Biomolecules

15 pages, 1581 KB

Open AccessArticle

Structural and Functional Characterization of LIMCH1 and Its Agmatinase-like Region: A Case of Catalysis in a Highly Disordered Protein

by María-Belén Reyes, Allison Fuentes, Diego Bustamante, Fernando Retamal, Ignacia Lillo, Cristián Villegas, Juan-Pablo Carrasco, Martin Pereira-Silva, Marcell Gatica, Juan Román, Maximiliano Figueroa, Yamil Neira, José Martínez-Oyanedel, Víctor Castro-Fernández and Elena Uribe

Biomolecules 2025, 15(11), 1620; https://doi.org/10.3390/biom15111620 - 18 Nov 2025

Abstract

Agmatine is a biogenic amine that functions as a neurotransmitter and exhibits anticonvulsant, antineurotoxic, and antidepressant properties. It can be metabolized into putrescine and urea by canonical agmatinases or by the agmatinase-like protein (ALP), which corresponds to the C-terminal region of the LIMCH1 [...] Read more.

Agmatine is a biogenic amine that functions as a neurotransmitter and exhibits anticonvulsant, antineurotoxic, and antidepressant properties. It can be metabolized into putrescine and urea by canonical agmatinases or by the agmatinase-like protein (ALP), which corresponds to the C-terminal region of the LIMCH1 protein. The amino acid sequence of ALP/LIMCH1 diverges significantly from that of canonical agmatinases and lacks the conserved residues typically required for coordination with Mn²⁺, an essential cofactor for ureohydrolase activity. The three-dimensional structure of ALP/LIMCH1 remains unresolved, and predictive artificial intelligence algorithms such as AlphaFold have failed to model it reliably. As a result, the configuration of its active site and the identity of potential metal-coordinating ligands remain elusive. In this study, we purified recombinant full-length rat LIMCH1 (119.5 kDa) and a truncated ALP variant, ΔLIM-ALP (51 kDa), and analyzed their secondary structures using circular dichroism spectroscopy. Our results indicate that both proteins differ markedly from known ureohydrolases, exhibiting a high proportion of disordered regions (~60%) and β-structures (~30%). In contrast, Escherichia coli agmatinase displays a well-defined α/β/α sandwich fold. Despite these structural differences, ALP/LIMCH1 remain the only known mammalian proteins exhibiting agmatinase activity. To gain insight into the putative active site of ALP, we proposed candidate Mn²⁺-binding residues and generated single-point mutants (N213A, Q215A, D217A, E288A, K290A). Although these mutations did not significantly alter Mn²⁺ binding or its overall content in the protein samples, four mutants exhibited a decreased K_m for agmatine and a reduced V_max when normalized to protein concentration. Full article

(This article belongs to the Section Molecular Biophysics: Structure, Dynamics, and Function)

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15 pages, 3168 KB

Open AccessArticle

Mechanistic and Kinetic Insights into the Acylation Reaction of Hepatitis C Virus NS3/NS4A Serine Protease with NS4B/5A Substrate

by José Ángel Martínez-González, Nuria Salazar-Sanchez, María Larriva-Hormigos, Rodrigo Martínez and Miguel González

Biomolecules 2025, 15(11), 1619; https://doi.org/10.3390/biom15111619 - 18 Nov 2025

Abstract

Reaction mechanisms and rate constants of the acylation reaction of the hepatitis C virus (HCV) NS3/NS4A serine protease with the NS4B/5A natural substrate were studied using SCC-DFTB/MM (self-consistent charge density functional tight binding/molecular mechanics) and EA-VTST/MT (ensemble-averaged variational transition state theory/multidimensional tunneling) methods, [...] Read more.

Reaction mechanisms and rate constants of the acylation reaction of the hepatitis C virus (HCV) NS3/NS4A serine protease with the NS4B/5A natural substrate were studied using SCC-DFTB/MM (self-consistent charge density functional tight binding/molecular mechanics) and EA-VTST/MT (ensemble-averaged variational transition state theory/multidimensional tunneling) methods, considering the isotope effect (H/D). This reaction is crucial in the HCV life cycle. The reaction follows an essentially concerted mechanism. Although two elementary steps are involved, no intermediate step has been found between them. Thus, the proposed general two-step serine protease acylation mechanism, which includes a tetrahedral intermediate, does not occur here. This finding aligns with our studies on another natural substrate (NS5A/5B), indicating a greater variety in mechanism than previously expected. Tunneling and recrossing play an intermediate role; the activation free energy barriers are in good agreement with the experimental value, and the kinetic isotope effect (k(H)/k(D)) is somewhat larger than one (1.3). The rate constant value is not reproduced due to the exponential dependence of the rate constant on the activation free energy. Full article

(This article belongs to the Section Chemical Biology)

20 pages, 6149 KB

Open AccessArticle

Multi-Omics Analysis Reveals 1-Propanol-Induced Pentadecanoic Acid Biosynthesis in Yarrowia lipolytica

by Jiahe Cong, Xin Hu, Dongsheng Lu, Sam C. Kollie, Ahmed A. Elolimy, Juan J. Loor, Zhendong Yang, Mingxun Li, Yongjiang Mao, Zhangping Yang and Huimin Zhang

Biomolecules 2025, 15(11), 1618; https://doi.org/10.3390/biom15111618 - 18 Nov 2025

Abstract

Pentadecanoic acid (C15:0) is an odd-chain fatty acid (OCFA) with significant health benefits, mainly produced by microbial fermentation. To improve C15:0 production, this study compared the effects of different alcohols on C15:0 production in Yarrowia lipolytica CICC1778, identified 1-propanol as the most effective [...] Read more.

