregulated the expression of transferrin receptor 1, ferroportin 1, and ferritin light chain in ferric-ammonium-citrate-treated or -untreated HepG2 cells. Moreover, intraperitoneal administration of rubiadin remarkably decreased serum iron and duodenal iron content and upregulated expression of hepcidin mRNA in the livers of high-iron-fed mice. Mechanistically, bone morphogenetic protein 6 (BMP6) inhibitor LDN-193189 completely reversed the hepcidin upregulation and suppressor of mother against decapentaplegic 1/5/9 (SMAD1/5/9) phosphorylation induced by rubiadin. These results suggested that rubiadin increased hepcidin expression through the BMP6/SMAD1/5/9-signaling pathway. Collectively, our findings uncover a crucial mechanism through which rubiadin modulates iron metabolism and highlight it as a potential natural compound for alleviating iron-overload-related diseases. Full article

(This article belongs to the Special Issue Recent Advances in Iron Metabolism, Ferritin and Hepcidin Research: 3rd Edition)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Rubiadin significantly upregulates hepcidin expression in HepG2 cells. (A) Effect of varying concentrations (0 to 40 μM) of rubiadin on the proliferation of HepG2 cells for 24 h. (B) Hepcidin expression was quantified in HepG2 cells that had been treated with the specified concentrations of rubiadin for 24 h. (C) The expression of hepcidin was quantified after treatment of HepG2 cells with 20 μM rubiadin at specific time points. (D) Hepcidin protein expression in HepG2 cells treated with the indicated concentrations of rubiadin for 24 h was evaluated using an immunofluorescence assay. (E) Hepcidin protein expression in HepG2 cells treated for 20 μM rubiadin for the indicated time points was evaluated via the immunofluorescence assay. Scale bars are 50 μm. Significance was calculated using a one-way ANOVA. ns, no significance, * p < 0.05, *** p < 0.001. Full article ">Figure 2
Rubiadin decreases the protein expression of TfR1, Fpn1 and FtL in FAC treated or untreated HepG2 cells. (A) Western blot analysis of proteins related to iron metabolism in HepG2 cells after rubiadin treatment for 24 h. Quantification of transferrin receptor 1 (TfR1) (B), ferroportin 1 (Fpn1) (C), ferritin light chain (FtL) (D), divalent metal transporter 1 (DMT1) (E), and ferritin heavy chain (FtH) (F) were shown. (G) HepG2 cells were loaded with iron by incubation with ferric ammonium citrate (FAC) (100 μM) for 24 h. After removing the medium, cells were washed and then treated with 20 μM rubiadin for 24 h. Western blot analysis of TfR1, DMT1, Fpn1, FtH, and FtL expression. Quantification of TfR1 (H), Fpn1 (I), FtL (J), DMT1 (K), and FtH (L) were shown. Significance was calculated using a one-way ANOVA. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 3
Rubiadin increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressor mothers against decapentaplegic 1/5/9 (SMAD1/5/9) in HepG2 cells (A,B) Interleukin 6 (IL-6) and bone morphogenetic protein 6 (BMP6) mRNA expression were measured in HepG2 cells that were treated with the specified concentrations of rubiadin for 24 h. (C–E) IL-6 and BMP6 protein expression were measured in HepG2 cells that were treated with the specified concentrations of rubiadin for 24 h. (F) Phosphorylation of STAT3 and SMAD1/5/9 in HepG2 cells analyzed by Western blot after rubiadin treatment for 24 h. (G,H) Quantification of p-STAT3 (G) and p-SMAD1/5/9 (H) were shown. Significance was calculated using a one-way ANOVA. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 4
Rubiadin remarkably reverses the abnormal elevation of serum iron (SI) and alleviates splenomegaly caused by iron overload. Six-week-old mice with a successful high-iron-diet-induced iron overload model were continuously fed the high-iron diet for 4 weeks and intraperitoneally injected with different doses of rubiadin (5 mg/kg, 20 mg/kg). (A) Daily weight changes were recorded. (B) The size of the spleen in wild-type mice, model group mice, and rubiadin-treated mice was compared. (C) Spleen weight index (mg/g) was calculated. (D) SI and (E) unsaturated iron-binding capacity (UIBC) were measured. (F) Total iron-binding capacity (TIBC) and (G) transferrin saturation (TF%) were calculated. Significance was calculated using the two-way ANOVA. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 5
Rubiadin remarkably reverses the elevation of duodenal iron content and further enhances the hepcidin mRNA and the phosphorylation of STAT3 and SMAD1/5/9 caused by iron overload. The tissue samples were isolated and tested for iron in each organ. (A) Duodenal iron content. (B) Liver iron content. (C) Heart iron content. (D) Spleen iron content. (E) Kidney iron content. (F) Hepcidin mRNA levels in the liver of high-iron-fed mice after rubiadin treatment were measured by RT-qPCR. (G) Liver sections from different groups were immunohistochemically stained for hepcidin. Scale bars are 100 μm. (H) Western blotting assessed the phosphorylation levels of STAT3 and SMAD1/5/9 in the liver of the treated mice. Quantification of p-STAT3 (I) and p-SMAD1/5/9 (J) were shown. Significance was calculated using a two-way ANOVA. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 6
Rubiadin upregulates hepcidin via BMP6/SMAD1/5/9 signaling pathway. (A,B) Hepcidin mRNA was assessed in HepG2 cells treated with 20 μM rubiadin for 24 h, either with or without stattic or LDN-193189. (C) Hepcidin protein expression in HepG2 cells treated for 20 μM rubiadin in the presence or absence of LDN-193189 for 24 h was evaluated via an immunofluorescence assay. Scale bars are 100 μm. (D) Western blot analysis of phosphorylated SMAD1/5/9 in HepG2 cells treated with 20 μM rubiadin for 3 h in the presence or absence of LDN-193189 for 24 h. (E) Quantification of p-SMAD1/5/9 was shown. Significance was calculated using a two-way ANOVA. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">

17 pages, 5427 KiB

Open AccessArticle

The NbCBP1-NbSAMS1 Module Promotes Ethylene Accumulation to Enhance Nicotiana benthamiana Resistance to Phytophthora parasitica Under High Potassium Status

by Sadia Noorin, Youwei Du, Yi Liu, Shuanghong Wang, Yan Wang, Hongchen Jia, Tom Hsiang, Rong Zhang and Guangyu Sun

Int. J. Mol. Sci. 2025, 26(3), 1384; https://doi.org/10.3390/ijms26031384 - 6 Feb 2025

Viewed by 419

Abstract

Potassium (K) fertilization is crucial for plant resistance to pathogens, but the underlying mechanisms remain unclear. Here, we investigate the molecular mechanism by which the addition of K promotes resistance in Nicotiana benthamiana to Phytophthora parasitica. We found that N. benthamiana with [...] Read more.

Potassium (K) fertilization is crucial for plant resistance to pathogens, but the underlying mechanisms remain unclear. Here, we investigate the molecular mechanism by which the addition of K promotes resistance in Nicotiana benthamiana to Phytophthora parasitica. We found that N. benthamiana with high K content (HK, 52.3 g/kg) produced more ethylene in response to P. parasitica infection, compared to N. benthamiana with low-K content (LK, 22.4 g/kg). An exogenous ethylene application effectively increased resistance in LK N. benthamiana to the level under HK status, demonstrating the involvement of ethylene in the HK-associated resistance in N. benthamiana. Further, transcriptome analysis showed that NbSAMS1, encoding ethylene biosynthesis, was induced to upregulate P. parasitica about five times higher in HK than in LK N. benthamiana. NbSAMS1 overexpression enhanced resistance in LK plants, whereas NbSAMS1 silencing reduced resistance in HK plants, confirming its importance in conferring resistance. Furthermore, we identified a calcium-binding protein, NbCBP1, which interacted with NbSAMS1, promoting its expression in HK N. benthamiana. Silencing NbCBP1 compromised resistance in HK N. benthamiana, whereas its overexpression improved resistance in LK N. benthamiana. Notably, NbCBP1 protected NbSAMS1 from degradation by the 26S proteasome, thereby sustaining ethylene accumulation in HK N. benthamiana in response to P. parasitica infection. Thus, our research elucidated some mechanisms of the NbCBP1-NbSAMS1 module associated with disease resistance in HK N. benthamiana. Full article

(This article belongs to the Section Molecular Plant Sciences)

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Ethylene is important in enhancing disease resistance. (a) Heatmap displays the expression variation in ethylene-synthesis and associated transcription factors in plants with high potassium content (HK), low potassium (LK), P. parasitica-infected HK (IHK), and P. parasitica-infected LK (ILK) shown in red and green. The scale bar represents the normalized FPKM of each gene. (b) Ethylene production in HK, LK, IHK, and ILK N. benthamiana. (c) Representative images and (d) comparative analysis of lesion diameter in HK plants treated with ethephon (HK-ETH), LK plants treated with ethephon (LK-ETH), HK plants without ethephon treatment (HK-MOCK), or LK plants without ethephon treatment (LK-MOCK). Data are shown as means ± SD and different letters represent significant differences among treatments in lesion diameter at p < 0.05 using Student’s t-tests. Full article ">Figure 2
NbSAMS1 plays a positive role in enhancing disease resistance in Nicotiana benthamiana. (a) NbSAMS1 expression in LK, HK, ILK, and IHK N. benthamiana. (b) Upregulation of PR genes in overexpressed NbSAMS1 in N. benthamiana inoculated with P. parasitica. (c) Lesion diameter analyses of NbSAMS1 overexpressing (OENbSAMS1) HK and LK N. benthamiana leaves (d) Lesion diameter analyses of NbSAMS1 silenced (VNbSAMS1) HK and LK N. benthamiana leaves. Data are shown as means ± SD using Student’s t-test analysis. *: p < 0.05; different letters denote significant differences at p < 0.05. Full article ">Figure 3
NbCBP1 interacts with NbSAMS1. (a) Coexpression network of NbCBP1 and NbSAMS1 with genes involved in the hub module. (b) Molecular docking analysis of NbSAMS1 and NbCBP1 interaction. (c) Yeast two-hybrid assay displays the interaction between NbCBP1 and NbSAMS1. (d) Co-immunoprecipitation (Co-IP) assay confirms the NbCBP1-NbSAMS1 interaction in vivo. Full article ">Figure 4
NbCBP1 enhances Nicotiana benthamiana resistance under high potassium conditions. (a) NbCBP1 expression in HK, LK, IHK, and ILK N. benthamiana. (b) Upregulation of PR genes in overexpressed NbCBP1 in N. benthamiana inoculated with P. parasitica. (c) Lesion diameter analyses of N. benthamiana leaves (HK or LK) with NbCBP1 overexpressing. (d) Lesion diameter analyses of NbCBP1 silenced HK N. benthamiana leaves. (e) Ethylene production in NbCBP1 overexpression and silenced IHK and ILK N. benthamiana. (f) Plant height of NbCBP1 silenced LK and HK N. benthamiana. Data are shown as means ± SD, *: p < 0.05; different letters denote significant differences at p < 0.05 from Student’s t-test. Full article ">Figure 5
NbCBP1 stabilizes NbSAMS1 and promotes ethylene accumulation in response to K availability and pathogen stress. Expression of NbSAMS1 in ILK and IHK N. benthamiana with (a) NbCBP1 overexpression or (b) NbCBP1 silencing. (c) Immunoblot analysis in NbSAMS1 overexpressing HK and LK N. benthamiana. (d) NbSAMS1 degradation assays performed with or without MG132 treatment in LK N. benthamiana. Data are shown as means ± SD; different letters denote significant differences at p < 0.05 from Student’s t-test. Full article ">Figure 6
Proposed Mechanism of high resistance in Nicotiana benthamiana with high potassium status. In low potassium N. benthamiana (left), the NbCBP1 shows low expression, leading to E3 ligase and SAMS1 interaction. SAMS1 is then degraded via 26S proteasome. This degradation results in lower ethylene production, weakening the plant’s defense response. In high potassium N. benthamiana (right), the NbCBP1 shows high expression; CBP1 competes with E3 ligase to interact with NbSAMS1, thereby protecting SAMS1 from degradation, enhancing the stability of SAMS1, resulting in increased ethylene production. The elevated ethylene levels contribute to the plant’s enhanced plant disease resistance. Full article ">

19 pages, 3754 KiB

Open AccessArticle

Differential Regulation of miRNA and Protein Profiles in Human Plasma-Derived Extracellular Vesicles via Continuous Aerobic and High-Intensity Interval Training

by Zhenghao Wang, Yiran Ou, Xinyue Zhu, Ye Zhou, Xiaowei Zheng, Meixia Zhang, Sheyu Li, Shao-Nian Yang, Lisa Juntti-Berggren, Per-Olof Berggren and Xiaofeng Zheng

Int. J. Mol. Sci. 2025, 26(3), 1383; https://doi.org/10.3390/ijms26031383 - 6 Feb 2025

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Abstract

Both continuous aerobic training (CAT) and high-intensity interval training (HIIT) are recommended to promote health and prevent diseases. Exercise-induced circulating extracellular vesicles (EX-EVs) have been suggested to play essential roles in mediating organ crosstalk, but corresponding molecular mechanisms remain unclear. To assess and [...] Read more.

