Biomolecules

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17 pages, 2702 KiB

Open AccessEditor’s ChoiceArticle

Exploring Novel Variants of the Cytochrome P450 Reductase Gene (POR) from the Genome Aggregation Database by Integrating Bioinformatic Tools and Functional Assays

by Maria Natalia Rojas Velazquez, Søren Therkelsen and Amit V. Pandey

Biomolecules 2023, 13(12), 1728; https://doi.org/10.3390/biom13121728 - 30 Nov 2023

Viewed by 1156

Abstract

Cytochrome P450 oxidoreductase (POR) is an essential redox partner for steroid and drug-metabolizing cytochromes P450 located in the endoplasmic reticulum. Mutations in POR lead to metabolic disorders, including congenital adrenal hyperplasia, and affect the metabolism of steroids, drugs, and xenobiotics. In this study, [...] Read more.

Cytochrome P450 oxidoreductase (POR) is an essential redox partner for steroid and drug-metabolizing cytochromes P450 located in the endoplasmic reticulum. Mutations in POR lead to metabolic disorders, including congenital adrenal hyperplasia, and affect the metabolism of steroids, drugs, and xenobiotics. In this study, we examined approximately 450 missense variants of the POR gene listed in the Genome Aggregation Database (gnomAD) using eleven different in silico prediction tools. We found that 64 novel variants were consistently predicted to be disease-causing by most tools. To validate our findings, we conducted a population analysis and selected two variations in POR for further investigation. The human POR wild type and the R268W and L577P variants were expressed in bacteria and subjected to enzyme kinetic assays using a model substrate. We also examined the activities of several cytochrome P450 proteins in the presence of POR (WT or variants) by combining P450 and reductase proteins in liposomes. We observed a decrease in enzymatic activities (ranging from 35% to 85%) of key drug-metabolizing enzymes, supported by POR variants R288W and L577P compared to WT-POR. These results validate our approach of curating a vast amount of data from genome projects and provide an updated and reliable reference for diagnosing POR deficiency. Full article

(This article belongs to the Special Issue Novel Insights into Cytochrome P450 Enzymes in Drug Metabolism)

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15 pages, 3677 KiB

Open AccessEditor’s ChoiceArticle

Nanoparticle-Mediated Radiotherapy: Unraveling Dose Enhancement and Apoptotic Responses in Cancer and Normal Cell Lines

by Maria Anthi Kouri, Ellas Spyratou, Maria-Eleni Kalkou, Georgios Patatoukas, Evangelia Angelopoulou, Ioanna Tremi, Sophia Havaki, Vassilis G. Gorgoulis, Vassilis Kouloulias, Kalliopi Platoni and Efstathios P. Efstathopoulos

Biomolecules 2023, 13(12), 1720; https://doi.org/10.3390/biom13121720 - 29 Nov 2023

Viewed by 1402

Abstract

Cervical cancer remains a pressing global health concern, necessitating advanced therapeutic strategies. Radiotherapy, a fundamental treatment modality, has faced challenges such as targeted dose deposition and radiation exposure to healthy tissues, limiting optimal outcomes. To address these hurdles, nanomaterials, specifically gold nanoparticles (AuNPs), [...] Read more.

Cervical cancer remains a pressing global health concern, necessitating advanced therapeutic strategies. Radiotherapy, a fundamental treatment modality, has faced challenges such as targeted dose deposition and radiation exposure to healthy tissues, limiting optimal outcomes. To address these hurdles, nanomaterials, specifically gold nanoparticles (AuNPs), have emerged as a promising avenue. This study delves into the realm of cervical cancer radiotherapy through the meticulous exploration of AuNPs’ impact. Utilizing ex vivo experiments involving cell lines, this research dissected intricate radiobiological interactions. Detailed scrutiny of cell survival curves, dose enhancement factors (DEFs), and apoptosis in both cancer and normal cervical cells revealed profound insights. The outcomes showcased the substantial enhancement of radiation responses in cancer cells following AuNP treatment, resulting in heightened cell death and apoptotic levels. Significantly, the most pronounced effects were observed 24 h post-irradiation, emphasizing the pivotal role of timing in AuNPs’ efficacy. Importantly, AuNPs exhibited targeted precision, selectively impacting cancer cells while preserving normal cells. This study illuminates the potential of AuNPs as potent radiosensitizers in cervical cancer therapy, offering a tailored and efficient approach. Through meticulous ex vivo experimentation, this research expands our comprehension of the complex dynamics between AuNPs and cells, laying the foundation for their optimized clinical utilization. Full article

(This article belongs to the Special Issue Drug Delivery Nanosystems for Biomedical Applications)

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18 pages, 2636 KiB

Open AccessEditor’s ChoiceArticle

ICF1-Syndrome-Associated DNMT3B Mutations Prevent De Novo Methylation at a Subset of Imprinted Loci during iPSC Reprogramming

by Ankit Verma, Varsha Poondi Krishnan, Francesco Cecere, Emilia D’Angelo, Vincenzo Lullo, Maria Strazzullo, Sara Selig, Claudia Angelini, Maria R. Matarazzo and Andrea Riccio

Biomolecules 2023, 13(12), 1717; https://doi.org/10.3390/biom13121717 - 28 Nov 2023

Cited by 1 | Viewed by 1184

Abstract

Parent-of-origin-dependent gene expression of a few hundred human genes is achieved by differential DNA methylation of both parental alleles. This imprinting is required for normal development, and defects in this process lead to human disease. Induced pluripotent stem cells (iPSCs) serve as a [...] Read more.

Parent-of-origin-dependent gene expression of a few hundred human genes is achieved by differential DNA methylation of both parental alleles. This imprinting is required for normal development, and defects in this process lead to human disease. Induced pluripotent stem cells (iPSCs) serve as a valuable tool for in vitro disease modeling. However, a wave of de novo DNA methylation during reprogramming of iPSCs affects DNA methylation, thus limiting their use. The DNA methyltransferase 3B (DNMT3B) gene is highly expressed in human iPSCs; however, whether the hypermethylation of imprinted loci depends on DNMT3B activity has been poorly investigated. To explore the role of DNMT3B in mediating de novo DNA methylation at imprinted DMRs, we utilized iPSCs generated from patients with immunodeficiency, centromeric instability, facial anomalies type I (ICF1) syndrome that harbor biallelic hypomorphic DNMT3B mutations. Using a whole-genome array-based approach, we observed a gain of methylation at several imprinted loci in control iPSCs but not in ICF1 iPSCs compared to their parental fibroblasts. Moreover, in corrected ICF1 iPSCs, which restore DNMT3B enzymatic activity, imprinted DMRs did not acquire control DNA methylation levels, in contrast to the majority of the hypomethylated CpGs in the genome that were rescued in the corrected iPSC clones. Overall, our study indicates that DNMT3B is responsible for de novo methylation of a subset of imprinted DMRs during iPSC reprogramming and suggests that imprinting is unstable during a specific time window of this process, after which the epigenetic state at these regions becomes resistant to perturbation. Full article

(This article belongs to the Special Issue Neurodevelopmental Disorders: Linking Genetics and Epigenetics to Disease-Related Pathways)

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20 pages, 12704 KiB

Open AccessEditor’s ChoiceArticle

Mechanisms of Fibroblast Growth Factor 21 Adsorption on Macroion Layers: Molecular Dynamics Modeling and Kinetic Measurements

by Monika Wasilewska, Maria Dąbkowska, Agata Pomorska, Piotr Batys, Bogusław Kowalski, Aneta Michna and Zbigniew Adamczyk

Biomolecules 2023, 13(12), 1709; https://doi.org/10.3390/biom13121709 - 26 Nov 2023

Viewed by 1835

Abstract

Molecular dynamic modeling and various experimental techniques, including multi-angle dynamic light scattering (MADLS), streaming potential, optical waveguide light spectroscopy (OWLS), quartz crystal microbalance with dissipation (QCM), and atomic force microscopy (AFM), were applied to determine the basic physicochemical parameters of fibroblast growth factor [...] Read more.

Molecular dynamic modeling and various experimental techniques, including multi-angle dynamic light scattering (MADLS), streaming potential, optical waveguide light spectroscopy (OWLS), quartz crystal microbalance with dissipation (QCM), and atomic force microscopy (AFM), were applied to determine the basic physicochemical parameters of fibroblast growth factor 21 in electrolyte solutions. The protein size and shape, cross-section area, dependence of the nominal charge on pH, and isoelectric point of 5.3 were acquired. These data enabled the interpretation of the adsorption kinetics of FGF 21 on bare and macrocation-covered silica investigated by OWLS and QCM. It was confirmed that the protein molecules irreversibly adsorbed on the latter substrate, forming layers with controlled coverage up to 0.8 mg m⁻², while their adsorption on bare silica was much smaller. The viability of two cell lines, CHO-K1 and L-929, on both bare and macrocation/FGF 21-covered substrates was also determined. It is postulated that the acquired results can serve as useful reference systems for designing complexes that can extend the half-life of FGF 21 in its active state. Full article

(This article belongs to the Special Issue Mechanisms and Kinetics of Interactions of Biomolecules at Interfaces II)

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21 pages, 3421 KiB

Open AccessEditor’s ChoiceArticle

Efficacy of HDAC Inhibitors in Driving Peroxisomal β-Oxidation and Immune Responses in Human Macrophages: Implications for Neuroinflammatory Disorders

by Andrea Villoria-González, Bettina Zierfuss, Patricia Parzer, Elisabeth Heuböck, Violetta Zujovic, Petra Waidhofer-Söllner, Markus Ponleitner, Paulus Rommer, Jens Göpfert, Sonja Forss-Petter, Johannes Berger and Isabelle Weinhofer

Biomolecules 2023, 13(12), 1696; https://doi.org/10.3390/biom13121696 - 23 Nov 2023

Cited by 1 | Viewed by 1596

Abstract

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by β-oxidation. Impaired VLCFA catabolism due to primary or secondary [...] Read more.

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by β-oxidation. Impaired VLCFA catabolism due to primary or secondary peroxisomal alterations is featured in neurodegenerative and neuroinflammatory disorders such as X-linked adrenoleukodystrophy and multiple sclerosis (MS). Here, we identified that healthy human macrophages upregulate the peroxisomal genes involved in β-oxidation during myelin phagocytosis and pro-inflammatory activation, and that this response is impaired in peripheral macrophages and phagocytes in brain white matter lesions in MS patients. The pharmacological targeting of VLCFA metabolism and peroxisomes in innate immune cells could be favorable in the context of neuroinflammation and neurodegeneration. We previously identified the epigenetic histone deacetylase (HDAC) inhibitors entinostat and vorinostat to enhance VLCFA degradation and pro-regenerative macrophage polarization. However, adverse side effects currently limit their use in chronic neuroinflammation. Here, we focused on tefinostat, a monocyte/macrophage-selective HDAC inhibitor that has shown reduced toxicity in clinical trials. By using a gene expression analysis, peroxisomal β-oxidation assay, and live imaging of primary human macrophages, we assessed the efficacy of tefinostat in modulating VLCFA metabolism, phagocytosis, chemotaxis, and immune function. Our results revealed the significant stimulation of VLCFA degradation with the upregulation of genes involved in peroxisomal β-oxidation and interference with immune cell recruitment; however, tefinostat was less potent than the class I HDAC-selective inhibitor entinostat in promoting a regenerative macrophage phenotype. Further research is needed to fully explore the potential of class I HDAC inhibition and downstream targets in the context of neuroinflammation. Full article

(This article belongs to the Special Issue The Role of Peroxisome in Cell Functions and Pathological Consequences of Its Dysfunction)

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21 pages, 3457 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

The Blocking of Drug Resistance Channels by Selected Hydrophobic Statins in Chemoresistance Human Melanoma

by Wojciech Placha, Piotr Suder, Agnieszka Panek, Patrycja Bronowicka-Adamska, Marta Zarzycka, Małgorzata Szczygieł, Jacek Zagajewski and Monika Weronika Piwowar

Biomolecules 2023, 13(12), 1682; https://doi.org/10.3390/biom13121682 - 21 Nov 2023

Viewed by 1419

Abstract

Despite the development of modern drugs, drug resistance in oncology remains the main factor limiting the curability of patients. This paper shows the use of a group of hydrophobic statins to inhibit drug resistance (Pgp protein). In a chemoresistance melanoma cell model, viability, [...] Read more.

Despite the development of modern drugs, drug resistance in oncology remains the main factor limiting the curability of patients. This paper shows the use of a group of hydrophobic statins to inhibit drug resistance (Pgp protein). In a chemoresistance melanoma cell model, viability, necroptosis with DNA damage, the absorption of the applied pharmaceuticals, and the functional activity of the ABCB1 drug transporter after administration of docetaxel or docetaxel with a selected hydrophobic statin were studied. Taxol-resistant human melanoma cells from three stages of development were used as a model: both A375P and WM239A metastatic lines and radial growth phase WM35 cells. An animal model (Mus musculus SCID) was developed for the A375P cell line. The results show that hydrophobic statins administered with docetaxel increase the accumulation of the drug in the tumor cell a.o. by blocking the ABCB1 channel. They reduce taxol-induced drug resistance. The tumor size reduction was observed after the drug combination was administrated. It was shown that the structural similarity of statins is of secondary importance, e.g., pravastatin and simvastatin. Using cytostatics in the presence of hydrophobic statins increases their effectiveness while reducing their overall toxicity. Full article

(This article belongs to the Special Issue Advances in Protein-Ligand Interactions: From Structure, Function to Applications)

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11 pages, 1741 KiB

Open AccessEditor’s ChoiceArticle

Characterization of Rickets Type II Model Rats to Reveal Functions of Vitamin D and Vitamin D Receptor

by Yuichiro Iwai, Ayano Iijima, Satoko Kise, Chika Nagao, Yuto Senda, Kana Yabu, Hiroki Mano, Miyu Nishikawa, Shinichi Ikushiro, Kaori Yasuda and Toshiyuki Sakaki

Biomolecules 2023, 13(11), 1666; https://doi.org/10.3390/biom13111666 - 19 Nov 2023

Viewed by 1163

Abstract

Vitamin D has been known to exert a wide range of physiological effects, including calcemic, osteogenic, anticancer, and immune responses. We previously generated genetically modified (GM) rats and performed a comparative analysis of their physiological properties to elucidate the roles of vitamin D [...] Read more.