Pentadecanoic acid (C15:0) is an odd-chain fatty acid (OCFA) with significant health benefits, mainly produced by microbial fermentation. To improve C15:0 production, this study compared the effects of different alcohols on C15:0 production in Yarrowia lipolytica CICC1778, identified 1-propanol as the most effective precursor, assessed its optimal concentration, and employed transcriptomic and metabolomic analyses to elucidate the regulatory mechanisms. The results showed that supplementation with 0.5% 1-propanol resulted in a total lipid production of 1.54 g/L in Y. lipolytica CICC1778, showing no differences compared with the negative control (NC) group, while C15:0 production increased to 76.68 mg/L, representing a 794.7% increase compared with the NC group. Integrated omics analysis showed that propionylcarnitine was positively correlated with ADH2, ADH1, ACADSB, ALDH6A1, and CAT2; O-methylmalonylcarnitine was positively correlated with IVD, MCCC2, ACADSB, and ALDH6A1; and (R)-leucic acid and 2-hydroxy-3-methylbutyric acid were positively correlated with IVD, BAT2, MCCC2, and ACADSB and ALDH6A1 and BAT2, respectively. All of these DEGs and DEMs were upregulated in the alcohol-treated (ALC; supplementation with 0.5% 1-propanol) group. Taken together, supplementation with 0.5% 1-propanol was an effective strategy for enhancing C15:0 production in Y. lipolytica CICC1778; 1-propanol underwent dehydrogenation-oxidation and promoted branched-chain amino acid degradation to expand the propionyl-CoA pool, thereby facilitating C15:0 synthesis. Full article

(This article belongs to the Section Lipids)

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19 pages, 1525 KB

Open AccessArticle

Screening and Validation of Functional Residues of the Antimicrobial Peptide PpRcys1

by Ming Tao, Zixun Fei, Aobo Sun, Guangming Yu, Huaiyuan Ye, Huishao Shi, Wei Zhang and Junjian Wang

Biomolecules 2025, 15(11), 1617; https://doi.org/10.3390/biom15111617 - 18 Nov 2025

Abstract

The excessive use of conventional antibiotics in aquaculture has created significant challenges, making it essential to explore and develop effective alternatives. Antimicrobial peptides (AMPs) have gained attention as potential therapeutic agents owing to their wide-ranging antibacterial effects and their ability to address pathogens [...] Read more.

The excessive use of conventional antibiotics in aquaculture has created significant challenges, making it essential to explore and develop effective alternatives. Antimicrobial peptides (AMPs) have gained attention as potential therapeutic agents owing to their wide-ranging antibacterial effects and their ability to address pathogens resistant to conventional drugs. PpRcys1 is an antimicrobial peptide that mainly targets bacterial cell membranes, exhibiting a minimum inhibitory concentration of 8–32 μM. Its antibacterial activity should be further optimized. Before such optimization, however, it is crucial to identify the key amino acid residues that determine its functional activity. In this study, molecular dynamics simulations indicated that arginine 40 (ARG40), lysine 55 (LYS55), lysine 90 (LYS90), and lysine 93 (LYS93) play critical roles in the interaction between PpRcys1 and bacterial membranes. To investigate this further, these residues were mutated to serine, producing the mutant peptide PpRcys1_RMRK. Compared with PpRcys1, the mutant peptide PpRcys1_RMRK showed a significant reduction in antibacterial activity. Results from molecular dynamics simulations, Western blot, and ELISA demonstrated a marked decrease in its ability to bind to bacterial cell membranes. Membrane permeation assays, cell membrane depolarization experiments, and scanning electron microscopy revealed that PpRcys1 could not compromise the integrity of the bacterial membrane after losing ARG40, LYS55, LYS90 and LYS93. These findings highlight the critical roles of ARG40, LYS55, LYS90, and LYS93 in sustaining the antibacterial activity of PpRcys1. This study provides important initial insights into the structure–activity relationship of PpRcys1 and establishes a theoretical foundation for its future optimization. Full article

(This article belongs to the Special Issue Recent Molecular Research on Protein Structure and Function)

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12 pages, 1203 KB

Open AccessReview

Amylase Binding to Oral Streptococci: A Key Interaction for Human Oral Microbial Ecology, Adaptation and Fitness

by Amarpreet Sabharwal, Elaine M. Haase and Frank A. Scannapieco

Biomolecules 2025, 15(11), 1616; https://doi.org/10.3390/biom15111616 - 18 Nov 2025

Abstract

The interaction between human salivary alpha-amylase (HSAmy) and amylase-binding oral streptococci (ABS) helps determine the bacteria that colonize the oral cavity by establishing dental biofilms. Streptococci are important pioneer species of the oral cavity and influence oral health as well as common diseases [...] Read more.

