Biomolecules

Article

15 pages, 2943 KiB

Open AccessEditor’s ChoiceArticle

An 11-mer Synthetic Peptide Suppressing Aggregation of Aβ25-35 and Resolving Its Aggregated Form Improves Test Performance in an Aβ25-35-Induced Alzheimer’s Mouse Model

by Rina Nakamura, Akira Matsuda, Youichirou Higashi, Yoshihiro Hayashi, Motomi Konishi, Motoaki Saito and Toshifumi Akizawa

Biomolecules 2024, 14(10), 1234; https://doi.org/10.3390/biom14101234 - 29 Sep 2024

Cited by 1 | Viewed by 1665

Abstract

There is a high demand for the development of drugs against Alzheimer’s disease (AD), which is related to the misfolding and aggregation of Amyloid-β (Aβ), due to the increasing number of patients with AD. In our present study, we aimed to assess the [...] Read more.

There is a high demand for the development of drugs against Alzheimer’s disease (AD), which is related to the misfolding and aggregation of Amyloid-β (Aβ), due to the increasing number of patients with AD. In our present study, we aimed to assess the aggregation inhibitory effect of various synthetic YS-peptides on Aβ25-35 to identify an applicable peptide for clinical use for AD treatment and prevention. Suppression and aggregate resolution activities of YS-peptides against Aβ25-35 were evaluated using a Thioflavin T assay and scanning electron microscopy (SEM). Structure–activity relationship studies revealed that YS-RD11 (RETLVYLTHLD) and YS-RE16 (RETLVYLTHLDYDDTE) showed suppression and aggregate-resolution activities. The effect of YS-peptides on phagocytosis in microglial cells (BV-2 cells) demonstrated that YS-RD11 and YS-RE16 activated the phagocytic ability of microglia. In the Aβ25-35-induced AD mouse model, YS-RD11 prevented and improved the deficits in short-term memory. In conclusion, YS-RD11 is a suitable candidate therapeutic drug against AD and uses a strategy similar to that used for antibodies. Full article

(This article belongs to the Special Issue Amyloid-Beta and Alzheimer’s Disease)

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16 pages, 7408 KiB

Open AccessEditor’s ChoiceArticle

Lactate Suppresses Growth of Esophageal Adenocarcinoma Patient-Derived Organoids through Alterations in Tumor NADH/NAD+ Redox State

by Steven H. Su, Yosuke Mitani, Tianxia Li, Uma Sachdeva, Samuel Flashner, Andres Klein-Szanto, Karen J. Dunbar, Julian Abrams, Hiroshi Nakagawa and Joel Gabre

Biomolecules 2024, 14(9), 1195; https://doi.org/10.3390/biom14091195 - 22 Sep 2024

Viewed by 2051

Abstract

Barrett’s esophagus (BE) is a common precancerous lesion that can progress to esophageal adenocarcinoma (EAC). There are significant alterations in the esophageal microbiome in the progression from healthy esophagus to BE to EAC, including an increased abundance of a variety of lactate-producing bacteria [...] Read more.

Barrett’s esophagus (BE) is a common precancerous lesion that can progress to esophageal adenocarcinoma (EAC). There are significant alterations in the esophageal microbiome in the progression from healthy esophagus to BE to EAC, including an increased abundance of a variety of lactate-producing bacteria and an increase of lactate in the tumor microenvironment, as predicted by metabolic modeling. The role of bacterial lactate in EAC is unknown. Here, we utilize patient-derived organoid (PDO) models of EAC and demonstrate that lactate inhibits the growth and proliferation of EAC PDOs through alterations in the tumor NADH/NAD+ redox state. Further RNA sequencing of EAC PDOs identifies ID1 and RSAD2 as potential regulatory molecules crucial in mediating lactate’s ability to suppress glycolysis and proliferation. Gene ontology analysis also identifies the activation of inflammatory and immunological pathways in addition to alterations in the metabolic pathways in EAC PDOs exposed to lactate, suggesting a multi-faceted role for lactate in the pathogenesis of EAC. Full article

(This article belongs to the Section Molecular Medicine)

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19 pages, 3791 KiB

Open AccessEditor’s ChoiceArticle

Synergistic Inhibition of Pancreatic Cancer Cell Growth and Migration by Gemcitabine and Withaferin A

by Renata Szydlak

Biomolecules 2024, 14(9), 1178; https://doi.org/10.3390/biom14091178 - 19 Sep 2024

Viewed by 1778

Abstract

Pancreatic cancer remains one of the most lethal malignancies due to its aggressive nature and resistance to conventional therapies. This study investigates the anti-proliferative, pro-apoptotic, and anti-migratory effects of Gemcitabine (GC) and Withaferin A (WFA) on pancreatic cancer cell lines PANC-1 and Hs766t. [...] Read more.

Pancreatic cancer remains one of the most lethal malignancies due to its aggressive nature and resistance to conventional therapies. This study investigates the anti-proliferative, pro-apoptotic, and anti-migratory effects of Gemcitabine (GC) and Withaferin A (WFA) on pancreatic cancer cell lines PANC-1 and Hs766t. The MTS assay revealed that both compounds effectively inhibit cell proliferation, with WFA showing a stronger effect in Hs766t cells. Flow cytometry analysis demonstrated that GC and WFA, particularly in combination, significantly induce apoptosis in both cell lines. Migration assays confirmed the potent inhibition of cell migration by both compounds, with the combination treatment being the most effective. Furthermore, actin cytoskeleton analysis indicated substantial changes in cell morphology and stiffness, suggesting that GC and WFA disrupt the structural integrity of cancer cells. Additionally, the study highlights a ROS-mediated mechanism underlying the effects of GC and WFA, as evidenced by increased ROS levels following treatment, which were attenuated by N-acetylcysteine. Importantly, NF-κB activity was significantly modulated, with WFA reducing NF-κB activation induced by GC, potentially contributing to the synergistic pro-apoptotic effect of the combination. These findings suggest that the combination of GC and WFA may offer a synergistic therapeutic approach for treating pancreatic cancer by targeting multiple aspects of tumor cell behavior. Full article

(This article belongs to the Topic Advances in Natural Products and Phytochemicals in Cancer Prevention and Therapeutics)

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12 pages, 3403 KiB

Open AccessEditor’s ChoiceArticle

L-Cysteine Upregulates Testosterone Biosynthesis and Blood–Testis Barrier Genes in Cultured Human Leydig Cells and THP-1 Monocytes and Increases Testosterone Secretion in Human Leydig Cells

by Jeffrey Justin Margret and Sushil K. Jain

Biomolecules 2024, 14(9), 1171; https://doi.org/10.3390/biom14091171 - 18 Sep 2024

Cited by 1 | Viewed by 1933

Abstract

Leydig cells are the primary source of testosterone or androgen production in male mammals. The blood–testis barrier (BTB) maintains structural integrity and safeguards germ cells from harmful substances by blocking their entry into the seminiferous tubules. L-cysteine is essential to the production of [...] Read more.

Leydig cells are the primary source of testosterone or androgen production in male mammals. The blood–testis barrier (BTB) maintains structural integrity and safeguards germ cells from harmful substances by blocking their entry into the seminiferous tubules. L-cysteine is essential to the production of glutathione, a powerful antioxidant crucial to protecting against oxidative stress-induced damage. Animal studies have demonstrated the protective effect of L-cysteine in preventing testicular damage caused by chemicals or radiation. This study examines whether L-cysteine enhances the expression of testosterone biosynthesis and the BTB genes in human Leydig cells and THP-1 monocytes. The Leydig cells and THP-1 monocytes were treated with L-cysteine for 24 h. RNA was extracted following treatment, and the gene expression was analyzed using quantitative RT-PCR. Testosterone levels in the cell supernatant were measured using an ELISA kit. L-cysteine treatment in Leydig cells significantly upregulated the expression of CYP11A1 (p = 0.03) and the BTB genes CLDN1 (p = 0.03), CLDN11 (p = 0.02), and TJP1 (p = 0.02). Similarly, L-cysteine significantly upregulated the expression of CYP11A1 (p = 0.03) and CYP19A1 (p < 0.01), and the BTB genes CLDN1 (p = 0.04), CLDN2 (p < 0.01), CLDN4 (p < 0.01), CLDN11 (p < 0.01), and TJP1 (p = 0.03) in THP-1 monocytes. Further, L-cysteine supplementation increased the testosterone secretion levels in human Leydig cells. The findings suggest that L-cysteine supplementation could be used as an adjuvant therapy to promote the integrity of the BTB genes, testosterone biosynthesis and secretion, and the maintenance of testicular functions, which in turn mitigates the risk of male infertility. Full article

(This article belongs to the Section Molecular Reproduction)

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17 pages, 13879 KiB

Open AccessEditor’s ChoiceArticle

Sirt2 Regulates Liver Metabolism in a Sex-Specific Manner

by Alexandra V. Schmidt, Sivakama S. Bharathi, Keaton J. Solo, Joanna Bons, Jacob P. Rose, Birgit Schilling and Eric S. Goetzman

Biomolecules 2024, 14(9), 1160; https://doi.org/10.3390/biom14091160 - 15 Sep 2024

Cited by 3 | Viewed by 1644

Abstract

Sirtuin-2 (Sirt2), an NAD+-dependent lysine deacylase enzyme, has previously been implicated as a regulator of glucose metabolism, but the specific mechanisms remain poorly defined. Here, we observed that Sirt2−/− males, but not females, have decreased body fat, moderate hypoglycemia upon fasting, and perturbed [...] Read more.

Sirtuin-2 (Sirt2), an NAD+-dependent lysine deacylase enzyme, has previously been implicated as a regulator of glucose metabolism, but the specific mechanisms remain poorly defined. Here, we observed that Sirt2−/− males, but not females, have decreased body fat, moderate hypoglycemia upon fasting, and perturbed glucose handling during exercise compared to wild type controls. Conversion of injected lactate, pyruvate, and glycerol boluses into glucose via gluconeogenesis was impaired, but only in males. Primary Sirt2−/− male hepatocytes exhibited reduced glycolysis and reduced mitochondrial respiration. RNAseq and proteomics were used to interrogate the mechanisms behind this liver phenotype. Loss of Sirt2 did not lead to transcriptional dysregulation, as very few genes were altered in the transcriptome. In keeping with this, there were also negligible changes to protein abundance. Site-specific quantification of the hepatic acetylome, however, showed that 13% of all detected acetylated peptides were significantly increased in Sirt2−/− male liver versus wild type, representing putative Sirt2 target sites. Strikingly, none of these putative target sites were hyperacetylated in Sirt2−/− female liver. The target sites in the male liver were distributed across mitochondria (44%), cytoplasm (32%), nucleus (8%), and other compartments (16%). Despite the high number of putative mitochondrial Sirt2 targets, Sirt2 antigen was not detected in purified wild type liver mitochondria, suggesting that Sirt2’s regulation of mitochondrial function occurs from outside the organelle. We conclude that Sirt2 regulates hepatic protein acetylation and metabolism in a sex-specific manner. Full article

(This article belongs to the Special Issue Molecular Mechanisms Underlying Liver Diseases)

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12 pages, 6206 KiB

Open AccessEditor’s ChoiceArticle

Prognostic Value of Circulating Fibrosis Biomarkers in Dilated Cardiomyopathy (DCM): Insights into Clinical Outcomes

by Elham Kayvanpour, Farbod Sedaghat-Hamedani, Daniel Tian Li, Tobias Miersch, Tanja Weis, Imo Hoefer, Norbert Frey and Benjamin Meder

Biomolecules 2024, 14(9), 1137; https://doi.org/10.3390/biom14091137 - 9 Sep 2024

Cited by 3 | Viewed by 2067

Abstract

Background: Dilated cardiomyopathy (DCM) involves myocardial remodeling, characterized by significant fibrosis and extracellular matrix expansion. These changes impair heart function, increasing the risk of heart failure and sudden cardiac death. This study investigates the prognostic value of circulating fibrosis biomarkers as a less [...] Read more.

Background: Dilated cardiomyopathy (DCM) involves myocardial remodeling, characterized by significant fibrosis and extracellular matrix expansion. These changes impair heart function, increasing the risk of heart failure and sudden cardiac death. This study investigates the prognostic value of circulating fibrosis biomarkers as a less invasive method in DCM patients. Methods: Plasma samples from 185 patients with confirmed DCM were analyzed to measure 13 circulating biomarkers using Luminex bead-based multiplex assays and ELISA. The prognostic value of these biomarkers was evaluated concerning heart failure-associated events and all-cause mortality. Results: Elevated MMP-2 levels (>1519.3 ng/mL) were linked to older age, higher diabetes prevalence, lower HDL, increased NT-proBNP and hs-TnT levels, and severe systolic dysfunction. High TIMP-1 levels (>124.9 ng/mL) correlated with elevated NT-proBNP, more atrial fibrillation, reduced exercise capacity, and larger right ventricles. Increased GDF-15 levels (>1213.9 ng/mL) were associated with older age, systemic inflammation, renal impairment, and poor exercise performance. Elevated OPN levels (>81.7 ng/mL) were linked to higher serum creatinine and NT-proBNP levels. Over a median follow-up of 32.4 months, higher levels of these biomarkers predicted worse outcomes, including increased risks of heart failure-related events and mortality. Conclusions: Circulating fibrosis biomarkers, particularly MMP-2, TIMP-1, GDF-15, and OPN, are valuable prognostic tools in DCM. They reflect the severity of myocardial remodeling and systemic disease burden, aiding in risk stratification and therapeutic intervention. Integrating these biomarkers into clinical practice could improve DCM management and patient prognosis. Full article

(This article belongs to the Special Issue Biomarkers of Cardiovascular and Cerebrovascular Diseases)

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11 pages, 2208 KiB

Open AccessEditor’s ChoiceArticle

AMPK Activation Serves as a Common Pro-Survival Pathway in Esophageal Adenocarcinoma Cells

by Niamh McNamee, Pavithra Rajagopalan, Aya Tal-Mason, Samuel Roytburd and Uma M. Sachdeva

Biomolecules 2024, 14(9), 1115; https://doi.org/10.3390/biom14091115 - 4 Sep 2024

Cited by 1 | Viewed by 2389

Abstract

Esophageal adenocarcinoma (EAC) is a subtype of esophageal cancer that is difficult to treat, with overall poor survival and frequent recurrence despite curative-intent treatment strategies. There is limited understanding of EAC resistance mechanisms to chemotherapy or radiation. We have found that the AMP-activated [...] Read more.

