Biomolecules

26 pages, 1923 KiB

Open AccessReview

tRNA and tsRNA: From Heterogeneity to Multifaceted Regulators

by Yun Li, Zongyu Yu, Wenlin Jiang, Xinyi Lyu, Ailian Guo, Xiaorui Sun, Yiting Yang and Yunfang Zhang

Biomolecules 2024, 14(10), 1340; https://doi.org/10.3390/biom14101340 - 21 Oct 2024

Viewed by 2283

Abstract

As the most ancient RNA, transfer RNAs (tRNAs) play a more complex role than their constitutive function as amino acid transporters in the protein synthesis process. The transcription and maturation of tRNA in cells are subject to stringent regulation, resulting in the formation [...] Read more.

As the most ancient RNA, transfer RNAs (tRNAs) play a more complex role than their constitutive function as amino acid transporters in the protein synthesis process. The transcription and maturation of tRNA in cells are subject to stringent regulation, resulting in the formation of tissue- and cell-specific tRNA pools with variations in tRNA overall abundance, composition, modification, and charging levels. The heterogeneity of tRNA pools contributes to facilitating the formation of histocyte-specific protein expression patterns and is involved in diverse biological processes. Moreover, tRNAs can be recognized by various RNase under physiological and pathological conditions to generate tRNA-derived small RNAs (tsRNAs) and serve as small regulatory RNAs in various biological processes. Here, we summarize these recent insights into the heterogeneity of tRNA and highlight the advances in the regulation of tRNA function and tsRNA biogenesis by tRNA modifications. We synthesize diverse mechanisms of tRNA and tsRNA in embryonic development, cell fate determination, and epigenetic inheritance regulation. We also discuss the potential clinical applications based on the new knowledge of tRNA and tsRNA as diagnostic and prognostic biomarkers and new therapeutic strategies for multiple diseases. Full article

(This article belongs to the Special Issue Advances in tRNA Biology)

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Figure 1

Figure 1
Transfer RNA (tRNA) heterogeneity is manifested in multiple aspects. tRNA heterogeneity is reflected in overall tRNA abundance; the composition of tRNA isotypes, isoacceptors, and isodecoders; the abundance of various RNA modifications; and the aminoacylation levels in cellular tRNA pools, which may be regulated by multiple effectors, such as chromatin accessibility, transcription factor binding, and RNA polymerase III (Pol III) activity, as well as the abundance and activity of tRNA-modifying enzymes and aminoacyl-tRNA synthetases (AARS). The tRNA isotypes are distinguished by different color schemes, and the varying shades within the same color range in-dicate different tRNA isoacceptors. The different colored dots on the tRNA sequence represent different types of RNA modifications. tRNAs are not always fully aminoacylated and that the level varies depending on cell type and growth conditions. Created in BioRender. Yang, Y. (2024) <a href="http://BioRen-der.com/a28u622" target="_blank">BioRen-der.com/a28u622</a> (accessed on 15 August 2024). Full article ">Figure 2
RNA modification on tRNAs and biogenesis of tsRNAs. (a) Possible tRNA modification types, sites, and corresponding enzymes. (b) 5′tsRNAs generate from D loop nuclease or anticodon loop nuclease; 3′tsRNAs generate from TΨC loop or anticodon loop nuclease; inner’tsRNAs generate from D loop and TΨC loop nuclease. Full article ">Figure 3
Neural network model for transmitting parental traits to offspring through the sperm RNA code. The paternally acquired phenotypes are encoded as the sperm RNA code, with various features. It is possible that these features may interact with each other, either directly or indirectly, to influence the biological process in the early embryo and subsequently affect the phenotypes of the offspring. Created in BioRender. Yang, Y. (2024) <a href="http://BioRender.com/x33h223" target="_blank">BioRender.com/x33h223</a> (accessed on 15 August 2024). Full article ">

23 pages, 1998 KiB

Open AccessReview

Astragalus membranaceus: A Traditional Chinese Medicine with Multifaceted Impacts on Breast Cancer Treatment

by Zhong Tang and Xuefei Tian

Biomolecules 2024, 14(10), 1339; https://doi.org/10.3390/biom14101339 - 21 Oct 2024

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Abstract

Breast cancer, the most prevalent malignant tumor among women globally, remains a critical area of focus for researchers striving to refine therapeutic approaches. As an important component of traditional Chinese medicine, Astragalus membranaceus (AM) has demonstrated potential for multifaceted impacts on breast cancer treatment [...] Read more.

Breast cancer, the most prevalent malignant tumor among women globally, remains a critical area of focus for researchers striving to refine therapeutic approaches. As an important component of traditional Chinese medicine, Astragalus membranaceus (AM) has demonstrated potential for multifaceted impacts on breast cancer treatment through various mechanisms. To guide clinical practice and further explore the under-researched field of AM in breast cancer treatment, this paper mainly reviews the regulatory roles of AM-derived compounds and extracts on breast cancer cell proliferation, migration, invasion, and chemoresistance. Furthermore, this study delves into the synergistic effects observed when AM is co-administered with chemotherapeutic agents, including the enhancement of chemosensitivity, mitigation of toxic side effects, and reversal of drug resistance. This review indicates that AM holds promise not only as a therapy in breast cancer treatment but also paves the way for innovative integrated treatment approaches that combine the benefits of traditional medicine with modern pharmaceuticals. Nevertheless, future research endeavors are also urged to elucidate the in vivo pharmacological effects and underlying mechanisms of AM to inform more effective clinical treatment strategies. Full article

(This article belongs to the Special Issue Antitumor Agents from Natural Sources 2024–2025)

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Figure 1
Signal pathways of calycosin in breast cancer. Calycosin can suppress EMT by inhibiting the BATF/TGF-β1 pathway. Calycosin targets the ER and downstream pathways to inhibit the proliferation of cancer cells. In addition, calycosin can promote apoptosis by inducing the Bax/BCL-2 pathway. Orange arrows indicate inhibitory effects, while green arrows represent inducing effects. Full article ">Figure 2
Signal pathways of formononetin in breast cancer. Formononetin can inhibit the proliferation, autophagy, invasion, and migration of breast cancer cells by suppressing the NF-κB p65, p38, Akt, and p53 signaling pathways. Formononetin can enhance autophagy through the MiR-199a-3p/mTOR pathway. Formononetin can modulate drug resistance by affecting the lncRNA AFAP1-AS1. Orange arrows indicate inhibitory effects, while green arrows represent inducing effects. Full article ">Figure 3
Signal pathways of astragaloside IV in breast cancer. Astragaloside IV can suppress cell invasion by inhibiting the MAPK signaling pathway and induce TRHDE-ASI to inhibit cell proliferation. Additionally, it can influence FOXO1, promoting the polarization of M2 macrophages. Orange arrows indicate inhibitory effects, while green arrows represent inducing effects. Full article ">Figure 4
Signal pathways of biochanin A in breast cancer. Biochanin A can suppress the viability, invasion, and growth of breast cancer cells by inhibiting the Erk1/2, mTOR, Akt, NF-κB, and p53 signaling pathway, while it can enhance cell proliferation through the MiR-199a-3p/Era pathway. Orange arrows indicate inhibitory effects, while green arrows represent inducing effects. Full article ">

16 pages, 2676 KiB

Open AccessArticle

Cooperative Substructure and Energetics of Allosteric Regulation of the Catalytic Core of the E3 Ubiquitin Ligase Parkin by Phosphorylated Ubiquitin

by Xiang Ye, Sravya Kotaru, Rosana Lopes, Shannen Cravens, Mauricio Lasagna and A. Joshua Wand

Biomolecules 2024, 14(10), 1338; https://doi.org/10.3390/biom14101338 - 21 Oct 2024

Viewed by 1266

Abstract

Mutations in the parkin gene product Parkin give rise to autosomal recessive juvenile parkinsonism. Parkin is an E3 ubiquitin ligase that is a critical participant in the process of mitophagy. Parkin has a complex structure that integrates several allosteric signals to maintain precise [...] Read more.

Mutations in the parkin gene product Parkin give rise to autosomal recessive juvenile parkinsonism. Parkin is an E3 ubiquitin ligase that is a critical participant in the process of mitophagy. Parkin has a complex structure that integrates several allosteric signals to maintain precise control of its catalytic activity. Though its allosterically controlled structural reorganization has been extensively characterized by crystallography, the energetics and mechanisms of allosteric regulation of Parkin are much less well understood. Allostery is fundamentally linked to the energetics of the cooperative (sub)structure of the protein. Herein, we examine the mechanism of allosteric activation by phosphorylated ubiquitin binding to the enzymatic core of Parkin, which lacks the antagonistic Ubl domain. In this way, the allosteric effects of the agonist phosphorylated ubiquitin can be isolated. Using native-state hydrogen exchange monitored by mass spectrometry, we find that the five structural domains of the core of Parkin are energetically distinct. Nevertheless, association of phosphorylated ubiquitin destabilizes structural elements that bind the ubiquitin-like domain antagonist while promoting the dissociation of the catalytic domain and energetically poises the protein for transition to the fully activated structure. Full article

(This article belongs to the Special Issue The Illumination of Biophysical Mechanisms by Hydrogen Exchange: A Themed Issue in Honor of Professor S. Walter Englander)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Cartoon representation of the structure of the Parkin(∆Ubl)·pUb complex. RING0 (residues 142–225, red), RING1 (residues 226–329, yellow), IBR (residues 330–378, magenta), REP (residues 379–410, cyan), RING2 (residues 411–465, blue), zinc atoms (wheat) and pUb (orange). Drawn using PyMol from the structure determined by Wauer et al. (PDB code: 5caw) [<a href="#B10-biomolecules-14-01338" class="html-bibr">10</a>]. Full article ">Figure 2
Parkin(∆Ubl) denaturation by urea followed by circular dichroism at 222 nm. The solid line results from fitting the solid circles to Equation (1). For reasons outlined in the main text, the fitted ∆Gunf and m values indicate a relatively uncooperative multistate unfolding of the protein, which is consistent with the hydrogen exchange studies described below. The hollow circles correspond to a separate transition likely involving unfolding of structure stabilized by high-affinity zinc fingers. See main text. Full article ">Figure 3
Structural fluctuations in Parkin(∆Ubl). Native-state hydrogen exchange time profiles representative of the various domains of Parkin(∆Ubl) measured by peptide fragmentation mass spectrometry (A–I). The domain and primary sequence residue numbers are shown in each panel. Fitting of these data (solid lines) gave average R2 and Sy.x values of 0.99 ± 0.02 and 0.16 ± 0.09 amu, respectively. Full article ">Figure 4
Structural fluctuations in the Parkin(∆Ubl)·pUb complex. Native-state hydrogen exchange time profiles representative of the various domains of Parkin(∆Ubl) in the Parkin(∆Ubl)·pUb complex measured by peptide fragmentation mass spectrometry (A–I). The domain and primary sequence residue numbers are shown in each panel. Fitting of these data (solid lines) gave average R2 and Sy.x values of 0.98 ± 0.02 and 0.16 ± 0.10 amu, respectively. Full article ">Figure 5
Comparison of the energetics of the protein ensemble revealed by native-state hydrogen exchange <math display="inline"><semantics> <mrow> <msubsup> <mrow> <mo>∆</mo> <mi>G</mi> </mrow> <mrow> <mi>H</mi> <mi>X</mi> </mrow> <mrow> <mi>d</mi> <mi>e</mi> <mi>p</mi> </mrow> </msubsup> </mrow> </semantics></math> in Parkin(∆Ubl) and the Parkin(∆Ubl)·pUb complex. Denaturant dependence of the apparent free energy of hydrogen exchange (∆GHX) of representative peptides derived from the domains of Parkin(∆Ubl) (A–I). The domain and primary sequence residue numbers are shown in each panel. fitted with one or two exponentials as statistically warranted (see <a href="#biomolecules-14-01338-t001" class="html-table">Table 1</a>). Fitting of these data (solid lines) gave average R2 and Sy.x values of 0.96 ± 0.05 and 0.32 ± 0.17 kJ mol−1, respectively. Full article ">Figure 6
Allosteric transition in Parkin(∆Ubl) upon binding of pUb. Cartoon representations of the structure of the RING1 domain (shown in yellow) of (A) hsParkin(∆Ubl) (PDB code: 4bm9) and (B) the PhParkin(∆Ubl)·pUb complex (PDB code: 5caw). pUb is colored orange. Shown in green are glutamate and arginine residues participating in an ion pair in hsParkin(∆Ubl), which is disrupted in the PhParkin(∆Ubl)·pUb complex. Note that the involved E and R side chains are ill defined (disordered) in the PhParkin(∆Ubl))·pUb complex. The urea dependence of the apparent free energy of the fluctuations leading to hydrogen exchange in the helix spanning residues 268–280 in hsParkin(∆Ubl) (C) and in the homologous region in the PhParkin(∆Ubl)·pUb complex (D). The HX time courses of this region in both the free and complexed Parkin(∆Ubl) are best fitted with two exponentials. Fitting of these data gave average R2 and Sy.x values of 0.94 ± 0.06 and 0.36 ± 0.11 kJ mol−1, respectively. Deconvolution using overlapping peptides indicates that the fast phase is associated with the C-terminus of this helix while the slower phase is associated with the N-terminal region. The C-terminus is destabilized by the binding of pUb. In contrast, the N-terminal region is stabilized by the binding of pUb. Cartoons drawn with PyMol. Full article ">Figure 7
Urea dependence of the activation free energy of dissociation of pUb from Parkin(∆Ubl). Rates of dissociation of pUb from Parkin(∆Ubl) were extracted from two population binomial fits of the m/z distribution HX time courses of the Parkin(∆Ubl)·pUb complex as a function of urea (see <a href="#app1-biomolecules-14-01338" class="html-app">Figure S1</a>). The fitted m-value was 0.83 ± 0.11 kJ mol−1 M−1 and R2 0.89. Full article ">