Both continuous aerobic training (CAT) and high-intensity interval training (HIIT) are recommended to promote health and prevent diseases. Exercise-induced circulating extracellular vesicles (EX-EVs) have been suggested to play essential roles in mediating organ crosstalk, but corresponding molecular mechanisms remain unclear. To assess and compare the systemic effects of CAT and HIIT, five healthy male volunteers were assigned to HIIT and CAT, with a 7-day interval between sessions. Plasma EVs were collected at rest or immediately after each training section, prior to proteomics and miRNA profile analysis. We found that the differentially expressed (DE) miRNAs in EX-EVs were largely involved in the regulation of transcriptional factors, while most of the DE proteins in EX-EVs were identified as non-secreted proteins. Both CAT and HIIT play common roles in neuronal signal transduction, autophagy, and cell fate regulation. Specifically, CAT showed distinct roles in cognitive function and substrate metabolism, while HIIT was more associated with organ growth, cardiac muscle function, and insulin signaling pathways. Interestingly, the miR-379 cluster within EX-EVs was specifically regulated by HIIT, involving several biological functions, including neuroactive ligand–receptor interaction. Furthermore, EX-EVs likely originate from various tissues, including metabolic tissues, the immune system, and the nervous system. Our study provides molecular insights into the effects of CAT and HIIT, shedding light on the roles of EX-EVs in mediating organ crosstalk and health promotion. Full article

(This article belongs to the Special Issue Molecular Insights into the Role of Exercise in Disease and Health)

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48 pages, 2940 KiB

Open AccessReview

Molecular Regulation of Palatogenesis and Clefting: An Integrative Analysis of Genetic, Epigenetic Networks, and Environmental Interactions

by Hyuna Im, Yujeong Song, Jae Kyeom Kim, Dae-Kyoon Park, Duk-Soo Kim, Hankyu Kim and Jeong-Oh Shin

Int. J. Mol. Sci. 2025, 26(3), 1382; https://doi.org/10.3390/ijms26031382 - 6 Feb 2025

Viewed by 520

Abstract

Palatogenesis is a complex developmental process requiring temporospatially coordinated cellular and molecular events. The following review focuses on genetic, epigenetic, and environmental aspects directing palatal formation and their implication in orofacial clefting genesis. Essential for palatal shelf development and elevation (TGF-β, BMP, FGF, [...] Read more.

Palatogenesis is a complex developmental process requiring temporospatially coordinated cellular and molecular events. The following review focuses on genetic, epigenetic, and environmental aspects directing palatal formation and their implication in orofacial clefting genesis. Essential for palatal shelf development and elevation (TGF-β, BMP, FGF, and WNT), the subsequent processes of fusion (SHH) and proliferation, migration, differentiation, and apoptosis of neural crest-derived cells are controlled through signaling pathways. Interruptions to these processes may result in the birth defect cleft lip and/or palate (CL/P), which happens in approximately 1 in every 700 live births worldwide. Recent progress has emphasized epigenetic regulations via the class of non-coding RNAs with microRNAs based on critically important biological processes, such as proliferation, apoptosis, and epithelial–mesenchymal transition. These environmental risks (maternal smoking, alcohol, retinoic acid, and folate deficiency) interact with genetic and epigenetic factors during palatogenesis, while teratogens like dexamethasone and TCDD inhibit palatal fusion. In orofacial cleft, genetic, epigenetic, and environmental impact on the complex epidemiology. This is an extensive review, offering current perspectives on gene-environment interactions, as well as non-coding RNAs, in palatogenesis and emphasizing open questions regarding these interactions in palatal development. Full article

(This article belongs to the Special Issue Gene Regulatory and Signaling Pathways in Palatogenesis)

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Figure 1
Developmental progression of secondary palate formation in mouse embryos from E11.5 to E15.5. (A–E) Frontal views show the developmental sequence of palatal shelf elevation and fusion. At E11.5 (A), the medial nasal process (MNP) and maxillary processes (MxP) are visible with the initial formation of the primary palate (PP). From E13.5 to E15.5 (B–E), the palatal shelves (PS) undergo vertical-to-horizontal elevation, with concurrent development of the nasal septum (NS). Progressive fusion of the PS occurs, resulting in the formation of the secondary palate (SP) by E15.5. (F–T) Frontal sections through the developing palate’s anterior, middle, and posterior regions at corresponding developmental stages. The sections demonstrate the progressive growth, elevation, and fusion of the PS around the tongue (T). The medial edge epithelium (MEE) is visible in anterior sections at early stages. The dynamic process of palatal shelf reorientation and fusion proceeds in an anterior-to-posterior sequence, with complete fusion achieved by E15.5. Color code: pink—medial nasal process (MNP); turquoise—maxillary process (MxP) (E11.5) and maxillary tissue (E13.5–E15.5) and lip; yellow—primary palate (PP); light blue—nasal septum (NS); light pink—palatal shelves (PS); black arrowheads indicate the gap between the primary and secondary palates, which will close following fusion between these tissues. Also, the black curved arrows indicate the direction of palatal growth. Full article ">Figure 2
The anatomical spectrum of normal and cleft lip and/or palate malformations in humans. (A–C) Frontal facial views show variations in lip formation. (A) Normal lip morphology with complete fusion. (B) The unilateral cleft lip is showing incomplete fusion on one side. (C) Bilateral cleft lip presenting incomplete fusion on both sides of the upper lip. (D–F) Oral views of the palatal region depicting normal and cleft phenotypes. (D) Normal palate showing complete fusion of palatal shelves. (E) Unilateral cleft lip and palate with the incomplete fusion of the palatal shelf on one side. (F) Bilateral cleft lip and palate showing incomplete fusion of palatal shelves on both sides. Color code: turquoise—maxillary tissue; yellow—primary palate; pink—palatal and soft tissues. Full article ">Figure 3
Complex molecular networks that coordinate palatal shelf growth, patterning, and morphogenesis along both the anterior–posterior and medial–lateral axes during palatogenesis. (A–D) Lateral and oral views of developing mouse embryos at E12.5 and E13.5 with corresponding schematic diagram The anterior and posterior orientation is indicated by dashed lines. (E–G) Schematic representations of molecular networks controlling palate development: (E) Key factors involved in palate growth and patterning along the anterior–posterior axis, showing interactions between epithelial and mesenchymal factors and their downstream targets. (F) Molecular regulation of anterior palatal shelf development. (G) Posterior palatal shelf patterning network showing interactions. Color code: pink—palatal epithelium; apricot—mesenchyme; blue—transcription factors; green—receptors; yellow—ligands; orange—other regulatory molecules. Full article ">Figure 4
Development and molecular regulation of palatal shelf adhesion and fusion. (A–C) Schematic representation of mouse embryo development from E14.5 to E15.5. (A) Lateral view of E12.5 mouse embryo showing the anterior–posterior axis of palatal development. (B) Oral view at E14.5 showing elevated palatal shelves before fusion. (C) Oral view at E15.5 showing palatal fusing. (D–F) Molecular pathways controlling three key stages of palatal fusion. (D) Epithelial differentiation and periderm maintenance pathway showing genetic interactions. (E) Palatal adhesion and medial edge epithelium (MEE) formation pathway involving β-catenin, Tgf-β3, and downstream effectors. (F) Midline epithelial seam (MES) degeneration process leading to palatal fusion. Color code: pink—epithelium; apricot—mesenchyme; blue—transcription factors; green—receptors; yellow—ligands; orange—other regulatory molecules; black dotted line—remaining MES during palatal fusion. Full article ">Figure 5
Epigenetic regulation during craniofacial development. (A,B) Schematic representation of early craniofacial development showing the morphological changes from initial facial prominences to their fusion. (A) The left panel shows the initial facial prominences, including midbrain, forebrain, lateral and medial nasal processes, nasal pit, maxillary and mandibular processes, and second arch. (B) The right panel shows the subsequent development of the frontonasal region, maxillary region, and the mandibular region. (C) Four major epigenetic mechanisms regulating craniofacial development: DNA methylation: addition of methyl groups to promoter regions controlling target gene expression. The red cross symbol means that the expression of the target gene is suppressed. Histone modification: post-translational modifications, including methylation (Me) and acetylation (Ac) of histone proteins. Non-coding RNAs: involvement of microRNAs (miRNA) and long non-coding RNAs (lncRNA) in gene regulation. Chromatin remodeling: ATP-dependent nucleosome ejection and sliding mediated by SWI/SNF complexes. Color code: dark blue—midbrain; light blue—forebrain; green—lateral nasal process; red—medial nasal process; navy blue—nasal pit; turquoise—maxillary process; purple—mandibular process and second arch; orange—frontonasal region; gray—other facial region behind maxillary/mandibular regions. Full article ">Figure 6
Summary of the miRNAs and genes associated with cleft lip and/or palate (CL/P) in mice and human. The complex miRNA-mediated regulatory networks involved in cleft lip and/or palate (CL/P). Environmental factors also affect cleft palate development by modulating miRNA activity, and how their dysregulation contributes to cleft formation through cell proliferation defects, differentiation defects, and cell death. Red arrows; graph showing genes going up, blue arrows; graph showing genes going down. CL, cleft lip; CP, cleft palate; Full article ">

22 pages, 13927 KiB

Open AccessArticle

Discovery of TRPV4-Targeting Small Molecules with Anti-Influenza Effects Through Machine Learning and Experimental Validation

by Yan Sun, Jiajing Wu, Beilei Shen, Hengzheng Yang, Huizi Cui, Weiwei Han, Rongbo Luo, Shijun Zhang, He Li, Bingshuo Qian, Lingjun Fan, Junkui Zhang, Tiecheng Wang, Xianzhu Xia, Fang Yan and Yuwei Gao

Int. J. Mol. Sci. 2025, 26(3), 1381; https://doi.org/10.3390/ijms26031381 - 6 Feb 2025

Viewed by 457

Abstract

Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable cation channel critical for maintaining intracellular Ca²⁺ homeostasis and is essential in regulating immune responses, metabolic processes, and signal transduction. Recent studies have shown that TRPV4 activation enhances influenza A virus infection, promoting [...] Read more.

Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable cation channel critical for maintaining intracellular Ca²⁺ homeostasis and is essential in regulating immune responses, metabolic processes, and signal transduction. Recent studies have shown that TRPV4 activation enhances influenza A virus infection, promoting viral replication and transmission. However, there has been limited exploration of antiviral drugs targeting the TRPV4 channel. In this study, we developed the first machine learning model specifically designed to predict TRPV4 inhibitory small molecules, providing a novel approach for rapidly identifying repurposed drugs with potential antiviral effects. Our approach integrated machine learning, virtual screening, data analysis, and experimental validation to efficiently screen and evaluate candidate molecules. For high-throughput virtual screening, we employed computational methods to screen open-source molecular databases targeting the TRPV4 receptor protein. The virtual screening results were ranked based on predicted scores from our optimized model and binding energy, allowing us to prioritize potential inhibitors. Fifteen small-molecule drugs were selected for further in vitro and in vivo antiviral testing against influenza. Notably, glecaprevir and everolimus demonstrated significant inhibitory effects on the influenza virus, markedly improving survival rates in influenza-infected mice (protection rates of 80% and 100%, respectively). We also validated the mechanisms by which these drugs interact with the TRPV4 channel. In summary, our study presents the first predictive model for identifying TRPV4 inhibitors, underscoring TRPV4 inhibition as a promising strategy for antiviral drug development against influenza. This pioneering approach lays the groundwork for future clinical research targeting the TRPV4 channel in antiviral therapies. Full article

(This article belongs to the Special Issue Machine Learning Applications in Bioinformatics and Biomedicine: 2nd Edition)

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29 pages, 12007 KiB

Open AccessArticle

Molecular Simulation of the Binding of Amyloid Beta to Apolipoprotein A-I in High-Density Lipoproteins

by Chris J. Malajczuk and Ricardo L. Mancera

Int. J. Mol. Sci. 2025, 26(3), 1380; https://doi.org/10.3390/ijms26031380 - 6 Feb 2025

Viewed by 337

Abstract

Disrupted clearance of amyloid beta (Aβ) from the brain enhances its aggregation and formation of amyloid plaques in Alzheimer’s disease. The most abundant protein constituent of circulating high-density lipoprotein (HDL) particles, apoA-I, readily crosses the blood–brain barrier from periphery circulation, exhibits low-micromolar binding [...] Read more.

Disrupted clearance of amyloid beta (Aβ) from the brain enhances its aggregation and formation of amyloid plaques in Alzheimer’s disease. The most abundant protein constituent of circulating high-density lipoprotein (HDL) particles, apoA-I, readily crosses the blood–brain barrier from periphery circulation, exhibits low-micromolar binding affinity for soluble, neurotoxic forms of Aβ, and modulates Aβ aggregation and toxicity in vitro. Its highly conserved N-terminal sequence, ⁴²LNLKLLD⁴⁸ (‘LN’), has been proposed as a binding region for Aβ. However, high-resolution structural characterisation of the mechanism of HDL–Aβ interaction is very difficult to attain. Molecular dynamics simulations were conducted to investigate for the first time the interaction of Aβ and the ‘LN’ segment of apoA-I. Favourable binding of Aβ by HDLs was found to be driven by hydrophobic and hydrogen-bonding interactions predominantly between the ‘LN’ segment of apoA-I and Aβ. Preferential binding of Aβ may proceed in small, protein-rich HDLs whereby solvent-exposed hydrophobic ‘LN’ segments of apoA-I interact specifically with Aβ, stabilising it on the HDL surface in a possibly non-amyloidogenic conformation, facilitating effective Aβ clearance. These findings rationalise the potentially therapeutic role of HDLs in reducing Aβ aggregation and toxicity, and of peptide mimics of the apoA-I interacting region in blocking Aβ aggregation. Full article

(This article belongs to the Special Issue Advances in Protein Dynamics)

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28 pages, 1219 KiB

Open AccessReview

Antioxidant and Anti-Inflammatory Effects of Bioactive Compounds in Atherosclerosis

by Ştefan Horia Roşian, Ioana Boarescu and Paul-Mihai Boarescu

Int. J. Mol. Sci. 2025, 26(3), 1379; https://doi.org/10.3390/ijms26031379 - 6 Feb 2025

Viewed by 549

Abstract

Atherosclerosis, a chronic inflammatory disease characterized by the accumulation of lipids and immune cells within arterial walls, remains a leading cause of cardiovascular morbidity and mortality worldwide. Oxidative stress and inflammation are central to its pathogenesis, driving endothelial dysfunction, foam cell formation, and [...] Read more.

Atherosclerosis, a chronic inflammatory disease characterized by the accumulation of lipids and immune cells within arterial walls, remains a leading cause of cardiovascular morbidity and mortality worldwide. Oxidative stress and inflammation are central to its pathogenesis, driving endothelial dysfunction, foam cell formation, and plaque instability. Emerging evidence highlights the potential of bioactive compounds with antioxidant and anti-inflammatory properties to mitigate these processes and promote vascular health. This review explores the mechanisms through which bioactive compounds—such as polyphenols, carotenoids, flavonoids, omega-3 fatty acids, coenzyme Q10, and other natural compounds—modulate oxidative stress and inflammation in atherosclerosis. It examines their effects on key molecular pathways, including the inhibition of reactive oxygen species (ROS) production, suppression of nuclear factor-κB (NF-κB), and modulation of inflammatory cytokines. By integrating current knowledge, this review underscores the therapeutic potential of dietary and supplemental bioactive compounds as complementary strategies for managing atherosclerosis, paving the way for future research and clinical applications. Full article

(This article belongs to the Special Issue Effects of Bioactive Compounds in Oxidative Stress and Inflammation)

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16 pages, 1891 KiB

Open AccessArticle

Mitochondrial COX3 and tRNA Gene Variants Associated with Risk and Prognosis of Idiopathic Pulmonary Fibrosis

by Li-Na Lee, I-Shiow Jan, Wen-Ru Chou, Wei-Lun Liu, Yen-Liang Kuo, Chih-Yueh Chang, Hsiu-Ching Chang, Jia-Luen Liu, Chia-Lin Hsu, Chia-Nan Lin, Ke-Yun Chao, Chi-Wei Tseng, I-Hsien Lee, Jann-Tay Wang and Jann-Yuan Wang

Int. J. Mol. Sci. 2025, 26(3), 1378; https://doi.org/10.3390/ijms26031378 - 6 Feb 2025

Viewed by 423

Abstract

Idiopathic pulmonary fibrosis (IPF) has been associated with mitochondrial dysfunction. We investigated whether mitochondrial DNA variants in peripheral blood leukocytes (PBLs), which affect proteins of the respiratory chain and mitochondrial function, could be associated with an increased risk and poor prognosis of IPF. [...] Read more.

Idiopathic pulmonary fibrosis (IPF) has been associated with mitochondrial dysfunction. We investigated whether mitochondrial DNA variants in peripheral blood leukocytes (PBLs), which affect proteins of the respiratory chain and mitochondrial function, could be associated with an increased risk and poor prognosis of IPF. From 2020 to 2022, we recruited 36 patients (age: 75.3 ± 8.5; female: 19%) with IPF, and 80 control subjects (age: 72.3 ± 9.0; female: 27%). The mitochondrial genome of peripheral blood leukocytes was determined using next-generation sequencing. During a 45-month follow-up, 10 (28%) patients with IPF remained stable and the other 26 (72%) progressed, with 12 (33%) mortalities. IPF patients had more non-synonymous (NS) variants (substitution/deletion/insertion) in mitochondrial COX3 gene (coding for subunit 3 of complex IV of the respiratory chain), and more mitochondrial tRNA variants located in the anticodon (AC) stem, AC loop, variable loop, T-arm, and T-loop of the tRNA clover-leaf structure in PBLs than the control group. The succumbed IPF patients were older, had lower initial diffusion capacity, and higher initial fibrosis score on high-resolution computerized tomography (HRCT) than the alive group. NS variants in mitochondrial COX3 gene and tRNA variants in PBLs were associated with shorter survival. Our study shows that (1) leukocyte mitochondrial COX3 NS variants are associated with risk and prognosis of IPF; (2) leukocyte mitochondrial tRNA variants located in the AC stem, AC loop, variable loop, T-arm, and T-loop of the tRNA clover-leaf structure are associated with risk, and the presence of tRNA variants is associated with poor prognosis of IPF. Full article

(This article belongs to the Special Issue Advanced Molecular Research in Lung Diseases)

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The map of human mitochondrial DNA, a double-stranded circular DNA with H- and L-strands. It has 37 genes coding for 13 proteins of the respiratory chain (complex I, III, IV, and V), 2 rRNAs, and 22 tRNAs. Full article ">Figure 2
Distribution of mitochondrial tRNA variants in regions of the secondary clover-leaf tRNA structure. Patients with IPF had more tRNA variants located in the right half of the clover-leaf structure (including anticodon loop, anticodon arm, variable loop, T-arm, and T loop) than the control subjects, with the majority of tRNA variants located in these regions (73% vs. 31% in the control group, p = 0.008). Numbering of nucleotides follows the standard system [<a href="#B19-ijms-26-01378" class="html-bibr">19</a>]. Full article ">Figure 3
Survival curves of patients with IPF, stratified by mitochondrial COX3 gene NS variants (substitution/deletion/insertion) (A), mitochondrial tRNA variants (B), the presence of neither, either, or both COX3 gene NS variants and mitochondrial tRNA variants (C), and anti-fibrotic treatment (D). For the 3 curves in (C): p = 0.127 when comparing between “Both mutated” and “Either one mutated”; p = 0.011 when comparing between “Either one mutated” and “Neither mutated”; p = 0.001 when comparing between “Both mutated” and “Neither mutated”. The four curves in (D) are: IPF patients treated with pirfenidone only, nintedanib only, pirfenidone and nintedanib sequentially (or vice versa), or not treated. Full article ">Figure 3 Cont.
Survival curves of patients with IPF, stratified by mitochondrial COX3 gene NS variants (substitution/deletion/insertion) (A), mitochondrial tRNA variants (B), the presence of neither, either, or both COX3 gene NS variants and mitochondrial tRNA variants (C), and anti-fibrotic treatment (D). For the 3 curves in (C): p = 0.127 when comparing between “Both mutated” and “Either one mutated”; p = 0.011 when comparing between “Either one mutated” and “Neither mutated”; p = 0.001 when comparing between “Both mutated” and “Neither mutated”. The four curves in (D) are: IPF patients treated with pirfenidone only, nintedanib only, pirfenidone and nintedanib sequentially (or vice versa), or not treated. Full article ">

28 pages, 3097 KiB

Open AccessReview

Epigenetic Regulation by lncRNA GAS5/miRNA/mRNA Network in Human Diseases

by Lam Ngoc Thao Nguyen, Jaeden S. Pyburn, Nhat Lam Nguyen, Madison B. Schank, Juan Zhao, Ling Wang, Tabitha O. Leshaodo, Mohamed El Gazzar, Jonathan P. Moorman and Zhi Q. Yao

Int. J. Mol. Sci. 2025, 26(3), 1377; https://doi.org/10.3390/ijms26031377 - 6 Feb 2025

Viewed by 369

Abstract

The interplay between long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) is crucial in the epigenetic regulation of mRNA and protein expression, impacting the development and progression of a plethora of human diseases, such as cancer, cardiovascular disease, inflammatory-associated diseases, and viral infection. Among [...] Read more.