Esophageal adenocarcinoma (EAC) is a subtype of esophageal cancer that is difficult to treat, with overall poor survival and frequent recurrence despite curative-intent treatment strategies. There is limited understanding of EAC resistance mechanisms to chemotherapy or radiation. We have found that the AMP-activated protein kinase (AMPK) can serve a pro-survival function in EAC cells in response to cytotoxic treatments. Treatment with the IL-6 inhibitor tocilizumab, which previously has been shown to inhibit EAC organoid growth, resulted in the activation of AMPK in the OE33 EAC cell line, which was accompanied by a decrease in MTORC1 signaling and an increase in oxidative mitochondrial metabolism, both known downstream effects of AMPK activation to promote cell survival under conditions of metabolic stress. This increase in oxidative metabolism was abrogated in cells with a genetic knockdown of AMPK expression. Furthermore, we found that AMPK was activated in OE33 cells following treatment with cisplatin or ionizing radiation. Treatment with the AMPK inhibitor Compound C or genetic knockdown of AMPK expression enhanced cell death in a synergistic manner with chemotherapeutics or ionizing radiation. These findings were recapitulated in human patient-derived EAC organoids, suggesting that AMPK may be a common pro-survival mechanism to confer treatment resistance in EAC and may serve as a novel target to enhance the efficacy of current and future treatment strategies. Full article

(This article belongs to the Special Issue Esophageal Diseases: Molecular Basis and Therapeutic Approaches)

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29 pages, 4521 KiB

Open AccessEditor’s ChoiceArticle

New BDNF and NT-3 Cyclic Mimetics Concur with Copper to Activate Trophic Signaling Pathways as Potential Molecular Entities to Protect Old Brains from Neurodegeneration

by Antonio Magrì, Barbara Tomasello, Irina Naletova, Giovanni Tabbì, Warren R. L. Cairns, Valentina Greco, Sebastiano Sciuto, Diego La Mendola and Enrico Rizzarelli

Biomolecules 2024, 14(9), 1104; https://doi.org/10.3390/biom14091104 - 2 Sep 2024

Cited by 1 | Viewed by 1629

Abstract

A low level of Neurotrophins (NTs), their Tyrosine Kinase Receptors (Trks), Vascular Endothelial Growth Factors (VEGFs) and their receptors, mainly VEGFR1 and VEGFR2, characterizes AD brains. The use of NTs and VEGFs as drugs presents different issues due to their low permeability of [...] Read more.

A low level of Neurotrophins (NTs), their Tyrosine Kinase Receptors (Trks), Vascular Endothelial Growth Factors (VEGFs) and their receptors, mainly VEGFR1 and VEGFR2, characterizes AD brains. The use of NTs and VEGFs as drugs presents different issues due to their low permeability of the blood−brain barrier, the poor pharmacokinetic profile, and the relevant side effects. To overcome these issues, different functional and structural NT mimics have been employed. Being aware that the N-terminus domain as the key domain of NTs for the binding selectivity and activation of Trks and the need to avoid or delay proteolysis, we herein report on the mimicking ability of two cyclic peptide encompassing the N-terminus of Brain Derived Growth Factor (BDNF), (c-[HSDPARRGELSV-]), cBDNF(1-12) and of Neurotrophin3 (NT3), (c-[YAEHKSHRGEYSV-]), cNT3(1-13). The two cyclic peptide features were characterized by a combined thermodynamic and spectroscopic approach (potentiometry, NMR, UV-vis and CD) that was extended to their copper(II) ion complexes. SH-SY5Y cell assays show that the Cu²⁺ present at the sub-micromolar level in the complete culture media affects the treatments with the two peptides. cBDNF(1-12) and cNT3(1-13) act as ionophores, induce neuronal differentiation and promote Trks and CREB phosphorylation in a copper dependent manner. Consistently, both peptide and Cu²⁺ stimulate BDNF and VEGF expression as well as VEGF release; cBDNF(1-12) and cNT3(1-13) induce the expression of Trks and VEGFRs. Full article

(This article belongs to the Special Issue The Role of Metals Ions in Neurodegenerative Diseases)

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13 pages, 790 KiB

Open AccessEditor’s ChoiceArticle

IL-4, IL-7, IL-9, NT, NRP1 May Be Useful Markers in the Diagnosis of Endometrial Cancer

by Mateusz Kozłowski, Dominika Borzyszkowska, Natalia Lerch, Agnieszka Turoń-Skrzypińska, Marta Tkacz, Jerzy Lubikowski, Maciej Tarnowski, Iwona Rotter and Aneta Cymbaluk-Płoska

Biomolecules 2024, 14(9), 1095; https://doi.org/10.3390/biom14091095 - 1 Sep 2024

Cited by 2 | Viewed by 1563

Abstract

The search for novel endometrial cancer diagnostic biomarkers is pertinent. The purpose of this study was to determine if IL-4, IL-7, IL-9, IL-10, NT, TSP-2, and NRP1 could be used as novel, helpful markers for the detection of endometrial cancer. Ninety-three women diagnosed [...] Read more.

The search for novel endometrial cancer diagnostic biomarkers is pertinent. The purpose of this study was to determine if IL-4, IL-7, IL-9, IL-10, NT, TSP-2, and NRP1 could be used as novel, helpful markers for the detection of endometrial cancer. Ninety-three women diagnosed with endometrial cancer (EC) and sixty-six patients with noncancerous endometrial lesions (NCEL) were included in this study. ELISA was used to measure the concentrations of the proteins tested. Median serum levels of IL-4, IL-7, IL-9, NT, and NRP1 were significantly higher in the EC group compared with NCEL. The cut-off level of IL-4 was set at 802.26 pg/mL with a sensitivity of 83.87% and a specificity of 50% (AUC = 0.7, p = 0.000023). The cut-off level of IL-7 was set at 133.63 ng/L with a sensitivity of 96.77% and a specificity of 75.76% (AUC = 0.91, p < 0.000001). The cut-off level of IL-9 was set at 228.79 pg/mL with a sensitivity of 69.89% and a specificity of 81.82% (AUC = 0.8, p < 0.000001). The cut-off level of NT was set at 275.43 pmol/L with a sensitivity of 94.62% and a specificity of 59.09% (AUC = 0.83, p < 0.000001). The cut-off level of NRP1 was set at 30.37 ng/mL with a sensitivity of 81.72% and a specificity of 57.58% (AUC = 0.71, p = 0.000004). This study suggests the clinical utility of IL-4, IL-7, IL-9, NT, and NRP1 in the diagnosis of endometrial cancer. Nevertheless, these biomarkers may also have prognostic or predictive value, which should be tested in future studies. Full article

(This article belongs to the Special Issue Molecular, Cellular, and Blood Biomarkers in Diagnosis, Prognosis, Monitoring, and Treatment)

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22 pages, 2538 KiB

Open AccessEditor’s ChoiceArticle

Cannabis sativa L. Extract Alleviates Neuropathic Pain and Modulates CB1 and CB2 Receptor Expression in Rat

by Joanna Bartkowiak-Wieczorek, Agnieszka Bienert, Kamila Czora-Poczwardowska, Radosław Kujawski, Michał Szulc, Przemysław Mikołajczak, Anna-Maria Wizner, Małgorzata Jamka, Marcin Hołysz, Karolina Wielgus, Ryszard Słomski and Edyta Mądry

Biomolecules 2024, 14(9), 1065; https://doi.org/10.3390/biom14091065 - 26 Aug 2024

Cited by 6 | Viewed by 3035

Abstract

Introduction: Cannabis sativa L. (CSL) extract has pain-relieving potential due to its cannabinoid content, so the effects of two CSL extracts on alleviating neuropathic pain were investigated in vivo. Methods and groups: Male Wistar rats (n = 130) were divided into groups and [...] Read more.

Introduction: Cannabis sativa L. (CSL) extract has pain-relieving potential due to its cannabinoid content, so the effects of two CSL extracts on alleviating neuropathic pain were investigated in vivo. Methods and groups: Male Wistar rats (n = 130) were divided into groups and received vincristine (0.1 mg/kg) and gabapentin (60 mg/kg) to induce and relieve neuropathic pain or CSL extracts (D and B). The mRNA and protein expression of the cannabinoid receptors type 1 and 2 (CB1R, CB2R) were evaluated in the cerebral cortex, hippocampus, and lymphocytes. Behavioural tests (Tail-Flick and von Frey) were performed on all animals. Results: VK-induced neuropathic pain was accompanied by decreased CB1R protein level and CB2R mRNA expression in the cortex. Gabapentin relieved pain and increased CB1R protein levels in the hippocampus compared to the vincristine group. Hippocampus CB1R protein expression increased with the administration of extract D (10 mg/kg, 40 mg/kg) and extract B (7.5 mg/kg, 10 mg/kg) compared to VK group. In the cerebral cortex CSL decreased CB1R protein expression (10 mg/kg, 20 mg/kg, 40 mg/kg of extract B) and mRNA level (5 mg/kg, 7.5 mg/kg of extract B; 20 mg/kg of extract D) compared to the VK-group.CB2R protein expression increased in the hippocampus after treatment with extract B (7.5 mg/kg) compared to the VK-group. In the cerebral cortex extract B (10 mg/kg, 20 mg/kg) increased CB2R protein expression compared to VK-group. Conclusion: Alterations in cannabinoid receptor expression do not fully account for the observed behavioural changes in rats. Therefore, additional signalling pathways may contribute to the initiation and transmission of neuropathic pain. The Cannabis extracts tested demonstrated antinociceptive effects comparable to gabapentin, highlighting the antinociceptive properties of Cannabis extracts for human use. Full article

(This article belongs to the Special Issue The Value of Natural Compounds as Therapeutic Agents: 2nd Edition)

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21 pages, 5025 KiB

Open AccessEditor’s ChoiceArticle

Targeting Grb2 SH3 Domains with Affimer Proteins Provides Novel Insights into Ras Signalling Modulation

by Anna A. S. Tang, Andrew Macdonald, Michael J. McPherson and Darren C. Tomlinson

Biomolecules 2024, 14(8), 1040; https://doi.org/10.3390/biom14081040 - 22 Aug 2024

Cited by 1 | Viewed by 2314

Abstract

Src homology 3 (SH3) domains play a critical role in mediating protein–protein interactions (PPIs) involved in cell proliferation, migration, and the cytoskeleton. Despite their abundance in the human proteome, the functions and molecular interactions of many SH3 domains remain unknown, and this is [...] Read more.

