International Journal of Molecular Sciences

4 pages, 181 KiB

Open AccessEditorial

Advanced Therapy Medicinal Products as Potential Therapeutic Strategy against COVID-19 and Immune-Related Disorders

by Panagiotis Mallis, Efstathios Michalopoulos and Catherine Stavropoulos-Giokas

Int. J. Mol. Sci. 2024, 25(5), 3079; https://doi.org/10.3390/ijms25053079 - 6 Mar 2024

Viewed by 1509

Advanced Therapy Medicinal Products (ATMPs) comprise a heterogenous class of innovative medicinal products, which further require extensive preclinical and clinical assessments before their broader use in the general population [...] Full article

(This article belongs to the Special Issue Advanced Therapy Medicinal Products as Potential Therapeutic Strategy against COVID-19 and Immune-Related Disorders)

21 pages, 3996 KiB

Open AccessArticle

Oxidative Stress-Mediated Repression of Virulence Gene Transcription and Biofilm Formation as Antibacterial Action of Cinnamomum burmannii Essential Oil on Staphylococcus aureus

by Lingling Shi, Wei Lin, Yanling Cai, Feng Chen, Qian Zhang, Dongcheng Liang, Yu Xiu, Shanzhi Lin and Boxiang He

Int. J. Mol. Sci. 2024, 25(5), 3078; https://doi.org/10.3390/ijms25053078 - 6 Mar 2024

Viewed by 1793

Abstract

This work aimed to identify the chemical compounds of Cinnamomum burmannii leaf essential oil (CBLEO) and to unravel the antibacterial mechanism of CBLEO at the molecular level for developing antimicrobials. CBLEO had 37 volatile compounds with abundant borneol (28.40%) and showed good potential [...] Read more.

This work aimed to identify the chemical compounds of Cinnamomum burmannii leaf essential oil (CBLEO) and to unravel the antibacterial mechanism of CBLEO at the molecular level for developing antimicrobials. CBLEO had 37 volatile compounds with abundant borneol (28.40%) and showed good potential to control foodborne pathogens, of which Staphylococcus aureus had the greatest inhibition zone diameter (28.72 mm) with the lowest values of minimum inhibitory concentration (1.0 μg/mL) and bactericidal concentration (2.0 μg/mL). To unravel the antibacterial action of CBLEO on S. aureus, a dynamic exploration of antibacterial growth, material leakage, ROS formation, protein oxidation, cell morphology, and interaction with genome DNA was conducted on S. aureus exposed to CBLEO at different doses (1/2–2×MIC) and times (0–24 h), indicating that CBLEO acts as an inducer for ROS production and the oxidative stress of S. aureus. To highlight the antibacterial action of CBLEO on S. aureus at the molecular level, we performed a comparative association of ROS accumulation with some key virulence-related gene (sigB/agrA/sarA/icaA/cidA/rsbU) transcription, protease production, and biofilm formation in S. aureus subjected to CBLEO at different levels and times, revealing that CBLEO-induced oxidative stress caused transcript suppression of virulence regulators (RsbU and SigB) and its targeted genes, causing a protease level increase destined for the biofilm formation and growth inhibition of S. aureus, which may be a key bactericidal action. Our findings provide valuable information for studying the antibacterial mechanism of essential oil against pathogens. Full article

(This article belongs to the Section Molecular Microbiology)

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The growth kinetics curve of Staphylococcus aureus affected by CBLEO. The data represent the mean value ± SD of three parallel replicates. Full article ">Figure 2
Effect of CBLEO on cell wall and cell membrane of S. aureus. (A) Extracellular activity of alkaline phosphatase (AKP); (B) relative electric conductivity; (C) leakage of protein; (D) release of 260 nm absorbing material. Data represent the value ± SD of three replicates. Full article ">Figure 3
Effect of CBLEO on cell morphologically of S. aureus by scanning electron microscope (SEM) assay. (A) SEM image of untreated S. aureus; (B) SEM image of S. aureus treated with CBLEO (1×MIC) for 2 h. Red arrows represent cell morphology change and cell membrane damage. Yellow boxes represent the details of cell morhology, bar = 2 μm. Yellow arrow represents the severely damaged cell. Full article ">Figure 4
Effect of CBLEO on cell lipid peroxidation and oxidative stress in response of S. aureus to different doses and times. (A) Intracellular mallondialdehyde (MDA) level; (B) intracellular ROS generation; (C) protein carbonyl content; (D) total cellular protein. Data represent mean value ± SD of three parallel replicates, and different letters denote significant differences (p < 0.05). Full article ">Figure 5
Effect of CBLEO on biofilm formation and protease activity in response of S. aureus cells to different doses and times. (A) Assessment of inhibitory capacity of CBLEO on biofilm formation by microtiter plate assay; (B) change in cellular protease activity. Values of biofilm formation and protease production in S. aureus cells from control and CBLEO-treated samples with different doses at 0 h were arbitrarily set to 1.00 for standardization. Data represent mean value ± SD of six parallel replicates, and different letters denote significant differences (p < 0.05). Full article ">Figure 6
Effect of CBLEO on transcriptions of virulence-associated regulators of S. aureus under exposure to different concentrations and times by qRT-PCR detection. (A) Relative transcription of agrA (accessory gene regulator A); (B) relative transcription of sarA gene (staphylococcal accessory regulator A); (C) relative transcription of sigB (sigma factor B); (D) relative transcription of icaA (intercellular adhesin A); (E) relative transcription of cidA gene (encoding for holin); (F) relative transcription of rsbU (SigB activator). Relative expression values were counted as 2−ΔΔCt, and 16S RNA was used as internal control. Transcription level in S. aureus cells from control and CBLEO-treated samples with different concentrations at 0 h was arbitrarily set to 1.00 for standardization. Data represent mean value ± SD of three parallel replicates, and different letters denote significant differences (p < 0.05). Full article ">Figure 7
The antibacterial acting mode of C. burmami leaf essential oil (CBLEO) on S. aureus. The identified antibacterial mechanism of CBLEO against S. aureus is based on the present work and is summarized from the two perspectives of virulence-related gene transcription regulation and cellular structure destruction. The abbreviations are shown as follows: agrA, accessory gene regulator A; sarA, staphylococcal accessory regulator A; sigB, sigma factor B; icaA, intercellular adhesin A; ROS, reactive oxygen species; MDA, mallondialdehyde. Full article ">

15 pages, 2220 KiB

Open AccessArticle

Tissue Hypoxia and Associated Innate Immune Factors in Experimental Autoimmune Optic Neuritis

by Zhiyuan Yang, Cristina Marcoci, Hatice Kübra Öztürk, Eleni Giama, Ayse Gertrude Yenicelik, Ondřej Slanař, Christopher Linington, Roshni Desai and Kenneth J. Smith

Int. J. Mol. Sci. 2024, 25(5), 3077; https://doi.org/10.3390/ijms25053077 - 6 Mar 2024

Cited by 2 | Viewed by 1986

Abstract

Visual loss in acute optic neuritis is typically attributed to axonal conduction block due to inflammatory demyelination, but the mechanisms remain unclear. Recent research has highlighted tissue hypoxia as an important cause of neurological deficits and tissue damage in both multiple sclerosis (MS) [...] Read more.

Visual loss in acute optic neuritis is typically attributed to axonal conduction block due to inflammatory demyelination, but the mechanisms remain unclear. Recent research has highlighted tissue hypoxia as an important cause of neurological deficits and tissue damage in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) and, here, we examine whether the optic nerves are hypoxic in experimental optic neuritis induced in Dark Agouti rats. At both the first and second peaks of disease expression, inflamed optic nerves labelled significantly for tissue hypoxia (namely, positive for hypoxia inducible factor-1α (HIF1α) and intravenously administered pimonidazole). Acutely inflamed nerves were also labelled significantly for innate markers of oxidative and nitrative stress and damage, including superoxide, nitric oxide and 3-nitrotyrosine. The density and diameter of capillaries were also increased. We conclude that in acute optic neuritis, the optic nerves are hypoxic and come under oxidative and nitrative stress and damage. Tissue hypoxia can cause mitochondrial failure and thus explains visual loss due to axonal conduction block. Tissue hypoxia can also induce a damaging oxidative and nitrative environment. The findings indicate that treatment to prevent tissue hypoxia in acute optic neuritis may help to restore vision and protect from damaging reactive oxygen and nitrogen species. Full article

(This article belongs to the Special Issue Molecular Mechanism in Multiple Sclerosis and Related Disorders: 2nd Edition)

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Identification of EAE-ON and EAE-NON. Examples of fluorescent images of optic nerve sections labelled with (a) ED1 and (c) IBA1 in an IFA animal (IFA; (a-i,c-i)) and in an inflamed optic nerve of an animal with EAE (EAE-ON; (a-ii,c-ii)). Both (b) ED1 and (d) IBA1 showed significantly greater labelling in EAE-ON than in EAE-NON and IFA nerves. Each data point in (b,d) represents one nerve. (e,f) show inflamed nerves (EAE-ON). In such nerves, the labelling for ED1 and more intense IBA1 commenced from the orbital end of the nerve (e), extending in some nerves towards the chiasm and involving the chiasm (f). Mean ± SEM, one-tailed independent t-test, * p < 0.05, ** p < 0.01, *** p < 0.001, bar = 100 µm. Full article ">Figure 2
The inflamed optic nerve was hypoxic. Photomicrographs of optic nerve sections labelled for (a) HIF1α and (b) pimonidazole in tissue from an IFA animal (IFA; (a-i,b-i)), and tissue representing EAE-NON (a-ii,b-ii) and EAE-ON (a-iii,b-iii) from animals immunised for EAE. The EAE-ON nerves showed significantly more intense labelling for (c) HIF1α compared with nerves from IFA animals, which was more intense in the most inflamed nerves (d). A reduction in absolute tissue oxygenation was confirmed in the EAE-ON nerves by significantly greater labelling for (e) pimonidazole compared with nerves from IFA animals. Each data point in (c–e) represents one nerve. Mean ± SEM, one-tailed independent t-test and linear regression (in EAE-ON group; (d)), * p < 0.05, ** p < 0.01, bar = 50 µm. Full article ">Figure 3
Vascular changes in the inflamed optic nerve. Fluorescent images of sections of optic nerves labelled for (a) GLUT1 in nerves from an IFA animal (IFA; (a-i)) and nerves identified as EAE-NON (a-ii) and EAE-ON (a-iii). The inflamed nerve showed a tendency for vessels to be less aligned with the length of the nerve, perhaps due to displacement by inflammatory cells. (b) Vessel number appeared significantly increased in the EAE-ON group, accompanied by significant (c) dilation compared with the EAE-NON and IFA groups. Each data point in (b,c) represents one nerve. Mean ± SEM, one-tailed independent t-test, ns: not significant, ** p < 0.01, *** p < 0.001, bar = 100 µm. Full article ">Figure 4
The inflamed optic nerve showed increased labelling for the presence of suspected superoxide (DHE), nitric oxide (iNOS) and peroxynitrite (3NT). Sections of optic nerves showing fluorescence resulting from (a) DHE and labelled for (c) iNOS; (e) 3NT in animals ‘immunised’ with IFA (a-i,c-i,e-i) and those immunised for EAE but without optic neuritis (EAE-NON; (a-ii,c-ii,e-ii)) or with optic neuritis (EAE-ON; (a-iii,c-iii,e-iii)). Inflamed optic nerves from animals with EAE showed significantly more intense fluorescence from (b) DHE and labelling for (b) iNOS, (c) 3NT and (f) DHE. The labelling for iNOS was highest for EAE-ON at peak of disease (day 2 after disease onset), but was significantly reduced when examined on day 4 (d). On day 2, the labelling for iNOS was positively correlated with the magnitude of inflammation (g), but labelling for 3NT was absent in the most inflamed nerves (h). Each data point in (b,d,f–h) represents one nerve. Mean ± SEM, one-way ANOVA, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, bar = (a,c) 100 µm or (e,g) 50 µm. Full article ">

19 pages, 4370 KiB

Open AccessArticle

Identification of Key Molecular Pathways and Associated Genes as Targets to Overcome Radiotherapy Resistance Using a Combination of Radiotherapy and Immunotherapy in Glioma Patients

by Tianqi Zhang, Qiao Zhang, Xinwei He, Yuting Lu, Andrew Shao, Xiaoqiang Sun and Yongzhao Shao

Int. J. Mol. Sci. 2024, 25(5), 3076; https://doi.org/10.3390/ijms25053076 - 6 Mar 2024

Cited by 1 | Viewed by 2085

Abstract

Recent mechanistic studies have indicated that combinations of radiotherapy (RT) plus immunotherapy (via CSF-1R inhibition) can serve as a strategy to overcome RT resistance and improve the survival of glioma mice. Given the high mortality rate for glioma, including low-grade glioma (LGG) patients, [...] Read more.