1 pages, 143 KiB

Open AccessCorrection

Correction: Choi et al. β-Ionone Attenuates Dexamethasone-Induced Suppression of Collagen and Hyaluronic Acid Synthesis in Human Dermal Fibroblasts. Biomolecules 2021, 11, 619

by Dabin Choi, Wesuk Kang, Soyoon Park, Bomin Son and Taesun Park

Biomolecules 2024, 14(10), 1337; https://doi.org/10.3390/biom14101337 - 21 Oct 2024

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Abstract

The authors would like to modify the Conflicts of Interest section of the published paper [...] Full article

20 pages, 1322 KiB

Open AccessArticle

Chemical Profiling of Polar Lipids and the Polyphenolic Fraction of Commercial Italian Phaseolus Seeds by UHPLC-HRMS and Biological Evaluation

by Vadym Samukha, Francesca Fantasma, Gilda D’Urso, Ester Colarusso, Anna Schettino, Noemi Marigliano, Maria Giovanna Chini, Gabriella Saviano, Vincenzo De Felice, Gianluigi Lauro, Francesco Maione, Giuseppe Bifulco, Agostino Casapullo and Maria Iorizzi

Biomolecules 2024, 14(10), 1336; https://doi.org/10.3390/biom14101336 - 20 Oct 2024

Viewed by 1218

Abstract

The common bean (Phaseolus vulgaris L.) is one of the oldest food crops in the world. In this study, the ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-MS/MS) technique was used to characterize the polar lipid composition and polyphenolic fraction of five bean [...] Read more.

The common bean (Phaseolus vulgaris L.) is one of the oldest food crops in the world. In this study, the ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-MS/MS) technique was used to characterize the polar lipid composition and polyphenolic fraction of five bean varieties commonly consumed in Italy: Cannellino (PVCA), Controne (PVCO), Borlotti (PVBO), Stregoni (PVST), and Vellutina (PVVE). Lipid content represents a minor fraction of the whole metabolome in dry beans, and little is known about their polar lipids, which could be potentially bioactive components. Thirty-three compounds were detected through UHPLC-MS/MS, including oxylipins, phospholipids, N-acyl glycerolipids, and several fatty acids. The dichloromethane extracts were subjected to principal component analysis (PCA), with the results showing greater differentiation for the Borlotti variety. Moreover, 27 components belonging to different polyphenol classes, such as phenolic acids, flavonoids, catechins, anthocyanins and their glycosides, and some saponins, were identified in the hydroalcoholic seed extracts. In addition, the mineral content of the beans was determined. Considering the high number of compounds in the five apolar seed extracts, all samples were examined to determine their in vitro inhibitory activity against the enzyme cyclooxygenase-2 (COX-2), which is inducible in inflammatory cells and mediates inflammatory responses. Only PVCO showed the best inhibition of the COX-2 enzyme with an IC₅₀ = 31.15 ± 2.16 µg/mL. In light of these results, the potential anti-inflammatory properties of PVCO were evaluated in the LPS-stimulated murine macrophage cell line J774A.1. Herein, we demonstrate, for the first time, that PVCO at 30 µg/mL can significantly reduce the release of TNF-α, with a less significant anti-inflammatory effect being observed in terms of IL-6 release. Full article

(This article belongs to the Special Issue Recent Advances in the Isolation, Chemical and Biological Characterization and Synthesis of Bioactive Metabolites from Plants)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Selected Italian commercial bean varieties: Vellutina (PVVE) (a); Borlotti (PVBO) (b); Stregoni (PVST) (c); Controne (PVCO) (d); Cannellino (PVCA) (e). Full article ">Figure 2
Score scatter plot of the partial least squares–discriminant analysis (PLS-DA) performed as a targeted approach on the detected compounds of the lipophilic extracts of five Italian commercial P. vulgaris varieties: Borlotti (PVBO) (green), Cannellino (PVCA) (blue), Controne (PVCO) (red), Stregoni (PVST) (yellow), and Vellutina (PVVE) (light blue). Full article ">Figure 3
Loading scatter plot of the targeted partial least squares–discriminant analysis (PLS-DA) performed on the lipophilic extracts of five Italian commercial P. vulgaris varieties: Borlotti (PVBO (B)), Cannellino (PVCA (C)), Controne (PVCO (O)), Stregoni (PVST (S)), and Vellutina (PVVE (V)). Full article ">Figure 4
(A,B) An in vitro cytotoxic examination, evaluated using an MTT assay, was performed on the murine macrophage J774A.1 cell line, following 4 (A) and 24 h (B) of treatment with selected concentrations of PVCO (3, 10, 30, and 100 μg/mL). The dotted lines indicate the 75% cell viability limit. Data are expressed as cell viability (% of control) and presented as the means ± S.D. of three independent experiments. Our statistical analysis was conducted using one-way ANOVA followed by Bonferroni correction for multiple comparisons. (C,F) Murine macrophages J774A.1 were stimulated with LPS (10 µg/mL) and treated with PVCO at the concentration of 30 µg/mL for 4 and 24 h (C). Thereafter, cell supernatants were assayed using ELISA to determine TNF-α and IL-6 levels (expressed as pg/mL) at both 4 (D,F, respectively) and 24 h (E,G, respectively). Data are presented as the means ± S.D. of three independent experiments. #### p ≤ 0.0001 vs. the Ctrl group; * p ≤ 0.05, ** p ≤ 0.01 vs. the LPS group. Full article ">

17 pages, 764 KiB

Open AccessArticle

Increased Cardiometabolic Risk in Men with Hypoprolactinemia: A Pilot Study

by Robert Krysiak, Karolina Kowalcze, Witold Szkróbka and Bogusław Okopień

Biomolecules 2024, 14(10), 1335; https://doi.org/10.3390/biom14101335 - 20 Oct 2024

Viewed by 1213

Abstract

Low prolactin levels in men predispose them to mood disturbances, sexual dysfunction, and diabetes. The purpose of the current study was to assess cardiometabolic risk in males with hypoprolactinemia. This prospective study included three age-matched groups of young and middle-aged men: individuals with [...] Read more.

Low prolactin levels in men predispose them to mood disturbances, sexual dysfunction, and diabetes. The purpose of the current study was to assess cardiometabolic risk in males with hypoprolactinemia. This prospective study included three age-matched groups of young and middle-aged men: individuals with cabergoline-induced hypoprolactinemia (n = 15), cabergoline-treated subjects with prolactin levels within the reference range (n = 20), and untreated men with normal prolactin levels (n = 31). In men with hypoprolactinemia, the cabergoline dose was reduced in order to normalize prolactin concentration. Anthropometric parameters, blood pressure, QRISK3 score; plasma concentrations of prolactin, glucose, insulin, lipids, uric acid, high-sensitivity C-reactive protein (hsCRP), fibrinogen, homocysteine, and testosterone; whole-blood levels of glycated hemoglobin (HbA_1C); urinary albumin-to-creatinine ratio (UACR); and carotid intima–media thickness were assessed at baseline and six months later. Men with hypoprolactinemia were characterized by higher body mass index, fat content, waist circumference, systolic blood pressure, fasting and 2 h post-load glucose, HbA_1C, HOMA1-IR, uric acid, hsCRP, fibrinogen, homocysteine, and UACR; by lower HDL cholesterol and testosterone; by greater intima–media thickness; and by a higher QRISK3 score than their peers with normal prolactin levels. There were no statistically significant differences in the measured parameters between both groups of men with normal prolactin levels. Normalization of prolactin concentration was accompanied by normalization of biochemical variables, systolic blood pressure, and QRISK3 score. Although cabergoline dose reduction did not cause statistically significant changes in the remaining anthropometric parameters and intima–media thickness, six months later, they did not differ from those observed in the remaining study groups. Our findings suggest that iatrogenic hypoprolactinemia is associated with increased cardiometabolic risk, which is reversible and resolves after the normalization of prolactin levels. Full article

(This article belongs to the Special Issue Advances in Cardiometabolic Health)

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19 pages, 1379 KiB

Open AccessReview

Potential Roles of IP₃ Receptors and Calcium in Programmed Cell Death and Implications in Cardiovascular Diseases

by Chanon Piamsiri, Nadezhda Fefelova, Sri Harika Pamarthi, Judith K. Gwathmey, Siriporn C. Chattipakorn, Nipon Chattipakorn and Lai-Hua Xie

Biomolecules 2024, 14(10), 1334; https://doi.org/10.3390/biom14101334 - 20 Oct 2024

Cited by 1 | Viewed by 2048

Abstract

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) play a crucial role in maintaining intracellular/cytosolic calcium ion (Ca²⁺_i) homeostasis. The release of Ca²⁺ from IP₃Rs serves as a second messenger and a modulatory factor influencing various intracellular and interorganelle [...] Read more.

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) play a crucial role in maintaining intracellular/cytosolic calcium ion (Ca²⁺_i) homeostasis. The release of Ca²⁺ from IP₃Rs serves as a second messenger and a modulatory factor influencing various intracellular and interorganelle communications during both physiological and pathological processes. Accumulating evidence from in vitro, in vivo, and clinical studies supports the notion that the overactivation of IP₃Rs is linked to the pathogenesis of various cardiac conditions. The overactivation of IP₃Rs results in the dysregulation of Ca²⁺ concentration ([Ca²⁺]) within cytosolic, mitochondrial, and nucleoplasmic cellular compartments. In cardiovascular pathologies, two isoforms of IP₃Rs, i.e., IP₃R1 and IP₃R2, have been identified. Notably, IP₃R1 plays a pivotal role in cardiac ischemia and diabetes-induced arrhythmias, while IP₃R2 is implicated in sepsis-induced cardiomyopathy and cardiac hypertrophy. Furthermore, IP₃Rs have been reported to be involved in various programmed cell death (PCD) pathways, such as apoptosis, pyroptosis, and ferroptosis underscoring their multifaceted roles in cardiac pathophysiology. Based on these findings, it is evident that exploring potential therapeutic avenues becomes crucial. Both genetic ablation and pharmacological intervention using IP₃R antagonists have emerged as promising strategies against IP₃R-related pathologies suggesting their potential therapeutic potency. This review summarizes the roles of IP₃Rs in cardiac physiology and pathology and establishes a foundational understanding with a particular focus on their involvement in the various PCD pathways within the context of cardiovascular diseases. Full article

(This article belongs to the Section Cellular Biochemistry)

