Biomolecules doi: 10.3390/biom16070936

Authors: Yingxin Zhou Leilei Shi Weichen Wang

Interleukin-33 (IL-33) is an IL-1 family cytokine that functions as an alarmin and contributes to inflammatory responses, immune regulation, and tumor-associated processes through the IL-33/ST2 signaling axis. In this study, IL-33-specific nanobodies were isolated from a synthetic phage display library and further engineered into bivalent tandem formats to improve their binding performance. Five representative monovalent nanobodies showed concentration-dependent binding to IL-33, with SPR-derived KD values ranging from 3.6 × 10−8 to 2.81 × 10−7 M. Among the engineered bivalent constructs, Nb1–Nb2 exhibited the strongest apparent binding affinity, mainly due to a markedly reduced dissociation rate. Competitive SPR analysis indicated that Nb1 and Nb2 show largely compatible binding to IL-33, consistent with distinct or minimally overlapping binding regions, supporting their selection as a hetero-bivalent pair. In a preliminary wound-healing assay using HT-29 colorectal cancer cells, Nb1–Nb2 attenuated IL-33-induced wound closure under low-serum conditions. These results indicate that hetero-bivalent engineering can enhance the apparent binding affinity of IL-33-targeting nanobodies and provide a useful molecular tool for further investigation of IL-33-associated biological responses.

Biomolecules doi: 10.3390/biom16070935

Authors: Maria Kirillova Dzhuliia Dzhalilova Natalia Zolotova Vladimir Kirillov Larisa Ogneva Mikhail Kirillov Tatiana Portnova Natalia Berlizeva Nikolai Fokichev Olga Makarova

Systemic hypoxia influences the state of the intestinal epithelial barrier and the microbiome; however, the role of the initial tolerance of the organism to oxygen deficiency in the development of these changes remains poorly studied. The aim of the study was to evaluate the colon histophysiological features and the gut microbiome in rats that were tolerant and susceptible to hypoxia under intermittent hypoxic exposure of varying severity. In male Wistar rats, tolerance to oxygen deficiency was determined according to the Hif1a, Epas1, and Hif3a expression levels in peripheral blood leukocytes, after which they were subjected to intermittent hypoxic exposure at an “altitude” of 5000 m or 7000 m for 1 h daily for 21 days. Subsequently, the state of the intestinal epithelial barrier was assessed using histological, histochemical, and immunohistochemical methods, and the microbiota composition was analyzed by PCR. Under normoxic conditions, in comparison with rats that are tolerant to hypoxia, susceptible animals demonstrated a greater volume fraction of goblet cells and a low abundance of Parabacteroides spp. Intermittent hypoxic exposure induced multidirectional changes depending on the initial tolerance and the severity of the regimen. In tolerant-to-hypoxia animals, an increase in the goblet cells volume fraction was detected after the exposure at the 5000 m “altitude”, while at an “altitude” of 7000 m, a decrease in the number of cells in the lamina propria of the mucosa and Clostridium perfringens gr. abundance, as well as a reduction in the Firmicutes/Bacteroidetes ratio, was observed. In susceptible-to-hypoxia animals, a higher abundance of Clostridium perfringens gr. in comparison with tolerant rats was revealed after the exposure at an “altitude” of 7000 m, with no structural changes in the intestinal wall. Thus, intermittent hypoxic exposure led to a rearrangement of the gut microbiome and the morphofunctional characteristics of the intestinal barrier, and the severity of these changes depended on the initial tolerance of the organism to oxygen deficiency and the severity of the hypoxic regime, which should be taken into account when conducting biomedical research.

Biomolecules doi: 10.3390/biom16070934

Authors: Annika Bender Laurine Schweizer Mirac Nur Musaoglu Sarah Pasche Ariane Wiegand Susanne Edelmann Vanessa Nieratschker

Adult mental disorders (aMD), including borderline personality disorder (BPD), major depressive disorder (MDD), and social anxiety disorder (SAD), share adverse childhood experiences (ACEs) as an environmental risk factor. Epigenetic mechanisms, including DNA methylation (DNAm), may mediate the biological link between early adversity and psychiatric risk. SIAH3, implicated in stress-related and mitochondrial pathways, has been previously associated with both ACE and aMD. This study examined SIAH3 DNAm in adults with BPD, MDD, or SAD, relative to healthy control participants (HC), testing effects of diagnosis, ACE exposure, and their interaction across the pooled sample and within each diagnostic group. Both aMD diagnosis and high ACE exposure showed trends toward SIAH3 hypomethylation, and a significant diagnosis × ACE interaction emerged, with inconclusive post-hoc tests. Disorder-specific analyses revealed heterogeneous patterns: in BPD, high ACE showed a trend toward hypermethylation in unadjusted models; in MDD, interaction effects were marginal and not robust to covariate adjustment; in SAD, significant main effects and a diagnosis × ACE interaction were observed, with high ACE associated with lower DNAm exclusively in HC. These findings suggest disorder-specific epigenetic responses to ACE, positioning SIAH3 as a potential molecular link between early life stress, mitochondrial function, and aMD.

Biomolecules doi: 10.3390/biom16070933

Authors: Narada Vicharnnikornkij Wanna Chaijaroenkul Kesara Na Bangchang

Background: Insomnia and stress-related sleep disorders are increasingly recognized as systemic conditions linked to the microbiota–gut–brain axis (MGBA). With growing clinical interest in natural products that modulate the gut environment, this systematic review evaluates the efficacy and mechanisms of non-pharmacological interventions, specifically probiotics, prebiotics, dietary indices, and botanicals, in alleviating insomnia, restoring circadian rhythms, and modulating neurochemical markers. Methods: In strict accordance with PRISMA 2020 guidelines, we searched PubMed, ScienceDirect, Scopus, and The Cochrane Library for English language studies published from inception to March 31, 2026. Eligibility was restricted to studies with rigorously controlled designs, specifically randomized controlled trials (RCTs) and controlled in vivo animal studies. Interventions had to target the gut microbiota, with primary outcomes measuring sleep quality (subjective or objective) or sleep-related neurochemical markers. We excluded uncontrolled, single-arm, or observational designs; in vitro studies; non-original research; and studies involving subjects with severe medical or psychiatric comorbidities (e.g., cancer, ADHD, severe psychiatric disorders) to prevent confounding variables, though mild-to-moderate anxiety and depression were permitted. Risk of bias was assessed using the Cochrane RoB 2.0 and SYRCLE tools. Due to significant methodological heterogeneity, a narrative synthesis stratified by intervention and population was conducted. This review was not registered in PROSPERO. Results: A total of 56 studies (33 humans, 23 animals) met the inclusion criteria. Taxonomic nomenclature was updated to reflect 2020 reclassifications (e.g., Lactiplantibacillus plantarum). In human trials, interventions significantly improved subjective sleep metrics (PSQI, ISI). Recent additions demonstrated the efficacy of the Dietary Index for Gut Microbiota (DI-GM) and the improvement in N3 sleep latency by yeast mannan. Furthermore, whole-food patterns (e.g., the MIND diet) and Traditional Chinese Medicine (TCM) decoctions successfully enriched beneficial taxa, such as Bacteroides coprophilus, and increased short-chain fatty acid (SCFA) production. Animal models demonstrated that “psychobiotic” strains (Bifidobacterium breve, Lacticaseibacillus paracasei), prebiotics (GOS/PDX), and TCM formulas effectively restored GABA/5-HT profiles, lowered morning cortisol, and facilitated REM rebound in PCPA-induced models, while also consolidating non-rapid eye movement (NREM) sleep and downregulating clock genes (Per1/Per2). Conclusions: Psychobiotics, prebiotics, and botanicals represent a highly viable non-pharmacological strategy for treating insomnia. However, current evidence is constrained by a heavy reliance on subjective human questionnaires, short follow-up durations limiting insight into long-term stability, and a substantial translational gap between mechanistic rodent models and human clinical outcomes.

Biomolecules doi: 10.3390/biom16070932

Authors: Keren Xu Yue Wang Hong Wang Xuanrong Sun Zhikun Yang

Celastrol, one of the top five traditional natural products with high potential for modern drug development, exerts potent broad-spectrum biological activities, yet its poor aqueous solubility, low bioavailability, potential toxicity, and limited selectivity severely compromise its drug-likeness. Advanced drug delivery strategies, mainly including multifunctional polymer/lipid/protein-based organic nanoparticles, metal/silica-based inorganic nanoparticles, vesicles represented by liposomes, and nanoemulsions, are expected to overcome these druggability hurdles of celastrol via oral, transdermal or intravenous administration. This review summarizes recent progress in a series of celastrol formulations, including novel dosage forms and delivery routes accompanied with consequential pharmacological effects and mechanisms of action, which have the potential to bring about better druggability conducive to future medical treatment.

Biomolecules doi: 10.3390/biom16070931

Authors: Richard G. Webster Susan Maxwell Yin Y. Dong

Fast-channel genetic myasthenic syndromes (FCGMSs) are caused by genetic variants in muscle nicotinic acetylcholine receptor (AChR) subunits that reduce channel open times and impair neuromuscular transmission. Among these, the CHRNE p.P141L variant (εP141L) is associated with particularly severe disease. Here, we characterized a knock-in mouse model harboring the homologous p.P141L variant in Chrne (εP141L)—C57BL/6J-Chrneem1H/H made by the MRC GEMM program. Homozygous mutant mice fail to thrive, with early lethality (median survival of 16 days), closely recapitulating the severity observed in patients. Despite a preserved neuromuscular junction (NMJ) morphology and robust AChR expression, electrophysiological analyses revealed marked reductions in miniature and evoked endplate potential amplitudes and areas, accompanied by prolonged depolarization kinetics (contrary to expectations for AChR with reduced open times) and increased quantal content, indicative of impaired post-synaptic function with compensatory pre-synaptic adaptation. Notably, disease severity exceeded that of Chrne null mice, likely through competition with more functional g-subunit-containing fetal AChRs. Consistent with this, crossing εP141L mice with CHRNG-expressing mice provided little survival benefit. These findings demonstrate that dysfunctional AChR incorporation is more deleterious than receptor absence and highlight the critical role of subunit composition in sustaining neuromuscular transmission. Pharmacological enhancement of pre-synaptic release with 3,4-diaminopyridine partially improved synaptic parameters. In addition, the AChR-positive allosteric modulator DC-98 modestly improved neurotransmission. Thus, this mouse model provides a faithful platform for mechanistic studies and therapeutic development in FCGMS.

Biomolecules doi: 10.3390/biom16070930

Authors: Remigiusz Olędzki Kristi Kerner

The aim of this review is to provide a narrative analysis of the role of Lactobacillus acidophilus as an active modulating factor in the prevention and treatment of cancer and allergic diseases. The paper discusses the molecular, metabolic, and bionanotechnological mechanisms of Lactobacillus acidophilus’s anticancer and immunomodulatory effects, which define this probiotic as an essential component of modern natural and functional medicine. A narrative review of the scientific literature was conducted, mainly from 2019–2026, focusing on the results of in vitro studies and studies on preclinical in vivo models, which analyzed the effect of live L. acidophilus strains, tyndallized bacteria (paraprobiotics) and cell-free supernatant from L. acidophilus cultures on, among others, immune system signaling pathways, tissue cytokine profile, and the integrity of the gastrointestinal epithelial cell barrier (enterocytes). Results indicate that L. acidophilus exerts significant antiallergic, antiproliferative, and proapoptotic effects against many types of cancer. Among other aspects, the ability of L. acidophilus to stimulate the production of anticancer exopolysaccharides and short-chain fatty acids, which directly influence the functioning of immune cells, is covered. The article thoroughly explains the immunomodulatory effects of L. acidophilus and the ability of this probiotic to regulate cytokine profiles, which helps promote an anti-inflammatory environment crucial for maintaining intestinal homeostasis. The article also discusses the direct interaction of L. acidophilus with immune cells, such as dendritic cells and macrophages, which leads to their activation and subsequent influence on the differentiation of T lymphocytes, which play a key role in the regulation of immune processes and in the development of immune tolerance. L. acidophilus is a universal mediator of immunological and metabolic homeostasis. Its ability to synergize with conventional therapies (chemotherapy, oncolytic virotherapy) and its innovative applications in the creation of postbiotics and paraprobiotics may provide a new approach to the treatment of inflammatory, allergic, and neoplastic diseases. Further clinical studies are necessary to assess the efficacy, safety, and optimal dose of this probiotic, which are essential for the widespread use of L. acidophilus in human therapy.

Biomolecules doi: 10.3390/biom16070928

Authors: Satoru Kaneko Yukako Kuroda Yuki Okada

In our previous studies, OptiPrep and Percoll density gradients separated human motile sperm without DNA fragmentation from immotile sperm with DNA damage. Even in normospermia, over half of the sperm were already immotile, and angle-modulated two-dimensional single-cell pulsed-field gel electrophoresis showed that these were at the end stage of fragmentation. We developed sperm-specific dye- and lectin-exclusion assays to evaluate plasma and acrosomal membranes, mitochondrial endogenous reactive oxygen species, and vacuole negative staining. Comprehensive analyses suggested that they corresponded to sperm that had not yet undergone apoptosis and to those that had undergone apoptotic denaturation. In ICSI, injectable motile sperm that fully meet criteria have an oval-shaped head, intact membranes on both the head and tail, and normal oxidative phosphorylation in cylindrical mitochondria, and they lack vacuoles and DNA damage. Conversely, sperm exhibiting apoptotic signs, such as immotility, plasma membrane damage, and DNA fragmentation, are not injectable. We must establish threshold criteria for injectable sperm; multiple impairments in sperm hinder the study of these issues. The topic of functional impairments in human sperm is too extensive to cover in a single review; for the full scope of the issue, technical guidance for DNA fragmentation analyses is presented in our previous review.

Biomolecules doi: 10.3390/biom16070929

Authors: Qiqi Wang Wenjuan Luo Qihang Wan Yuying Li Diane Latawiec Robert Sutton John Windsor Wei Huang Peter Szatmary Tingting Liu

Acute inflammatory disorders, including acute lung injury, acute pancreatitis, ischaemia–reperfusion injury, and sepsis, are major clinical challenges characterised by rapid progression, a characteristic cytokine storm, and high mortality rates. The receptor for advanced glycation end-products (RAGE) serves as a pivotal multi-ligand pattern recognition receptor that integrates PAMPs and DAMPs. Excessive RAGE engagement triggers detrimental signalling cascades, notably NF-κB and MAPKs, which exacerbate hyperinflammation and lead to progressive organ dysfunction. Consequently, the RAGE axis represents a potent therapeutic target for mitigating hyperinflammation and improving clinical outcomes in acute inflammatory disorders. While initial pharmacological efforts focused on synthetic inhibitors and biologics, there is a shifting focus toward bioactive alternatives with high safety profiles. Here, we present recent molecular insights into RAGE-mediated pathogenesis in acute inflammatory disorders and evaluate current therapeutic strategies. Furthermore, we emphatically summarise the bioactive natural products, including terpenoids, flavonoids, alkaloids, and a xanthone, that prevent and treat acute inflammatory disorders by disrupting RAGE–ligand interactions and suppressing downstream oxidative stress and cytokine release. Integrating these molecular mechanisms with the pharmacological profiling of natural RAGE modulators provides a robust foundation for the development of next-generation therapeutic strategies to improve clinical outcomes in acute inflammatory disorders.

Biomolecules doi: 10.3390/biom16070927

Authors: Jose Cuenca-Alcocel Lorena Villalba-Heredia Daiana Ibarretxe Jose A. Casajús Jose M. Arbonés-Mainar Pilar Calmarza

Background: Childhood obesity and overweight have increased considerably in recent years, representing a major global public health problem. This was a comparative study between a group of overweight or obese children and a group of normal-weight children, within an observational setting, performed in a previously studied cohort in which, in the present work, the objective was specifically to evaluate lipoprotein subclasses, particle size, particle number and lipid composition. Methods: We studied the different lipoprotein particles using the Liposcale test. The number of particles of each lipoprotein subclass was quantified by 1H-NMR. This method measures the signals emitted by the protons of the terminal methyl group of the four types of lipids present in the lipoprotein particles. Results: It was found that the concentrations of VLDL-C, VLDL-TG, IDL-TG, and HDL-TG, as well as the number of VLDL-Ps and all their subclasses, were statistically higher in the overweight/obese children group. REM-C was also higher in overweight/obese children, and they had a smaller mean LDL-Z. Conclusions: These results support the presence, already in prepubertal childhood, of early metabolic alterations, associated with excess weight, and show that advanced lipoprotein profiling may provide additional information beyond the conventional lipid profile.

Biomolecules doi: 10.3390/biom16070926

Authors: Jingbo Wang Boyu Liao Yijing Ma Yihan Yang Yiyang Cao Xin Huang Tianxin Wen Hai-Shu Lin

Burn wound healing is a complex and dynamic process involving coordinated regulation of inflammation, oxidative stress, angiogenesis, and tissue remodeling. Polygonum cuspidatum, a traditional Chinese medicinal herb widely used for trauma- and inflammation-related disorders, represents an important source of bioactive compounds for tissue repair. Piceatannol (PIC), a naturally occurring stilbene constituent of P. cuspidatum, possesses potent anti-inflammatory and antioxidant activities; however, its therapeutic potential in burn wound healing remains insufficiently understood. In the present study, the therapeutic effects and underlying mechanisms of topical PIC were investigated using a murine deep second-degree burn model combined with multiple skin-related cellular models, including keratinocytes, fibroblasts, endothelial cells, and macrophages. PIC markedly accelerated wound closure and improved histological architecture, as evidenced by reduced inflammatory infiltration, enhanced collagen organization, and increased neovascularization. Mechanistically, PIC suppressed NF-κB activation and modulated KEAP1/NRF2-associated redox signaling, thereby alleviating inflammation–oxidative stress crosstalk during wound healing. In keratinocyte–fibroblast co-culture systems, PIC inhibited fibroblast-to-myofibroblast transition, reduced α-smooth muscle actin (α-SMA) expression, and attenuated excessive collagen deposition, suggesting anti-fibrotic activity. In addition, PIC promoted endothelial tube formation through activation of the STAT3–VEGF signaling axis. Collectively, these findings demonstrate that PIC facilitates burn wound repair through coordinated anti-inflammatory, antioxidative, pro-angiogenic, and anti-fibrotic effects. This study provides pharmacological support for the therapeutic potential of P. cuspidatum-derived compounds in burn management and highlights PIC as a promising candidate for topical treatment of burn injuries.

Biomolecules doi: 10.3390/biom16070925

Authors: Chavee Laomeephol Helena Ferreira Supansa Yodmuang Rui L. Reis Siriporn Damrongsakkul Nuno M. Neves

In the original publication [...]