Vitamin D has been known to exert a wide range of physiological effects, including calcemic, osteogenic, anticancer, and immune responses. We previously generated genetically modified (GM) rats and performed a comparative analysis of their physiological properties to elucidate the roles of vitamin D and vitamin D receptor (VDR). In this study, our primary goal was to investigate the manifestations of type II rickets in rats with the VDR(H301Q) mutation, analogous to the human VDR(H305Q). Additionally, we created a double-mutant rat with the VDR(R270L/H301Q) mutation, resulting in almost no affinity for 1,25-dihydroxy-vitamin D3 (1,25D3) or 25-hydroxy-vitamin D3 (25D3). Notably, the plasma calcium concentration in Vdr(R270L/H301Q) rats was significantly lower than in wild-type (WT) rats. Meanwhile, Vdr(H301Q) rats had calcium concentrations falling between those of Vdr(R270L/H301Q) and WT rats. GM rats exhibited markedly elevated plasma parathyroid hormone and 1,25D3 levels compared to those of WT rats. An analysis of bone mineral density in the cortical bone of the femur in both GM rats revealed significantly lower values than in WT rats. Conversely, the bone mineral density in the trabecular bone was notably higher, indicating abnormal bone formation. This abnormal bone formation was more pronounced in Vdr(R270L/H301Q) rats than in Vdr(H301Q) rats, highlighting the critical role of the VDR-dependent function of 1,25D3 in bone formation. In contrast, neither Vdr(H301Q) nor Vdr(R270L/H301Q) rats exhibited symptoms of alopecia or cyst formation in the skin, which were observed in the Vdr-KO rats. These findings strongly suggest that unliganded VDR is crucial for maintaining the hair cycle and normal skin. Our GM rats hold significant promise for comprehensive analyses of vitamin D and VDR functions in future research. Full article

(This article belongs to the Special Issue Biochemistry and Molecular Biology of Vitamin D and Its Analog II)

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24 pages, 38016 KiB

Open AccessEditor’s ChoiceArticle

Dithiothreitol Affects the Fertilization Response in Immature and Maturing Starfish Oocytes

by Nunzia Limatola, Jong Tai Chun, Kazuyoshi Chiba and Luigia Santella

Biomolecules 2023, 13(11), 1659; https://doi.org/10.3390/biom13111659 - 17 Nov 2023

Viewed by 1312

Abstract

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the [...] Read more.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca²⁺ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca²⁺ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca²⁺ response at fertilization and the subsequent embryonic development. Full article

(This article belongs to the Special Issue Gametogenesis and Gamete Interaction)

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13 pages, 3086 KiB

Open AccessEditor’s ChoiceArticle

Furin Regulates the Alveolarization of Neonatal Lungs in a Mouse Model of Hyperoxic Lung Injury

by Shin Kato, Osuke Iwata, Hiroyuki Kato, Satoko Fukaya, Yukari Imai and Shinji Saitoh

Biomolecules 2023, 13(11), 1656; https://doi.org/10.3390/biom13111656 - 16 Nov 2023

Viewed by 1087

Abstract

Despite advances in treatment options, such as corticosteroid administration and less invasive respiratory support, bronchopulmonary dysplasia (BPD) remains an important prognostic factor in preterm infants. We previously reported that furin regulates changes in lung smooth muscle cell phenotypes, suggesting that it plays a [...] Read more.

Despite advances in treatment options, such as corticosteroid administration and less invasive respiratory support, bronchopulmonary dysplasia (BPD) remains an important prognostic factor in preterm infants. We previously reported that furin regulates changes in lung smooth muscle cell phenotypes, suggesting that it plays a critical role in BPD pathogenesis. Therefore, in this study, we aimed to evaluate whether it regulates the alveolarization of immature lungs through activating alveolarization-driving proteins. We first examined furin expression levels, and its functions, using an established hyperoxia-induced BPD mouse model. Thereafter, we treated mice pups, as well as primary myofibroblast cell cultures, with furin inhibitors. Finally, we administered the hyperoxia-exposed mice pups with recombinant furin. Immunofluorescence revealed the co-expression of furin with alpha-smooth muscle actin. Hyperoxia exposure for 10 d decreased alveolar formation, as well as the expression of furin and its target, IGF-1R. Hexa-D-arginine administration also significantly inhibited alveolar formation. Another furin inhibitor, decanoyl-RVKR-chloromethylketone, accumulated pro-IGF-1R, and decreased IGF-1R phosphorylation in myofibroblast primary cultures. Finally, recombinant furin treatment significantly improved alveolarization in hyperoxia-exposed mice pups. Furin regulates alveolarization in immature lungs. Therefore, this study provides novel insights regarding the involvement of furin in BPD pathogenesis, and highlights a potential treatment target for ameliorating the impact of BPD. Full article

(This article belongs to the Section Molecular Medicine)

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20 pages, 5728 KiB

Open AccessEditor’s ChoiceArticle

Sexually Dimorphic Effects of Histamine Degradation by Enteric Glial Histamine N-Methyltransferase (HNMT) on Visceral Hypersensitivity

by Jonathon L. McClain, Wilmarie Morales-Soto, Jacques Gonzales, Visha Parmar, Elena Y. Demireva and Brian D. Gulbransen

Biomolecules 2023, 13(11), 1651; https://doi.org/10.3390/biom13111651 - 14 Nov 2023

Cited by 2 | Viewed by 1552

Abstract

Histamine is a neuromodulator that affects gut motility and visceral sensitivity through intrinsic and extrinsic neural pathways, yet the mechanisms regulating histamine availability in these pathways remain poorly understood. Here, we show that enteric glia contribute to histamine clearance in the enteric nervous [...] Read more.

Histamine is a neuromodulator that affects gut motility and visceral sensitivity through intrinsic and extrinsic neural pathways, yet the mechanisms regulating histamine availability in these pathways remain poorly understood. Here, we show that enteric glia contribute to histamine clearance in the enteric nervous system (ENS) through their expression of the enzyme histamine N-methyltransferase (HNMT). Glial HNMT expression was initially assessed using immunolabeling and gene expression, and functionally tested using CRISPR-Cas9 to create a Cre-dependent conditional Hnmt ablation model targeting glia. Immunolabeling, calcium imaging, and visceromotor reflex recordings were used to assess the effects on ENS structure and visceral hypersensitivity. Immunolabeling and gene expression data show that enteric neurons and glia express HNMT. Deleting Hnmt in Sox10+ enteric glia increased glial histamine levels and altered visceromotor responses to colorectal distension in male mice, with no effect in females. Interestingly, deleting glial Hnmt protected males from histamine-driven visceral hypersensitivity. These data uncover a significant role for glial HNMT in histamine degradation in the gut, which impacts histamine-driven visceral hypersensitivity in a sex-dependent manner. Changes in the capacity of glia to clear histamines could play a role in the susceptibility to developing visceral pain in disorders of the gut–brain interaction. Full article

(This article belongs to the Special Issue Pathogenesis and Potential Treatments of Neurointestinal Diseases)

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14 pages, 956 KiB

Open AccessEditor’s ChoiceArticle

Plasma Androstenedione Concentration Can Discriminate Frail versus Non-Frail Men with Prostate Cancer under Androgen Deprivation Therapy

by Mayra Alejandra Mafla-España, María Dolores Torregrosa, Manel Beamud-Cortés, Lorena Bermell-Marco, José Rubio-Briones and Omar Cauli

Biomolecules 2023, 13(11), 1642; https://doi.org/10.3390/biom13111642 - 13 Nov 2023

Viewed by 1588

Abstract

Background: Androgen deprivation therapy (ADT) is a mainstay of prostate cancer in both adjuvant and palliative settings. Since androgens are crucial for functional status and psychological functions, we evaluated whether blood testosterone, androstenedione, or DHEA concentrations were associated with functional status and psychological [...] Read more.

Background: Androgen deprivation therapy (ADT) is a mainstay of prostate cancer in both adjuvant and palliative settings. Since androgens are crucial for functional status and psychological functions, we evaluated whether blood testosterone, androstenedione, or DHEA concentrations were associated with functional status and psychological alterations in patients with localised (PCa) or metastatic prostate cancer (mPCa) receiving ADT with analogues of luteinising hormone-releasing hormone (LHRH). Methods: The five Fried criteria were considered to identify frailty syndrome. In addition, complementary evaluations were carried out to measure other variables of interest. Sleep quality was assessed using the Athens Insomnia Scale, cognitive functions were assessed using the Mini-Mental State Examination, and symptoms of depression were measured using the Yesavage Geriatric Depression Scale. Logistic regression analysis was performed to determine if the androgens level could be related to frailty syndrome, sleep impairment, depressive symptoms, and cognitive functions. Results: The results of the multivariate analyses show that high concentrations of androstenedione were significantly associated with frailty syndrome in both groups (p = 0.018; odds ratio = 4.66, 95% confidence interval [1.30–16.6]). There were significant relationships between frailty syndrome and the systemic concentration of androstenedione (p = 0.01), but not the concentration of testosterone (p = 0.60) or DHEA (p = 0.42). In addition, the results of the non-parametric tests show significant results between a decreased gait speed in the two groups (metastatic and localised) and the concentration of androstenedione (p = 0.015). High androstenedione levels were associated with a slow walking speed in the mCaP group (p = 0.016), while high testosterone levels were associated with a better walking speed in the localised CaP group (p = 0.03). For the concentration of androstenedione in plasma, the area under the curve was 0.72, with a 95% CI of 0.55–0.88 with acceptable values, and with a cut-off point of 4.51 pg/mL, a sensitivity of 82.9%, and specificity of 53.8%. No relationships between the concentration of androgens in plasma and sleep quality, cognitive functions, or symptoms of depression suggest that the changes were specific to frailty syndrome. Conclusions: Further research into the role of androstenedione should be evaluated in follow-up studies in order to recommend its use as a suitable biomarker of frailty syndrome in prostate cancer patients. Full article

(This article belongs to the Section Molecular Biomarkers)

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19 pages, 4672 KiB

Open AccessEditor’s ChoiceArticle

Lenvatinib-Loaded Poly(lactic-co-glycolic acid) Nanoparticles with Epidermal Growth Factor Receptor Antibody Conjugation as a Preclinical Approach to Therapeutically Improve Thyroid Cancer with Aggressive Behavior

by Giovanna Revilla, Nuseibah Al Qtaish, Pablo Caruana, Myriam Sainz-Ramos, Tania Lopez-Mendez, Francisco Rodriguez, Verónica Paez-Espinosa, Changda Li, Núria Fucui Vallverdú, Maria Edwards, Antonio Moral, José Ignacio Pérez, Juan Carlos Escolà-Gil, José Luis Pedraz, Idoia Gallego, Rosa Corcoy, María Virtudes Céspedes, Gustavo Puras and Eugènia Mato

Biomolecules 2023, 13(11), 1647; https://doi.org/10.3390/biom13111647 - 13 Nov 2023

Cited by 2 | Viewed by 1730

Abstract

Background: Lenvatinib, a tyrosine kinase inhibitor (TKI) approved for the treatment of progressive and radioactive iodine (RAI)-refractory differentiated thyroid cancer (DTC), is associated with significant adverse effects that can be partially mitigated through the development of novel drug formulations. The utilization of nanoparticles [...] Read more.

Background: Lenvatinib, a tyrosine kinase inhibitor (TKI) approved for the treatment of progressive and radioactive iodine (RAI)-refractory differentiated thyroid cancer (DTC), is associated with significant adverse effects that can be partially mitigated through the development of novel drug formulations. The utilization of nanoparticles presents a viable option, as it allows for targeted drug delivery, reducing certain side effects and enhancing the overall quality of life for patients. This study aimed to produce and assess, both in vitro and in vivo, the cytotoxicity, biodistribution, and therapeutic efficacy of lenvatinib-loaded PLGA nanoparticles (NPs), both with and without decoration using antibody conjugation (cetuximab), as a novel therapeutic approach for managing aggressive thyroid tumors. Methods: Poly(lactic-co-glycolic acid) nanoparticles (NPs), decorated with or without anti-EGFR, were employed as a lenvatinib delivery system. These NPs were characterized for size distribution, surface morphology, surface charge, and drug encapsulation efficiency. Cytotoxicity was evaluated through MTT assays using two cellular models, one representing normal thyroid cells (Nthy-ori 3-1) and the other representing anaplastic thyroid cells (CAL-62). Additionally, an in vivo xenograft mouse model was established to investigate biodistribution and therapeutic efficacy following intragastric administration. Results: The NPs demonstrated success in terms of particle size, polydispersity index (PDI), zeta potential, morphology, encapsulation efficiency, and cetuximab distribution across the surface. In vitro analysis revealed cytotoxicity in both cellular models with both formulations, but only the decorated NPs achieved an ID50 value in CAL-62 cells. Biodistribution analysis following intragastric administration in xenografted thyroid mice demonstrated good stability in terms of intestinal barrier function and tumor accumulation. Both formulations were generally well tolerated without inducing pathological effects in the examined organs. Importantly, both formulations increased tumor necrosis; however, decorated NPs exhibited enhanced parameters related to apoptotic/karyolytic forms, mitotic index, and vascularization compared with NPs without decoration. Conclusions: These proof-of-concept findings suggest a promising strategy for administering TKIs in a more targeted and effective manner. Full article

(This article belongs to the Special Issue Advance in Nanoparticles for Tumor Targeting)

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15 pages, 4655 KiB

Open AccessEditor’s ChoiceArticle

Placentas from Women with Late-Onset Preeclampsia Exhibit Increased Expression of the NLRP3 Inflammasome Machinery

by Luis M. Garcia-Puente, Oscar Fraile-Martinez, Cielo García-Montero, Julia Bujan, Juan A. De León-Luis, Coral Bravo, Patrocinio Rodríguez-Benitez, Pilar Pintado, Francisco Javier Ruiz-Labarta, Melchor Álvarez-Mon, Natalio García-Honduvilla, María J. Cancelo, Miguel A. Saez and Miguel A. Ortega

Biomolecules 2023, 13(11), 1644; https://doi.org/10.3390/biom13111644 - 13 Nov 2023

Cited by 5 | Viewed by 1188

Abstract

Pre-eclampsia is a harmful and potentially lethal medical condition during pregnancy clinically diagnosed by hypertension and commonly accompanied by proteinuria and multiorgan affections. According to the time of diagnosis, it is differentiated between early-onset (EO-PE) and late-onset preeclampsia (LO-PE). Despite being less dangerous [...] Read more.