Recent mechanistic studies have indicated that combinations of radiotherapy (RT) plus immunotherapy (via CSF-1R inhibition) can serve as a strategy to overcome RT resistance and improve the survival of glioma mice. Given the high mortality rate for glioma, including low-grade glioma (LGG) patients, it is of critical importance to investigate the mechanism of the combination of RT and immunotherapy and further translate the mechanism from mouse studies to improve survival of RT-treated human glioma patients. Using the RNA-seq data from a glioma mouse study, 874 differentially expressed genes (DEGs) between the group of RT-treated mice at glioma recurrence and the group of mice with combination treatment (RT plus CSF-1R inhibition) were translated to the human genome to identify significant molecular pathways using the KEGG enrichment analysis. The enrichment analysis yields statistically significant signaling pathways, including the phosphoinositide 3-kinase (PI3K)/AKT pathway, Hippo pathway, and Notch pathway. Within each pathway, a candidate gene set was selected by Cox regression models as genetic biomarkers for resistance to RT and response to the combination of RT plus immunotherapies. Each Cox model is trained using a cohort of 295 RT-treated LGG patients from The Cancer Genome Atlas (TCGA) database and validated using a cohort of 127 RT-treated LGG patients from the Chinese Glioma Genome Atlas (CGGA) database. A four-DEG signature (ITGB8, COL9A3, TGFB2, JAG1) was identified from the significant genes within the three pathways and yielded the area under time-dependent ROC curve AUC = 0.86 for 5-year survival in the validation set, which indicates that the selected DEGs have strong prognostic value and are potential intervention targets for combination therapies. These findings may facilitate future trial designs for developing combination therapies for glioma patients. Full article

(This article belongs to the Special Issue Molecular Mechanism of Anti-cancer Drugs)

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19 pages, 2696 KiB

Open AccessReview

Pathophysiology and Main Molecular Mechanisms of Urinary Stone Formation and Recurrence

by Flavia Tamborino, Rossella Cicchetti, Marco Mascitti, Giulio Litterio, Angelo Orsini, Simone Ferretti, Martina Basconi, Antonio De Palma, Matteo Ferro, Michele Marchioni and Luigi Schips

Int. J. Mol. Sci. 2024, 25(5), 3075; https://doi.org/10.3390/ijms25053075 - 6 Mar 2024

Cited by 13 | Viewed by 8888

Abstract

Kidney stone disease (KSD) is one of the most common urological diseases. The incidence of kidney stones has increased dramatically in the last few decades. Kidney stones are mineral deposits in the calyces or the pelvis, free or attached to the renal papillae. [...] Read more.

Kidney stone disease (KSD) is one of the most common urological diseases. The incidence of kidney stones has increased dramatically in the last few decades. Kidney stones are mineral deposits in the calyces or the pelvis, free or attached to the renal papillae. They contain crystals and organic components, and they are made when urine is supersaturated with minerals. Calcium-containing stones are the most common, with calcium oxalate as the main component of most stones. However, many of these form on a calcium phosphate matrix called Randall’s plaque, which is found on the surface of the kidney papilla. The etiology is multifactorial, and the recurrence rate is as high as 50% within 5 years after the first stone onset. There is a great need for recurrence prevention that requires a better understanding of the mechanisms involved in stone formation to facilitate the development of more effective drugs. This review aims to understand the pathophysiology and the main molecular mechanisms known to date to prevent recurrences, which requires behavioral and nutritional interventions, as well as pharmacological treatments that are specific to the type of stone. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Renal Diseases)

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19 pages, 4431 KiB

Open AccessArticle

The CHD Protein Kismet Restricts the Synaptic Localization of Cell Adhesion Molecules at the Drosophila Neuromuscular Junction

by Ireland R. Smith, Emily L. Hendricks, Nina K. Latcheva, Daniel R. Marenda and Faith L. W. Liebl

Int. J. Mol. Sci. 2024, 25(5), 3074; https://doi.org/10.3390/ijms25053074 - 6 Mar 2024

Cited by 1 | Viewed by 1884

Abstract

The appropriate expression and localization of cell surface cell adhesion molecules must be tightly regulated for optimal synaptic growth and function. How neuronal plasma membrane proteins, including cell adhesion molecules, cycle between early endosomes and the plasma membrane is poorly understood. Here we [...] Read more.

The appropriate expression and localization of cell surface cell adhesion molecules must be tightly regulated for optimal synaptic growth and function. How neuronal plasma membrane proteins, including cell adhesion molecules, cycle between early endosomes and the plasma membrane is poorly understood. Here we show that the Drosophila homolog of the chromatin remodeling enzymes CHD7 and CHD8, Kismet, represses the synaptic levels of several cell adhesion molecules. Neuroligins 1 and 3 and the integrins αPS2 and βPS are increased at kismet mutant synapses but Kismet only directly regulates transcription of neuroligin 2. Kismet may therefore regulate synaptic CAMs indirectly by activating transcription of gene products that promote intracellular vesicle trafficking including endophilin B (endoB) and/or rab11. Knock down of EndoB in all tissues or neurons increases synaptic FasII while knock down of EndoB in kis mutants does not produce an additive increase in FasII. In contrast, neuronal expression of Rab11, which is deficient in kis mutants, leads to a further increase in synaptic FasII in kis mutants. These data support the hypothesis that Kis influences the synaptic localization of FasII by promoting intracellular vesicle trafficking through the early endosome. Full article

(This article belongs to the Special Issue Neural Adhesion Molecules—from Development to Adult Synaptic Plasticity)

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Kismet restricts the synaptic localization of Nlgs and regulates nlg2 transcription. High-resolution confocal micrographs of 6/7 NMJ terminal boutons showing presynaptic motor neurons (magenta, HRP) and either Nlg1 (green, (A)) or Nlg3 (green, (B)). Scale bar = 5 µm. * p < 0.05. Right panels show quantification relative to the control, w1118. (C) Nlg transcript levels in the CNS (left) and muscle (right) of kis mutants. Points represent technical replicates of two or three biological replicates. (D) Kismet enrichment within the promoter (pro) or transcription start sites (TSS) of gene regions listed. Data shown represent two ChIP-qPCR biological replicates from CNS. Full article ">Figure 2
Kismet restricts the synaptic localization of αPS2 and βPS but does not influence their transcripts. High-resolution confocal micrographs of 6/7 NMJ terminal boutons showing presynaptic motor neurons (magenta, HRP) and either αPS2 (green, (A)) or βPS (green, (B)). Scale bar = 5 µm. * p < 0.05. Right panels show quantification relative to the control, w1118. Integrin subunit transcript levels in the CNS (C) or muscle (D) of kis mutants. Points represent technical replicates of two to three biological replicates. Full article ">Figure 3
Knock down of EndoB in all tissues or neurons increases synaptic FasII. Left panels: EndoB was knocked down in all tissues (using the Actin5c-Gal4 driver), in neurons (using the elav-Gal4 driver), in postsynaptic muscle (using the 24B-Gal4 driver), or glial cells (using the repo-Gal4 driver). High-resolution confocal micrographs show terminal boutons of 6/7 NMJs labeled with HRP (magenta) and FasII (green) in animals where EndoB was knocked down in all tissues or neurons. Scale bar = 5 µm. Right histogram: quantification of synaptic FasII relative to the outcrossed control, UAS-EndoBRNAi/+. * p < 0.05. Full article ">Figure 4
Knock down of EndoB in kis mutant motor neurons or muscles does not further increase synaptic FasII. (A) EndoB was knocked down by expressing UAS-EndoBRNAi in all tissues (using the Actin5c-Gal4 driver), in motor neurons (using the D42-Gal4 driver), or in postsynaptic muscle (using the 24B-Gal4 driver) of kis mutants. High-resolution confocal micrographs depict terminal boutons of 6/7 NMJs labeled with HRP (magenta) and FasII (green). Scale bar = 5 µm. (B) Histogram of synaptic FasII relative to the outcrossed control, kisLM27/+; UAS-EndoBRNAi/+. (C) Histogram of synaptic FasII relative to the outcrossed control, kisLM27/+; UAS-AP-1σRNAi/+. Full article ">Figure 5
Synaptic FasII is increased when expressing a constitutively active Rab11 in all tissues but decreased when expressing a dominant negative Rab11 in motor neurons. (A) A constitutively active Rab11, Rab11Q70L, was expressed in all tissues (using the Actin5c-Gal4 driver), in motor neurons (using the D42-Gal4 driver), or in postsynaptic muscle (using the 24B-Gal4 driver). High-resolution confocal micrographs depict terminal boutons of 6/7 NMJs labeled with HRP (magenta) and FasII (green). Scale bar = 5 µm. Histogram of synaptic FasII relative to the outcrossed control, UAS-Rab11Q70L/+. ** p = 0.0046. (B) Histogram of synaptic FasII relative to the outcrossed control, UAS-Rab11S25N/+. * p = 0.011. (C) A dominant negative Rab11, Rab11S25N, was expressed in motor neurons (using the D42-Gal4 driver) or in postsynaptic muscle (using the 24B-Gal4 driver). High-resolution confocal micrographs depict terminal boutons of 6/7 NMJs labeled with HRP (magenta) and FasII (green). Scale bar = 5 µm. Full article ">Figure 6
Expression of Rab11Q70L and Rab11WT in neurons of kis heterozygous mutants increases locomotion and synaptic FasII levels, respectively. (A) Rab11Q70L was expressed in neurons of kisk13416 mutants using the elav-Gal4 driver. Histograms show larval crawling behavior on agar for 30 s quantified by wrMTrck and normalized to body lengths per second. Left histogram: ** p = 0.0045; *** p = 0.0001. Right histogram: *** p < 0.0001. (B) Quantification of synaptic FasII relative to the outcrossed control, UAS-Rab11WT/+. * p = 0.046. Rab11 was expressed in kisLM27/+ heterozygous mutants by expressing UAS-Rab11eYFP in neurons using the elav-Gal4 driver. (C) High-resolution confocal micrographs of 6/7 NMJ terminal boutons showing presynaptic motor neurons (magenta, HRP) and FasII (green). Scale bar = 5 µm. Full article ">

21 pages, 22219 KiB

Open AccessArticle

Scutellaria baicalensis Induces Cell Apoptosis and Elicits Mesenchymal–Epithelial Transition to Alleviate Metastatic Hepatocellular Carcinoma via Modulating HSP90β

by Tung-Ho Wu, Tung-Yi Lin, Pei-Ming Yang, Wen-Tai Li, Chau-Ting Yeh and Tai-Long Pan

Int. J. Mol. Sci. 2024, 25(5), 3073; https://doi.org/10.3390/ijms25053073 - 6 Mar 2024

Cited by 4 | Viewed by 1927

Abstract

Hepatocellular carcinoma is one of the most common malignant tumors in the world and shows strong metastatic potential. Current medicine for hepatocellular carcinoma therapy is invalid, while Scutellaria baicalensis Georgi exhibits the pharmaceutical potential to treat liver diseases and liver cancer. Herein, we [...] Read more.