The interaction between human salivary alpha-amylase (HSAmy) and amylase-binding oral streptococci (ABS) helps determine the bacteria that colonize the oral cavity by establishing dental biofilms. Streptococci are important pioneer species of the oral cavity and influence oral health as well as common diseases such as dental caries. Various oral streptococcal species express distinct amylase-binding proteins, among which amylase-binding protein A (AbpA), encoded by the abpA gene in Streptococcus gordonii and several other species, which is the most extensively studied. Amylase binding facilitates microbial adhesion to host surfaces and biofilm formation and enables bacteria to harness the host’s amylase enzymatic activity at their cell surface, enhancing their capacity to metabolize dietary starch for nutritional gain. Additionally, amylase binding may also influence bacterial cell division and stress tolerance by engaging novel bacterial signaling pathways. From an evolutionary perspective, both Neanderthals and modern humans exhibit functional adaptations in nutrient metabolism, including selection for salivary amylase-binding oral streptococci, highlighting the importance of microbial co-adaptation in response to host diet. Further research is warranted to elucidate the broader roles of amylase binding to bacteria in host-bacterial signaling, bacterial cell division and fitness and the evolutionary trajectory of the oral microbiome. Full article

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

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27 pages, 5073 KB

Open AccessArticle

Activity of Serpins in Context to Hydrophobic Interaction

by Irena Roterman, Katarzyna Stapor, Grzegorz Zemanek, Dawid Dulak and Leszek Konieczny

Biomolecules 2025, 15(11), 1615; https://doi.org/10.3390/biom15111615 - 18 Nov 2025

Abstract

The activity of serpins uses a specific mechanism or process. This process comprises several steps and is related to significant structural changes that involve significant displacement of chain fragments and whole molecules of protease. An important role is played by a segment of [...] Read more.

The activity of serpins uses a specific mechanism or process. This process comprises several steps and is related to significant structural changes that involve significant displacement of chain fragments and whole molecules of protease. An important role is played by a segment of the serpin chain called the Reactive Central Loop (RCL), which interacts with the protease by inhibiting its activity. For the covalent binding of the protease to serpin, the movement of the protease molecule is an effect of splicing the RCL segment into beta-sheet A of serpin. There are structural forms—native, latent, Michaelis complex (non-covalent enzyme-inhibitor complex prior to RCL cleavage), covalent serpin–protease complex, and cleaved—associated with serpin activity. In this work, all these structural forms are discussed using the fuzzy oil drop (FOD-M) model, where the assessment criterion of structuring is based on identifying the type of hydrophobicity distribution. The analysis reveals the specificity of the inhibition mechanism, including the specific action of the RCL. The structural changes involved in this process have been shown to preserve the distribution of hydrophobicity in the form preferred by the aqueous environment in which serpins are active. The disorder (according to FOD-M model) in two complexes (Michaelis and covalent) is hypothetically treated as code for degradation factors. The applied model assesses the function-related structures using the hydrophobicity distribution as the criterion in contrast to many publications based on energetic aspects of serpin activity. Structural changes appear appropriate for water environments—the environment of serpin activity. Full article

(This article belongs to the Special Issue Protein Biophysics)

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14 pages, 5073 KB

Open AccessArticle

Citronellol Reduces Sepsis-Induced Renal Inflammation via AP-1/NF-κB/TNF-α Pathway

by Huda Rashid Atiyah, Sarmed H. Kathem and Surya M. Nauli

Biomolecules 2025, 15(11), 1614; https://doi.org/10.3390/biom15111614 - 17 Nov 2025

Abstract

Sepsis is characterized by the over-production of pro-inflammatory cytokines. Cecal ligation and puncture (CLP) is a well-accepted model for recreating sepsis-induced renal injury in mice. The current study investigates how citronellol, a naturally occurring substance with a variety of biological characteristics, can prevent [...] Read more.

Sepsis is characterized by the over-production of pro-inflammatory cytokines. Cecal ligation and puncture (CLP) is a well-accepted model for recreating sepsis-induced renal injury in mice. The current study investigates how citronellol, a naturally occurring substance with a variety of biological characteristics, can prevent acute kidney inflammation brought on by CLP. In the CLP mouse model, citronellol was administered orally at doses of 50 and 100 mg/kg. Serum levels of creatinine and urea were used as markers of renal function, and the Murine Sepsis Score (MSS) was used to assess the severity of sepsis. According to our findings, CLP caused a decline in renal function, as shown by higher serum urea and creatinine levels in comparison to control mice. Nevertheless, administering citronellol as pretreatment at doses of 50 and 100 mg/kg alleviated the deterioration in renal functions. Citronellol decreased levels of serum urea and creatinine. Citronellol demonstrated an anti-inflammatory effect by reducing pro-inflammatory cytokines (TNF-α, NF-κB, AP-1) and KIM-1. Overall, our study suggests that citronellol holds a promise as a potential therapeutic agent for mitigating kidney inflammation. Full article

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

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39 pages, 2165 KB

Open AccessReview

Antimicrobial Peptides for Skin Wound Healing

by Yifan Wu, Tingting Liu, Lili Jin, Chuyuan Wang and Dianbao Zhang

Biomolecules 2025, 15(11), 1613; https://doi.org/10.3390/biom15111613 - 17 Nov 2025

Abstract

Skin wound healing is a highly regulated biological process that requires the coordinated activity of multiple cell types. However, this process can be significantly impaired by factors such as metabolic diseases and infections, posing ongoing challenges for current treatment strategies. As a critical [...] Read more.