The interplay between long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) is crucial in the epigenetic regulation of mRNA and protein expression, impacting the development and progression of a plethora of human diseases, such as cancer, cardiovascular disease, inflammatory-associated diseases, and viral infection. Among the many lncRNAs, growth arrest-specific 5 (GAS5) has garnered substantial attention for its evident role in the regulation of significant biological processes such as proliferation, differentiation, senescence, and apoptosis. Through miRNA-mediated signaling pathways, GAS5 modulates disease progression in a cell-type-specific manner, typically by influencing proteins involved in inflammation and cell death. While GAS5 is recognized as a tumor suppressor in cancer, recent reports highlight its broader regulatory capacity in non-cancerous diseases. Its modulation of protein expression through the GAS5/miRNA network has been shown to both mitigate and exacerbate disease, depending on the specific context. Furthermore, the therapeutic potential of GAS5 manipulation, via knockdown or overexpression, offers promising avenues for targeted interventions across human diseases. This review explores the dualistic impacts of the GAS5/miRNA network in conditions such as cancer, cardiovascular disease, viral infections, and inflammatory disorders. Through the evaluation of current evidence, we aim to provide insight into GAS5’s biological functions and its implications for future research and therapeutic development. Full article

(This article belongs to the Special Issue Role of MicroRNAs in Human Diseases)

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The GAS5/miRNA-regulated pathways in cancers. This figure depicts the pathways modulated by the GAS5/miRNA-mediated mechanisms that regulate the progression of cancers. The upregulation of GAS5 results in the downregulation of its target miRNAs, thereby regulating downstream proteins involved in cellular activation, proliferation, differentiation or apoptosis, and cell cycle arrest. Thus, increasing GAS5 levels is considered a promising therapeutic approach for suppressing the progression of various cancers. PTEN, phosphatase and tensin homolog; AKT, protein kinase B; mTOR, mammalian target of rapamycin; SPRY1, Sprouty RTK signaling antagonist 1; RAF, rapidly accelerated fibrosarcoma; ERK, extracellular signal-regulated kinase; PDCD4, programmed cell death 4; P21, cyclin-dependent kinase inhibitor 1; SMAD7, SMAD family member 7; BIM, Bcl-2-like protein 11; BAX, BCL2-associated X; FOXO1, forkhead box protein O1; SOCS3, suppressor of cytokine signaling 3; ARHI, aplasia Ras homolog member I; DKK2, Dickkopf 2; ATG12, autophagy related 12; ATG3, autophagy related 3; ATG8, autophagy related 8; LC3II, LC3-phosphatidylethanolamine conjugate. Full article ">Figure 2
The GAS5/miRNA-regulated pathways in cardiovascular diseases. This figure depicts the pathways modulated by GAS5/miRNA-mediated mechanisms that have either debilitating or protective effects on cardiovascular diseases. The pathways involved in modulating cell activity in a debilitating manner exert their effects, in general, through the suppression of cellular proliferation and differentiation while promoting cell death and apoptosis. The pathways involved in modulating cell activity in a protective manner exert their effects, in general, through the suppression of senescence, inflammation, or pyroptosis while promoting cellular proliferation. These pathways are largely cell-type- and context-dependent. PDCD4, programmed cell death protein 4; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; MEK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; ROCK1, rho-associated coiled-coil containing protein kinase 1; GSK-3β, glycogen synthase kinase 3 beta; TXNIP, thioredoxin-interacting protein; NAMPT, nicotinamide phosphoribosyltransferase; NLRP3, NOD-, LRR-, and pyrin domain-containing protein 3; TLR4, toll-like receptor 4; MI, myocardial infarction. Full article ">Figure 3
The GAS5/miRNA-regulated pathways with debilitating roles in inflammation-associated diseases. This figure depicts the pathways modulated by GAS5/miRNA-mediated mechanisms that exert debilitating effects on inflammation-associated diseases. The proteins involved in these pathways are known for their roles in cell activation, proliferation, differentiation, apoptosis, and cell cycle arrest. By inhibiting specific miRNAs and their target proteins, elevated GAS5 levels promote apoptosis and cell death, thereby promoting inflammation and apoptosis, increasing the severity of inflammatory diseases. TSP-1, thrombospondin-1; GSK-3β, glycogen synthase kinase 3; PTEN, phosphatase and tensin homolog; NLRP3, NOD-like receptor family pyrin domain containing 3; BDNF, brain-derived neurotrophic factor; SMAD1, SMAD family member 1; FOXO3, forkhead box protein O3; PUMA, p53-upregulated modulator of apoptosis; JNK, c-Jun N-terminal kinase; γH2AX, phosphorylated histone H2AX; AKT, protein kinase B; Notch-1, neurogenic locus notch homolog protein 1; KCNQ3, potassium voltage-gated channel subfamily Q member 3. Full article ">Figure 4
The GAS5/miRNA-regulated pathways with protective roles in inflammation-associated diseases. This figure depicts the pathways modulated by GAS5/miRNA-mediated mechanisms that exert protective effects and differential effects (sepsis) on inflammation-associated diseases. For sepsis, miRNAs appear to have a stage-dependent role in inflammation, where early sepsis relies on mitochondrial autophagy for antioxidant defense and intracellular stability, while GAS5 depletion in late-stage sepsis exacerbates inflammation, increasing tissue damage. Regarding the other disease models displayed, the overexpression of GAS5 leads to the repression of miRNAs displaying roles in inflammation, pyroptosis, proliferation, and apoptosis. This suppression of inflammation, apoptosis, and pyroptosis is significant in reducing the progression of these inflammation-associated diseases. MITF, microphthalmia-associated transcription factor; NRF2, nuclear factor erythroid 2–related factor 2; TLR4, toll-like receptor 4; SIRT1, Sirtuin 1; HMGB1, high mobility group box 1; MARCH7, membrane-associated ring-CH-type finger 7; NLRP3, NOD-like receptor family pyrin domain containing 3; PDK4, pyruvate dehydrogenase kinase 4; SMAD4, SMAD family member 4; GSDMD-N, gasdermin D N-terminal fragment; NAFLD, nonalcoholic fatty liver disease; OA, osteoarthritis; DN; diabetic nephropathy. Full article ">Figure 5
The GAS5/miRNA-mediated regulation of viral infections. This figure depicts the mechanisms by which GAS5 regulates the progression of infectious diseases caused by viral infections. GAS5 plays a critical role in regulating infectious diseases associated with HCV and HIV infections by directly binding to viral proteins or miRNAs that promote viral replication. GAS5 also demonstrates therapeutic potential in PLWH, displaying an ability to improve the activity and longevity of CD4 T cells in ART-treated PLWH. Finally, GAS5 serves as an important biomarker of severe SARS-CoV-2 infection through its ability to function as a ceRNA for miR-200. Unbound miR-200 can inhibit ACE-2 expression, a phenomenon which is linked to increased inflammation and cytokine storms in SARS-CoV-2 infection. HCV, hepatitis C virus; HIV, human immunodeficiency virus; PLWH, patients living with HIV; ART, antiretroviral therapy; ACE-2, angiotensin-converting enzyme 2. Full article ">

32 pages, 1452 KiB

Open AccessReview

Modification in Structures of Active Compounds in Anticancer Mitochondria-Targeted Therapy

by Agnieszka Pyrczak-Felczykowska and Anna Herman-Antosiewicz

Int. J. Mol. Sci. 2025, 26(3), 1376; https://doi.org/10.3390/ijms26031376 - 6 Feb 2025

Viewed by 522

Abstract

Cancer is a multifaceted disease characterised by uncontrolled cellular proliferation and metastasis, resulting in significant global mortality. Current therapeutic strategies, including surgery, chemotherapy, and radiation therapy, face challenges such as systemic toxicity and tumour resistance. Recent advancements have shifted towards targeted therapies that [...] Read more.

Cancer is a multifaceted disease characterised by uncontrolled cellular proliferation and metastasis, resulting in significant global mortality. Current therapeutic strategies, including surgery, chemotherapy, and radiation therapy, face challenges such as systemic toxicity and tumour resistance. Recent advancements have shifted towards targeted therapies that act selectively on molecular structures within cancer cells, reducing off-target effects. Mitochondria have emerged as pivotal targets in this approach, given their roles in metabolic reprogramming, retrograde signalling, and oxidative stress, all of which drive the malignant phenotype. Targeting mitochondria offers a promising strategy to address these mechanisms at their origin. Synthetic derivatives of natural compounds hold particular promise in mitochondrial-targeted therapies. Innovations in drug design, including the use of conjugates and nanotechnology, focus on optimizing these compounds for mitochondrial specificity. Such advancements enhance therapeutic efficacy while minimizing systemic toxicity, presenting a significant step forward in modern anticancer strategies. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Bioactive Compounds Against Cancer—From Natural Sources to Drug Candidates)

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21 pages, 5178 KiB

Open AccessArticle

The Disruptions of Sphingolipid and Sterol Metabolism in the Short Fiber of Ligon-Lintless-1 Mutant Revealed Obesity Impeded Cotton Fiber Elongation and Secondary Cell Wall Deposition

by Huidan Tian, Qiaoling Wang, Xingying Yan, Hongju Zhang, Zheng Chen, Caixia Ma, Qian Meng, Fan Xu and Ming Luo

Int. J. Mol. Sci. 2025, 26(3), 1375; https://doi.org/10.3390/ijms26031375 - 6 Feb 2025

Viewed by 433

Abstract

Boosting evidence indicated lipids play important roles in plants. To explore lipid function in cotton fiber development, the lipid composition and content were detected by untargeted and targeted lipidomics. Compared with rapid elongation fibers, the lipid intensity of 16 sub-classes and 56 molecular [...] Read more.

Boosting evidence indicated lipids play important roles in plants. To explore lipid function in cotton fiber development, the lipid composition and content were detected by untargeted and targeted lipidomics. Compared with rapid elongation fibers, the lipid intensity of 16 sub-classes and 56 molecular species decreased, while only 7 sub-classes and 26 molecular species increased in the fibers at the stage of secondary cell wall deposition. Unexpectedly, at the rapid elongation stage, 20 sub-classes and 60 molecular species increased significantly, while only 5 sub-classes and 8 molecular species decreased in the ligon lintless-1 (li-1) mutant compared with its wild-type Texas Maker-1 (TM-1). Particularly, campesteryl, sitosteryl, and total steryl ester increased by 21.8-, 48.7-, and 45.5-fold in the li-1 fibers, respectively. All the molecular species of sphingosine-1-P, phytoceramide-OHFA, and glucosylceramide increased while all sphingosine, phytosphingosine, and glycosyl inositol phospho ceramides decreased in the li-1 fibers. Similarly, the different expression genes between the mutant and wild type were enriched in many pathways involved in the lipid metabolism. Furthermore, the number of lipid droplets also increased in the li-1 leaf and fiber cells when compared with the wild type. These results illuminated that fiber cell elongation being blocked in the li-1 mutant was not due to a lack of lipids, but rather lipid over-accumulation (obesity), which may result from the disruption of sphingolipid and sterol metabolism. This study provides a new perspective for further studying the regulatory mechanisms of fiber development. Full article

(This article belongs to the Section Molecular Plant Sciences)