Hepatocellular carcinoma is one of the most common malignant tumors in the world and shows strong metastatic potential. Current medicine for hepatocellular carcinoma therapy is invalid, while Scutellaria baicalensis Georgi exhibits the pharmaceutical potential to treat liver diseases and liver cancer. Herein, we verified the inhibitory properties and the pivotal molecules regimented by Scutellaria baicalensis on advanced hepatocellular carcinoma. At first, the viability of SK-Hep-1 cells was significantly reduced under treatment of Scutellaria baicalensis extract in a dose-dependent manner without affecting the growth of normal hepatocyte. Scutellaria baicalensis extract application could remarkably cause apoptosis of SK-Hep-1 cells through p53/cytochrome C/poly-ADP ribose polymerase cascades and arrest the cell cycle at the G1/S phase by downregulating cyclin-dependent kinases. Meanwhile, administration of Scutellaria baicalensis extract remarkably attenuated the migration capability as well as suppressed matrix metalloproteinase activity of advanced hepatocellular carcinoma cells. The proteome profiles and network analysis particularly implied that exposure to Scutellaria baicalensis extract downregulated the expression of HSP90β, and the clinical stage of hepatocellular carcinoma is also positively correlated with the HSP90β level. Combined treatment of Scutellaria baicalensis extract and HSP90β siRNAs could markedly enhance the ubiquitination activity and the degradation of vimentin to subsequently inhibit the metastatic property of SK-Hep-1 cells. Moreover, application of Scutellaria baicalensis extract and HSP90β siRNAs depleted phosphorylation of AKT, which stimulated the expression of p53 and consecutively triggered cell apoptosis. These findings suggest that HSP90β may be a prospective target for the effective therapy of advanced hepatocellular carcinoma via accelerating apoptosis of hepatocellular carcinoma cells and eliciting mesenchymal–epithelial transition with the administration of Scutellaria baicalensis extract. Full article

(This article belongs to the Special Issue Advance in Cancer Biomarker: From Molecular Mechanisms to Potential Therapy)

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20 pages, 2795 KiB

Open AccessArticle

Genome-Wide Identification, Characterization, and Expression Analysis of the BES1 Family Genes under Abiotic Stresses in Phoebe bournei

by Jingshu Li, Honggang Sun, Yanhui Wang, Dunjin Fan, Qin Zhu, Jiangyonghao Zhang, Kai Zhong, Hao Yang, Weiyin Chang and Shijiang Cao

Int. J. Mol. Sci. 2024, 25(5), 3072; https://doi.org/10.3390/ijms25053072 - 6 Mar 2024

Cited by 1 | Viewed by 1591

Abstract

The BRI1 EMS suppressor 1(BES1) transcription factor is a crucial regulator in the signaling pathway of Brassinosteroid (BR) and plays an important role in plant growth and response to abiotic stress. Although the identification and functional validation of BES1 genes have been extensively [...] Read more.

The BRI1 EMS suppressor 1(BES1) transcription factor is a crucial regulator in the signaling pathway of Brassinosteroid (BR) and plays an important role in plant growth and response to abiotic stress. Although the identification and functional validation of BES1 genes have been extensively explored in various plant species, the understanding of their role in woody plants—particularly the endangered species Phoebe bournei (Hemsl.) Yang—remains limited. In this study, we identified nine members of the BES1 gene family in the genome of P. bournei; these nine members were unevenly distributed across four chromosomes. In our further evolutionary analysis of PbBES1, we discovered that PbBES1 can be divided into three subfamilies (Class I, Class II, and Class IV) based on the evolutionary tree constructed with Arabidopsis thaliana, Oryza sativa, and Solanum lycopersicum. Each subfamily contains 2–5 PbBES1 genes. There were nine pairs of homologous BES1 genes in the synteny analysis of PbBES1 and AtBES1. Three segmental replication events and one pair of tandem duplication events were present among the PbBES1 family members. Additionally, we conducted promoter cis-acting element analysis and discovered that PbBES1 contains binding sites for plant growth and development, cell cycle regulation, and response to abiotic stress. PbBES1.2 is highly expressed in root bark, stem bark, root xylem, and stem xylem. PbBES1.3 was expressed in five tissues. Moreover, we examined the expression profiles of five representative PbBES1 genes under heat and drought stress. These experiments preliminarily verified their responsiveness and functional roles in mediating responses to abiotic stress. This study provides important clues to elucidate the functional characteristics of the BES1 gene family, and at the same time provides new insights and valuable information for the regulation of resistance in P. bournei. Full article

(This article belongs to the Special Issue Genes Function and Mechanism Identification in Plant Stress Resistance 2.0)

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19 pages, 6374 KiB

Open AccessArticle

Profiling Cell Heterogeneity and Fructose Transporter Expression in the Rat Nephron by Integrating Single-Cell and Microdissected Tubule Segment Transcriptomes

by Ronghao Zhang, Darshan Aatmaram Jadhav, Najeong Kim, Benjamin Kramer and Agustin Gonzalez-Vicente

Int. J. Mol. Sci. 2024, 25(5), 3071; https://doi.org/10.3390/ijms25053071 - 6 Mar 2024

Cited by 1 | Viewed by 1956

Abstract

Single-cell RNA sequencing (scRNAseq) is a crucial tool in kidney research. These technologies cluster cells based on transcriptome similarity, irrespective of the anatomical location and order within the nephron. Thus, a transcriptome cluster may obscure the heterogeneity of the cell population within a [...] Read more.

Single-cell RNA sequencing (scRNAseq) is a crucial tool in kidney research. These technologies cluster cells based on transcriptome similarity, irrespective of the anatomical location and order within the nephron. Thus, a transcriptome cluster may obscure the heterogeneity of the cell population within a nephron segment. Elevated dietary fructose leads to salt-sensitive hypertension, in part, through fructose reabsorption in the proximal tubule (PT). However, the organization of the four known fructose transporters in apical PTs (SGLT4, SGLT5, GLUT5, and NaGLT1) remains poorly understood. We hypothesized that cells within each subsegment of the proximal tubule exhibit complex, heterogeneous fructose transporter expression patterns. To test this hypothesis, we analyzed rat kidney transcriptomes and proteomes from publicly available scRNAseq and tubule microdissection databases. We found that microdissected PT-S1 segments consist of 81% ± 12% cells with scRNAseq-derived transcriptional characteristics of S1, whereas PT-S2 express a mixture of 18% ± 9% S1, 58% ± 8% S2, and 19% ± 5% S3 transcripts, and PT-S3 consists of 75% ± 9% S3 transcripts. The expression of all four fructose transporters was detectable in all three PT segments, but key fructose transporters SGLT5 and GLUT5 progressively increased from S1 to S3, and both were significantly upregulated in S3 vs. S1/S2 (Slc5a10: 1.9 log2FC, p < 1 × 10⁻²⁹⁹; Scl2a5: 1.4 log2FC, p < 4 × 10⁻¹⁰⁵). A similar distribution was found in human kidneys. These data suggest that S3 is the primary site of fructose reabsorption in both humans and rats. Finally, because of the multiple scRNAseq transcriptional phenotypes found in each segment, our findings also imply that anatomical labels applied to scRNAseq clusters may be misleading. Full article

(This article belongs to the Special Issue Renal Dysfunction, Uremic Compounds, and Other Factors 2.0)

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3 pages, 474 KiB

Open AccessEditorial

Molecular World Today and Tomorrow: Recent Trends in Biological Sciences 2.0

by Wajid Zaman

Int. J. Mol. Sci. 2024, 25(5), 3070; https://doi.org/10.3390/ijms25053070 - 6 Mar 2024

Viewed by 3522

Abstract

Molecular techniques have become influential instruments in biological study, transforming our comprehension of life at the cellular and genetic levels [...] Full article

(This article belongs to the Special Issue Molecular World Today and Tomorrow: Recent Trends in Biological Sciences 2.0)

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15 pages, 5957 KiB

Open AccessArticle

Toxicity of Water-Soluble D-g-PNIPAM Polymers in a Complex with Chemotherapy Drugs and Mechanism of Their Action In Vitro

by Svitlana Prylutska, Anna Grebinyk, Stanislav Ponomarenko, Defne Gövem, Vasyl Chumachenko, Nataliya Kutsevol, Mykola Petrovsky, Uwe Ritter, Marcus Frohme, Jacek Piosik and Yuriy Prylutskyy

Int. J. Mol. Sci. 2024, 25(5), 3069; https://doi.org/10.3390/ijms25053069 - 6 Mar 2024

Cited by 1 | Viewed by 1442

Abstract

The application of a biocompatible polymer nanocarrier can provide target delivery to tumor tissues, improved pharmacokinetics, controlled drug release, etc. Therefore, the proposed strategy was to use the water-soluble star-like copolymers with a Dextran core and Poly(N-isopropylacrylamide) grafts (D-g-PNIPAM) for conjugation with the [...] Read more.

The application of a biocompatible polymer nanocarrier can provide target delivery to tumor tissues, improved pharmacokinetics, controlled drug release, etc. Therefore, the proposed strategy was to use the water-soluble star-like copolymers with a Dextran core and Poly(N-isopropylacrylamide) grafts (D-g-PNIPAM) for conjugation with the widely used chemotherapy drugs in oncology–Cisplatin (Cis-Pt) and Doxorubicin (Dox). The molecular characteristics of the copolymer were received using size-exclusion chromatography. The physicochemical characterization of the D-g-PNIPAM-Cis-Pt (or Dox) nanosystem was conducted using dynamic light scattering and FTIR spectroscopy. Using traditional biochemical methods, a comparative analysis of the enhancement of the cytotoxic effect of free Cis-Pt and Dox in combination with D-g-PNIPAM copolymers was performed in cancer cells of the Lewis lung carcinoma line, which are both sensitive and resistant to Dox; in addition, the mechanism of their action in vitro was evaluated. Full article

(This article belongs to the Special Issue Development, Characterization and Applications of Novel Polymeric Materials and Composites)

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3 pages, 173 KiB

Open AccessEditorial

Molecular World Today and Tomorrow: Recent Trends in Biological Sciences

by Wajid Zaman

Int. J. Mol. Sci. 2024, 25(5), 3068; https://doi.org/10.3390/ijms25053068 - 6 Mar 2024

Viewed by 1664

Abstract

Various molecular techniques based on omics (transcriptomics, proteomics, genomics) and phylogenetics have been applied in the field of biological sciences [...] Full article

(This article belongs to the Special Issue Molecular World Today and Tomorrow: Recent Trends in Biological Sciences)

12 pages, 435 KiB

Open AccessArticle

OPRM1 Gene Polymorphism in Women with Alcohol Use Disorder

by Agnieszka Boroń, Aleksandra Suchanecka, Krzysztof Chmielowiec, Małgorzata Śmiarowska, Jolanta Chmielowiec, Aleksandra Strońska-Pluta, Remigiusz Recław and Anna Grzywacz

Int. J. Mol. Sci. 2024, 25(5), 3067; https://doi.org/10.3390/ijms25053067 - 6 Mar 2024

Viewed by 1290

Abstract

The main aims of the present study were to explore the relationship of the OPRM1 gene rs1074287 polymorphism in alcohol-dependent women with their personality traits and to try to find out whether any specific features may influence alcohol cravings and be a prognostic [...] Read more.