Biomolecules doi: 10.3390/biom16060924

Authors: Zhiyuan Bian Huanhuan Gao Haijun Wu Tao Yang

The Arabidopsis thaliana Heat Shock Protein-Related (AtHSPR) gene participates in plant growth and abiotic stress tolerance, while its role in biotic stress resistance remains unclear. Here, we report that the athspr mutant is sensitive to Pseudomonas syringae pv. tomato (Pst) DC3000, whereas over-expression of AtHSPR enhances the defense of Arabidopsis against the pathogen. AtHSPR expression was induced by treatment with Pst DC3000, flg22, or salicylic acid (SA). Transcriptome analysis showed that mutation of AtHSPR changed the expression patterns of genes associated with defense response, oxidation–reduction, and SA responses, as well as transcription factors. The biochemical evidence revealed that AtHSPR interacted with Thimet Oligopeptidase 1 (TOP1), which modulated the SA-mediated immune response. Co-expression of AtHSPR and TOP1 showed that the TOP1 protein, normally located in the chloroplasts, gathered around the nucleus in response to a pathogen. After pathogen treatment, dynamic tubular projections (stromules) were present, extending from the chloroplasts toward the nucleus, and TOP1 was observed in the nucleus, together with AtHSPR. The top1athspr double mutant had lower SA levels and was more sensitive to pathogens than the top1 and athspr single mutants. Taken together, our results demonstrated that the interaction between AtHSPR and TOP1 plays a positive role in SA-mediated plant resistance against Pst DC3000.

Biomolecules doi: 10.3390/biom16060923

Authors: Ying Dong Yingying Su Damu Tang

Cytohesin-4 (CYTH4), an ARF guanine nucleotide exchange factor, remains unknown in RCC pathogenesis. We report that CYTH4 was dramatically upregulated in clear cell renal cell carcinoma (ccRCC) and following ccRCC progression. CYTH4 was strongly associated with ccRCC’s immune-suppressive features and stratified ccRCC poor outcome. From CYTH4′s network/NW, a multigene panel, SigCYTH4NW, was derived. In retrospective studies, (1) SigCYTH4NW effectively predicted ccRCC’s inferior prognosis, was strongly associated with the well-validated poor risk ccB signature in four independent ccRCC cohorts (n = 1132), was significantly upregulated in ccB compared to ccA (favorable risk) tumors, was robustly correlated with an immune checkpoint signature (SigIC), and was predominantly expressed in tumor-associated macrophages, and (2) SigCYTH4NW effectively predicted poor prognosis and correlated with SigIC across 21 other cancer types. CYTH4 was expressed at low levels in 786-0 ccRCC cells; its stable expression promoted 786-0 cell proliferation in vitro and xenograft formation in vivo. CYTH4 bound PPP1R9B, which maintains pRb’s hypophosphorylation. 786-0 CYTH4 cells displayed intensive pRb hyperphosphorylation, suggesting that CYTH4 enhances cell proliferation partially by pRb inhibition. Gene expression profiling by RNA-seq revealed a 786-0 CYTH4 network that was relevant to primary ccRCC, particularly in the aspect of immune evasion. Collectively, this study supports CYTH4’s promoting ccRCC.

Biomolecules doi: 10.3390/biom16060922

Authors: Laura-Mihaela Ion Carmen Pavelescu Denisa Maria Canut Mihaela Oros Gheorghita Jugulete Smaranda Diaconescu

The clinical relevance of food-specific IgG antibodies in pediatric gastrointestinal disorders remains controversial. Although current international guidelines discourage their use as standalone diagnostic tools, their significance within a broader immune–gut inflammatory framework has not been sufficiently explored. This study aimed to investigate associations between food-specific IgG reactivity, inflammatory and permeability biomarkers, microbiological findings, and abdominal ultrasound abnormalities in children with chronic gastrointestinal symptoms. Methods: (1) Children presenting chronic gastrointestinal symptoms associated with food-specific IgG polysensitization, elevated inflammatory and permeability biomarkers, and abdominal ultrasound abnormalities (number (n) = 196); (2) a symptomatic gastrointestinal group without the complete multimodal profile (n = 146); and (3) a control group with normal abdominal ultrasound findings and biomarkers within reference ranges (n = 210). All participants underwent food-specific IgG testing using a 216-antigen ELISA panel, abdominal ultrasound examination, and assessment of intestinal inflammatory and permeability biomarkers. Food-specific IgG antibodies were not interpreted as diagnostic markers of food allergy or food intolerance. Comparative analyses, correlation analyses, multivariable logistic regression, and receiver operating characteristic (ROC) analyses were performed. Results: Food-specific IgG polysensitization was significantly more frequent among children presenting the multimodal inflammatory profile compared with symptomatic and control groups (all p < 0.001). Reactivity predominantly involved gluten-containing cereals, dairy proteins, and mixed gluten–dairy patterns. Elevated fecal calprotectin, zonulin, and fecal histamine concentrations were more frequently observed in this subgroup, together with a higher prevalence of ultrasound abnormalities, including bowel wall thickening and mesenteric lymphadenopathy. Correlation analyses demonstrated significant associations between cumulative IgG burden and bowel wall thickness (r = 0.48, p < 0.001), while fecal calprotectin showed the strongest association with ultrasound abnormalities (r = 0.62, p < 0.0001). Multivariable logistic regression identified elevated calprotectin, increased zonulin, IgG polysensitization, and mixed gluten–dairy reactivity as independent predictors of pathological ultrasound findings. The integrated multimodal model demonstrated higher classification performance than isolated biomarkers. Conclusions: Children presenting chronic gastrointestinal symptoms, food-specific IgG polysensitization, inflammatory biomarker abnormalities, and ultrasound changes represented a multimodal clinical subgroup within the study population. These findings support evaluating food-specific IgG reactivity within a broader immune–gut assessment framework rather than as a standalone diagnostic biomarker. The observed associations should be considered exploratory and hypothesis-generating, requiring prospective validation and mechanistic investigation.

Biomolecules doi: 10.3390/biom16060921

Authors: Hans-Werner Denker Evdokia Dimitriadis Lois A. Salamonsen

Embryo implantation within the uterus is critical for the establishment of pregnancy and is an important issue in infertility [...]

Biomolecules doi: 10.3390/biom16060920

Authors: Ayumi Matsushita Maki Kimura Naoko Tajima Tsuyoshi Yamanaka Masato Inazu

Zinc deficiency is increasingly recognized as a risk factor for neurodegenerative diseases, yet the underlying molecular mechanisms remain incompletely understood. In this study, we investigated the impact of intracellular zinc depletion on oxidative stress and inflammasome activation in microglial (SIM-A9) and neuronal (SH-SY5Y) cell models, and evaluated the protective effects of polyphenolic compounds. Intracellular zinc chelation with the membrane-permeable chelator TPEN markedly increased reactive oxygen species (ROS) production, reduced cell viability, and upregulated the mRNA expression of NLRP3 inflammasome-related genes and pro-inflammatory cytokines. In contrast, extracellular zinc chelation had no effect, highlighting the critical role of intracellular zinc homeostasis in maintaining redox balance. Zinc supplementation significantly attenuated these responses. Among 32 polyphenols screened by DPPH radical scavenging assay, caffeic acid derivatives—chicoric acid (ChA), rosmarinic acid (RA), and caffeic acid phenethyl ester (CAPE)—exhibited the most potent antioxidant activity, surpassing that of edaravone. These compounds suppressed ROS production and differentially protected against zinc deficiency-induced cellular damage. ChA showed the strongest ROS inhibitory activity (IC50: 1.9 µM in SIM-A9), RA provided robust cytoprotection even at low concentrations, and CAPE most effectively suppressed inflammasome-related gene expression and inhibited aggregation of both Aβ1–42 and the highly neurotoxic pyroglutamate-modified variant pEAβ3–42. These findings demonstrate that intracellular zinc deficiency drives ROS-dependent upregulation of NLRP3 inflammasome-related genes, and suggest that caffeic acid derivative polyphenols may serve as complementary agents for mitigating neuroinflammatory and amyloidogenic processes relevant to Alzheimer’s disease.

Biomolecules doi: 10.3390/biom16060919

Authors: Boglárka Schilling-Tóth Daiana Alymbaeva Krisztián Németh Dávid Sándor Kiss István Tóth Gábor Andócs Ondrašovičová Silvia Brigitta Tagscherer-Micska Gergely Jócsák Tibor Bartha

Extracellular vesicles (EVs) are key mediators of intercellular communication across biological kingdoms, with central roles in immune regulation and disease processes. Despite shared structural features, EVs derived from bacteria, plants, and mammalian cells differ substantially in their biogenesis, molecular composition, and immunological functions. EV formation pathways generate vesicles with distinct cargo profiles, including pathogen-associated molecular patterns (PAMPs) in bacterial EVs, regulatory small RNAs in plant-derived vesicles, and cytokines, microRNAs, and antigen-presenting complexes in mammalian EVs. Differences in cargo result in divergent immune outcomes. Bacterial EVs predominantly activate innate immunity via pattern recognition receptors such as Toll-like receptors, whereas plant-derived EVs exhibit low immunogenicity and mediate cross-kingdom RNA interference. In contrast, mammalian EVs primarily regulate immune responses by modulating antigen presentation and cytokine signaling. These findings support a framework in which EV origin determines immunological function and therapeutic applicability. This perspective highlights the importance of selecting appropriate EV sources for vaccine development, regenerative medicine, and targeted delivery strategies, while addressing current challenges related to heterogeneity, standardization, and safety.

Biomolecules doi: 10.3390/biom16060918

Authors: Jada L. Brown Padmanabhan Mahadevan Michael Middlebrooks

Certain sacoglossan sea slugs, often known as “solar-powered sea slugs”, are a group of marine gastropods that have the unique ability to photosynthesize by stealing functional chloroplasts from algae. The sacoglossan Elysia papillosa can maintain functional chloroplasts for up to two weeks after feeding. The microbiome of these slugs may play a crucial role in their metabolism, immunity, development, but more importantly their photosynthesis. Shotgun metagenomic sequencing was conducted on four samples of E. papillosa in order to characterize their microbiome. Sequences were classified and relative abundance was quantified with Centrifuger and functional data was examined using SqueezeMeta. Bacteria were analyzed by taxonomic groups and hypothesized function to the sea slug was determined with literature analysis. All samples were dominated by phyla Actinomycetota, Bacillota, Patescibacteriota, and Pseudomonadota. The presence of the phyla Bacteroidota and Bacillota was notable in all samples, which contain species known to produce enzymes that break down polysaccharides. It is possible that these bacteria could assist in degradation of the polysaccharide xylan found in the cell walls of Penicillus, the algal food source of E. papillosa. One species that was found in all samples was Cutibacterium acnes which has been shown to be an important component of the gut microbiota in other marine invertebrates and may provide the host with vitamin B12 and other beneficial nutrients. Many of these bacteria may be opportunistic rather than commensal. As a result, more research is required to describe the interactions between the slug and its microbiome, but this preliminary report provides a valuable starting point for identifying the microbiome make-up to further understanding of these relationships.

Biomolecules doi: 10.3390/biom16060917

Authors: Xianglong Ou Yi Dai Xiangyue Hu Yuan Liu Shibin Yuan Le Wang Bangyuan Wu Tingting Fang

Severe acute pancreatitis (SAP) is a life-threatening inflammatory disorder characterized by high mortality and limited therapeutic options. Alginate oligosaccharide (AOS), a marine-derived bioactive polysaccharide, exhibits prebiotic, anti-inflammatory and antioxidant properties that are effective against various inflammatory diseases. In this study, a mouse model of SAP was established by intraperitoneal injection of cerulein (100 μg/kg) and lipopolysaccharide (5 mg/kg), and the mice were pretreated with AOS (200 mg/kg) by gavage for 4 consecutive weeks to explore the potential protective efficacy and underlying mechanisms. The results shown that AOS attenuated the severity of SAP, as evidenced by reduced serum amylase and lipase levels, as well as alleviated histopathological injury in both pancreatic and ileal tissues. AOS suppressed the overproduction of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in serum, pancreas, and ileum at protein or mRNA levels. Moreover, AOS effectively diminished pancreatic and ileal inflammatory infiltration and oxidative stress in SAP mice, accompanied by inhibited the TLR4/MyD88/NF-κB pathway and activated the Nrf2/HO-1 antioxidant axis. Furthermore, AOS restored intestinal barrier integrity, as manifested by upregulated expression of tight junction proteins (claudin-1, occludin, ZO-1), reduced serum diamine oxidase, and decreased bacterial translocation from the gut to the pancreas. It was revealed by 16S rRNA sequencing that AOS ameliorated SAP-induced gut dysbiosis by restoring microbial diversity, normalizing the Firmicutes/Bacteroidetes ratio, enriching beneficial genera (Lactobacillus, Blautia), and enhancing cecal short-chain fatty acid (acetic, propionic, butyric acid) production. Collectively, our findings demonstrate that AOS exerts comprehensive protective effects against SAP through suppression of inflammatory signaling and oxidative stress, as well as restoring gut homeostasis. These results suggest that AOS may serve as a promising prebiotic-based nutritional strategy for the management of SAP.

Biomolecules doi: 10.3390/biom16060916

Authors: Jae Jin Jeong Mauli Maniar Shahrzad Ghane Sakshi Deshpande Claire Ellis Ashakumary Lakshmikuttyamma

Protein arginine methyltransferase 5 (PRMT5) mediates arginine methylation of a wide range of proteins and plays context-dependent oncogenic or tumor-suppressive roles. In cancer, PRMT5 represses several tumor suppressor genes, including E-cadherin, TP53BP1, ST7, PTEN, and RB (retinoblastoma). Elevated PRMT5 expression has been reported across multiple cancer types, notably triple-negative breast cancer (TNBC). In TNBC, high PRMT5 levels are associated with enhanced cancer stem cell self-renewal, increased tumor growth and metastasis, and reduced patient survival. Mechanistically, PRMT5 promotes breast cancer stem cell maintenance and proliferation through stabilization of the transcription factors KLF4 and KLF5. Disruption of the PRMT5–KLF4 axis results in significant tumor reduction in TNBC models. Moreover, increased PRMT5 expression has been linked to resistance to chemotherapy and immunotherapy in TNBC. Notably, PRMT5 inhibitors demonstrate synergistic anticancer activity when combined with inhibitors of key oncogenic signaling pathways, including EGFR, PARP, and AKT. While several PRMT5 inhibitors are currently being evaluated in clinical trials for other malignancies, no clinical trials have yet been initiated specifically for TNBC.

Biomolecules doi: 10.3390/biom16060914

Authors: Larisa Dobrynina Alexandra Byrochkina Kamila Shamtieva Elena Kremneva Maryam Zabitova Alla Shabalina

A key mechanism in the pathogenesis of cerebral small vessel disease (CSVD) is endothelial dysfunction associated with impaired metabolism of nitric oxide (NO) and its main substrate, L-arginine. The aim of the study was to assess parameters of L-arginine metabolism and their association with MRI-defined brain damage in CSVD patients. A total of 100 CSVD patients (according to MRI STRIVE standards) and cognitive impairment (CI) of varying severity, as well as 20 healthy volunteers, were analyzed. Levels of L-arginine and its metabolites—L-ornithine, L-citrulline, and asymmetric dimethylarginine (ADMA)—were measured; diffusion tensor MRI, MRI volumetry, and morphometry were performed. A threshold level of L-arginine (51.25 μmol/L) was identified, above which an association with CI was observed. Patients with L-arginine ≥ 51.25 μmol/L demonstrated poorer performance on cognitive tests (Stroop test, trail-making test (TMT)-B, TMT B–A, 10-word test) and more severe brain damage, reflected by greater severity of MRI markers (white matter hyperintensities, microbleeds), changes in brain component volumes, cortical atrophy in specific regions, and impairment of white matter microstructural integrity. The obtained data indicate a pathogenetic link between disturbances in L-arginine homeostasis and the development of CSVD with CI and support the need for further studies aimed at refining approaches to their correction.

Biomolecules doi: 10.3390/biom16060915

Authors: Mircea Tampa Ilinca Nicolae Madalina Irina Mitran Cristina Iulia Mitran Clara Matei Milena Tocut Simona Roxana Georgescu Cosmin Ene Cristina Capusa Corina Daniela Ene

The skin serves as the body’s first line of defense against environmental threats, acting as a barrier between external aggressors and internal systems. Current evidence regarding the roles of sulfur dioxide (SO2) in biology and medicine is limited. Environmental pollutants, including SO2, can increase the production of reactive oxygen species in the skin, leading to oxidative damage that may worsen various dermatological conditions. Endogenous SO2, proposed as the fourth member of the gasotransmitter family, functions as a biological signaling molecule. It is generated in various human skin cells, including vascular smooth muscle cells, endothelial cells, mast cells, keratinocytes, macrophages, adipocytes, fibroblasts, dermal immune cell population, etc, where it performs multiple functions at physiologically relevant concentrations. Endogenous SO2 plays a crucial role in regulating cell signaling and maintaining skin homeostasis through its antioxidant, anti-inflammatory, and cytoprotective effects. Abnormal generation and metabolism of SO2 are linked to several critical processes in the skin, including vascular biology, immune response, cell proliferation, pigmentation, malignancy, protective barriers, senescence, and resistance to stress. This paper provides a narrative review of the significant roles of SO2 in skin health and disease. A comprehensive understanding of the complex molecular effects and mechanisms mediated by SO2 in human skin, along with the development of gas therapy, will be essential for translating fundamental research into clinical applications.

Biomolecules doi: 10.3390/biom16060913

Authors: Fabio Ferrini Giosuè Annibalini Michela Battistelli SeyedehMahboobeh Moosavi Osman Riham Fabiana Fanelli Italo Capparucci Piero Sestili Elena Barbieri

Hyaluronic acid (HA), a major component of the glycome and a non-sulfated glycosaminoglycan, plays a crucial role in regulating stem cell behavior and function, thereby supporting skeletal muscle repair under inflammatory conditions. In this study, we investigated the effects of a mixture of HA fractions with different molecular weights (M-HA; 2–1000 kDa) on the repair capacity and myogenic potential of C2C12 murine myoblasts exposed to inflammatory stimuli. C2C12 cells were cultured, induced to differentiate, and treated with M-HA (1 mg/mL) under either physiological or inflammatory conditions (LPS, 10 µg/mL; IL-1β, 20 ng/mL). M-HA exhibited no cytotoxic effects, even at the highest concentration tested (1.0 mg/mL), and significantly enhanced scratch wound closure. Moreover, M-HA improved the myogenic index at day 5 of differentiation, promoted the expression of myogenic markers, preserved myosin heavy chain (MHC) levels under inflammatory stress, and reduced the expression of autophagy-related genes. Ultrastructural analyses revealed that untreated myotubes displayed swollen mitochondria, disrupted cristae architecture, and numerous autophagic vacuoles, whereas M-HA-treated cells exhibited well-preserved mitochondrial morphology, intact cristae organization, reduced cytoplasmic damage, and maintained myofibrillar structure. Taken together, the functional, molecular, and ultrastructural findings demonstrate that M-HA protects myoblasts from inflammation-induced cellular damage and supports their regenerative capacity. These results underscore the potential of glycomics-based strategies to enhance myogenic differentiation and promote skeletal muscle regeneration in inflammatory microenvironments.