Src homology 3 (SH3) domains play a critical role in mediating protein–protein interactions (PPIs) involved in cell proliferation, migration, and the cytoskeleton. Despite their abundance in the human proteome, the functions and molecular interactions of many SH3 domains remain unknown, and this is in part due to the lack of SH3-domain-specific reagents available for their study. Affimer proteins have been developed as affinity reagents targeting a diverse range of targets, including those involved in PPIs. In this study, Affimer proteins were isolated against both the N- and C-terminal SH3 domains (NSH3 and CSH3) of growth-factor-receptor-bound protein 2 (Grb2), an adapter protein that provides a critical link between cell surface receptors and Ras signalling pathways. Targeting the CSH3 alone for the inhibition of PPIs appeared sufficient for curtailing Ras signalling in mammalian cell lines stimulated with human epidermal growth factor (EGF), which conflicts with the notion that the predominant interactions with Ras activating Son of sevenless (SOS) occur via the NSH3 domain. This result supports a model in which allosteric mechanisms involved in Grb2-SOS1 interaction modulate Ras activation. Full article

(This article belongs to the Special Issue The Role of Scaffold Proteins in Human Diseases)

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24 pages, 34001 KiB

Open AccessEditor’s ChoiceArticle

Pyrimidine Triones as Potential Activators of p53 Mutants

by Maryam M. Jebril Fallatah, Özlem Demir, Fiona Law, Linda Lauinger, Roberta Baronio, Linda Hall, Elodie Bournique, Ambuj Srivastava, Landon Tyler Metzen, Zane Norman, Rémi Buisson, Rommie E. Amaro and Peter Kaiser

Biomolecules 2024, 14(8), 967; https://doi.org/10.3390/biom14080967 - 8 Aug 2024

Cited by 1 | Viewed by 2573

Abstract

p53 is a crucial tumor suppressor in vertebrates that is frequently mutated in human cancers. Most mutations are missense mutations that render p53 inactive in suppressing tumor initiation and progression. Developing small-molecule drugs to convert mutant p53 into an active, wild-type-like conformation is [...] Read more.

p53 is a crucial tumor suppressor in vertebrates that is frequently mutated in human cancers. Most mutations are missense mutations that render p53 inactive in suppressing tumor initiation and progression. Developing small-molecule drugs to convert mutant p53 into an active, wild-type-like conformation is a significant focus for personalized cancer therapy. Prior research indicates that reactivating p53 suppresses cancer cell proliferation and tumor growth in animal models. Early clinical evidence with a compound selectively targeting p53 mutants with substitutions of tyrosine 220 suggests potential therapeutic benefits of reactivating p53 in patients. This study identifies and examines the UCI-1001 compound series as a potential corrector for several p53 mutations. The findings indicate that UCI-1001 treatment in p53 mutant cancer cell lines inhibits growth and reinstates wild-type p53 activities, including DNA binding, target gene activation, and induction of cell death. Cellular thermal shift assays, conformation-specific immunofluorescence staining, and differential scanning fluorometry suggest that UCI-1001 interacts with and alters the conformation of mutant p53 in cancer cells. These initial results identify pyrimidine trione derivatives of the UCI-1001 series as candidates for p53 corrector drug development. Full article

(This article belongs to the Special Issue Recent Advances in p53)

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16 pages, 3486 KiB

Open AccessEditor’s ChoiceArticle

Effects of Synthetic Toll-Like Receptor 9 Ligand Molecules on Pulpal Immunomodulatory Response and Repair after Injuries

by Angela Quispe-Salcedo, Tomohiko Yamazaki and Hayato Ohshima

Biomolecules 2024, 14(8), 931; https://doi.org/10.3390/biom14080931 - 1 Aug 2024

Cited by 2 | Viewed by 1494

Abstract

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine–phosphate–guanine (CpG) motifs (CpG-ODNs) are ligand molecules for Toll-like receptor 9 (TLR9), which is expressed by odontoblasts in vitro and dental pulp cells. This study determined the effects of CpG-ODNs on pulpal immunomodulatory response and repair following injury. [...] Read more.

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine–phosphate–guanine (CpG) motifs (CpG-ODNs) are ligand molecules for Toll-like receptor 9 (TLR9), which is expressed by odontoblasts in vitro and dental pulp cells. This study determined the effects of CpG-ODNs on pulpal immunomodulatory response and repair following injury. Briefly, the upper right first molars of three-week-old mice were extracted, immersed in Type A (D35) or B (K3) CpG-ODN solutions (0.1 or 0.8 mM) for 30 min, and then replanted. Pulpal healing and immunomodulatory activity were assessed by hematoxylin–eosin and AZAN staining, as well as immunohistochemistry. One week following the operation, inflammatory reactions occurred in all of the experimental groups; however, re-revascularization and newly formed hard tissue deposition were observed in the pulp chamber of all groups at week 2. A positive trend in the expression of immune cell markers was observed toward the CpG-ODN groups at 0.1 mM. Our data suggest that synthetic CpG-ODN solutions at low concentrations may evoke a long-lasting macrophage–TLR9-mediated pro-inflammatory, rather than anti-inflammatory, response in the dental pulp to modulate the repair process and hard tissue formation. Further studies are needed to determine the effects of current immunomodulatory agents in vitro and in vivo and develop treatment strategies for dental tissue regeneration. Full article

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22 pages, 2920 KiB

Open AccessEditor’s ChoiceArticle

Integrated Multi-Omics Analysis of Cerebrospinal Fluid in Postoperative Delirium

by Bridget A. Tripp, Simon T. Dillon, Min Yuan, John M. Asara, Sarinnapha M. Vasunilashorn, Tamara G. Fong, Sharon K. Inouye, Long H. Ngo, Edward R. Marcantonio, Zhongcong Xie, Towia A. Libermann and Hasan H. Otu

Biomolecules 2024, 14(8), 924; https://doi.org/10.3390/biom14080924 - 30 Jul 2024

Cited by 2 | Viewed by 2320

Abstract

Preoperative risk biomarkers for delirium may aid in identifying high-risk patients and developing intervention therapies, which would minimize the health and economic burden of postoperative delirium. Previous studies have typically used single omics approaches to identify such biomarkers. Preoperative cerebrospinal fluid (CSF) from [...] Read more.

Preoperative risk biomarkers for delirium may aid in identifying high-risk patients and developing intervention therapies, which would minimize the health and economic burden of postoperative delirium. Previous studies have typically used single omics approaches to identify such biomarkers. Preoperative cerebrospinal fluid (CSF) from the Healthier Postoperative Recovery study of adults ≥ 63 years old undergoing elective major orthopedic surgery was used in a matched pair delirium case–no delirium control design. We performed metabolomics and lipidomics, which were combined with our previously reported proteomics results on the same samples. Differential expression, clustering, classification, and systems biology analyses were applied to individual and combined omics datasets. Probabilistic graph models were used to identify an integrated multi-omics interaction network, which included clusters of heterogeneous omics interactions among lipids, metabolites, and proteins. The combined multi-omics signature of 25 molecules attained an AUC of 0.96 [95% CI: 0.85–1.00], showing improvement over individual omics-based classification. We conclude that multi-omics integration of preoperative CSF identifies potential risk markers for delirium and generates new insights into the complex pathways associated with delirium. With future validation, this hypotheses-generating study may serve to build robust biomarkers for delirium and improve our understanding of its pathophysiology. Full article

(This article belongs to the Special Issue The Next Generation of Proteomics for Precision Medicine: Second Edition)

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18 pages, 2718 KiB

Open AccessEditor’s ChoiceArticle

Markers of Mitochondrial Function and DNA Repair Associated with Physical Function in Centenarians

by Ines Sanchez-Roman, Beatriz Ferrando, Camilla Myrup Holst, Jonas Mengel-From, Signe Hoei Rasmussen, Mikael Thinggaard, Vilhelm A. Bohr, Kaare Christensen and Tinna Stevnsner

Biomolecules 2024, 14(8), 909; https://doi.org/10.3390/biom14080909 - 26 Jul 2024

Viewed by 2545

Abstract

Mitochondrial dysfunction and genomic instability are key hallmarks of aging. The aim of this study was to evaluate whether maintenance of physical capacities at very old age is associated with key hallmarks of aging. To investigate this, we measured mitochondrial bioenergetics, mitochondrial DNA [...] Read more.

Mitochondrial dysfunction and genomic instability are key hallmarks of aging. The aim of this study was to evaluate whether maintenance of physical capacities at very old age is associated with key hallmarks of aging. To investigate this, we measured mitochondrial bioenergetics, mitochondrial DNA (mtDNA) copy number and DNA repair capacity in peripheral blood mononuclear cells from centenarians. In addition, circulating levels of NAD+/NADH, brain-derived neurotrophic factor (BDNF) and carbonylated proteins were measured in plasma and these parameters were correlated to physical capacities. Centenarians without physical disabilities had lower mitochondrial respiration values including ATP production, reserve capacity, maximal respiration and non-mitochondrial oxygen-consumption rate and had higher mtDNA copy number than centenarians with moderate and severe disabilities (p < 0.05). In centenarian females, grip strength had a positive association with mtDNA copy number (p < 0.05), and a borderline positive trend for activity of the central DNA repair enzyme, APE 1 (p = 0.075), while a negative trend was found with circulating protein carbonylation (p = 0.07) in the entire cohort. Lastly, a trend was observed for a negative association between BDNF and activity of daily living disability score (p = 0.06). Our results suggest that mechanisms involved in maintaining mitochondrial function and genomic stability may be associated with maintenance of physical function in centenarians. Full article

(This article belongs to the Special Issue Mechanisms and Novel Biomarkers Underlying Aging and Longevity)

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35 pages, 5909 KiB

Open AccessEditor’s ChoiceArticle

CPT2 Deficiency Modeled in Zebrafish: Abnormal Neural Development, Electrical Activity, Behavior, and Schizophrenia-Related Gene Expression

by Carly E. Baker, Aaron G. Marta, Nathan D. Zimmerman, Zeljka Korade, Nicholas W. Mathy, Delaney Wilton, Timothy Simeone, Andrew Kochvar, Kenneth L. Kramer, Holly A. F. Stessman and Annemarie Shibata

Biomolecules 2024, 14(8), 914; https://doi.org/10.3390/biom14080914 - 26 Jul 2024

Viewed by 2098

Abstract

Carnitine palmitoyltransferase 2 (CPT2) is an inner mitochondrial membrane protein of the carnitine shuttle and is involved in the beta-oxidation of long chain fatty acids. Beta-oxidation provides an alternative pathway of energy production during early development and starvation. CPT2 deficiency is a genetic [...] Read more.

Carnitine palmitoyltransferase 2 (CPT2) is an inner mitochondrial membrane protein of the carnitine shuttle and is involved in the beta-oxidation of long chain fatty acids. Beta-oxidation provides an alternative pathway of energy production during early development and starvation. CPT2 deficiency is a genetic disorder that we recently showed can be associated with schizophrenia. We hypothesize that CPT2 deficiency during early brain development causes transcriptional, structural, and functional abnormalities that may contribute to a CNS environment that is susceptible to the emergence of schizophrenia. To investigate the effect of CPT2 deficiency on early vertebrate development and brain function, CPT2 was knocked down in a zebrafish model system. CPT2 knockdown resulted in abnormal lipid utilization and deposition, reduction in body size, and abnormal brain development. Axonal projections, neurotransmitter synthesis, electrical hyperactivity, and swimming behavior were disrupted in CPT2 knockdown zebrafish. RT-qPCR analyses showed significant increases in the expression of schizophrenia-associated genes in CPT2 knockdown compared to control zebrafish. Taken together, these data demonstrate that zebrafish are a useful model for studying the importance of beta-oxidation for early vertebrate development and brain function. This study also presents novel findings linking CPT2 deficiency to the regulation of schizophrenia and neurodegenerative disease-associated genes. Full article

(This article belongs to the Section Synthetic Biology and Bioengineering)

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16 pages, 5071 KiB

Open AccessEditor’s ChoiceArticle

The Autophagic Activator GHF-201 Can Alleviate Pathology in a Mouse Model and in Patient Fibroblasts of Type III Glycogenosis

by Kumudesh Mishra, Sahar Sweetat, Saja Baraghithy, Uri Sprecher, Monzer Marisat, Sultan Bastu, Hava Glickstein, Joseph Tam, Hanna Rosenmann, Miguel Weil, Edoardo Malfatti and Or Kakhlon

Biomolecules 2024, 14(8), 893; https://doi.org/10.3390/biom14080893 - 24 Jul 2024

Cited by 1 | Viewed by 2052

Abstract

Glycogen storage disease type III (GSDIII) is a hereditary glycogenosis caused by deficiency of the glycogen debranching enzyme (GDE), an enzyme, encoded by Agl, enabling glycogen degradation by catalyzing alpha-1,4-oligosaccharide side chain transfer and alpha-1,6-glucose cleavage. GDE deficiency causes accumulation of phosphorylase-limited [...] Read more.

Glycogen storage disease type III (GSDIII) is a hereditary glycogenosis caused by deficiency of the glycogen debranching enzyme (GDE), an enzyme, encoded by Agl, enabling glycogen degradation by catalyzing alpha-1,4-oligosaccharide side chain transfer and alpha-1,6-glucose cleavage. GDE deficiency causes accumulation of phosphorylase-limited dextrin, leading to liver disorder followed by fatal myopathy. Here, we tested the capacity of the new autophagosomal activator GHF-201 to alleviate disease burden by clearing pathogenic glycogen surcharge in the GSDIII mouse model Agl^−/−. We used open field, grip strength, and rotarod tests for evaluating GHF-201’s effects on locomotion, a biochemistry panel to quantify hematological biomarkers, indirect calorimetry to quantify in vivo metabolism, transmission electron microscopy to quantify glycogen in muscle, and fibroblast image analysis to determine cellular features affected by GHF-201. GHF-201 was able to improve all locomotion parameters and partially reversed hypoglycemia, hyperlipidemia and liver and muscle malfunction in Agl^−/− mice. Treated mice burnt carbohydrates more efficiently and showed significant improvement of aberrant ultrastructural muscle features. In GSDIII patient fibroblasts, GHF-201 restored mitochondrial membrane polarization and corrected lysosomal swelling. In conclusion, GHF-201 is a viable candidate for treating GSDIII as it recovered a wide range of its pathologies in vivo, in vitro, and ex vivo. Full article

(This article belongs to the Topic Bioactive Compounds and Therapeutics: Molecular Aspects, Metabolic Profiles, and Omics Studies)

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21 pages, 6756 KiB

Open AccessEditor’s ChoiceArticle

Plasma and Myocardial miRNomes Similarities and Differences during Cardiac Remodelling and Reverse Remodelling in a Murine Model of Heart Failure with Preserved Ejection Fraction

by Sara-Ève Thibodeau, Emylie-Ann Labbé, Élisabeth Walsh-Wilkinson, Audrey Morin-Grandmont, Marie Arsenault and Jacques Couet

Biomolecules 2024, 14(8), 892; https://doi.org/10.3390/biom14080892 - 24 Jul 2024

Cited by 1 | Viewed by 1635

Abstract

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome characterised by multiple risk factors touching various organs outside the heart. Using a murine HFpEF model, we studied cardiac reverse remodelling (RR) after stopping the causing metabolic-hypertensive stress (MHS; Angiotensin II [AngII] [...] Read more.