The main aims of the present study were to explore the relationship of the OPRM1 gene rs1074287 polymorphism in alcohol-dependent women with their personality traits and to try to find out whether any specific features may influence alcohol cravings and be a prognostic for alcohol dependency and treatment in AUD women. Our study found a notable correlation between openness and the interaction of the ORIM1 gene and AUD. The alcohol use disorder subjects with genotype AG showed a higher level of openness compared to the control group with genotypes AG (p = 0.0001) and AA (p = 0.0125). The alcohol use disorder subjects with the AA genotype displayed higher levels of openness than the control group with genotype AG (p = 0.0271). However, the alcohol use disorder subjects with the AA genotype displayed lower levels of openness than the control group with genotype GG (p = 0.0212). Our study indicates that openness as a personality trait is correlated with the OPRM1 gene rs1074287 polymorphism in alcohol-dependent women. These are the first data and results exploring such a relationship between opioid and alcohol pathways and the mental construction of AUD women. Personality traits such as openness to experience and neuroticism might play major roles in the addiction mechanism, especially in genetically predisposed females, independent of the reward system involved in the emotional disturbances that coexist with anxiety and depression. Full article

(This article belongs to the Special Issue Recent Progress of Opioid Research, 2nd Edition)

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14 pages, 2538 KiB

Open AccessArticle

Combination Treatment with EGFR Inhibitor and Doxorubicin Synergistically Inhibits Proliferation of MCF-7 Cells and MDA-MB-231 Triple-Negative Breast Cancer Cells In Vitro

by Beynon Abrahams, Anthonie Gerber and Donavon Charles Hiss

Int. J. Mol. Sci. 2024, 25(5), 3066; https://doi.org/10.3390/ijms25053066 - 6 Mar 2024

Cited by 1 | Viewed by 2781

Abstract

The role of the epidermal growth factor receptor (EGFR) in tumor progression and survival is often underplayed. Its expression and/or dysregulation is associated with disease advancement and poor patient outcome as well as drug resistance in breast cancer. EGFR is often overexpressed in [...] Read more.

The role of the epidermal growth factor receptor (EGFR) in tumor progression and survival is often underplayed. Its expression and/or dysregulation is associated with disease advancement and poor patient outcome as well as drug resistance in breast cancer. EGFR is often overexpressed in breast cancer and particularly triple-negative breast cancer (TNBC), which currently lacks molecular targets. We examined the synergistic potential of an EGFR inhibitor (EGFRi) in combination with doxorubicin (Dox) in estrogen-positive (ER+) MCF-7 and MDA-MB-231 TNBC cell lines. The exposure of MDA-MB-231 and MCF-7 to EGFRi produced an IC_50s of 6.03 µM and 3.96 µM, respectively. Dox induced MDA-MB-231 (IC₅₀ 9.67 µM) and MCF-7 (IC₅₀ 1.4 µM) cytotoxicity. Combinations of EGFRi-Dox significantly reduced the IC₅₀ in MCF-7 (0.46 µM) and MBA-MB 231 (0.01 µM). Synergistic drug interactions in both cell lines were confirmed using the Bliss independence model. Pro-apoptotic Caspase-3/7 activation occurred in MCF-7 at 0.1–10 µM of EGFRi and Dox single treatments, whilst 1 μM Dox yielded a more potent effect on MDA-MB-231. EGFRi and Dox individually and in combination downregulated the EGFR gene expression in MCF-7 and MDA-MB-231 (p < 0.001). This study demonstrates EGFRi’s potential for eliciting synergistic interactions with Dox, causing enhanced growth inhibition, apoptosis induction, and downregulation of EGFR in both cell lines. Full article

(This article belongs to the Special Issue Recent Advances in Breast Cancer Research, 2nd Edition)

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Dose-response curves of the effects of 24, 48 and 72 h exposure of MCF-7 (A) and MDA-MB-231 cells (B) to EGFRi and Dox, individually and in combination. IC50 estimates and corresponding 95% confidence intervals (95% CI) for EGFRi and Dox and combinations were determined via non-linear regression analyses of dose-response data using the variable slope model of GraphPad Prism v10. Full article ">Figure 2
(A–F): Bliss independence response surface reference model for the dual agent combination effects of 24 h–72 h treatment of MCF-7 (A–C) and MDA-MB-231 (D–F) cells with EGFRi and Dox. Each figure (A–F) is represented as follows—Top panel left: Bliss independence mapping of the synergy levels on the experimental combination dose-response surface | Top Middle pannel: Bliss synergy and antagonism levels visualized as a surface | Top panel right: Contour map of isoboles (iso-effect lines) of Bliss synergy and/or antagonism | Bottom panel left: Bliss model reference of dose-response surface | Bottom panel middle: Bliss synergy and antagonism matrix | Bottom panel right: Bliss model reference of dose-response contour map. Full article ">Figure 3
(A–D): Caspase-3/7 activity following 24 h and 48 h exposure of MCF-7 and MDA-MB-231 cells to EGFRi and Dox. Data were analyzed via one-way ANOVA. All multiple comparisons were performed according to Dunnet’s method, and the overall significance level was set at p < 0.05. Significant differences are indicated by (ns = non-significant, * indicates p value ≤ 0.05, ** indicates p value < 0.01, *** indicates p value < 0.001, **** indicates p value < 0.0001). Values are means ± SEM (n = 3). Full article ">Figure 4
(A,B): Effects of 48 h pairwise EGFRi and Dox treatments on the expression levels of the EGFR gene in MCF-7 (A) and MDA-MB-231 TNBC (B) breast carcinoma cells. All data are representative of at least three independent experiments and are presented as means ± SEM (n = 3) for cells treated with single agent drugs and combination as indicated. One-way ANOVA analysis was also used to determine significant differences among treatments compared with their respective untreated controls), followed by a Dunnett’s post hoc test to compare all pairs of data sets. Data were considered statistically significant when p < 0.05. (ns = non-significant, * indicates p value ≤ 0.05, *** indicates p value < 0.001, **** indicates p value < 0.0001) Full article ">

13 pages, 1335 KiB

Open AccessArticle

Early Increase in Blood–Brain Barrier Permeability in a Murine Model Exposed to Fifteen Days of Intermittent Hypoxia

by Frederic Roche, Anne Briançon-Marjollet, Maurice Dematteis, Marie Baldazza, Brigitte Gonthier, Frederique Bertholon, Nathalie Perek and Jean-Louis Pépin

Int. J. Mol. Sci. 2024, 25(5), 3065; https://doi.org/10.3390/ijms25053065 - 6 Mar 2024

Cited by 1 | Viewed by 1280

Abstract

Obstructive sleep apnea (OSA) is characterized by intermittent repeated episodes of hypoxia–reoxygenation. OSA is associated with cerebrovascular consequences. An enhanced blood–brain barrier (BBB) permeability has been proposed as a marker of those disorders. We studied in mice the effects of 1 day and [...] Read more.

Obstructive sleep apnea (OSA) is characterized by intermittent repeated episodes of hypoxia–reoxygenation. OSA is associated with cerebrovascular consequences. An enhanced blood–brain barrier (BBB) permeability has been proposed as a marker of those disorders. We studied in mice the effects of 1 day and 15 days intermittent hypoxia (IH) exposure on BBB function. We focused on the dorsal part of the hippocampus and attempted to identify the molecular mechanisms by combining in vivo BBB permeability (Evans blue tests) and mRNA expression of several junction proteins (zona occludens (ZO-1,2,3), VE-cadherin, claudins (1,5,12), cingulin) and of aquaporins (1,4,9) on hippocampal brain tissues. After 15 days of IH exposure we observed an increase in BBB permeability, associated with increased mRNA expressions of claudins 1 and 12, aquaporins 1 and 9. IH seemed to increase early for claudin-1 mRNA expression as it doubled with 1 day of exposure and returned near to its base level after 15 days. Claudin-1 overexpression may represent an immediate response to IH exposure. Then, after 15 days of exposure, an increase in functional BBB permeability was associated with enhanced expression of aquaporin. These BBB alterations are possibly associated with a vasogenic oedema that may affect brain functions and accelerate neurodegenerative processes. Full article

(This article belongs to the Collection Feature Papers in Molecular Pathology, Diagnostics, and Therapeutics)

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Blue Evans experiments. Increase in blood-brain barrier permeability in mice exposed to 15 days of intermittent hypoxia (IH) compared to control mice exposed to normoxia (NO) and to those exposed to 1 day of IH. The results are expressed as the concentration of Evans blue in the ground brain tissue ([EB] µg/mg of tissue). n = 12 in each group, Student t-test ** p < 0.01 IH/15 d vs. NO/15 d; ### p < 0.001 IH/15d vs. IH/1 d. Full article ">Figure 2
Aquaporins. Expression of mRNAs of aquaporins (Aqp) 1 (A), 4 (B) and 9 (C) in the dorsal hippocampus of mice exposed to 1 d and 15 d of intermittent hypoxia (IH), compared to control mice exposed to normoxia (NO). The results are expressed in arbitrary units (a.u.). n = 6 in each group, mean ± SEM (* p < 0.05 IH vs. NO). All values were normalized to Normoxia 1 d. Full article ">Figure 3
Claudin Occludin VE cadh. Claudins 1, 5 and 12 (Cldn1, Cldn5 and Cldn12), occludin (Ocln) and VE-cadherin (Cdh5) mRNA expression in the dorsal hippocampus of mice exposed to 1 d and 15 d of intermittent hypoxia (IH) compared to control mice exposed to normoxia (NO). The results are expressed in arbitrary units (a.u.). n = 6 in each group, mean ± SEM * p < 0.05 IH/15 d vs. NO/15 d; *** p < 0.001 IH/15 d vs. NO/15 d; # p < 0.05 IH/15 d vs. IH/1 d. Full article ">Figure 4
ZO. Zonula Occludens (ZO-1, ZO-2, ZO-3) and Cingulin mRNA expressions in the dorsal hippocampus of mice exposed to 1 d and 15 d of intermittent hypoxia (IH) compared to control mice exposed to normoxia (NO). The results are expressed in arbitrary units (a.u.). n = 6 in each group, mean ± SEM. Full article ">Figure 5
Flowchart of the experiments. (NO: normoxia, IH: intermittent hypoxia, BBB: blood brain, barrier). Full article ">

14 pages, 2133 KiB

Open AccessArticle

The Potential of Twendee X^® as a Safe Antioxidant Treatment for Systemic Sclerosis

by Fukka You, Carole Nicco, Yoshiaki Harakawa, Toshikazu Yoshikawa and Haruhiko Inufusa

Int. J. Mol. Sci. 2024, 25(5), 3064; https://doi.org/10.3390/ijms25053064 - 6 Mar 2024

Viewed by 2101

Abstract

Systemic sclerosis (SSc) is an autoimmune disease characterized by systemic skin hardening, which combines Raynaud’s phenomenon and other vascular disorders, skin and internal organ fibrosis, immune disorders, and a variety of other abnormalities. Symptoms vary widely among individuals, and personalized treatment is sought [...] Read more.