Biomolecules doi: 10.3390/biom16060912

Authors: Tianzhu Zhang Yufei Li Jiahui Pei Qingxia Zhang Fengyun Lin Shuzhen Li

The gut microbiota and its metabolites, as components of the gut–bone axis, play a pivotal role in regulating skeletal homeostasis through the bidirectional communication network. In this systematic review, evidence was collected from mainstream databases following standardized inclusion/exclusion criteria for screening, to comprehensively retrieve and screen eligible studies from multiple mainstream databases according to standardized inclusion and exclusion criteria, and systematically summarize current research progress on plant-derived bioactive compounds targeting the gut–bone axis for skeletal health regulation. This review systematically explores the underlying mechanisms of the gut–bone axis and critically evaluates the regulatory effects and therapeutic potential of plant-derived bioactive compounds. Particular attention is given to targeted interventions involving prebiotics, probiotics, synbiotics, and plant-rich diets or functional foods. Among these interventions, synbiotics represent the most successful strategy and show the most prominent therapeutic possibilities in bone-related disorders. Different from single prebiotics (only nourish endogenous intestinal microbes), individual probiotics (easy to be degraded in gastrointestinal tract with poor colonization) and ordinary plant-rich diets (unfixed effective dosage and weak targeting property), synbiotics combine prebiotic carriers and viable probiotic strains to produce complementary advantages, which is the core reason for its outstanding therapeutic prospect against bone diseases. Synbiotics exert synergistic effects on gut microecology, mineral absorption, and immune regulation, leading to more robust and consistent improvements in bone health than single prebiotics, probiotics, or general plant-rich diets. They have been verified in preclinical and clinical studies to ameliorate osteoporosis and related skeletal diseases via the gut–bone axis. These strategies offer novel insights into the prevention and treatment of bone metabolic disorders, such as osteoporosis, by targeting the gut–bone axis with phytochemicals. Key outcomes of this review include that synbiotics, soy isoflavones, naringin, curcumin, and resveratrol effectively improve bone mineral density, restore gut microbiota balance, and inhibit pathological bone resorption via the gut–bone axis. Collectively, the above bioactive substances realize bone protection mainly by reshaping gut flora, elevating mineral uptake and suppressing excessive osteoclast activity. Representative cases include soy isoflavones mitigating estrogen-deficient bone loss in OVX models, naringin improving the trabecular microarchitecture, and probiotic BL-11 promoting longitudinal bone growth in children. Future directions will focus on clarifying dose–response relationships, developing standardized synbiotic formulations, constructing microbiome-guided precision diets, and conducting large-sample randomized controlled trials to translate plant-derived compounds into clinical therapies.

Biomolecules doi: 10.3390/biom16060911

Authors: Walter Giuseppe Giordano Ludovica Pepe Canio Martinelli Valeria Zuccalà Giuliana Ciappina Massimiliano Berretta Giuseppe Giuffrè Vincenzo Fiorentino Antonio Ieni

Endometrial cancer is the most common gynecologic malignancy in developed countries and remains challenging in terms of risk stratification, treatment monitoring, and early detection of recurrence. Liquid biopsy provides a minimally invasive approach for the dynamic assessment of tumor-derived biomarkers and may complement tissue-based diagnosis and molecular classification. This narrative review summarizes current evidence on circulating biomarkers in endometrial cancer, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), extracellular vesicles (EVs), circulating microRNAs, and tumor-educated platelets, with attention to validity, applicability, and implementation barriers. Among these biomarkers, ctDNA currently has the strongest evidence base, especially for longitudinal monitoring, prognostic stratification, molecular residual disease assessment, and early detection of relapse in high-risk or recurrent disease. However, its sensitivity remains limited in early-stage, low-volume, and low-shedding tumors. CTCs, EVs, microRNAs, and platelet-derived signatures are promising but still largely investigational. Artificial intelligence may support multimodal biomarker validation, although clinical adoption will require external validation, locked algorithms, standardized workflows, and prospective utility trials. Overall, liquid biopsy represents a promising adjunct to tissue-based diagnosis and molecular classification in endometrial cancer, particularly for monitoring and follow-up. Prospective studies are now needed to demonstrate whether liquid-biopsy-informed decisions can improve outcomes or safely reduce overtreatment.

Biomolecules doi: 10.3390/biom16060910

Authors: Henrietta Cserne Szappanos László Zsolt Szabó Ildikó Balatoni Martin F. Schneider László Csernoch Péter Szentesi

Calcium sparks can arise as both voltage-dependent and voltage-independent ligand-activated release events in amphibian skeletal muscle. To assess their gating behavior, calcium sparks were recorded from intact frog skeletal muscle fibers using high-temporal-resolution confocal microscopy (line scans: 15 and 50 µs/line). Sparks were triggered by 1 mmol/L caffeine to open ryanodine receptors (RyRs) or by subthreshold depolarization to a −65 mV membrane potential to activate dihydropyridine receptors (DHPRs). Both treatments increased the frequency of sparks and altered their morphology. The sparks were significantly greater after caffeine treatment than in depolarized cells. The signal mass of sparks (i.e., the amount of calcium released) resembled the amplitude in shape. Additionally, the calcium release flux followed a staggered function during the activation of sparks. The detailed analysis of the sparks’ time profile revealed that the events were activated in a stepwise manner. The average step size (in F/F0; 0.071 ± 0.003) remained constant regardless of the scanning speed. The number of steps during the activation of sparks followed a linear function based on the spark’s amplitude. Our results suggest that the activation of neighboring release units may occur sequentially, and the amplitude of the sparks depends linearly on the number of activated RyR channels.

Biomolecules doi: 10.3390/biom16060909

Authors: Mohamed S. Gad Samar Habib Khaled Elmasry

Diabetes mellitus represents a major global health challenge with rapidly increasing prevalence and substantial morbidity driven by metabolic and vascular complications. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and are increasingly implicated in the pathogenesis and progression of diabetes. This review summarizes current knowledge on EV biology, including their classification, cellular sources, biogenesis, uptake mechanisms, and molecular cargo. We discuss the contribution of EV-associated microRNAs to immune dysregulation and β-cell damage in type 1 diabetes mellitus (T1DM), as well as the role of EVs in insulin resistance, metabolic signaling, and vascular dysfunction in type 2 diabetes mellitus (T2DM). Particular emphasis is placed on EV-mediated modulation of endothelial function, angiogenesis, and tissue repair, alongside their involvement in the impairment of insulin receptor integrity. We further explore how lifestyle factors may influence EV composition and function, highlighting their potential integration into preventive strategies. Finally, we evaluate the emerging therapeutic potential of EVs as biomarkers and delivery systems, while addressing current limitations and future directions. Collectively, EVs represent a promising frontier in understanding diabetes pathophysiology and developing innovative diagnostic and therapeutic approaches. Unlike previous reviews that examine EVs separately as biomarkers or therapeutic vehicles, this review integrates emerging evidence supporting EVs as mediators of systemic communication linking pancreatic islets, adipose tissue, immune cells, vascular endothelium, kidney, heart, and retina throughout diabetes progression. We further critically evaluate translational barriers that currently limit clinical implementation of EV-based diagnostics and therapeutics.

Biomolecules doi: 10.3390/biom16060908

Authors: Lin Xie Hongmei Xu Pinglu Zhang Jianshe Xiong Jing Li

Existing drug–target interaction (DTI) prediction methods still face challenges caused by sparse interaction data, complex multi-source relationships, and imbalanced information contributions among different nodes. In this study, we propose NAFF-DTI, a node-level adaptive feature fusion network based on multi-view graphs. The model uniformly represents drug similarity, target similarity, and known drug–target interactions as multiple relational views, and learns node representations through graph encoding and cross-view representation learning. To more effectively utilize heterogeneous relational information, NAFF-DTI introduces cross-view feature discrepancy modeling and a node-level adaptive fusion mechanism to dynamically adjust the contribution of different views according to node structural characteristics. Experimental results show that NAFF-DTI achieves the best AUC and AUPR on all five benchmark datasets. Compared with the strongest baseline for each dataset and metric, NAFF-DTI achieves average relative improvements of 3.81% in AUC and 3.23% in AUPR. It can also improve the utilization of multi-source information, maintain relatively stable prediction under different data distributions, and prioritize biologically plausible candidate drug–target associations from the unannotated candidate space. These results indicate that NAFF-DTI can provide computational support for DTI candidate prioritization and repurposing-oriented hypothesis generation.

Biomolecules doi: 10.3390/biom16060907

Authors: Pnina Fishman

The A3 adenosine receptor (A3AR) is a stress-inducible G-protein-coupled receptor that is selectively upregulated in inflamed, hypoxic, and fibrotic tissues as well as in many malignancies, while remaining weakly expressed in most normal organs. This distinctive expression pattern provides a strong biological basis for pathology-selective pharmacology. Activation of A3AR by highly selective agonists, including piclidenoson (IB-MECA) and namodenoson (Cl-IB-MECA), initiates signaling through Gi proteins and phospholipase C (PLC), which in turn regulate a coordinated network of downstream intracellular pathways, including PI3K/Akt, NF-κB, MAPKs, and Wnt/β-catenin, resulting in suppression of inflammation, inhibition of pathological cell survival, and protection of metabolically stressed tissues. Over the three decades, extensive preclinical studies have demonstrated that A3AR agonism exerts anti-cancer, anti-fibrotic, immunomodulatory, neuroprotective, and organ-protective effects across diverse disease models, including hepatocellular carcinoma, pancreatic cancer, psoriasis, osteoarthritis, metabolic dysfunction-associated steatohepatitis, ischemic stroke, neurodegeneration, ophthalmic disorders, and inherited metabolic syndromes. Importantly, these mechanistic insights have been translated into clinical programs, with piclidenoson and namodenoson demonstrating favorable safety profiles and disease-modifying activity in inflammatory, fibrotic, and oncologic indications. This review integrates molecular, cellular, and translational evidence to highlight A3AR activation as a unifying therapeutic principle for diseases driven by inflammation, oxidative stress, hypoxia, and dysregulated cell survival, positioning selective A3AR agonists as first-in-class agents targeting the A3AR, with broad clinical applicability across multiple disease domains.

Biomolecules doi: 10.3390/biom16060906

Authors: Qiang Li Jianglong Xu Yuhao Zhang Junbing Qian Diana Bee-Lan Ong Kein Seong Mun Yiping Tang Xiuchao Geng Kean Chang Phang

Background: Acquired tolerance to temozolomide (TMZ) remains one of the main obstacles to enduring therapeutic success in glioblastoma (GBM). While tumor-derived extracellular vesicles are known to orchestrate therapy evasion by horizontally transferring molecules across the tumor microenvironment, the precise regulatory roles of specific exosomal circular RNAs (circRNAs) in establishing this refractory state require further elucidation. Methods: The expression of circ_0050688 in TMZ-resistant GBM clinical tissues and cell lines was evaluated. Exosomes derived from resistant cells were isolated and confirmed via transmission electron microscopy (TEM) and marker analysis. PKH67 fluorescent tracking was utilized to visually demonstrate exosome internalization by sensitive recipient cells. Biological functions, including the expression of the multidrug resistance protein P-glycoprotein (P-gp) and the proliferation marker Ki-67, were evaluated. The competing endogenous RNA mechanism was validated using RNA FISH, dual-luciferase reporters, and functional rescue experiments. In vivo efficacy was determined using subcutaneous xenograft mouse models. Results: Clinical and in vitro analyses revealed that circ_0050688 is upregulated in TMZ-refractory GBM, predicting adverse patient survival. Through PKH67-based tracing, we confirmed that resistant cells actively secrete circ_0050688-enriched exosomes, which are subsequently engulfed by drug-sensitive bystander cells. This vesicular transfer directly instigates a chemoresistant and highly proliferative phenotype, marked by elevated P-gp and Ki-67 levels. At the molecular level, circ_0050688 operates as a molecular decoy for miR-508-5p, thereby preventing the suppression of its downstream target, MDM2. Functionally, circ_0050688 depletion eradicated these aggressive traits and restored TMZ vulnerability across both cellular and murine xenograft models. Furthermore, rescue assays confirmed that this circ_0050688-driven chemoresistance is fundamentally dependent on the miR-508-5p/MDM2 signaling axis. Conclusions: Current data uncover an intercellular signaling network driven by vesicular circ_0050688, which functions as a mobile oncogene to reshape the TMZ-refractory microenvironment. Targeting this exosomal circ_0050688/miR-508-5p/MDM2 network to suppress P-gp and Ki-67 expression represents a highly promising therapeutic strategy for refractory GBM.

Biomolecules doi: 10.3390/biom16060905

Authors: Tarek Gheith Sherihan M. Rohayem Atef Taha El Bahrawy Amira E. Mesbah Safaa Gaber Aly Salem Noha M. Hammad

Background: Sublingual immunotherapy (SLIT) is an effective treatment for house dust mite (HDM)-induced allergic rhinitis (AR); however, the significance of qualitative changes in specific IgE (sIgE) remains unclear. This study evaluated post-treatment changes in sIgE reactivity and compared the performance of three immunoassays. Methods: In this prospective study, monosensitized patients with HDM-induced AR were identified using skin prick testing and followed for 12 months after SLIT. Serum sIgE levels were assessed at baseline and after treatment using immunoblot, chemiluminescent immunoassay (CLIA), and ImmunoCAP as the reference method. Qualitative changes in sIgE reactivity were analyzed. Clinical response was assessed using the total nasal symptom score (TNSS), and total IgE (tIgE) levels were measured. Results: At baseline, HDM-sIgE reactivity was detected in 85.7%, 82.1%, and 92.9% of patients by immunoblot, CLIA, and ImmunoCAP, respectively. Following SLIT, a significant qualitative conversion to non-reactive status was observed across all assays (p < 0.001). Conversion rates were 94.0% for immunoblot, 83.3% for CLIA, and 100.0% for ImmunoCAP. Significant improvements in TNSS and reductions in tIgE were also observed. Conclusions: SLIT induces a marked qualitative reduction in HDM-sIgE reactivity, with complete serological conversion detected by ImmunoCAP. Although the immunoassays showed comparable rates of HDM-sIgE detection, their agreement in classifying individual patients differed, indicating variability in assay performance. Qualitative assessment of sIgE may provide a clinically meaningful approach for monitoring immunotherapy response.

Biomolecules doi: 10.3390/biom16060904

Authors: Zhiyuan Liu Xia Li Hanwen Hu Shangyu Xiao Jianli Tao Jing Yang

O’nyong-nyong virus (ONNV) is a mosquito-borne alphavirus responsible for large-scale epidemics in sub-Saharan Africa. As the closest evolutionary relative of Chikungunya virus (CHIKV), ONNV shares substantial genetic similarity and overlapping clinical manifestations with CHIKV. Mechanistic understanding of ONNV infection has therefore largely been extrapolated from CHIKV rather than directly established. However, ONNV exhibits distinct biological features, including predominant transmission by Anopheles mosquitoes and a clinical presentation characterized by prominent lymphadenopathy with limited acute joint edema. These distinctions underscore the need for an integrated synthesis of experimentally validated determinants of ONNV infection. In this review, we summarize current evidence on molecular and immunological factors regulating ONNV infection in mammalian hosts and mosquito vectors. We first discuss species-specific viral clearance, host dependency factors, intrinsic antiviral restriction mechanisms, protective innate immunity, inflammatory pathology, and mechanism-informed therapeutic strategies in mammalian hosts. We then examine stage-specific immune regulation in Anopheles mosquitoes, emphasizing mechanisms that constrain viral replication while permitting persistent infection and transmission. Finally, we discuss nsP3-dependent vector specificity and the potential contribution of alternative mosquito species to ONNV ecology. Together, this review provides an integrated framework for understanding how host factors, immune responses, and vector-specific adaptations shape ONNV infection, pathogenesis, and transmission.

Biomolecules doi: 10.3390/biom16060900

Authors: Alexandre Alberto Barros Duarte Danielle Lopes Teixeira Ferdinando Vânia Belintani Piatto Heloísa Cristina Caldas

Necrotizing enterocolitis (NEC) is a multifactorial disease associated with prematurity, intestinal hypoperfusion, dysbiosis, and oxidative stress. Interindividual variability in disease occurrence suggests a role for genetic susceptibility. Null genotypes of the GSTM1 and GSTT1 genes result in absent glutathione S-transferase activity and may impair antioxidant defenses. This study investigated whether GSTM1 and GSTT1 null genotypes are associated with NEC development and severity in preterm newborns. This single-center case–control pilot study included 100 preterm newborns (50 NEC and 50 controls). Genotyping was performed by multiplex polymerase chain reaction. Baseline characteristics were comparable between groups (p > 0.05). Stages II-A and II-B accounted for 82% of NEC cases. A significant inverse correlation was observed between gestational age and postnatal age at NEC diagnosis (r = −0.5994; p < 0.0001). The GSTM1-null genotype was more frequent in the NEC group (60% vs. 36%) and was associated with increased disease risk in both unadjusted (OR = 2.667; 95%CI: 1.188–5.986; p = 0.027) and adjusted analyses (aOR = 3.09; 95%CI: 1.29–7.40; p = 0.011). No significant associations were observed for GSTT1, combined genotypes, or disease severity. These findings provide preliminary evidence of an association between the GSTM1-null genotype and NEC susceptibility. Given the exploratory pilot design, these results should be considered hypothesis-generating and require confirmation in larger prospective studies.

Biomolecules doi: 10.3390/biom16060903

Authors: Adheip Monikantan Nair Leonardo Tullo Kenneth Andrei Espinosa Siu Lun Terrence Tong Rongmin Zhao

The plastid stroma-localized chaperone HSP90C is essential for maintaining chloroplast proteostasis and facilitating protein translocation. Prior research has established HSP90C’s imperative role in the SEC translocase-dependent transport of the photosystem II subunit PsbO1 and its interaction with the SEC1 translocase motor protein SecA1. However, the exact mechanism of this interaction remains to be explored. In this study, we delineated the interactional mode of HSP90C and SecA1 with the model client protein. Yeast two-hybrid and in vitro ATPase activity analyses with purified proteins revealed PsbO1 may bind to HSP90C at multiple sites, including the DPW motif within the C-terminal extension (CTE) region, suggesting a possible client-loading mechanism unique to plastid orthologs. We also confirmed that glycine-646 is important in mediating substrate interaction, though it conferred a much weaker binding than the CTE region, thereby elucidating a critical role for the amino acid whose mutation resulted in visible plant phenotypes. Our in vitro biochemical assays also demonstrated that the stromal intermediate form of PsbO1 with the thylakoid signal peptide (tSP) significantly enhanced SecA1 ATPase activity, suggesting a preferential binding to the motor protein. On the other hand, the mature domain of the PsbO1, excluding the tSP sequence, inhibited HSP90C ATPase activity. We also observed the HSP90C-PsbO1-SecA1 ternary complex was stabilized by the presence of the client tSP. This work therefore provides new insights into the functional mechanisms of HSP90C and its contribution to chloroplast stromal protein stabilization and thylakoid protein transport.

Biomolecules doi: 10.3390/biom16060902

Authors: Yingnan Yue Naoyuki Chado Rike Rachmayati Rie Wakabayashi Noriho Kamiya Shinichi Aishima Hiroyuki Ijima Yasuhiro Ikegami

Diabetic chronic wounds are often accompanied by excessive wound exudate maceration, which prolongs the inflammatory phase and increases the risk of infection. Such a complex wound microenvironment imposes more stringent requirements on multifunctional wound dressings. A multifunctional Cur Janus nanofibrous dressing is developed by integrating an electrospun poly(ε-caprolactone)/gelatin hydrophilic layer with a curcumin (Cur)-loaded PCL hydrophobic layer. Janus structure with asymmetric wettability, which exhibited unidirectional liquid transport properties both in vitro and in vivo. Its unique structure also makes it possible to carry both hydrophilic and hydrophobic drugs at the same time. The incorporation of curcumin endows the dressing with antibacterial and antioxidant functionalities, offering the potential to modulate the inflammatory microenvironment of diabetic chronic wounds. Furthermore, the wound healing ability and anti-inflammatory effects of Cur Janus nanofibers were evaluated in a diabetic mouse model. The results showed that Cur Janus nanofibers significantly reduced wound area, increased the proportion of pro-healing M2 macrophages, shortened the inflammatory phase, and ultimately accelerated diabetic wound healing. This work provides a multifunctional and scalable platform for advanced wound dressing design. Its excellent antibacterial, antioxidant (ROS scavenging) and anti-inflammatory (macrophage phenotype M1 to M2) properties, combined with the unidirectional fluid transport and dual-release potential of hydrophilic and hydrophobic drugs, demonstrate broad prospects in the management of diabetic wounds.