Pre-eclampsia is a harmful and potentially lethal medical condition during pregnancy clinically diagnosed by hypertension and commonly accompanied by proteinuria and multiorgan affections. According to the time of diagnosis, it is differentiated between early-onset (EO-PE) and late-onset preeclampsia (LO-PE). Despite being less dangerous and presenting distinct pathophysiological signatures, LO-PE has a greater prevalence than EO-PE, both having significant consequences on the placenta. Previous works have evidenced that exacerbated inflammation in this organ might play a potential pathogenic role in the development of pre-eclampsia, and there is some preliminary evidence that the hyperactivation of inflammasomes can be related to the altered immunoinflammatory responses observed in the placentas of these patients. However, the precise role of inflammasomes in the placentas of women with LO-PE remains to be fully understood. In this work, we have studied the gene and protein expression of the main components related to the canonical and non-canonical pathways of the inflammasome NLRP3 (NLRP3, ASC, caspase 1, caspase 5, caspase 8, interleukin 1β, and interleukin 18) in the placental tissue of women with LO-PE. Our results show a marked increase in all these components in the placentas of women who have undergone LO-PE, suggesting that NLRP3 inflammasome plays a potentially pathophysiological role in the development of this entity. Future works should aim to evaluate possible translational approaches to this dysregulation in these patients. Full article

(This article belongs to the Special Issue Placental-Related Disorders of Pregnancy)

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22 pages, 5322 KiB

Open AccessEditor’s ChoiceArticle

Proteome Profiling of the Dystrophic mdx Mice Diaphragm

by Olga Mucha, Małgorzata Myszka, Paulina Podkalicka, Bianka Świderska, Agata Malinowska, Józef Dulak and Agnieszka Łoboda

Biomolecules 2023, 13(11), 1648; https://doi.org/10.3390/biom13111648 - 13 Nov 2023

Cited by 2 | Viewed by 1482

Abstract

Mdx mice with a spontaneous mutation in exon 23 of the Dmd gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The disease, caused by the lack of functional dystrophin, is characterized by irreversible impairment of muscle [...] Read more.

Mdx mice with a spontaneous mutation in exon 23 of the Dmd gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The disease, caused by the lack of functional dystrophin, is characterized by irreversible impairment of muscle functions, with the diaphragm affected earlier and more severely than other skeletal muscles. We applied a label-free (LF) method and the more thorough tandem mass tag (TMT)-based method to analyze differentially expressed proteins in the diaphragm of 6-week-old mdx mice. The comparison of both methods revealed 88 commonly changed proteins. A more in-depth analysis of the TMT-based method showed 953 significantly changed proteins, with 867 increased and 86 decreased in dystrophic animals (q-value < 0.05, fold-change threshold: 1.5). Consequently, several dysregulated processes were demonstrated, including the immune response, fibrosis, translation, and programmed cell death. Interestingly, in the dystrophic diaphragm, we found a significant decrease in the expression of enzymes generating hydrogen sulfide (H₂S), suggesting that alterations in the metabolism of this gaseous mediator could modulate DMD progression, which could be a potential target for pharmacological intervention. Full article

(This article belongs to the Special Issue Muscular Dystrophy: From Molecular Basis to Therapies)

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14 pages, 1351 KiB

Open AccessEditor’s ChoiceArticle

Dynamics of Docosahexaenoic Acid Utilization by Mouse Peritoneal Macrophages

by Patricia Monge, Alma M. Astudillo, Laura Pereira, María A. Balboa and Jesús Balsinde

Biomolecules 2023, 13(11), 1635; https://doi.org/10.3390/biom13111635 - 10 Nov 2023

Cited by 1 | Viewed by 1336

Abstract

In this work, the incorporation of docosahexaenoic acid (DHA) in mouse resident peritoneal macrophages and its redistribution within the various phospholipid classes were investigated. Choline glycerophospholipids (PC) behaved as the major initial acceptors of DHA. Prolonged incubation with the fatty acid resulted in [...] Read more.

In this work, the incorporation of docosahexaenoic acid (DHA) in mouse resident peritoneal macrophages and its redistribution within the various phospholipid classes were investigated. Choline glycerophospholipids (PC) behaved as the major initial acceptors of DHA. Prolonged incubation with the fatty acid resulted in the transfer of DHA from PC to ethanolamine glycerophospholipids (PE), reflecting phospholipid remodeling. This process resulted in the cells containing similar amounts of DHA in PC and PE in the resting state. Mass spectrometry-based lipidomic analyses of phospholipid molecular species indicated a marked abundance of DHA in ether phospholipids. Stimulation of the macrophages with yeast-derived zymosan resulted in significant decreases in the levels of all DHA-containing PC and PI species; however, no PE or PS molecular species were found to decrease. In contrast, the levels of an unusual DHA-containing species, namely PI(20:4/22:6), which was barely present in resting cells, were found to markedly increase under zymosan stimulation. The levels of this phospholipid also significantly increased when the calcium-ionophore A23187 or platelet-activating factor were used instead of zymosan to stimulate the macrophages. The study of the route involved in the synthesis of PI(20:4/22:6) suggested that this species is produced through deacylation/reacylation reactions. These results define the increases in PI(20:4/22:6) as a novel lipid metabolic marker of mouse macrophage activation, and provide novel information to understand the regulation of phospholipid fatty acid turnover in activated macrophages. Full article

(This article belongs to the Collection Feature Papers in Biomacromolecules: Lipids)

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20 pages, 4921 KiB

Open AccessEditor’s ChoiceArticle

Assessing Genetic Algorithm-Based Docking Protocols for Prediction of Heparin Oligosaccharide Binding Geometries onto Proteins

by Samuel G. Holmes and Umesh R. Desai

Biomolecules 2023, 13(11), 1633; https://doi.org/10.3390/biom13111633 - 9 Nov 2023

Viewed by 2835

Abstract

Although molecular docking has evolved dramatically over the years, its application to glycosaminoglycans (GAGs) has remained challenging because of their intrinsic flexibility, highly anionic character and rather ill-defined site of binding on proteins. GAGs have been treated as either fully “rigid” or fully [...] Read more.

Although molecular docking has evolved dramatically over the years, its application to glycosaminoglycans (GAGs) has remained challenging because of their intrinsic flexibility, highly anionic character and rather ill-defined site of binding on proteins. GAGs have been treated as either fully “rigid” or fully “flexible” in molecular docking. We reasoned that an intermediate semi-rigid docking (SRD) protocol may be better for the recapitulation of native heparin/heparan sulfate (Hp/HS) topologies. Herein, we study 18 Hp/HS–protein co-complexes containing chains from disaccharide to decasaccharide using genetic algorithm-based docking with rigid, semi-rigid, and flexible docking protocols. Our work reveals that rigid and semi-rigid protocols recapitulate native poses for longer chains (5→10 mers) significantly better than the flexible protocol, while 2→4-mer poses are better predicted using the semi-rigid approach. More importantly, the semi-rigid docking protocol is likely to perform better when no crystal structure information is available. We also present a new parameter for parsing selective versus non-selective GAG–protein systems, which relies on two computational parameters including consistency of binding (i.e., RMSD) and docking score (i.e., GOLD Score). The new semi-rigid protocol in combination with the new computational parameter is expected to be particularly useful in high-throughput screening of GAG sequences for identifying promising druggable targets as well as drug-like Hp/HS sequences. Full article

(This article belongs to the Special Issue Computer Aided Drug Discovery)

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15 pages, 1038 KiB

Open AccessEditor’s ChoiceArticle

Bioactive Compounds from an Endophytic Pezicula sp. Showing Antagonistic Effects against the Ash Dieback Pathogen

by Özge Demir, Haoxuan Zeng, Barbara Schulz, Hedda Schrey, Michael Steinert, Marc Stadler and Frank Surup

Biomolecules 2023, 13(11), 1632; https://doi.org/10.3390/biom13111632 - 8 Nov 2023

Cited by 4 | Viewed by 1472

Abstract

A fungal endophyte originating from the Canary Islands was identified as a potent antagonist against the fungal phytopathogen Hymenoscyphus fraxineus, which causes the devastating ash dieback disease. This endophyte was tentatively identified as Pezicula cf. ericae, using molecular barcoding. Isolation of [...] Read more.

A fungal endophyte originating from the Canary Islands was identified as a potent antagonist against the fungal phytopathogen Hymenoscyphus fraxineus, which causes the devastating ash dieback disease. This endophyte was tentatively identified as Pezicula cf. ericae, using molecular barcoding. Isolation of secondary metabolites by preparative high-performance liquid chromatography (HPLC) yielded the known compounds CJ-17,572 (1), mycorrhizin A (3) and cryptosporioptides A–C (4–6), besides a new N-acetylated dihydroxyphenylalanin derivative 2, named peziculastatin. Planar structures were elucidated by NMR and HRMS data, while the relative stereochemistry of 2 was assigned by H,H and C,H coupling constants. The assignment of the unknown stereochemistry of CJ-17,572 (1) was hampered by the broadening of NMR signals. Nevertheless, after semisynthetic conversion of 1 into its methyl derivatives 7 and 8, presumably preventing tautomeric effects, the relative configuration could be assigned, whereas comparison of ECD data to those of related compounds determined the absolute configuration. Metabolites 1 and 3 showed significant antifungal effects in vitro against H. fraxineus. Furthermore, 4–6 exhibited significant dispersive effects on preformed biofilms of S. aureus at concentrations up to 2 µg/mL, while the biofilm formation of C. albicans was also inhibited. Thus, cryptosporioptides might constitute a potential source for the development of novel antibiofilm agents. Full article

(This article belongs to the Topic Natural Products and Drug Discovery)

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14 pages, 1854 KiB

Open AccessEditor’s ChoiceArticle

Identification of a Novel Acid Sphingomyelinase Activity Associated with Recombinant Human Acid Ceramidase

by Xingxuan He and Edward H. Schuchman

Biomolecules 2023, 13(11), 1623; https://doi.org/10.3390/biom13111623 - 6 Nov 2023

Viewed by 1561

Abstract

Acid ceramidase (AC) is a lysosomal enzyme required to hydrolyze ceramide to sphingosine by the removal of the fatty acid moiety. An inherited deficiency in this activity results in two disorders, Farber Lipogranulomatosis and spinal muscular atrophy with myoclonic epilepsy, leading to the [...] Read more.

Acid ceramidase (AC) is a lysosomal enzyme required to hydrolyze ceramide to sphingosine by the removal of the fatty acid moiety. An inherited deficiency in this activity results in two disorders, Farber Lipogranulomatosis and spinal muscular atrophy with myoclonic epilepsy, leading to the accumulation of ceramides and other sphingolipids in various cells and tissues. In addition to ceramide hydrolysis, several other activities have been attributed to AC, including a reverse reaction that synthesizes ceramide from free fatty acids and sphingosine, and a deacylase activity that removes fatty acids from complex lipids such as sphingomyelin and glycosphingolipids. A close association of AC with another important enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM), has also been observed. Herein, we used a highly purified recombinant human AC (rhAC) and novel UPLC-based assay methods to investigate the recently described deacylase activity of rhAC against three sphingolipid substrates, sphingomyelin, galactosyl- and glucosylceramide. No deacylase activities were detected using this method, although we did unexpectedly identify a significant ASM activity using natural (C-18) and artificial (Bodipy-C12) sphingomyelin substrates as well as the ASM-specific fluorogenic substrate, hexadecanoylamino-4-methylumbelliferyl phosphorylcholine (HMU-PC). We showed that this ASM activity was not due to contaminating, hamster-derived ASM in the rhAC preparation, and that the treatment of ASM-knockout mice with rhAC significantly reduced sphingomyelin storage in the liver. However, unlike the treatment with rhASM, this did not lead to elevated ceramide or sphingosine levels. Full article

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12 pages, 1287 KiB

Open AccessEditor’s ChoiceArticle

Association of Asymmetric and Symmetric Dimethylarginine with Inflammation in the Population-Based Study of Health in Pomerania

by Martin Sebastian Winkler, Martin Bahls, Rainer H. Böger, Till Ittermann, Marcus Dörr, Nele Friedrich and Edzard Schwedhelm

Biomolecules 2023, 13(11), 1612; https://doi.org/10.3390/biom13111612 - 4 Nov 2023

Cited by 1 | Viewed by 1222

Abstract

The amino acids arginine (Arg), asymmetric (ADMA) and symmetric dimethylarginine (SDMA) are related to nitric oxide (NO) metabolism and potential markers of two different disease entities: cardiovascular disease such as atherosclerosis and systemic inflammation in critically ill patients with sepsis. Although very different [...] Read more.

The amino acids arginine (Arg), asymmetric (ADMA) and symmetric dimethylarginine (SDMA) are related to nitric oxide (NO) metabolism and potential markers of two different disease entities: cardiovascular disease such as atherosclerosis and systemic inflammation in critically ill patients with sepsis. Although very different in their pathophysiological genesis, both entities involve the functional integrity of blood vessels. In this context, large population-based data associating NO metabolites with proinflammatory markers, e.g., white blood cell count (WBC), high-sensitivity C-reactive protein (hsCRP), and fibrinogen, or cytokines are sparse. We investigated the association of Arg, ADMA and SDMA with WBC, hsCRP, and fibrinogen in 3556 participants of the Study of Health in Pomerania (SHIP)-TREND study. Furthermore, in a subcohort of 456 subjects, 31 inflammatory markers and cytokines were analyzed. We identified Arg and SDMA to be positively associated with hsCRP (β coefficient 0.010, standard error (SE) 0.002 and 0.298, 0.137, respectively) as well as fibrinogen (β 5.23 × 10⁻³, SE 4.75 × 10⁻⁴ and 0.083, 0.031, respectively). ADMA was not associated with WBC, hsCRP, or fibrinogen. Furthermore, in the subcohort, Arg was inversely related to a proliferation-inducing ligand (APRIL). SDMA was positively associated with osteocalcin, tumor necrosis factor receptor 1 and 2, and soluble cluster of differentiation 30. Our findings provide new insights into the involvement of Arg, ADMA, and SDMA in subclinical inflammation in the general population. Full article

(This article belongs to the Special Issue Immune-Related Biomarkers: 2nd Edition)

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19 pages, 1844 KiB

Open AccessEditor’s ChoiceArticle

Sweet Orange Juice Processing By-Product Extracts: A Caries Management Alternative to Chlorhexidine

by Suvro Saha, Christine Boesch, Joanne Maycock, Simon Wood and Thuy Do

Biomolecules 2023, 13(11), 1607; https://doi.org/10.3390/biom13111607 - 2 Nov 2023

Viewed by 1327

Abstract

Dental caries is one of the most prevalent chronic diseases globally in both children and adults. This study investigated the potential of industrial sweet orange waste extracts (ISOWE) as a substitute for chlorhexidine (CHX) in managing dental caries. First, the cytotoxicity of ISOWE [...] Read more.