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome characterised by multiple risk factors touching various organs outside the heart. Using a murine HFpEF model, we studied cardiac reverse remodelling (RR) after stopping the causing metabolic-hypertensive stress (MHS; Angiotensin II [AngII] and a high-fat diet [HFD]) after 28 days and introducing voluntary exercise (VE) for four more weeks. We measured the effects of MHS and RR on the plasma and myocardial microRNA (miR) profile (miRNome) to characterise better cardiac and non-cardiac responses to HFpEF-inducing risk factors and their reversibility. AngII alone, the HFD or the MHS caused cardiac hypertrophy (CH), left ventricular (LV) concentric remodelling and left atrial enlargement in females. Only AngII and the MHS, but not HFD, did in males. After RR, CH, LV concentric remodelling and atrial enlargement were normalised. Among the 25 most abundant circulating miRs, 10 were modulated by MHS. Plasma miRNomes from AngII, HFD or MHS mice shared 31 common significantly modulated miRs (24 upregulated and 7 downregulated), suggesting that the response of organs producing the bulk of those circulating miRs was similar even for seemingly different stress. In the LV, 19 out of 25 most expressed miRs were modulated. RR restored normality for the plasma miRNome but not for the LV miRNome, which remained mostly unchanged. Our results suggest that abnormalities persist in the myocardium of the HFpEF mice and that the normalisation of circulatory markers may be falsely reassuring after recovery. Full article

(This article belongs to the Special Issue New Insights into Cardiometabolic Diseases)

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24 pages, 4736 KiB

Open AccessEditor’s ChoiceArticle

Biophysical Analysis of a Minimalistic Kidney Model Expressing SGLT1 Reveals Crosstalk between Luminal and Lateral Membranes and a Plausible Mechanism of Isosmotic Transport

by Erik Hviid Larsen and Jens Nørkær Sørensen

Biomolecules 2024, 14(8), 889; https://doi.org/10.3390/biom14080889 - 23 Jul 2024

Cited by 1 | Viewed by 1313

Abstract

We extended our model of the S1 tubular segment to address the mechanisms by which SGLT1 interacts with lateral Na/K pumps and tight junctional complexes to generate isosmotic fluid reabsorption via tubular segment S3. The strategy applied allowed for simulation of laboratory experiments. [...] Read more.

We extended our model of the S1 tubular segment to address the mechanisms by which SGLT1 interacts with lateral Na/K pumps and tight junctional complexes to generate isosmotic fluid reabsorption via tubular segment S3. The strategy applied allowed for simulation of laboratory experiments. Reproducing known experimental results constrained the range of acceptable model outputs and contributed to minimizing the free parameter space. (1) In experimental conditions, published Na and K concentrations of proximal kidney cells were found to deviate substantially from their normal physiological levels. Analysis of the mechanisms involved suggested insufficient oxygen supply as the cause and, indirectly, that a main function of the Na/H exchanger (NHE3) is to extrude protons stemming from mitochondrial energy metabolism. (2) The water path from the lumen to the peritubular space passed through aquaporins on the cell membrane and claudin-2 at paracellular tight junctions, with an additional contribution to water transport by the coupling of 1 glucose:2 Na:400 H₂O in SGLT1. (3) A Na-uptake component passed through paracellular junctions via solvent drag in Na- and water-permeable claudin-2, thus bypassing the Na/K pump, in agreement with the findings of early studies. (4) Electrical crosstalk between apical rheogenic SGLT1 and lateral rheogenic Na/K pumps resulted in tight coupling of luminal glucose uptake and transepithelial water flow. (5) Isosmotic transport was achieved by Na-mediated ion recirculation at the peritubular membrane. Full article

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10 pages, 4642 KiB

Open AccessEditor’s ChoiceArticle

Rational Design of Chimeric Antisense Oligonucleotides on a Mixed PO–PS Backbone for Splice-Switching Applications

by Bao T. Le, Suxiang Chen and Rakesh N. Veedu

Biomolecules 2024, 14(7), 883; https://doi.org/10.3390/biom14070883 - 22 Jul 2024

Cited by 1 | Viewed by 2157

Abstract

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson–Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate [...] Read more.

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson–Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO’s pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO–PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs. Full article

(This article belongs to the Special Issue RNA Therapeutics)

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18 pages, 3903 KiB

Open AccessEditor’s ChoiceArticle

Leucine-Rich Repeat Kinase-2 Controls the Differentiation and Maturation of Oligodendrocytes in Mice and Zebrafish

by Alice Filippini, Elena Cannone, Valentina Mazziotti, Giulia Carini, Veronica Mutti, Cosetta Ravelli, Massimo Gennarelli, Marco Schiavone and Isabella Russo

Biomolecules 2024, 14(7), 870; https://doi.org/10.3390/biom14070870 - 19 Jul 2024

Cited by 1 | Viewed by 1899

Abstract

Leucine-rich repeat kinase-2 (LRRK2), a gene mutated in familial and sporadic Parkinson’s disease (PD), controls multiple cellular processes important for GLIA physiology. Interestingly, emerging studies report that LRRK2 is highly expressed in oligodendrocyte precursor cells (OPCs) compared to the pathophysiology of [...] Read more.

Leucine-rich repeat kinase-2 (LRRK2), a gene mutated in familial and sporadic Parkinson’s disease (PD), controls multiple cellular processes important for GLIA physiology. Interestingly, emerging studies report that LRRK2 is highly expressed in oligodendrocyte precursor cells (OPCs) compared to the pathophysiology of other brain cells and oligodendrocytes (OLs) in PD. Altogether, these observations suggest crucial function(s) of LRRK2 in OPCs/Ols, which would be interesting to explore. In this study, we investigated the role of LRRK2 in OLs. We showed that LRRK2 knock-out (KO) OPC cultures displayed defects in the transition of OPCs into OLs, suggesting a role of LRRK2 in OL differentiation. Consistently, we found an alteration of myelin basic protein (MBP) striosomes in LRRK2 KO mouse brains and reduced levels of oligodendrocyte transcription factor 2 (Olig2) and Mbp in olig2:EGFP and mbp:RFP transgenic zebrafish embryos injected with lrrk2 morpholino (MO). Moreover, lrrk2 knock-down zebrafish exhibited a lower amount of nerve growth factor (Ngf) compared to control embryos, which represents a potent regulator of oligodendrogenesis and myelination. Overall, our findings indicate that LRRK2 controls OL differentiation, affecting the number of mature OLs. Full article

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13 pages, 1739 KiB

Open AccessEditor’s ChoiceArticle

Reversible Histone Modifications Contribute to the Frozen and Thawed Recovery States of Wood Frog Brains

by Tighe Bloskie, Olawale O. Taiwo and Kenneth B. Storey

Biomolecules 2024, 14(7), 839; https://doi.org/10.3390/biom14070839 - 12 Jul 2024

Cited by 3 | Viewed by 2042

Abstract

Epigenetic regulation, notably histone post-translational modification (PTM), has emerged as a major transcriptional control of gene expression during cellular stress adaptation. In the present study, we use an acid extraction method to isolate total histone protein and investigate dynamic changes in 23 well-characterized [...] Read more.

Epigenetic regulation, notably histone post-translational modification (PTM), has emerged as a major transcriptional control of gene expression during cellular stress adaptation. In the present study, we use an acid extraction method to isolate total histone protein and investigate dynamic changes in 23 well-characterized histone methylations/acetylations in the brains of wood frogs subject to 24-h freezing and subsequent 8-h thawed recovery conditions. Our results identify four histone PTMs (H2BK5ac, H3K14ac, H3K4me3, H3K9me2) and three histone proteins (H1.0, H2B, H4) that were significantly (p < 0.05) responsive to freeze-thaw in freeze-tolerant R. sylvatica brains. Two other permissive modifications (H3R8me2a, H3K9ac) also trended downwards following freezing stress. Together, these data are strongly supportive of the proposed global transcriptional states of hypometabolic freeze tolerance and rebounded thawed recovery. Our findings shed light on the intricate interplay between epigenetic regulation, gene transcription and energy metabolism in wood frogs’ adaptive response to freezing stress. Full article

(This article belongs to the Special Issue Exploring the Dynamics of Protein Lysine Methylation and Its Implication in Disease)

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13 pages, 1530 KiB

Open AccessEditor’s ChoiceArticle

Zinc Protects against Swine Barn Dust-Induced Cilia Slowing

by Christopher D. Bauer, Deanna D. Mosley, Derrick R. Samuelson, Jill A. Poole, Deandra R. Smith, Daren L. Knoell and Todd A. Wyatt

Biomolecules 2024, 14(7), 843; https://doi.org/10.3390/biom14070843 - 12 Jul 2024

Cited by 4 | Viewed by 1825

Abstract

Agricultural workers exposed to organic dust from swine concentrated animal feeding operations (CAFOs) have increased chances of contracting chronic lung disease. Mucociliary clearance represents a first line of defense against inhaled dusts, but organic dust extracts (ODEs) from swine barns cause cilia slowing, [...] Read more.

Agricultural workers exposed to organic dust from swine concentrated animal feeding operations (CAFOs) have increased chances of contracting chronic lung disease. Mucociliary clearance represents a first line of defense against inhaled dusts, but organic dust extracts (ODEs) from swine barns cause cilia slowing, leading to decreased bacterial clearance and increased lung inflammation. Because nutritional zinc deficiency is associated with chronic lung disease, we examined the role of zinc supplementation in ODE-mediated cilia slowing. Ciliated mouse tracheal epithelial cells were pretreated with 0–10 µg/mL ZinPro^TM for 1 h, followed by treatment with 5% ODE for 24 h. Cilia beat frequency (CBF) and protein kinase C epsilon (PKCε) activity were assayed. ODE treatment resulted in cilia slowing after 24 h, which was reversed with 0.5 and 1.0 µg/mL ZinPro pre-treatment. No zinc protection was observed at 50 ng/mL, and ciliated cells detached at high concentrations (100 µg/mL). ZinPro alone produced no changes in the baseline CBF and showed no toxicity to the cells at concentrations of up to 10 µg/mL. Pre-treatment with ZinPro inhibited ODE-stimulated PKCε activation in a dose-dependent manner. Based on ZinPro’s superior cell permeability compared to zinc salts, it may be therapeutically more effective at reversing ODE-mediated cilia slowing through a PKCε pathway. These data demonstrate that zinc supplementation may support the mucociliary transport apparatus in the protection of CAFO workers against dust-mediated chronic lung disease. Full article

(This article belongs to the Special Issue Zinc in Health and Disease Conditions: 2nd Edition)

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17 pages, 9279 KiB

Open AccessEditor’s ChoiceArticle

Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche

by Shankar Suman, Wendy K. Nevala, Alexey A. Leontovich, Caitlin Ward, James W. Jakub, Yohan Kim, Liyi Geng, Noah A. Stueven, Chathu L. Atherton, Raymond M. Moore, Jill M. Schimke, Fabrice Lucien-Matteoni, Sarah A. McLaughlin and Svetomir N. Markovic

Biomolecules 2024, 14(7), 837; https://doi.org/10.3390/biom14070837 - 11 Jul 2024

Cited by 3 | Viewed by 2264

Abstract

CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To [...] Read more.

CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To understand the role of CD36 in melanoma, we first analyzed the SKCM dataset for clinical prognosis, evaluated the percentage of CD36 in lymphatic fluid-derived EVs (LEVs), and tested whether melanoma-derived EVs increase CD36 expression and induce M2-macrophage-like characteristics. Furthermore, we performed a multiplex immunofluorescence (MxIF) imaging analysis to evaluate the CD36 expression and its colocalization with various other cells in the lymph node (LN) of patients and control subjects. Our findings show that cutaneous melanoma patients have a worse clinical prognosis with high CD36 levels, and a higher percentage of CD36 in total LEVs were found at baseline in melanoma patients compared to control. We also found that monocytic and endothelial cells treated with melanoma EVs expressed more CD36 than untreated cells. Furthermore, melanoma-derived EVs can regulate immunosuppressive macrophage-like characteristics by upregulating CD36. The spatial imaging data show that cells in tumor-involved sentinel LNs exhibit a higher probability of CD36 expression than cells from control LNs, but this was not statistically significant. Conclusively, our findings demonstrated that CD36 plays a vital role in controlling the immunosuppressive microenvironment in the LN, which can promote the formation of a protumorigenic niche. Full article

(This article belongs to the Special Issue The Role of CD36 in Human Health and Disease)

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15 pages, 4465 KiB

Open AccessEditor’s ChoiceArticle

Glial Response and Neuronal Modulation Induced by Epidural Electrode Implant in the Pilocarpine Mouse Model of Epilepsy

by Giulia Spagnoli, Edoardo Parrella, Sara Ghazanfar Tehrani, Francesca Mengoni, Valentina Salari, Cristina Nistreanu, Ilaria Scambi, Andrea Sbarbati, Giuseppe Bertini and Paolo Francesco Fabene

Biomolecules 2024, 14(7), 834; https://doi.org/10.3390/biom14070834 - 11 Jul 2024

Viewed by 2426

Abstract

In animal models of epilepsy, cranial surgery is often required to implant electrodes for electroencephalography (EEG) recording. However, electrode implants can lead to the activation of glial cells and interfere with physiological neuronal activity. In this study, we evaluated the impact of epidural [...] Read more.