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Figure 1

Figure 1
A schematic diagram illustrating the regulatory mechanisms of IP3Rs in cardiomyocytes under both physiological and pathological conditions. (A) During physiological conditions, external stimuli such as adenosine, angiotensin II, bradykinin, endothelin, vasopressin, dopamine, epinephrine, norepinephrine, etc., bind and activate GPCRs on the plasma membrane. Subsequently, the Gqα-subunit dissociates from the trimeric GPCR structure, activating PLC to produce IP3 from PIP2. IP3 then binds to the IP3 binding core domain of IP3Rs leading to the release of Ca2+ from the SR. However, the amount of Ca2+ flux via IP3Rs is relatively lower compared to the Ca2+ transients generated by CICR and other major voltage-operated channels and ligand-gated channels. IP3R-mediated Ca2+ release causes Ca2+ translocation from the SR to the cytosol, mitochondria, and nucleus. In the cytosol, Ca2+ serves as a secondary messenger playing modulatory roles. Ca2+i binds to cTnC and initiates cardiac contraction. In mitochondria, Ca2+Mito influences mitochondrial energy production and metabolic activity. Mitochondria also act as secondary storage sites and modulate [Ca2+]i levels. In the nucleus, Ca2+Nuc regulates gene expression and transcription. (B) During cardiac pathologies (e.g., ischemia, I/R injuries, arrhythmias, sepsis-induced cardiomyopathy, cardiac hypertrophy, and heart failure), neurohormonal hyperactivation leads to exacerbated IP3 production from GPCR. Additionally, these pathological conditions cause IP3Rs overexpression leading to Ca2+i, Ca2+Mito, and Ca2+Nuc overload. Increased Ca2+i binds with calmodulin thus leading to CaMKII overactivation. Ca2+i overload causes cardiac hypercontracture, contractile dysfunction, cardiac cell injuries, cardiac cell death, and arrhythmias. Ca2+Mito overload causes mitochondrial dysfunction and impaired mitochondrial ATP production. Ca2+Nuc overload enhances the function of transcription factors that control the expression of pro-hypertrophic and pro-arrhythmic genes. It should be noted that the remodeling of IP3Rs and Ca2+ signaling contribute to different cardiovascular diseases and pathophysiological conditions. Endothelin-induced excessive IP3R1 expression and overactivation lead to Ca2+i and Ca2+Nuc overload, subsequently contributing to the development of atrial arrhythmias [<a href="#B6-biomolecules-14-01334" class="html-bibr">6</a>,<a href="#B10-biomolecules-14-01334" class="html-bibr">10</a>,<a href="#B21-biomolecules-14-01334" class="html-bibr">21</a>]. In sepsis-induced cardiomyopathy, the LPS-induced overexpression of IP3R2 leads to Ca2+i overload triggering myocardial cell death through pyroptosis and apoptosis [<a href="#B14-biomolecules-14-01334" class="html-bibr">14</a>]. In ischemic heart disease, the upregulation of IP3R1 in ventricular cardiomyocytes results in Ca2+i and Ca2+Mito overload leading to myocardial cell death through pyroptosis [<a href="#B6-biomolecules-14-01334" class="html-bibr">6</a>,<a href="#B13-biomolecules-14-01334" class="html-bibr">13</a>]. In the failing heart, persistent increased cardiac workload leads to chronic neurohormonal hyperactivation thereby triggering IP3R2/calcineurin/CaMKII-mediated NFAT nuclear translocation, which contributes to the transcription of hypertrophic genes [<a href="#B6-biomolecules-14-01334" class="html-bibr">6</a>,<a href="#B22-biomolecules-14-01334" class="html-bibr">22</a>,<a href="#B23-biomolecules-14-01334" class="html-bibr">23</a>]. This figure was created using BioRender. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CICR, calcium-induced calcium release; cTnC, cardiac troponin C; GPCR, G protein-coupled receptors; IP3, inositol 1,4,5-trisphosphate; IP3Rs, inositol 1,4,5-trisphosphate receptors; LTCC, L-type calcium channel; PIP2, phosphatidylinositol 4,5-bisphosphate (PIP2); PLB, phospholamban; PLC, phospholipase C; MCU, mitochondrial calcium uniporter; mPTP, mitochondrial permeability transition pore; mtROS, mitochondrial ROS production; NCX, sodium–calcium exchanger; RyR, ryanodine receptors; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; [Ca2+], calcium ion concentration; [Ca2+]i, calcium ion concentration in the cytosol; [Ca2+]Mito, calcium ion concentration in mitochondria; [Ca2+]Nuc calcium ion concentration in the nucleus; ΔΨm mitochondrial membrane potential. Full article ">Figure 2
A schematic diagram illustrating the roles of IP3Rs in apoptosis, pyroptosis, and ferroptosis. (A) Apoptosis: Several stress conditions (e.g., high glucose condition) mediate ER stress resulting in the upregulation of IP3Rs and GRP75 expression. Formation of the IP3Rs-GRP75-VDAC complex facilitates ER-mediated Ca2+Mito overload. Excessive [Ca2+]Mito causes mitochondrial dysfunction and facilitates mPTP opening, leading to Cytochrome C being released. The access of Cytochrome C to the cytosol initiates mitochondrial-mediated apoptosis by activating caspase cascades, including the cleavage of caspase 9 and caspase 3. (B) Pyroptosis: Stressors such as I/R or H/R induce the overexpression of IP3Rs and the downregulation of ERP44, thereby mediating Ca2+Mito and Ca2+i overload. Ca2+Mito overload impairs mitochondrial functions, disrupts oxidative phosphorylation, and increases mtROS production. The resultant overproduction of mtROS and Ca2+i overload lead to NLRP3 activation and inflammasome assembly. Once activated, the NLRP3 inflammasome cleaves pro-caspase 1 to its activated form. Then, cleaved caspase 1 further cleaves and activates GSDMD into its N-terminal fragment (GSDMD-NT). As a result, GSDMD-NT translocates and oligomerizes at the inner leaflet of the plasma membrane resulting in the formation of transmembrane pores. (C) Ferroptosis: It is proposed that ER stress could mediate ferroptosis through the overactivation of IP3Rs, thereby, leading to Ca2+Mito overload and mtROS overproduction. The administration of ferroptosis activator RSL3 inhibited the antioxidant property of GPX4 exacerbating mitochondrial and intracellular ROS levels. The subsequent augmentation of ROS leads to membrane lipid peroxidation and ultimately causes membrane rupture. In addition, Ca2+i overload caused by IP3Rs also enhances the catalytic activity of LOX, further promoting lipid membrane peroxidation. However, the underlying mechanism of whether and how Ca2+i impacts LOX remains unclear. This figure was created using BioRender. Abbreviations: BAX, Bcl-2-associated protein X; Bcl-2, B-cell lymphoma 2; GSDMD, gasdermin D; GSDMD-NT, gasdermin D N-terminus; H/R, hypoxia-reoxygenation; IP3Rs, inositol 1,4,5-trisphosphate receptors; I/R, ischemia–reperfusion; LOX, lipoxygenases; MCU, mitochondrial calcium uniporter; MFRN, mitoferrin 2; mPTP, mitochondrial permeability transition pore; mtROS, mitochondrial ROS; NLRP3, nod-like receptor protein-3; RSL3, RAS-selective lethal 3; [Ca2+], calcium ion concentration; VDAC, voltage-dependent anion channel; [Ca2+]i, calcium ion concentration in the cytosol; [Ca2+]Mito, calcium ion concentration in mitochondria; [Ca2+]Nuc calcium ion concentration in the nucleus; ΔΨm, mitochondrial membrane potential. Full article ">

15 pages, 1695 KiB

Open AccessReview

Stress-Induced Evolution of the Nucleolus: The Role of Ribosomal Intergenic Spacer (rIGS) Transcripts

by Anastasia A. Gavrilova, Margarita V. Neklesova, Yuliya A. Zagryadskaya, Irina M. Kuznetsova, Konstantin K. Turoverov and Alexander V. Fonin

Biomolecules 2024, 14(10), 1333; https://doi.org/10.3390/biom14101333 - 20 Oct 2024

Viewed by 1681

Abstract

It became clear more than 20 years ago that the nucleolus not only performs the most important biological function of assembling ribonucleic particles but is also a key controller of many cellular processes, participating in cellular adaptation to stress. The nucleolus’s multifunctionality is [...] Read more.

It became clear more than 20 years ago that the nucleolus not only performs the most important biological function of assembling ribonucleic particles but is also a key controller of many cellular processes, participating in cellular adaptation to stress. The nucleolus’s multifunctionality is due to the peculiarities of its biogenesis. The nucleolus is a multilayered biomolecular condensate formed by liquid–liquid phase separation (LLPS). In this review, we focus on changes occurring in the nucleolus during cellular stress, molecular features of the nucleolar response to abnormal and stressful conditions, and the role of long non-coding RNAs transcribed from the intergenic spacer region of ribosomal DNA (IGS rDNA). Full article

(This article belongs to the Section Biological Factors)

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The ribosomal DNA cassette (rDNA cassette). The rDNA cassette contains sequences encoding the pre-RNA and the ribosomal intergenic spacer (rIGS). In humans, the cassette size is approximately 43 kb; they are located on the p-arms of five chromosomes. Distal Junction is a sequence approximately 400 kb long that flanks the ribosomal gene repeat. Polymerase I transcribes several functional noncoding RNAs from rIGS. rIGS 16 RNA and rIGS 22 RNA are synthesized during heat shock, and rIGS 28 RNA is synthesized under acidosis conditions. pRNA is transcribed from spacer promoters upstream of the pre-rRNA transcription start site. PAPAS are a set of antisense RNAs that are synthesized by Pol II. PAPAS transcripts do not have a common promoter; their transcription begins at random sites and can span both pre-rRNA coding and IGS regions longer than 10 kb. Full article ">Figure 2
Nucleolar transformation in response to stress. The interphase nucleolus with characteristic tripartite structure is shown in the center. Panels (A–D) show structures that arise under different stressful influences. (A) Nucleolar segregation or nucleolar caps are formed when RNA pol I transcription is inactivated. Actinomycin D or ionizing radiation induces rDNA double-strand breaks (DSBs), resulting in the formation of nucleolar capsules adjacent to their DJ anchor. (B) Upon DRB treatment, RNA pol I transcription is active, but rRNA processing is converted to form a nucleolar necklace. (C) When exposed to heat shock and acidosis, the nucleolus transforms into an electron-dense fibrillar organization, the A-body. The fibers contain immobilized proteins in an amyloid-like state. (D) Nucleolar aggresomes are formed upon proteotoxic insults such as proteasome inhibition and heat shock. This may or may not involve inhibited RNA pol I activity. Full article ">Figure 3
Stress-induced transcription of non-coding RNAs from the IGS region. (A) pRNA recruits the Nucleolar Remodeling Complex (NoRC) to the promoter and, due to its hairpin structure, binds to one of the NoRC proteins, TIP5, thereby mediating the nucleolar localization of the entire complex. NoRC moves the nucleosome to a repressive position, preventing transcription initiation. (B) During cellular quiescence and starvation, the amount of PAPAS increases. The transcripts recruit the histone methyltransferase Suv4-20h2 to the rDNA locus, which installs the repressive H4K20me3 mark on the rDNA, resulting in immediate suppression of rDNA expression. (C) During heat shock and acidosis, rIGS 16 RNA, rIGS 22 RNA, and rIGS 28 RNA are transcribed, respectively. These transcripts likely mediate the nucleolar sequestration of VHL, CDC73, MDM2, POLD1, and many other proteins possessing amyloid-converting motifs. The local concentration of proteins with an amyloidogenic propensity in the foci triggers physiological amyloidogenesis and generates nascent amyloid bodies (A-bodies). Full article ">

22 pages, 7650 KiB

Open AccessArticle

Identification of Multifunctional Putative Bioactive Peptides in the Insect Model Red Palm Weevil (Rhynchophorus ferrugineus)

by Carmen Scieuzo, Roberta Rinaldi, Fabiana Giglio, Rosanna Salvia, Mohammed Ali AlSaleh, Jernej Jakše, Arnab Pain, Binu Antony and Patrizia Falabella

Biomolecules 2024, 14(10), 1332; https://doi.org/10.3390/biom14101332 - 19 Oct 2024

Cited by 1 | Viewed by 1631

Abstract

Innate immunity, the body’s initial defense against bacteria, fungi, and viruses, heavily depends on antimicrobial peptides (AMPs), which are small molecules produced by all living organisms. Insects, with their vast biodiversity, are one of the most abundant and innovative sources of AMPs. In [...] Read more.

Innate immunity, the body’s initial defense against bacteria, fungi, and viruses, heavily depends on antimicrobial peptides (AMPs), which are small molecules produced by all living organisms. Insects, with their vast biodiversity, are one of the most abundant and innovative sources of AMPs. In this study, AMPs from the red palm weevil (RPW) Rhynchophorus ferrugineus (Coleoptera: Curculionidae), a known invasive pest of palm species, were examined. The AMPs were identified in the transcriptomes from different body parts of male and female adults, under different experimental conditions, including specimens collected from the field and those reared in the laboratory. The RPW transcriptomes were examined to predict antimicrobial activity, and all sequences putatively encoding AMPs were analyzed using several machine learning algorithms available in the CAMP_R3 database. Additionally, anticancer, antiviral, and antifungal activity of the peptides were predicted using iACP, AVPpred, and Antifp server tools, respectively. Physicochemical parameters were assessed using the Antimicrobial Peptide Database Calculator and Predictor. From these analyses, 198 putatively active peptides were identified, which can be tested in future studies to validate the in silico predictions. Genome-wide analysis revealed that several AMPs have predominantly emerged through gene duplication. Noticeably, we detect a newly originated defensin allele from an ancestral defensin via the deletion of two amino acids following gene duplication in RPW, which may confer an enhanced resilience to microbial infection. Our study shed light on AMP gene families and shows that high duplication and deletion rates are essential to achieve a diversity of antimicrobial mechanisms; hence, we propose the RPW AMPs as a model for exploring gene duplication and functional variations against microbial infection. Full article

(This article belongs to the Special Issue State of the Art and Perspectives in Antimicrobial Peptides)

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Heatmap showing the expression levels of duplicated genes in the different body parts of Rhynchophorus ferrugineus lab-reared and field-collected male and female adults. The heatmap colors represent transcript abundance in transcripts per million (TPM) from highest (red) to lowest (blue) expression levels. The data represented as log-transformed TPM values were tabulated and converted into heatmaps using R and R Studio software (version 2023.06.2+561). (A) Wings; (B) Legs; (C) Abdomen; (D) Thorax; (E) Head; (F) Antennae; (G) Snout; (H) Gut; (I) Fat Body. Full article ">Figure 2
Classification of the 198 total AMPs detected in adult transcriptomes related to their family classification. Full article ">Figure 3
Graphical display for the cecropin genes mapped in scaffold_405 (NCBI acc no. JAACXV010000404.1) showing functional cecropins gene length, CDS (coding region) length, and protein length, which was generated using the NCBI graphical sequence viewer available in the Genome workbench. The visual code shows green, red, and purple, indicating gene, coding region, and mRNA, respectively. The line shows the introns and boxes for the exons. Full article ">Figure 4
Graphical display for the defensin genes mapped in the six scaffolds (NCBI acc no. JAACXV010014362.1, JAACXV010000413.1, JAACXV010014484.1, JAACXV010014200.1, JAACXV010014575.1, and JAACXV010014362.1) showing functional gene length, CDS length, and protein length generated using the NCBI graphical sequence viewer available in the Genome workbench. The visual code shows green, red, and purple, indicating gene, coding region, and mRNA. Full article ">Figure 5
Two allelic variants in the locus tag “GWI33_019784” and deduced amino acids predict 82aa and 84-aa proteins (NCBI acc nos. KAF7266949 and KAF7266950), with a conserved DEFL_defensin-like domain at the C-terminal region. Dots denote identical amino acid residues, and conserved DEFL_defensin-like domain at the C-terminal region are underlined. Full article ">Figure 6
Graphical display for the hypothetical AMP genes mapped in the two scaffolds (66363 and 66088) (NCBI acc nos. JAACXV010014549.1 and JAACXV010014301.1) showing the functional AMP gene length, CDS length, and protein length generated using the NCBI graphical sequence viewer available in the Genome workbench. The visual code shows green, red, and purple, indicating gene, coding region, and mRNA, respectively. The line shows the introns and boxes for the exons. Full article ">Figure 7
Graphical display for the lysozyme genes mapped in the two scaffolds (66335 and 66281) (NCBI acc nos. JAACXV010014523.1 and JAACXV010014472.1) showing functional lysozyme gene length, CDS length, and protein length generated using the NCBI graphical sequence viewer available in the Genome workbench. The visual code shows green, red, and purple, indicating gene, coding region, and mRNA, respectively. The line shows the introns and boxes for the exons. Full article ">

17 pages, 4282 KiB

Open AccessArticle

A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses

by Xiaojing Lin, Qiang Sun, Yang Cao, Zi Li, Cuiling Xu, Jun Liu, Jingdong Song, Kun Qin, Yong Zhang and Jianfang Zhou

Biomolecules 2024, 14(10), 1331; https://doi.org/10.3390/biom14101331 - 19 Oct 2024

Viewed by 1219

Abstract

Enteroviruses have been a historical concern since the identification of polioviruses in humans. Wild polioviruses have almost been eliminated, while multiple species of non-polio enteroviruses and their variants co-circulate annually. To date, at least 116 types have been found in humans and are [...] Read more.