Biomolecules doi: 10.3390/biom16060901

Authors: Beatrice Radu Bogdan Amuzescu

Neuropharmacology has emerged in recent years as a field of active research, driven by the need to address increasing challenges posed by clinical conditions such as neurodegenerative disorders, which affect more and more elderly people worldwide as the average life expectancy extends progressively [...]

Biomolecules doi: 10.3390/biom16060899

Authors: Dafni F. T. Frohman Stella E. Tsirka

Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system characterized by demyelination, neuroinflammation, and progressive neurodegeneration. While there is a small component of genetic susceptibility to MS risk, environmental factors, including infectious exposures, are gaining increased recognition as playing a critical role in MS initiation and progression. Viral infections, especially by Epstein–Barr virus (EBV), have emerged as strong candidates and triggers of MS symptoms, through antibody-mediated molecular mimicry and B-cell dysregulation. In contrast, parasitic infections, including helminths and select protozoa, appear to exert neuroprotective effects by skewing immune responses toward regulation and tolerance. In this review, we examine antibody-driven mechanisms by which viral pathogens promote autoimmunity in MS and contrast these with parasite-induced immunoregulatory pathways that suppress pathogenic inflammation. We further discuss diagnostic and therapeutic implications, highlighting how insights from infectious immunology may inform novel strategies for MS treatment.

Biomolecules doi: 10.3390/biom16060898

Authors: Cesare Mancuso Rosaria Santangelo

Carbon monoxide (CO) is a gasotransmitter generated by heme oxygenase (HO) isoforms during heme catabolism. The inducible HO-1 produces CO under conditions of redox imbalance, such as oxidative stress and inflammation. On the other hand, HO-2 constitutively generates CO, primarily during the physiological turnover of heme. Extensive evidence indicates that CO exerts autocrine effects by targeting hemoproteins, including soluble guanylyl cyclase, cyclooxygenase, and cytochromes. Furthermore, CO regulates many biological processes within the brain, including mitochondrial biogenesis, potassium channel activity, mitogen-activated protein kinase and phosphatidylinositol-3-kinase/Akt signaling. It also controls the activity of transcription factors, such as hypoxia-inducible factor-1 and peroxisome proliferator-activated receptor-γ. Through these mechanisms, CO modulates inflammatory gene expression, promotes anti-apoptotic signaling, and contributes to local stress responses. Conversely, CO produced in the hypothalamus inhibits the stress-induced release of corticotropin-releasing hormone and arginine vasopressin under pro-inflammatory conditions, resulting in reduced adrenocorticotropin hormone release and cortisol secretion from the anterior pituitary and adrenal cortex, respectively. Moreover, hypothalamic CO acts in a paracrine manner to modulate glucocorticoid release during psychological stress, including restraint or water deprivation. Together, these findings support the view that endogenous CO is a key modulator of the stress axis, exerting pleiotropic effects that integrate neuroendocrine, immune, and metabolic responses.

Biomolecules doi: 10.3390/biom16060897

Authors: Shirui Bai Tao Lin Haoxia Li Bo Han John P. Kastelic Tao Zhang Hao Shi Gang Liu Yipeng Jin

Cutaneous leishmaniasis (CL) is the most common clinical form of leishmaniasis, characterized by persistent skin ulcers and nodules. Standard chemotherapeutic agents have substantial toxicity and do nothing to repair the damaged tissue, an unmet need that motivates the search for adjunctive strategies. Mesenchymal stem cells (MSCs) can modulate macrophage activity and support tissue regeneration, yet their role in CL has received limited attention. In this study, we tested whether bone marrow-derived MSCs (BM-MSCs) could attenuate Leishmania mexicana-induced inflammation and facilitate skin repair. Indirect co-culture of BM-MSCs with infected RAW264.7 macrophages shifted the macrophage phenotype from M1 toward M2, with higher IL-10 and Arg-1 expression and lower iNOS and IL-1β. In BALB/c mice with established CL, three weekly intravenous injections of BM-MSCs reduced paw swelling, improved skin histology, decreased type I collagen deposition, lowered Integrin β1 and Cytokeratin 17 expression, and reduced tissue parasite load. Immunofluorescence confirmed a predominantly M2 macrophage distribution in treated lesions. We inferred that BM-MSCs acted on both the immune and reparative aspects of the disease process, supporting their potential as an adjunct to conventional anti-leishmanial therapy.

Biomolecules doi: 10.3390/biom16060896

Authors: Saltanat Nayekova Vladimir Kiyan Zhanar Tulegenova Timur Savin Evgeniy Ten Zerekbay Alikulov

Freezing stress is one of the major abiotic factors limiting plant growth and productivity. This study evaluates the effects of diatomite (DTM) as a natural silicon-rich amendment on growth performance, physiological responses, and cold stress tolerance in barley (Hordeum vulgare L.). Seed priming and substrate application of DTM at different concentrations (5–20%) were used to assess morphological, biochemical, and ultrastructural changes under normal and low-temperature conditions. Results showed that DTM significantly enhanced root growth and biomass accumulation, with the most pronounced effect at 10% concentration. Treated plants exhibited improved survival under freezing stress, along with better preservation of leaf cellular structure and photosynthetic pigments. Biochemical analyses revealed reduced proline accumulation and decreased activity of key antioxidant enzymes, indicating alleviation of oxidative stress and improved redox balance. Electron microscopy confirmed the integration of diatomite particles into seed and tissue structures, providing physical reinforcement and thermal protection. Overall, diatomite acts as a multifunctional, environmentally safe soil amendment that enhances plant growth and improves tolerance to cold stress through combined physical and physiological mechanisms.

Biomolecules doi: 10.3390/biom16060895

Authors: Carolina Hogerty Yantao Zhao Weiran Wang Steven A. Weinman Wei Zhong

Peroxisomes are essential metabolic organelles that support core aspects of cellular homeostasis. In the hepatocytes, peroxisomes govern key aspects of cellular homeostasis, including processing lipid substrates that are inadequately handled by mitochondria, controlling hydrogen peroxide metabolism, and regulating bile acid synthesis. Increasing evidence indicates that these organelles are not merely auxiliary metabolic compartments but active contributors to the development and progression of liver disease. Dynamic alterations in peroxisomal proteins and function are being noted. Across metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, cholestatic disorders, fibrosis, and hepatocellular carcinoma, peroxisomes undergo remodeling that shows a change from adaptive reactions to maladaptive states. These changes perturb signaling pathways that regulate inflammation, stress responses, and cell fate. In addition, because peroxisomes operate within an interconnected organelle network, their dysfunction propagates to mitochondria, endoplasmic reticulum, and other cellular systems, amplifying metabolic and cellular stress. This review summarizes current understanding of how peroxisomal pathways contribute to liver disease, highlighting mechanisms involving lipid accumulation, oxidative stress, and disrupted organelle crosstalk. How peroxisome-dependent control of circulating metabolites links hepatic injury to extrahepatic organ systems is further discussed. At the end, emerging therapeutic strategies for liver disease targeting peroxisomal pathways are discussed. Together, the emerging understanding of peroxisomal remodeling, metabolic regulation, organelle crosstalk, and inter-organ communication positions peroxisomes as active and dynamic regulators of liver disease and potential targets for therapeutic intervention.

Biomolecules doi: 10.3390/biom16060894

Authors: Takayuki Miyazawa

Essential host functions are often maintained by conserved molecular systems, but in biological contexts shaped by evolutionary conflict, the genes that execute such functions may be unstable, replaceable, or repeatedly recruited from different evolutionary sources. Mammalian placentation provides a striking example of this principle. Trophoblast cell fusion is essential for placental development, yet this function is mediated in different mammalian lineages by distinct endogenous retrovirus-derived envelope proteins, including syncytin-1, syncytin-2, and other lineage-specific Env-derived fusogens. Here, I propose the Baton Pass model as a conceptual framework for explaining how host-level biological functions can be maintained despite turnover of the molecular agents that execute them. This model differs from conventional examples of antagonistic coevolution, which often emphasize recurrent mutations within the same interacting genes, and from non-orthologous gene displacement, which generally concerns replacement among cellular genes. In the syncytin paradigm, the molecular executors are repeatedly supplied by exogenous retroviral env genes that become endogenized, domesticated, and incorporated into host developmental programs. I further discuss how receptor compatibility, placental expression control, and host–virus evolutionary conflict may together destabilize individual Env–receptor systems while allowing the host-level function of trophoblast fusion to persist. Analogous functional reassignment is also observed in primate lentiviruses, where antagonism of BST-2 shifts among distinct viral genes. The Baton Pass model therefore describes a testable evolutionary principle: essential host functions can be preserved not only through conservation of specific genes, but also through dynamic succession of genes of distinct evolutionary origins.

Biomolecules doi: 10.3390/biom16060891

Authors: Innokentii E. Vishnyakov Alexey D. Vedyaykin

Small heat shock proteins (sHSPs) serve as “first aid” stress-response proteins in both eukaryotes and prokaryotes. Their holdase activity enables binding to partially denatured proteins, maintaining them in a folding-competent state under stress. The sHSP IbpA from the mycoplasma Acholeplasma laidlawii is a unique member of its family, combining the functions of two Escherichia coli sHSPs that typically act in tandem. In this study, we demonstrate for the first time that IbpA forms distinct supramolecular structures under contrasting temperature stresses in crowded environments without any artificial truncations or mutations at the protein termini. Upon cooling, IbpA in vitro forms long fibril bundles, whereas heating induces the formation of large, rounded agglomerates. At the temperature optimal for culture growth, the protein exists as a mixture of short fibrils and small globules, with the latter predominating. IbpA’s cellular localization mirrors in vitro properties, with an increased proportion of surface-associated proteins among the sHSP partners during cold shock. We also report, for the first time, a rapid and reversible transition of IbpA to a fibrillar form in response to cold. We propose hypotheses regarding potential roles of IbpA in the mycoplasma cell. IbpA from A. laidlawii appears to act as a “molecular equilibrist,” protecting the cell against damage under opposing stresses, though the precise mechanism of its action during cold shock remains to be elucidated.

Biomolecules doi: 10.3390/biom16060893

Authors: Bing-Chang Lee Juey-Jen Hwang Huai-Jen Tsai

Amyotrophic lateral sclerosis (ALS) remains an intractable motor neuron (MN) disease with a growing patient population and few effective treatments. Here, we review how extracellular phosphoglycerate kinase 1 (ePgk1) improves neurite outgrowth of MNs (NOMN) and axonal growth, both in vitro and in vivo. Our group first elucidated a novel non-canonical function of ePgk1 as a cross-tissue mediator between nerve and muscle tissues. We then discovered that neural membranous Enolase 2 (Eno2) serves as a receptor of ligand ePgk1 and that ePgk1-Eno2 interaction suppresses the Rac1-GTP/p-Pak1-T423/p-P38-T180/pMK2-T334/p-Limk1-S323 axis, reducing p-Cofilin and promoting NOMN and axonal growth, finally suggesting that the 419th aspartic acid residue of Eno2 mediates this interaction. In a crucial preclinical step, we truncated two short 16-amino-acid derivatives from Pgk1, FD-1/-2, each mediating neuroprotection comparable to that of full-length 417-amino-acid Pgk1 in ALS animal models, in terms of improvements of innervated neuromuscular junction, MN cell bodies, motor performance, and endpoint prolongation. In this context, we also discuss the opposite function driven by Eno1-plasminogen interaction and by Eno2-ePgk1 interaction; the latter results in unfavorable for tumorigenesis. Unlike intracellular Pgk1 roles, ePgk1 is an extracellular factor with anti-angiogenic properties, further positioning ePgk1 and its FD-1/-2 as promising protein/peptide drugs for ALS treatment.

Biomolecules doi: 10.3390/biom16060892

Authors: Ben Li Hamzah Khan Farah Shaikh Abdelrahman Zamzam Ravel Raphael Muzammil H. Syed Rawand Abdin Mohammad Qadura

Background: While trefoil factor 3 (TFF3) has been linked to cardiovascular disease, its role in peripheral artery disease (PAD) remains largely unexplored. In this prospective study, we assessed three pre-selected circulating biomarkers and found that TFF3 demonstrated the strongest association with the presence of PAD. Building on this finding, we integrated plasma TFF3 concentrations with clinical characteristics to construct predictive models aimed at identifying individuals with PAD and estimating their risk of major adverse limb events (MALE) over a two-year follow-up period. Methods: A total of 476 individuals were prospectively recruited, including 312 patients with PAD and 164 controls without PAD. At study entry, circulating concentrations of TFF3, oncostatin M (OSM), and brain-derived neurotrophic factor (BDNF) were quantified, and all participants were subsequently monitored for a two-year period. The primary endpoint was the occurrence of MALE within two years, comprising acute limb ischemia, major amputation, or lower extremity revascularization by either open surgical or endovascular approaches. PAD diagnosis served as the secondary outcome and was established by an ankle–brachial index (ABI) ≤ 0.9 or toe–brachial index (TBI) ≤ 0.67 in the presence of reduced or absent pedal pulses. For predictive model development, the cohort was randomly divided into training (70%) and testing (30%) sets. A random forest algorithm incorporating clinical variables and plasma TFF3 levels was developed and optimized using 10-fold cross-validation. Model discrimination was quantified using the area under the receiver operating characteristic curve (AUROC). For prognostic evaluation, patients were classified into low- and high-risk groups based on the optimal ROC-derived probability threshold of 0.60, and MALE-free survival between groups was assessed using Cox proportional hazards regression. Results: Among the three candidate biomarkers evaluated, only TFF3 demonstrated a significant association with PAD. Patients with PAD exhibited higher circulating TFF3 concentrations than those without PAD (7.27 ± 3.36 vs. 5.89 ± 2.67 pg/mL; p < 0.001), whereas OSM and BDNF showed no significant differences between groups. Over the two-year follow-up period, MALE occurred in 28 patients (9%). Predictive models combining plasma TFF3 measurements with clinical variables achieved strong performance for both PAD detection and 2-year MALE risk estimation, yielding AUROCs of 0.79 and 0.85, respectively. Furthermore, patients classified as high risk by the model experienced a significantly increased hazard of MALE during follow-up (HR 1.12, 95% CI 1.10–1.19; p = 0.003). Variable importance analysis revealed that TFF3 was the most influential predictor of MALE, followed by age and smoking history. Conclusions: Combining plasma TFF3 levels with readily available clinical characteristics enabled the development of a predictive model with good discriminatory ability for both PAD diagnosis and estimation of 2-year MALE risk. Such an approach may enhance risk stratification by identifying patients at elevated risk earlier in their disease course, thereby informing decisions related to vascular testing, referral for specialist evaluation, and implementation of targeted treatment strategies.

Biomolecules doi: 10.3390/biom16060890

Authors: Xiaoyu Sun Chunguang Cui Guangrong Huang Xiaoli Zou Shaofang Yu Xin Du Xia Xu Jine Chen Xingjian He Yongqiang Wang Linbao Zhu

C-type lectins (CTLs) are proteins with carbohydrate-recognition domains. These macromolecules interact with pathogen components, thereby playing important roles in the immune system. Current studies indicate that silkworm CTLs are involved in Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Nevertheless, the molecular mechanisms through which these CTLs affect viral infection remain unclear. In this study, B. mori C-type lectin 16 (BmCTL16) was identified in the silkworm. Its expression was significantly downregulated upon BmNPV infection. Functional assays showed that BmCTL16 overexpression suppressed BmNPV proliferation, whereas its knockdown enhanced BmNPV proliferation. Protein–protein interaction assays confirmed that BmCTL16 interacts with BmNPV protein Bm9 in the cytoplasm. Notably, BmCTL16 promoted the degradation of Bm9 via the ubiquitin–proteasome system. Knockdown of Bm9 by siRNA significantly reduced BmNPV proliferation, confirming that Bm9 is the key target for BmCTL16 to exert its antiviral function. Collectively, this study reveals a novel CTL-mediated antiviral mechanism. BmCTL16 interacts with Bm9 and promotes its ubiquitin–proteasome degradation, thereby inhibiting viral proliferation. Furthermore, BmNPV evades this host defense by downregulating BmCTL16 expression. These findings enhance our understanding of silkworm CTL-mediated antiviral defense and offer novel perspectives on host–virus interactions in B. mori.

Biomolecules doi: 10.3390/biom16060889

Authors: Ehab Marwan-Abdelbaset Xiaoyun Lu Dan Tan

This study presents a transformative “one-pot” biorefinery approach for the simultaneous production of hyaluronic acid (HA) and polyhydroxybutyrate (PHB) using an engineered, non-pathogenic Halomonas bluephagenesis TD01 chassis. By leveraging the principles of Next-Generation Industrial Biotechnology (NGIB), a one-step fermentation process was developed in nutrient-rich 40-LBG-Y medium, achieving a balanced metabolic flux that yielded 1.99 g/L and high-molecular-weight (HMw) HA (9.6 × 106 Da) as the highest HA-Mw reported by heterogeneous bacteria, alongside intracellular PHB (0.68 to 1.6 g/L). A bioactive HA-PHB nanoparticle scaffold was fabricated, exhibiting a highly porous, interconnected 3D sponge-like architecture with a significant particle size shift from 12 nm to 450 nm, confirming successful polymer complexation. Antimicrobial evaluations revealed that the scaffold exhibited preliminary antimicrobial potential against representative Gram-positive and Gram-negative strains against Staphylococcus aureus, Klebsiella variicola, and Candida albicans. Notably, while Pseudomonas aeruginosa metabolically exploited purified HA, the integrated scaffold reversed this effect, providing preliminary antimicrobial potential by sterically hindering bacterial hyaluronidases. Furthermore, Halomonas-derived HA consistently outperformed Moringa oil and complex emulsions in preliminary tests against a wide range of pathogenic microbes. These results demonstrate that this dual-product platform provides a sustainable, cost-effective source of high-performance functional materials for advanced antimicrobial coatings and clinical wound management.

Biomolecules doi: 10.3390/biom16060888

Authors: Wenjing Zhang Jinjie Liang Yiping Li Yong Yang Haiying Chen Liansheng Qiao Lingzhi Wang

Hypertension is one major risk factor of cardiovascular diseases, and RAS plays vital role during the development of hypertension. To obtain a novel antihypertensive peptide, Coix glutelin was hydrolyzed by trypsin and further separated by Sephadex G10. Based on 751 identified sequences, pharmacophore mapping, molecular docking, and in silico proteolysis were applied to screen and optimize the candidate sequence. Finally, a novel peptide, ENWAAL, was generated with IC50 of 210.57 μM, which acted with ACE in a competitively inhibitory pattern. The in vivo antihypertensive effect was evaluated in SHRs. Significant improvements were observed in hypertension-related characteristics, including blood pressure, cardiac structure and function, and serum angiotensin II (Ang II) level. In the brain, quantitative real-time PCR analysis revealed significant downregulation of angiotensin II type 1 receptor (AT1R) mRNA expression, concomitant with upregulation of angiotensin-converting enzyme 2 (ACE2) and MAS receptor. The protein expression of ACE and AT1R in the ENWAAL group also significantly decreased. This study can provide a candidate antihypertensive drug targeting RAS.