Dental caries is one of the most prevalent chronic diseases globally in both children and adults. This study investigated the potential of industrial sweet orange waste extracts (ISOWE) as a substitute for chlorhexidine (CHX) in managing dental caries. First, the cytotoxicity of ISOWE (40, 80, 120 mg/mL) and CHX (0.1 and 0.2%) on buccal epithelial cells was determined. ISOWE exhibited no overall toxicity, whereas CHX strongly affected cell viability. The combination of ISOWE and CHX significantly enhanced cell proliferation compared to CHX alone. Next, the antimicrobial efficacy of ISOWE, CHX, and their combination was assessed against a 7-day complex biofilm model inoculated with oral samples from human volunteers. CHX exhibited indiscriminate antimicrobial action, affecting both pathogenic and health-associated oral microorganisms. ISOWE demonstrated lower antimicrobial efficacy than CHX but showed enhanced efficacy against pathogenic species while preserving the oral microbiome’s balance. When applied to a cariogenic biofilm, the combined treatment of ISOWE with 0.1% CHX showed similar efficacy to 0.2% CHX treatment alone. Overall, the findings suggest that ISOWE is a promising natural anti-cariogenic agent with lower toxicity and enhanced selectivity for pathogenic species compared to CHX. Full article

(This article belongs to the Special Issue Recent Advances in Antimicrobial Agents)

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21 pages, 6484 KiB

Open AccessEditor’s ChoiceArticle

Untargeted Lipidomic Approach for Studying Different Nervous System Tissues of the Murine Model of Krabbe Disease

by Husam B. R. Alabed, Ambra Del Grosso, Valeria Bellani, Lorena Urbanelli, Sara Carpi, Miriam De Sarlo, Lorenzo Bertocci, Laura Colagiorgio, Sandra Buratta, Luca Scaccini, Dorotea Frongia Mancini, Ilaria Tonazzini, Marco Cecchini, Carla Emiliani and Roberto Maria Pellegrino

Biomolecules 2023, 13(10), 1562; https://doi.org/10.3390/biom13101562 - 23 Oct 2023

Cited by 4 | Viewed by 1761

Abstract

Krabbe disease is a rare neurodegenerative disease with an autosomal recessive character caused by a mutation in the GALC gene. The mutation leads to an accumulation of psychosine and a subsequent degeneration of oligodendrocytes and Schwann cells. Psychosine is the main biomarker of [...] Read more.

Krabbe disease is a rare neurodegenerative disease with an autosomal recessive character caused by a mutation in the GALC gene. The mutation leads to an accumulation of psychosine and a subsequent degeneration of oligodendrocytes and Schwann cells. Psychosine is the main biomarker of the disease. The Twitcher mouse is the most commonly used animal model to study Krabbe disease. Although there are many references to this model in the literature, the lipidomic study of nervous system tissues in the Twitcher model has received little attention. This study focuses on the comparison of the lipid profiles of four nervous system tissues (brain, cerebellum, spinal cord, and sciatic nerve) in the Twitcher mouse compared to the wild-type mouse. Altogether, approximately 230 molecular species belonging to 19 lipid classes were annotated and quantified. A comparison at the levels of class, molecular species, and lipid building blocks showed significant differences between the two groups, particularly in the sciatic nerve. The in-depth study of the lipid phenotype made it possible to hypothesize the genes and enzymes involved in the changes. The integration of metabolic data with genetic data may be useful from a systems biology perspective to gain a better understanding of the molecular basis of the disease. Full article

(This article belongs to the Special Issue Recent Advances in Neurological Diseases)

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15 pages, 4548 KiB

Open AccessEditor’s ChoiceArticle

Early Resveratrol Treatment Mitigates Joint Degeneration and Dampens Pain in a Mouse Model of Pseudoachondroplasia (PSACH)

by Jacqueline T. Hecht, Alka C. Veerisetty, Debabrata Patra, Mohammad G. Hossain, Frankie Chiu, Claire Mobed, Francis H. Gannon and Karen L. Posey

Biomolecules 2023, 13(10), 1553; https://doi.org/10.3390/biom13101553 - 20 Oct 2023

Cited by 2 | Viewed by 1488

Abstract

Pseudoachondroplasia (PSACH), a severe dwarfing condition associated with early-onset joint degeneration and lifelong joint pain, is caused by mutations in cartilage oligomeric matrix protein (COMP). The mechanisms underlying the mutant-COMP pathology have been defined using the MT-COMP mouse model of PSACH that has [...] Read more.

Pseudoachondroplasia (PSACH), a severe dwarfing condition associated with early-onset joint degeneration and lifelong joint pain, is caused by mutations in cartilage oligomeric matrix protein (COMP). The mechanisms underlying the mutant-COMP pathology have been defined using the MT-COMP mouse model of PSACH that has the common D469del mutation. Mutant-COMP protein does not fold properly, and it is retained in the rough endoplasmic reticulum (rER) of chondrocytes rather than being exported to the extracellular matrix (ECM), driving ER stress that stimulates oxidative stress and inflammation, driving a self-perpetuating cycle. CHOP (ER stress signaling protein) and TNFα inflammation drive high levels of mTORC1 signaling, shutting down autophagy and blocking ER clearance, resulting in premature loss of chondrocytes that negatively impacts linear growth and causes early joint degeneration in MT-COMP mice and PSACH. Previously, we have shown that resveratrol treatment from birth to 20 weeks prevents joint degeneration and decreases the pathological processes in articular chondrocytes. Resveratrol’s therapeutic mechanism of action in the mutant-COMP pathology was shown to act by primarily stimulating autophagy and reducing inflammation. Importantly, we demonstrated that MT-COMP mice experience pain consistent with PSACH joint pain. Here, we show, in the MT-COMP mouse, that resveratrol treatment must begin within 4 weeks to preserve joint health and reduce pain. Resveratrol treatment started at 6 or 8 weeks (to 20 weeks) was not effective in preventing joint degeneration. Collectively, our findings in MT-COMP mice show that there is a postnatal resveratrol treatment window wherein the inevitable mutant-COMP joint degeneration and pain can be prevented. Full article

(This article belongs to the Special Issue The Value of Natural Compounds as Therapeutic Agents)

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31 pages, 5364 KiB

Open AccessEditor’s ChoiceArticle

DUBing Primary Tumors of the Central Nervous System: Regulatory Roles of Deubiquitinases

by Thomas Klonisch, Susan E. Logue, Sabine Hombach-Klonisch and Jerry Vriend

Biomolecules 2023, 13(10), 1503; https://doi.org/10.3390/biom13101503 - 10 Oct 2023

Cited by 1 | Viewed by 1657

Abstract

The ubiquitin proteasome system (UPS) utilizes an orchestrated enzymatic cascade of E1, E2, and E3 ligases to add single or multiple ubiquitin-like molecules as post-translational modification (PTM) to proteins. Ubiquitination can alter protein functions and/or mark ubiquitinated proteins for proteasomal degradation but deubiquitinases [...] Read more.

The ubiquitin proteasome system (UPS) utilizes an orchestrated enzymatic cascade of E1, E2, and E3 ligases to add single or multiple ubiquitin-like molecules as post-translational modification (PTM) to proteins. Ubiquitination can alter protein functions and/or mark ubiquitinated proteins for proteasomal degradation but deubiquitinases (DUBs) can reverse protein ubiquitination. While the importance of DUBs as regulatory factors in the UPS is undisputed, many questions remain on DUB selectivity for protein targeting, their mechanism of action, and the impact of DUBs on the regulation of diverse biological processes. Furthermore, little is known about the expression and role of DUBs in tumors of the human central nervous system (CNS). In this comprehensive review, we have used publicly available transcriptional datasets to determine the gene expression profiles of 99 deubiquitinases (DUBs) from five major DUB families in seven primary pediatric and adult CNS tumor entities. Our analysis identified selected DUBs as potential new functional players and biomarkers with prognostic value in specific subtypes of primary CNS tumors. Collectively, our analysis highlights an emerging role for DUBs in regulating CNS tumor cell biology and offers a rationale for future therapeutic targeting of DUBs in CNS tumors. Full article

(This article belongs to the Special Issue Deubiquitinating Enzymes in Health and Disease)

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13 pages, 1969 KiB

Open AccessEditor’s ChoiceArticle

Outcomes and Adverse Effects of Voretigene Neparvovec Treatment for Biallelic RPE65-Mediated Inherited Retinal Dystrophies in a Cohort of Patients from a Single Center

by Peter Kiraly, Charles L. Cottriall, Laura J. Taylor, Jasleen K. Jolly, Jasmina Cehajic-Kapetanovic, Imran H. Yusuf, Cristina Martinez-Fernandez de la Camara, Morag Shanks, Susan M. Downes, Robert E. MacLaren and M. Dominik Fischer

Biomolecules 2023, 13(10), 1484; https://doi.org/10.3390/biom13101484 - 5 Oct 2023

Cited by 5 | Viewed by 1730

Abstract

Our study evaluated the morphological and functional outcomes, and the side effects, of voretigene neparvovec (VN) gene therapy for RPE65-mediated inherited retinal dystrophies (IRDs) in 12 eyes (six patients) at the Oxford Eye Hospital with a mean follow-up duration of 8.2 (range 1–12) [...] Read more.

Our study evaluated the morphological and functional outcomes, and the side effects, of voretigene neparvovec (VN) gene therapy for RPE65-mediated inherited retinal dystrophies (IRDs) in 12 eyes (six patients) at the Oxford Eye Hospital with a mean follow-up duration of 8.2 (range 1–12) months. All patients reported a subjective vision improvement 1 month after gene therapy. Best-corrected visual acuity (BCVA) remained stable (baseline: 1.28 (±0.71) vs. last follow-up: 1.46 (±0.60); p = 0.25). Average white Full-Field Stimulus Testing (FST) showed a trend towards improvement (baseline: −4.41 (±10.62) dB vs. last follow-up: −11.98 (±13.83) dB; p = 0.18). No changes in central retinal thickness or macular volume were observed. The side effects included mild intraocular inflammation (two eyes) and cataracts (four eyes). Retinal atrophy occurred in 10 eyes (eight mild, two severe) but did not impact FST measurements during the follow-up period. Increased intraocular pressure (IOP) was noted in three patients (six eyes); four eyes (two patients) required glaucoma surgery. The overall safety and effectiveness of VN treatment in our cohort align with previous VN clinical trials, except for the higher occurrence of retinal atrophy and increased IOP in our cohort. This suggests that raised IOP and retinal atrophy may be more common than previously reported. Full article

(This article belongs to the Special Issue Molecular Pathology, Diagnostics and Therapeutics of Retinal Diseases)

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25 pages, 8851 KiB

Open AccessEditor’s ChoiceArticle

Newborns with Favourable Outcomes after Perinatal Asphyxia Have Upregulated Glucose Metabolism-Related Proteins in Plasma

by Ping K. Yip, Michael Bremang, Ian Pike, Vennila Ponnusamy, Adina T. Michael-Titus and Divyen K. Shah

Biomolecules 2023, 13(10), 1471; https://doi.org/10.3390/biom13101471 - 30 Sep 2023

Viewed by 1257

Abstract

Hypoxic-ischaemic encephalopathy (HIE) is an important cause of morbidity and mortality globally. Although mild therapeutic hypothermia (TH) may improve outcomes in selected babies, the mechanism of action is not fully understood. A proteomics discovery study was carried out to analyse proteins in the [...] Read more.

Hypoxic-ischaemic encephalopathy (HIE) is an important cause of morbidity and mortality globally. Although mild therapeutic hypothermia (TH) may improve outcomes in selected babies, the mechanism of action is not fully understood. A proteomics discovery study was carried out to analyse proteins in the plasma of newborns with HIE. Proteomic analysis of plasma from 22 newborns with moderate-severe HIE that had initially undergone TH, and relative controls including 10 newborns with mild HIE who did not warrant TH and also cord blood from 10 normal births (non-HIE) were carried out using the isobaric Tandem Mass Tag (TMT^®) 10plex^TM labelling with tandem mass spectrometry. A total of 7818 unique peptides were identified in all TMT10plex^TM samples, translating to 3457 peptides representing 405 proteins, after applying stringent filter criteria. Apart from the unique protein signature from normal cord blood, unsupervised analysis revealed several significantly regulated proteins in the TH-treated moderate-severe HIE group. GO annotation and functional clustering revealed various proteins associated with glucose metabolism: the enzymes fructose-bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase 1, phosphoglycerate kinase 1, and pyruvate kinase PKM were upregulated in newborns with favourable (sHIE+) outcomes compared to newborns with unfavourable (sHIE−) outcomes. Those with favourable outcomes had normal MR imaging or mild abnormalities not predictive of adverse outcomes. However, in comparison to mild HIE and the sHIE− groups, the sHIE+ group had the additional glucose metabolism-related enzymes upregulated, including triosephosphate isomerase, α-enolase, 6-phosphogluconate dehydrogenase, transaldolase, and mitochondrial glutathione reductase. In conclusion, our plasma proteomic study demonstrates that TH-treated newborns with favourable outcomes have an upregulation in glucose metabolism. These findings may open new avenues for more effective neuroprotective therapy. Full article

(This article belongs to the Collection Feature Papers in 'Biomacromolecules: Proteins')

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14 pages, 1609 KiB

Open AccessEditor’s ChoiceArticle

Circulating Small Extracellular Vesicles Reflect the Severity of Myocardial Damage in STEMI Patients

by Marta Zarà, Andrea Baggiano, Patrizia Amadio, Jeness Campodonico, Sebastiano Gili, Andrea Annoni, Gianluca De Dona, Maria Ludovica Carerj, Francesco Cilia, Alberto Formenti, Laura Fusini, Cristina Banfi, Paola Gripari, Calogero Claudio Tedesco, Maria Elisabetta Mancini, Mattia Chiesa, Riccardo Maragna, Francesca Marchetti, Marco Penso, Luigi Tassetti, Alessandra Volpe, Alice Bonomi, Giancarlo Marenzi, Gianluca Pontone and Silvia Stella Barbieri Show full author list Hide full author list

Biomolecules 2023, 13(10), 1470; https://doi.org/10.3390/biom13101470 - 29 Sep 2023

Cited by 1 | Viewed by 1266

Abstract

Circulating small extracellular vesicles (sEVs) contribute to inflammation, coagulation and vascular injury, and have great potential as diagnostic markers of disease. The ability of sEVs to reflect myocardial damage assessed by Cardiac Magnetic Resonance (CMR) in ST-segment elevation myocardial infarction (STEMI) is unknown. [...] Read more.