Enteroviruses have been a historical concern since the identification of polioviruses in humans. Wild polioviruses have almost been eliminated, while multiple species of non-polio enteroviruses and their variants co-circulate annually. To date, at least 116 types have been found in humans and are grouped into the species Enterovirus A–D and Rhinovirus A–C. However, there are few available antiviral drugs, especially with a universal pharmaceutical effect. Here, we demonstrate that peptide P25 from EV-D68 has broad antiviral activity against EV A–D enteroviruses in vitro. P25, derived from the HI loop and β-I sheet of VP1, operates through a conserved hydrophilic motif -R---K-K--K- and the hydrophobic F near the N-terminus. It could prevent viral infection of EV-A71 by competing for the heparan sulfate (HS) receptor, binding and stabilizing virions by suppressing the release of the viral genome. P25 also inhibited the generation of infectious viral particles by reducing viral protein synthesis. The molecular docking revealed that P25 might bind to the pocket opening area, a potential target for broad-spectrum antivirals. Our findings implicate the multiple antiviral effects of peptide P25, including blocking viral binding to the HS receptor, impeding viral genome release, and reducing progeny particles, which could be a novel universal anti-enterovirus drug candidate. Full article

(This article belongs to the Section Biomacromolecules: Proteins)

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26 pages, 1530 KiB

Open AccessReview

A Survey on Computational Methods in Drug Discovery for Neurodegenerative Diseases

by Caterina Vicidomini, Francesco Fontanella, Tiziana D’Alessandro and Giovanni N. Roviello

Biomolecules 2024, 14(10), 1330; https://doi.org/10.3390/biom14101330 - 19 Oct 2024

Cited by 4 | Viewed by 1907

Abstract

Currently, the age structure of the world population is changing due to declining birth rates and increasing life expectancy. As a result, physicians worldwide have to treat an increasing number of age-related diseases, of which neurological disorders represent a significant part. In this [...] Read more.

Currently, the age structure of the world population is changing due to declining birth rates and increasing life expectancy. As a result, physicians worldwide have to treat an increasing number of age-related diseases, of which neurological disorders represent a significant part. In this context, there is an urgent need to discover new therapeutic approaches to counteract the effects of neurodegeneration on human health, and computational science can be of pivotal importance for more effective neurodrug discovery. The knowledge of the molecular structure of the receptors and other biomolecules involved in neurological pathogenesis facilitates the design of new molecules as potential drugs to be used in the fight against diseases of high social relevance such as dementia, Alzheimer’s disease (AD) and Parkinson’s disease (PD), to cite only a few. However, the absence of comprehensive guidelines regarding the strengths and weaknesses of alternative approaches creates a fragmented and disconnected field, resulting in missed opportunities to enhance performance and achieve successful applications. This review aims to summarize some of the most innovative strategies based on computational methods used for neurodrug development. In particular, recent applications and the state-of-the-art of molecular docking and artificial intelligence for ligand- and target-based approaches in novel drug design were reviewed, highlighting the crucial role of in silico methods in the context of neurodrug discovery for neurodegenerative diseases. Full article

(This article belongs to the Special Issue Experimental and Theoretical Approaches to Protein-Targeting Drug Discovery: 2nd Edition)

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17 pages, 971 KiB

Open AccessReview

Aspirin Hypersensitivity in Patients with Coronary Artery Disease: An Updated Review and Practical Recommendations

by Luigi Cappannoli, Stefania Colantuono, Francesco Maria Animati, Francesco Fracassi, Mattia Galli, Cristina Aurigemma, Enrico Romagnoli, Rocco Antonio Montone, Mattia Lunardi, Lazzaro Paraggio, Carolina Ierardi, Ilaria Baglivo, Cristiano Caruso, Carlo Trani and Francesco Burzotta

Biomolecules 2024, 14(10), 1329; https://doi.org/10.3390/biom14101329 - 19 Oct 2024

Viewed by 1822

Abstract

Acetylsalicylic acid (ASA) represents a cornerstone of antiplatelet therapy for the treatment of atherosclerotic coronary artery disease (CAD). ASA is in fact indicated in case of an acute coronary syndrome or after a percutaneous coronary intervention with stent implantation. Aspirin hypersensitivity is frequently [...] Read more.

Acetylsalicylic acid (ASA) represents a cornerstone of antiplatelet therapy for the treatment of atherosclerotic coronary artery disease (CAD). ASA is in fact indicated in case of an acute coronary syndrome or after a percutaneous coronary intervention with stent implantation. Aspirin hypersensitivity is frequently reported by patients, and this challenging situation requires a careful evaluation of the true nature of the presumed sensitivity and of its mechanisms, as well as to differentiate it from a more frequent (and more easily manageable) aspirin intolerance. Two main strategies are available to allow ASA administration for patients with CAD and suspected ASA hypersensitivity: a low-dose ASA challenge, aimed at assessing the tolerability of ASA at the antiplatelet dose of 100 mg, and desensitization, a therapeutic procedure which aims to induce tolerance to ASA. For those patients who cannot undergo ASA challenge and desensitization due to previous serious adverse reactions, or for those in whom desensitization was unsuccessful, a number of further alternative strategies are available, even if these have not been validated and approved by guidelines. The aim of this state-of-the-art review is therefore to summarize the established evidence regarding pathophysiology, clinical presentation, diagnosis, and management of aspirin hypersensitivity and to provide a practical guide for cardiologists (and clinicians) who have to face the not uncommon situation of a patient with concomitant coronary artery disease and aspirin hypersensitivity. Full article

(This article belongs to the Special Issue New Discoveries in Biological Functions of Platelet)

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17 pages, 2211 KiB

Open AccessArticle

The Biological Effect of Enriching the Plasma Content in Platelet-Rich Plasma: An In Vitro Study

by Eduardo Anitua, Mar Zalduendo, Roberto Prado, María Troya, Roberto Tierno, María de la Fuente and Mohammad H. Alkhraisat

Biomolecules 2024, 14(10), 1328; https://doi.org/10.3390/biom14101328 - 18 Oct 2024

Viewed by 1006

Abstract

BACKGROUND: Platelet-rich plasma (PRP) formulations have become valuable therapeutic tools in regenerative medicine. In addition, these blood derivates have been successfully included in cell therapy as fetal bovine serum substitutes, due to the real need to avoid the risk of host immunologic reactions [...] Read more.

BACKGROUND: Platelet-rich plasma (PRP) formulations have become valuable therapeutic tools in regenerative medicine. In addition, these blood derivates have been successfully included in cell therapy as fetal bovine serum substitutes, due to the real need to avoid the risk of host immunologic reactions and the animal disease transmission associated with reagents from animal origin. However, the protocols for obtaining them should be optimized to improve their biological potential. METHODS: PRP-derived preparations with different concentrations of the platelet and plasma components were obtained from the blood of five donors by freeze-drying. Measurements of the pH, protein, and growth factor concentration were performed. Moreover, their biological effects on cell proliferation and migration and their angiogenic potential were assessed. RESULTS: An increased plasma component concentration resulted in an augmented quantity of the total protein content, a significative variation in the hepatocyte growth factor concentration, and an experimental but clinically irrelevant alteration of the pH value. No significant changes were induced in their potential to enhance proliferative and migratory responses in epithelial cells, with the latter being reduced for dermal fibroblasts. The endothelial cell capacity for tube formation was significatively reduced. CONCLUSIONS: An increased blood plasma content did not improve the biological potential of the formulations. However, they have emerged as a promising approach for regenerative therapies where neovascularization must be avoided. Full article

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15 pages, 2592 KiB

Open AccessArticle

Pulling Forces Differentially Affect Refolding Pathways Due to Entangled Misfolded States in SARS-CoV-1 and SARS-CoV-2 Receptor Binding Domain

by Pham Dang Lan, Edward P. O’Brien and Mai Suan Li

Biomolecules 2024, 14(10), 1327; https://doi.org/10.3390/biom14101327 - 18 Oct 2024

Viewed by 1335

Abstract

Single-molecule force spectroscopy (SMFS) experiments can monitor protein refolding by applying a small force of a few piconewtons (pN) and slowing down the folding process. Bell theory predicts that in the narrow force regime where refolding can occur, the folding time should increase [...] Read more.

Single-molecule force spectroscopy (SMFS) experiments can monitor protein refolding by applying a small force of a few piconewtons (pN) and slowing down the folding process. Bell theory predicts that in the narrow force regime where refolding can occur, the folding time should increase exponentially with increased external force. In this work, using coarse-grained molecular dynamics simulations, we compared the refolding pathways of SARS-CoV-1 RBD and SARS-CoV-2 RBD (RBD refers to the receptor binding domain) starting from unfolded conformations with and without a force applied to the protein termini. For SARS-CoV-2 RBD, the number of trajectories that fold is significantly reduced with the application of a 5 pN force, indicating that, qualitatively consistent with Bell theory, refolding is slowed down when a pulling force is applied to the termini. In contrast, the refolding times of SARS-CoV-1 RBD do not change meaningfully when a force of 5 pN is applied. How this lack of a Bell response could arise at the molecular level is unknown. Analysis of the entanglement changes of the folded conformations revealed that in the case of SARS-CoV-1 RBD, an external force minimizes misfolding into kinetically trapped states, thereby promoting efficient folding and offsetting any potential slowdown due to the external force. These misfolded states contain non-native entanglements that do not exist in the native state of either SARS-CoV-1-RBD or SARS-CoV-2-RBD. These results indicate that non-Bell behavior can arise from this class of misfolding and, hence, may be a means of experimentally detecting these elusive, theoretically predicted states. Full article

(This article belongs to the Section Biomacromolecules: Proteins)

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Representative native entanglements in the native structures of SARS-CoV-1 RBD and SARS-CoV-2 RBD. These structures were obtained using AlphaFold2 and MD simulations. The loops (colored red) are closed by native contacts (colored yellow). (Left) The loop is closed by the native contact between residue 99 and 136, with both N and C termini threading through the loop. Note that only the C-terminal thread is colored blue for more visibility. (Right) The loop is closed by the native contact between residue 108 and 146, with the C-terminal thread highlighted in blue. Full article ">Figure 2
The root mean square deviation (RMSD) and the radius of gyration (Rg) of the backbone from three 500 ns all-atom MD simulations for (a) SARS-CoV-1 RBD and (b) SARS-CoV-2 RBD. Full article ">Figure 3
Dependence of −ln(P) on Q, where P is the probability of sampling a particular Q value of (a) SARS-CoV-1 RBD and (b) SARS-CoV-2 RBD. An additional minimum is observed at Q~0.2, which occurs in the presence of the external force. Full article ">Figure 4
Misfolded states were observed during the folding of the SARS-CoV-1 RBD. Force may promote the folding process of the SARS-CoV-1 RBD, resulting in an increase in the percentage of trajectories that follow paths leading to correct folding. (a,b) represent −ln(P) surfaces, where P is the probability of sampling Q and G values during refolding simulations with F = 0 and F = 5 pN, respectively. (c,d) are transition networks of discrete trajectories along metastable states represented by nodes. Blue, green and red nodes correspond to folded, intermediate and misfolded states, respectively, that trajectories visited at the end of simulations. Red numbers indicate the number of transitions between states, and black numbers represent nodes. Full article ">Figure 5
Misfolded states were also observed in the folding of the SARS-CoV-2 RBD. The force effect results in 43% of trajectories remaining in the unfolded state 1 at the end of the simulation. (a,b) represent the −ln(P) surfaces, where P is the probability of sampling certain values of Q and G from refolding simulations with F = 0 and F = 5 pN, respectively. (c,d) are transition networks of discrete trajectories along metastable states represented by nodes. Blue, green and red nodes correspond to folded, intermediate and misfolded states, respectively, that trajectories visited at the end of the simulations. The black node indicates that trajectories remain in the unfolded state 1 at the end of the simulations. Red numbers indicate the number of transitions between states, and black numbers represent nodes. Full article ">Figure 6
Conformational distribution along the G parameter of (a) SARS-CoV-1 RBD and (b) SARS-CoV-2 RBD with peaks are concise with characteristics of (Q, G) log probability surfaces, indicating the presence of structures with change in entanglement at different levels, namely G~0.05–0.1, 0.15–0.2, 0.2–0.25. Full article ">