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Figure 1
OPLS-DA score plot and the number of lipid species in each detected lipid class. (A): The OPLS-DA score plot between the 10-DPA fiber group and 20-DPA fiber group, * represents the multiplication sign. (B): The OPLS-DA score plot between the 10-DPA fiber group of wild type (TM-1) and the 10-DPA fiber group of li-1 mutant. (C): Thirty-three lipid classes detected in three samples and the number of lipid molecule species in each detected lipid class. The number on each column represents the number of molecular species in each lipid class. 10 D: 10-DPA fibers of TM-1 (wild type); 20 D: 20-DPA fibers of TM-1 (wild type); 10 D-li: 10-DPA fibers of li-1 mutant. AGlcSiE, AcylGlcSitosterol ester; Cer, ceramides; CerG1, glucocerebroside; CerP, ceramides phosphate; CL, cardiolipin; Co, coenzyme; DG, diglyceride; DGMG, Digalactosylmonoacylglycerol; DGDG, digalactosyldiacylglycerol; FA, fatty acid; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; MG, monoglyceride; MGDG, monogalactosyldiacylglycerol; MGMG, Monogalactosylmonoacylglycerol; OAHFA, O-Acetylated Hydroxy Fatty Acid. PA, phosphatidic acid; PC, phosphatidylserine; PE, phosphatidylcholine; PG, phosphatidylglycerol; phSM, phytosphingosine; PI, phosphatidylinositol; PS, phosphatidylserine; SiE, sitosterol ester; SM, sphingomyelin; So, sphingosine; MG, monoglyceride; SQDG, Sulfoquinovosyldiacylglycerol; StE, stigmasterol ester; TG, triglyceride; WE, wax esters. Full article ">Figure 2
The lipid intensity of various lipid classes. The intensity of 7 classes of lipids including GP, SP, GL, SL, ST, FA, and PL and 33 kinds of lipid sub-classes in the fiber cell. GP, glycerophospholipid; SP, sphingolipid; GL, glycerolipid; ST, sterol lipids; PL, prenol lipid; FA, fatty acid; SL, saccharolipid; AGlcSiE, AcylGlcSitosterol ester; Cer, ceramides; CerG1, glucocerebroside; CerP, ceramides phosphate; DG, diglyceride; CL, cardiolipin; DGMG, Digalactosylmonoacylglycerol; DGDG, digalactosyldiacylglycerol; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; MG, monoglyceride; MGDG, monogalactosyldiacylglycerol; MGMG, Monogalactosylmonoacylglycerol; OAHFA, O-Acetylated Hydroxy Fatty Acid; PA, phosphatidic acid; PC, phosphatidylserine; PE, phosphatidylcholine; PG, phosphatidylglycerol; phSM, phytosphingosine; PI, phosphatidylinositol; PS, phosphatidylserine; SiE, sitosterol ester; SM, sphingomyelin; So, sphingosine; SQDG, Sulfoquinovosyldiacylglycerol; StE, stigmasterol ester; TG, triglyceride; WE, wax esters; Co, coenzyme. Full article ">Figure 3
The intensity difference in lipid sub-classes and lipid molecule species between 10-DPA fiber cells and 20-DPA fiber cells. (A): The fold changes in various lipid sub-classes between 10-DPA fiber cells and 20-DPA fiber cells; (B): the fold changes in various lipid molecule species between 10-DPA fiber cells and 20-DPA fiber cells; (C): the proportion of various lipid compounds in the total significantly changed lipids; (D): the proportion of various lipid molecule species in the total significantly changed lipids. 10 DPA: 10-DPA fibers of TM-1 (wild type); 20 DPA: 20-DPA fibers of TM-1 (wild type). Full article ">Figure 4
The intensity difference in lipid sub-classes and lipid molecule species between 10-DPA fiber cells of TM-1 (wild type) and 10-DPA fiber cells of li-1 mutant. (A): The fold changes in various lipid sub-classes between 10-DPA fiber cells of TM-1 and 10-DPA fiber cells of li-1 mutant; (B): the fold changes in various lipid molecule species between 10-DPA fiber cells of TM-1 and 10-DPA fiber cells of li-1 mutant; (C): the proportion of various lipid compounds in the total significantly changed lipids; (D): the proportion of various lipid molecule species in the total significantly changed lipids. 10 DPA: 10-DPA fibers of TM-1 (wild type); 10 li-1: 10-DPA fibers of li-1 mutant. Full article ">Figure 5
Sphingolipid classes in cotton fiber cells and their alteration in li-1 mutant fiber cells compared with its wild-type TM-1. The change ratio represents the percentage of increase and decrease in sphingolipid classes and molecular species in li-1 fiber cells compared to wild-type fiber cells. (A): The number of classes and molecular species of sphingolipids detected in fiber cells of li-1 and TM-1; (B): the change percentage of sphingolipid content in li-1 fiber cells; (C): the change percentage of molecular species of t-S1P and Sph; (D): the change percentage of molecular species of Cer; (E): the change percentage of molecular species of Phyto-Cer; (F): the change percentage of molecular species of GIPC; (G): the change percentage of molecular species of PhytoCer-OHFA; (H): the change percentage of molecular species of GluCer. Cer, ceramides; PhytoCer, phytoceramides; PhytoCer-OHFA, phytoceramides with hydroxylated fatty acyls; S1P, sphingosine-1-phosphate; t-S1P, phytosphingosine-1-phosphate; Sph, sphingosines; PhytoSph, phytosphingosines; GluCer, glucosylceramides; Phyto-GluCer, phyto-glucosylceramides; GIPC, glycosyl inositol phospho ceramides. Full article ">Figure 6
The content changes in sterol and steryl ester in li-1 fiber cells. (A) The content changes in total sterol and various sterol classes in li-1 fiber cells. (B) The content changes in total steryl ester and two steryl esters in li-1 fiber cells. (C) The ratio of stigmasterol to sitosterol (St/Si) and campesterol to sitosterol (C/S) in 10-DPA fibers of mutant and wild type. Full article ">Figure 7
GO annotations and KEGG enrichment analysis for the differentially expressed genes in 10-DPA fiber cells between li-1 mutant and TM-1 wild type. Red arrows indicated the pathway involved in lipid metabolism. Full article ">Figure 8
The expression changes in selected genes in li-1 mutant fiber cells. Gh_D12G0217, LAG1 homologue 2; Gh_A07G0513, LAG1 longevity assurance homolog 3; Gh_D10G0211, Lactosylceramide 4-alpha-galactosyltransferase; Gh_A06G0144, phospholipase A 2A; Gh_A02G0884, GDSL-like Lipase/Acylhydrolase superfamily protein; Gh_D03G1074 and Gh_A05G3810, HXXXD-type acyl-transferase family protein; Gh_A08G1600, phospholipase D beta 1; Gh_A01G1605, alcohol dehydrogenase 1; Gh_D06G2376, 3-ketoacyl-CoA synthase 19. Error bars represent the SD for three independent experiments and asterisks indicate statistically significant differences between li-1 and TM-1 fiber cells, as determined by Student’s t-test (** p < 0.01). Full article ">Figure 9
Oil bodies in the leaf and fiber cell of TM-1 and li-1 mutant. (A) Oil bodies in the leaf of TM-1 and li-1 mutant; (B) oil bodies in the fiber cell of TM-1 and li-1 mutant. Li-1, li-1 mutant; TM-1, wild type; UV, ultraviolet light; BF, bright light. Full article ">

11 pages, 4355 KiB

Open AccessCase Report

Peripheral Blood Mononuclear Cells Cytokine Profile in a Patient with Toxic Epidermal Necrolysis Triggered by Lamotrigine and COVID-19: A Case Study

by Margarita L. Martinez-Fierro, Idalia Garza-Veloz, Sidere Monserrath Zorrilla-Alfaro, Andrés Eduardo Campuzano-Garcia and Monica Rodriguez-Borroel

Int. J. Mol. Sci. 2025, 26(3), 1374; https://doi.org/10.3390/ijms26031374 - 6 Feb 2025

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Abstract

Stevens–Johnson Syndrome (SJS)/toxic epidermal necrolysis (TEN) is a severe mucocutaneous reaction often induced by medications. The co-occurrence of SJS/TEN and COVID-19 presents a unique challenge due to overlapping inflammatory pathways. This case study examined the cytokine profile of a patient with both TEN [...] Read more.

Stevens–Johnson Syndrome (SJS)/toxic epidermal necrolysis (TEN) is a severe mucocutaneous reaction often induced by medications. The co-occurrence of SJS/TEN and COVID-19 presents a unique challenge due to overlapping inflammatory pathways. This case study examined the cytokine profile of a patient with both TEN (triggered by lamotrigine) and COVID-19. The clinical history of the patient, including lamotrigine exposure and COVID-19 diagnosis, was documented. A 13-cytokine profile assessment was performed in peripheral blood mononuclear cells from the patient and their parents by using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). A 6-year-old male patient developed lamotrigine-induced TEN with concomitant COVID-19 affecting 90% of the body surface area. Compared with their parents, who were positive for COVID-19, IL-6, IL-4, and IL-12 were modulated (downregulated) by TEN. The cytokine profile showed elevated levels of IL-1α, IL-1β, IL-5, IL-8, NF-κβ, and interferons (IFN; α, β, and γ), indicating a robust antiviral response. The immune profile suggested a hyperactivated immune state that contributed to the severity of the patient’s clinical manifestations, leading to death 18 days after hospitalization. Understanding the immune response is important for developing future targeted therapeutic strategies and improving patient outcomes. Further research is needed to explore the interaction between drug-induced SJS/TEN and infections. Full article

(This article belongs to the Special Issue Targeted Therapy for Immune Diseases)

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Figure 1
Toxic epidermal necrolysis clinical spectrum. (A–D): exanthematous rash. Lesions start on the face and thorax before spreading to other areas and are symmetrically distributed. Early lesions typically begin with ill-defined, coalescing, erythematous macules; (E–H): extensive, sheet-like detachment and erosions, and Nikolsky sign is present. Full article ">Figure 2
Expression profile of evaluated cytokines. Figure shows the expression level of a 13-citokine panel for the patient with toxic epidermal necrolysis (TEN) and concurrent COVID-19, and for his parents, grouped by immune response in which they participate (A); and according with their status of overexpression and underexpression profile (B). Expression levels were calculated by quantitative real-time polymerase chain reaction by using GAPDH as endogenous control and RNA of peripheral blood mononuclear cell obtained from healthy controls (with negative qRT-PCR for SARS-CoV-2) as calibrator. Full article ">Figure 3
Cell and immune pathophysiology of TEN. Infiltration of the epidermis by activated T lymphocytes (CD8+ epidermis; CD4+ dermis) and natural killer cells induce an immune response against the drug-reactive metabolites. TCRs recognize the molecules and produce interleukins (mainly TNFα) which cause epidermal detachment secondary to keratinocyte apoptosis induced by granzymes, perforins, and Fas/Fas ligand. MHC-II: major histocompatibility complex class II; TCR: T lymphocyte receptor; IFNγ: interferon gamma; TNFα: tumor necrosis factor alpha; IL: interleukin. Full article ">

26 pages, 2623 KiB

Open AccessReview

The Role of Gut Microbiota-Derived Trimethylamine N-Oxide in the Pathogenesis and Treatment of Mild Cognitive Impairment

by Haihua Xie, Jia Jiang, Sihui Cao, Xuan Xu, Jingyin Zhou, Ruhan Zhang, Bo Huang, Penghui Lu, Liang Peng and Mi Liu

Int. J. Mol. Sci. 2025, 26(3), 1373; https://doi.org/10.3390/ijms26031373 - 6 Feb 2025

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Abstract

Mild cognitive impairment (MCI) represents a transitional stage between normal aging and dementia, often considered critical for dementia prevention. Despite its significance, no effective clinical treatment for MCI has yet been established. Emerging evidence has demonstrated a strong association between trimethylamine-N-oxide (TMAO), a [...] Read more.