Systemic sclerosis (SSc) is an autoimmune disease characterized by systemic skin hardening, which combines Raynaud’s phenomenon and other vascular disorders, skin and internal organ fibrosis, immune disorders, and a variety of other abnormalities. Symptoms vary widely among individuals, and personalized treatment is sought for each patient. Since there is no fundamental cure for SSc, it is designated as an intractable disease with patients receiving government subsidies for medical expenses in Japan. Oxidative stress (OS) has been reported to play an important role in the cause and symptoms of SSc. HOCl-induced SSc mouse models are known to exhibit skin and visceral fibrosis, vascular damage, and autoimmune-like symptoms observed in human SSc. The antioxidant combination Twendee X^® (TwX) is a dietary supplement consisting of vitamins, amino acids, and CoQ10. TwX has been proven to prevent dementia in humans with mild cognitive impairment and significantly improve cognitive impairment in an Alzheimer’s disease mouse model by regulating OS through a strong antioxidant capacity that cannot be achieved with a single antioxidant ingredient. We evaluated the effectiveness of TwX on various symptoms of HOCl-induced SSc mice. TwX-treated HOCl-induced SSc mice showed significantly reduced lung and skin fibrosis compared to untreated HOCl-induced SSc mice. TwX also significantly reduced highly oxidized protein products (AOPP) in serum and suppressed Col-1 gene expression and activation of B cells involved in autoimmunity. These findings suggest that TwX has the potential to be a new antioxidant treatment for SSc without side effects. Full article

(This article belongs to the Special Issue Oxidative Stress and Antioxidants in Human Diseases)

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Effect of Twendee X® on sera redox status. Concentrations of advanced oxidation protein products (AOPP) in the sera from mice (mM of chloramine T equivalent). Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for statistical analysis). * p < 0.05. Full article ">Figure 2
Effect of Twendee X® on fibrosis parameters. (A) Change in skin fold thickness in millimeters from day 1 to day 36, measured weekly (n = 10). **: p < 0.01, ***: p < 0.001 (PBS vs. HOCl), †: p < 0.05 (HOCl vs. HOCl + TwX). (B) Collagen type I levels in skin (mg/punch biopsy) and (C) in lung (mg/lobe biopsy) were evaluated by hydroxyproline dosage. Each box represents mean ± SD from n = 10 individual mice. * p < 0.05; ** p < 0.01; *** p < 0.001. (D) Relative mRNA level of Collagen-1. Results were standardized by GAPDH. Each box represents mean ± SD from n = 10 individual mice. ** p < 0.01; *** p < 0.001. (E) Representative H&E dyed skin sections of 6 μm, showing enhanced fibrosis in mice. Photographs were taken with a Nikon Eclipse 80i microscope (Nikon Instruments, Inc., Melville, NY, USA). Original magnification ×20. The scale represents 500 μm. All data are expressed as the mean ± SD. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis. Full article ">Figure 3
Effect of Twendee X® on fibroblast differentiation. (A) α-SMA and (B) H-Ras in skin were evaluated by Western blot. Results were normalized to tubulin. Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis. * p < 0.05. Full article ">Figure 4
Effect of Twendee X® on Collagen and cytokines expression. Relative mRNA level of (A) Il-6, (B) Il-33, and (C) Il-17 mRNA levels in skin evaluated by qRT-PCR. Results were normalized to GAPDH. Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis. No significant difference was observed. Full article ">Figure 5
Effects of Twendee X® on B and T CD4+ cells activation assessed by flow cytometry in SSc mice. The side-scatter (SSC) and the forward-scatter channels (FSC) were used to gate the leukocytes. A total of 100,000 events were accumulated for each sample. Doublets were excluded with FSC-A and FSC-H channels. (A,B) Activation of B220+ cells assessed by CD40 expression (A) and MHC II expression (B). (C,D) Activation of CD4+ T cells assessed by CD69 (C) and CD44 (D) expression. Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001. Full article ">Figure 6
Flow cytometric analysis of the effects of Twendee X® on splenic macrophage phenotype in SSc mice. Splenic macrophages were gated on CD11b- and F4/80-positive cells among CD45-positive. A total of 100,000 events were accumulated for each sample. Doublets were excluded with FSC-A and FSC-H channels. (A) Percentage of splenic macrophages among total splenic cells. Data represent the percentage and SD. (B–D) Flow cytometric analysis of CD86, Ly6C, and CD206 expression on splenic macrophages. (E) Ratio of M1/M2 macrophages’ frequency. M1 macrophages were defined as B220-F4/80+CD11b+Ly6CHighCD206- and M2 macrophages as B220-F4/80+CD11b+Ly6cLowCD206+. Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001. Full article ">

19 pages, 2537 KiB

Open AccessArticle

Evaluating Temperature Effects on Bluetongue Virus Serotype 10 and 17 Coinfection in Culicoides sonorensis

by Molly Carpenter, Jennifer Kopanke, Justin Lee, Case Rodgers, Kirsten Reed, Tyler J. Sherman, Barbara Graham, Lee W. Cohnstaedt, William C. Wilson, Mark Stenglein and Christie Mayo

Int. J. Mol. Sci. 2024, 25(5), 3063; https://doi.org/10.3390/ijms25053063 - 6 Mar 2024

Cited by 1 | Viewed by 1384

Abstract

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an [...] Read more.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host–pathogen interactions with implications for transmission and evolution. Full article

(This article belongs to the Special Issue Host-Pathogen Interaction 5.0)

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Temperature and BTV infection status impacts C. sonorensis lifespan. Plots include all groups from both survival assay trials (experiment 2 and 3) with parametric survival regression models fit to each temperature separately (20 °C, 25 °C or 30 °C) with default Weibull distribution and robust sandwich error calculation. The plots consists of smooth lines for the survival regression curves of each infection group (BTV-10 in purple, BTV-17 in blue, BTV 10:BTV17 coinfected in green, and negative control in yellow) with the y-axis representing probability of survival and the x-axis representing days post infection. A Kaplan–Meier step curve is included for each group with a separate step curve for the repeated coinfected and negative control groups from experiment 3. Results are shown in associated tables that provide the mean survival days post infection, standard error (SE), and 95% confidence interval (CI) for individual curves and survival odds (Hazard ratio), SE, statistic (t.ratio), and p-value for comparisons between curves. Full article ">Figure 2
BTV Detected in all pools of the coinfected group. Pool size = 5 C. sonorensis. BTV detection was determined by pan BTV qRT-PCR. Black indicates BTV detected and gray indicates BTV not detected. Full article ">Figure 3
BTV virogenesis is similar across infection groups. Normalization of BTV Ct values calculated for each sample was accomplished using the ∆Ct method based on mean Ct values for BTV and cox1 (i.e., Pan BTV Ct−cox1 Ct). Due to the decreased survival of C. sonorensis at higher temperatures, data collection was limited for the 30 °C collection group. A linear model was fitted to each temperature (20 °C, 25 °C or 30 °C) separately with robust linear models applied to account for outliers. Some pools did not have detectable BTV and are represented by imputed values using multivariate imputation by chained equation with a Bayesian linear regression method and represented by triangles. Circles indicates data points. A third-degree polynomial was fit to Ct values for the 20 °C plot and second-degree polynomials were fit to Ct values for the 25 °C and 30 °C plots. Solid lines indicate the model curve, the shaded region represents the standard error, and the dash line is a curve based on the mean of the data at each day post infection. Pairwise comparisons were performed on the linear portions of the curves with a Tukey HSD adjustment. Estimates of the differences in linear trends, standard error, z-ratio statistic, and p-value are given for all comparisons. Full article ">Figure 4
BTV-17 is the predominant plaque genotype. Plaque genotypes that were sequenced from coinfected C. sonorensis are depicted by heat maps. (A) Represents plaque genotypes from pools that had qRT-PCR detection of only BTV-17 serotype. (B) Represents plaque genotypes from pools that had qRT-PCR detection of both BTV-17 and BTV10 serotypes. Each column represents an individual plaque with pools separated by white margins and labeled by dpi. Rows represents the ten BTV segments. Blue indicates that 100% of the sequencing reads for that segment aligned to BTV-10. Orange indicates that 100% of the sequencing reads for that segment aligned to BTV-17. A mixed color gradient indicates that the segment contains reads from both parental strains. X indicates insufficient reads. Full article ">Figure 5
Positive controls of BTV-10, BTV-17, and BTV-10:BTV-17 (at 50:50 ratio) were library prepped and sequenced with genotypes depicted by heatmaps. Rows represents the ten BTV segments. Blue indicates that 100% of the sequencing reads for that segment aligned to BTV-10. Orange indicates that 100% of the sequencing reads for that segment aligned to BTV-17. A mixed color gradient indicates that the segment contains reads from both parental strains. Full article ">

17 pages, 3111 KiB

Open AccessArticle

Proteomic Analysis Highlights the Impact of the Sphingolipid Metabolizing Enzyme β-Galactosylceramidase on Mitochondrial Plasticity in Human Melanoma

by Davide Capoferri, Luca Mignani, Marcello Manfredi and Marco Presta

Int. J. Mol. Sci. 2024, 25(5), 3062; https://doi.org/10.3390/ijms25053062 - 6 Mar 2024

Cited by 1 | Viewed by 1335

Abstract

Mitochondrial plasticity, marked by a dynamism between glycolysis and oxidative phosphorylation due to adaptation to genetic and microenvironmental alterations, represents a characteristic feature of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations [...] Read more.

Mitochondrial plasticity, marked by a dynamism between glycolysis and oxidative phosphorylation due to adaptation to genetic and microenvironmental alterations, represents a characteristic feature of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations have shown that the lysosomal sphingolipid-metabolizing enzyme β-galactosylceramidase (GALC) exerts pro-oncogenic functions in melanoma. Here, mining the cBioPortal for a Cancer Genomics data base identified the top 200 nuclear-encoded genes whose expression is negatively correlated with GALC expression in human melanoma. Their categorization indicated a significant enrichment in Gene Ontology terms and KEGG pathways related to mitochondrial proteins and function. In parallel, proteomic analysis by LC-MS/MS of two GALC overexpressing human melanoma cell lines identified 98 downregulated proteins when compared to control mock cells. Such downregulation was confirmed at a transcriptional level by a Gene Set Enrichment Analysis of the genome-wide expression profiling data obtained from the same cells. Among the GALC downregulated proteins, we identified a cluster of 42 proteins significantly associated with GO and KEGG categorizations related to mitochondrion and energetic metabolism. Overall, our data indicate that changes in GALC expression may exert a significant impact on mitochondrial plasticity in human melanoma cells. Full article

(This article belongs to the Special Issue New Advances in Proteomics in Disease)

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Full article ">Figure 1
Gene Ontology and KEGG categorization of the top 200 genes whose expression levels are negatively correlated with GALC expression in human melanoma specimens following data mining on the cBioPortal for Cancer Genomics platform. Full article ">Figure 2
STRING analysis of the top 200 genes whose expression levels are negatively correlated with GALC expression in human melanoma following data mining on the cBioPortal for Cancer Genomics platform. The two clusters are defined by the GO terms “Oxidative phosphorylation” (in red) and “Structural constituent of ribosome” (in green). Full article ">Figure 3
GO categorization of the genes negatively correlated to GALC expression in human cancers. GO categorization was performed on the top 200 genes whose expression levels are negatively correlated with GALC expression in tumor cell lines (Cancer Cell Line Encyclopedia) and human tumors (TCGA, Firehose Legacy) following data mining on the cBioPortal for Cancer Genomics platform. Arrows highlight enriched GO Cellular Component terms related to mitochondrial structure and function. Full article ">Figure 4
GSEA of GEP data from GALC-overexpressing melanoma cells. The expression levels of the gene encoding for the 98 proteins downregulated in A258-upGALC and A375-upGALC vs. mock cells were calculated from GEP data by GSEA. Full article ">Figure 5
STRING analysis of the proteins downregulated in upGALC melanoma cells when compared to mock cells. The two clusters are defined by the GO terms “TCA cycle” and “Mitochondrion” (in red) and “Ribonucleoprotein complex biogenesis” and “RNA binding” (in green). Full article ">

18 pages, 4241 KiB

Open AccessArticle

Mitotic Spindle Positioning (MISP) Facilitates Colorectal Cancer Progression by Forming a Complex with Opa Interacting Protein 5 (OIP5) and Activating the JAK2-STAT3 Signaling Pathway

by Koki Hiura, Masaki Watanabe, Naoki Hirose, Kenta Nakano, Tadashi Okamura, Hayato Sasaki and Nobuya Sasaki

Int. J. Mol. Sci. 2024, 25(5), 3061; https://doi.org/10.3390/ijms25053061 - 6 Mar 2024

Cited by 2 | Viewed by 1574

Abstract

Patients with inflammatory bowel disease (IBD) who experience long-term chronic inflammation of the colon are at an increased risk of developing colorectal cancer (CRC). Mitotic spindle positioning (MISP), an actin-binding protein, plays a role in mitosis and spindle positioning. MISP is found on [...] Read more.