23 pages, 3110 KiB

Open AccessArticle

From Organotypic Mouse Brain Slices to Human Alzheimer’s Plasma Biomarkers: A Focus on Nerve Fiber Outgrowth

by Sakir Necat Yilmaz, Katharina Steiner, Josef Marksteiner, Klaus Faserl, Mathias Villunger, Bettina Sarg and Christian Humpel

Biomolecules 2024, 14(10), 1326; https://doi.org/10.3390/biom14101326 - 18 Oct 2024

Viewed by 968

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by memory loss and progressive deterioration of cognitive functions. Being able to identify reliable biomarkers in easily available body fluids such as blood plasma is vital for the disease. To achieve this, we used a [...] Read more.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by memory loss and progressive deterioration of cognitive functions. Being able to identify reliable biomarkers in easily available body fluids such as blood plasma is vital for the disease. To achieve this, we used a technique that applied human plasma to organotypic brain slice culture via microcontact printing. After a 2-week culture period, we performed immunolabeling for neurofilament and myelin oligodendrocyte glycoprotein (MOG) to visualize newly formed nerve fibers and oligodendrocytes. There was no significant change in the number of new nerve fibers in the AD plasma group compared to the healthy control group, while the length of the produced fibers significantly decreased. A significant increase in the number of MOG+ dots around these new fibers was detected in the patient group. According to our hypothesis, there are factors in the plasma of AD patients that affect the growth of new nerve fibers, which also affect the oligodendrocytes. Based on these findings, we selected the most promising plasma samples and conducted mass spectrometry using a differential approach and we identified three putative biomarkers: aldehyde-dehydrogenase 1A1, alpha-synuclein and protein S100-A4. Our method represents a novel and innovative approach for translating research findings from mouse models to human applications. Full article

(This article belongs to the Section Molecular Biomarkers)

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Experimental design of this study. Plasma samples from Alzheimer’s disease (AD) patients, mild cognitive impairment (MCI) patients and healthy controls (Co) were used for experiments. Mouse organotypic brain slices were coupled with plasma microcontact (µC) printed membranes and cultured for 2 weeks. At the end of the culture period, brain slices were fixed and labeled for neurofilament (NF), microtubule-associated protein-2 (MAP2) or myelin oligodendrocyte glycoprotein (MOG). For mass spectrometry analysis, selected plasma samples were used. Full article ">Figure 2
Immunolabeling of neurofilament (NF) and microtubule-associated protein-2 (MAP2). (A) Organotypic brain slices from the hippocampal level were cultured on an extra membrane in semipermeable inserts. (B) The scheme shows that these slices were connected to microcontact prints loaded with plasma, allowing nerve fibers to grow along them. (C) The microcontact prints can be visualized by additionally loading a red-fluorescent Alexa 546 antibody to assess the efficiency, appearing as red lines perpendicular to the border of the slice (dashed line). (D) Nerve fibers are stained with neurofilament (green, Alexa 488) and (E) with MAP2 (red, Alexa 546). (F) This panel shows the merged staining of NF and MAP2. The slices were counterstained with nuclear DAPI (blue). Scale bar = 1 cm (A), 75 µm (C), 25 µm (D–F). Full article ">Figure 3
Immunolabeling of neurofilament (NF)-positive nerve fibers and myelin oligodendrocyte glycoprotein (MOG)-positive dots. Organotypic brain slices were cultured on plasma microcontact prints for 2 weeks. (A) Following fixation, they were stained for NF (green, Alexa 488) and (B) MOG (red, Alexa 546). (C) This panel shows the merged staining of NF and MOG. The slices were counterstained with nuclear DAPI (blue). The dashed white line indicates the border of the brain slices. The arrows point to specific examples of MOG-positive dots (bottom arrow) and negative staining (top arrow). (D,E) This panel shows an example at higher magnification of MOG+ dots located along NF+ nerve fibers. Scale bar = 75 µm (A–C), 100 µm (D), 20 µm (E). Full article ">Figure 4
Immunolabeling of neurofilament (NF)-positive nerve fibers and myelin oligodendrocyte glycoprotein (MOG)-positive staining on healthy control plasma microcontact prints compared to those from Alzheimer’s samples. Organotypic brain slices were cultured on plasma microcontact prints for 2 weeks. (A,D) Following fixation, they were stained for NF (green, Alexa 488) and (B,E) MOG (red, Alexa 546). (C,F) These panels show the merged staining of NF and MOG. Note the MOG+ dots along the nerve fibers (indicated by arrows), which are more prominent and larger in the Alzheimer’s group. Scale bar = 20 µm (A–F). Full article ">Figure 5
The best six plasma samples (three healthy controls and three Alzheimer’s disease) were selected based on the results of measurements and counting on NF+ axons and MOG+ dot structures. Proteins identified after depletion of 14 proteins are shown. The keywords “oligodendrocyte”, or “axon” or “myelin” were searched in combination with differential mass spectrometry results and ultimately three possible biomarkers were identified. Full article ">

10 pages, 1292 KiB

Open AccessArticle

Endometrial Dysbiosis: A Possible Association with Estrobolome Alteration

by Giorgia Scarfò, Simona Daniele, Elisa Chelucci, Francesca Papini, Francesco Epifani, Maria Ruggiero, Vito Cela, Ferdinando Franzoni and Paolo Giovanni Artini

Biomolecules 2024, 14(10), 1325; https://doi.org/10.3390/biom14101325 - 18 Oct 2024

Viewed by 984

Abstract

Background/Objectives: Microbiota modification at the endometrial level can favor gynecological diseases and impair women’s fertility. The overgrowth of pathogen microorganisms is related to the contemporary alteration of estrogen-metabolizing bacteria, including β-glucuronidase, thereby enhancing estrogen-related inflammatory states and decreasing anti-inflammatory cells. The possible connection [...] Read more.

Background/Objectives: Microbiota modification at the endometrial level can favor gynecological diseases and impair women’s fertility. The overgrowth of pathogen microorganisms is related to the contemporary alteration of estrogen-metabolizing bacteria, including β-glucuronidase, thereby enhancing estrogen-related inflammatory states and decreasing anti-inflammatory cells. The possible connection between estrobolome impairment and gynecological diseases has been suggested in animal models. Nevertheless, in humans, coherent evidence on the estrobolome alteration and functionality of the female reproductive tract is still lacking. The objective of this study was to explore alterations in estrogen-related signaling and the putative link with endometrial dysbiosis. Methods: Women with infertility and repeated implantation failure (RIF, N = 40) were enrolled in order to explore the putative link between estrogen metabolism and endometrial dysbiosis. Endometrial biopsies were used to measure inflammatory and growth factor molecules. β-glucuronidase enzyme activity and estrogen receptor (ER) expression were also assessed. Results: Herein, increased levels of inflammatory molecules (i.e., IL-1β and HIF-1α) and decreased levels of the growth factor IGF-1 were found in the endometrial biopsies of patients presenting dysbiosis compared to eubiotic ones. β-glucuronidase activity and the expression of ERβ were significantly enhanced in patients in the dysbiosis group. Interestingly, Lactobacilli abundance was inversely related to β-glucuronidase activity and to ERβ expression, thus suggesting that an alteration of the estrogen-activating enzyme may affect the expression of ERs as well. Conclusions. Overall, these preliminary data suggested a link between endometrial dysbiosis and estrobolome impairment as possible synergistic contributing factors to women infertility and RIF. Full article

(This article belongs to the Special Issue Molecular Aspects of Female Infertility)

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15 pages, 577 KiB

Open AccessReview

Targeting Protein Aggregation in ALS

by Michele Perni and Benedetta Mannini

Biomolecules 2024, 14(10), 1324; https://doi.org/10.3390/biom14101324 - 18 Oct 2024

Viewed by 1598

Abstract

Proteinopathies involve the abnormal accumulation of specific proteins. Maintaining the balance of the proteome is a finely regulated process managed by a complex network of cellular machinery responsible for protein synthesis, folding, and degradation. However, stress and ageing can disrupt this balance, leading [...] Read more.

Proteinopathies involve the abnormal accumulation of specific proteins. Maintaining the balance of the proteome is a finely regulated process managed by a complex network of cellular machinery responsible for protein synthesis, folding, and degradation. However, stress and ageing can disrupt this balance, leading to widespread protein aggregation. Currently, several therapies targeting protein aggregation are in clinical trials for ALS. These approaches mainly focus on two strategies: addressing proteins that are prone to aggregation due to mutations and targeting the cellular mechanisms that maintain protein homeostasis to prevent aggregation. This review will cover these emerging drugs. Advances in ALS research not only offer hope for better outcomes for ALS patients but also provide valuable insights and methodologies that can benefit the broader field of neurodegenerative disease drug discovery. Full article

(This article belongs to the Special Issue The Role of Amyloid in Neurological Disorders)

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12 pages, 687 KiB

Open AccessReview

The Emerging Role of Immunoglobulins and Complement in the Stimulation of Neuronal Activity and Repair: Not as Simple as We Thought

by Tatyana Veremeyko, Natasha S. Barteneva, Ivan Vorobyev and Eugene D. Ponomarev

Biomolecules 2024, 14(10), 1323; https://doi.org/10.3390/biom14101323 - 18 Oct 2024

Viewed by 1156

Abstract

Neurologic disorders such as traumatic brain injury, multiple sclerosis, Alzheimer’s disease, and drug-resistant epilepsy have a high socioeconomic impact around the world. Current therapies for these disorders are often not effective. This creates a demand for the development of new therapeutic approaches to [...] Read more.

Neurologic disorders such as traumatic brain injury, multiple sclerosis, Alzheimer’s disease, and drug-resistant epilepsy have a high socioeconomic impact around the world. Current therapies for these disorders are often not effective. This creates a demand for the development of new therapeutic approaches to treat these disorders. Recent data suggest that autoreactive naturally occurring immunoglobulins produced by subsets of B cells, called B1 B cells, combined with complement, are actively involved in the processes of restoration of neuronal functions during pathological conditions and remyelination. The focus of this review is to discuss the possibility of creating specific therapeutic antibodies that can activate and fix complement to enhance neuronal survival and promote central nervous system repair after injuries associated with many types of neurodegenerative diseases. Full article

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

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20 pages, 1446 KiB

Open AccessArticle

The Role of Keeving in Modulating Fermentation and the Flavour Profiles of Apple Brandy

by Magdalena Januszek, Paweł Satora, Aneta Pater and Łukasz Wajda

Biomolecules 2024, 14(10), 1322; https://doi.org/10.3390/biom14101322 - 18 Oct 2024

Cited by 1 | Viewed by 834

Abstract

Keeving is the removal of nutrients from apple musts due to their binding to pectin, resulting in a slower fermentation and spontaneous arrest. The aim of this study was to determine the effect of keeving on the chemical composition of fermented apple must [...] Read more.