Mild cognitive impairment (MCI) represents a transitional stage between normal aging and dementia, often considered critical for dementia prevention. Despite its significance, no effective clinical treatment for MCI has yet been established. Emerging evidence has demonstrated a strong association between trimethylamine-N-oxide (TMAO), a prominent metabolite derived from the gut microbiota, and MCI, highlighting its potential as a biomarker and therapeutic target. TMAO has been implicated in increasing MCI risk through its influence on factors such as hypertension, cardiovascular disease, depression, diabetes, and stroke. Moreover, it contributes to MCI by promoting oxidative stress, disrupting the blood–brain barrier, impairing synaptic plasticity, inducing inflammation, causing mitochondrial metabolic disturbances, and facilitating abnormal protein aggregation. This review further explores therapeutic strategies targeting TMAO to mitigate MCI progression. Full article

(This article belongs to the Section Molecular Microbiology)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Origins and Excretion of TMAO. TMAO is absorbed directly from dietary sources through the intestines. Exogenous TMAO is subsequently produced via oxidation by the gut microbiome and the liver. The primary excretion pathways for TMAO include urine, feces, and respiration. Abbreviations: TMA, trimethylamine; TMAO, trimethylamine N-oxide; FMO, flavin-containing monooxygenase; OCT, organic cation transporter. Full article ">Figure 2
Contributions of TMAO to the pathogenesis of MCI. TMAO potentially contributes to the pathogenesis of MCI by promoting oxidative stress, neuroinflammation, and abnormal protein accumulation. TMAO induces oxidative stress by enhancing the production of reactive oxygen species (ROS) and reducing antioxidant activity. It also triggers neuroinflammation by activating NF-κβ and the NLRP3 inflammasome. Furthermore, TMAO exacerbates the formation of amyloid plaques and neurofibrillary tangles by impairing the intracellular ubiquitin–proteasome system. Abbreviations: TMAO, trimethylamine N-oxide; GSH, glutathione; GPX, glutathione peroxidase; SOD, superoxide dismutase; MsrA, methionine sulfoxide reductase A; NF-κB, nuclear factor kappa B; NLRP3, NOD-like receptor family pyrin domain containing 3; Sirt3, sirtuin 3; mtROS, mitochondrial reactive oxygen species; IL, interleukin; TXNIP, thioredoxin-interacting protein; NFTs, neurofibrillary tangles. Full article ">Figure 3
Effects of TMAO on the blood–brain barrier and synaptic plasticity. TMAO impairs the structural integrity and function of the blood–brain barrier (BBB) and reduces synaptic plasticity, contributing to the pathogenesis of MCI. It reduces hippocampal synaptic plasticity by activating the PI3K/Akt/mTOR and PERK signaling pathways. Simultaneously, TMAO disrupts the BBB, facilitating the accumulation of neurotoxic molecules in the brain and inducing oxidative stress and neuroinflammation. Abbreviations: SYN, synaptophysin; NMDAR, N-methyl-D-aspartate receptor; GluA1, glutamate receptor ionotropic AMPA 1; GluN2A, glutamate receptor ionotropic NMDA 2A; PSD95, postsynaptic density protein 95; PERK, protein kinase R-like endoplasmic reticulum kinase; ATF4, activating transcription factor 4; CREB, cAMP response element-binding protein; p-PI3K, phosphorylated phosphoinositide 3-kinase; p-Akt, phosphorylated Akt protein; p-mTOR, phosphorylated mammalian target of rapamycin; ZO-1, zonula occludens-1; PDGFRβ, platelet-derived growth factor receptor beta. Full article ">Figure 4
Effects of TMAO on mitochondrial metabolism. TMAO adversely affects mitochondrial metabolism, contributing to the pathogenesis of MCI. It significantly inhibits the oxidation of pyruvate and fatty acids in mitochondria, leading to energy metabolism disorders. Abbreviations: CAT, carnitine acylcarnitine translocase; CPT2, carnitine palmitoyl transferase II; TCA cycle, tricarboxylic acid cycle; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; ATP, adenosine triphosphate. Full article ">Figure 5
Treatment Strategy for improving MCI by affecting TMAO. Full article ">

24 pages, 1083 KiB

Open AccessReview

Correlations Between Gut Microbiota Composition, Medical Nutrition Therapy, and Insulin Resistance in Pregnancy—A Narrative Review

by Robert-Mihai Enache, Oana Alexandra Roşu, Monica Profir, Luciana Alexandra Pavelescu, Sanda Maria Creţoiu and Bogdan Severus Gaspar

Int. J. Mol. Sci. 2025, 26(3), 1372; https://doi.org/10.3390/ijms26031372 - 6 Feb 2025

Viewed by 480

Abstract

Many physiological changes accompany pregnancy, most of them involving metabolic perturbations. Alterations in microbiota composition occur both before and during pregnancy and have recently been correlated with an important role in the development of metabolic complications, such as insulin resistance and gestational diabetes [...] Read more.

Many physiological changes accompany pregnancy, most of them involving metabolic perturbations. Alterations in microbiota composition occur both before and during pregnancy and have recently been correlated with an important role in the development of metabolic complications, such as insulin resistance and gestational diabetes mellitus (GDM). These changes may be influenced by physiological adaptations to pregnancy itself, as well as by dietary modifications during gestation. Medical nutritional therapy (MNT) applied to pregnant women at risk stands out as one of the most important factors in increasing the microbiota’s diversity at both the species and genus levels. In this review, we discuss the physiological changes during pregnancy and their impact on the composition of the intestinal microbiota, which may contribute to GDM. We also discuss findings from previous studies regarding the effectiveness of MNT in reducing insulin resistance. In the future, additional studies should aim to identify specific gut microbial profiles that serve as early indicators of insulin resistance during gestation. Early diagnosis, achievable through stool analysis or metabolite profiling, may facilitate the timely implementation of dietary or pharmaceutical modifications, thereby mitigating the development of insulin resistance and its associated sequelae. Full article

(This article belongs to the Special Issue Molecular Insight into Gestational Diabetes Mellitus)

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14 pages, 4642 KiB

Open AccessArticle

Dynamics of Immune Cell Infiltration and Fibroblast-Derived IL-33/ST2 Axis Induction in a Mouse Model of Post-Surgical Lymphedema

by Kazuhisa Uemura, Kei-ichi Katayama, Toshihiko Nishioka, Hikaru Watanabe, Gen Yamada, Norimitsu Inoue and Shinichi Asamura

Int. J. Mol. Sci. 2025, 26(3), 1371; https://doi.org/10.3390/ijms26031371 - 6 Feb 2025

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Abstract

Lymphedema is an intractable disease most commonly associated with lymph node dissection for cancer treatment and can lead to a decreased quality of life. Type 2 T helper (Th2) lymphocytes have been shown to be important in the progression of lymphedema. The activation [...] Read more.

Lymphedema is an intractable disease most commonly associated with lymph node dissection for cancer treatment and can lead to a decreased quality of life. Type 2 T helper (Th2) lymphocytes have been shown to be important in the progression of lymphedema. The activation of IL-33 and its receptor, the suppression of tumorigenicity 2 (ST2) signaling pathway, induces the differentiation of Th2 cells, but its involvement in lymphedema remains unclear. In the present study, we analyzed the dynamics of immune cell infiltration, including the IL-33/ST2 axis, in a mouse tail lymphedema model. Neutrophil infiltration was first detected in the lymphedema tissue on postoperative day (POD) 2. Macrophage infiltration increased from POD 2 to 5. The number of CD4⁺ T cells, including 50% Tregs, gradually increased from POD 14. The mRNA expression of ll13 and Ifng increased on POD 21. The expression of IL-33 was induced in fibroblast nuclei within dermal and subcutaneous tissues from POD 2, and the expression of the Il1rl1 gene encoding ST2 increased from POD 7. We demonstrated the infiltration process from innate to acquired immune cells through the development of a mouse tail lymphedema. The IL-33/ST2 axis was found to be induced during the transition from innate to acquired immunity. Full article

(This article belongs to the Special Issue Lymphedema: From Mechanism to Treatment)

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20 pages, 10581 KiB

Open AccessArticle

Phylogeny of Camphora and Cinnamomum (Lauraceae) Based on Plastome and Nuclear Ribosomal DNA Data

by Jian Xu, Haorong Zhang, Fan Yang, Wen Zhu, Qishao Li, Zhengying Cao, Yu Song and Peiyao Xin

Int. J. Mol. Sci. 2025, 26(3), 1370; https://doi.org/10.3390/ijms26031370 - 6 Feb 2025

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Abstract

Camphora Fabr. is a genus in the family Lauraceae, comprising over 20 tropical and subtropical tree species. Since the genera Camphora and Cinnamomum Schaeff. were described, there has been a long-lasting controversy regarding the phylogenetic relationships among taxa in both genera. In particular, [...] Read more.

Camphora Fabr. is a genus in the family Lauraceae, comprising over 20 tropical and subtropical tree species. Since the genera Camphora and Cinnamomum Schaeff. were described, there has been a long-lasting controversy regarding the phylogenetic relationships among taxa in both genera. In particular, phylogenetic inferences derived from plastid data remain debated, with varying hypotheses proposed and occasional disputes concerning the monophyly of Camphora taxa. To further investigate the relationships, We analyzed plastomes and nuclear ribosomal cistron sequences (nrDNA) of 22 Camphora taxa, 15 Cinnamomum taxa, and 13 representative taxa of related genera. The Camphora plastomes range from 152,745 to 154,190 bp, with a GC content of 39.1% to 39.2%. A total of 128 genes were identified in the Camphora plastomes, including 84 protein-coding genes, 8 rRNA genes, and 36 tRNA genes. A total of 1130 SSR loci were detected from plastomes of Camphora, and A/T base repeats looked like the most common. Comparative analyses revealed that the plastomes of Camphora exhibit high similarity in overall structure. The loci ycf1, ycf2, trnK (UUU), psbJ-psbL, and ccsA-ndhD were identified as candidate DNA barcodes for these taxa. Plastome phylogenetic analysis revealed that Camphora is not monophyletic, whereas the nrDNA dataset supported the monophyly of Camphora. We propose that intergeneric hybridization may underlie the observed discordance between plastid and nuclear data in Camphora, and we recommend enhanced taxonomic sampling and precise species identification to improve phylogenetic resolution and accuracy. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

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20 pages, 11711 KiB

Open AccessArticle

CITE-Seq Analysis Reveals a Differential Natural Killer Cell SPON2 Expression in Cardiovascular Disease Patients Impacted by Human-Cytomegalovirus Serostatus and Diabetes

by Sujit Silas Armstrong, Daniel G. Chen, Sunil Kumar, James R. Heath, Matthew J. Feinstein, John R. Greenland, Daniel R. Calabrese, Lewis L. Lanier, Klaus Ley and Avishai Shemesh

Int. J. Mol. Sci. 2025, 26(3), 1369; https://doi.org/10.3390/ijms26031369 - 6 Feb 2025

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Abstract

Coronary artery disease (CAD) is linked to atherosclerosis plaque formation. In pro-inflammatory conditions, human Natural Killer (NK) cell frequencies in blood or plaque decrease; however, NK cells are underexplored in CAD pathogenesis, inflammatory mechanisms, and CAD comorbidities, such as human cytomegalovirus (HCMV) infection [...] Read more.