Skin wound healing is a highly regulated biological process that requires the coordinated activity of multiple cell types. However, this process can be significantly impaired by factors such as metabolic diseases and infections, posing ongoing challenges for current treatment strategies. As a critical defense mechanism for cells and organisms against external threats, antimicrobial peptides (AMPs) hold great potential to enhance both the rate and quality of healing in both acute and chronic wounds. AMPs play a crucial role in promoting skin wound healing through mechanisms such as keratinocyte migration and proliferation, collagen synthesis and tissue remodeling, promotion of angiogenesis, immunomodulatory effects and broad-spectrum antimicrobial activity. Moreover, structural modifications and optimized delivery systems have further enhanced the stability and efficacy of AMPs. This paper explores the mechanisms by which AMPs aid in the healing of damaged skin and reviews the types of AMPs in clinical trials, providing a foundation for their development and clinical application. Full article

(This article belongs to the Special Issue Advances in Antimicrobial Peptides (AMPs))

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23 pages, 3479 KB

Open AccessArticle

Altered Short Non-Coding RNA Landscape in the Hippocampus of a Mouse Model of CDKL5 Deficiency Disorder

by Bilal El-Mansoury, Adrian Hayes, Samuel Egan, Jordan Higgins, Stephen B. Keane, Elena Langa, Erva Ghani, Morten T. Venø, Mona Heiland, David C. Henshall and Omar Mamad

Biomolecules 2025, 15(11), 1612; https://doi.org/10.3390/biom15111612 - 17 Nov 2025

Abstract

CDKL5 deficiency disorder (CDD) is a rare developmental epileptic encephalopathy (DEE) caused by mutations in cyclin-dependent kinase-like 5 (CDKL5). The clinical manifestations include early and severe epilepsy, intellectual disability, motor abnormalities, and cortical visual impairments. The pathophysiological mechanisms underlying CDD are [...] Read more.

CDKL5 deficiency disorder (CDD) is a rare developmental epileptic encephalopathy (DEE) caused by mutations in cyclin-dependent kinase-like 5 (CDKL5). The clinical manifestations include early and severe epilepsy, intellectual disability, motor abnormalities, and cortical visual impairments. The pathophysiological mechanisms underlying CDD are not fully understood, and current treatments are limited to symptomatic management and do not target the underlying cause. Characterizing the downstream molecular pathways that are disrupted by CDKL5 deficiency may provide a more complete understanding of the underlying molecular mechanisms and yield therapeutic strategies. Previous studies have focused on mapping the differential expression of protein-coding genes and post-translational modifications of CDKL5 targets, but the role of non-coding RNAs (ncRNAs) in CDD is unknown. Here we performed small RNA sequencing to define the short non-coding RNA landscape in the hippocampus of mice in the Cdkl5 exon 6 deletion mouse model (12-week-old heterozygous mice). Our findings catalog extensive bi-directional alterations in the expression of multiple ncRNA species including microRNAs, tRNAs, piwi-RNAs, snoRNAs, and snRNAs. We further validated two dysregulated miRNAs, namely, miRNA-200c-3p and miRNA-384-3p, in CDD mice. The findings reveal that the loss of this single gene has an extensive impact on the non-coding transcriptional landscape in CDD. Such dysregulated ncRNAs may hold potential as biomarkers and could provide valuable insights into underlying disease mechanisms. Full article

(This article belongs to the Section Biological Factors)

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20 pages, 3718 KB

Open AccessArticle

Modulation of Spliceosomal Proteins hnRNPH1 and H2 Increases Melanoma Cell Pro-Inflammatory Signaling In Vitro

by Maab Sultan, Shuai Ma, Juan Diez, Sadeeshkumar Velayutham, Yousef Al-Harbi, Jun Yong Choi, Keiran S. M. Smalley, Lubov Nathanson, Vladimir Beljanski and Dmitriy Minond

Biomolecules 2025, 15(11), 1611; https://doi.org/10.3390/biom15111611 - 17 Nov 2025

Abstract

Melanoma is the most aggressive and deadliest form of skin cancer, and the current treatments of melanoma have many limitations, which necessitate discovering new compounds and targets for melanoma. Two probes, 2155-14 and 2155-18, were identified to induce apoptotic cell death, autophagy, and [...] Read more.