In animal models of epilepsy, cranial surgery is often required to implant electrodes for electroencephalography (EEG) recording. However, electrode implants can lead to the activation of glial cells and interfere with physiological neuronal activity. In this study, we evaluated the impact of epidural electrode implants in the pilocarpine mouse model of temporal lobe epilepsy. Brain neuroinflammation was assessed 1 and 3 weeks after surgery by cytokines quantification, immunohistochemistry, and western blotting. Moreover, we investigated the effect of pilocarpine, administered two weeks after surgery, on mice mortality rate. The reported results indicate that implanted mice suffer from neuroinflammation, characterized by an early release of pro-inflammatory cytokines, microglia activation, and subsequent astrogliosis, which persists after three weeks. Notably, mice subjected to electrode implants displayed a higher mortality rate following pilocarpine injection 2 weeks after the surgery. Moreover, the analysis of EEGs recorded from implanted mice revealed a high number of single spikes, indicating a possible increased susceptibility to seizures. In conclusion, epidural electrode implant in mice promotes neuroinflammation that could lower the seizure thresholds to pilocarpine and increase the death rate. An improved protocol considering the persistent neuroinflammation induced by electrode implants will address refinement and reduction, two of the 3Rs principles for the ethical use of animals in scientific research. Full article

(This article belongs to the Special Issue Role of Neuron-Glia and Glia-Glia Communications in the Brain Function: Recent Advances in the Central Nervous System Cellular Crosstalk and Signaling)

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16 pages, 3963 KiB

Open AccessEditor’s ChoiceArticle

Combined Metabolomic and NIRS Analyses Reveal Biochemical and Metabolite Changes in Goat Milk Kefir under Different Heat Treatments and Fermentation Times

by Rubén Sánchez-Rodríguez, Carlos Terriente-Palacios, Juan García-Olmo, Sonia Osorio and Manuel J. Rodríguez-Ortega

Biomolecules 2024, 14(7), 816; https://doi.org/10.3390/biom14070816 - 9 Jul 2024

Cited by 1 | Viewed by 1877

Abstract

Dairy products are an important source of protein and other nutrients in the Mediterranean diet. In these countries, the most common sources of milk for producing dairy products are cow, goat, sheep, and buffalo. Andalusia is traditionally the largest producer of goat milk [...] Read more.

Dairy products are an important source of protein and other nutrients in the Mediterranean diet. In these countries, the most common sources of milk for producing dairy products are cow, goat, sheep, and buffalo. Andalusia is traditionally the largest producer of goat milk in Spain. Kefir is a fermented product made from bacteria and yeasts and has health benefits beyond its nutritional properties. There is a lack of knowledge about the molecular mechanisms and metabolites that bring about these benefits. In this work, the combination of analytical techniques (GC-FID, UHPLC-MS-QToF, GC-QqQ-MS, and GC-ToF-MS) resulted in the detection of 105 metabolites in kefir produced with goat milk from two different thermal treatments (raw and pasteurized) fermented at four time points (12, 24, 36, and 48 h, using 0 h as the control). Of these, 27 metabolites differed between kefir produced with raw and pasteurized milk. These changes could possibly be caused by the effect of pasteurization on the microbial population in the starting milk. Some interesting molecules were identified, such as shikimic acid, dehydroabietic acid, GABA, and tyramine, which could be related to antibacterial properties, strengthening of the immune system, and arterial pressure. Moreover, a viability assay of the NIRS technique was performed to evaluate its use in monitoring the fermentation and classification of samples, which resulted in a 90% accuracy in comparison to correctly classified samples according to their fermentation time. This study represents the most comprehensive metabolomic analysis of goat milk kefir so far, revealing the intricate changes in metabolites during fermentation and the impact of milk treatment. Full article

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16 pages, 3708 KiB

Open AccessEditor’s ChoiceArticle

Isolation and Characterization of Milk Exosomes for Use in Advanced Therapies

by Ana Medel-Martinez, Ana Redrado-Osta, Alejandra Crespo-Barreda, Maria Sancho-Albero, Lourdes Sánchez, Víctor Sebastián, María Pardo, Antonio de la Vieja and Pilar Martín-Duque

Biomolecules 2024, 14(7), 810; https://doi.org/10.3390/biom14070810 - 8 Jul 2024

Cited by 8 | Viewed by 4068

Abstract

Exosomes are cell-derived extracellular vesicles (EVs) with diameters between 30 and 120 nm. In recent years, several studies have evaluated the therapeutic potential of exosomes derived from different fluids due to their low immunogenicity and high biocompatibility. However, producing exosomes on a large [...] Read more.

Exosomes are cell-derived extracellular vesicles (EVs) with diameters between 30 and 120 nm. In recent years, several studies have evaluated the therapeutic potential of exosomes derived from different fluids due to their low immunogenicity and high biocompatibility. However, producing exosomes on a large scale is still challenging. One of the fluids from which they could be isolated in large quantities is milk. Moreover, regeneration is a well-known property of milk. The present work seeks to optimize a method for isolating exosomes from bovine and human milk, comparing different storage conditions and different extraction protocols. We found differences in the yield extraction associated with pre-storage milk conditions and observed some differences according to the processing agent. When we removed milk fat globules and added rennet before freezing, we obtained a cleaner final fraction. In summary, we attempted to optimize a rennet-based new milk–exosome isolation method and concluded that pre-treatment, followed by freezing of samples, yielded the best exosome population. Full article

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18 pages, 6547 KiB

Open AccessEditor’s ChoiceArticle

West Nile Virus-Induced Expression of Senescent Gene Lgals3bp Regulates Microglial Phenotype within Cerebral Cortex

by Artem Arutyunov, Violeta Durán-Laforet, Shenjian Ai, Loris Ferrari, Robert Murphy, Dorothy P. Schafer and Robyn S. Klein

Biomolecules 2024, 14(7), 808; https://doi.org/10.3390/biom14070808 - 8 Jul 2024

Cited by 3 | Viewed by 2685

Abstract

Microglia, the resident macrophages of the central nervous system, exhibit altered gene expression in response to various neurological conditions. This study investigates the relationship between West Nile Virus infection and microglial senescence, focusing on the role of LGALS3BP, a protein implicated in both [...] Read more.

Microglia, the resident macrophages of the central nervous system, exhibit altered gene expression in response to various neurological conditions. This study investigates the relationship between West Nile Virus infection and microglial senescence, focusing on the role of LGALS3BP, a protein implicated in both antiviral responses and aging. Using spatial transcriptomics, RNA sequencing and flow cytometry, we characterized changes in microglial gene signatures in adult and aged mice following recovery from WNV encephalitis. Additionally, we analyzed Lgals3bp expression and generated Lgals3bp-deficient mice to assess the impact on neuroinflammation and microglial phenotypes. Our results show that WNV-activated microglia share transcriptional signatures with aged microglia, including upregulation of genes involved in interferon response and inflammation. Lgals3bp was broadly expressed in the CNS and robustly upregulated during WNV infection and aging. Lgals3bp-deficient mice exhibited reduced neuroinflammation, increased homeostatic microglial numbers, and altered T cell populations without differences in virologic control or survival. These data indicate that LGALS3BP has a role in regulating neuroinflammation and microglial activation and suggest that targeting LGALS3BP might provide a potential route for mitigating neuroinflammation-related cognitive decline in aging and post-viral infections. Full article

(This article belongs to the Special Issue The Role of Microglia in Aging and Neurodegenerative Disease)

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20 pages, 2246 KiB

Open AccessEditor’s ChoiceArticle

DNA Base Damage Repair Crosstalks with Chromatin Structures to Contract Expanded GAA Repeats in Friedreich’s Ataxia

by Yanhao Lai, Nicole Diaz, Rhyisa Armbrister, Irina Agoulnik and Yuan Liu

Biomolecules 2024, 14(7), 809; https://doi.org/10.3390/biom14070809 - 8 Jul 2024

Viewed by 2244

Abstract

Trinucleotide repeat (TNR) expansion is the cause of over 40 neurodegenerative diseases, including Huntington’s disease and Friedreich’s ataxia (FRDA). There are no effective treatments for these diseases due to the poor understanding of molecular mechanisms underlying somatic TNR expansion and contraction in neural [...] Read more.

Trinucleotide repeat (TNR) expansion is the cause of over 40 neurodegenerative diseases, including Huntington’s disease and Friedreich’s ataxia (FRDA). There are no effective treatments for these diseases due to the poor understanding of molecular mechanisms underlying somatic TNR expansion and contraction in neural systems. We and others have found that DNA base excision repair (BER) actively modulates TNR instability, shedding light on the development of effective treatments for the diseases by contracting expanded repeats through DNA repair. In this study, temozolomide (TMZ) was employed as a model DNA base damaging agent to reveal the mechanisms of the BER pathway in modulating GAA repeat instability at the frataxin (FXN) gene in FRDA neural cells and transgenic mouse mice. We found that TMZ induced large GAA repeat contraction in FRDA mouse brain tissue, neurons, and FRDA iPSC-differentiated neural cells, increasing frataxin protein levels in FRDA mouse brain and neural cells. Surprisingly, we found that TMZ could also inhibit H3K9 methyltransferases, leading to open chromatin and increasing ssDNA breaks and recruitment of the key BER enzyme, pol β, on the repeats in FRDA neural cells. We further demonstrated that the H3K9 methyltransferase inhibitor BIX01294 also induced the contraction of the expanded repeats and increased frataxin protein in FRDA neural cells by opening the chromatin and increasing the endogenous ssDNA breaks and recruitment of pol β on the repeats. Our study provides new mechanistic insight illustrating that inhibition of H3K9 methylation can crosstalk with BER to induce GAA repeat contraction in FRDA. Our results will open a new avenue for developing novel gene therapy by targeting histone methylation and the BER pathway for repeat expansion diseases. Full article

(This article belongs to the Special Issue Molecular Mechanisms in DNA and RNA Damage and Repair)

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21 pages, 5750 KiB

Open AccessEditor’s ChoiceArticle

Anti-Biofilm Perspectives of Propolis against Staphylococcus epidermidis Infections

by Virginia Vadillo-Rodríguez, Irene Fernández-Babiano, Ciro Pérez-Giraldo and María Coronada Fernández-Calderón

Biomolecules 2024, 14(7), 779; https://doi.org/10.3390/biom14070779 - 29 Jun 2024

Cited by 2 | Viewed by 2650

Abstract

Staphylococcus epidermis has emerged as the main causative agent of medical device-related infections. Their major pathogenicity factor lies in its ability to adhere to surfaces and proliferate into biofilms, which increase their resistance to antibiotics. The main objective of this study was to [...] Read more.

Staphylococcus epidermis has emerged as the main causative agent of medical device-related infections. Their major pathogenicity factor lies in its ability to adhere to surfaces and proliferate into biofilms, which increase their resistance to antibiotics. The main objective of this study was to evaluate the use and the mechanism of action of an ethanolic extract of Spanish propolis (EESP) as a potential alternative for preventing biofilm-related infections caused by S. epidermidis. The chemical composition of propolis is reported and its antibacterial activity against several strains of S. epidermidis with different biofilm-forming capacities evaluated. The influence of sub-inhibitory concentrations (sub-MICs) of EESP on their growth, physicochemical surface properties, adherence, and biofilm formation were studied. EESP interferes with planktonic cells, homogenizing their physicochemical surface properties and introducing a significant delay in their growth. The adherence and biofilms at the EESP concentrations investigated were decreased up to 90.5% among the strains. Microscopic analysis indicated that the planktonic cells that survived the treatment were the ones that adhere and proliferate on the surfaces. The results obtained suggest that the EESP has a high potential to be used as an inhibitor of both the adhesion and biofilm formation of S. epidermidis. Full article

(This article belongs to the Special Issue Research into Bioactive Natural Compounds: New Molecules, Bioinspired Syntheses, Biotechnological Methods and Bioassays—Celebrating the Pioneering Work of Prof. Alejandro Fernandez Barrero)

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25 pages, 27606 KiB

Open AccessEditor’s ChoiceArticle

Structural Analysis of the Drosophila melanogaster GSTome

by Nicolas Petiot, Mathieu Schwartz, Patrice Delarue, Patrick Senet, Fabrice Neiers and Adrien Nicolaï

Biomolecules 2024, 14(7), 759; https://doi.org/10.3390/biom14070759 - 26 Jun 2024

Cited by 2 | Viewed by 1951

Abstract

Glutathione transferase (GST) is a superfamily of ubiquitous enzymes, multigenic in numerous organisms and which generally present homodimeric structures. GSTs are involved in numerous biological functions such as chemical detoxification as well as chemoperception in mammals and insects. GSTs catalyze the conjugation of [...] Read more.