Biomolecules doi: 10.3390/biom16060887

Authors: Diana-Antonia Costea Valentina-Alexandra Badaluta Ioana Zachia-Zlatea Alina-Maria Holban Lia-Mara Ditu Veronica Lazar

Biofilms are structured communities of microorganisms embedded in a self-produced extracellular polymeric substance (EPS) matrix, whose development significantly enhances microbial resistance to antibiotics, disinfectants, and host immune defenses, posing major challenges in clinical, industrial, and environmental settings. Compared with planktonic cells, biofilm-associated microorganisms can exhibit up to 10- to 1000-fold increased tolerance to antimicrobial agents, contributing to the persistence of biofilm-associated infections (BAIs). These infections remain difficult to eradicate due to reduced penetration, altered metabolic states, and the presence of dormant or persister cells. Anti-biofilm strategies can be broadly classified into physical approaches (e.g., ultrasound, mechanical stress, and light-based approaches) that target biofilm structure; chemical and enzymatic methods (e.g., EPS-degrading enzymes) that destabilize the matrix; and biological and molecular strategies (e.g., quorum-sensing (QS) inhibitors, anti-virulence agents, bacteriophages, phage-derived antimicrobial molecules, antimicrobial peptides, and natural bioactive compounds) that modulate biofilm development and integrity by targeting regulatory pathways and matrix stability through distinct mechanisms of action. Natural compounds, including lactoferrin, lactoferrin-derived peptides, and probiotic and postbiotic fractions of lactic acid bacteria (LAB), as well as plant-derived metabolites, have shown promising anti-biofilm effects, with efficacy often enhanced through complementary or potentially synergistic interactions. However, despite these advancements, clinical translation remains limited. For example, BAIs account for approximately 80% of chronic infections, with high recurrence rates and therapeutic failure reported in device-associated infections and chronic wounds. These limitations highlight the need for clinically translatable, multimodal approaches that integrate structural biofilm disruption, antimicrobial targeting, and host response modulation to design more effective and sustainable anti-biofilm strategies.

Biomolecules doi: 10.3390/biom16060886

Authors: Eman Samy Shalaby Mohamed Azab El-Liethy Sherif Abd-Elmaksoud Corrado Tagliati Rawia Mohamed Khalil Said Ibrahim Shalaby

Effective hand washing takes time and hand sanitizers that contain alcohol have a number of drawbacks, and frequent use of alcohol may cause skin damage. The objective of this study is to formulate nanostructured lipid carrier systems containing chlorhexidine digluconate to be applied topically for hand hygiene, especially for people sensitive to alcohol. A cytotoxicity experiment was conducted to ascertain the safe dosage for each of the three nano-cream formulas (F1, F2 and F3). Following each treatment, the viral titer was assessed using tissue culture infectious dose50 and standard plaque assays. The selected formulation was characterized rheologically. Furthermore, fifteen volunteers of various ages and genders participated in the vivo antimicrobial test of the selected formulation as a hand sanitizer. All of the formulas were found to be safe. Using the disc diffusion method, the three formulations exhibited in vitro antimicrobial effects against different microbes. F1 showed biphasic release, reasonable skin deposition and spherical droplets under a microscope. F1 exhibited a non-Newtonian shear thinning flow behavior. After 30 min, the reduction values for rotavirus and Phix-174 were 21 and 4%, respectively. Additionally, the impact of F1 was assessed on the infectivity of simian rotavirus sa-11 (ds RNA) and Phix-174 (ss DNA) bacteriophage. According to the findings of the in vivo study, the percentage of total bacterial counts that were removed varied from 91 to 100%. Moreover, the range of the removal percentage of total fungi was 95.38 to 100%. In summary, F1 can be used as an economic, safe, and effective hand antiseptic. It can also completely replace alcohol in the market.

Biomolecules doi: 10.3390/biom16060885

Authors: Rohit Sai Reddy Konada James Osborn Sharmistha Mitra

Glycosylation, glycogen metabolism, and ubiquitination represent three fundamental cellular processes that are traditionally studied as distinct aspects of biology. Glycosylation and glycogen metabolism are unique carbohydrate-based pathways. The process of glycosylation generates structurally diverse glycans that regulate protein folding, cell signaling, and host–pathogen interactions, while glycogen serves as a glucose reserve essential for energy homeostasis. Emerging evidence reveals a deep mechanistic connection between these pathways, particularly in the context of brain biology and inherited metabolic diseases. Here, we present recent research linking glycosylation defects with glycogen metabolism, highlighting how changes in the shared metabolites and enzymatic pathways contribute to human health and disease. We then discuss the overlapping disease symptoms of congenital disorders of glycosylation and glycogen storage diseases, with particular emphasis on polyglucosan body-forming diseases. We also highlight the role of non-canonical ubiquitin ligase complexes such as laforin–malin and LUBAC and present emerging evidence for their potential role in the glycogen quality-control mechanism. Finally, we review current therapeutic strategies for CDGs and GSDs, including monosaccharide supplementation, glycogen synthase modulation, and gene therapy. Together, this review underscores glycogen as more than an energy store—as a key contributor to glycosylation homeostasis and cellular regulation in health and disease.

Biomolecules doi: 10.3390/biom16060884

Authors: Cristina Benavides Pepa Kecheva Fernando Solano Olga Martínez-Arroyo Juan V. Esplugues Ana Blas-García Nadezda Apostolova

Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) that occurs in most types of chronic liver diseases (CLDs) as a response to sustained liver injury. While the ECM comprises different proteins, collagen being the most abundant, the term matrisome refers to a plethora of ECM-related molecules, including collagen-associated proteins, growth factors, cytokines, enzymes and their endogenous inhibitors. The hepatic matrisome undergoes significant qualitative and quantitative changes during liver fibrosis. Despite intense research over recent years, our understanding of the matrisome in the liver—both in health and disease—and particularly of its function beyond its conventional structural role, remains poor. This review highlights how comprehending hepatic matrisome responses to liver injury can yield novel insights into disease progression and regression and could be exploited as a potential antifibrotic strategy. The antifibrotic potency of drugs that interfere with the matrisome at different levels has been demonstrated in preclinical studies, but translation to clinical trials remains still limited. So far, simtuzumab (LOXL2 inhibitor antibody), imatinib (small-molecule inhibitor against discoidin domain receptors—DDRs), bexotegrast (integrin inhibitor), GR-MD-02 (galectin 3 inhibitor), and BMS-986263 (siRNA-targeting HSP47) have been or are being evaluated in clinical trials related to CLD, and some of them have shown promising results.

Biomolecules doi: 10.3390/biom16060883

Authors: Onur Konukcu Mehmet Argun Semra Acer Ömer Çelik Özlem Tök Levent Tök Mustafa Nazıroğlu

Anti-vascular endothelial growth factor agents (anti-VEGFs) are the cornerstone of treatment for neovascular age-related macular degeneration (AMD). Resveratrol is a natural polyphenol with well-established antioxidant and anti-apoptotic properties. This study investigated whether resveratrol exerts cytoprotective effects when combined with anti-VEGFs on ARPE-19 cells in vitro. Cells were treated with ranibizumab (RNZ), aflibercept (AFL), or ziv-aflibercept (ZFL), either alone or in combination with resveratrol. Mitochondrial and cytosolic reactive oxygen species (MitROS and CytROS), mitochondrial membrane depolarization (MitDep), caspase-3, -8 and -9 activities, cell viability, apoptosis, and VEGF-A levels were evaluated using confocal microscopy, plate reader-based assays, and ELISA techniques. Anti-VEGFs induced tolerable oxidative or apoptotic stress in ARPE-19 cells but did not exhibit intrinsic antioxidant and cytoprotective effects. The addition of resveratrol significantly enhanced beneficial effects by reducing oxidative stress, preserving mitochondrial integrity, and suppressing intrinsic apoptotic signalling, while increasing cell viability. VEGF-A levels were effectively reduced by anti-VEGF treatment, and this suppression was further augmented by resveratrol without compromising cellular survival. These findings indicate that resveratrol acts as an additive modulator that strengthens the cellular effects of anti-VEGFs on ARPE-19 cells. The combination strategy may represent a supportive approach to optimize long-term anti-VEGF therapy in AMD.

Biomolecules doi: 10.3390/biom16060882

Authors: Kelsey Abernathy Sheng Wang Chiguang Feng Justin Mancini Guanghui Zong Nuria González-Montalbán Lai-Xi Wang Gerardo R. Vasta

Galectins are β-galactosyl-binding lectins with key roles in immune regulation and as pattern recognition receptors. To address their potential role(s) in viral infection of mucosal epithelia we currently investigate adhesion and entry mechanisms of the infectious hematopoietic necrosis virus (IHNV) using the zebrafish (Danio rerio) model system. We previously reported the recognition of IHNV envelope glycoprotein by the zebrafish galectin Drgal1-L2 and its inhibitory activity for viral adhesion to epithelial cells. Subsequently, we determined the structure of Drgal1-L2 and proposed a mechanism for Drgal1-mediated inhibition of IHNV spike fusion to the host epithelial cell. We now show that Drgal1 can also hinder viral adhesion and infection by binding to glycans on the host cell surface and epidermal mucus. We identified fibronectin, the reported IHNV receptor, as the cell surface glycoprotein recognized by Drgal1-L2. Surprisingly, IHNV also adhered in vitro to purified β1integrin, and pre-exposure of either IHNV or the immobilized β1integrin to Drgal1-L2 hindered IHNV adhesion. Binding of either anti-fibronectin or anti-β1integrin antibodies to the cell surface partially inhibited IHNV adherence. Drgal1-L2 also hindered IHNV adhesion by binding to mucus glycans. Taken together, our results suggest complementary mechanisms by which Drgal1-L2 may protect mucosal epithelial cells against IHNV infection and tentatively identify β1integrin as a novel receptor for IHNV.

Biomolecules doi: 10.3390/biom16060881

Authors: Menna Hamdy Dina M. Khodeer Mayada E. Elsakka Ali M. Alaseem Yasser M. Mostafa Afaf Alharthi Mohammad El-Nablaway Mohamed M. Tawfik

Diabetic neuropathy (DN) is a multifactorial complication of diabetes mellitus driven by chronic hyperglycemia, insulin resistance, and disturbed metabolic homeostasis, leading to progressive injury of both the peripheral and central nervous systems. This study investigated whether L-serine supplementation could attenuate DN through dose-dependent metabolic and neuroprotective mechanisms in a high-fat diet (HFD) plus streptozotocin (STZ)-induced diabetic rat model. Male Wistar rats (n = 8 per group) were allocated to five groups: normal control (NC), diabetic control (DC), pioglitazone (PIO; 1.5 mg/kg/day), low-dose L-serine (S1; 200 mg/kg/day), and high-dose L-serine (S2; 400 mg/kg/day). After 60 days of oral gavage, behavioural testing, glucose and insulin profiling, HOMA-IR calculation, brain histopathology, nerve growth factor (NGF) immunohistochemistry, and LC–MS/MS-based proteomic analysis of cerebral tissue were performed. Diabetic rats exhibited marked hyperglycaemia (355.33 ± 4.72 mg/dL), hyperinsulinaemia, severe insulin resistance (HOMA-IR 16.8 ± 3.2; a 14-fold increase), impaired thermal nociception, motor dysfunction, and pronounced neuronal degeneration. L-serine supplementation significantly improved metabolic status: S1 reduced HOMA-IR by 77.4% and S2 by 87.5% relative to diabetic controls (p < 0.001). High-dose L-serine produced greater improvements in thermal sensitivity, motor coordination (rotarod latency 26.67 ± 1.52 s vs. 16.1 ± 0.85 s in DC; p < 0.05), and NGF expression (8.6-fold increase vs. DC). Histopathology confirmed attenuation of neuronal injury and gliosis in both treatment groups. Exploratory, group-level proteomic profiling identified dose-specific molecular signatures: S1 was predominantly associated with carbohydrate, lipid, and biosynthetic pathways, whereas S2 was associated with synaptic, neurotransmission-related, and proteostasis pathways. Within the constraints of an exploratory design—group-level pooled proteomics, analysis of cerebral rather than peripheral-nerve tissue, and only two doses—these findings indicate that L-serine attenuates the metabolic and behavioural features of experimental diabetic neuropathy and generates the testable hypothesis of dose-dependent neuro-metabolic remodelling. The proteomic signatures are hypothesis-generating and require orthogonal validation before any mechanistic or translational inference can be drawn.

Biomolecules doi: 10.3390/biom16060880

Authors: Gong Chen Weiping Wei Dan Li Shanshan Han Michael Schäfer Xiaoyan Huang

Gastric adenocarcinoma (STAD) exhibits extensive intratumoral heterogeneity that contributes to tumor progression and therapeutic resistance. In this study, we integrated single-cell RNA sequencing and bulk transcriptomic analyses to characterize malignant epithelial subtypes in STAD. Among seven identified tumor subtypes, the OLFM4-associated C3 subtype exhibited enriched palmitoylation-related signatures and altered metabolic activities, particularly glycolysis-related pathways. Functional enrichment analyses further supported the enrichment of multiple energy metabolism pathways. To evaluate the association between OLFM4 and metabolic regulation, recombinant OLFM4 treatment and siRNA-mediated OLFM4 knockdown were performed in gastric cancer cell lines. OLFM4 upregulation increased the expression of ZDHHC2 and GLUT1, accompanied by enhanced glucose uptake and elevated ATP production, whereas OLFM4 silencing reduced ZDHHC2 and GLUT1 expression. In addition, a prognostic risk model derived from C3 subtype-associated genes (MUC16, RALA, and PCBD1) effectively stratified STAD patients and was associated with immune checkpoint expression and immune infiltration. Collectively, our findings identify an OLFM4-associated gastric cancer cell state with palmitoylation-related signatures and altered metabolic activities, highlighting its potential relevance to metabolic heterogeneity in gastric adenocarcinoma.

Biomolecules doi: 10.3390/biom16060879

Authors: Hao Zhou Wenjia Guo Liang He

Metastasis remains a major cause of cancer mortality, making reliable primary–metastatic state prediction from somatic genomic alterations clinically important yet technically difficult. We present TF-GateNet, a biologically constrained neural network that combines TF-aware feature integration based on TRRUST and DoRothEA TF–gene regulatory priors with sample-specific dynamic gating on a Reactome-defined hierarchical sparse backbone. The model was evaluated on multi-center prostate and breast-cancer cohorts using mutation and copy-number features across 10 repeated runs on a fixed 80/10/10 split, together with independent prostate external validation, and was compared with biologically informed neural-network baselines (P-NET, BKGNet-Pathway, and BKGNet-Protein), a dense feed-forward neural network (FNN), and conventional machine-learning baselines (LR, SVM, RF, DT, and XGBoost). On prostate, TF-GateNet achieved the best internal performance (AUROC 0.954 ± 0.005; AUPRC 0.925 ± 0.007) and the best combined external performance (AUROC 0.952 ± 0.009; AUPRC 0.898 ± 0.018). On breast, TF-GateNet achieved the strongest internal ranking performance, reaching AUROC 0.893 ± 0.004 and AUPRC 0.835 ± 0.006. Ablation analysis indicated that TF-aware integration accounted for the larger prostate gain, whereas within the TF-GateNet family on breast, both TF-aware integration and dynamic gating contributed positively. Interpretability analysis further supported a cross-level route from TF-related genomic perturbation cues to genes, pathways, and phenotype-associated predictions. These results position TF-GateNet as a biologically grounded and interpretable framework for primary–metastatic state prediction, with the strongest overall evidence in prostate cancer and favorable internal evidence in breast cancer.

Biomolecules doi: 10.3390/biom16060878

Authors: Rosanna Squitti Anastasia De Luca Altea Severino Gianluca Rizzo Luca Emanuele Amodio Federica Marzi Gabriella Vicano Mauro Cozzolino Angela Lombardi Mauro Rongioletti Vincenzo Tondolo

Copper (Cu)–zinc (Zn) imbalance has been implicated in colorectal cancer (CRC). Exchangeable copper (exCu), the labile circulating Cu fraction, may better reflect functionally relevant metal dysregulation than total Cu. We investigated sex-specific associations between systemic Cu–Zn indices, tumor burden, and epithelial–mesenchymal transition (EMT)-related tissue remodeling in CRC. We studied 152 CRC patients and 140 healthy controls. Serum Cu, Zn, and exCu were measured using validated analytical methods; circulating gonadotropins, sex steroids, and carcinoembryonic antigen were also assessed. EMT-related proteins (E-cadherin, vimentin, fibronectin, vinculin, MEMO1) were quantified by Western blot in paired tumor and adjacent mucosa. Analyses were sex-stratified and age-adjusted. CRC patients exhibited higher serum Cu and exCu and lower Zn than controls, resulting in a marked increase in the exCu:Zn ratio in both sexes. In patients, exCu:Zn was associated with tumor burden and pathological stage, with stronger associations with tumor size and pT stage in women and with metastatic status in men. Serum exCu:Zn was associated with tumor − normal differences in EMT-related proteins, particularly ΔE-cadherin, in both sexes. Systemic Cu–Zn disequilibrium, summarized by the exCu:Zn ratio, was associated with tumor burden and epithelial remodeling in CRC in a sex-specific manner, suggesting its potential as a biologically informative biomarker warranting further validation.

Biomolecules doi: 10.3390/biom16060877

Authors: Jiazhen Zou Huihui Gao Qingdan Gu Peng Zhang Heran Cao

Azoospermia represents the most severe manifestation of male infertility and is classified into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). NOA patients experience a lack of sperm due to testicular dysfunction, posing significant challenges in clinical diagnosis and treatment. Recent advancements in molecular biology and high-throughput technologies have led to the discovery and validation of numerous biomarkers, including proteins, non-coding RNAs, genetic polymorphisms, and imaging indicators, which have greatly enhanced the understanding of the pathophysiological mechanisms of azoospermia and facilitated non-invasive diagnostic approaches. This review aims to systematically summarize the pathogenesis of azoospermia and critically evaluate the latest advancements in diagnostic and prognostic biomarkers, including small RNAs, proteomic profiles, genetic markers, and imaging features. The overarching goal is to synthesize this knowledge toward the development of integrated, biomarker-guided strategies for precise diagnosis, prognosis prediction, and improved clinical management of azoospermia, particularly NOA.