Melanoma is the most aggressive and deadliest form of skin cancer, and the current treatments of melanoma have many limitations, which necessitate discovering new compounds and targets for melanoma. Two probes, 2155-14 and 2155-18, were identified to induce apoptotic cell death, autophagy, and immune signaling modulation through hnRNPH1/H2-dependent mechanisms. RNA sequencing following the siRNA-mediated knockdown of hnRNPH2 in melanoma cells revealed an enrichment of immune-related signaling pathways. The present study investigated the effect of genetic and pharmacologic downregulation of hnRNPH1/H2 on melanoma immunogenicity in vitro. Our results indicated that treating melanoma cell lines with 2155-14 and 2155-18 led to hnRNPH1/H2 downregulation, whereas hnRNPH2 siRNA treatment led to only hnRNPH2 downregulation. Both types of treatment resulted in a significant upregulation of pro-inflammatory pathways and simultaneous downregulation of anti-inflammatory pathways. These findings provide the first insight into the role of hnRNPH1/H2 as critical drivers of melanoma immunogenicity and suggest their potential as novel therapeutic targets for enhancing melanoma treatment outcomes. This study underscores the impact of post-transcriptional regulation on the immune environment in melanoma and in cancer in general. Full article

(This article belongs to the Section Molecular Medicine)

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18 pages, 7696 KB

Open AccessArticle

Interactive Role of the DHPR β_1a SH3 Domain in Skeletal Muscle Excitation–Contraction Coupling

by Yamuna Karunasekara, Shouvik Aditya, Nicole C. Norris, Jean Cappello, Angela F. Dulhunty, Philip G. Board, Jose M. Eltit, Claudio F. Perez and Marco G. Casarotto

Biomolecules 2025, 15(11), 1610; https://doi.org/10.3390/biom15111610 - 17 Nov 2025

Abstract

Excitation–contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel, dihydropyridine receptor (DHPR), and the ryanodine receptor (RyR1) Ca²⁺ release channel. Although the exact mode of communication that links these two membrane proteins remains to be fully [...] Read more.

Excitation–contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel, dihydropyridine receptor (DHPR), and the ryanodine receptor (RyR1) Ca²⁺ release channel. Although the exact mode of communication that links these two membrane proteins remains to be fully resolved, both the α_1s and β_1a subunits of DHPR are two of a select number of critical proteins involved in this process. A detailed in vitro interaction study of these two proteins reveals that their association occurs between the β_1a SH3 domain and the polyproline motifs located in a critical region of the α_1s II-III loop. We demonstrate that subtle changes in the composition of the β_1a SH3 domain influences the ability of β proteins to bind to II-III loop proteins and investigate the effect of these changes on EC skeletal coupling. Furthermore, investigation into the composition of the II-III loop shows that previously identified amino acids demonstrated to be important in EC coupling are implicated in in vitro binding. In summary, we ascribe a role for the DHPR β_1a which involves the engagement of its SH3 domain with the α_1s II-III loop and propose a scenario whereby this interaction may facilitate skeletal muscle EC coupling. Full article

(This article belongs to the Special Issue The Role of Calcium Signaling in Cardiac and Skeletal Muscle)

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44 pages, 3780 KB

Open AccessReview

Lactobacilli-Derived Microbe-Associated Molecular Patterns (MAMPs) in Host Immune Modulation

by Salvatore Furnari, Ruben Ciantia, Adriana Garozzo, Pio Maria Furneri and Virginia Fuochi

Biomolecules 2025, 15(11), 1609; https://doi.org/10.3390/biom15111609 - 17 Nov 2025

Abstract

Although traditionally sidelined by live probiotic effects, Lactobacilli-derived Microbe-Associated Molecular Patterns (MAMPs) are emerging as potent modulators of innate and adaptive immune responses, capable of acting independently of bacterial viability. However, the underlying mechanisms remain incompletely understood. These MAMPs, such as peptidoglycan (PGN), [...] Read more.

Although traditionally sidelined by live probiotic effects, Lactobacilli-derived Microbe-Associated Molecular Patterns (MAMPs) are emerging as potent modulators of innate and adaptive immune responses, capable of acting independently of bacterial viability. However, the underlying mechanisms remain incompletely understood. These MAMPs, such as peptidoglycan (PGN), lipoteichoic acid (LTA), and exopolysaccharides (EPSs), interact with pattern recognition receptors (PRRs) like Toll-like receptors (TLRs), initiating immune-signaling cascades that regulate cytokine production and inflammation. Lactobacilli-derived MAMPs exhibit dual immunomodulatory effects: they can enhance pro-inflammatory responses, e.g., interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) under inflammatory contexts, while enhancing regulatory pathways via IL-10 and regulatory T-cell (T_regs) induction in anti-inflammatory settings. Importantly, these immunomodulatory properties persist in the absence of bacterial viability, making MAMPs promising candidates for postbiotic therapies. This opens new avenues for MAMP-based strategies to target inflammation, overcoming the risks associated with live bacterial administration. This review examines the therapeutic relevance of non-viable MAMPs, particularly in inflammatory diseases where they have demonstrated benefits in reducing tissue damage, enhancing gut barrier function, and alleviating disease symptoms. Additionally, we discuss regulatory and translational challenges hindering their clinical implementation, highlighting the need for standardized characterization, a clear safety framework, and strain-specific profiling. Given their ability to fine-tune immune responses, MAMPs represent an emerging strategy for innovative treatments aimed at restoring immune balance and reinforcing host–microbe interactions. Full article