Patients with inflammatory bowel disease (IBD) who experience long-term chronic inflammation of the colon are at an increased risk of developing colorectal cancer (CRC). Mitotic spindle positioning (MISP), an actin-binding protein, plays a role in mitosis and spindle positioning. MISP is found on the apical membrane of the intestinal mucosa and helps stabilize and elongate microvilli, offering protection against colitis. This study explored the role of MISP in colorectal tumorigenesis using a database, human CRC cells, and a mouse model for colitis-induced colorectal tumors triggered by azoxymethane (AOM)/dextran sodium sulfate (DSS) treatment. We found that MISP was highly expressed in colon cancer patient tissues and that reduced MISP expression inhibited cell proliferation. Notably, MISP-deficient mice showed reduced colon tumor formation in the AOM/DSS-induced colitis model. Furthermore, MISP was found to form a complex with Opa interacting protein 5 (OIP5) in the cytoplasm, influencing the expression of OIP5 in a unidirectional manner. We also observed that MISP increased the levels of phosphorylated STAT3 in the JAK2-STAT3 signaling pathway, which is linked to tumorigenesis. These findings indicate that MISP could be a risk factor for CRC, and targeting MISP might provide insights into the mechanisms of colitis-induced colorectal tumorigenesis. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Inhibition of Colorectal Cancer)

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19 pages, 1513 KiB

Open AccessReview

Oxy-Inflammation in Humans during Underwater Activities

by Alessandra Vezzoli, Simona Mrakic-Sposta, Andrea Brizzolari, Costantino Balestra, Enrico Maria Camporesi and Gerardo Bosco

Int. J. Mol. Sci. 2024, 25(5), 3060; https://doi.org/10.3390/ijms25053060 - 6 Mar 2024

Cited by 3 | Viewed by 3313

Abstract

Underwater activities are characterized by an imbalance between reactive oxygen/nitrogen species (RONS) and antioxidant mechanisms, which can be associated with an inflammatory response, depending on O₂ availability. This review explores the oxidative stress mechanisms and related inflammation status (Oxy-Inflammation) in underwater activities [...] Read more.

Underwater activities are characterized by an imbalance between reactive oxygen/nitrogen species (RONS) and antioxidant mechanisms, which can be associated with an inflammatory response, depending on O₂ availability. This review explores the oxidative stress mechanisms and related inflammation status (Oxy-Inflammation) in underwater activities such as breath-hold (BH) diving, Self-Contained Underwater Breathing Apparatus (SCUBA) and Closed-Circuit Rebreather (CCR) diving, and saturation diving. Divers are exposed to hypoxic and hyperoxic conditions, amplified by environmental conditions, hyperbaric pressure, cold water, different types of breathing gases, and air/non-air mixtures. The “diving response”, including physiological adaptation, cardiovascular stress, increased arterial blood pressure, peripheral vasoconstriction, altered blood gas values, and risk of bubble formation during decompression, are reported. Full article

(This article belongs to the Special Issue Oxygen Variations, 2nd Edition)

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18 pages, 5884 KiB

Open AccessArticle

Computational Modeling of the Interactions between DPP IV and Hemorphins

by Priya Antony, Bincy Baby, Amie Jobe and Ranjit Vijayan

Int. J. Mol. Sci. 2024, 25(5), 3059; https://doi.org/10.3390/ijms25053059 - 6 Mar 2024

Cited by 2 | Viewed by 1361

Abstract

Type 2 diabetes is a chronic metabolic disorder characterized by high blood glucose levels due to either insufficient insulin production or ineffective utilization of insulin by the body. The enzyme dipeptidyl peptidase IV (DPP IV) plays a crucial role in degrading incretins that [...] Read more.

Type 2 diabetes is a chronic metabolic disorder characterized by high blood glucose levels due to either insufficient insulin production or ineffective utilization of insulin by the body. The enzyme dipeptidyl peptidase IV (DPP IV) plays a crucial role in degrading incretins that stimulate insulin secretion. Therefore, the inhibition of DPP IV is an established approach for the treatment of diabetes. Hemorphins are a class of short endogenous bioactive peptides produced by the enzymatic degradation of hemoglobin chains. Numerous in vitro and in vivo physiological effects of hemorphins, including DPP IV inhibiting activity, have been documented in different systems and tissues. However, the underlying molecular binding behavior of these peptides with DPP IV remains unknown. Here, computational approaches such as protein–peptide molecular docking and extensive molecular dynamics (MD) simulations were employed to identify the binding pose and stability of peptides in the active site of DPP IV. Findings indicate that hemorphins lacking the hydrophobic residues LVV and VV at the N terminal region strongly bind to the conserved residues in the active site of DPP IV. Furthermore, interactions with these critical residues were sustained throughout the duration of multiple 500 ns MD simulations. Notably, hemorphin 7 showed higher binding affinity and sustained interactions by binding to S1 and S2 pockets of DPP IV. Full article

(This article belongs to the Special Issue Advances in Molecular Modeling, Docking and Simulations of Protein Structure)

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Figure 1
Structure of human DPP IV (PDB ID:1WCY) [<a href="#B6-ijms-25-03059" class="html-bibr">6</a>]. (A) Structure of DPP IV monomer with bond ligand diprotin A. The α/β hydrolase domain is brown, the β-propeller domain is lilac, and the ligand is green. The interface between these two domains forms a central cavity within the black box. (B) Active site residues of the protein. Full article ">Figure 2
Interactions between DPP IV and hemorphins obtained from molecular docking. (A) DPP IV-H7, (B) DPP IV-H6, (C) DPP IV-H5, (D) DPP IV-H4. Full article ">Figure 3
Interactions between DPP IV and camel hemorphins obtained from molecular docking. (A) DPP IV-Camel H7, (B) DPP IV-Camel H6, (C) DPP IV-Camel H5, (D) DPP IV-Diprotin A. Full article ">Figure 4
Root mean square deviation (RMSD) of protein Cα atoms obtained from 500 ns simulations of (A) DPP IV-H7, (B) DPP IV-Camel H7, (C) DPP IV-H6, (D) DPP IV-Camel H6, (E) DPP IV-H5, (F) DPP IV-Camel H5, (G) DPP IV-H4, (H) DPP IV-Diprotin A. Full article ">Figure 5
Root mean square fluctuation (RMSF) of protein residues obtained from 500 ns simulations. (A) DPP IV-H7, (B) DPP IV-Camel H7, (C) DPP IV-H6, (D) DPP IV-Camel H6, (E) DPP IV-H5 (F) DPP IV-Camel H5, (G) DPP IV-H4, (H) DPP IV-Diprotin A. Full article ">Figure 6
Percentage of simulation time during which intermolecular polar and hydrophobic contacts were retained between DPP IV and non-camel hemorphin peptides in the 500 ns systems. (A) Polar interaction of H7 with DPP IV, (B) polar interaction of H6 with DPP IV, (C) polar interaction of H5 with DPP IV, (D) polar interaction of H4 with DPP IV, (E) hydrophobic interaction of H7 with DPP IV, (F) hydrophobic interaction of H6 with DPP IV, (G) hydrophobic interaction of H5 with DPP IV, (H) hydrophobic interaction of H4 with DPP IV. Full article ">Figure 7
Percentage of simulation time during which intermolecular polar and hydrophobic contacts were retained between DPP IV and camel hemorphin peptides in the 500 ns systems. (A) Polar interaction of Camel H7 with DPP IV, (B) polar interaction of Camel H6 with DPP IV, (C) polar interaction of Camel H5 with DPP IV, (D) hydrophobic interaction of Camel H7 with DPP IV, (E) hydrophobic interaction of Camel H6 with DPP IV, (F) hydrophobic interaction of Camel H5 with DPP IV. Full article ">

15 pages, 2984 KiB

Open AccessArticle

MicroRNA-133b Dysregulation in a Mouse Model of Cervical Contusion Injury

by James Young Ho Yu, Thomas C. Chen and Camelia A. Danilov

Int. J. Mol. Sci. 2024, 25(5), 3058; https://doi.org/10.3390/ijms25053058 - 6 Mar 2024

Viewed by 1024

Abstract

Our previous research studies have demonstrated the role of microRNA133b (miR133b) in healing the contused spinal cord when administered either intranasally or intravenously 24 h following an injury. While our data showed beneficial effects of exogenous miR133b delivered within hours of a spinal [...] Read more.

Our previous research studies have demonstrated the role of microRNA133b (miR133b) in healing the contused spinal cord when administered either intranasally or intravenously 24 h following an injury. While our data showed beneficial effects of exogenous miR133b delivered within hours of a spinal cord injury (SCI), the kinetics of endogenous miR133b levels in the contused spinal cord and rostral/caudal segments of the injury were not fully investigated. In this study, we examined the miR133b dysregulation in a mouse model of moderate unilateral contusion injury at the fifth cervical (C5) level. Between 30 min and 7 days post-injury, mice were euthanized and tissues were collected from different areas of the spinal cord, ipsilateral and contralateral prefrontal motor cortices, and off-targets such as lung and spleen. The endogenous level of miR133b was determined by RT-qPCR. We found that after SCI, (a) most changes in miR133b level were restricted to the injured area with very limited alterations in the rostral and caudal parts relative to the injury site, (b) acute changes in the endogenous levels were predominantly specific to the lesion site with delayed miR133b changes in the motor cortex, and (c) ipsilateral and contralateral hemispheres responded differently to unilateral SCI. Our results suggest that the therapeutic window for exogenous miR133b therapy begins earlier than 24 h post-injury and potentially lasts longer than 7 days. Full article

(This article belongs to the Special Issue Comprehensive Insights into Molecular Mechanisms and Pathophysiology of Spinal Cord Injury)

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Figure 1
Schematic diagram of the research study design. Mice received unilateral spinal cord injury at the cervical 5th level. At different time points between 0.5 h and 7 days post-injury, mice were euthanized followed by the tissue harvesting as follows: spinal cord (lesion area, medulla and thoracic segments), brain (ipsilateral and contralateral hemispheres), lung and spleen. The level of miR133b in different tissue samples was determined by RT-qPCR. H = hours and d = days post-injury. Full article ">Figure 2
The effect of spinal cord injury on the endogenous level of miR133b at the site of injury. Between 0.5 h and 7 days after receiving a cervical contusion injury, mice were euthanized and spinal cords that included the lesion scar were collected and processed for microRNA isolation. The level of miR133b was assessed by RT-qPCR analysis. One-way ANOVA was used for analysis, with Tukey’s post hoc test for multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 when compared to 0.5 h injury group; # p < 0.05 when compared to 1.5 h injury group; & p < 0.0004 when compared to uninjured group. The circles, triangles, squares, and stars in the graph represent the individual mouse miR level per group. The red line shows the mean with SEM. Full article ">Figure 3
The effect of spinal cord injury on the endogenous level of miR133b in the rostral and caudal parts of the spinal cord. The rostral and caudal parts were collected from mice that received cervical contusion injury at the indicated time points. Panel (A) represents the level of miR measured in the medulla–cervical 1st (C1) segment (rostral part). Panel (B) shows the level of miR in the thoracic 5th (T5) –thoracic 7th (T7) segment of the spinal cord (caudal part). One-way ANOVA was used for analysis, with Dunnett’s multiple comparisons test. Ns = not significant; p = 0.45 and F = 1.00 (rostral part) and p = 0.093 and F = 2.18 (caudal part). The circles, triangles, squares, and stars in the graph represent the individual mouse miR level per group. The red line shows the mean with SEM. Full article ">Figure 4
The effect of cervical contusion injury on the endogenous level of miR133b in the prefrontal cortex. Between 0.5 h and 7 days after receiving a unilateral contusion injury at the C5th level, mice were euthanized and pre-frontal cortices harvested and processed for miR isolation. The graphs represent the level of miR133b in the ipsilateral (panel (A)) and contralateral (panel (B)) hemispheres. One-way ANOVA was used for analysis, with Dunnett’s for multiple comparisons test. * p < 0.05 when compared to untreated group; # p < 0.05 when compared to 0.5 h group. The circles, triangles, squares, and stars in the graph represent the individual mouse miR level per group. The red line shows the mean with SEM. Full article ">Figure 5
Heatmap of the Pearson correlation coefficient matrix between lesion and prefrontal cortex. The heatmap shows the correlation analysis (r values) between levels of miR133b measured in the lesion and ipsilateral and contralateral hemispheres. The r values were obtained by performing a Pearson correlation coefficient test. Full article ">Figure 6
The endogenous levels of miR133b in the lung and spleen after cervical contusion injury. The graph represents the level of miR133b in off-targets at different time points following the injury. Two-way ANOVA was used for analysis, with Tukey’s for multiple comparison test. Ns = not statistically significant. Full article ">

11 pages, 12151 KiB

Open AccessBrief Report

Familial Dilated Cardiomyopathy: A Novel MED9 Short Isoform Identification

by Monica Franzese, Mario Zanfardino, Andrea Soricelli, Annapaola Coppola, Ciro Maiello, Marco Salvatore, Concetta Schiano and Claudio Napoli

Int. J. Mol. Sci. 2024, 25(5), 3057; https://doi.org/10.3390/ijms25053057 - 6 Mar 2024

Cited by 1 | Viewed by 1352

Abstract

Familial dilated cardiomyopathy (DCM) is among the leading indications for heart transplantation. DCM alters the transcriptomic profile. The alteration or activation/silencing of physiologically operating transcripts may explain the onset and progression of this pathological state. The mediator complex (MED) plays a fundamental role [...] Read more.