Biomolecules doi: 10.3390/biom16060876

Authors: Zixuan Cao Yibin Jia Zhuoyuan Zhang Hanjiang Xue Hanwei Yu Xin Li Peng Luo

SYNGAP1-related intellectual disability presents a therapeutic paradox where genetic rescue is highly effective in neonates but limited in adults, suggesting that deficiency represents a developmental trajectory violation rather than a static biochemical defect. By synthesizing molecular, biophysical, and clinical evidence, this review proposes the “Synaptic Clock” framework, redefining SynGAP1 as a critical developmental regulator. We hypothesize that SynGAP1 operates through a strictly ordered temporal sequence: Phase I (Scaffold Assembly) utilizes the α1 isoform and phase separation to establish the structural postsynaptic density, while Phase II (Catalytic Refinement) involves isoform switching to enable activity-dependent plasticity and homeostatic scaling. This model characterizes synaptic maturation as a biophysical transition from a fluid scaffold to a consolidated gel, potentially marking the biological closure of structural rescue windows. Based on this hypothesized temporal mapping, we establish a phase-stratified therapeutic roadmap—transitioning from early-stage “reset” strategies like gene replacement to late-stage “refinement” and “compensation” via pharmacological and neuromodulatory interventions. Ultimately, validating phase-specific biomarkers, including gamma oscillations and isoform stoichiometry, is essential for shifting from generic interventions toward precision, phase-matched medicine for neurodevelopmental timing.

Biomolecules doi: 10.3390/biom16060875

Authors: Andrea Ižarik Verešpejová Marián Grendár Martin Kertys Natália Huňarová Li Sheng Chien Milan Grofik Michaela Škorvanová Jakub Šofranko Nela Žideková Egon Kurča Martin Kolísek

Bile acids (BA) are increasingly recognized as signaling molecules involved in metabolic regulation and inflammatory processes, both of which are relevant to Parkinson’s disease (PD). However, their role in PD and disease progression remains unclear. In this study, plasma BA profiles were analyzed in 113 participants, including early- and advanced-stage PD patients and age- and sex-matched controls, across three time points over three years. Targeted metabolomics using LC-MS was applied to quantify 20 BA, complemented by analyses of functional ratios, including unconjugated/conjugated and hydrophobic/hydrophilic BA ratios and correlation patterns between BA species. Although most individual BA did not show consistent longitudinal changes, pooled analysis identified significant differences in the unconjugated/conjugated BA ratio between PD patients and controls. In contrast, the hydrophobic/hydrophilic ratio did not differ significantly between groups. Correlation analysis revealed differences in selected BA interrelationships, particularly involving primary and secondary BA, while the overall network structure remained largely preserved. These results indicate that BA metabolism in PD might be characterized rather by subtle, distributed alterations than pronounced changes in individual metabolites. BA profiling may therefore contribute to a broader metabolic characterization of PD, but its utility as a standalone biomarker appears limited.

Biomolecules doi: 10.3390/biom16060874

Authors: Sukjoo Cho Katherine Shelmidine Jason T. Yustein

Osteosarcoma (OS) is the most common primary bone cancer in adolescents and young adults. Despite tremendous preclinical and clinical efforts to advance therapy for OS, the standard of care, consisting of surgical resection and pre- and postoperative chemotherapy, has remained unchanged for over 40 years. Growing molecular understanding of OS highlights tumor heterogeneity as a major obstacle to therapeutic advances. In this narrative review, we comprehensively discuss current evidence of OS heterogeneity and strategies to overcome the barrier. Evidence shows that OS heterogeneity is multifactorial: it retains complex and dynamic somatic genomics, including genomic instability, alterations in tumor suppressors, and amplification/overexpression of oncogenes such as MYC. The tumor is associated with various germline vulnerabilities. OS’s tumor microenvironment has intense cellular and spatial diversity, which significantly shapes its heterogeneity. The effects of lineage plasticity, as well as epigenetic and metabolomic mechanisms, on OS heterogeneity are under study. To overcome this extreme heterogeneity, the therapeutic strategies for OS must be comprehensive and diversified. While surgical resection remains a mainstay of treatment, efforts to identify actionable biomarkers that guide risk stratification and therapy are ongoing. Diverse preclinical models offer insights into OS biology and novel therapeutics. To enhance combinational therapy for OS, various agents, including multi-targeted receptor tyrosine kinase inhibitors, immunotherapies, and epigenetic and metabolic modifiers, are being investigated. Distinctive efforts are continuing to establish maintenance therapy for OS. In summary, elucidating the complex drivers of OS heterogeneity, together with the development of multifaceted strategies to address them, is critical to accelerating therapeutic progress in OS.

Biomolecules doi: 10.3390/biom16060873

Authors: Khrystyna Ryabenko Valérie Schini-Kerth Patrick Ohlmann Elena Galli

Hypertrophic cardiomyopathy (HCM) is the most common inherited myocardial disorder and a major cause of heart failure (HF) and sudden cardiac death. Although sarcomeric gene mutations initiate the disease, increasing evidence identifies oxidative stress, mitochondrial dysfunction, and maladaptive nutrient signaling as key drivers of disease progression. Enhanced reactive oxygen species (ROS) production in HCM promotes energetic impairment, calcium mishandling, fibrosis, and the activation of pro-hypertrophic pathways, while disrupting protein quality control and endothelial function. Despite recent therapeutic advances, effective disease-modifying strategies targeting these molecular mechanisms remain limited. Sodium–glucose cotransporter 2 inhibitors (SGLT2i), originally developed for type 2 diabetes, have demonstrated robust cardioprotective effects in HF independent of glycemic control. Beyond their renal actions, SGLT2i modulate myocardial metabolism, reduce oxidative stress, improve mitochondrial function, restore sodium and calcium homeostasis, and attenuate inflammation and maladaptive mTOR activation. Emerging preclinical and translational data suggest that these pleiotropic mechanisms may counteract key pathophysiological processes underlying HCM. This review summarizes the molecular interplay between oxidative stress and hypertrophic remodeling in HCM and explores the rationale for SGLT2 inhibition as a potential disease-modifying therapeutic strategy.

Biomolecules doi: 10.3390/biom16060872

Authors: Linzhu Fang Pengfei Zhi Jiao Liu Haoyu Li Xiaoyu Wang Cheng Chang

Cuticular wax mixtures are the major components of the lipophilic cuticle coating of plant aerial organs during primary growth and they protect plants from environmental stresses. Decoding cuticular wax biosynthesis in bread wheat (Triticum aestivum L.) could contribute to the genetic improvement of this agriculturally important crop. Herein, we revealed that the wheat MYB46-like transcription factor TaMYB46 positively regulates cuticular wax by activating transcription of the long-chain acyl-CoA synthetase 1 (TaLACS1) gene. Knockdown of the wheat TaMYB46 gene resulted in significantly reduced cuticular wax loads and increased permeability of the wheat leaf cuticle. Furthermore, wheat long-chain acyl-CoA synthetase TaLACS1 was identified as a core component of the cuticular lipid biosynthetic machinery. Knockdown of the TaLACS1 gene led to reduced cuticular wax accumulation and increased leaf cuticle permeability. Moreover, the transcription factor TaMYB46 was found to enrich at the TaLACS1 promoter regions and activate TaLACS1 gene transcription. These findings collectively support the conclusion that the transcription factor TaMYB46 stimulates cuticular wax biosynthesis, likely by activating TaLACS1 transcription.

Biomolecules doi: 10.3390/biom16060871

Authors: Wesam Abd El-Fattah Ahlem Guesmi Naoufel Ben Hamadi Khulud M. Alshehri Ehab Mohamed Abdella Rehab R. Mohamed Reda F. M. Elshaarawy Hani S. Hafez

Chronic depression is associated with oxidative stress, neuroinflammation, neurotransmitter imbalance, and Alzheimer’s-like changes. Current monoamine oxidase inhibitors have limited cognitive benefits and disease-modifying properties. A new nanotherapeutic, combining chitosan nanoparticles, propargylamino-1-(4-methylthiophenyl) propane (PAMTP), and L-tyrosine (En@PAMTP_Tyr), was developed. En@PAMTP_Tyr nanoparticles were ~140 nm in diameter, with a zeta potential of +27 mV and entrapment efficiencies of 73.45% for PAMTP and 90.85% for L-tyrosine. Drug release was pH-sensitive, favoring acidity. Intraperitoneal administration of En@PAMTP_Tyr reduced anhedonia, despair, cognitive deficits, and neuromuscular weakness, with efficacy matching or exceeding that of selegiline. In treated rats’ hippocampal tissue, En@PAMTP_Tyr increased superoxide dismutase and glutathione, normalized MAO and acetylcholinesterase activities, and corrected CUSD-induced TNF-α and IL-10 changes, showing antioxidant and anti-inflammatory effects. Histological analyses revealed that En@PAMTP_Tyr preserved CA1 pyramidal neurons, reduced β-amyloid levels, restored tau protein, and improved brain-derived neurotrophic factor levels, indicating reduced neurodegeneration. Molecular docking studies showed that PAMTP had high affinity for monoamine oxidase and acetylcholinesterase, supporting its role as an MAO-B inhibitor and cholinergic modulator. These findings suggest that En@PAMTP_Tyr is a promising nanoplatform for targeting MAO-B in depression, addressing mood, cognitive function, oxidative stress, inflammation, and Alzheimer-like pathology in the hippocampus.

Biomolecules doi: 10.3390/biom16060870

Authors: José Ángel Moreno-Cabezuelo Allanah Booth Da Lin Kiran Gathani David S. Kim Uma Shankar Sagaram

The fast-growing cyanobacterium Synechococcus sp. PCC 11901 is emerging as a promising chassis for photosynthetic biomanufacturing. Here we report recombinant protein production in PCC 11901 via signal peptide-mediated secretion, enabling direct recovery of target proteins from the culture medium without cell disruption. Seven signal peptides spanning both Sec and Tat pathways are screened using eYFP as a reporter, with secretion quantified daily over seven days by fluorescence measurements. FutA, belonging to the Tat pathway from Synechocystis sp. PCC 6803, achieves 92.2% extracellular export by day 7, substantially outperforming all Sec candidates, including the best Sec signal peptide thermitase from Cyanobacterium aponinum PCC 10605 (55.7%). Signal peptide-bearing strains exhibit growth reductions of up to 26% relative to the wild-type, with FutA most affected, indicating a general metabolic cost correlated with secretion efficiency. The best-performing signal peptides from both pathways, FutA and thermitase, are validated with secretion of lichenase. Notably, the rank order of signal peptide performance is reversed for lichenase: thermitase demonstrates 2.6-fold higher extracellular activity than FutA, indicating that optimal signal peptide selection is cargo-dependent. These results establish PCC 11901 as a secretion-competent chassis and provide a rational framework for matching signal peptide pathways to target protein properties.

Biomolecules doi: 10.3390/biom16060869

Authors: Marwa Shekfeh Mariam M. Konaté Hari Sankaran Ming-Chung Li Yingdong Zhao

Background: Age at diagnosis and histologic differentiation are clinically relevant in early gastric cancer (GC), as poorly differentiated tumors and those diagnosed in younger patients often demonstrate more aggressive characteristics. Serum microRNAs (miRNAs) may provide insights into the molecular basis of these features. Methods: We compared expression profiles between undifferentiated and differentiated early GC cases to identify differentially expressed miRNAs (DEmiRNAs) and associated enriched pathways. Using Lasso regression, we developed and cross-validated a histologic differentiation classifier based on miRNA profiles from 1399 early GC serum samples. Finally, cancer-specific miRNA differences between adolescent and young adult (AYA) and non-AYA patients were evaluated using samples from cancer cases and normal controls. Results: We identified 75 differentiation-associated DEmiRNAs targeting genes enriched in cancer hallmark pathways such as TP53 and PI3K/AKT/mTOR signaling. In the validation set, the combined Lasso model predicted differentiation status with a sensitivity of 69.2%, specificity of 75.3%, positive predictive value (PPV) of 66.9%, negative predictive value (NPV) of 77.2%, an overall accuracy of 73.1%, and an area under the curve (AUC) of 79.7%. Comparison of AYA and non-AYA groups identified 52 cancer-specific and age-related miRNAs. Notably, three components of a previously reported four-miRNA GC diagnostic signature were significantly associated with age. Conclusions: Age-related variation in miRNA expression suggests that patient age may influence the performance of the existing four-miRNA diagnostic signature in early GC. Overall, our findings demonstrate the utility of miRNA profiling for predicting differentiation status in early GC and reveal age-associated variation in cancer-specific miRNAs.

Biomolecules doi: 10.3390/biom16060868

Authors: Junpeng Yao Wen Wang Wei Zhang Hang Dong Yujun Hou Qianhua Zheng Ying Li Fang Zeng

Mitochondrial dysfunction in colonic smooth muscle cells (SMCs) is closely associated with impaired gut motility in functional constipation (FC), but the underlying molecular mechanisms remain incompletely understood. The mitochondrial unfolded protein response (UPRmt) is a critical pathway for maintaining mitochondrial proteostasis, and heat shock factor 1 (HSF1) acts as an important upstream regulator of this response. In the present study, we employed a loperamide-induced FC mouse model, combined with single-cell transcriptomic, molecular, and functional analyses to characterize the HSF1-UPRmt pathway in colonic SMCs and to investigate its role in FC. Single-cell transcriptomic analysis of colon tissue from FC mice revealed marked downregulation of UPRmt-associated genes in colonic SMCs. Immunofluorescence, Western blotting, and RT-qPCR analyses of colonic tissue confirmed that HSF1 expression was reduced in colonic SMCs, along with the downregulation of the UPRmt components, including HSP60, mtHSP70, and LONP1. These molecular changes were accompanied by mitochondrial structural damage, seen by transmission electron microscopy, and by functional impairments, including reduced mitochondrial membrane potential, elevated mtROS production, decreased ATP levels, and diminished activities of respiratory chain complexes I–V. AAV9-mediated overexpression of HSF1 reactivated the UPRmt pathway, improved mitochondrial function, and ameliorated constipation, whereas shRNA-mediated knockdown of HSF1 further suppressed UPRmt activity and aggravated mitochondrial damage, indicating that HSF1 bidirectionally regulates this pathway. Complementary experiments in primary colonic SMCs confirmed that this regulatory mechanism operates in a cell-autonomous manner, as modulation of HSF1 expression produced corresponding changes in the UPRmt pathway, in the expression of mitochondrial respiratory chain complex subunits (ATP5A, NDUFA9, COX1, SDHA, UQCRC1), and in ATP production, mirroring the in vivo findings. Collectively, these results demonstrate that HSF1 plays a pivotal role in maintaining mitochondrial homeostasis in colonic SMCs through regulation of the UPRmt pathway and that HSF1 dysfunction is closely associated with slowed gut motility in FC. These findings offer a new mechanistic perspective on FC and point to the HSF1–UPRmt axis as a potential therapeutic target.

Biomolecules doi: 10.3390/biom16060867

Authors: Evelina Charidemou Eleni Andreou Christos Papaneophytou

Mitochondrial reactive oxygen species (mtROS) are central regulators of cellular function, yet their biological roles are often reduced to an oxidative-stress/antioxidant dichotomy. This review reframes mtROS through the concept of mitohormesis, in which outcomes are neither inherently harmful nor beneficial but are determined by a defined set of contextual variables. We present a mechanistic framework in which mtROS effects depend on chemical species identity, sub-mitochondrial site of production, temporal dynamics, redox-buffering capacity, and metabolic state; together, these variables determine whether mtROS promote adaptive eustress or pathological distress. We then show that, across polyphenols, isothiocyanates, terpenoids, alkaloids, and quinones, the biologically relevant effects of natural redox-modulating compounds are mediated less by direct radical scavenging than by pro-hormetic mechanisms, including mild electron transport chain perturbation, nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (NRF2/KEAP1) activation, modulation of mitochondrial membrane potential, mitochondrial quality control, and NAD+/NADPH regulation. Applying this framework to disease reveals strong tissue and state dependence: neurodegeneration favors buffering expansion and mitophagy; metabolic disease may benefit from exercise-mimetic and NRF2-activating strategies; cardiovascular disease illustrates mitohormesis through ischemic preconditioning and CoQ10 supplementation; and cancer requires distinction between prevention and therapy because redox buffering can either protect normal tissue or support tumor survival. Finally, we argue that the failure of non-specific antioxidant supplementation is mechanistically predictable and propose context-aware, biomarker-guided, temporally optimized, and compartment-targeted redox interventions as a more rational translational path.

Biomolecules doi: 10.3390/biom16060866

Authors: Oluwatosin Daramola Judith Nwaiwu Odunayo Oluokun Mojibola Fowowe Alexandra Lux Isaac Lopez Andrew I. Bennett Yehia Mechref

Parkinson’s disease (PD) is a progressive neurodegenerative disorder traditionally defined by dopaminergic neuronal loss and Lewy body pathology; however, increasing evidence indicates that metabolic dysfunction contributes to both motor and non-motor manifestations of disease. While metabolomics studies in PD have largely focused on peripheral biofluids or subcortical brain regions, metabolic remodeling within cortical regions critical for cognition remains poorly characterized. Here, we applied LC-MS/MS-based untargeted metabolomics to post-mortem frontal cortex tissue from PD and neurologically normal control donors, with statistical models adjusted for age, sex, and post-mortem interval. A total of 893 metabolites were quantified, of which 234 exhibited significant differential abundance following false discovery rate correction. Pathway enrichment and network-based integration revealed coordinated metabolic remodeling characterized by predicted inhibition of β-alanine metabolism and pantothenate-dependent coenzyme A biosynthesis alongside activation of amino acid, vitamin B-dependent, cofactor-related, redox-associated, oxidative stress, and inflammatory pathways. Recurrent alterations in pantothenic acid, β-alanine-related intermediates, arginine- and histidine-derived metabolites, lumichrome, and vitamin B6-associated species may reflect cortical metabolic perturbations associated with mitochondrial bioenergetic vulnerability and oxidative stress. Together, these findings indicate selective metabolic vulnerability in the PD frontal cortex rather than diffuse metabolic collapse.

Biomolecules doi: 10.3390/biom16060865

Authors: Katalin Kuffa Tamás Langó András Czirók Júlia Tárnoki-Zách Szilvia Bősze Loretta László Virág Vas Zoltán Szabó Gábor E. Tusnády

Colorectal cancer (CRC) is the third most common malignancy worldwide. Detailed characterization of cell surface proteins (CSPs) is essential for the identification of prognostic biomarkers and the development of novel therapeutic strategies. Cancer progression and epithelial cell polarity influence the expression levels and subcellular localization of these proteins. However, quantitative information on the distribution of CSPs between the apical and basolateral membranes remains limited, particularly in CRC cells. Here, we developed a rapid, high-throughput method based on the enrichment of biotinylated peptides and proteins from the apical and basolateral surfaces of polarized CRC epithelial cells (HT29 and HCT116), followed by LC-MS/MS analysis. This approach enables the simultaneous identification of the side-specific distribution of ~1200 CSPs. In addition, almost 500 potential N-glycosylation sites with the canonical consensus sequence of these proteins were identified, which may serve as targets for future site-specific glycosylation analyses. To evaluate the sensitivity of the method, we altered the surface proteome by generating TKS4-knockout cells and identified several surface markers whose expression levels differed significantly from those of wild-type cells. Overall, our findings provide new insights into the role of CSPs in CRC cells and gene-edited models, particularly in the context of TKS4-dependent epithelial-to-mesenchymal transition (EMT)-like phenotypes that model cancer metastasis.