(This article belongs to the Section Biomacromolecules: Proteins, Nucleic Acids and Carbohydrates)

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13 pages, 6821 KB

Open AccessArticle

Rapid In Situ Coating of Covered Stents with Highly Tough, Biocompatible Membrane for Emergency Coronary Artery Perforation

by Yuan Ji, Mingyue Fan, Bing Li, Guolin Gao and Zaixing Jiang

Biomolecules 2025, 15(11), 1608; https://doi.org/10.3390/biom15111608 - 17 Nov 2025

Abstract

Covered stents have made a significant contribution to managing coronary artery perforation (CAP). Biocompatibility and toughness are critical properties for the covering membrane of covered stents. The mismatch between covered stents and patient coronary arteries in the clinic restricts the application of covered [...] Read more.

Covered stents have made a significant contribution to managing coronary artery perforation (CAP). Biocompatibility and toughness are critical properties for the covering membrane of covered stents. The mismatch between covered stents and patient coronary arteries in the clinic restricts the application of covered stents for emergency CAP. The ability to rapidly in situ coating of the stent at the rescue scene has so far been elusive, especially for small-diameter coronary artery covered stents. Here, we investigate a rapid coating technology of covered stents with polyvinylidene fluoride (PVDF)/dibutyl phthalate (DBP) covering membrane for CAP. The highly tough membrane and the short coating timeframe make it possible to prepare the covered stent suitable for patients with emergency CAP. In vitro cell assays demonstrated the excellent biocompatibility of the covering membrane. Moreover, in vivo evaluation in a rabbit model demonstrated successful delivery of the covered stent through the sheath system and effective sealing of vascular perforation. Full article

(This article belongs to the Special Issue Applications of Biomaterials in Medicine and Healthcare)

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17 pages, 609 KB

Open AccessReview

RhoA/Rho-Kinase Signaling in Vascular Smooth Muscle and Endothelium: Mechanistic Insights and Translational Implications in Hypertension

by Stephanie Randar, Diana L. Silva-Velasco, Fernanda Priviero and R. Clinton Webb

Biomolecules 2025, 15(11), 1607; https://doi.org/10.3390/biom15111607 - 16 Nov 2025

Abstract

The small GTPase RhoA and its downstream effector Rho-kinase (ROCK) have emerged as pivotal regulators of vascular smooth muscle cell (VSMC) contraction, endothelial function, and vascular remodeling. Activation of the RhoA/ROCK pathway enhances calcium (Ca²⁺) sensitivity by inhibiting myosin light chain [...] Read more.

The small GTPase RhoA and its downstream effector Rho-kinase (ROCK) have emerged as pivotal regulators of vascular smooth muscle cell (VSMC) contraction, endothelial function, and vascular remodeling. Activation of the RhoA/ROCK pathway enhances calcium (Ca²⁺) sensitivity by inhibiting myosin light chain phosphatase (MLCP), thereby promoting sustained vascular tone independent of intracellular Ca²⁺ levels. In endothelial cells (ECs), RhoA/ROCK signaling contributes to nitric oxide (NO) dysregulation, oxidative stress, cytoskeletal reorganization, and inflammatory activation. Cumulative evidence implicates this pathway in the development and progression of hypertension and other cardiovascular diseases, where maladaptive vascular remodeling, VSMC proliferation, and endothelial dysfunction drive increased vascular resistance. Translational studies have identified ROCK inhibitors and indirect modulators such as statins as promising therapeutic strategies. This review integrates recent mechanistic insights into RhoA/ROCK regulation of vascular function with clinical and translational perspectives on targeting this pathway in hypertension. Full article

(This article belongs to the Special Issue Calcium Signaling and Transport in Health and Disease: Recent Developments and New Insights)

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18 pages, 1880 KB

Open AccessArticle

Evaluations of Quinone/Hydroquinone Couples Acting as Two Hydrogen Atoms Antioxidants, Radical Quenchers, and Hydrogen Atom Abstractors

by Xiaotang Chen, Jun-Ke Wang, Xiao-Qing Zhu and Guang-Bin Shen

Biomolecules 2025, 15(11), 1606; https://doi.org/10.3390/biom15111606 - 15 Nov 2025

Abstract

Quinone/hydroquinone couples play a crucial role in a variety of biochemical processes and chemical syntheses. Extending from our previous work, a practical dataset including the thermodynamic driving forces of 12 chemical processes for 118 quinone/hydroquinone couples accepting or releasing two hydrogen atoms in [...] Read more.