Familial dilated cardiomyopathy (DCM) is among the leading indications for heart transplantation. DCM alters the transcriptomic profile. The alteration or activation/silencing of physiologically operating transcripts may explain the onset and progression of this pathological state. The mediator complex (MED) plays a fundamental role in the transcription process. The aim of this study is to investigate the MED subunits, which are altered in DCM, to identify target crossroads genes. RNA sequencing allowed us to identify specific MED subunits that are altered during familial DCM, transforming into human myocardial samples. N = 13 MED subunits were upregulated and n = 7 downregulated. MED9 alone was significantly reduced in patients compared to healthy subjects (HS) (FC = −1.257; p < 0.05). Interestingly, we found a short MED9 isoform (MED9s) (ENSG00000141026.6), which was upregulated when compared to the full-transcript isoform (MED9f). Motif identification analysis yielded several significant matches (p < 0.05), such as GATA4, which is downregulated in CHD. Moreover, although the protein–protein interaction network showed FOG2/ZFPM2, FOS and ID2 proteins to be the key interacting partners of GATA4, only FOG2/ZFPM2 overexpression showed an interaction score of “high confidence” ≥ 0.84. A significant change in the MED was observed during HF. For the first time, the MED9 subunit was significantly reduced between familial DCM and HS (p < 0.05), showing an increased MED9s isoform in DCM patients with respect to its full-length transcript. MED9 and GATA4 shared the same sequence motif and were involved in a network with FOG2/ZFPM2, FOS, and ID2, proteins already implicated in cardiac development. Full article

(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

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Figure 1
MED subunits alteration into familial DCM patients. (A) The heatmap represents MED subunit alteration, combining the MED DEGs into DCM and HS groups. (B) MED9 expression levels by RNA-seq. (C) Different expression levels of MED9 full (MED9f) and short (MED9s) isoforms. Statistically significant differences between HS and DCM groups were evaluated via the Mann–Whitney U test. * p < 0.05 vs. HS. Sample size: n = 22 subjects included, of which n = 11 were healthy subjects (HS), such as organ donors, and n = 11 were DCM patients undergoing heart transplantation. Full article ">Figure 2
Motif enrichment analysis in familial DCM patients. Motif enrichment analysis identifies candidate TF regulators of tissue-specific DCM development and function. MEME online tool identified more than 30 enriched motifs in DCM group (<a href="#app1-ijms-25-03057" class="html-app">Supplementary Data</a>). Among these, GATA4 was identified as a fundamental target in heart development. Sample size: n = 22 subjects included, of which n = 11 were healthy subjects (HS), such as organ donors, and n = 11 were DCM patients undergoing heart transplantation. Full article ">Figure 3
PPI network; GO; STRING analyses and validation data. The figure shows the (A) GATA4 PPI network, of which only ZFPM2 showed high-confidence interaction (score > 0.8). The color of nodes indicates the query proteins and the first shell of interactors. The high-confidence interaction for ZFPM2 was indicated with a thicker line. (B) GATA4 interactions. GO results were analyzed using STRING tool. (C) Quantitative reverse transcriptase PCR (qRT-PCR) was performed to validate data obtained. Bar graph of normalized gene expression show GATA4, FOG2/ZFPM2, FOS, and ID2 analysis in DCM and HS groups. The relative expression was calculated as fold change (FC) between DCM and HS groups and we assigned a value = 1 for the healthy subjects’ (HS) group. Statistically significant difference was evaluated via Mann–Whitney U test for comparison between two groups. * p < 0.05 vs. HS. Sample size: n = 22 subjects included, of which n = 11 were healthy subjects (HS), such as organ donors, and n = 11 were DCM patients undergoing heart transplantation. Full article ">

14 pages, 1372 KiB

Open AccessReview

The Incredible Adventure of Omalizumab

by Christian Domingo, Daniel R. Monserrate, Ana Sogo and Rosa M. Mirapeix

Int. J. Mol. Sci. 2024, 25(5), 3056; https://doi.org/10.3390/ijms25053056 - 6 Mar 2024

Cited by 1 | Viewed by 2337

Abstract

The basis of our current understanding of allergies begins with the discovery of IgE in the mid-1960s. The whole theory of the physiology and pathophysiology of allergic diseases, including rhinitis and asthma, dates from that period. Among the key regions of IgE identified [...] Read more.

The basis of our current understanding of allergies begins with the discovery of IgE in the mid-1960s. The whole theory of the physiology and pathophysiology of allergic diseases, including rhinitis and asthma, dates from that period. Among the key regions of IgE identified were the FAB (fragment antigen binding) portion that has the ability to capture allergens, and the Cε3 domain, through which IgE binds to its membrane receptor. It was then postulated that blocking IgE at the level of the Cε3 domain would prevent it from binding to its receptor and thus set in motion the allergic cascade. This was the beginning of the development of omalizumab, a monoclonal antibody with an anti-IgE effect. In this article, we review the pathophysiology of allergic disease and trace the clinical development of omalizumab. We also review the benefits of omalizumab treatment that are apparently unrelated to allergies, such as its effect on immunity and bronchial remodeling. Full article

(This article belongs to the Special Issue 55th Anniversary of IgE Discovery: Pros and Cons of Anti-IgE Treatment)

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Structure of IgE (modified from [<a href="#B2-ijms-25-03056" class="html-bibr">2</a>]). The Cε3 domain is the fraction of the IgE molecule that binds to membrane receptors. The key epitope is the Cε3 domain of human IgE. Full article ">Figure 2
Types of IgE. There are two types: those secreted into the medium that bind to the cell receptor through the Cε3 domain (a) and the non-secreted (or membrane) IgE molecules, which are synthesized and expressed by the cell at the level of its cell membrane (b). Full article ">Figure 3
Acute allergic reaction: cross-linking phenomenon (taken from [<a href="#B5-ijms-25-03056" class="html-bibr">5</a>]). The individual is sensitized, produces IgE against the allergen to which s/he is sensitized, and these IgE molecules bind to their membrane receptors on mast cells and basophils. When a new allergen appears, two IgE molecules block the allergen, leading to changes in the intracellular component of the IgE receptor. This will cause mediator-loaded vesicles (in this case histamine) to be released into the environment. Full article ">Figure 4
Effects of omalizumab. The steps of the allergic process are shown in black,. The steps blocked by the direct immunoglobulin E (IgE) blocking effect of omalizumab are shown in red. Blue indicates indirect immunomodulation mediated by the action of omalizumab, which causes the down-regulation of the cellular expression of FcεRI, and of FcεRII at different levels, the secretion of interleukin IL-4 and IL-5, and lowers eosinophil and B-lymphocyte levels, as well as IgE production. APC: antigen-presenting cell (modifed from [<a href="#B3-ijms-25-03056" class="html-bibr">3</a>], Domingo, C. Drugs 2014, 74, 521–533). Full article ">Figure 5
Modified from Lynch et al. [<a href="#B61-ijms-25-03056" class="html-bibr">61</a>]. The figure shows how IgE molecules bind to their receptors and are ready for the cross-linking phenomenon to occur in the presence of allergens. This will activate the cascade that blocks the command sent from the toll-like receptors for the nucleus to synthesize interferon. Full article ">

17 pages, 4762 KiB

Open AccessArticle

iBio-GATS—A Semi-Automated Workflow for Structural Modelling of Insect Odorant Receptors

by Vaanathi Chidambara Thanu, Amara Jabeen and Shoba Ranganathan

Int. J. Mol. Sci. 2024, 25(5), 3055; https://doi.org/10.3390/ijms25053055 - 6 Mar 2024

Cited by 2 | Viewed by 1366

Abstract

Insects utilize seven transmembrane (7TM) odorant receptor (iOR) proteins, with an inverted topology compared to G-protein coupled receptors (GPCRs), to detect chemical cues in the environment. For pest biocontrol, chemical attractants are used to trap insect pests. However, with the influx of invasive [...] Read more.

Insects utilize seven transmembrane (7TM) odorant receptor (iOR) proteins, with an inverted topology compared to G-protein coupled receptors (GPCRs), to detect chemical cues in the environment. For pest biocontrol, chemical attractants are used to trap insect pests. However, with the influx of invasive insect pests, novel odorants are urgently needed, specifically designed to match 3D iOR structures. Experimental structural determination of these membrane receptors remains challenging and only four experimental iOR structures from two evolutionarily distant organisms have been solved. Template-based modelling (TBM) is a complementary approach, to generate model structures, selecting templates based on sequence identity. As the iOR family is highly divergent, a different template selection approach than sequence identity is needed. Bio-GATS template selection for GPCRs, based on hydrophobicity correspondence, has been morphed into iBio-GATS, for template selection from available experimental iOR structures. This easy-to-use semi-automated workflow has been extended to generate high-quality models from any iOR sequence from the selected template, using Python and shell scripting. This workflow was successfully validated on Apocrypta bakeri Orco and Machilis hrabei OR5 structures. iBio-GATS models generated for the fruit fly iOR, OR59b and Orco, yielded functional ligand binding results concordant with experimental mutagenesis findings, compared to AlphaFold2 models. Full article

(This article belongs to the Topic The 22nd International Conference on Bioinformatics (InCoB 2023): Translational Bioinformatics Transforming Life)

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14 pages, 2476 KiB

Open AccessArticle

A Combination of Library Screening and Rational Mutagenesis Expands the Available Color Palette of the Smallest Fluorogen-Activating Protein Tag nanoFAST

by Nadezhda S. Baleeva, Yulia A. Bogdanova, Marina V. Goncharuk, Anatolii I. Sokolov, Ivan N. Myasnyanko, Vadim S. Kublitski, Alexander Yu. Smirnov, Aidar R. Gilvanov, Sergey A. Goncharuk, Konstantin S. Mineev and Mikhail S. Baranov

Int. J. Mol. Sci. 2024, 25(5), 3054; https://doi.org/10.3390/ijms25053054 - 6 Mar 2024

Cited by 1 | Viewed by 1321

Abstract

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro [...] Read more.