Coronary artery disease (CAD) is linked to atherosclerosis plaque formation. In pro-inflammatory conditions, human Natural Killer (NK) cell frequencies in blood or plaque decrease; however, NK cells are underexplored in CAD pathogenesis, inflammatory mechanisms, and CAD comorbidities, such as human cytomegalovirus (HCMV) infection and diabetes. Analysis of PBMC CITE-seq data from sixty-one CAD patients revealed higher blood NK cell SPON2 expression in CAD patients with higher stenosis severity. Conversely, NK cell SPON2 expression was lower in pro-inflammatory atherosclerosis plaque tissue with an enriched adaptive NK cell gene signature. In CAD patients with higher stenosis severity, peripheral blood NK cell SPON2 expression was lower in patients with high HCMV-induced adaptive NK cell frequencies and corresponded to lower PBMC TGFβ transcript expression with dependency on diabetes status. These results suggest that high NK cell SPON2 expression is linked to atherosclerosis pro-homeostatic status and may have diagnostic and prognostic implications in cardiovascular disease. Full article

(This article belongs to the Special Issue Newer Pharmacological Treatment Concepts for Patients with Acute Coronary Syndrome)

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Figure 1
NK cell SPON2 expression significantly increases in CAD patients with high stenosis. (A) PBMC from 61 CAD patients were clustered by CITE-seq protein expression. UMAP of PBMC clusters based on CITE-seq protein expression. NK cells (purple cluster, black arrow). (B) Differential gene expression (DGE) analysis of NK cell gene expression between CADlow vs. CADhigh at the single-cell level (red: upregulated in CADhigh; blue: upregulated in CADlow). (C) Patients’ mean NK cell SPON2 expression in CADlow vs. CADhigh patients (dot = patient). (D) UMAP of SPON2 RNA expression relative to PBMC clusters. (E) NK cell SPON2 expression in patients grouped by CAD stenosis severity score. Patients were grouped the patients by stenosis severity score (combined percent stenosis of each artery segment score): I, 0–6 [n = 29, CADlow]; II, 30–48 [n = 13]; III, 49–67.5 [n = 11]; IV, 77–150 [n = 8]. Mean +/− S.D.; Mann–Whitney test; one-tail; * p < 0.05; ** p < 0.01; *** p < 0.001. Full article ">Figure 2
NK cell SPON2 expression is higher in pro-homeostatic plaque tissue. (A) NK cell gene expression between carotid plaques (pro-inflammatory, n =3) relative to the femoral plaques (pro-homeostatic, n =7). Gene expression is displayed as mean z-score. (B) Mean NK cell SPON2 expression in femoral relative to carotid plaque tissue. (C) Pearson correlation (one-tail) between NK cell SPON2 and IFNG expression in atherosclerosis plaques (n = 10). (D) NK cell SPON2/IFNG ratio in carotid or femoral plaques. (E) Pearson correlation (one-tail) between NK cell SPON2 and IFNG expression in CAD patients. NK cell SPON2/IFNG ratio in (F) CADlow vs. CADhigh patients or (G) relative to stenosis severity. (H) Pearson correlation (one-tail) between NK cell IFNG expression and hs-CRP levels in CAD patients. # To avoid misinterpretation of the data, one patient outlier (hs-CRP (mg/L) = 150) was removed from the analysis. (I) NK cell SPON2/ hs-CRP ratio relative to stenosis severity. (J) Schematic representation of findings. Mean +/− S.D.; Mann–Whitney test; one-tail; * p < 0.05; ** p < 0.01; dot = patient. Full article ">Figure 3
HCMV serostatus and diabetes impact on NK cell SPON2 expression. Mean NK cell SPON2 expression per patient grouped by CADlow vs. CADhigh; (A) HCMV seronegative (HCMV−) or (B) HCMV seropositive (HCMV+). NK cell SPON2 expression relative to stenosis severity in (C) HCMV− or (D) HCMV+ patients. (E) NK cell SPON2 expression in patients, grouped by CAD, diabetes, and HCMV status (black: HCMV−; red: HCMV+); (E.i) all patients, (E.ii) HCMV− patients, or (E.iii) HCMV+ patients. (F) Schematic representation of figure findings. Mean+/− S.D.; Mann–Whitney test; one-tail; * p < 0.05; ** p < 0.01; dot = patient. Full article ">Figure 4
SPON2low HCMV-induced NK cell frequencies do not decrease with stenosis severity. (A) UMAP of NK cell clusters. The NK cell cluster (Cluster 4, <a href="#ijms-26-01369-f001" class="html-fig">Figure 1</a>A) was re-clustered based on CITE-seq protein expression of CD56, CD16, CD25, CD27, CD2, or HLA-DR. (B) Heatmap of mean NK cell gene expression for the indicated genes. (C) density plots of NK cell clusters in HCMV− or HCMV+ patients. C2 frequencies (%) between (D) HCMV− and HCMV+ patients, or patients grouped by (E) CAD low vs. high status, (F) CAD and HCMV serostatus, or (G) CAD, diabetes, and HCMV status. (H) Density plot of NK cell clusters in patients grouped by CAD, diabetes, and HCMV status. (I) Schematic representation of findings. (J) Blood NK cell frequencies in patients grouped by CAD, diabetes, and HCMV status. (K) C2 frequencies (%) (upper panels), C3/C2 ratio (middle panels), or NKG2C−/NKG2C+ ratio (lower panels) relative to stenosis severity. Patient groups (not all patients) are shown as black: HCMV− or red: HCMV+). Mean+/− S.D.; Mann–Whitney test; one-tail; * p < 0.05; ** p < 0.01; *** p < 0.001; dot = patient. Full article ">Figure 5
Higher NK cell SPON2 expression corresponds to lower C2 frequencies and higher PBMC TGFβ expression in CADhigh patients. (A) C2 frequencies in CADhigh patients with low (C2low) or high (C2high) frequencies (left), and relative to diabetes status (right: black, diabetes−; blue, diabetes+). (B) Number of HCMV− or HCMV+ cases in C2low vs. C2high patients: left, diabetes−; right, diabetes+. (C) Mean NK cell SPON2 expression in CADhigh C2low or C2high patients (left), and relative to diabetes status (right: black, diabetes−; blue, diabetes+). (D) PBMC TGFβ expression in CAD low vs. high patients. Right: expression of PBMC TGFβ in patients grouped by CAD, diabetes, and HCMV status, or (E) in CADhigh C2low or C2high patients, and relative to diabetes status (right: black, diabetes−; blue, diabetes+). (F) PBMC IL15/TGFβ ratio in CADhigh C2low or C2high patients (left), and relative to diabetes status (right: black, diabetes−; blue, diabetes+). (G) PBMC IL15/TGFβ ratio (G.i) or mean NK cell SPON2 expression (G.ii), relative to stenosis severity in (left to right) diabetes−HCMV−, diabetes−HCMV+, diabetes+HCMV−, or diabetes+HCMV+ patients. Patient groups are shown as black: HCMV− or red: HCMV+). Mean +/− S.D.; Mann–Whitney test; one-tail; * p < 0.05; ** p < 0.01; *** p < 0.001; dot = patient. Full article ">Figure 6
Differential upregulation of Spondin-2 by IL-15 and TGFβ in primary NK cells. (A) Dot plots of intracellular Spondin-2 (X-axis) vs. IFNγ (Y-axis) expression in isolated human primary NK cells (n = 2) following 24 h of CD16 stimulation with media without cytokines or with IL-12 (1 ng/mL), IL-2 (300 U/mL), IL-15 (50 ng/mL), or IFNα (50 ng/mL). (B) Histograms of Spondin-2 intracellular expression (red) relative to media without IL-15 control (black) in isolated primary NK cells following 3 days of stimulation with IL-15 (50 ng/mL). (C) Histograms of Spondin-2 intracellular expression between the defined NK cell subsets (color-coded) after 3 days of stimulation with IL-15 (50 ng/mL) or media without cytokines. The right dot plot represents the gating of mature CD56dimCD16+NK cells to identify adaptive NK cell subsets by NKG2C vs. FcεR1γ protein expression. Right panel: IL-2 or IL-15 concentration-dependent expression of Spondin-2 between the defined NK cell subsets (n = 5). IL-2 (300, 30, and 3 U/mL), IL-15 (50 and 0.5 ng/mL), or media (media without cytokines). (D) FcεR1γ (left) or Spondin-2 (right) intracellular expression or (E) cell trace violet (left) or relative NK cell numbers/well (right) in purified primary non-adaptive NK cells (n = 4) stimulated for 6 days with IL-15 (10 ng/mL), with or without glucose (16 or 4 g/L) or TGFβ (5 ng/mL) relative to media without cytokines. (F) Expression of FcεR1γ (left) or Spondin-2 (right) in purified primary non-adaptive NK cells stimulated for 6 days with IL-15 (50 ng/mL) with or without rapamycin (RAPA, 10 nM) or FOXO1 inhibitor (50 nM) relative to media without cytokines. (G) Schematic representation of findings. Mean+/− S.D.; Mann–Whitney test; one-tail; * p < 0.05; ** p < 0.01; *** p < 0.001; dot = donor. Full article ">

17 pages, 9918 KiB

Open AccessArticle

Aspirin Foliar Spray-Induced Changes in Light Energy Use Efficiency, Chloroplast Ultrastructure, and ROS Generation in Tomato

by Julietta Moustaka, Ilektra Sperdouli, Emmanuel Panteris, Ioannis-Dimosthenis S. Adamakis and Michael Moustakas

Int. J. Mol. Sci. 2025, 26(3), 1368; https://doi.org/10.3390/ijms26031368 - 6 Feb 2025

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Abstract

Aspirin (Asp) is extensively used in human health as an anti-inflammatory, antipyretic, and anti-thrombotic drug. In this study, we investigated if the foliar application of Asp on tomato plants has comparable beneficial effects on photosynthetic function to that of salicylic acid (SA), with [...] Read more.

Aspirin (Asp) is extensively used in human health as an anti-inflammatory, antipyretic, and anti-thrombotic drug. In this study, we investigated if the foliar application of Asp on tomato plants has comparable beneficial effects on photosynthetic function to that of salicylic acid (SA), with which it shares similar physiological characteristics. We assessed the consequences of foliar Asp-spray on the photosystem II (PSII) efficiency of tomato plants, and we estimated the reactive oxygen species (ROS) generation and the chloroplast ultrastructural changes. Asp acted as an osmoregulator by increasing tomato leaf water content and offering antioxidant protection. This protection kept the redox state of plastoquinone (PQ) pull (qp) more oxidized, increasing the fraction of open PSII reaction centers and enhancing PSII photochemistry (Φ_PSII). In addition, Asp foliar spray decreased reactive oxygen species (ROS) formation, decreasing the excess excitation energy on PSII. This resulted in a lower singlet oxygen (¹O₂) generation and a lower quantum yield for heat dissipation (Φ_NPQ), indicating the photoprotective effect provided by Asp, especially under excess light illumination. Simultaneously, we observed a decrease in stomatal opening by Asp, which reduced the transpiration. Chloroplast ultrastructural data revealed that Asp, by offering a photoprotective effect, decreased the need for the photorespiration process, which reduces photosynthetic performance. It is concluded that Asp shares similar physiological characteristics with SA, having an equivalent beneficial impact to SA by acting as a biostimulant of the photosynthetic function for an enhanced crop yield. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Plant Abiotic Stress Tolerance)

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28 pages, 7769 KiB

Open AccessArticle

Impact of African-Specific ACE2 Polymorphisms on Omicron BA.4/5 RBD Binding and Allosteric Communication Within the ACE2–RBD Protein Complex

by Victor Barozi and Özlem Tastan Bishop

Int. J. Mol. Sci. 2025, 26(3), 1367; https://doi.org/10.3390/ijms26031367 - 6 Feb 2025

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Abstract

Severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) infection occurs via the attachment of the spike (S) protein’s receptor binding domain (RBD) to human ACE2 (hACE2). Natural polymorphisms in hACE2, particularly at the interface, may alter RBD–hACE2 interactions, potentially affecting viral infectivity across populations. [...] Read more.

Severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) infection occurs via the attachment of the spike (S) protein’s receptor binding domain (RBD) to human ACE2 (hACE2). Natural polymorphisms in hACE2, particularly at the interface, may alter RBD–hACE2 interactions, potentially affecting viral infectivity across populations. This study identified the effects of six naturally occurring hACE2 polymorphisms with high allele frequency in the African population (S19P, K26R, M82I, K341R, N546D and D597Q) on the interaction with the S protein RBD of the BA.4/5 Omicron sub-lineage through post-molecular dynamics (MD), inter-protein interaction and dynamic residue network (DRN) analyses. Inter-protein interaction analysis suggested that the K26R variation, with the highest interactions, aligns with reports of enhanced RBD binding and increased SARS-CoV-2 susceptibility. Conversely, S19P, showing the fewest interactions and largest inter-protein distances, agrees with studies indicating it hinders RBD binding. The hACE2 M82I substitution destabilized RBD–hACE2 interactions, reducing contact frequency from 92 (WT) to 27. The K341R hACE2 variant, located distally, had allosteric effects that increased RBD–hACE2 contacts compared to WThACE2. This polymorphism has been linked to enhanced affinity for Alpha, Beta and Delta lineages. DRN analyses revealed that hACE2 polymorphisms may alter the interaction networks, especially in key residues involved in enzyme activity and RBD binding. Notably, S19P may weaken hACE2–RBD interactions, while M82I showed reduced centrality of zinc and chloride-coordinating residues, hinting at impaired communication pathways. Overall, our findings show that hACE2 polymorphisms affect S BA.4/5 RBD stability and modulate spike RBD–hACE2 interactions, potentially influencing SARS-CoV-2 infectivity—key insights for vaccine and therapeutic development. Full article

(This article belongs to the Section Biochemistry)

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16 pages, 1841 KiB

Open AccessArticle

AgNP-Containing Niosomes Functionalized with Fucoidan Potentiated the Intracellular Killing of Mycobacterium abscessus in Macrophages

by Nereyda Niño-Martínez, Kayla Audreyartha, Kaitlyn Cheung, Sol Melchor Parra, Gabriel Martínez-Castañón and Horacio Bach

Int. J. Mol. Sci. 2025, 26(3), 1366; https://doi.org/10.3390/ijms26031366 - 6 Feb 2025

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Abstract

Intracellular pathogens represent a challenge for therapy because the antibiotics used need to diffuse into the cytoplasm to target the pathogens. The situation is more complicated in the mycobacteria family because members of this family infect and multiply within macrophages, the cells responsible [...] Read more.

Intracellular pathogens represent a challenge for therapy because the antibiotics used need to diffuse into the cytoplasm to target the pathogens. The situation is more complicated in the mycobacteria family because members of this family infect and multiply within macrophages, the cells responsible for clearing microorganisms in the body. In addition, mycobacteria members are enclosed inside pathogen-containing vesicles or phagosomes. The treatments of these pathogens are aggravated when these pathogens acquire resistance to antibiotic molecules. As a result, new antimicrobial alternatives are needed. Niosomes are vesicles composed of cholesterol and nonionic surfactants that can be used for antibiotic encapsulation and delivery. The current study developed a systematic formulation of niosomes to determine the best option for niosome functionalizing for precise delivery to the intracellular pathogen Mycobacterium abscessus. Silver nanoparticles (AgNPs) were synthesized using gallic acid as an antibacterial agent. Then, niosomes were prepared and characterized, following the encapsulation of AgNPs functionalized with a single-chain antibody screened against the cell wall glycopeptidolipid of Mycobacterium abscessus. For a precise delivery of the cargo into macrophages, the niosomes were also functionalized with the polysaccharide fucoidan, taken specifically by the scavenger receptor class A expressed on the surface of macrophages. Results of the study showed a steady decrease in the intracellular pathogen load after 48 h post-infection. In conclusion, this system could be developed into a platform to target other types of intracellular pathogens and as an option for antimicrobial therapy. Full article

(This article belongs to the Special Issue Advances in Antimicrobial Nanomaterials 2.0)

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(A) Structure of GPL. (B). The antibody scFv recognizes M. abscessus. Bacteria were labeled with Rhodamine B (10 μg/mL, MilliporeSigma, Burlington, MA USA) and exposed to M. abscessus, which had previously flamed on a microscope slide. The scFv-51 (not co-expressed with the red fluorescent protein) and bacteria were incubated for 30 min, washed with PBS (×3), and incubated with FITC-mouse-anti-his tag (Thermo Fisher, Waltham, MA USA) for 30 min. The slides were analyzed by fluorescence microscopy after being washed with PBS (×3). The yellow color indicates the co-localization of scFv-51 to the pathogen. ABS, M. abscessus; BCG, M. bovis strain BCG; MAA, M. avium avium. Full article ">Figure 2
Effect of the sonication on the niosome size. NA4 niosomes were subjected to sonication during their last step of synthesis. (A) Impact of the sonicator power on the niosome sizes. (B) Impact of the sonication time on the niosome size using a probe power between 12 and 15 W. The numbers at each time point represent the highest percentage of the main peak, as measured by PDI in the DLS device. Full article ">Figure 3
Scheme showing the synthesis of AgNP-scFv-51 with aminated fucoidan. Created with <a href="http://Biorender.com" target="_blank">Biorender.com</a>. Full article ">Figure 4
Cytotoxicity of niosomes. Differentiated THP-1 was treated with different concentrations of NC3 AgNP-scFv-51 and evaluated by MTT. Untreated and SDS-treated cells were used as negative and positive controls, respectively. OD, optical density. * p-value < 0.05. Experiments were performed in triplicate. Full article ">Figure 5
Survival of M. abscessus within macrophages. Differentiated THP-1 cells were infected with M. abscessus at a multiplicity of infection of 1. The macrophages were treated with the NB5 5 μg/mL AgNP-scFv-51 + 10% cholesteryl acetate:fucoidan. Untreated cells were used as a negative control, whereas AgNPs were used as a positive control. Shown is the median ± SD of three independent experiments. CFU, colony-forming units. * p-value <0.05. Full article ">

14 pages, 7571 KiB

Open AccessArticle

Sterol Regulatory Element-Binding Protein Sre1 Mediates the Development and Pathogenicity of the Grey Mould Fungus Botrytis cinerea

by Ye Yuan, Shengnan Cao, Jiao Sun, Jie Hou, Mingzhe Zhang, Qingming Qin and Guihua Li

Int. J. Mol. Sci. 2025, 26(3), 1365; https://doi.org/10.3390/ijms26031365 - 6 Feb 2025

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Abstract

The grey mould fungus Botrytis cinerea is a dangerous plant pathogen responsible for substantial agricultural losses worldwide. The pathogenic mechanisms still have many unclear aspects, and numerous new pathogenic genes remain to be identified. Here, we show that the sterol regulatory element-binding protein [...] Read more.

The grey mould fungus Botrytis cinerea is a dangerous plant pathogen responsible for substantial agricultural losses worldwide. The pathogenic mechanisms still have many unclear aspects, and numerous new pathogenic genes remain to be identified. Here, we show that the sterol regulatory element-binding protein Sre1 plays an important role in the development and pathogenicity of B. cinerea. We identified a homologue of gene SRE1 in the B. cinerea genome and utilized a reverse genetics approach to create the knockout mutant Δsre1. Our results demonstrate that SRE1 is essential for conidiation, as Δsre1 produced only 3% of the conidia compared to the wild-type strain. Conversely, Δsre1 exhibited increased sclerotium production, indicating a negative regulatory role of SRE1 in sclerotium formation. Furthermore, ergosterol biosynthesis was significantly reduced in the Δsre1 mutant, correlating with increased sensitivity to low-oxygen conditions. Pathogenicity assays revealed that Δsre1 had significantly reduced virulence, although it maintained normal infection cushion formation and penetration capabilities. Additionally, SRE1 was found to be crucial for hypoxia adaptation, as Δsre1 showed abnormal germination and reduced growth under low-oxygen conditions. These findings suggest that SRE1 mediates the development and pathogenicity of B. cinerea by regulating lipid homeostasis and facilitating adaptation to host tissue environments. Full article

(This article belongs to the Special Issue Plant Responses to Biotic and Abiotic Stresses)

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Figure 1
Generations of Botrytis cinerea SRE1 knockout mutants and its genetic complemented strains. (a) Strategy for generation of SRE1 knockout strain Δsre1 via gene replacement approach. WT, the wild-type strain B05.10; pSRE1-ko, SRE1 knockout vector. HPH, the hygromycin resistance gene. (b) Screening of Δsre1 strains. Numbers 1–23 indicate partial selected transformants. PCR amplifications were used for detecting HPH recombination (rec) and SRE1 loss in transformants, respectively, with indicated primers. Up-rec, upstream recombination; down-rec, downstream recombination. (c) Verification of the complemented strain Δsre1-c. (d) Relative SRE1 expression level in the indicated strains determined by quantitative reverse transcription PCR. M, DNA marker D2000. ND, not detected. Data represent means ± standard deviations (SD) from at least three independent experiments. *, ***, significance at p < 0.05, 0.001, respectively. Full article ">Figure 2
SRE1 is required for B. cinerea conidiation but dispensable for conidial morphogenesis and germination. (a) Colony of tested strains cultured on PDA at 3 days post inoculation (DPI). (b) Quantification of the colony sizes (determined by the relative colony diameter). (c) Conidiation on CM plates at 12 DPI. (d) Quantification of the relative conidiation of the indicated strains (determined by the relative conidial number per plate). (e) Conidial morphology of tested strains. Bar = 10 μm. (f) Conidial germination of tested strains at 4 h post inoculation (HPI). Bar = 10 μm. Data represent means ± standard deviations (SD) from at least three independent experiments. ***, significance at p < 0.001. Full article ">Figure 3
SRE1 mediates sclerotium production and ergosterol biosynthesis in B. cinerea. (a) Deletion of SRE1 in B. cinerea increases sclerotial production. The tested strains were cultured on CM at 20 °C in darkness and observed at 15 DPI. (b) Quantification of sclerotium production (determined by the relative sclerotial number per plate). (c) Quantification of ergosterol content (determined by the relative ergosterol content per gram of mycelium fresh weight). Data represent means ± standard deviations (SDs) from at least three independent experiments. *, **, ***, significance at p < 0.05, 0.01, 0.001, respectively. Full article ">Figure 4
Deletion of SRE1 increases B. cinerea resistance to antifungal drug imidazole. Conidial suspensions (containing 50 mM glucose) of each strain were treated with 0, 2, 5, and 10 mM imidazole, respectively. Conidial germinations were observed at 4 h post inoculation (HPI). Full article ">Figure 5
SRE1 is required for B. cinerea virulence but dispensable for its infection cushion formation and penetration. (a) Mycelial plugs of each strain were inoculated on green bean leaves and the lesions were photographically documented at 3 DPI. (b) Conidial suspensions of each strain were inoculated on green bean leaves and the lesions were photographically documented at 3 DPI. (c) Infection cushion formation of tested strains at 20 HPI. (d) Onion epidermis penetration of the test strains at 12 HPI. Successful penetrations are indicated by red arrows. (e) Quantification of lesion size (determined by the relative lesion area per inoculation) caused by the indicated strains on green bean leaves shown in (a). (f) Quantification of penetration (determined per conidium) by the indicated strains on onion epidermis shown in (d). Data represent means ± standard deviations (SDs) from at least three independent experiments. *, ***, significance at p < 0.05, 0.001, respectively. Full article ">Figure 6
SRE1 is involved in hypoxia adaptation. (a) Conidial suspensions (containing 50 mM glucose) of each strain were treated with 100 or 200 μM cobalt chloride (CoCl2), a hypoxia-mimicking agent. Conidial germinations were observed at 4 HPI. Germ tubes are indicated by red arrows. (b) Quantification of abnormal conidial germination in hypoxia-mimic with 100 μM CoCl2. (c) Quantification of mycelial biomass cultured in normoxia or in hypoxia-mimic with 100 μM CoCl2. Data represent means ± standard deviations (SD) from at least three independent experiments. **, ***, significance at p < 0.01, 0.001, respectively. Full article ">

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