Quinone/hydroquinone couples play a crucial role in a variety of biochemical processes and chemical syntheses. Extending from our previous work, a practical dataset including the thermodynamic driving forces of 12 chemical processes for 118 quinone/hydroquinone couples accepting or releasing two hydrogen atoms in DMSO is established. The dataset serves as a foundation for assessing and discussing the thermodynamic capabilities of hydroquinones acting as two-hydrogen-atoms antioxidants or radical quenchers, quinones and semiquinone radicals acting as hydrogen atoms abstractors, and quinone/hydroquinone couples acting as dehydrogenation and hydrogenation reagents. The fundamental thermodynamic knowledge is expected to further promote the broader application of quinone/hydroquinone couples in the field of chemical antioxidation and redox reactions. Full article

(This article belongs to the Section Lipids)

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24 pages, 4208 KB

Open AccessArticle

Nanoparticle-Based Delivery of Resveratrol Suppresses Ehrlich Ascites Carcinoma and Protects Testicular Function via Antioxidant, Anti-Angiogenic, Anti-Inflammatory, and Pro-Apoptotic Mechanisms

by M. Alfawaz, Ekramy M. Elmorsy, Ahmad Najem Alshammari, Marwa Nagy Emam, Islam Ibrahim Hegab, Aly A. M. Shaalan, Manal S. Fawzy and Lina Abdelhady Mohammed

Biomolecules 2025, 15(11), 1605; https://doi.org/10.3390/biom15111605 - 15 Nov 2025

Abstract

This study, for the first time, evaluated the therapeutic potential of resveratrol-loaded phytosome nanoparticles (RES-PNPs) against Ehrlich ascites carcinoma (EAC) and associated testicular dysfunction, compared with free resveratrol (RES). Ninety male Swiss albino mice were divided into six groups, (1) control; (2) RES [...] Read more.

This study, for the first time, evaluated the therapeutic potential of resveratrol-loaded phytosome nanoparticles (RES-PNPs) against Ehrlich ascites carcinoma (EAC) and associated testicular dysfunction, compared with free resveratrol (RES). Ninety male Swiss albino mice were divided into six groups, (1) control; (2) RES (10 mg/kg/day, orally); (3) RES-PNPs (10 mg/kg/day, orally); (4) EAC, induced by intraperitoneal injection of 2.5 × 10⁶ cells; (5) EAC + RES; and (6) EAC + RES-PNPs, treated for 20 days post-tumor inoculation. Tumor growth parameters, reproductive function, antioxidant enzyme activities, inflammatory mediators, apoptotic markers, and histopathological features were assessed. Additionally, in silico docking was performed to identify molecular targets mediating RES effects. RES-PNPs markedly reduced tumor volume, ascitic cell viability, and body weight gain while significantly prolonging survival compared with free RES. Molecular assays revealed enhanced pro-apoptotic signaling (increased Bax and Caspase-3, decreased Bcl-2), suppression of vascular endothelial growth factor (VEGF), and inhibition of COX-2 with reduced TNF-α, IFN-γ, and IL-1β levels. RES-PNPs also restored semen quality, normalized reproductive hormones, elevated antioxidant enzyme activities, and reduced lipid and protein oxidation, corroborated by notable testicular histological protection. In conclusion, Resveratrol-loaded phytosome nanoparticles provide superior anti-tumor, antioxidant, anti-inflammatory, and pro-apoptotic benefits compared with free RES. These findings highlight RES-PNPs as a potent and stable nanoformulation for effective EAC suppression and preservation of male reproductive integrity. Full article

(This article belongs to the Special Issue The Role of Nanoparticles in Tumor Treatment)

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20 pages, 3608 KB

Open AccessArticle

Toll-like Receptor 7 Deficiency Attenuates Platelet Dysfunction in Sepsis

by Rashida Mohamed-Hinds, Arijit Dutta, Chanhee Park, Xiaomei Yang, Lin Zou, Wei Chao and Brittney Williams

Biomolecules 2025, 15(11), 1604; https://doi.org/10.3390/biom15111604 - 15 Nov 2025

Abstract

Sepsis is a clinical syndrome caused by abnormal host response to infection. Thrombocytopenia and platelet dysfunction are common findings in sepsis and associated with worse outcomes. The innate immune single-stranded RNA sensor, Toll-like Receptor-7 (TLR7), plays a key role in thrombocytopenia in sepsis. [...] Read more.

Sepsis is a clinical syndrome caused by abnormal host response to infection. Thrombocytopenia and platelet dysfunction are common findings in sepsis and associated with worse outcomes. The innate immune single-stranded RNA sensor, Toll-like Receptor-7 (TLR7), plays a key role in thrombocytopenia in sepsis. This study investigated whether TLR7 signaling also contributes to platelet dysfunction in sepsis, and whether the bioactivity of downstream inflammatory mediators, specifically extracellular vesicles (EVs), is impacted by the TLR7 signaling pathway. Sepsis was induced in wild-type (WT) and TLR7-deficient (TLR7^−/−) mice by cecal ligation and puncture. Blood was collected at twenty-four hours for platelet and plasma isolation, and platelet function was assessed using aggregation, adhesion, and calcium flux assays. EVs were isolated from plasma and used in vitro to evaluate their impact on platelet–leukocyte aggregate (PLA) formation. We found that septic platelets are highly activated and more adhesive, yet show markedly impaired aggregation and reduced calcium signaling, indicating functional exhaustion despite activation. Notably, mice lacking TLR7 maintained stronger platelet aggregation, enhanced adhesion, and preserved calcium release in the septic state compared to wild-type controls, suggesting a protective effect of TLR7 deficiency. Plasma EVs increased in abundance and size during sepsis and promoted clot and PLA formation in vitro. Notably, EV-mediated platelet activation was reduced with EVs derived from TLR7-deficient mice. Our results demonstrate that while sepsis drives persistent platelet activation and dysfunction, TLR7 deficiency preserves platelet function and modulates the pathogenic activity of EV-mediated platelet activation, highlighting TLR7 as a key regulator and potential therapeutic target in sepsis-induced platelet dysfunction. Full article