Biomolecules doi: 10.3390/biom16060864

Authors: Zainab R. Abdelrahman Amani A. Harb Shtaywy S. Abdalla

Astragalus membranaceus, a medicinal plant used in traditional Chinese medicine for centuries, has attracted growing scientific attention for its potential anti-aging and anticancer properties, particularly for skin and bone health. Its key bioactive compounds like astragalosides, cycloastragenol, and its commercial derivative TA-65, have been associated with telomerase activation and telomere maintenance, suggesting a possible role in modulating cellular senescence and tissue repair processes. In addition to the claimed telomere maintenance, A. membranaceus exhibits antioxidant, anti-inflammatory, and DNA-protective activities, properties that contribute to its anti-aging effects. Emerging evidence also suggests that telomerase modulation by A. membranaceus influences cancer cell dynamics, either suppressing tumor progression through immune regulation and apoptosis induction or, in some contexts, potentially promoting tumor growth. This duality highlights the importance of dose, formulation, and targeted application. Clinically, TA-65 has been reported to improve vascular health, bone mineral density, and skin elasticity in aging individuals. Preclinical studies further support its protective effects against osteoporotic bone loss and photoaging-induced dermal degeneration. This review summarizes the phytochemical composition of A. membranaceus and critically evaluates the mechanistic and therapeutic evidence underlying its anti-aging and anticancer actions on skin and bone tissues. It also discusses the pharmacokinetic properties of A. membranaceus, including its absorption, bioavailability, and safety profile. The integration of A. membranaceus into evidence-based senile therapeutic strategies holds promise, but further mechanistic and clinical studies are required to optimize its safety and efficacy.

Biomolecules doi: 10.3390/biom16060863

Authors: Sonia Terriaca Maria Giovanna Scioli Fabio Bertoldo Paolo Nardi Gian Paolo Novelli Beatrice Belmonte Tommaso D’Anna Carmela Rita Balistreri Calogera Pisano Amedeo Ferlosio Augusto D’Onofrio Augusto Orlandi

Background: Marfan syndrome (MFS) is a connective tissue disorder caused by FBN1 mutations, leading to elastic fiber disarray and early thoracic aortic aneurysm (TAA) formation. Currently, pharmacological treatments lack specificity and only delay progression. We previously reported a specific TGFβ-driven miR-632 upregulation in MFS TAA tissues and blood causing smooth muscle cell dedifferentiation and aortic wall degeneration. This study evaluated the effects of three conventional antihypertensive drugs (β-blocker, ACE inhibitor, and sartan) on parietal remodeling, comparing them with a miR-632 inhibitor in an ex vivo TGFβ1-induced model of MFS TAA. Methods and Results: Using an ex vivo paired experimental framework based on independent biological pools of human TAA tissue, gene expression and Western blot analyses demonstrated that only losartan significantly reduced miR-632 and vascular degeneration markers. Notably, combined treatment with ramipril and carvedilol compromised losartan’s efficacy, highlighting the need for careful therapeutic selection. In this ex vivo setting, miR-632 inhibition demonstrated a promising capacity to counteract aortic remodeling, serving as a mechanistic proof-of-concept that warrants further preclinical in vivo validation. Conclusions: Our data emphasize that choosing the right treatment in MFS aortopathy requires understanding its specific impact on cellular pathways. Our findings identify losartan as the most effective standard drug in this model, while suggesting miR-632 as a potential future target to stabilize the aortic wall and, prospectively, delay surgery.

Biomolecules doi: 10.3390/biom16060862

Authors: Kevin M. Truong-Balderas Rachel C. Chang Claudia Lasalle Yi Gao Nicole C. Nowak Kyle T. Amber Adrian P. Mansini

Melanoma treatment has been transformed by immune checkpoint blockade, yet many patients still experience primary resistance, limited durability of response, or acquired resistance. These limitations underscore the need for additional targets that reflect melanoma biology while enabling new therapeutic strategies, particularly in biologically defined settings of immune escape such as checkpoint-resistant, HLA-low, dedifferentiated, or stress-adapted melanoma. The B7-H6/NKp30 axis has gained attention as a link between tumor cell stress, immune recognition, and therapy-related adaptation. B7-H6 (NCR3LG1), an inducible ligand for NKp30, has been detected in melanoma cell lines and tumor specimens, and soluble B7-H6 has been identified in a subset of patients. Membrane-bound B7-H6 may support NK-cell activation, whereas ligand shedding and accumulation of soluble B7-H6 may reduce effective antitumor recognition and promote immune evasion. Emerging evidence further suggests that B7-H6 expression may be linked to tumor-intrinsic programs relevant to melanoma cell survival, migration, and adaptation to therapeutic stress. However, B7-H6 is not yet a validated predictive biomarker or an established therapeutic target in melanoma, and current evidence remains limited by small melanoma-specific datasets, incomplete information on spatial and temporal heterogeneity, and the absence of melanoma-focused clinical validation. In this review, we examine the role of the B7-H6/NKp30 axis in immune surveillance, tumor escape, biomarker development, and therapeutic targeting, and discuss its translational potential in melanoma as an emerging but incompletely validated pathway that warrants focused investigation in melanoma states where conventional immune control is limited.

Biomolecules doi: 10.3390/biom16060861

Authors: Jamila Aminu Amina Jega Yusuf Bor-Jang Hwang Sonia Kamran Nasiru Abdullahi Adamu Jibril Alhassan John Obadipe Valerie Odero-Marah Hajjagana Hamza Abdullahi Ibrahim Uba James Wachira Jiangnan Peng

Triple-negative breast cancer (TNBC) is an aggressive subtype lacking effective targeted therapies, highlighting the need for new anticancer agents. Natural products remain a valuable source of bioactive compounds with diverse mechanisms of action. In this study, a pyrone glucoside, 7-hydroxymaltol-3-O-β-D-glucoside, was isolated from the methanolic leaf extract of Maerua angolensis and evaluated for its anticancer activity against TNBC cells. Structural elucidation was achieved using NMR and LC–MS analyses. Both the crude extract and the isolated compound exhibited dose-dependent cytotoxicity against MDA-MB-468 cells, with IC50 values of 2.94 and 0.78 µg/mL, respectively, while showing reduced toxicity toward MCF10A normal cells. Mechanistic studies revealed induction of apoptosis, evidenced by activation of caspase-9 and caspase-7 and PARP cleavage. Confocal imaging further demonstrated lysosomal disruption and nuclear morphological alterations consistent with stress-associated cell death. Gene expression analysis indicated minimal involvement of the PI3K/AKT/mTOR pathway. Molecular docking showed favorable binding of the compound to AKT1, PARP-1, and caspase-7, suggesting a multi-target mode of action. ADMET analysis indicated low oral bioavailability but a favorable safety profile. These findings highlight the potential of this compound as a lead for TNBC therapy.

Biomolecules doi: 10.3390/biom16060860

Authors: Nikoleta Kircheva Vladislava Petkova Silvia Angelova Todor Dudev

Protein phosphatase PPM1A plays a critical role in cellular signaling by dephosphorylating key regulatory proteins. According to experimental data, the enzyme requires either Mn2+ or Mg2+ bound in the active center(s), hence its catalytic activity strongly depends on the chelated metal ions. In this study, the metal ion selectivity of PPM1A is investigated using DFT calculations on active site constructs of bi- and trinuclear metal centers and protein ligands from the first and second metal coordination shells. Binuclear Mn-Mn and trinuclear Mn-Mn-Mn sites show poor resistance to substitution by biogenic Fe2+ and Zn2+, with Gibbs energies of the Mn2+ → Fe2+/Zn2+ exchange being consistently negative in both the gas phase and condensed media. In contrast, Mg-Mg and Mg-Mg-Mg centers are substantially more robust, with a thermodynamically unfavorable Mg2+ → Fe2+/Zn2+ substitution—except in the case of the Mg-Mg-Zn complex. The primary factors governing this metal competition in the modeled structures are the nature of the competing cation and the solvation properties of its aqua complexes, while solvent exposure of the binding site and the number of metal cations in the catalytic center exert a comparatively minor effect. Overall, these findings demonstrate that Mg2+-loaded active sites offer considerably greater protection against biogenic metal displacement than their Mn2+ counterparts, thus shedding light on the metalloprotein stability and enzyme fidelity of PPM1A.

Biomolecules doi: 10.3390/biom16060858

Authors: Liujie Long Yi Yang Chufang Zheng Kang Kang

Cardiovascular remodeling, encompassing vascular remodeling, myocardial remodeling, and fibrosis-associated tissue remodeling, underlies atherosclerosis, pulmonary hypertension, myocardial infarction, myocardial fibrosis, and other cardiovascular diseases. Its regulation has traditionally been studied through transcriptional, inflammatory, metabolic, mechanical, and intercellular signaling mechanisms. Recent advances in epitranscriptomics have identified N6-methyladenosine (m6A) RNA methylation as an additional post-transcriptional layer that interacts with microRNA (miRNA) pathways during cardiovascular disease progression. This review summarizes current evidence for m6A-miRNA crosstalk in cardiovascular remodeling, focusing on epitranscriptomic checkpoints that regulate miRNA fate, feedback-like regulatory circuits involving miRNAs and the m6A machinery, and cell-type-specific programs across endothelial cells, vascular smooth muscle cells, fibroblasts, and cardiomyocytes. We further discuss emerging analytical technologies and translational implications of this regulatory axis. Future studies should clarify causal mechanisms, cell-type and disease-stage specificity, and translational feasibility. Together, this multilayered framework provides a systems-level perspective on how RNA regulatory networks may shape pathological remodeling in cardiovascular disease.

Biomolecules doi: 10.3390/biom16060859

Authors: Alessandro Ravoni Veronica Paparozzi Tiziana Guarnieri Cecilia Sanzini Luigi Manni Christine Nardini

The ability of cells to translate optical radiation into biochemical signals, i.e., optotransduction, plays an important role in the life sciences, including the development of emerging therapeutic strategies, with a relevant influence on inflammation. However, a systemic understanding of the molecular pathways underlying the transduction of these physical stimuli is still lacking. In this work, we present a molecular map of optotransduction reconstructed from the literature and provide its representation as pathway, using the standard Systems Biology Markup Language. This representation enables network-based analyses and allows us to explore the differential effect of stimuli wavelengths and, for the first time, the systematic overlap with other forms of physical transduction, namely mechanotransduction.

Biomolecules doi: 10.3390/biom16060857

Authors: Irina-Georgeta Sufaru Bogdan Constantin Vasiliu Monica Hancianu Stefan-Ioan Stratul Monica Silvia Tatarciuc Gianina Iovan Diana Tatarciuc Ioana Rudnic Diana Hanu Sorina Paduraru Sorina Mihaela Solomon

GLP-1 receptor agonists (GLP-1RAs) are widely used in the treatment of type 2 diabetes and obesity and are increasingly relevant in periodontal and implant practice. This review covers mechanisms, preclinical and early human evidence, and practical periodontal considerations; the structured database search is conducted in accordance with the Scale for the Assessment of Narrative Review Articles (SANRA) and the International Committee of Medical Journal Editors (ICMJE) principles. Two pathways explain GLP-1RAs’ relevance: indirect effects from better glycemic control, weight loss, and reduced inflammation; and direct tissue effects involving GLP-1R signaling and the GLP-1/dipeptidyl peptidase-4 (DPP-4) axis. Preclinical studies show reduced inflammation, osteoclast activity, and alveolar bone loss, along with improved periodontal stem cell function under hyperglycemia or inflammation via Nuclear Factor-kappaB (NF-kappaB), Wingless-related integration site (Wnt)/beta-catenin, and Mitogen-Activated Protein Kinase (MAPK) pathways. Animal studies on implants and local delivery, including exendin-4 platforms, suggest osteometabolic benefits. Human data are limited and mostly observational, and confounders include metabolic status, smoking, medication, and nutrition. Oral side effects such as xerostomia and dehydration are also noted. At present, GLP-1RA therapy should be regarded as a contextual modifier of periodontal risk and healing capacity rather than as a stand-alone periodontal therapy.

Biomolecules doi: 10.3390/biom16060856

Authors: Shinjini Basu Asitha Premaratne Scott Widmann Esther A. Olaleye Chin-Yo Lin

Endocrine therapy is an effective and common treatment strategy for estrogen receptor (ER)-positive breast cancers. However, the development of endocrine resistance, through genetic mutations and epigenetic alterations, in about 40% of treated patients remains a significant therapeutic challenge. Liver X receptors (LXRs) are nuclear receptors that regulate lipid metabolism and cholesterol homeostasis and have been implicated in metabolic reprogramming in breast cancers and other malignancies. We previously identified a novel LXR ligand GAC0001E5 (1E5), with potent antiproliferative activity across breast cancer subtypes. Here, we investigate its mechanisms of action in responsive (MCF-7) and endocrine-resistant (MCF-7-TamR) ER-positive breast cancer cells. Treatment with 1E5 resulted in the downregulation of LXR and its target genes, and significantly reduced ERα expression and the expression of ER-responsive genes. Aberrant expression of androgen receptor (AR) and human epidermal growth factor receptor 2 (HER2), both implicated in endocrine resistance, were downregulated following 1E5 treatment. siRNA-mediated knockdown of LXR expression only partially recapitulated the actions of 1E5, suggesting the involvement of LXR-dependent and independent mechanisms. Collectively, these findings reveal potential crosstalk between LXR and the genetic and epigenetic regulation of pathways involved in endocrine response and alternative signaling mechanisms, highlighting potential targets in endocrine-resistant breast cancer.

Biomolecules doi: 10.3390/biom16060855

Authors: José Joaquín Merino José Julio Rodríguez-Arellano Xavier Busquets Isabel Álvarez-Vicente María Eugenia Cabaña-Muñoz Ana Isabel Flores Adolfo Toledano Gasca

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder characterized by amyloid-β (Aβ) and the accumulation of tau in the brain, which triggers robust innate immune responses. Growing evidence indicates that neuroinflammation contributes to AD progression by overactivating microglia through the release of cytokines and chemokines. In general, chemokines can disrupt neuronal communication and promote blood–brain barrier permeability. Peripheral immune cells are mobilized into the brain by a gradient of chemokines. These processes link peripheral immune responses with substantial T-cell infiltration into the CNS parenchyma, leptomeninges and cerebrospinal fluid of both AD mice and AD patients. This finding underscores the relevance of the adaptive immune system, particularly T and B cells, in AD neuropathology. T-cell infiltration into the brain can influence amyloid clearance through chemokine signalling. However, chemokines play a critical role in AD by either promoting or suppressing disease progression. The infiltration of peripheral T and B cells into the brain parenchyma can exacerbate neuronal loss, yet it may also exert neuroprotective effects. Despite the presence of CD4+ and CD8+ T cells in postmortem brains of AD patients, debate continues about their role in AD brains, in terms of whether they are protective or detrimental. Understanding the complex role of chemokines in controlling innate and adaptive immune responses by modulating neuron–glia interactions (involving astrocytes and microglia) may provide novel therapeutic approaches for AD. Targeting chemokine signalling or treating with drugs that can prevent the recruitment of immune cells may be promising strategies for treating AD neuropathology. Therapies that prevent the overactivation of T cells in the brain could lead to protective strategies against AD. In fact, regulatory T cells (Tregs) could delay the onset of cognitive symptoms, because they suppress inflammation and slow the accumulation of Aβ plaques and p-Tau in the brain. Complementary strategies, such as photobiomodulation, nanoparticle, and T-cell-based approaches, could mitigate AD progression in patients.

Biomolecules doi: 10.3390/biom16060854

Authors: Shizhong Liu Xianghong Hou Daiqianhui Li Zhenli Guo Xin Zhou Yan Wang Ketao Ma Rui Yang Xinzhi Li

Carfilzomib (CFZ) is a proteasome inhibitor primarily used to treat relapsed and refractory multiple myeloma. However, its clinical application is limited by significant cardiotoxicity, the underlying mechanisms of which remain incompletely understood. In this study, we aimed to elucidate the pathogenic pathways involved. In vitro, CFZ induced mitochondrial dysfunction and apoptosis in AC16 cardiomyocytes in a concentration- and time-dependent manner. Transcriptomic analysis revealed enrichment in pathways related to autophagy and endoplasmic reticulum stress. Mechanistically, CFZ promoted autophagosome formation but downregulated the SNARE proteins STX17, SNAP29, and VAMP8, thereby impairing autophagosome–lysosome fusion and blocking autophagic flux. This disruption was associated with the activation of the cGAS-STING signaling pathway. In vivo, CFZ administration resulted in cardiac dysfunction and apoptosis in mice, both of which were attenuated by the STING inhibitor C-176. Consistently, STING knockdown restored autophagic flux and reduced cardiomyocyte injury in vitro. In conclusion, CFZ induces cardiotoxicity by activating the cGAS-STING pathway, which disrupts the autophagic clearance of damaged mitochondria and promotes cardiomyocyte apoptosis. Targeting STING may represent a promising therapeutic strategy to mitigate CFZ-induced cardiotoxicity.

Biomolecules doi: 10.3390/biom16060853

Authors: Xiaozhe Liu Likun Cheng Mingcheng Liu Mingzhu Zhou Bingze Jiao Xuehan Liu Jianhe Hu Yanwei Li Xiaojing Xia

Regulated cell death is essential for tissue homeostasis, immune defense, and disease progression, yet the lipid-based regulatory mechanisms that coordinate cell death signaling remain incompletely understood. Protein palmitoylation is a dynamic and reversible lipid post-translational modification that controls protein membrane association, trafficking, stability, and signaling complex assembly. This review summarizes the regulatory roles of palmitoylation and depalmitoylation in major forms of regulated cell death, including apoptosis, necroptosis, pyroptosis, ferroptosis, and autophagy-related cell death. Particular attention is given to representative palmitoylated substrates, including Fas cell surface death receptor (Fas), receptor-interacting protein kinase 1 (RIPK1), NLR family pyrin domain containing 3 (NLRP3), gasdermin D (GSDMD), glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), autophagy-related 16 like 1 (ATG16L1), and Beclin1. These substrates illustrate how palmitoylation links membrane organization, metabolic status, inflammatory signaling, and cell fate decisions. Disease-oriented evidence further indicates that dysregulated palmitoylation contributes to cancer, neurodegenerative diseases, and inflammatory or immune-related disorders by modulating cell death resistance, inflammatory amplification, immune evasion, or impaired proteostasis. Current challenges include limited quantitative information on palmitoylation dynamics, incomplete evidence for some enzyme–substrate relationships, and insufficient distinction between disease-driving and secondary palmitoylation events. Targeting zinc finger Asp-His-His-Cys (zDHHC) palmitoyl acyltransferases, depalmitoylating enzymes, or specific palmitoylated substrates may provide new therapeutic opportunities. Overall, this review positions protein palmitoylation as a dynamic molecular switch linking lipid metabolism, membrane signaling, regulated cell death, and disease remodeling.

Biomolecules doi: 10.3390/biom16060852

Authors: Yanqi Zhao Tutu Wang Xinuan Zhang Wange Wang Yu Fu Ismail Laher Shunchang Li

Exerkines are bioactive molecules released in response to physical exercise and are considered important mediators of systemic adaptations. While previous research has largely focused on the effects of exercise modalities, the role of exercise intensity in regulating exerkine responses remains unclear. This narrative review summarizes findings on the effects of different exercise intensities on nine circulating exerkines with sufficient available data in healthy populations, including irisin, follistatin-like 1, myostatin, fibroblast growth factor 21, follistatin, leptin, adiponectin, apelin and brain-derived neurotrophic factor, without systematically covering all known exercise-responsive molecules. Given the narrative design of this review, the findings should be interpreted as descriptive and hypothesis-generating rather than as definitive evidence of intensity-dependent effects. The included studies show that acute exercise is associated with changes in several exerkines, with some direct within-study comparisons reporting larger responses under higher-intensity exercise conditions, whereas others exhibit increases, decreases, or no measurable changes across intensities. In contrast, studies examining chronic exercise interventions report changes in some studies and no measurable differences in others. Overall, the current evidence in this review suggests that exercise intensity may influence exerkine responses under some conditions, particularly during acute exercise, although the available findings remain limited and inconsistent across studies.