Glutathione transferase (GST) is a superfamily of ubiquitous enzymes, multigenic in numerous organisms and which generally present homodimeric structures. GSTs are involved in numerous biological functions such as chemical detoxification as well as chemoperception in mammals and insects. GSTs catalyze the conjugation of their cofactor, reduced glutathione (GSH), to xenobiotic electrophilic centers. To achieve this catalytic function, GSTs are comprised of a ligand binding site and a GSH binding site per subunit, which is very specific and highly conserved; the hydrophobic substrate binding site enables the binding of diverse substrates. In this work, we focus our interest in a model organism, the fruit fly Drosophila melanogaster (D. mel), which comprises 42 GST sequences distributed in six classes and composing its GSTome. The goal of this study is to describe the complete structural GSTome of D. mel to determine how changes in the amino acid sequence modify the structural characteristics of GST, particularly in the GSH binding sites and in the dimerization interface. First, we predicted the 3D atomic structures of each GST using the AlphaFold (AF) program and compared them with X-ray crystallography structures, when they exist. We also characterized and compared their global and local folds. Second, we used multiple sequence alignment coupled with AF-predicted structures to characterize the relationship between the conservation of amino acids in the sequence and their structural features. Finally, we applied normal mode analysis to estimate thermal B-factors of all GST structures of D. mel. Particularly, we extracted flexibility profiles of GST and identify key residues and motifs that are systematically involved in the ligand binding/dimerization processes and thus playing a crucial role in the catalytic function. This methodology will be extended to guide the in silico design of synthetic GST with new/optimal catalytic properties for detoxification applications. Full article

(This article belongs to the Special Issue The Multiple Roles of Glutathione-Dependent Enzymes: A Themed Issue in Honor of Prof. Bengt Mannervik)

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15 pages, 2548 KiB

Open AccessEditor’s ChoiceArticle

Extracellular Vesicle-Mediated Modulation of Stem-like Phenotype in Breast Cancer Cells under Fluid Shear Stress

by Spenser R. Brown, Margaret E. Radcliffe, Joseph T. Danner, Wilmer J. Andújar Cruz, Kimberly H. Lackey, Han-A Park, Steven T. Weinman and Yonghyun Kim

Biomolecules 2024, 14(7), 757; https://doi.org/10.3390/biom14070757 - 25 Jun 2024

Cited by 1 | Viewed by 2456

Abstract

Circulating tumor cells (CTCs) are some of the key culprits that cause cancer metastasis and metastasis-related deaths. These cells exist in a dynamic microenvironment where they experience fluid shear stress (FSS), and the CTCs that survive FSS are considered to be highly metastatic [...] Read more.

Circulating tumor cells (CTCs) are some of the key culprits that cause cancer metastasis and metastasis-related deaths. These cells exist in a dynamic microenvironment where they experience fluid shear stress (FSS), and the CTCs that survive FSS are considered to be highly metastatic and stem cell-like. Biophysical stresses such as FSS are also known to cause the production of extracellular vesicles (EVs) that can facilitate cell–cell communication by carrying biomolecular cargos such as microRNAs. Here, we hypothesized that physiological FSS will impact the yield of EV production, and that these EVs will have biomolecules that transform the recipient cells. The EVs were isolated using direct flow filtration with and without FSS from the MDA-MB-231 cancer cell line, and the expression of key stemness-related genes and microRNAs was characterized. There was a significantly increased yield of EVs under FSS. These EVs also contained significantly increased levels of miR-21, which was previously implicated to promote metastatic progression and chemotherapeutic resistance. When these EVs from FSS were introduced to MCF-7 cancer cells, the recipient cells had a significant increase in their stem-like gene expression and CD44⁺/CD24⁻ cancer stem cell-like subpopulation. There was also a correlated increased proliferation along with an increased ATP production. Together, these findings indicate that the presence of physiological FSS can directly influence the EVs’ production and their contents, and that the EV-mediated transfer of miR-21 can have an important role in FSS-existing contexts, such as in cancer metastasis. Full article

(This article belongs to the Special Issue Advances in Nano-Based Drug Delivery: Unveiling the Next Frontier)

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22 pages, 24995 KiB

Open AccessEditor’s ChoiceArticle

Expression Proteomics and Histone Analysis Reveal Extensive Chromatin Network Changes and a Role for Histone Tail Trimming during Cellular Differentiation

by Giorgio Oliviero, Kieran Wynne, Darrell Andrews, John Crean, Walter Kolch and Gerard Cagney

Biomolecules 2024, 14(7), 747; https://doi.org/10.3390/biom14070747 - 24 Jun 2024

Cited by 2 | Viewed by 2555

Abstract

In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins [...] Read more.

In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification. Full article

(This article belongs to the Section Molecular Biology)

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17 pages, 2727 KiB

Open AccessEditor’s ChoiceArticle

Concept of Normativity in Multi-Omics Analysis of Axon Regeneration

by Isabella Moceri, Sean Meehan, Emily Gonzalez, Kevin K. Park, Abigail Hackam, Richard K. Lee and Sanjoy Bhattacharya

Biomolecules 2024, 14(7), 735; https://doi.org/10.3390/biom14070735 - 21 Jun 2024

Cited by 2 | Viewed by 1732

Abstract

Transcriptomes and proteomes can be normalized with a handful of RNAs or proteins (or their peptides), such as GAPDH, β-actin, RPBMS, and/or GAP43. Even with hundreds of standards, normalization cannot be achieved across different molecular mass ranges for small molecules, such as lipids [...] Read more.

Transcriptomes and proteomes can be normalized with a handful of RNAs or proteins (or their peptides), such as GAPDH, β-actin, RPBMS, and/or GAP43. Even with hundreds of standards, normalization cannot be achieved across different molecular mass ranges for small molecules, such as lipids and metabolites, due to the non-linearity of mass by charge ratio for even the smallest part of the spectrum. We define the amount (or range of amounts) of metabolites and/or lipids per a defined amount of a protein, consistently identified in all samples of a multiple-model organism comparison, as the normative level of that metabolite or lipid. The defined protein amount (or range) is a normalized value for one cohort of complete samples for which intrasample relative protein quantification is available. For example, the amount of citrate (a metabolite) per µg of aconitate hydratase (normalized protein amount) identified in the proteome is the normative level of citrate with aconitase. We define normativity as the amount of metabolites (or amount range) detected when compared to normalized protein levels. We use axon regeneration as an example to illustrate the need for advanced approaches to the normalization of proteins. Comparison across different pharmacologically induced axon regeneration mouse models entails the comparison of axon regeneration, studied at different time points in several models designed using different agents. For the normalization of the proteins across different pharmacologically induced models, we perform peptide doping (fixed amounts of known peptides) in each sample to normalize the proteome across the samples. We develop Regen V peptides, divided into Regen III (SEB, LLO, CFP) and II (HH4B, A1315), for pre- and post-extraction comparisons, performed with the addition of defined, digested peptides (bovine serum albumin tryptic digest) for protein abundance normalization beyond commercial labeled relative quantification (for example, 18-plex tandem mass tags). We also illustrate the concept of normativity by using this normalization technique on regenerative metabolome/lipidome profiles. As normalized protein amounts are different in different biological states (control versus axon regeneration), normative metabolite or lipid amounts are expected to be different for specific biological states. These concepts and standardization approaches are important for the integration of different datasets across different models of axon regeneration. Full article

(This article belongs to the Special Issue Advances in Neuroproteomics)

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12 pages, 1884 KiB

Open AccessEditor’s ChoiceArticle

Macrophage Phenotype Induced by Circulating Small Extracellular Vesicles from Women with Endometriosis

by María Angeles Martínez-Zamora, Olga Armengol-Badia, Lara Quintas-Marquès, Francisco Carmona and Daniel Closa

Biomolecules 2024, 14(7), 737; https://doi.org/10.3390/biom14070737 - 21 Jun 2024

Cited by 3 | Viewed by 1998

Abstract

Evidence suggests that immune system dysfunction and macrophages are involved in the disease establishment and progression of endometriosis. Among the factors involved in this alteration in macrophage activity, Small Extracellular Vesicles (sEVs) have been described to play a role favoring the switch to [...] Read more.

Evidence suggests that immune system dysfunction and macrophages are involved in the disease establishment and progression of endometriosis. Among the factors involved in this alteration in macrophage activity, Small Extracellular Vesicles (sEVs) have been described to play a role favoring the switch to a specific phenotype with controversial results. This study aims to investigate the potential effect of circulating sEVs in the plasma of well-characterized patients with endometriosis on the polarization of macrophages. sEVs were isolated from the plasma of patients diagnosed with endometriosis confirmed by histopathological analysis. Two groups of patients were recruited: the endometriosis group consisted of patients diagnosed with endometriosis by imaging testing (gynecological ultrasonography and/or magnetic resonance imaging), confirmed by histopathologic study (n = 12), and the control group included patients who underwent laparoscopy for tubal sterilization without presurgical suspicion of endometriosis and without endometriosis or signs of any inflammatory pelvic condition during surgery (n = 12). Human THP1 monocytic cells were differentiated into macrophages, and the effect of sEVs on cell uptake and macrophage polarization was evaluated by fluorescent labeling and measurement of the IL1B, TNF, ARG1, and MRC1 expression, respectively. Although no changes in cell uptake were detected, sEVs from endometriosis induced a polarization of macrophages toward an M2 phenotype, characterized by lower IL1B and TNF expression and a tendency to increase MRC1 and ARG1 levels. When macrophages were stimulated with lipopolysaccharides, less activation was also detected after treatment with endometriosis sEVs. Finally, endometriosis sEVs also induced the expression of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARG); however, treatment with rosiglitazone, a PPARG agonist, had no effect on the change in macrophage phenotype. We conclude that circulating sEVs in women with endometriosis have a certain capacity to shift the activation state of macrophages toward an M2 phenotype, but this does not modify the uptake level or the response to PPARG ligands. Full article

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

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15 pages, 3824 KiB

Open AccessEditor’s ChoiceArticle

The Structural Basis of the Activity Cliff in Modafinil-Based Dopamine Transporter Inhibitors

by Kuo-Hao Lee, Gisela Andrea Camacho-Hernandez, Amy Hauck Newman and Lei Shi

Biomolecules 2024, 14(6), 713; https://doi.org/10.3390/biom14060713 - 17 Jun 2024

Cited by 2 | Viewed by 2380

Abstract

Modafinil analogs with either a sulfoxide or sulfide moiety have improved binding affinities at the human dopamine transporter (hDAT) compared to modafinil, with lead sulfoxide-substituted analogs showing characteristics of atypical inhibition (e.g., JJC8-091). Interestingly, the only distinction between sulfoxide and sulfide substitution is [...] Read more.

Modafinil analogs with either a sulfoxide or sulfide moiety have improved binding affinities at the human dopamine transporter (hDAT) compared to modafinil, with lead sulfoxide-substituted analogs showing characteristics of atypical inhibition (e.g., JJC8-091). Interestingly, the only distinction between sulfoxide and sulfide substitution is the presence of one additional oxygen atom. To elucidate why such a subtle difference in ligand structure can result in different typical or atypical profiles, we investigated two pairs of analogs. Our quantum mechanical calculations revealed a more negatively charged distribution of the electrostatic potential surface of the sulfoxide substitution. Using molecular dynamics simulations, we demonstrated that sulfoxide-substituted modafinil analogs have a propensity to attract more water into the binding pocket. They also exhibited a tendency to dissociate from Asp79 and form a new interaction with Asp421, consequently promoting an inward-facing conformation of hDAT. In contrast, sulfide-substituted analogs did not display these effects. These findings elucidate the structural basis of the activity cliff observed with modafinil analogs and also enhance our understanding of the functionally relevant conformational spectrum of hDAT. Full article

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

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18 pages, 2574 KiB

Open AccessEditor’s ChoiceArticle

Aromatic Characterisation of Moscato Giallo by GC-MS/MS and Validation of Stable Isotopic Ratio Analysis of the Major Volatile Compounds

by Mauro Paolini, Alberto Roncone, Lorenzo Cucinotta, Danilo Sciarrone, Luigi Mondello, Federica Camin, Sergio Moser, Roberto Larcher and Luana Bontempo

Biomolecules 2024, 14(6), 710; https://doi.org/10.3390/biom14060710 - 16 Jun 2024

Cited by 4 | Viewed by 2057

Abstract

Among the Moscato grapes, Moscato Giallo is a winegrape variety characterised by a high content of free and glycosylated monoterpenoids, which gives wines very intense notes of ripe fruit and flowers. The aromatic bouquet of Moscato Giallo is strongly influenced by the high [...] Read more.

Among the Moscato grapes, Moscato Giallo is a winegrape variety characterised by a high content of free and glycosylated monoterpenoids, which gives wines very intense notes of ripe fruit and flowers. The aromatic bouquet of Moscato Giallo is strongly influenced by the high concentration of linalool, geraniol, linalool oxides, limonene, α-terpineol, citronellol, hotrienol, diendiols, trans/cis-8-hydroxy linalool, geranic acid and myrcene, that give citrus, rose, and peach notes. Except for quali-quantitative analysis, no investigations regarding the isotopic values of the target volatile compounds in grapes and wines are documented in the literature. Nevertheless, the analysis of the stable isotope ratio represents a modern and powerful tool used by the laboratories responsible for official consumer protection, for food quality and genuineness assessment. To this aim, the aromatic compounds extracted from grapes and wine were analysed both by GC-MS/MS, to define the aroma profiles, and by GC-C/Py-IRMS, for a preliminary isotope compound-specific investigation. Seventeen samples of Moscato Giallo grapes were collected during the harvest season in 2021 from two Italian regions renowned for the cultivation of this aromatic variety, Trentino Alto Adige and Veneto, and the corresponding wines were produced at micro-winery scale. The GC-MS/MS analysis confirmed the presence of the typical terpenoids both in glycosylated and free forms, responsible for the characteristic aroma of the Moscato Giallo variety, while the compound-specific isotope ratio analysis allowed us to determine the carbon (δ¹³C) and hydrogen (δ²H) isotopic signatures of the major volatile compounds for the first time. Full article

(This article belongs to the Special Issue 2nd Edition: Biochemistry of Wine and Beer)

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14 pages, 2204 KiB

Open AccessEditor’s ChoiceArticle

Haloperidol, Olanzapine, and Risperidone Induce Morphological Changes in an In Vitro Model of Human Hippocampal Neurogenesis

by Bálint Jezsó, Sára Kálmán, Kiara Gitta Farkas, Edit Hathy, Katalin Vincze, Dzsenifer Kovács-Schoblocher, Julianna Lilienberg, Csongor Tordai, Zsófia Nemoda, László Homolya, Ágota Apáti and János M. Réthelyi

Biomolecules 2024, 14(6), 688; https://doi.org/10.3390/biom14060688 - 13 Jun 2024

Cited by 2 | Viewed by 2146

Abstract

Background: Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs). Methods: Proliferation and [...] Read more.