(This article belongs to the Special Issue Innate Immunomodulation and Inflammation: From Molecular Mechanisms to Pre-Clinical Insights)

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14 pages, 5161 KB

Open AccessArticle

The Synaptic and Intrinsic Cellular Mechanisms of Persistent Firing in Neurogliaform Cells

by Shiyuan Chen, Xiaoshan Chen, Jianwen Zhou, Jinzhao Wang, Kaiyuan Li, Wenyuan Xie, Cheng Long and Gangyi Wu

Biomolecules 2025, 15(11), 1603; https://doi.org/10.3390/biom15111603 - 15 Nov 2025

Abstract

While persistent firing in glutamatergic neurons has been well-characterized, the intrinsic and synaptic mechanisms driving this phenomenon in neurogliaform cells (NGFCs), a subtype of GABAergic interneurons, remain unclear. This study investigates the mechanisms underlying persistent firing in hippocampal NGFCs. Whole-cell current-clamp recordings were [...] Read more.

While persistent firing in glutamatergic neurons has been well-characterized, the intrinsic and synaptic mechanisms driving this phenomenon in neurogliaform cells (NGFCs), a subtype of GABAergic interneurons, remain unclear. This study investigates the mechanisms underlying persistent firing in hippocampal NGFCs. Whole-cell current-clamp recordings were performed on acute brain slices from C57BL/6J mice to examine the electrophysiological properties of NGFCs in the hippocampal stratum lacunosum-moleculare (SLM). Pharmacological interventions, including T-type calcium channel blocker ML218 and 5-hydroxytryptamine (5-HT) receptor antagonist olanzapine, were used to dissect the mechanisms of persistent firing. Biocytin labeling and confocal microscopy were employed to confirm neuronal morphology and location. The study revealed that persistent firing in NGFCs is induced by a long-lasting delayed afterdepolarization (L-ADP), which depends on T-type calcium channels (intrinsic mechanism) and is modulated by 5-HT receptors (synaptic mechanism). Persistent firing was observed in 62.96% of SLM neurons and was abolished by ML218 or olanzapine. The findings bridge a gap in understanding how inhibitory interneurons contribute to memory processes. The dual-mechanism framework (T-type channels and 5-HT receptors) aligns with prior work on glutamatergic systems but highlights unique features of GABAergic persistent firing. These insights advance the understanding of inhibitory circuit dynamics and their potential role in cognitive functions, paving the way for further research into interneuron-specific memory encoding. Full article

(This article belongs to the Special Issue Inflammatory Mechanisms and Molecular Targets in Neurological Disorders: Effects of Neuroprotective Drugs)

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24 pages, 2282 KB

Open AccessArticle

ScFv-h3D6 Prevents Bapineuzumab-Induced Hemorrhagic Events in the APP23 Mouse Model of Alzheimer’s Disease

by Silvia Lope-Piedrafita, Gabriel Serra-Mir, Paula Melón, Anna Bonaterra, Mar Hernández-Guillamon and Sandra Villegas

Biomolecules 2025, 15(11), 1602; https://doi.org/10.3390/biom15111602 - 15 Nov 2025

Abstract

The occurrence of amyloid-related imaging abnormalities (ARIAs), found in clinical trials for Aβ-immunotherapy, has been related to the antibody’s effector function on glial activation by the Fc portion of the antibody. The use of single-chain variable fragments (scFv) has been proposed as a [...] Read more.

The occurrence of amyloid-related imaging abnormalities (ARIAs), found in clinical trials for Aβ-immunotherapy, has been related to the antibody’s effector function on glial activation by the Fc portion of the antibody. The use of single-chain variable fragments (scFv) has been proposed as a safer therapeutic strategy. Here, the effects of the mice format of bapineuzumap (mAb-m3D6) and its scFv (scFv-h3D6) on the occurrence of ARIAs in the APP23 mouse model of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA) have been addressed by magnetic resonance imaging (MRI). Results are supported by histological and/or biochemical determinations. Aged APP23 mice showed a significantly higher number of microhemorrhages than non-transgenic mice. mAb-m3D6 produced an increase in the number of new hemorrhagic events, mainly in the cortex, whereas scFv-h3D6 did not. Both mAb-m3D6 and scFv-h3D6 reduced Aβ levels by the same extent. Axonal/myelin damage was found in the frontal corpus callosum of APP23 mice, which did not recover after treatment. In conclusion, the scFv-h3D6 format appears safer than the full-length mAb in the APP23 model of AD and CAA. This finding is highly relevant in light of the new FDA- and EMA-approved mAbs, which exclude APOEε4 allele carriers due to the occurrence of hemorrhages. Full article

(This article belongs to the Special Issue Molecular Mechanisms and Drug Research in Alzheimer’s Disease)

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