Circulating small extracellular vesicles (sEVs) contribute to inflammation, coagulation and vascular injury, and have great potential as diagnostic markers of disease. The ability of sEVs to reflect myocardial damage assessed by Cardiac Magnetic Resonance (CMR) in ST-segment elevation myocardial infarction (STEMI) is unknown. To fill this gap, plasma sEVs were isolated from 42 STEMI patients treated by primary percutaneous coronary intervention (pPCI) and evaluated by CMR between days 3 and 6. Nanoparticle tracking analysis showed that sEVs were greater in patients with anterior STEMI (p = 0.0001), with the culprit lesion located in LAD (p = 0.045), and in those who underwent late revascularization (p = 0.038). A smaller sEV size was observed in patients with a low myocardial salvage index (MSI, p = 0.014). Patients with microvascular obstruction (MVO) had smaller sEVs (p < 0.002) and lower expression of the platelet marker CD41–CD61 (p = 0.039). sEV size and CD41–CD61 expression were independent predictors of MVO/MSI (OR [95% CI]: 0.93 [0.87–0.98] and 0.04 [0–0.61], respectively). In conclusion, we provide evidence that the CD41–CD61 expression in sEVs reflects the CMR-assessed ischemic damage after STEMI. This finding paves the way for the development of a new strategy for the timely identification of high-risk patients and their treatment optimization. Full article

(This article belongs to the Special Issue Extracellular Vesicles as Biomarkers of Diseases)

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21 pages, 5889 KiB

Open AccessEditor’s ChoiceArticle

Variations in O-Glycosylation Patterns Influence Viral Pathogenicity, Infectivity, and Transmissibility in SARS-CoV-2 Variants

by Sherifdeen Onigbinde, Cristian D. Gutierrez Reyes, Mojibola Fowowe, Oluwatosin Daramola, Mojgan Atashi, Andrew I. Bennett and Yehia Mechref

Biomolecules 2023, 13(10), 1467; https://doi.org/10.3390/biom13101467 - 29 Sep 2023

Cited by 5 | Viewed by 2200

Abstract

The highly glycosylated S protein plays a vital role in host cell invasion, making it the principal target for vaccine development. Differences in mutations observed on the spike (S) protein of SARS-CoV-2 variants may result in distinct glycosylation patterns, thus influencing immunological evasion, [...] Read more.

The highly glycosylated S protein plays a vital role in host cell invasion, making it the principal target for vaccine development. Differences in mutations observed on the spike (S) protein of SARS-CoV-2 variants may result in distinct glycosylation patterns, thus influencing immunological evasion, infectivity, and transmissibility. The glycans can mask key epitopes on the S1 protein and alter its structural conformation, allowing the virus to escape the immune system. Therefore, we comprehensively characterize O-glycosylation in eleven variants of SARS-CoV-2 S1 subunits to understand the differences observed in the biology of the variants. In-depth characterization was performed with a double digestion strategy and an efficient LC-MS/MS approach. We observed that O-glycosylation is highly conserved across all variants in the region between the NTD and RBD, whereas other domains and regions exhibit variation in O-glycosylation. Notably, omicron has the highest number of O-glycosylation sites on the S1 subunit. Also, omicron has the highest level of sialylation in the RBD and RBM functional motifs. Our findings may shed light on how differences in O-glycosylation impact viral pathogenicity in variants of SARS-CoV-2 and facilitate the development of a robust vaccine with high protective efficacy against the variants of concern. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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30 pages, 4569 KiB

Open AccessEditor’s ChoiceArticle

New Insights on Sperm Function in Male Infertility of Unknown Origin: A Multimodal Approach

by Rita I. Pacheco, Maria I. Cristo, Sandra I. Anjo, Andreia F. Silva, Maria Inês Sousa, Renata S. Tavares, Ana Paula Sousa, Teresa Almeida Santos, Mariana Moura-Ramos, Francisco Caramelo, Bruno Manadas, João Ramalho-Santos and Sandra Gomes Amaral

Biomolecules 2023, 13(10), 1462; https://doi.org/10.3390/biom13101462 - 27 Sep 2023

Viewed by 2377

Abstract

The global trend of rising (male) infertility is concerning, and the unidentifiable causes in half of the cases, the so-called unknown origin male infertility (UOMI), demands a better understanding and assessment of both external/internal factors and mechanisms potentially involved. In this work, it [...] Read more.

The global trend of rising (male) infertility is concerning, and the unidentifiable causes in half of the cases, the so-called unknown origin male infertility (UOMI), demands a better understanding and assessment of both external/internal factors and mechanisms potentially involved. In this work, it was our aim to obtain new insight on UOMI, specifically on idiopathic (ID) and Unexplained male infertility (UMI), relying on a detailed evaluation of the male gamete, including functional, metabolic and proteomic aspects. For this purpose, 1114 semen samples, from males in couples seeking infertility treatment, were collected at the Reproductive Medicine Unit from the Centro Hospitalar e Universitário de Coimbra (CHUC), from July 2018–July 2022. Based on the couples’ clinical data, seminal/hormonal analysis, and strict eligibility criteria, samples were categorized in 3 groups, control (CTRL), ID and UMI. Lifestyle factors and anxiety/depression symptoms were assessed via survey. Sperm samples were evaluated functionally, mitochondrially and using proteomics. The results of Assisted Reproduction Techniques were assessed whenever available. According to our results, ID patients presented the worst sperm functional profile, while UMI patients were similar to controls. The proteomic analysis revealed 145 differentially expressed proteins, 8 of which were specifically altered in ID and UMI samples. Acrosin (ACRO) and sperm acrosome membrane-associated protein 4 (SACA4) were downregulated in ID patients while laminin subunit beta-2 (LAMB2), mannose 6-phosphate isomerase (MPI), ATP-dependent 6-phosphofructokinase liver type (PFKAL), STAR domain-containing protein 10 (STA10), serotransferrin (TRFE) and exportin-2 (XPO2) were downregulated in UMI patients. Using random forest analysis, SACA4 and LAMB2 were identified as the sperm proteins with a higher chance of distinguishing ID and UMI patients, and their function and expression variation were in accordance with the functional results. No alterations were observed in terms of lifestyle and psychological factors among the 3 groups. These findings obtained in an experimental setting based on 3 well-defined groups of subjects, might help to validate new biomarkers for unknown origin male infertility (ID and UMI) that, in the future, can be used to improve diagnostics and treatments. Full article

(This article belongs to the Collection Feature Papers in Molecular Reproduction)

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25 pages, 3209 KiB

Open AccessEditor’s ChoiceArticle

Decanoic Acid Rescues Differences in AMPA-Mediated Calcium Rises in Hippocampal CA1 Astrocytes and Neurons in the 5xFAD Mouse Model of Alzheimer’s Disease

by Mina Abghari, Jenny Thythy Cecilia Mai Vu, Ninna Eckberg, Blanca I. Aldana and Kristi A. Kohlmeier

Biomolecules 2023, 13(10), 1461; https://doi.org/10.3390/biom13101461 - 27 Sep 2023

Cited by 1 | Viewed by 1419

Abstract

Alzheimer’s disease (AD), a devastating neurodegenerative disease characterized by cognitive dysfunctions, is associated with high levels of amyloid beta 42 (Aβ₄₂), which is believed to play a role in cellular damage and signaling changes in AD. Decanoic acid has been shown [...] Read more.

Alzheimer’s disease (AD), a devastating neurodegenerative disease characterized by cognitive dysfunctions, is associated with high levels of amyloid beta 42 (Aβ₄₂), which is believed to play a role in cellular damage and signaling changes in AD. Decanoic acid has been shown to be therapeutic in AD. Glutamatergic signaling within neurons and astrocytes of the CA1 region of the hippocampus is critical in cognitive processes, and previous work has indicated deficiencies in this signaling in a mouse model of AD. In this study, we investigated glutamate-mediated signaling by evaluating AMPA-mediated calcium rises in female and male CA1 neurons and astrocytes in a mouse model of AD and examined the potential of decanoic acid to normalize this signaling. In brain slices from 5xFAD mice in which there are five mutations leading to increasing levels of Aβ₄₂, AMPA-mediated calcium transients in CA1 neurons and astrocytes were significantly lower than that seen in wildtype controls in both females and males. Interestingly, incubation of 5xFAD slices in decanoic acid restored AMPA-mediated calcium levels in neurons and astrocytes in both females and males to levels indistinguishable from those seen in wildtype, whereas similar exposure to decanoic acid did not result in changes in AMPA-mediated transients in neurons or astrocytes in either sex in the wildtype. Our data indicate that one mechanism by which decanoic acid could improve cognitive functioning is through normalizing AMPA-mediated signaling in CA1 hippocampal cells. Full article

(This article belongs to the Special Issue Role of Amyloid Protein in Neurological Diseases)

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17 pages, 1085 KiB

Open AccessEditor’s ChoiceArticle

Neonatal Maternal Separation Induces Sexual Dimorphism in Brain Development: The Influence on Amino Acid Levels and Cognitive Disorders

by Jolanta H. Kotlinska, Pawel Grochecki, Agnieszka Michalak, Anna Pankowska, Katarzyna Kochalska, Piotr Suder, Joanna Ner-Kluza, Dariusz Matosiuk and Marta Marszalek-Grabska

Biomolecules 2023, 13(10), 1449; https://doi.org/10.3390/biom13101449 - 26 Sep 2023

Cited by 3 | Viewed by 1508

Abstract

Repeated maternal separation (MS) is a useful experimental model in rodents for studying the long-term influence of early-life stress on brain neurophysiology. In our work, we assessed the effect of repeated MS (postnatal day (PND)1–21, 180 min/day) on the postnatal development of rat [...] Read more.

Repeated maternal separation (MS) is a useful experimental model in rodents for studying the long-term influence of early-life stress on brain neurophysiology. In our work, we assessed the effect of repeated MS (postnatal day (PND)1–21, 180 min/day) on the postnatal development of rat brain regions involved in memory using proton magnetic resonance spectroscopy (¹HMRS) for tissue volume and the level of amino acids such as glutamate, aspartate, glutamine, glycine and gamma-aminobutyric acid (GABA) in the hippocampus. We assessed whether these effects are sex dependent. We also use novel object recognition (NOR) task to examine the effect of MS on memory and the effect of ethanol on it. Finally, we attempted to ameliorate postnatal stress-induced memory deficits by using VU-29, a positive allosteric modulator (PAM) of the metabotropic glutamate type 5 (mGlu5) receptor. In males, we noted deficits in the levels of glutamate, glycine and glutamine and increases in GABA in the hippocampus. In addition, the values of perirhinal cortex, prefrontal cortex and insular cortex and CA3 were decreased in these animals. MS females, in contrast, demonstrated significant increase in glutamate levels and decrease in GABA levels in the hippocampus. Here, the CA1 values alone were increased. VU-29 administration ameliorated these cognitive deficits. Thus, MS stress disturbs amino acids levels mainly in the hippocampus of adult male rats, and enhancement of glutamate neurotransmission reversed recognition memory deficits in these animals. Full article

(This article belongs to the Special Issue Glutamate and Glutamate Receptors in Health and Diseases)

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18 pages, 7637 KiB

Open AccessEditor’s ChoiceArticle

A Structural Model for the Core Nup358-BicD2 Interface

by James M. Gibson, Xiaoxin Zhao, M. Yusuf Ali, Sozanne R. Solmaz and Chunyu Wang

Biomolecules 2023, 13(10), 1445; https://doi.org/10.3390/biom13101445 - 26 Sep 2023

Cited by 1 | Viewed by 1689

Abstract

Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to several cellular cargo for transport, including Nup358, which facilitates a nuclear positioning pathway that is essential for the differentiation of distinct [...] Read more.

Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to several cellular cargo for transport, including Nup358, which facilitates a nuclear positioning pathway that is essential for the differentiation of distinct brain progenitor cells. Previously, we showed that Nup358 forms a “cargo recognition α-helix” upon binding to BicD2; however, the specifics of the BicD2-Nup358 interface are still not well understood. Here, we used AlphaFold2, complemented by two additional docking programs (HADDOCK and ClusPro) as well as mutagenesis, to show that the Nup358 cargo-recognition α-helix binds to BicD2 between residues 747 and 774 in an anti-parallel manner, forming a helical bundle. We identified two intermolecular salt bridges that are important to stabilize the interface. In addition, we uncovered a secondary interface mediated by an intrinsically disordered region of Nup358 that is directly N-terminal to the cargo-recognition α-helix and binds to BicD2 between residues 774 and 800. This is the same BicD2 domain that binds to the competing cargo adapter Rab6, which is important for the transport of Golgi-derived and secretory vesicles. Our results establish a structural basis for cargo recognition and selection by the dynein adapter BicD2, which facilitates transport pathways that are important for brain development. Full article

(This article belongs to the Collection Molecular Biology: Feature Papers)

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20 pages, 5133 KiB

Open AccessEditor’s ChoiceArticle

Functional, Morphological and Molecular Changes Reveal the Mechanisms Associated with Age-Related Vestibular Loss

by Vasiliki Georgia Paplou, Nick M. A. Schubert, Marcel van Tuinen, Sarath Vijayakumar and Sonja J. Pyott

Biomolecules 2023, 13(9), 1429; https://doi.org/10.3390/biom13091429 - 21 Sep 2023

Viewed by 1693

Abstract

Age-related loss of vestibular function and hearing are common disorders that arise from the loss of function of the inner ear and significantly decrease quality of life. The underlying pathophysiological mechanisms are poorly understood and difficult to investigate in humans. Therefore, our study [...] Read more.