Background: Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs). Methods: Proliferation and neurite outgrowth were measured by live cell imaging, and gene expression levels related to neuronal identity were analyzed by RT-QPCR and immunocytochemistry during differentiation into hippocampal dentate gyrus granule cells following treatment of low- and high-dose antipsychotics (haloperidol, olanzapine, and risperidone). Results: Antipsychotics did not modify the growth properties of NPCs after 3 days of treatment. However, the characteristics of neurite outgrowth changed significantly in response to haloperidol and olanzapine. After three weeks of differentiation, mRNA expression levels of the selected neuronal markers increased (except for MAP2), while antipsychotics caused only subtle changes. Additionally, we found no changes in MAP2 or GFAP protein expression levels as a result of antipsychotic treatment. Conclusions: Altogether, antipsychotic medications promoted neurogenesis in vitro by influencing neurite outgrowth rather than changing cell survival or gene expression. This study provides insights into the effects of antipsychotics on neuronal differentiation and highlights the importance of considering neurite outgrowth as a potential target of action. Full article

(This article belongs to the Special Issue Molecular Insights into the Mechanism of Antipsychotic Drugs)

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37 pages, 9291 KiB

Open AccessEditor’s ChoiceArticle

New Heterostilbene and Triazole Oximes as Potential CNS-Active and Cholinesterase-Targeted Therapeutics

by Milena Mlakić, Tena Čadež, Goran Šinko, Irena Škorić and Zrinka Kovarik

Biomolecules 2024, 14(6), 679; https://doi.org/10.3390/biom14060679 - 11 Jun 2024

Viewed by 1978

Abstract

New furan, thiophene, and triazole oximes were synthesized through several-step reaction paths to investigate their potential for the development of central nervous systems (CNS)-active and cholinesterase-targeted therapeutics in organophosphorus compound (OP) poisonings. Treating patients with acute OP poisoning is still a challenge despite [...] Read more.

New furan, thiophene, and triazole oximes were synthesized through several-step reaction paths to investigate their potential for the development of central nervous systems (CNS)-active and cholinesterase-targeted therapeutics in organophosphorus compound (OP) poisonings. Treating patients with acute OP poisoning is still a challenge despite the development of a large number of oxime compounds that should have the capacity to reactivate acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The activity of these two enzymes, crucial for neurotransmission, is blocked by OP, which has the consequence of disturbing normal cholinergic nerve signal transduction in the peripheral and CNS, leading to a cholinergic crisis. The oximes in use have one or two pyridinium rings and cross the brain–blood barrier poorly due to the quaternary nitrogen. Following our recent study on 2-thienostilbene oximes, in this paper, we described the synthesis of 63 heterostilbene derivatives, of which 26 oximes were tested as inhibitors and reactivators of AChE and BChE inhibited by OP nerve agents–sarin and cyclosarin. While the majority of oximes were potent inhibitors of both enzymes in the micromolar range, we identified several oximes as BChE or AChE selective inhibitors with the potential for drug development. Furthermore, the oximes were poor reactivators of AChE; four heterocyclic derivatives reactivated cyclosarin-inhibited BChE up to 70%, and cis,trans-5 [2-((Z)-2-(5-((E)-(hydroxyimino)methyl)thiophen-2-yl)vinyl)benzonitrile] had a reactivation efficacy comparable to the standard oxime HI-6. In silico analysis and molecular docking studies, including molecular dynamics simulation, connected kinetic data to the structural features of these oximes and confirmed their productive interactions with the active site of cyclosarin-inhibited BChE. Based on inhibition and reactivation and their ADMET properties regarding lipophilicity, CNS activity, and hepatotoxicity, these compounds could be considered for further development of CNS-active reactivators in OP poisoning as well as cholinesterase-targeted therapeutics in neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Full article

(This article belongs to the Special Issue The Role of Cholinesterases in Alzheimer’s Disease and Other Neurodegenerative Disorders)

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15 pages, 1446 KiB

Open AccessEditor’s ChoiceArticle

Perimenopause Decreases SERCA2a Activity in the Hearts of a Mouse Model of Ovarian Failure

by Ciara Barry, Sarah Rouhana, Jessica L. Braun, Mia S. Geromella, Val A. Fajardo and W. Glen Pyle

Biomolecules 2024, 14(6), 675; https://doi.org/10.3390/biom14060675 - 9 Jun 2024

Cited by 3 | Viewed by 2329

Abstract

Risk of cardiovascular disease mortality rises in women after menopause. While increased cardiovascular risk is largely attributed to postmenopausal declines in estrogens, the molecular changes in the heart that contribute to risk are poorly understood. Disruptions in intracellular calcium handling develop in ovariectomized [...] Read more.

Risk of cardiovascular disease mortality rises in women after menopause. While increased cardiovascular risk is largely attributed to postmenopausal declines in estrogens, the molecular changes in the heart that contribute to risk are poorly understood. Disruptions in intracellular calcium handling develop in ovariectomized mice and have been implicated in cardiac dysfunction. Using a mouse model of menopause in which ovarian failure occurs over 120 days, we sought to determine if perimenopause impacted calcium removal mechanisms in the heart and identify the molecular mechanisms. Mice were injected with 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure over 120 days, mimicking perimenopause. Hearts were removed at 60 and 120 days after VCD injections, representing the middle and end of perimenopause. SERCA2a function was significantly diminished at the end of perimenopause. Neither SERCA2a nor phospholamban expression changed at either time point, but phospholamban phosphorylation at S16 and T17 was dynamically altered. Intrinsic SERCA inhibitors sarcolipin and myoregulin increased >4-fold at day 60, as did the native activator DWORF. At the end of perimenopause, sarcolipin and myoregulin returned to baseline levels while DWORF was significantly reduced below controls. Sodium–calcium exchanger expression was significantly increased at the end of perimenopause. These results show that the foundation for increased cardiovascular disease mortality develops in the heart during perimenopause and that regulators of calcium handling exhibit significant fluctuations over time. Understanding the temporal development of cardiovascular risk associated with menopause and the underlying mechanisms is critical to developing interventions that mitigate the rise in cardiovascular mortality that arises after menopause. Full article

(This article belongs to the Special Issue Heart Diseases: Molecular Mechanisms and New Therapies)

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18 pages, 3012 KiB

Open AccessEditor’s ChoiceArticle

Smoking-Induced DNA Hydroxymethylation Signature Is Less Pronounced than True DNA Methylation: The Population-Based KORA Fit Cohort

by Liye Lai, Pamela R. Matías-García, Anja Kretschmer, Christian Gieger, Rory Wilson, Jakob Linseisen, Annette Peters and Melanie Waldenberger

Biomolecules 2024, 14(6), 662; https://doi.org/10.3390/biom14060662 - 5 Jun 2024

Cited by 1 | Viewed by 1853

Abstract

Despite extensive research on 5-methylcytosine (5mC) in relation to smoking, there has been limited exploration into the interaction between smoking and 5-hydroxymethylcytosine (5hmC). In this study, total DNA methylation (5mC+5hmC), true DNA methylation (5mC) and hydroxymethylation (5hmC) levels were profiled utilizing conventional bisulphite [...] Read more.

Despite extensive research on 5-methylcytosine (5mC) in relation to smoking, there has been limited exploration into the interaction between smoking and 5-hydroxymethylcytosine (5hmC). In this study, total DNA methylation (5mC+5hmC), true DNA methylation (5mC) and hydroxymethylation (5hmC) levels were profiled utilizing conventional bisulphite (BS) and oxidative bisulphite (oxBS) treatment, measured with the Illumina Infinium Methylation EPIC BeadChip. An epigenome-wide association study (EWAS) of 5mC+5hmC methylation revealed a total of 38,575 differentially methylated positions (DMPs) and 2023 differentially methylated regions (DMRs) associated with current smoking, along with 82 DMPs and 76 DMRs associated with former smoking (FDR-adjusted p < 0.05). Additionally, a focused examination of 5mC identified 33 DMPs linked to current smoking and 1 DMP associated with former smoking (FDR-adjusted p < 0.05). In the 5hmC category, eight DMPs related to current smoking and two DMPs tied to former smoking were identified, each meeting a suggestive threshold (p < 1 × 10⁻⁵). The substantial number of recognized DMPs, including 5mC+5hmC (7069/38,575, 2/82), 5mC (0/33, 1/1), and 5hmC (2/8, 0/2), have not been previously reported. Our findings corroborated previously established methylation positions and revealed novel candidates linked to tobacco smoking. Moreover, the identification of hydroxymethylated CpG sites with suggestive links provides avenues for future research. Full article

(This article belongs to the Special Issue DNA Methylation in Human Diseases)

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19 pages, 5414 KiB

Open AccessEditor’s ChoiceArticle

Application of Graph Models to the Identification of Transcriptomic Oncometabolic Pathways in Human Hepatocellular Carcinoma

by Sergio Barace, Eva Santamaría, Stefany Infante, Sara Arcelus, Jesus De La Fuente, Enrique Goñi, Ibon Tamayo, Idoia Ochoa, Miguel Sogbe, Bruno Sangro, Mikel Hernaez, Matias A. Avila and Josepmaria Argemi

Biomolecules 2024, 14(6), 653; https://doi.org/10.3390/biom14060653 - 3 Jun 2024

Cited by 2 | Viewed by 1839

Abstract

Whole-tissue transcriptomic analyses have been helpful to characterize molecular subtypes of hepatocellular carcinoma (HCC). Metabolic subtypes of human HCC have been defined, yet whether these different metabolic classes are clinically relevant or derive in actionable cancer vulnerabilities is still an unanswered question. Publicly [...] Read more.

Whole-tissue transcriptomic analyses have been helpful to characterize molecular subtypes of hepatocellular carcinoma (HCC). Metabolic subtypes of human HCC have been defined, yet whether these different metabolic classes are clinically relevant or derive in actionable cancer vulnerabilities is still an unanswered question. Publicly available gene sets or gene signatures have been used to infer functional changes through gene set enrichment methods. However, metabolism-related gene signatures are poorly co-expressed when applied to a biological context. Here, we apply a simple method to infer highly consistent signatures using graph-based statistics. Using the Cancer Genome Atlas Liver Hepatocellular cohort (LIHC), we describe the main metabolic clusters and their relationship with commonly used molecular classes, and with the presence of TP53 or CTNNB1 driver mutations. We find similar results in our validation cohort, the LIRI-JP cohort. We describe how previously described metabolic subtypes could not have therapeutic relevance due to their overall downregulation when compared to non-tumoral liver, and identify N-glycan, mevalonate and sphingolipid biosynthetic pathways as the hallmark of the oncogenic shift of the use of acetyl-coenzyme A in HCC metabolism. Finally, using DepMap data, we demonstrate metabolic vulnerabilities in HCC cell lines. Full article

(This article belongs to the Special Issue Signal Transduction to Transcription Factors in Health and Disease—Honorary Special Issue Commemorating the Work of Prof. Athanasios G. Papavassiliou)

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23 pages, 4279 KiB

Open AccessEditor’s ChoiceArticle

Disruption of Transmembrane Phosphatidylserine Asymmetry by HIV-1 Incorporated SERINC5 Is Not Responsible for Virus Restriction

by Gokul Raghunath, Elizabeth H. Abbott, Mariana Marin, Hui Wu, Judith Mary Reyes Ballista, Melinda A. Brindley and Gregory B. Melikyan

Biomolecules 2024, 14(5), 570; https://doi.org/10.3390/biom14050570 - 10 May 2024

Cited by 3 | Viewed by 1784

Abstract

Host restriction factor SERINC5 (SER5) incorporates into the HIV-1 membrane and inhibits infectivity by a poorly understood mechanism. Recently, SER5 was found to exhibit scramblase-like activity leading to the externalization of phosphatidylserine (PS) on the viral surface, which has been proposed to be [...] Read more.