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein. Full article

(This article belongs to the Special Issue Advanced Fluorescence Methodologies: Focus on Macromolecules Research 2.0)

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Figure 1
Positions for the rational single-point mutations (purple) and the mutations adapted from the tFAST protein (red) [<a href="#B26-ijms-25-03054" class="html-bibr">26</a>] are shown on the spatial structure of nanoFAST in complex with HBR-DOM2 (PDB code A8O0) [<a href="#B19-ijms-25-03054" class="html-bibr">19</a>]. Full article ">Figure 2
(A). Design of the presented study. (B). The normalized absorption (dashed lines) and emission (solid lines) spectra of various fluorogens in complexes with nanoFAST and nano-tFAST-E46Q proteins. Sign "?" means various substituents in the molecule of the possible fluorogen arylidene-azolone. Full article ">Figure 3
Live-cell imaging with a set of fluorogens in different filter sets. HeLa Kyoto cells transiently transfected with nano-tFast-E46Q-H2B (A–C), nano-tFast-E46Q-LifeAct (D–F), and nano-tFast-E46Q-GAP43 (G–I). Cells were imaged using GFP filter in the presence of 5 μM HBR-2,5-DM (A,D,G), TRITC filter in the presence of 5 μM HBR-DOM2 (B,E,H), TxRed filter in the presence of 5 μM HBR-DOM (C,F,I). Scale bar is 10 μm. Full article ">Scheme 1
Fluorogens library tested in the present study (See <a href="#app1-ijms-25-03054" class="html-app">Supplementary Materials</a> for more details <a href="#app1-ijms-25-03054" class="html-app">Table S2.1 and Part S7</a>). The numbers on the structure of the fluorogen indicate the numbering of substituents in the arylidene fragment, discussed in the text. Full article ">Scheme 2
Results of nanoFAST variants screening and design (F.E.—fluorescent enhancement revealed by plate reader screening; n.m.—not measured). Amino acid substitutions taken from the tFAST and redFAST proteins, are marked in green and red respectively. Full article ">

20 pages, 9164 KiB

Open AccessArticle

New Biocides Based on N⁴-Alkylcytidines: Effects on Microorganisms and Application for the Protection of Cultural Heritage Objects of Painting

by Liudmila A. Alexandrova, Ivan A. Oskolsky, Dmitry A. Makarov, Maxim V. Jasko, Inna L. Karpenko, Olga V. Efremenkova, Byazilya F. Vasilyeva, Darya A. Avdanina, Anna A. Ermolyuk, Elizaveta E. Benko, Stanislav G. Kalinin, Tat’yana V. Kolganova, Maria Ya. Berzina, Irina D. Konstantinova, Alexander O. Chizhov, Sergey N. Kochetkov and Alexander A. Zhgun

Int. J. Mol. Sci. 2024, 25(5), 3053; https://doi.org/10.3390/ijms25053053 - 6 Mar 2024

Cited by 2 | Viewed by 1427

Abstract

The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this [...] Read more.

The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this study, a series of N⁴-alkylcytidines, including 5- and 6-methylcytidine derivatives, with extended alkyl substituents, were obtained in order to develop a new generation of antibacterial and antifungal biocides based on nucleoside derivatives. It has been shown that N⁴-alkyl 5- or 6-methylcytidines effectively inhibit the growth of molds, isolated from the paintings in the halls of the Ancient Russian Paintings of the State Tretyakov Gallery, Russia, Moscow. The novel compounds showed activity similar to antiseptics commonly used to protect works of art, such as benzalkonium chloride, to which a number of microorganisms have acquired resistance. It was also shown that the activity of N⁴-alkylcytidines is comparable to that of some antibiotics used in medicine to fight Gram-positive bacteria, including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis. N⁴-dodecyl-5- and 6-methylcytidines turned out to be the best. This compound seems promising for expanding the palette of antiseptics used in painting, since quite often the destruction of painting materials is caused by joint fungi and bacteria infection. Full article

(This article belongs to the Special Issue New Types of Antibacterial Biocides 2.0)

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Figure 1
Synthesized N4-alkylcytidines with antibacterial and antifungal activity. Full article ">Figure 2
The inhibitory effect (MIC, μg/mL) of the most active compounds and the antibiotic amikacin against a number of Gram-positive bacteria. Full article ">Figure 3
The phenotype of fungi strains on CDA medium, supplemented with the 200 µM addition of 2b, or without additives (control). Petri dishes were captured in 5 days (for STG-30 and STG-143B) or in 12 days (all other strains) after inoculation. Full article ">Figure 4
The dynamics of the relative growth inhibition (%) for STG strains on the CDA medium with the synthesized compounds, BAC, and NaPCP (all tested at concentrations of 200 μM and 1000 μM). Data were collected within 3–27 days after inoculation every 3 days. Full article ">Figure 5
The effect of the size of the alkyl group at N4 on the fungicidal activity of compounds: (a) the phenotype of fungi strains on CDA medium, supplemented with the 200 µM addition of 2a–c or without additives (control); Petri dishes were captured in 12 days after inoculation; (b) antifungal activity related to the activity of 2b. Full article ">Figure 6
The relative antifungal activity of 200 μM synthetized compounds, BAC, and NaPCP at 5, 12, and 27 days after inoculation. Grown as the ratio of the size of the colonies of all fungal strains on the medium supplemented with compound in relation to the size of the colonies in the control. The compounds are arranged in order of increasing antifungal activity on day 27. Full article ">Scheme 1
Reagents and conditions: (i) 1,2,4-triazole, 2-chlorophenyl dichlorophosphate, pyridine, RT, overnight, (ii) CnH2n+1NH2, DIPEA, EtOH, RT, overnight, (iii) NH3, H2O, EtOH, RT, overnight. Full article ">Scheme 2
Reagents and conditions: (i) bis(trimethylsilyl)-acetamide, DCE or AN, Δ, 15 min; (ii) 1′,2′,3′,5′-tetra-O-acetylribose, TMSOTf, DCE or AN, Δ, 3 h; (iii) 1,2,4-triazole, 2-chlorophenyl dichlorophosphate, pyridine, RT, overnight, (iv) Lawesson’s reagent, dioxane, Δ, 3 h; (v) CnH2n+1NH2, DIPEA, dioxane (for 9), EtOH (for 10), RT, overnight; (vi) NH3, H2O, EtOH, RT, overnight. Full article ">Scheme 3
Reagents and conditions: (i) Lawesson’s reagent, dioxane, pyridine, Δ, 3 h; (ii) CnH2n+1NH2, 7-methylquinoline, ethylene glycol, Δ, 2-3 h; (iii) (1) bis(trimethylsilyl)acetamide, AN, RT, 15 min; (2) 1′,2′,3′,5′-tetra-O-acetylribose, TMSOTf, AN, Δ, 3 h; (iv) NH3, H2O, EtOH, RT, overnight. Full article ">Scheme 4
Enzymes: purine nucleoside phosphorylase (PNP), uridine phosphorylase (UP), thymidine phosphorylase (TP). Full article ">

13 pages, 584 KiB

Open AccessReview

TGF-β Signaling Pathways in the Development of Diabetic Retinopathy

by Andrew Callan, Sonal Jha, Laura Valdez, Lois Baldado and Andrew Tsin

Int. J. Mol. Sci. 2024, 25(5), 3052; https://doi.org/10.3390/ijms25053052 - 6 Mar 2024

Cited by 4 | Viewed by 2654

Abstract

Diabetic retinopathy (DR), a prevalent complication of diabetes mellitus affecting a significant portion of the global population, has long been viewed primarily as a microvascular disorder. However, emerging evidence suggests that it should be redefined as a neurovascular disease with multifaceted pathogenesis rooted [...] Read more.

Diabetic retinopathy (DR), a prevalent complication of diabetes mellitus affecting a significant portion of the global population, has long been viewed primarily as a microvascular disorder. However, emerging evidence suggests that it should be redefined as a neurovascular disease with multifaceted pathogenesis rooted in oxidative stress and advanced glycation end products. The transforming growth factor-β (TGF-β) signaling family has emerged as a major contributor to DR pathogenesis due to its pivotal role in retinal vascular homeostasis, endothelial cell barrier function, and pericyte differentiation. However, the precise roles of TGF-β signaling in DR remain incompletely understood, with conflicting reports on its impact in different stages of the disease. Additionally, the BMP subfamily within the TGF-β superfamily introduces further complexity, with BMPs exhibiting both pro- and anti-angiogenic properties. Furthermore, TGF-β signaling extends beyond the vascular realm, encompassing immune regulation, neuronal survival, and maintenance. The intricate interactions between TGF-β and reactive oxygen species (ROS), non-coding RNAs, and inflammatory mediators have been implicated in the pathogenesis of DR. This review delves into the complex web of signaling pathways orchestrated by the TGF-β superfamily and their involvement in DR. A comprehensive understanding of these pathways may hold the key to developing targeted therapies to halt or mitigate the progression of DR and its devastating consequences. Full article

(This article belongs to the Section Molecular Immunology)

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4 pages, 197 KiB

Open AccessEditorial

Molecular Pharmacology in Diabetes

by Flávio Reis and Rosa Fernandes

Int. J. Mol. Sci. 2024, 25(5), 3051; https://doi.org/10.3390/ijms25053051 - 6 Mar 2024

Viewed by 1001

Abstract

This Special Issue highlights the key molecules and molecular signaling pathways associated with diabetes and its multifaceted complications [...] Full article

(This article belongs to the Special Issue Molecular Pharmacology in Diabetes)

14 pages, 1624 KiB

Open AccessReview

Astrocyte–Neuron Interaction via the Glutamate–Glutamine Cycle and Its Dysfunction in Tau-Dependent Neurodegeneration

by Marta Sidoryk-Węgrzynowicz, Kamil Adamiak and Lidia Strużyńska

Int. J. Mol. Sci. 2024, 25(5), 3050; https://doi.org/10.3390/ijms25053050 - 6 Mar 2024

Cited by 5 | Viewed by 2521

Abstract

Astroglia constitute the largest group of glial cells and are involved in numerous actions that are critical to neuronal development and functioning, such as maintaining the blood–brain barrier, forming synapses, supporting neurons with nutrients and trophic factors, and protecting them from injury. These [...] Read more.

Astroglia constitute the largest group of glial cells and are involved in numerous actions that are critical to neuronal development and functioning, such as maintaining the blood–brain barrier, forming synapses, supporting neurons with nutrients and trophic factors, and protecting them from injury. These properties are deeply affected in the course of many neurodegenerative diseases, including tauopathies, often before the onset of the disease. In this respect, the transfer of essential amino acids such as glutamate and glutamine between neurons and astrocytes in the glutamate–glutamine cycle (GGC) is one example. In this review, we focus on the GGC and the disruption of this cycle in tau-dependent neurodegeneration. A profound understanding of the complex functions of the GGC and, in the broader context, searching for dysfunctions in communication pathways between astrocytes and neurons via GGC in health and disease, is of critical significance for the development of novel mechanism-based therapies for neurodegenerative disorders. Full article

(This article belongs to the Special Issue Advances in Aging and Alzheimer’s Disease Research: From Molecular Mechanisms to Therapeutics)

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Figure 1

Figure 1
Schematic representation of the Gln and Glu transport and metabolism in astrocytes and neurons. Glu released from synaptic terminals is taken up by surrounding astrocytes via Glu transporters and converted to Gln in GS-mediated reaction. A proportion of Gln is released into the extracellular space by Gln carriers, with a predominant role in the N system. In addition to N system, the release of Gln from astrocytes is mediated by transporters belonging to systems L and ASC. Extracellular Gln is taken up into GABAergic and glutamatergic neurons by the unidirectional system A transporters. Once in neurons, Gln serves as a substrate for the mitochondrial enzyme PAG for the synthesis of Glu that can supply the neurotransmission pool of Glu or can be converted to GABA by GAD or to αKG by GDH. Full article ">Figure 2
Schematic representation of the disturbed astrocyte–neuron network in tau-dependent neurodegeneration based on the previously described results [<a href="#B81-ijms-25-03050" class="html-bibr">81</a>]. Astroglia after exposure to the mutant tau-expressing neurons, acquire functional deficit resulting in loss of neurosupportive properties and/or gain of neurotoxic function. One of the pathological events is associated with overexpression of GFAP and S100β protein as a consequence of the astrocytic phenotype reactivity. Moreover, astrocytes became deprived of key molecules that support glutamate (GLAST, GLT-1) and glutamine homeostasis (System N, System A carries) and synaptogenesis relevant processes (TSP-1, PSD95, SNP). Up arrows indicate targets that are increased; down arrows represent declined targets; lightning arrows mean deregulation of the glutamine transporting systems function in the astrocytic and neuronal tau-mediated neurodegeneration. For more details, see <a href="#sec5-ijms-25-03050" class="html-sec">Section 5</a> and <a href="#sec6-ijms-25-03050" class="html-sec">Section 6</a>. Full article ">

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Int. J. Mol. Sci., Volume 25, Issue 5 (March-1 2024) – 629 articles

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