Age-related loss of vestibular function and hearing are common disorders that arise from the loss of function of the inner ear and significantly decrease quality of life. The underlying pathophysiological mechanisms are poorly understood and difficult to investigate in humans. Therefore, our study examined young (1.5-month-old) and old (24-month-old) C57BL/6 mice, utilizing physiological, histological, and transcriptomic methods. Vestibular sensory-evoked potentials revealed that older mice had reduced wave I amplitudes and delayed wave I latencies, indicating reduced vestibular function. Immunofluorescence and image analysis revealed that older mice exhibited a significant decline in type I sensory hair cell density, particularly in hair cells connected to dimorphic vestibular afferents. An analysis of gene expression in the isolated vestibule revealed the upregulation of immune-related genes and the downregulation of genes associated with ossification and nervous system development. A comparison with the isolated cochlear sensorineural structures showed similar changes in genes related to immune response, chondrocyte differentiation, and myelin formation. These findings suggest that age-related vestibular hypofunction is linked to diminished peripheral vestibular responses, likely due to the loss of a specific subpopulation of hair cells and calyceal afferents. The upregulation of immune- and inflammation-related genes implies that inflammation contributes to these functional and structural changes. Furthermore, the comparison of gene expression between the vestibule and cochlea indicates both shared and distinct mechanisms contributing to age-related vestibular and hearing impairments. Further research is necessary to understand the mechanistic connection between inflammation and age-related balance and hearing disorders and to translate these findings into clinical treatment strategies. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms in Vestibular Disorders)

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16 pages, 750 KiB

Open AccessEditor’s ChoiceArticle

Plasma Brain-Derived Neurotrophic Factor Levels in First-Episode and Recurrent Major Depression and before and after Bright Light Therapy in Treatment-Resistant Depression

by Biljana Kosanovic Rajacic, Marina Sagud, Drazen Begic, Matea Nikolac Perkovic, Anja Dvojkovic, Lana Ganoci and Nela Pivac

Biomolecules 2023, 13(9), 1425; https://doi.org/10.3390/biom13091425 - 20 Sep 2023

Cited by 2 | Viewed by 1372

Abstract

Brain-derived neurotrophic factor (BDNF) is implicated in the etiology and treatment response in major depressive disorder (MDD). However, peripheral BDNF concentrations have not been compared across different MDD stages. Bright light therapy (BLT) offers some potential in treatment-resistant depression (TRD), but its effects [...] Read more.

Brain-derived neurotrophic factor (BDNF) is implicated in the etiology and treatment response in major depressive disorder (MDD). However, peripheral BDNF concentrations have not been compared across different MDD stages. Bright light therapy (BLT) offers some potential in treatment-resistant depression (TRD), but its effects on BDNF levels are unknown. This study included a cross-sectional analysis of plasma BDNF concentration in females with TRD, unmedicated MDD patients, and healthy controls (HC), and measurements of longitudinal BLT effects on plasma BDNF levels in TRD patients. The present study included 55 drug-naïve, first-episode patients, 25 drug-free recurrent-episode MDD patients, 71 HC participants, and 54 TRD patients. Patients were rated by Hamilton Depression Rating Scale (HAMD)-17 and the Montgomery–Åsberg Depression Rating Scale (MADRS). Patients with TRD received BLT during 4 weeks. The total HAMD-17 and MADRS scores decreased following BLT. All patient groups had lower plasma BDNF than HC, but BDNF levels did not differ between first- and recurrent-episode BDNF patients and TRD patients before or after BLT. However, responders and remitters to BLT had higher post-treatment plasma BDNF concentrations than patients who did not achieve response or remission. The changes in plasma BDNF levels may be candidates for biomarkers of treatment response to BLT in TRD patients. Full article

(This article belongs to the Special Issue Brain-Derived Neurotrophic Factor in Health and Diseases)

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15 pages, 2277 KiB

Open AccessEditor’s ChoiceArticle

Conservation of Glutathione Transferase mRNA and Protein Sequences Similar to Human and Horse Alpha Class GST A3-3 across Dog, Goat, and Opossum Species

by Shawna M. Hubert, Paul B. Samollow, Helena Lindström, Bengt Mannervik and Nancy H. Ing

Biomolecules 2023, 13(9), 1420; https://doi.org/10.3390/biom13091420 - 20 Sep 2023

Cited by 2 | Viewed by 1242

Abstract

The glutathione transferase A3-3 (GST A3-3) homodimeric enzyme is the most efficient enzyme that catalyzes isomerization of the precursors of testosterone, estradiol, and progesterone in the gonads of humans and horses. However, the presence of GST A3-3 orthologs with equally high ketosteroid isomerase [...] Read more.

The glutathione transferase A3-3 (GST A3-3) homodimeric enzyme is the most efficient enzyme that catalyzes isomerization of the precursors of testosterone, estradiol, and progesterone in the gonads of humans and horses. However, the presence of GST A3-3 orthologs with equally high ketosteroid isomerase activity has not been verified in other mammalian species, even though pig and cattle homologs have been cloned and studied. Identifying GSTA3 genes is a challenge because of multiple GSTA gene duplications (e.g., 12 in the human genome); consequently, the GSTA3 gene is not annotated in most genomes. To improve our understanding of GSTA3 gene products and their functions across diverse mammalian species, we cloned homologs of the horse and human GSTA3 mRNAs from the testes of a dog, goat, and gray short-tailed opossum, the genomes of which all currently lack GSTA3 gene annotations. The resultant novel GSTA3 mRNA and inferred protein sequences had a high level of conservation with human GSTA3 mRNA and protein sequences (≥70% and ≥64% identities, respectively). Sequence conservation was also apparent for the 12 residues of the “H-site” in the 222 amino acid GSTA3 protein that is known to interact with the steroid substrates. Modeling predicted that the dog GSTA3-3 may be a more active ketosteroid isomerase than the corresponding goat or opossum enzymes. However, expression of the GSTA3 gene was higher in liver than in other dog tissue. Our results improve understanding of the active sites of mammalian GST A3-3 enzymes, inhibitors of which might be useful for reducing steroidogenesis for medical purposes, such as fertility control or treatment of steroid-dependent diseases. Full article

(This article belongs to the Special Issue Versatility of Glutathione Transferase Proteins)

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17 pages, 6318 KiB

Open AccessEditor’s ChoiceArticle

N-Glycome Profile of the Spike Protein S1: Systemic and Comparative Analysis from Eleven Variants of SARS-CoV-2

by Cristian D. Gutierrez Reyes, Sherifdeen Onigbinde, Akeem Sanni, Andrew I. Bennett, Peilin Jiang, Oluwatosin Daramola, Parisa Ahmadi, Mojibola Fowowe, Mojgan Atashi, Vishal Sandilya, Md Abdul Hakim and Yehia Mechref

Biomolecules 2023, 13(9), 1421; https://doi.org/10.3390/biom13091421 - 20 Sep 2023

Cited by 4 | Viewed by 1593

Abstract

The SARS-CoV-2 virus rapidly spread worldwide, threatening public health. Since it emerged, the scientific community has been engaged in the development of effective therapeutics and vaccines. The subunit S1 in the spike protein of SARS-CoV-2 mediates the viral entry into the host and [...] Read more.

The SARS-CoV-2 virus rapidly spread worldwide, threatening public health. Since it emerged, the scientific community has been engaged in the development of effective therapeutics and vaccines. The subunit S1 in the spike protein of SARS-CoV-2 mediates the viral entry into the host and is therefore one of the major research targets. The S1 protein is extensively glycosylated, and there is compelling evidence that glycans protect the virus’ active site from the human defense system. Therefore, investigation of the S1 protein glycome alterations in the different virus variants will provide a view of the glycan evolution and its relationship with the virus pathogenesis. In this study, we explored the N-glycosylation expression of the S1 protein for eleven SARS-CoV-2 variants: five variants of concern (VOC), including alpha, beta, gamma, delta, and omicron, and six variants of interest (VOI), including epsilon, eta, iota, lambda, kappa, and mu. The results showed significant differences in the N-glycome abundance of all variants. The N-glycome of the VOC showed a large increase in the abundance of sialofucosylated glycans, with the greatest abundance in the omicron variant. In contrast, the results showed a large abundance of fucosylated glycans for most of the VOI. Two glycan compositions, GlcNAc₄,Hex₅,Fuc,NeuAc (4-5-1-1) and GlcNAc₆,Hex₈,Fuc,NeuAc (6-8-1-1), were the most abundant structures across all variants. We believe that our data will contribute to understanding the S1 protein’s structural differences between SARS-CoV-2 mutations. Full article

(This article belongs to the Section Biomacromolecules: Carbohydrates)

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15 pages, 6689 KiB

Open AccessEditor’s ChoiceArticle

The Effects of an Osteoarthritic Joint Environment on ACL Damage and Degeneration: A Yucatan Miniature Pig Model

by Elias Schwartz, Kenny Chang, Changqi Sun, Fei Zhang, Guoxuan Peng, Brett Owens and Lei Wei

Biomolecules 2023, 13(9), 1416; https://doi.org/10.3390/biom13091416 - 20 Sep 2023

Viewed by 1110

Abstract

Posttraumatic osteoarthritis (PTOA) arises secondary to joint injuries and is characteristically driven by inflammatory mediators. PTOA is often studied in the setting of ACL tears. However, a wide range of other injuries also lead to PTOA pathogenesis. The purpose of this study was [...] Read more.

Posttraumatic osteoarthritis (PTOA) arises secondary to joint injuries and is characteristically driven by inflammatory mediators. PTOA is often studied in the setting of ACL tears. However, a wide range of other injuries also lead to PTOA pathogenesis. The purpose of this study was to characterize the morphological changes in the uninjured ACL in a PTOA inflammatory environment. We retrospectively reviewed 14 ACLs from 13 Yucatan minipigs, 7 of which had undergone our modified intra-articular drilling (mIAD) procedure, which induced PTOA through inflammatory mediators. Seven ACLs were harvested from mIAD minipigs (PTOA) and seven ACLs from control minipigs with no cartilage degeneration (non-PTOA). ACL degeneration was evaluated using histological scoring systems. IL-1β, NF-κB, and TNF-α mRNA expression in the synovium was measured using qRT-PCR. PTOA minipigs demonstrated significant ACL degeneration, marked by a disorganized extracellular matrix, increased vascularity, and changes in cellular shape, density, and alignment. Furthermore, IL-1β, NF-κB, and TNF-α expression was elevated in the synovium of PTOA minipigs. These findings demonstrate the potential for ACL degeneration in a PTOA environment and emphasize the need for anti-inflammatory disease-modifying therapies following joint injury. Full article

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

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16 pages, 2055 KiB

Open AccessEditor’s ChoiceArticle

Eicosapentaenoic Acid Influences the Lipid Profile of an In Vitro Psoriatic Skin Model Produced with T Cells

by Sophie Morin, Andréa Tremblay, Elizabeth Dumais, Pierre Julien, Nicolas Flamand and Roxane Pouliot

Biomolecules 2023, 13(9), 1413; https://doi.org/10.3390/biom13091413 - 19 Sep 2023

Cited by 2 | Viewed by 1612

Abstract

Psoriasis is a skin disease characterized by epidermal hyperplasia and an inappropriate activation of the adaptive immunity. A dysregulation of the skin’s lipid mediators is reported in the disease with a predominance of the inflammatory cascade derived from n-6 polyunsaturated fatty acids (n-6 [...] Read more.

Psoriasis is a skin disease characterized by epidermal hyperplasia and an inappropriate activation of the adaptive immunity. A dysregulation of the skin’s lipid mediators is reported in the disease with a predominance of the inflammatory cascade derived from n-6 polyunsaturated fatty acids (n-6 PUFAs). Bioactive lipid mediators derived from arachidonic acid (AA) are involved in the inflammatory functions of T cells in psoriasis, whereas n-3 PUFAs’ derivatives are anti-inflammatory metabolites. Here, we sought to evaluate the influence of a supplementation of the culture media with eicosapentaenoic acid (EPA) on the lipid profile of a psoriatic skin model produced with polarized T cells. Healthy and psoriatic skin substitutes were produced following the auto-assembly technique. Psoriatic skin substitutes produced with or without T cells presented increased epidermal and dermal linolenic acid (LA) and AA levels. N-6 PUFA lipid mediators were strongly measured in psoriatic substitutes, namely, 13-hydroxyoctadecadienoic acid (13-HODE), prostaglandin E₂ (PGE₂) and 12-hydroxyeicosatetraenoic acid (12-HETE). The added EPA elevated the amounts of EPA, n-3 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in the epidermal and dermal phospholipids. The EPA supplementation balanced the production of epidermal lipid mediators, with an increase in prostaglandin E₃ (PGE₃), 12-hydroxyeicosapentaenoic acid (12-HEPE) and N-eicosapentaenoyl-ethanolamine (EPEA) levels. These findings show that EPA modulates the lipid composition of psoriatic skin substitutes by encouraging the return to a cutaneous homeostatic state. Full article

(This article belongs to the Special Issue Lipid Metabolism in Health and Disease 2023)

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14 pages, 2356 KiB

Open AccessEditor’s ChoiceArticle

Phytocannabinoids Reduce Seizures in Larval Zebrafish and Affect Endocannabinoid Gene Expression

by Roshni Kollipara, Evan Langille, Cameron Tobin and Curtis R. French

Biomolecules 2023, 13(9), 1398; https://doi.org/10.3390/biom13091398 - 16 Sep 2023

Cited by 2 | Viewed by 1341

Abstract

Cannabis has demonstrated anticonvulsant properties, and about thirty percent of epileptic patients do not have satisfactory seizure management with standard treatment and could potentially benefit from cannabis-based intervention. Here, we report the use of cannabinoids to treat pentylenetetrazol (PTZ)-induced convulsions in a zebrafish [...] Read more.