Host restriction factor SERINC5 (SER5) incorporates into the HIV-1 membrane and inhibits infectivity by a poorly understood mechanism. Recently, SER5 was found to exhibit scramblase-like activity leading to the externalization of phosphatidylserine (PS) on the viral surface, which has been proposed to be responsible for SER5’s antiviral activity. This and other reports that document modulation of HIV-1 infectivity by viral lipid composition prompted us to investigate the role of PS in regulating SER5-mediated HIV-1 restriction. First, we show that the level of SER5 incorporation into virions correlates with an increase in PS levels in the outer leaflet of the viral membrane. We developed an assay to estimate the PS distribution across the viral membrane and found that SER5, but not SER2, which lacks antiviral activity, abrogates PS asymmetry by externalizing this lipid. Second, SER5 incorporation diminished the infectivity of pseudoviruses produced from cells lacking a flippase subunit CDC50a and, therefore, exhibited a higher baseline level of surface-accessible PS. Finally, exogenous manipulation of the viral PS levels utilizing methyl-alpha-cyclodextrin revealed a lack of correlation between external PS and virion infectivity. Taken together, our study implies that the increased PS exposure to SER5-containing virions itself is not directly linked to HIV-1 restriction. Full article

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23 pages, 1045 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Risk and Resilience Variants in the Retinoic Acid Metabolic and Developmental Pathways Associated with Risk of FASD Outcomes

by Leo McKay, Berardino Petrelli, Molly Pind, James N. Reynolds, Richard F. Wintle, Albert E. Chudley, Britt Drögemöller, Abraham Fainsod, Stephen W. Scherer, Ana Hanlon-Dearman and Geoffrey G. Hicks

Biomolecules 2024, 14(5), 569; https://doi.org/10.3390/biom14050569 - 10 May 2024

Cited by 1 | Viewed by 2159

Abstract

Fetal Alcohol Spectrum Disorder (FASD) is a common neurodevelopmental disorder that affects an estimated 2–5% of North Americans. FASD is induced by prenatal alcohol exposure (PAE) during pregnancy and while there is a clear genetic contribution, few genetic factors are currently identified or [...] Read more.

Fetal Alcohol Spectrum Disorder (FASD) is a common neurodevelopmental disorder that affects an estimated 2–5% of North Americans. FASD is induced by prenatal alcohol exposure (PAE) during pregnancy and while there is a clear genetic contribution, few genetic factors are currently identified or understood. In this study, using a candidate gene approach, we performed a genetic variant analysis of retinoic acid (RA) metabolic and developmental signaling pathway genes on whole exome sequencing data of 23 FASD-diagnosed individuals. We found risk and resilience alleles in ADH and ALDH genes known to normally be involved in alcohol detoxification at the expense of RA production, causing RA deficiency, following PAE. Risk and resilience variants were also identified in RA-regulated developmental pathway genes, especially in SHH and WNT pathways. Notably, we also identified significant variants in the causative genes of rare neurodevelopmental disorders sharing comorbidities with FASD, including STRA6 (Matthew–Wood), SOX9 (Campomelic Dysplasia), FDG1 (Aarskog), and 22q11.2 deletion syndrome (TBX1). Although this is a small exploratory study, the findings support PAE-induced RA deficiency as a major etiology underlying FASD and suggest risk and resilience variants may be suitable biomarkers to determine the risk of FASD outcomes following PAE. Full article

(This article belongs to the Special Issue New Insight into the Molecular Genetics of Neurodevelopmental Disorders)

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12 pages, 1148 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Synthesis of the Antimicrobial Peptide Murepavadin Using Novel Coupling Agents

by Júlia García-Gros, Yolanda Cajal, Ana Maria Marqués and Francesc Rabanal

Biomolecules 2024, 14(5), 526; https://doi.org/10.3390/biom14050526 - 27 Apr 2024

Cited by 2 | Viewed by 3566

Abstract

The problem of antimicrobial resistance is becoming a daunting challenge for human society and healthcare systems around the world. Hence, there is a constant need to develop new antibiotics to fight resistant bacteria, among other important social and economic measures. In this regard, [...] Read more.

The problem of antimicrobial resistance is becoming a daunting challenge for human society and healthcare systems around the world. Hence, there is a constant need to develop new antibiotics to fight resistant bacteria, among other important social and economic measures. In this regard, murepavadin is a cyclic antibacterial peptide in development. The synthesis of murepavadin was undertaken in order to optimize the preparative protocol and scale-up, in particular, the use of new activation reagents. In our hands, classical approaches using carbodiimide/hydroxybenzotriazole rendered low yields. The use of novel carbodiimide and reagents based on OxymaPure^® and Oxy-B is discussed together with the proper use of chromatographic conditions for the adequate characterization of peptide crudes. Higher yields and purities were obtained. Finally, the antimicrobial activity of different synthetic batches was tested in three Pseudomonas aeruginosa strains, including highly resistant ones. All murepavadin batches yielded the same highly active MIC values and proved that the chiral integrity of the molecule was preserved throughout the whole synthetic procedure. Full article

(This article belongs to the Special Issue Development, Characterization, and Application of Bioactive Peptides, Diagnostic Biomarkers, and Pharmaceutical Proteins)

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17 pages, 3825 KiB

Open AccessEditor’s ChoiceArticle

Metformin Induces MeCP2 in the Hippocampus of Male Mice with Sex-Specific and Brain-Region-Dependent Molecular Impact

by Khatereh Saei Arezoumand, Chris-Tiann Roberts and Mojgan Rastegar

Biomolecules 2024, 14(4), 505; https://doi.org/10.3390/biom14040505 - 21 Apr 2024

Cited by 3 | Viewed by 2701

Abstract

Rett Syndrome (RTT) is a progressive X-linked neurodevelopmental disorder with no cure. RTT patients show disease-associated symptoms within 18 months of age that include developmental regression, progressive loss of useful hand movements, and breathing difficulties, along with neurological impairments, seizures, tremor, and mental [...] Read more.

Rett Syndrome (RTT) is a progressive X-linked neurodevelopmental disorder with no cure. RTT patients show disease-associated symptoms within 18 months of age that include developmental regression, progressive loss of useful hand movements, and breathing difficulties, along with neurological impairments, seizures, tremor, and mental disability. Rett Syndrome is also associated with metabolic abnormalities, and the anti-diabetic drug metformin is suggested to be a potential drug of choice with low or no side-effects. Previously, we showed that in vitro exposure of metformin in a human brain cell line induces MECP2E1 transcripts, the dominant isoform of the MECP2 gene in the brain, mutations in which causes RTT. Here, we report the molecular impact of metformin in mice. Protein analysis of specific brain regions in the male and female mice by immunoblotting indicated that metformin induces MeCP2 in the hippocampus, in a sex-dependent manner. Additional experiments confirm that the regulatory role of metformin on the MeCP2 target “BDNF” is brain region-dependent and sex-specific. Measurement of the ribosomal protein S6 (in both phosphorylated and unphosphorylated forms) confirms the sex-dependent role of metformin in the liver. Our results can help foster a better understanding of the molecular impact of metformin in different brain regions of male and female adult mice, while providing some insight towards its potential in therapeutic strategies for the treatment of Rett Syndrome. Full article

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

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14 pages, 2283 KiB

Open AccessEditor’s ChoiceArticle

Conformational Modulation of Tissue Transglutaminase via Active Site Thiol Alkylating Agents: Size Does Not Matter

by Pauline Navals, Alana M. M. Rangaswamy, Petr Kasyanchyk, Maxim V. Berezovski and Jeffrey W. Keillor

Biomolecules 2024, 14(4), 496; https://doi.org/10.3390/biom14040496 - 19 Apr 2024

Cited by 1 | Viewed by 1742

Abstract

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme’s dysregulated activity has been implicated in a variety of pathologies (e.g., celiac [...] Read more.

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme’s dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2’s conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters k_inact and K_I. Their impact on the enzyme’s ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity. Full article

(This article belongs to the Special Issue Transglutaminases: Regulation, Imaging, and Applications)

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17 pages, 3733 KiB

Open AccessEditor’s ChoiceArticle

Mitochondrial Transplantation’s Role in Rodent Skeletal Muscle Bioenergetics: Recharging the Engine of Aging

by Tasnim Arroum, Gerald A. Hish, Kyle J. Burghardt, James D. McCully, Maik Hüttemann and Moh H. Malek

Biomolecules 2024, 14(4), 493; https://doi.org/10.3390/biom14040493 - 18 Apr 2024

Cited by 6 | Viewed by 4360

Abstract

Background: Mitochondria are the ‘powerhouses of cells’ and progressive mitochondrial dysfunction is a hallmark of aging in skeletal muscle. Although different forms of exercise modality appear to be beneficial to attenuate aging-induced mitochondrial dysfunction, it presupposes that the individual has a requisite level [...] Read more.

Background: Mitochondria are the ‘powerhouses of cells’ and progressive mitochondrial dysfunction is a hallmark of aging in skeletal muscle. Although different forms of exercise modality appear to be beneficial to attenuate aging-induced mitochondrial dysfunction, it presupposes that the individual has a requisite level of mobility. Moreover, non-exercise alternatives (i.e., nutraceuticals or pharmacological agents) to improve skeletal muscle bioenergetics require time to be effective in the target tissue and have another limitation in that they act systemically and not locally where needed. Mitochondrial transplantation represents a novel directed therapy designed to enhance energy production of tissues impacted by defective mitochondria. To date, no studies have used mitochondrial transplantation as an intervention to attenuate aging-induced skeletal muscle mitochondrial dysfunction. The purpose of this investigation, therefore, was to determine whether mitochondrial transplantation can enhance skeletal muscle bioenergetics in an aging rodent model. We hypothesized that mitochondrial transplantation would result in sustained skeletal muscle bioenergetics leading to improved functional capacity. Methods: Fifteen female mice (24 months old) were randomized into two groups (placebo or mitochondrial transplantation). Isolated mitochondria from a donor mouse of the same sex and age were transplanted into the hindlimb muscles of recipient mice (quadriceps femoris, tibialis anterior, and gastrocnemius complex). Results: The results indicated significant increases (ranging between ~36% and ~65%) in basal cytochrome c oxidase and citrate synthase activity as well as ATP levels in mice receiving mitochondrial transplantation relative to the placebo. Moreover, there were significant increases (approx. two-fold) in protein expression of mitochondrial markers in both glycolytic and oxidative muscles. These enhancements in the muscle translated to significant improvements in exercise tolerance. Conclusions: This study provides initial evidence showing how mitochondrial transplantation can promote skeletal muscle bioenergetics in an aging rodent model. Full article

(This article belongs to the Special Issue Skeletal Muscle Homeostasis and Regeneration)

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9 pages, 820 KiB

Open AccessEditor’s ChoiceArticle

Development of Improved Spectrophotometric Assays for Biocatalytic Silyl Ether Hydrolysis

by Yuqing Lu, Chisom S. Egedeuzu, Peter G. Taylor and Lu Shin Wong

Biomolecules 2024, 14(4), 492; https://doi.org/10.3390/biom14040492 - 18 Apr 2024

Cited by 2 | Viewed by 1884

Abstract

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon−oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with [...] Read more.

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon−oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis−Menten kinetic parameters for silicatein. Full article

(This article belongs to the Section Enzymology)

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13 pages, 3079 KiB

Open AccessEditor’s ChoiceArticle

Radiolabelled FGF-2 for Imaging Activated Fibroblasts in the Tumor Micro-Environment

by Valeria Bentivoglio, Filippo Galli, Michela Varani, Danilo Ranieri, Pallavi Nayak, Annunziata D’Elia, Andrea Soluri, Roberto Massari, Chiara Lauri and Alberto Signore

Biomolecules 2024, 14(4), 491; https://doi.org/10.3390/biom14040491 - 18 Apr 2024

Cited by 2 | Viewed by 2195

Abstract

Tumor associated fibroblasts (TAFs) play a key role in tumor growth and metastatization. TAFs overexpress different biomarkers that are usually expressed at low levels in physiological conditions. Among them are the fibroblast growth factor receptors (FGFRs) that bind the fibroblast growth factors (FGFs). [...] Read more.

Tumor associated fibroblasts (TAFs) play a key role in tumor growth and metastatization. TAFs overexpress different biomarkers that are usually expressed at low levels in physiological conditions. Among them are the fibroblast growth factor receptors (FGFRs) that bind the fibroblast growth factors (FGFs). In particular, the overexpression of FGFR-2c in tumors has been associated with advanced clinical stages and increased metastatization. Here, we developed a non-invasive tool to evaluate, in vivo, the expression of FGFR-2c in metastatic cancer. This is based on ^99mTc-labelled FGF-2. Methods: ^99mTc-FGF-2 was tested in vitro and in vivo in mice bearing allografts of sarcoma cells. Images of ^99mTc-FGF-2 were acquired using a new portable high-resolution ultra-sensitive gamma camera for small animal imaging. Results: FGF-2 was labeled with high specific activity but low labelling efficiency, thus requiring post-labeling purification by gel-filtration chromatography. In vitro binding to 2C human keratinocytes showed a Kd of 3.36 × 10⁻⁹ M. In mice bearing J774A.1 cell allografts, we observed high and rapid tumor uptake of ^99mTc-FGF-2 with a high Tumor/Blood ratio at 24 h post-injection (26.1 %ID/g and 12.9 %ID) with low kidney activity and moderate liver activity. Conclusions: we labeled FGF-2 with ^99mTc and showed nanomolar Kd in vitro with human keratinocytes expressing FGF-2 receptors. In mice, ^99mTc-FGF-2 rapidly and efficiently accumulated in tumors expressing FGF-2 receptors. This new radiopharmaceutical could be used in humans to image TAFs. Full article

(This article belongs to the Section Molecular Medicine)

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