Keeving is the removal of nutrients from apple musts due to their binding to pectin, resulting in a slower fermentation and spontaneous arrest. The aim of this study was to determine the effect of keeving on the chemical composition of fermented apple must and on the volatile profile and sensory analysis of apple brandies. We compared the application of keeving during spontaneous fermentation with fermentation carried out by Saccharomyces cerevisiae (SafSpirit HG-1). We evaluated the impact of adding different doses of calcium chloride on various parameters of fermented musts and distillates. Calcium chloride had a greater effect on the ethanol concentration, total extract, and fermentation efficiency than on the type of fermentation used. However, a different phenomenon was observed with respect to the volatiles. The concentration of most of the higher alcohols, acetaldehyde, dodecanal, and geranylaceton, decreased after spontaneous fermentation and increased during the fermentation carried out with Saccharomyces cerevisiae SafSpirit HG-1. In general, the application of keeving contributed to a decrease in the concentration of ethyl and methyl esters, but caused an increase in the concentration of all acetate esters and terpenoids. When the amount of nutrients in the environment is limited and starvation occurs, microorganisms use the available nutrients for basic metabolic processes that allow them to survive and limit the formation of side metabolites such as volatiles. However, most of the samples fermented after the faecal depletion achieved high scores for the floral, fruity, and “overall note” parameters in the sensory analysis. This means that this method, carried out with a properly selected yeast strain, could be feasible for the distilling industry. Full article

(This article belongs to the Section Natural and Bio-derived Molecules)

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Dissolution of the gel during the fermentation process after keeving. Full article ">Figure 2
Principal component analysis showing the effect of different CaCl2 additions and types of yeast on the chemical composition of apple musts. SF—spontaneous fermentation; Sc—Saccharomyces cerevisiae SafSpirit HG-1. Doses of CaCl2: 0—0 g/L; 01—0.1 g/L; 02—0.2 g/L; and 04—0.4 g/L. Full article ">Figure 3
Principal component analysis for oenological parameters of fermented apple musts. Full article ">Figure 4
Principal component analysis showing the effect of different CaCl2 additions and types of yeast on the volatile compounds present in apple spirits. SF—spontaneous fermentation; Sc—Saccharomyces cerevisiae SafSpirit HG-1. Doses of CaCl2: 0—0 g/L; 01—0.1 g/L; 02—0.2 g/L; 04—0.4 g/L. Full article ">Figure 5
Principal component analysis showing the effect of different CaCl2 additions and types of yeast on the volatile compounds present in apple spirits. Esters (E); Methanol (M); Higher Alcohols (A); Aldehydes and ketones (Al), Terpenoids (T); Other compounds (O). The reference of the compound name to the symbol is summarized in <a href="#biomolecules-14-01322-t003" class="html-table">Table 3</a>. Full article ">Figure 6
Characteristic aroma features of apple brandies obtained from musts fermented with the addition of CaCl2; n = 5, STD < 5%. Sp—spontaneous fermentation; Sc—Saccharomyces cerevisiae SafSpirit HG-1 (0; 0.1; 0.2; 0.4 g/L—doses of CaCl2 added); *, **, and ***—the significance at 0.05, 0.01, and 0.005 by least significant difference, respectively. Full article ">

22 pages, 7042 KiB

Open AccessArticle

Glucocorticoid-Induced Leucine Zipper Protein and Yeast-Extracted Compound Alleviate Colitis and Reduce Fungal Dysbiosis

by Marco Gentili, Samuele Sabbatini, Emilia Nunzi, Eleonora Lusenti, Luigi Cari, Antonella Mencacci, Nathalie Ballet, Graziella Migliorati, Carlo Riccardi, Simona Ronchetti and Claudia Monari

Biomolecules 2024, 14(10), 1321; https://doi.org/10.3390/biom14101321 - 17 Oct 2024

Viewed by 1014

Abstract

Inflammatory bowel diseases (IBD) have a complex, poorly understood pathogenesis and lack long-lasting effective treatments. Recent research suggests that intestinal fungal dysbiosis may play a role in IBD development. This study investigates the effects of the glucocorticoid-induced leucine zipper protein (GILZp)”, known for [...] Read more.

Inflammatory bowel diseases (IBD) have a complex, poorly understood pathogenesis and lack long-lasting effective treatments. Recent research suggests that intestinal fungal dysbiosis may play a role in IBD development. This study investigates the effects of the glucocorticoid-induced leucine zipper protein (GILZp)”, known for its protective role in gut mucosa, and a yeast extract (Py) with prebiotic properties, either alone or combined, in DSS-induced colitis. Both treatments alleviated symptoms via overlapping or distinct mechanisms. In particular, they reduced the transcription levels of pro-inflammatory cytokines IL-1β and TNF-α, as well as the expression of the tight junction protein Claudin-2. Additionally, GILZp increased MUC2 transcription, while Py reduced IL-12p40 and IL-6 levels. Notably, both treatments were effective in restoring the intestinal burden of clinically important Candida and related species. Intestinal mycobiome analysis revealed that they were able to reduce colitis-associated fungal dysbiosis, and this effect was mainly the result of a decreased abundance of the Meyerozima genus, which was dominant in colitic mice. Overall, our results suggest that combined treatment regimens with GILZp and Py could represent a new strategy for the treatment of IBD by targeting multiple mechanisms, including the fungal dysbiosis. Full article

(This article belongs to the Section Molecular Medicine)

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36 pages, 3132 KiB

Open AccessReview

The Ambivalence of Post COVID-19 Vaccination Responses in Humans

by Radha Gopalaswamy, Vivekanandhan Aravindhan and Selvakumar Subbian

Biomolecules 2024, 14(10), 1320; https://doi.org/10.3390/biom14101320 - 17 Oct 2024

Cited by 1 | Viewed by 2617

Abstract

The Coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has prompted a massive global vaccination campaign, leading to the rapid development and deployment of several vaccines. Various COVID-19 vaccines are under different phases of clinical trials and include [...] Read more.

The Coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has prompted a massive global vaccination campaign, leading to the rapid development and deployment of several vaccines. Various COVID-19 vaccines are under different phases of clinical trials and include the whole virus or its parts like DNA, mRNA, or protein subunits administered directly or through vectors. Beginning in 2020, a few mRNA (Pfizer-BioNTech BNT162b2 and Moderna mRNA-1273) and adenovirus-based (AstraZeneca ChAdOx1-S and the Janssen Ad26.COV2.S) vaccines were recommended by WHO for emergency use before the completion of the phase 3 and 4 trials. These vaccines were mostly administered in two or three doses at a defined frequency between the two doses. While these vaccines, mainly based on viral nucleic acids or protein conferred protection against the progression of SARS-CoV-2 infection into severe COVID-19, and prevented death due to the disease, their use has also been accompanied by a plethora of side effects. Common side effects include localized reactions such as pain at the injection site, as well as systemic reactions like fever, fatigue, and headache. These symptoms are generally mild to moderate and resolve within a few days. However, rare but more serious side effects have been reported, including allergic reactions such as anaphylaxis and, in some cases, myocarditis or pericarditis, particularly in younger males. Ongoing surveillance and research efforts continue to refine the understanding of these adverse effects, providing critical insights into the risk-benefit profile of COVID-19 vaccines. Nonetheless, the overall safety profile supports the continued use of these vaccines in combating the pandemic, with regulatory agencies and health organizations emphasizing the importance of vaccination in preventing COVID-19’s severe outcomes. In this review, we describe different types of COVID-19 vaccines and summarize various adverse effects due to autoimmune and inflammatory response(s) manifesting predominantly as cardiac, hematological, neurological, and psychological dysfunctions. The incidence, clinical presentation, risk factors, diagnosis, and management of different adverse effects and possible mechanisms contributing to these effects are discussed. The review highlights the potential ambivalence of human response post-COVID-19 vaccination and necessitates the need to mitigate the adverse side effects. Full article

(This article belongs to the Special Issue Viral Respiratory Infection including COVID-19: From Molecular Mechanisms to Therapies Strategies)

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Summary of host response to COVID-19 vaccines. The COVID-19 vaccination-induced host responses can be broadly divided into immediate or delayed hypersensitivity. While the former response elicits allergic reactions and anaphylaxis, the latter response results in mild, moderate, or severe adverse events. The immediate hypersensitivity response is caused either by a classical, IgE-mediated activation of mast cells and basophils or an alternative non-classical pathway involving IgG and other antibodies activating neutrophils and basophils. Autoimmunity due to COVID-19 vaccination can be caused by molecular mimicry, bystander activation of immune cells, viral epitope spreading, or adjuvant-mediated immune response. The overall magnitude and durability of immune response as well as adverse effects mediated by COVID-19 vaccination are determined by several factors, including the age, sex, genetic makeup, immune status, and underlying health conditions of the host as well as the nature of the vaccine used. Image created in Biorender. Full article ">Figure 2
Effects of COVID-19 vaccination-induced immunity. Following vaccination, the immune response against COVID-19 is mediated mainly by the development of Abs against SARS-CoV-2 proteins. The magnitude of immune response developed and its impact on the host protection is determined by the nature of Ab response elicited. An effective neutralizing Ab response neutralizes the virus, controls the infecting viral load and protects the vaccinated host against severe disease and/or death due to infection. However, a sub-optimal non-neutralizing Ab response leads to poor neutralization of the virus and ineffective control of viral load in the organs and may also contribute to Ab-mediated adverse effects (AE), which may enhance the disease manifestations. Image created in Biorender. Full article ">Figure 3
Key pathways of COVID-19 vaccine-induced adverse immune reactions. The COVID-19 vaccine is comprised of the SARS-CoV-2 S protein (either as mRNA or protein) combined with an adjuvant such as polyethylene glycol (PEG). In the classical pathway, internalization of the viral and adjuvant-derived antigens (Ag) in the vaccine by antigen-presenting cells (APC) results in the presentation of antigenic epitopes to the T helper (Th) cells, which produces cytokines and activates Ag-specific B cells to produce various antibodies, such as IgG, IgE, IgM, etc. The Ag-specific IgE Abs binds to the FcεR1 and activates basophils and mast cells to produce histamine, which leads to allergy and/or anaphylaxis reactions. In the non-classical pathway, the antigens were taken up directly by the MRGPRX2 receptor on mast cells, which results in the induction of histamine and allergic responses. In addition, the immune complex formation by the Ag-specific and/or anti-idiotypic IgG, IgE, IgM Abs activates the C3a and C5a complement components, which ultimately results in complement activation-related pseudo-allergic reaction (CARPA). Finally, in the alternative/additional pathway, the antigen–IgG complex is taken up by neutrophils through FcγRs, which activates these polymorphonuclear cells to produce reactive oxygen species (ROS), proteases such as neutrophil-elastases (NE), Protease-3 (PR3), cathepsin G (CatG), and the formation of neutrophil extracellular traps (NETosis). The combined action of these pathways may contribute to the overall allergy and anaphylactic response due to COVID-19 vaccination. Image created in Biorender. Full article ">Figure 4
Various mechanisms of adverse immune activation by COVID-19 vaccines. The viral S protein, either as mRNA or recombinant, adenovector-DNA, is endocytosed through Toll-like receptors (TLR) present on antigen-presenting cells (APCs). These endosomes trigger intracellular signaling pathways that result in the activation of Interferon regulatory factor-7 (IRF-7) and nuclear factor k B (NFkB) networks. Activated IRF7 and NFkB upregulate the production of proinflammatory cytokines IL-6 and TNFα. Alternatively, the viral components can escape from the endosome and trigger the cGAS signaling pathway, which activates STING/IRF3 network that ultimately results in the upregulation of proinflammatory type I interferons (IFN) response. Finally, the viral nucleic acids are translated into peptides and presented by the APC to activate T cells through the T cell receptor (TcR). Activation of naïve T cells results in the production of cytokines. Exposure to IL-4 skews the naïve T cells to an anti-inflammatory, Th2-type T cells that produce IL-3, IL-5, and IL-9, all of which can activate mast cells to elicit an allergic/anaphylactic reaction. In contrast, exposure to IL-12 and IFNγ polarizes the naïve T cells into Th1-type cells, which contributes to the proinflammatory response. Apart from the viral-derived molecules, vaccine adjuvants, such as CpG, can be recognized by TLR on the APC, with further activation of the NFkB pathway, leading to the production of inflammatory response. The viral nucleic acids also form a complex with platelet factor-4 (PF4) produced by the blood platelets. This complex activates Ag-specific B cells to produce anti-DNA/PF4 complex IgG, which binds with the FCγRIIa receptor on the platelets and activates these cells to form aggregates, leading to vaccine-induced thrombotic thrombocytopenia (VITT). Thus, both APCs and platelets play divergent roles in mounting immune dysregulation upon exposure to viral antigens and/or adjuvants. Image created in Biorender. Full article ">

12 pages, 1430 KiB

Open AccessReview

N6-Methyladenosine Methyltransferase Component KIAA1429 Is a Potential Target of Cancer Therapy

by Junjun Huang, Jihua Guo and Rong Jia

Biomolecules 2024, 14(10), 1319; https://doi.org/10.3390/biom14101319 - 17 Oct 2024

Viewed by 1147

Abstract

N6-methyladenosine (m⁶A), the most abundant RNA modification in eukaryotes, has a crucial impact on tumorigenesis. KIAA1429 is the key component of the m⁶A methyltransferase complex, in which KIAA1429 functions as a scaffold to bridge the catalytic core proteins. KIAA1429 [...] Read more.

N6-methyladenosine (m⁶A), the most abundant RNA modification in eukaryotes, has a crucial impact on tumorigenesis. KIAA1429 is the key component of the m⁶A methyltransferase complex, in which KIAA1429 functions as a scaffold to bridge the catalytic core proteins. KIAA1429 is often overexpressed in malignances, associated with patient prognosis, and required for tumorigenesis. KIAA1429 regulates the expression of a number of tumor-associated genes in an m⁶A -dependent manner, and thus, contributes to cell proliferation, migration, drug resistance, tumor formation and metastasis. This review focuses on recent progress in the understanding of roles and mechanisms of KIAA1429 in cancers, and offers ideas for potential anti-cancer therapeutic methods by targeting KIAA1429. Full article

(This article belongs to the Section Molecular Medicine)

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Gene structure and isoforms of the VIRMA gene encoding the KIAA1429 protein. (A) The gene structure of the VIRMA gene. (B) The mRNA structures of the KIAA1429 long isoform (KIAA1429-L) and the KIAA1429 short isoform (KIAA1429-S). (C) The structures of proteins encoded by KIAA1429 isoforms. Full article ">Figure 2
Roles of KIAA1429 in tumorigenesis. Full article ">Figure 3
Molecular mechanisms of KIAA1429 in cancers. KIAA1429 destabilizes the mRNAs of some tumor-suppressive genes, including GATA3, RND3, BTG2, and DAPK3, but stabilizes the mRNAs of some oncogenic genes, such as RAB27B, HAS2, FOXM1, SMC1A, and JUN. In addition, KIAA1429 can stabilize the mRNAs of the SLC7A11 gene, a ferroptosis inhibitor, and some enzymes for aerobic glycolysis, like HK2 and ENO1. The down-arrow represents the downregulation of the protein level. The up-arrow represents the upregulation of the protein level. Full article ">Figure 4
The regulation of KIAA1429 expression. Full article ">

19 pages, 1565 KiB

Open AccessReview

The Role of Mitochondrial Permeability Transition in Bone Metabolism, Bone Healing, and Bone Diseases

by Xiting Zhu, Ziqi Qin, Min Zhou, Chen Li, Junjun Jing, Wushuang Ye and Xueqi Gan

Biomolecules 2024, 14(10), 1318; https://doi.org/10.3390/biom14101318 - 17 Oct 2024

Viewed by 1589

Abstract

Bone is a dynamic organ with an active metabolism and high sensitivity to mitochondrial dysfunction. The mitochondrial permeability transition pore (mPTP) is a low-selectivity channel situated in the inner mitochondrial membrane (IMM), permitting the exchange of molecules of up to 1.5 kDa in [...] Read more.