Cannabis has demonstrated anticonvulsant properties, and about thirty percent of epileptic patients do not have satisfactory seizure management with standard treatment and could potentially benefit from cannabis-based intervention. Here, we report the use of cannabinoids to treat pentylenetetrazol (PTZ)-induced convulsions in a zebrafish model, their effect on gene expression, and a simple assay for assessing their uptake in zebrafish tissues. Using an optimized behavioral assay, we show that cannabidiol (CBD) and cannabichromene (CBC) and cannabinol (CBN) are effective at reducing seizures at low doses, with little evidence of sedation, and our novel HPLC assay indicates that CBC is effective with the lowest accumulation in larval tissues. All cannabinoids tested were effective at higher concentrations. Pharmacological manipulation of potential receptors demonstrates that Gpr55 partially mediates the anticonvulsant effects of CBD. Treatment of zebrafish larvae with endocannabinoids, such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA), altered larvae movement, and the expression of genes that regulate their metabolism was affected by phytocannabinoid treatment, highlighting the possibility that changes to endocannabinoid levels may represent one facet of the anticonvulsant effect of phytocannabinoids. Full article

(This article belongs to the Section Molecular Genetics)

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17 pages, 2847 KiB

Open AccessEditor’s ChoiceArticle

Patients with Systemic Juvenile Idiopathic Arthritis (SJIA) Show Differences in Autoantibody Signatures Based on Disease Activity

by Julie Krainer, Michaela Hendling, Sandra Siebenhandl, Sabrina Fuehner, Christoph Kessel, Emely Verweyen, Klemens Vierlinger, Dirk Foell, Silvia Schönthaler and Andreas Weinhäusel

Biomolecules 2023, 13(9), 1392; https://doi.org/10.3390/biom13091392 - 15 Sep 2023

Viewed by 3232

Abstract

Systemic juvenile idiopathic arthritis (SJIA) is a severe rheumatic disease in children. It is a subgroup of juvenile idiopathic arthritis (JIA; MIM #604302), which is the most common rheumatic disease in children. The diagnosis of SJIA often comes with a significant delay, and [...] Read more.

Systemic juvenile idiopathic arthritis (SJIA) is a severe rheumatic disease in children. It is a subgroup of juvenile idiopathic arthritis (JIA; MIM #604302), which is the most common rheumatic disease in children. The diagnosis of SJIA often comes with a significant delay, and the classification between autoinflammatory and autoimmune disease is still discussed. In this study, we analyzed the immunological responses of patients with SJIA, using human proteome arrays presenting immobilized recombinantly expressed human proteins, to analyze the involvement of autoantibodies in SJIA. Results from group comparisons show several differentially reactive antigens involved in inflammatory processes. Intriguingly, many of the identified antigens had a high reactivity against proteins involved in the NF-

κ

B pathway, and it is also notable that many of the detected DIRAGs are described as dysregulated in rheumatoid arthritis. Our data highlight novel proteins and pathways potentially dysregulated in SJIA and offer a unique approach to unraveling the underlying disease pathogenesis in this chronic arthropathy. Full article

(This article belongs to the Special Issue Novel Insights into the Role of Autoantibodies in Diseases)

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14 pages, 1815 KiB

Open AccessEditor’s ChoiceArticle

Changes in Expression in BMP2 and Two Closely Related Genes in Guinea Pig Retinal Pigment Epithelium during Induction and Recovery from Myopia

by So Goto, Yan Zhang, Sonal Aswin Vyas, Qiurong Zhu and Christine F. Wildsoet

Biomolecules 2023, 13(9), 1373; https://doi.org/10.3390/biom13091373 - 11 Sep 2023

Viewed by 1324

Abstract

Purpose: We previously reported differential gene expression of the bone morphogenetic protein 2 (Bmp2) in guinea pig retinal pigment epithelium (RPE) after 1 day of hyperopic defocus, imposed with a negative contact lens (CLs). The study reported here sought to obtain [...] Read more.

Purpose: We previously reported differential gene expression of the bone morphogenetic protein 2 (Bmp2) in guinea pig retinal pigment epithelium (RPE) after 1 day of hyperopic defocus, imposed with a negative contact lens (CLs). The study reported here sought to obtain insights into the temporal profiles of gene expression changes in Bmp2, as well as those of two closely related genes, the inhibitor of DNA binding 3 (Id3) and Noggin (Nog), both during myopia induction and when the CL treatment was terminated to allow recovery from induced myopia. Methods: To induce myopia, 2-week-old pigmented guinea pigs (New Zealand strain, n = 8) wore monocular −10 diopter (D) rigid gas-permeable (RGP) CLs for one week, while the other eye served as a control. Ocular measurements were made at baseline, 3 days, and 7 days after the initiation of CL wear, with treatment then being terminated and additional measurements being made after a further 3 days, 1 week, and 2 weeks. Spherical equivalent refractive errors (SERs), axial length (AL), choroidal thickness (ChT), and scleral thickness (ScT) data were collected using retinoscopy, optical biometry (Lenstar), and spectral domain optical coherence tomography (SD-OCT), respectively. RPE samples were collected from both eyes of the guinea pigs after either 1 day or 1 week of CL wear or 1 day or 2 weeks after its termination, and RNA was subsequently isolated and subjected to quantitative real-time PCR (qRT-PCR) analyses, targeting the Bmp2, Id3, and Nog genes. Results: Mean interocular differences (treated—control) in AL and SER were significantly different from baseline after 3 and 7 days of CL wear, consistent with induced myopia (p < 0.001 for all cases). Termination of CL wear resulted in the normalization (i.e., recovery) of the ALs and SERs of the treated eyes within 7 days, and the earlier significant ChT thinning with CL wear (p = 0004, day 7) was replaced by rapid thickening, which remained significant on day 7 (p = 0.009) but had normalized by day 14. The ChT changes were much smaller in magnitude than the AL changes in both phases. Interocular differences in the ScT showed no significant changes. The Bmp2 and Id3 genes were both significantly downregulated with CL wear, after 1 day (p = 0.012 and 0.016) and 7 days (p = 0.002 and 0.005), while Bmp2 gene expression increased and Nog gene expression decreased after the termination of CL wear, albeit transiently, which was significant on 1 day (p = 0.004 and 0.04) but not 2 weeks later. No change in Id3 gene expression was observed over the latter period. Conclusions: The above patterns of myopia induction and recovery validate this negative RGP-CL model as an alternative to traditional spectacle lens models for guinea pigs. The defocus-driven, sign-dependent changes in the expression of the Bmp2 gene in guinea pig RPE are consistent with observations in chicks and demonstrate the important role of BMP2 in eye growth regulation. Full article

(This article belongs to the Special Issue New Insights into the Molecular Mechanisms of Myopia and Glaucoma)

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17 pages, 2229 KiB

Open AccessEditor’s ChoiceArticle

The Properties and Domain Requirements for Phase Separation of the Sup35 Prion Protein In Vivo

by Bryan Grimes, Walter Jacob, Amanda R. Liberman, Nathan Kim, Xiaohong Zhao, Daniel C. Masison and Lois E. Greene

Biomolecules 2023, 13(9), 1370; https://doi.org/10.3390/biom13091370 - 10 Sep 2023

Cited by 1 | Viewed by 1768

Abstract

The Sup35 prion protein of budding yeast has been reported to undergo phase separation to form liquid droplets both at low pH in vitro and when energy depletion decreases the intracellular pH in vivo. It also has been shown using purified proteins that [...] Read more.

The Sup35 prion protein of budding yeast has been reported to undergo phase separation to form liquid droplets both at low pH in vitro and when energy depletion decreases the intracellular pH in vivo. It also has been shown using purified proteins that this phase separation is driven by the prion domain of Sup35 and does not re-quire its C-terminal domain. In contrast, we now find that a Sup35 fragment consisting of only the N-terminal prion domain and the M-domain does not phase separate in vivo; this phase separation of Sup35 requires the C-terminal domain, which binds Sup45 to form the translation termination complex. The phase-separated Sup35 not only colocalizes with Sup45 but also with Pub1, a stress granule marker protein. In addition, like stress granules, phase separation of Sup35 appears to require mRNA since cycloheximide treatment, which inhibits mRNA release from ribosomes, prevents phase separation of Sup35. Finally, unlike Sup35 in vitro, Sup35 condensates do not disassemble in vivo when the intracellular pH is increased. These results suggest that, in energy-depleted cells, Sup35 forms supramolecular assemblies that differ from the Sup35 liquid droplets that form in vitro. Full article

(This article belongs to the Section Molecular Structure and Dynamics)

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17 pages, 2907 KiB

Open AccessEditor’s ChoiceArticle

Structure–Activity Relationships of Low Molecular Weight Alginate Oligosaccharide Therapy against Pseudomonas aeruginosa

by Manon F. Pritchard, Lydia C. Powell, Jennifer Y. M. Adams, Georgina Menzies, Saira Khan, Anne Tøndervik, Håvard Sletta, Olav Aarstad, Gudmund Skjåk-Bræk, Stephen McKenna, Niklaas J. Buurma, Damian J. J. Farnell, Philip D. Rye, Katja E. Hill and David W. Thomas

Biomolecules 2023, 13(9), 1366; https://doi.org/10.3390/biom13091366 - 8 Sep 2023

Cited by 1 | Viewed by 1561

Abstract

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa. Previous studies suggested that the disruption of calcium (Ca²⁺)–DNA binding within bacterial biofilms and dysregulation of quorum sensing [...] Read more.

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa. Previous studies suggested that the disruption of calcium (Ca²⁺)–DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca²⁺ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca²⁺ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca²⁺-dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures. Full article

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17 pages, 3685 KiB

Open AccessEditor’s ChoiceArticle

AAV-Mediated Targeting of the Activin A-ACVR1^R206H Signaling in Fibrodysplasia Ossificans Progressiva

by Yeon-Suk Yang, Chujiao Lin, Hong Ma, Jun Xie, Frederick S. Kaplan, Guangping Gao and Jae-Hyuck Shim

Biomolecules 2023, 13(9), 1364; https://doi.org/10.3390/biom13091364 - 8 Sep 2023

Cited by 5 | Viewed by 2115

Abstract

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by progressive disabling heterotopic ossification (HO) at extra-skeletal sites. Here, we developed adeno-associated virus (AAV)-based gene therapy that suppresses trauma-induced HO in FOP mice harboring a heterozygous allele of human ACVR1^R206H ( [...] Read more.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by progressive disabling heterotopic ossification (HO) at extra-skeletal sites. Here, we developed adeno-associated virus (AAV)-based gene therapy that suppresses trauma-induced HO in FOP mice harboring a heterozygous allele of human ACVR1^R206H (Acvr1^R206H/+) while limiting the expression in non-skeletal organs such as the brain, heart, lung, liver, and kidney. AAV gene therapy carrying the combination of codon-optimized human ACVR1 (ACVR1^opt) and artificial miRNAs targeting Activin A and its receptor ACVR1^R206H ablated the aberrant activation of BMP-Smad1/5 signaling and the osteogenic differentiation of Acvr1^R206H/+ skeletal progenitors. The local delivery of AAV gene therapy to HO-causing cells in the skeletal muscle resulted in a significant decrease in endochondral bone formation in Acvr1^R206H/+ mice. These mice showed little to no expression in a major AAV-targeted organ, the liver, due to liver-abundant miR-122-mediated repression. Thus, AAV gene therapy is a promising therapeutic strategy to explore in suppressing HO in FOP. Full article

(This article belongs to the Special Issue Fibrodysplasia Ossificans Progressiva (FOP): From Molecular Mechanisms to Therapeutic Strategies)

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20 pages, 2953 KiB

Open AccessEditor’s ChoiceArticle

The Potential Mechanisms behind Loperamide-Induced Cardiac Arrhythmias Associated with Human Abuse and Extreme Overdose

by Hua Rong Lu, Bruce P. Damiano, Mohamed Kreir, Jutta Rohrbacher, Henk van der Linde, Tamerlan Saidov, Ard Teisman and David J. Gallacher

Biomolecules 2023, 13(9), 1355; https://doi.org/10.3390/biom13091355 - 6 Sep 2023

Cited by 1 | Viewed by 2556

Abstract

Loperamide has been a safe and effective treatment for diarrhea for many years. However, many cases of cardiotoxicity with intentional abuse of loperamide ingestion have recently been reported. We evaluated loperamide in in vitro and in vivo cardiac safety models to understand the [...] Read more.

Loperamide has been a safe and effective treatment for diarrhea for many years. However, many cases of cardiotoxicity with intentional abuse of loperamide ingestion have recently been reported. We evaluated loperamide in in vitro and in vivo cardiac safety models to understand the mechanisms for this cardiotoxicity. Loperamide slowed conduction (QRS-duration) starting at 0.3 µM [~1200-fold (×) its human Free Therapeutic Plasma Concentration; FTPC] and reduced the QT-interval and caused cardiac arrhythmias starting at 3 µM (~12,000× FTPC) in an isolated rabbit ventricular-wedge model. Loperamide also slowed conduction and elicited Type II/III A-V block in anesthetized guinea pigs at overdose exposures of 879× and 3802× FTPC. In ion-channel studies, loperamide inhibited hERG (I_Kr)_, I_Na, and I_Ca currents with IC₅₀ values of 0.390 µM, 0.526 µM, and 4.091 µM, respectively (i.e., >1560× FTPC). Additionally, in silico trials in human ventricular action potential models based on these IC50s confirmed that loperamide has large safety margins at therapeutic exposures (≤600× FTPC) and confirmed repolarization abnormalities in the case of extreme doses of loperamide. The studies confirmed the large safety margin for the therapeutic use of loperamide but revealed that at the extreme exposure levels observed in human overdose, loperamide can cause a combination of conduction slowing and alterations in repolarization time, resulting in cardiac proarrhythmia. Loperamide’s inhibition of the I_Na channel and hERG-mediated IKr are the most likely basis for this cardiac electrophysiological toxicity at overdose exposures. The cardiac toxic effects of loperamide at the overdoses could be aggravated by co-medication with other drug(s) causing ion channel inhibition. Full article

(This article belongs to the Special Issue Pharmacology of Cardiovascular Disease)

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22 pages, 26292 KiB

Open AccessEditor’s ChoiceArticle

ABCD1 Transporter Deficiency Results in Altered Cholesterol Homeostasis

by Agnieszka Buda, Sonja Forss-Petter, Rong Hua, Yorrick Jaspers, Mark Lassnig, Petra Waidhofer-Söllner, Stephan Kemp, Peter Kim, Isabelle Weinhofer and Johannes Berger

Biomolecules 2023, 13(9), 1333; https://doi.org/10.3390/biom13091333 - 31 Aug 2023

Cited by 3 | Viewed by 2145

Abstract

X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the peroxisomal transporter ABCD1, resulting in the accumulation of very long-chain fatty acids (VLCFA). Strongly affected cell types, such as oligodendrocytes, adrenocortical cells and macrophages, exhibit high cholesterol turnover. Here, [...] Read more.