Bone is a dynamic organ with an active metabolism and high sensitivity to mitochondrial dysfunction. The mitochondrial permeability transition pore (mPTP) is a low-selectivity channel situated in the inner mitochondrial membrane (IMM), permitting the exchange of molecules of up to 1.5 kDa in and out of the IMM. Recent studies have highlighted the critical role of the mPTP in bone tissue, but there is currently a lack of reviews concerning this topic. This review discusses the structure and function of the mPTP and its impact on bone-related cells and bone-related pathological states. The mPTP activity is reduced during the osteogenic differentiation of mesenchymal stem cells (MSCs), while its desensitisation may underlie the mechanism of enhanced resistance to apoptosis in neoplastic osteoblastic cells. mPTP over-opening triggers mitochondrial swelling, regulated cell death, and inflammatory response. In particular, mPTP over-opening is involved in dexamethasone-induced osteoblast dysfunction and bisphosphonate-induced osteoclast apoptosis. In vivo, the mPTP plays a significant role in maintaining bone homeostasis, with many bone disorders linked to its excessive opening. Genetic deletion or pharmacological inhibition of the over-opening of mPTP has shown potential in enhancing bone injury recovery and alleviating bone diseases. Here, we review the findings on the relationship of the mPTP and bone at both the cellular and disease levels, highlighting novel avenues for pharmacological approaches targeting mitochondrial function to promote bone healing and manage bone-related disorders. Full article

(This article belongs to the Section Cellular Biochemistry)

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mPTP structure and the consequences of mPTP opening. The mitochondrial permeability transition pore (mPTP) has different conductance states. In its low-conductance state, the mPTP, potentially formed by the adenine nucleotide translocator (ANT), allows for the passage of ions and small metabolites. Ca2+ efflux in this state limits the Ca2+-dependent tricarboxylic acid (TCA) cycle. In its high-conductance state, the mPTP is formed by a rearrangement of the F1Fo ATP synthase complex. This state has more detrimental effects on cellular function. The release of mitochondrial DNA (mtDNA) through the mPTP triggers inflammatory responses. Extensive water influx causes mitochondrial swelling and subsequently induces outer membrane permeabilisation and the release of pro-apoptotic cofactors, leading to apoptosis or necrosis and TCA cycle collapse. ↓(orange downward arrow) represents downregulation of the TCA cycle. Full article ">Figure 2
Diverse roles of mPTP opening in bone-related cells. The mitochondrial permeability transition pore (mPTP) is crucial in various functions within bone-related cells. In bone marrow-derived mesenchymal stem cells (BMSCs), cyclophilin D (CypD) downregulation and subsequent mPTP activity decline are required for osteogenic differentiation. In osteoblasts, mPTP over-opening is involved in several drug-induced regulated cell deaths (RCDs) in an ROS-dependent or ROS-independent manner. The mechanism by which osteosarcoma cells are resistant to apoptosis is, at least in part, due to desensitisation to the mPTP. Several studies have indicated that the mPTP is also involved in osteoclast apoptosis. ↑(black upward arrow) and ↓(black downward arrow) represents upregulation and downregulation of mPTP opening, respectively. Full article ">

16 pages, 2015 KiB

Open AccessReview

The Roles of Mitochondria in Human Being’s Life and Aging

by Hiroko P. Indo, Moragot Chatatikun, Ikuo Nakanishi, Ken-ichiro Matsumoto, Motoki Imai, Fumitaka Kawakami, Makoto Kubo, Hiroshi Abe, Hiroshi Ichikawa, Yoshikazu Yonei, Hisashi J. Beppu, Yukiko Minamiyama, Takuro Kanekura, Takafumi Ichikawa, Atthaphong Phongphithakchai, Lunla Udomwech, Suriyan Sukati, Nurdina Charong, Voravuth Somsak, Jitbanjong Tangpong, Sachiyo Nomura and Hideyuki J. Majima Show full author list Hide full author list

Biomolecules 2024, 14(10), 1317; https://doi.org/10.3390/biom14101317 - 17 Oct 2024

Viewed by 2314

Abstract

The universe began 13.8 billion years ago, and Earth was born 4.6 billion years ago. Early traces of life were found as soon as 4.1 billion years ago; then, ~200,000 years ago, the human being was born. The evolution of life on earth [...] Read more.

The universe began 13.8 billion years ago, and Earth was born 4.6 billion years ago. Early traces of life were found as soon as 4.1 billion years ago; then, ~200,000 years ago, the human being was born. The evolution of life on earth was to become individual rather than cellular life. The birth of mitochondria made this possible to be the individual life. Since then, individuals have had a limited time of life. It was 1.4 billion years ago that a bacterial cell began living inside an archaeal host cell, a form of endosymbiosis that is the development of eukaryotic cells, which contain a nucleus and other membrane-bound compartments. The bacterium started to provide its host cell with additional energy, and the interaction eventually resulted in a eukaryotic cell, with both archaeal (the host cell) and bacterial (mitochondrial) origins still having genomes. The cells survived high concentrations of oxygen producing more energy inside the cell. Further, the roles of mitochondria in human being’s life and aging will be discussed. Full article

(This article belongs to the Special Issue Mitochondrial Quality Control in Aging and Neurodegeneration)

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Full article ">Figure 1
Schema of the electron transport chain. ATP production by oxidative phosphorylation with an electron transport chain (ETC). Full article ">Figure 2
A scheme of mitochondrial generation of ROS. O2•− generated from mitochondria binds with NO and further produces peroxynitrous acid (ONOOH), and further ONOOH produces NO2• and •OH. In the figure red arrow of up means up-regulations, and stable means no expression change. (Figure transferred from Reference [<a href="#B1-biomolecules-14-01317" class="html-bibr">1</a>]). Full article ">Figure 3
Illustration of the 4.6-billion-year-old Earth, acknowledging “Andrée Valley, University of Wisconsin—Madison”. (Reference [<a href="#B6-biomolecules-14-01317" class="html-bibr">6</a>]). The author, H.J.M. added mitochondria to the figure. Full article ">Figure 4
Increase of atmospheric oxygen in the history of Earth through 4.6 billion years. The figure was transferred from Reference [<a href="#B11-biomolecules-14-01317" class="html-bibr">11</a>] with a license. Full article ">Figure 5
Conclusion: relationship between aging and mitochondria. Upon aging, arrow stable means no change, arrow down means down regulation, and arrow up means up regulation. Full article ">

13 pages, 1968 KiB

Open AccessArticle

Gintonin Stimulates Glucose Uptake in Myocytes: Involvement of Calcium and Extracellular Signal-Regulated Kinase Signaling

by Rami Lee, Kyung-Jong Won, Ji-Hun Kim, Byung-Hwan Lee, Sung-Hee Hwang and Seung-Yeol Nah

Biomolecules 2024, 14(10), 1316; https://doi.org/10.3390/biom14101316 - 17 Oct 2024

Viewed by 1009

Abstract

Ginseng has anti-hyperglycemic effects. Gintonin, a glycolipoprotein derived from ginseng, also stimulates insulin release from pancreatic beta cells. However, the role of gintonin in glucose metabolism within skeletal muscle is unknown. Here, we showed the effect of gintonin on glucose uptake, glycogen content, [...] Read more.

Ginseng has anti-hyperglycemic effects. Gintonin, a glycolipoprotein derived from ginseng, also stimulates insulin release from pancreatic beta cells. However, the role of gintonin in glucose metabolism within skeletal muscle is unknown. Here, we showed the effect of gintonin on glucose uptake, glycogen content, glucose transporter (GLUT) 4 expression, and adenosine triphosphate (ATP) content in C2C12 myotubes. Gintonin (3–30 μg/mL) dose-dependently stimulated glucose uptake in myotubes. The expression of GLUT4 on the cell membrane was increased by gintonin treatment. Treatment with 1–3 μg/mL of gintonin increased glycogen content in myotubes, but the content was decreased at 30 μg/mL of gintonin. The ATP content in myotubes increased following treatment with 10–100 μg/mL gintonin. Gintonin transiently elevated intracellular calcium concentrations and increased the phosphorylation of extracellular signal-regulated kinase (ERK). Gintonin-induced transient calcium increases were inhibited by treatment with the lysophosphatidic acid receptor inhibitor Ki16425, the phospholipase C inhibitor U73122, and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Gintonin-stimulated glucose uptake was decreased by treatment with U73122, the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester, and the ERK inhibitor PD98059. These results show that gintonin plays a role in glucose metabolism by increasing glucose uptake through transient calcium increases and ERK signaling pathways. Thus, gintonin may be beneficial for glucose metabolism control. Full article

(This article belongs to the Special Issue Therapeutic Potential of Natural Products in Metabolic Diseases)

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14 pages, 2418 KiB

Open AccessReview

Fungal L-Methionine Biosynthesis Pathway Enzymes and Their Applications in Various Scientific and Commercial Fields

by Kamila Rząd, Aleksandra Kuplińska and Iwona Gabriel

Biomolecules 2024, 14(10), 1315; https://doi.org/10.3390/biom14101315 - 17 Oct 2024

Viewed by 1336

Abstract

L-methionine (L-Met) is one of the nine proteinogenic amino acids essential for humans since, in human cells, there are no complete pathways for its biosynthesis from simple precursors. L-Met plays a crucial role in cellular function as it is required for proper protein [...] Read more.

L-methionine (L-Met) is one of the nine proteinogenic amino acids essential for humans since, in human cells, there are no complete pathways for its biosynthesis from simple precursors. L-Met plays a crucial role in cellular function as it is required for proper protein synthesis, acting as an initiator. Additionally, this amino acid participates in various metabolic processes and serves as a precursor for the synthesis of S-adenosylmethionine (AdoMet), which is involved in the methylation of DNA molecules and phospholipids, as well as in maintaining genome stability. Due to its importance, fungal L-methionine biosynthesis pathway enzymes are being intensively studied. This review presents the current state of the art in terms of their cellular function, usefulness as molecular markers, antifungal targets, or industrial approaches. Full article

(This article belongs to the Section Enzymology)

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12 pages, 2239 KiB

Open AccessArticle

Some Glycoproteins Expressed on the Surface of Immune Cells and Cytokine Plasma Levels Can Be Used as Potential Biomarkers in Patients with Colorectal Cancer

by Tsvetelina Batsalova, Denitsa Uzunova, Gergana Chavdarova, Tatyana Apostolova and Balik Dzhambazov

Biomolecules 2024, 14(10), 1314; https://doi.org/10.3390/biom14101314 - 16 Oct 2024

Viewed by 1124

Abstract

Colorectal cancer (CRC) is a leading cause of mortality worldwide. Its incidence holds a major position among the most common life-threatening diseases. Hence, the early identification and precise characterization of disease activity based on proper biomarkers are of utmost importance for therapeutic strategy [...] Read more.