X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the peroxisomal transporter ABCD1, resulting in the accumulation of very long-chain fatty acids (VLCFA). Strongly affected cell types, such as oligodendrocytes, adrenocortical cells and macrophages, exhibit high cholesterol turnover. Here, we investigated how ABCD1 deficiency affects cholesterol metabolism in human X-ALD patient-derived fibroblasts and CNS tissues of Abcd1-deficient mice. Lipidome analyses revealed increased levels of cholesterol esters (CE), containing both saturated VLCFA and mono/polyunsaturated (V)LCFA. The elevated CE(26:0) and CE(26:1) levels remained unchanged in LXR agonist-treated Abcd1 KO mice despite reduced total C26:0. Under high-cholesterol loading, gene expression of SOAT1, converting cholesterol to CE and lipid droplet formation were increased in human X-ALD fibroblasts versus healthy control fibroblasts. However, the expression of NCEH1, catalysing CE hydrolysis and the cholesterol transporter ABCA1 and cholesterol efflux were also upregulated. Elevated Soat1 and Abca1 expression and lipid droplet content were confirmed in the spinal cord of X-ALD mice, where expression of the CNS cholesterol transporter Apoe was also elevated. The extent of peroxisome-lipid droplet co-localisation appeared low and was not impaired by ABCD1-deficiency in cholesterol-loaded primary fibroblasts. Finally, addressing steroidogenesis, progesterone-induced cortisol release was amplified in X-ALD fibroblasts. These results link VLCFA to cholesterol homeostasis and justify further consideration of therapeutic approaches towards reducing VLCFA and cholesterol levels in X-ALD. Full article

(This article belongs to the Special Issue The Role of Peroxisome in Cell Functions and Pathological Consequences of Its Dysfunction)

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21 pages, 3006 KiB

Open AccessEditor’s ChoiceArticle

Neuroactive Steroid–Gut Microbiota Interaction in T2DM Diabetic Encephalopathy

by Silvia Diviccaro, Lucia Cioffi, Rocco Piazza, Donatella Caruso, Roberto Cosimo Melcangi and Silvia Giatti

Biomolecules 2023, 13(9), 1325; https://doi.org/10.3390/biom13091325 - 29 Aug 2023

Viewed by 1378

Abstract

The pathological consequences of type 2 diabetes mellitus (T2DM) also involve the central nervous system; indeed, T2DM patients suffer from learning and memory disabilities with a higher risk of developing dementia. Although several factors have been proposed as possible contributors, how neuroactive steroids [...] Read more.

The pathological consequences of type 2 diabetes mellitus (T2DM) also involve the central nervous system; indeed, T2DM patients suffer from learning and memory disabilities with a higher risk of developing dementia. Although several factors have been proposed as possible contributors, how neuroactive steroids and the gut microbiome impact brain pathophysiology in T2DM remain unexplored. On this basis, in male Zucker diabetic fatty (ZDF) rats, we studied whether T2DM alters memory abilities using the novel object recognition test, neuroactive steroid levels by liquid chromatography–tandem mass spectrometry, hippocampal parameters using molecular assessments, and gut microbiome composition using 16S next-generation sequencing. Results obtained reveal that T2DM worsens memory abilities and that these are correlated with increased levels of corticosterone in plasma and with a decrease in allopregnanolone in the hippocampus, where neuroinflammation, oxidative stress, and mitochondrial dysfunction were reported. Interestingly, our analysis highlighted a small group of taxa strictly related to both memory impairment and neuroactive steroid levels. Overall, the data underline an interesting role for allopregnanolone and microbiota that may represent candidates for the development of therapeutic strategies. Full article

(This article belongs to the Special Issue Role of Neuroactive Steroids in Health and Disease)

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15 pages, 2820 KiB

Open AccessEditor’s ChoiceArticle

Chronic Aripiprazole and Trazodone Polypharmacy Effects on Systemic and Brain Cholesterol Biosynthesis

by Zeljka Korade, Allison Anderson, Marta Balog, Keri A. Tallman, Ned A. Porter and Karoly Mirnics

Biomolecules 2023, 13(9), 1321; https://doi.org/10.3390/biom13091321 - 28 Aug 2023

Cited by 1 | Viewed by 2719

Abstract

The concurrent use of several medications is a common practice in the treatment of complex psychiatric conditions. One such commonly used combination is aripiprazole (ARI), an antipsychotic, and trazodone (TRZ), an antidepressant. In addition to their effects on dopamine and serotonin systems, both [...] Read more.

The concurrent use of several medications is a common practice in the treatment of complex psychiatric conditions. One such commonly used combination is aripiprazole (ARI), an antipsychotic, and trazodone (TRZ), an antidepressant. In addition to their effects on dopamine and serotonin systems, both of these compounds are inhibitors of the 7-dehydrocholesterol reductase (DHCR7) enzyme. To evaluate the systemic and nervous system distribution of ARI and TRZ and their effects on cholesterol biosynthesis, adult mice were treated with both ARI and TRZ for 21 days. The parent drugs, their metabolites, and sterols were analyzed in the brain and various organs of mice using LC-MS/MS. The analyses revealed that ARI, TRZ, and their metabolites were readily detectable in the brain and organs, leading to changes in the sterol profile. The levels of medications, their metabolites, and sterols differed across tissues with notable sex differences. Female mice showed higher turnover of ARI and more cholesterol clearance in the brain, with several post-lanosterol intermediates significantly altered. In addition to interfering with sterol biosynthesis, ARI and TRZ exposure led to decreased ionized calcium-binding adaptor molecule 1 (IBA1) and increased DHCR7 protein expression in the cortex. Changes in sterol profile have been also identified in the spleen, liver, and serum, underscoring the systemic effect of ARI and TRZ on sterol biosynthesis. Long-term use of concurrent ARI and TRZ warrants further studies to fully evaluate the lasting consequences of altered sterol biosynthesis on the whole body. Full article

(This article belongs to the Special Issue Brain Sterols: Biosynthesis and Physiology in Health and Disease)

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18 pages, 4099 KiB

Open AccessEditor’s ChoiceArticle

Pegylated Liposomal Alendronate Biodistribution, Immune Modulation, and Tumor Growth Inhibition in a Murine Melanoma Model

by Md. Rakibul Islam, Jalpa Patel, Patricia Ines Back, Hilary Shmeeda, Raja Reddy Kallem, Claire Shudde, Maciej Markiewski, William C. Putnam, Alberto A. Gabizon and Ninh M. La-Beck

Biomolecules 2023, 13(9), 1309; https://doi.org/10.3390/biom13091309 - 26 Aug 2023

Cited by 2 | Viewed by 1940

Abstract

While tumor-associated macrophages (TAM) have pro-tumoral activity, the ablation of macrophages in cancer may be undesirable since they also have anti-tumoral functions, including T cell priming and activation against tumor antigens. Alendronate is a potent amino-bisphosphonate that modulates the function of macrophages in [...] Read more.

While tumor-associated macrophages (TAM) have pro-tumoral activity, the ablation of macrophages in cancer may be undesirable since they also have anti-tumoral functions, including T cell priming and activation against tumor antigens. Alendronate is a potent amino-bisphosphonate that modulates the function of macrophages in vitro, with potential as an immunotherapy if its low systemic bioavailability can be addressed. We repurposed alendronate in a non-leaky and long-circulating liposomal carrier similar to that of the clinically approved pegylated liposomal doxorubicin to facilitate rapid clinical translation. Here, we tested liposomal alendronate (PLA) as an immunotherapeutic agent for cancer in comparison with a standard of care immunotherapy, a PD-1 immune checkpoint inhibitor. We showed that the PLA induced bone marrow-derived murine non-activated macrophages and M2-macrophages to polarize towards an M1-functionality, as evidenced by gene expression, cytokine secretion, and lipidomic profiles. Free alendronate had negligible effects, indicating that liposome encapsulation is necessary for the modulation of macrophage activity. In vivo, the PLA showed significant accumulation in tumor and tumor-draining lymph nodes, sites of tumor immunosuppression that are targets of immunotherapy. The PLA remodeled the tumor microenvironment towards a less immunosuppressive milieu, as indicated by a decrease in TAM and helper T cells, and inhibited the growth of established tumors in the B16-OVA melanoma model. The improved bioavailability and the beneficial effects of PLA on macrophages suggest its potential application as immunotherapy that could synergize with T-cell-targeted therapies and chemotherapies to induce immunogenic cell death. PLA warrants further clinical development, and these clinical trials should incorporate tumor and blood biomarkers or immunophenotyping studies to verify the anti-immunosuppressive effect of PLA in humans. Full article

(This article belongs to the Special Issue Liposomes for Drug Delivery: Recent Advances and Discoveries)

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17 pages, 2326 KiB

Open AccessEditor’s ChoiceArticle

Peroxisomal NAD(H) Homeostasis in the Yeast Debaryomyces hansenii Depends on Two Redox Shuttles and the NAD⁺ Carrier, Pmp47

by Selva Turkolmez, Serhii Chornyi, Sondos Alhajouj, Lodewijk IJlst, Hans R. Waterham, Phil J. Mitchell, Ewald H. Hettema and Carlo W. T. van Roermund

Biomolecules 2023, 13(9), 1294; https://doi.org/10.3390/biom13091294 - 24 Aug 2023

Cited by 4 | Viewed by 1687

Abstract

Debaryomyces hansenii is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this [...] Read more.

Debaryomyces hansenii is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid β-oxidation pathway in D. hansenii. To this end, we employed recently developed methods to generate multiple gene deletions and tag open reading frames with GFP in their chromosomal context in this yeast. We found that, similar as in other yeasts, the β-oxidation of fatty acids in D. hansenii was restricted to peroxisomes. We report a series of experiments in D. hansenii and the well-studied yeast Saccharomyces cerevisiae that show that the homeostasis of NAD⁺ in D. hansenii peroxisomes is dependent upon the peroxisomal membrane protein Pmp47 and two peroxisomal dehydrogenases, Mdh3 and Gpd1, which both export reducing equivalents produced during β-oxidation to the cytosol. Pmp47 is the first identified NAD⁺ carrier in yeast peroxisomes. Full article

(This article belongs to the Special Issue The Role of Peroxisome in Cell Functions and Pathological Consequences of Its Dysfunction)

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16 pages, 4982 KiB

Open AccessEditor’s ChoiceArticle

Hybrid Material Based on Vaccinium myrtillus L. Extract and Gold Nanoparticles Reduces Oxidative Stress and Inflammation in Hepatic Stellate Cells Exposed to TGF-β

by Mara Filip, Ioana Baldea, Luminita David, Bianca Moldovan, Gabriel Cristian Flontas, Sergiu Macavei, Dana Maria Muntean, Nicoleta Decea, Adrian Bogdan Tigu and Simona Valeria Clichici

Biomolecules 2023, 13(8), 1271; https://doi.org/10.3390/biom13081271 - 20 Aug 2023

Cited by 2 | Viewed by 1273

Abstract

(1) Background: The study aimed to investigate the impact of gold nanoparticles capped with Cornus sanguinea (NPCS) and mixed with a fruit extract (Vaccinum myrtillus L.—VL) on human hepatic stellate cells (LX-2) exposed to TGF-β. (2) Methods: NPCS were characterized by UV-Vis, [...] Read more.

(1) Background: The study aimed to investigate the impact of gold nanoparticles capped with Cornus sanguinea (NPCS) and mixed with a fruit extract (Vaccinum myrtillus L.—VL) on human hepatic stellate cells (LX-2) exposed to TGF-β. (2) Methods: NPCS were characterized by UV-Vis, transmission electron microscopy (TEM), zeta potential measurement, X-ray diffraction (XRD) and energy dispersive spectroscopy (EDX). The cytotoxic effects of VL, NPCS and of the hybrid compounds obtained by mixing the two components in variable proportions (NPCS-VL) were assessed. LDH activity, MDA levels, secretion of inflammation markers, the expression of fibrogenesis markers and collagen I synthesis were estimated after treating the cells with a mixture of 25:25 μg/mL NPCS and VL. (3) Results: TEM analysis showed that NPCS had spherical morphology and homogenous distribution, while their formation and elemental composition were confirmed by XRD and EDX analysis. TGF-β increased cell membrane damage as well as secretion of IL-1β, IL-1α and TLR4. It also amplified the expression of α-SMA and type III collagen and induced collagen I deposition. NPCS administration reduced the inflammation caused by TGF-β and downregulated α-SMA expression. VL diminished LDH activity and the secretion of proinflammatory cytokines. The NPCS-VL mixture maintained IL-1β, IL-1α, TLR4 and LDH at low levels after TGF-β exposure, but it enhanced collagen III expression. (4) Conclusions: The mixture of NPCS and VL improved cell membrane damage and inflammation triggered by TGF-β and mitigated collagen I deposition, but it increased the expression of collagen III, suggestive of a fibrogenetic effect of the hybrid material. Full article

(This article belongs to the Special Issue Bionanomaterials: Synthesis, Characterization, Toxicity and Biomedical Applications (Volume II))

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