Colorectal cancer (CRC) is a leading cause of mortality worldwide. Its incidence holds a major position among the most common life-threatening diseases. Hence, the early identification and precise characterization of disease activity based on proper biomarkers are of utmost importance for therapeutic strategy and patient survival. The identification of new biomarkers for colorectal cancer or disease-specific levels/combinations of biomarkers will significantly contribute to precise diagnosis and improved personalized treatment of patients. Therefore, the present study aims to identify colorectal cancer-specific immunological biomarkers. The plasma levels of several cytokines (interleukin-1β /IL-1β/, IL-2, IL-4, IL-10, IL-12, IL-15, TGFβ and IFNγ) of 20 patients with colorectal cancer and 21 healthy individuals were determined by ELISA. The expression of several types of glycoproteins on the surface of peripheral blood leukocytes isolated from CRC patients and healthy volunteers was evaluated by flow cytometry. Correlations between cytokine levels and cell surface glycoprotein expression were analyzed. The obtained results demonstrated significantly elevated levels of CD80, CD86, CD279 and CD274 expressing leukocyte populations in the cancer patient group, while the numbers of NK cells and CD8- and CD25-positive cells were decreased. Based on these data and the correlations with cytokine levels, it can be concluded that CD25, CD80, CD86, CD274 and CD279 glycoproteins combined with specific plasma levels of IL-1β, IL-2, IL-15 and TGFβ could represent potential biomarkers for colorectal cancer. Full article

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

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Plasma concentrations of inflammatory cytokines in stage IV CRC patients and control healthy volunteers. Levels of IL-1β (A), IL-2 (B), IL-4 (C), IL-10 (D), IL-12 (E), IL-13 (F), IL-15 (G), TGFβ (H) and IFNγ (I). Data represent ± standard error of the mean (±SEM). A Mann–Whitney U test was used for statistical analyses. ** p < 0.01, *** p < 0.001. Full article ">Figure 2
Flow cytometry analyses of the expression of T-cell-specific markers on peripheral blood leukocytes derived from stage IV CRC patients and healthy individuals. CD4+ T cells (A), CD8+ T cells (B), CD25+ T cells (C), CD28+ T cells (D), CD152+ T cells (E), and CD279+ T cells (F). The results are presented as ±SEM. Statistical significance was defined by the Mann–Whitney U test. * p < 0.05, *** p < 0.001. Full article ">Figure 3
Levels of CD20+ B cells, NK cells and expression of some B7 family molecules in the peripheral blood of stage IV CRC patients and healthy controls. Percentage of CD20+ B cells (A), CD80+ B cells (B), CD86+ B cells (C), CD273+ lymphocytes (D), CD274+ lymphocytes (E), and CD56+/CD16+ NK cells (F). Data are shown as ±SEM. Statistical analyses were performed using the Mann–Whitney U test; ** p < 0.01, *** p < 0.001. Full article ">Figure 4
Correlations between IL-2 and IL-15 plasma concentrations with other cytokines. First column: IL-2 vs. IL-1β (A), IL-2 vs. TGFβ (C), IL-2 vs. IFNγ (E), IL-2 vs. IL-15 (G), IL-2 vs. IL-12 (I); Second column: IL-15 vs. IL-12 (B), IL-15 vs. IL-1β (D), IL-15 vs. IL-13 (F), IL-15 vs. TGFβ (H), and IL-2 vs. IL-12 only for CRC patients (J). The charts indicate Spearman’s rank correlation coefficient (ρ). * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 5
Correlations between circulating cytokines and immunomodulatory molecule expression levels determined for the stage IV CRC patient group. IL-15 vs. CD279+ cells (A), IL-1β vs. CD86+ cells (B) and CD8+ vs. CD279+ cells (C). The graphs show Spearman’s rank correlation coefficient (ρ). * p < 0.05. Full article ">

16 pages, 685 KiB

Open AccessArticle

Altered Gut Microbiota Patterns in Young Children with Recent Maltreatment Exposure

by Gergana Karaboycheva, Melanie L. Conrad, Peggy Dörr, Katja Dittrich, Elena Murray, Karolina Skonieczna-Żydecka, Mariusz Kaczmarczyk, Igor Łoniewski, Heiko Klawitter, Claudia Buss, Sonja Entringer, Elisabeth Binder, Sibylle M. Winter and Christine Heim

Biomolecules 2024, 14(10), 1313; https://doi.org/10.3390/biom14101313 - 16 Oct 2024

Viewed by 1247

Abstract

Background: The brain and the intestinal microbiota are highly interconnected and especially vulnerable to disruptions in early life. Emerging evidence indicates that psychosocial adversity detrimentally impacts the intestinal microbiota, affecting both physical and mental health. This study aims to investigate the gut microbiome [...] Read more.

Background: The brain and the intestinal microbiota are highly interconnected and especially vulnerable to disruptions in early life. Emerging evidence indicates that psychosocial adversity detrimentally impacts the intestinal microbiota, affecting both physical and mental health. This study aims to investigate the gut microbiome in young children in the immediate aftermath of maltreatment exposure. Methods: Maltreatment exposure was assessed in 88 children (ages 3–7) using the Maternal Interview for the Classification of Maltreatment [MICM]. Children were allocated to three groups according to the number of experienced maltreatment categories: no maltreatment, low maltreatment, and high maltreatment exposures. Stool samples were collected and analyzed by 16S rRNA sequencing. Results: Children subjected to high maltreatment exposure exhibited lower alpha diversity in comparison to those with both no and low maltreatment exposure (Simpson Index, Tukey post hoc, p = 0.059 and p = 0.007, respectively). No significant distinctions in beta diversity were identified. High maltreatment exposure was associated with the enrichment of several genera from the class Clostridia (Clostridium, Intestinibacter, Howardella and Butyrivibrio) and the depletion of the genus Phocaeicola (class Bacteriodia). Conclusions: Severe maltreatment exposure is associated with alterations in the gut microbiota of young children. Longitudinal trajectories of intestinal microbiota composition in the context of maltreatment may reveal important insights related to psychiatric and somatic health outcomes. Full article

(This article belongs to the Special Issue Early Adversity and the Immune Hypothesis: Call for a Dialogue between Basic and Clinical Neuroscience)

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16 pages, 2591 KiB

Open AccessArticle

Short Link N Modulates Inflammasome Activity in Intervertebral Discs Through Interaction with CD14

by Muskan Alad, Michael P. Grant, Laura M. Epure, Sunny Y. Shih, Geraldine Merle, Hee-Jeong Im, John Antoniou and Fackson Mwale

Biomolecules 2024, 14(10), 1312; https://doi.org/10.3390/biom14101312 - 16 Oct 2024

Cited by 1 | Viewed by 1104

Abstract

Intervertebral disc degeneration and pain are associated with the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome activation and the processing of interleukin-1 beta (IL-1β). Activation of thehm inflammasome is triggered by Toll-like receptor stimulation and requires the cofactor receptor cluster [...] Read more.

Intervertebral disc degeneration and pain are associated with the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome activation and the processing of interleukin-1 beta (IL-1β). Activation of thehm inflammasome is triggered by Toll-like receptor stimulation and requires the cofactor receptor cluster of differentiation 14 (CD14). Short Link N (sLN), a peptide derived from link protein, has been shown to modulate inflammation and pain in discs in vitro and in vivo; however, the underlying mechanisms remain elusive. This study aims to assess whether sLN modulates IL-1β and inflammasome activity through interaction with CD14. Disc cells treated with lipopolysaccharides (LPS) with or without sLN were used to assess changes in Caspase-1, IL-1β, and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Peptide docking of sLN to CD14 and immunoprecipitation were performed to determine their interaction. The results indicated that sLN inhibited LPS-induced NFκB and Caspase-1 activation, reducing IL-1β maturation and secretion in disc cells. A significant decrease in inflammasome markers was observed with sLN treatment. Immunoprecipitation studies revealed a direct interaction between sLN and the LPS-binding pocket of CD14. Our results suggest that sLN could be a potential therapeutic agent for discogenic pain by mitigating IL-1β and inflammasome activity within discs. Full article

(This article belongs to the Section Molecular Biology)

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Figure 1
Immunohistochemistry of NLRP3 expression in a degenerative IVD model. Rabbit IVDs from a puncture model were given a single injection of either saline or sLN. Sham animals were used as controls. Twelve weeks following injection, IVDs were processed for immunohistochemistry and the detection of NLRP3. Higher-magnification inset images are included and demarcated to highlight specific areas of interest. Immunohistochemistry of NLRP3 in (A) sham and degenerative IVDS treated with (B) saline or (C) sLN. (D) Densitometry of NLRP3 expression in IVDs presented in (A). Statistical significance was assessed using Student’s t-test (comparison between saline and sLN); ***, p < 0.001; n = 4. (IVD—intervertebral disc, NLRP3—nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing 3, yellow dotted line—endplate). Full article ">Figure 2
Evaluation of NF-κB activation in human nucleus pulposus cells treated with sLN and LPS. (A) Human nucleus pulposus (NP) cells were treated with LPS alone or in combination with sLN with the indicated concentrations (0.05, 0.5, or 5.0 μg/mL) for 45 min. Western blotting was performed to detect activation of P-NF-κB. Blots were normalized to GAPDH for loading by densitometry and calculated as a fold increase over control. The densitometry of the blots is presented in the graph below. (B) HNP cells were incubated with LPS alone or (C) LPS and 0.5 μg/mL sLN for the indicated times (0–180 min). P-NF-κB signal was normalized to GAPDH and calculated as a fold-over control. Statistical significance was assessed using ANOVA and post hoc Dunnett’s test (comparison to control); ****, p < 0.0001; n = 4. Original Western blot images are available in <a href="#app1-biomolecules-14-01312" class="html-app">Supplementary Materials</a>. Full article ">Figure 3
Suppression of inflammasome markers by sLN. HNP cells were incubated for 48 h in medium supplemented with LPS or LPS and 0.5 μg/mL sLN. Control samples were incubated with medium alone. RNA expression is shown for (A) NLRP3, (B) PYC, (C) CASP1, (D) IL1B, and (E) TNFA. Treatments were compared to controls. ANOVA and post hoc Dunnett’s test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n = 4. Full article ">Figure 4
Assessment of Caspase-1 activation and IL-1β secretion in hNP cells. HNP cells were treated with LPS alone or in combination with sLN at varying concentrations (0.05, 0.5 and 5.0 μg/mL) for 48 h. (A) Western blotting and densitometry of Caspase-1 demonstrating Pro-Caspase-1, Caspase-1, and GAPDH as loading control. (B) Western blotting and densitometry of pro- and mature forms of IL-1β. Data are expressed as fold-over controls. Statistical significance was assessed using ANOVA and post hoc Dunnett’s test (treatments compared to LPS alone); **, p < 0.01; ***, p < 0.001; n = 4. Original Western blot images are available in <a href="#app1-biomolecules-14-01312" class="html-app">Supplementary Materials</a>. Full article ">Figure 5
Inflammasome activation and hNP-induced macrophage polarization. RAW macrophages and hNP cells were co-cultured to determine the effects of hNP on macrophage polarization. HNP cells were stimulated with LPS and incubated for 48 hrs with RAW cells. Expression of M1 (A–C) and M2 (D–F) markers in RAW macrophages were measured by qPCR. Plots represent fold-over control. ANOVA and post hoc Dunnett’s test (treatments compared to control); *, p < 0.05; **, p < 0.01; ***, p < 0.001; n = 4. Full article ">Figure 6
Interaction and immunoprecipitation of sLN and CD14. (A) Schematic on the co-immunoprecipitation of CD14 and sLN and dot-blot showing enrichment of sLN following pull-down with CD14. (B) Schematic demonstrating the competitive co-immunoprecipitation of biotinylated LPS for CD14 in the presence of unlabeled LPS and sLN. Western blot showing the detection of CD14 following the indicated pull-downs. Lanes from left to right: control (CD14 only), LPS-treated, sLN-treated. Original Western blot images are available in <a href="#app1-biomolecules-14-01312" class="html-app">Supplementary Materials</a>. Full article ">Figure 7
Mechanism of sLN inhibition of inflammasome activation in disc cells. This schematic illustrates the process by which sLN inhibits inflammasome activation in nucleus pulposus cells, detailing the interactions and molecular pathways involved. Full article ">

17 pages, 3440 KiB

Open AccessArticle

Caution for Multidrug Therapy: Significant Baroreflex Afferent Neuroexcitation Coordinated by Multi-Channels/Pumps Under the Threshold Concentration of Yoda1 and Dobutamine Combination

by Yin-zhi Xu, Zhao-yuan Xu, Hui-xiao Fu, Mao Yue, Jia-qun Li, Chang-peng Cui, Di Wu and Bai-yan Li

Biomolecules 2024, 14(10), 1311; https://doi.org/10.3390/biom14101311 - 16 Oct 2024

Viewed by 796

Abstract

Multi-drug therapies are common in cardiovascular disease intervention; however, io channel/pump coordination has not been tested electrophysiologically. Apparently, inward currents were not elicited by Yoda1/10 nM or Dobutamine/100 nM alone in Ah-type baroreceptor neurons, but were by their combination. To verify this, electroneurography [...] Read more.

Multi-drug therapies are common in cardiovascular disease intervention; however, io channel/pump coordination has not been tested electrophysiologically. Apparently, inward currents were not elicited by Yoda1/10 nM or Dobutamine/100 nM alone in Ah-type baroreceptor neurons, but were by their combination. To verify this, electroneurography and the whole-cell patch-clamp technique were performed. The results showed that Ah- and C-volley were dramatically increased by the combination at 0.5 V and 5 V, in contrast to A-volley, as consistent with repetitive discharge elicited by step and ramp with markedly reduced current injection/stimulus intensity. Notably, a frequency-dependent action potential (AP) duration was increased with Iberiotoxin-sensitive K⁺ component. Furthermore, an increased peak in AP measured in phase plots suggested enhanced Na⁺ influx, cytoplasmic Ca²⁺ accumulation through reverse mode of Na⁺/Ca²⁺ exchanger, and, consequently, functional KCa1.1 up-regulation. Strikingly, the Yoda1- or Dbtm-mediated small/transient Na⁺/K⁺-pump currents were robustly increased by their combination, implying a quick ion equilibration that may also be synchronized by hyperpolarization-induced voltage-sag, enabling faster repetitive firing. These novel findings demonstrate multi-channel/pump collaboration together to integrate neurotransmission at the cellular level for baroreflex, providing an afferent explanation in sexual dimorphic blood pressure regulation, and raising the caution regarding the individual drug concentration in multi-drug therapies to optimize efficacy and minimize toxicity. Full article

(This article belongs to the Section Molecular Medicine)

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