Biomolecules doi: 10.3390/biom16060851

Authors: Muhammad Tufail Ryotaro Kawasumi Sangita Dattatray Shinde Sivapriya Kirubakaran Kouji Hirota

Targeting checkpoints is one of the most promising strategies in cancer chemotherapy. Leukemia, in particular, is expected to yield high therapeutic efficacy due to its high replication stress. However, the DNA damage response factors involved in the vulnerability to checkpoint inhibitors of these hematopoietic cancers remain elusive. In this study, we reveal common factors required for cellular resistance to ATR inhibition in hematopoietic cancer cells. We explored the DNA damage response pathways contributing to cellular tolerance to three types of ATR inhibitors using an isogenic DNA repair factor mutant collection derived from the chicken lymphoma cell line, DT40. We first demonstrated significant ATR inhibition activity of the recently developed Torin2 analogous compounds, SPK67 and SPK98, under stressed replication conditions. We then compared cellular sensitivity patterns of the known ATR inhibitor, VE-821, and the potential ATR inhibitors, SPK67 and SPK98, in 24 types of mutants deficient in genome maintenance systems and found that RAD17/−, FEN1−/−, and POLB−/− cells exhibited hypersensitivity to all these drugs. Consistently, these mutant cells exhibited increased chromosome instability upon treatment with VE-821, SPK67, and SPK98, resulting in apoptosis. These results suggest that Rad17, Fen1, and Polymerase β play roles in responding to DNA damage caused by these drugs. However, ATR inhibition did not result in cell-cycle arrest, Chk1 phosphorylation, or increased γH2AX levels. These results suggest that, although ATR inhibition causes DNA damage, impaired checkpoint function suppresses the appropriate activation of DNA damage signaling pathways, thereby leading to cell death. This study is the first to demonstrate the importance of Rad17, Fen1, and Polymerase β in cellular tolerance to ATR inhibition in hematopoietic cells.

Biomolecules doi: 10.3390/biom16060850

Authors: Stephen M. Cornish Jose Peralta-Huertas

Sarcopenia is defined as the age-related loss of skeletal muscle strength, power, and size. Understanding the fundamental mechanisms whereby sarcopenia occurs is an area of research that has received much attention due to the aging population. Skeletal muscle tissue is used for locomotion and acts as a major site aiding the regulation of metabolism. Myokines are cytokines released from skeletal muscle tissue that act in an autocrine, paracrine, or endocrine manner. Myokines have been termed the ‘exercise factor’ or ‘work factor’ that scientists have long thought communicate between skeletal muscle and various physiological systems, including muscle-to-muscle cross-talk. One area of research that has been underexplored is the effect that myokines may have in an autocrine manner on skeletal muscle tissue itself. Although the myokine role in skeletal muscle hypertrophy and atrophy has been somewhat elucidated in rodent models, relatively little research has been performed in human models to understand the role myokines have on anabolic and catabolic metabolism in an autocrine manner. This review will provide an overview of myokine function within a biological context, some molecular pathways involved in skeletal muscle anabolism, a mechanistic understanding of myokine autocrine action, key evidence in relation to skeletal muscle satellite cell interaction with myokines, how myokines may be involved in skeletal muscle regeneration, and an outline of some key myokines that have the potential to act in an anabolic fashion within skeletal muscle. The review will then emphasize some important areas of research that are needed to understand the role of myokines in maintaining or improving skeletal muscle mass within an aging context.

Biomolecules doi: 10.3390/biom16060849

Authors: Daniel P. Reis Beatriz Domingues Cátia Fidalgo Rui L. Reis Luca Gasperini Alexandra P. Marques

In the original publication [...]

Biomolecules doi: 10.3390/biom16060848

Authors: Udayakumar Karunakaran Suma Elumalai

Reductive stress, characterized by excessive reducing equivalents such as NADH, NADPH, and reduced glutathione (GSH), is increasingly recognized as a pathophysiological counterpart to oxidative stress. Chronic hyperinsulinemia and insulin resistance promote this over-reduced state by increasing glucose flux, pentose phosphate pathway activity and de novo lipogenesis, thereby elevating NADPH pools and reshaping cellular lipid composition. While reducing equivalents are essential for biosynthesis and antioxidant defense, persistent over-reduction disrupts redox balance, mitochondrial function and metabolic flexibility. Paradoxically, this reductive metabolic environment may increase susceptibility to ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation and failure of glutathione peroxidase 4 (GPX4). Here, we define how reductive stress becomes deregulated in the context of insulin signaling and insulin resistance, and assess whether antioxidant interventions can mitigate ferroptosis, providing a framework for therapeutic strategies to restore redox balance in metabolic disease.

Biomolecules doi: 10.3390/biom16060847

Authors: Lucia De Salvo Cinzia Franchin

In recent years, rapid technological progress in high-throughput sequencing, advanced mass spectrometry, and quantitative imaging has profoundly reshaped biomedical and clinical research [...]

Biomolecules doi: 10.3390/biom16060846

Authors: Ehab Marwan-Abdelbaset Xiaoyun Lu Dan Tan

This study evaluates the development of Halomonas bluephagenesis TD01 as a novel, sustainable microbial platform for the production of hyaluronic acid (HA). Three distinct hyaluronan synthase genes (sezHasA and spHasA "Class I" from the Streptococcal group and pmHasA "class II") were heterologously expressed and compared, with the Class II synthase from Pasteurella multocida (pmHasA) emerging as the superior variant in rich media 60-LBG, achieving significantly higher titers of 0.88 g/L and molecular weight (Mw) of 1.15 MDa (Mega Daltons). Using a combination of Plackett–Burman design and Response Surface Methodology (RSM), the fermentation process was optimized, identifying initial pH, nitrogen source, and NaCl concentration as critical factors. These optimizations led to a maximum HA yield from 0.88 to 2.38 g/L (265% improvement) and Mw from 1.15 to 9.67 MDa. Furthermore, the study demonstrates precise tuning of HA molecular weight, ranging from 2.04 MDa to 9.67 MDa in a modified medium (40LBG-Y), by modulating L-arabinose induction levels. The structural integrity of the purified HA was confirmed via ESI-MS and 1H-NMR. These findings establish H. bluephagenesis TD01 as a robust Next-Generation Industrial Biotechnology (NGIB) chassis for the scalable and customizable production of HA with a minimal cost and high-molecular-weight HA for medical applications.

Biomolecules doi: 10.3390/biom16060845

Authors: Maria Gabriela Álvarez-Rodríguez Sonia Vega Felipe Hornos Adrian Velazquez-Campoy Bruno Rizzuti José L. Neira

Protein kinases have key roles in cells as they regulate diverse signal transduction pathways. Mitogen-activated protein kinase (MAPK) signaling route modulates several processes, such as cell proliferation, cell programming, metabolic changes and stress responses. Within the group of proteins participating in this pathway, the MAPK kinase (MEK1) is a dimeric, 393-residue-long, dual-specificity protein kinase that phosphorylates both tyrosine and threonine residues. In this study, we explored the conformational changes occurring during the unfolding of MEK1, by using orthogonal biophysical techniques. Intrinsic fluorescence, extrinsic 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, dynamic light scattering (DLS), and far-ultraviolet (UV) circular dichroism (CD) showed that the protein acquired a native-like conformation within a narrow pH range (8.0 to 9.0). Urea and guanidinium hydrochloride (GdmCl) denaturations followed by intrinsic and ANS fluorescence and far-UV CD, at pH 8.1, where the protein acquired a native-like conformation, showed that: (i) the apparent conformational stability of isolated MEK1 was low; and (ii) the unfolding occurred through the presence of intermediates. The presence of several unfolding intermediates was also evidenced through: (i) differential scanning calorimetry (DSC) in the absence of the ligand ATP; and (ii) unfolding simulations with the help of computational techniques based on constraint network analysis (CNA). We propose that the apparent low stability of this protein was related to its flexibility and modulates its ability to interact with diverse molecular partners.

Biomolecules doi: 10.3390/biom16060844

Authors: Yuting Wang Dianpeng Wang Xinyue Ma Yuqing Cui Jing Liu Wenyuan Fang

Biomedical materials, which are engineered to interact safely and effectively with biological systems, serve as the foundation of modern medicine. They facilitate precise diagnostics, targeted therapies, tissue regeneration, and the functional restoration of damaged organs and tissues. Propelled by advancements in materials science, nanotechnology, and clinical understanding, this field is rapidly evolving from passive implants to intelligent, responsive, and bioactive systems. This review summarize recent breakthroughs in four crucial domains: hard-tissue repair, dynamic wound healing, spatiotemporally controlled drug delivery, and advanced surface engineering. This article rigorously assesses the persistent translational barriers, particularly the disparity between in vitro biocompatibility assays and clinical performance, the scalability constraints in manufacturing, and the fragmentation in regulatory frameworks and international standards. By connecting fundamental innovation with real-world clinical needs, this review functions as both a strategic reference for researchers and a practical resource for clinicians exploring the next generation of biomedical materials.

Biomolecules doi: 10.3390/biom16060843

Authors: Seo Hyeong Kim Ji Hye Kim Ji Min Shin Yoon Mi Choi Da Som Kim Su Min Seo Eun Young Jang Sung Jae Lee Jin-Muk Lim Minsoo Han Do Hyeon Jeong Kwang Hoon Lee

Sensitive skin is characterized by hypersensitivity to normal stimuli, and objective diagnostic tools and treatments are still limited. Currently, cosmetics for sensitive skin are developed through the exclusion of known irritants rather than investigation into the underlying mechanisms of sensitivity. In this study, we developed an integrated pipeline combining transcriptome analysis via microneedle-based skin sampling (MISSM), bioinformatics, in vitro validation, and clinical assessment to identify sensitive skin-associated inflammatory biomarkers and cosmetic ingredients that regulate them. Candidate biomarkers and matched cosmetic ingredients were identified from transcriptomic data and validated in lactic acid-stimulated HaCaT and human dermal fibroblasts via qRT-PCR. A prototype emulsion was developed and evaluated in a 4-week open-label pilot clinical trial with longitudinal molecular monitoring via MISSM. After lactic acid stimulation, sensitive skin-associated biomarkers (MCOLN1, CYR61, PMAIP1, PTGS2, and HMGB2) were significantly upregulated in both cell types, and cosmetic ingredients that regulate these biomarkers were confirmed in vitro. The emulsion prototype demonstrated hypoallergenicity in a primary irritation test. In the pilot clinical trial, target biomarker expression was significantly reduced in MISSM-derived samples, with improvements in skin hydration, barrier function, redness, and sensory reactivity also observed. This integrated pipeline will enable the discovery of inflammatory biomarker-regulating cosmetic ingredients, with potential applicability to various inflammatory skin conditions.

Biomolecules doi: 10.3390/biom16060842

Authors: Giampaolo Morciano Ruggiero Gorgoglione Vito Porcelli Amer Ahmed Pasquale Scarcia Angelo Vozza Francesco Massimo Lasorsa Giuseppe Fiermonte Luigi Palmieri

Neurodegenerative diseases are increasingly recognized as disorders of due to disrupted cellular homeostasis, with mitochondrial dysfunction playing a central and early role in disease progression. This review explores the intricate relationship between mitochondrial function and neuronal health, emphasizing the pivotal role of the solute carrier family 25 (SLC25) transporters in maintaining mitochondrial homeostasis. We provide a comprehensive overview of mitochondrial biology in the central nervous system, including energy metabolism, calcium signaling, redox regulation, organelle interactions and mitochondrial dynamics. We delve into the SLC25 transporter family, highlighting their transport mechanisms, substrates and roles in brain metabolism and neuroprotection. SLC25 on one hand and proteins involved in the regulation of mitochondrial morphology and calcium signaling on the other hand are two sides of the same coin influencing each other. A critical analysis follows, examining how mitochondrial dysfunction contributes to mitochondrial abnormalities in a spectrum of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, ALS and rare mitochondrial encephalopathies. Finally, we assess emerging therapeutic strategies targeting mitochondrial pathways and SLC25 function, including metabolic modulation, gene therapies, antioxidants and pharmacological agents. This review underscores mitochondria and the SLC25 transporters as promising targets for disease-modifying interventions in neurodegeneration and raises key questions about the causality between mitochondrial failure and neuronal death.

Biomolecules doi: 10.3390/biom16060841

Authors: Meike Meinzer Markus Bassler Franziska Kessemeier Corinna Webering Detlef Neumann Heike Bähre Ralf Lichtinghagen Mathias Rhein Johannes Achenbach Christoph Gutenbrunner Matthias Karst

Background: Chronic pain is a multifactorial condition for which interdisciplinary multimodal rehabilitation is guideline-recommended, yet the biological mechanisms underlying treatment response remain incompletely understood and validated predictive biomarkers have not been established. Objective: This exploratory prospective cohort study examined clinical outcomes and circulating biomarker changes, encompassing the endocannabinoid system (ECS), inflammatory mediators, stress-regulatory markers, and metabolic parameters, in 410 patients with chronic pain of predominantly musculoskeletal etiology undergoing a standardized five-week rehabilitation program. Materials and Methods: Pain intensity and affective pain were assessed at baseline and end of rehabilitation; global performance of treatment (GPT) was additionally recorded. Serum analyses included anandamide (AEA), 2-arachidonoylglycerol (2-AG), IL-6, cortisol, IGF-1, BDNF, and leptin. Biomarker-outcome associations were examined via multiple regression analyses adjusted for demographics and biological and clinical confounders. Results: Statistically significant reductions were observed in pain intensity (−0.785 points, NRS; p < 0.001) and affective pain (−0.750 points; p < 0.001). IL-6 was associated with pain outcomes across time points. Higher baseline 2-AG independently predicted lower end-of-rehabilitation pain intensity, affective pain, and more favorable GPT. Greater AEA reductions were associated with favorable GPT. Conclusions: Baseline 2-AG emerges as a candidate predictor of treatment response, with lower pre-treatment levels potentially reflecting reduced stress-adaptive capacity, supporting inclusion of ECS markers in future controlled biomarker studies.

Biomolecules doi: 10.3390/biom16060840

Authors: Béatrice Vibert Faustine Henot Oriane Frances Jérôme Boisbouvier

Monoclonal antibodies (mAbs) have been the subject of extensive study in recent years due to their recognition as highly promising therapeutic molecules offering high specificity and a low risk of side effects. Monitoring the structure of these molecules is crucial for developing new therapeutics, characterizing interactions with antigens or receptors, and explaining potential changes in activity between antibody production batches. However, commonly used biophysical approaches provide only low-spatial-resolution information, and conventional structural biology techniques such as crystallography and cryo-electron microscopy (cryo-EM) are difficult to apply to these highly dynamic proteins. Solution nuclear magnetic resonance (NMR) spectroscopy is the method of choice for structural studies of flexible proteins at atomic resolution; however, it has traditionally been limited to low-molecular-weight biological systems. In this review, we present recent advances in NMR spectroscopy and advanced isotopic labeling methods that have enabled the atomic-resolution study of both the crystallizable (Fc) and antigen-binding (Fab) fragments of antibodies. We show how NMR is becoming a powerful tool for investigating full-length mAbs at an atomic level, opening up new possibilities for the characterization and in-depth quality control of therapeutic antibodies in solution.

Biomolecules doi: 10.3390/biom16060839

Authors: Ren-Lei Ji Ya-Xiong Tao

Understanding and regulating fish growth is vital for the economic sustainability of aquaculture. The melanocortin-3 and -4 receptors (MC3R/MC4R, known as neural MCRs), integral components of the leptin–melanocortin circuit, play crucial roles in vertebrate energy homeostasis and growth. Abnormal neural MCR signaling contributes to human obesity. In teleosts, Mc4r was first comprehensively studied in goldfish in 2003. Since then, Mc4r has been characterized in various teleosts. Genetic and pharmacological reduction of neural Mcr signaling can increase feeding or growth in several fish models, although its aquaculture value must be evaluated using production endpoints such as feed conversion, body composition, reproduction, welfare, and biosafety. Furthermore, neural Mcrs also play a role in modulating reproductive processes and sexual function in teleosts. This review systematically examines recent progress on the roles of fish neural Mcrs, offering an overview of basic molecular characteristics, tissue distribution, and pharmacology. Physiological roles and mechanisms in growth regulation are reviewed. Finally, the potential and limitations of targeting neural Mcrs for aquaculture-relevant traits are discussed. This work contributes to our understanding of the evolution of energy homeostasis regulation in vertebrates, providing a foundation for healthier and more efficient aquaculture practices.

Biomolecules doi: 10.3390/biom16060838

Authors: Catherine C. Y. Chang Toyoshi Fujimoto Yoshio Yamauchi Yasuomi Urano Ta Yuan Chang

Caveolin-1 is a scaffolding protein of caveolae, flask-shaped membrane microdomains involved in diverse cellular processes. Caveolae are primarily localized to the plasma membrane, the trans-Golgi network, and mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs). Most enzymes involved in cholesterol biosynthesis reside in the ER, and although caveolin-1 avidly binds cholesterol, its role in cholesterol trafficking remains unclear. Acyl-coenzyme A:cholesterol acyltransferases (ACAT1 and ACAT2) convert free cholesterol into cholesteryl esters for storage, with ACAT1 serving as the predominant isoenzyme in most cell types. ACAT1 is an ER-resident protein, with a fraction associated with specialized ER subdomains, including the MAM. Here, we report that a subset of caveolin-1 molecules appears to be associated with a fraction of ACAT1 in ER subdomains. Using immunoprecipitation under detergent conditions, immunoadsorption of MAM-enriched membranes under detergent-free conditions, and electron microscopy, we provide evidence consistent with an association between a subset of caveolin-1 molecules and ACAT1. Functionally, in mouse embryonic fibroblasts, we show that genetic ablation of caveolin-1 significantly increases the esterification of low-density lipoprotein-derived cholesterol, suggesting that caveolin-1 may attenuate ACAT1 activity. Collectively, these findings indicate that caveolin-1 may modulate cholesterol esterification and contribute to the regulation of cholesterol distribution among cellular membranes.

Biomolecules doi: 10.3390/biom16060837

Authors: Xiaoyun Pang Dong Zhang Hongwei Duan Zhenxing Yan Xianghong Du Lujie Zhao Jincheng Yang Li Xue Yanyan Wang Yuxuan He

Ovarian granulosa cells (GCs) ensure proper follicular development and oocyte maturation through gap-junction-mediated intercellular communication. Zearalenone (ZEA), a mycotoxin with estrogen-like activity, specifically targets and impairs ovarian function. Most existing studies have focused on ZEA-induced apoptosis in GCs, but whether ZEA disrupts gap junctions in ovarian GCs remains unclear. Therefore, the aim of this study was to investigate whether and how ZEA induces gap junction injury in ovine ovarian GCs, with a particular focus on the roles of G protein-coupled receptor 30 (GPR30), oxidative stress, and the NLRP3 inflammasome. In the present study, primary ovine ovarian GCs were isolated, cultured, and treated with different concentrations of ZEA to establish a gap junction injury model, and specific inhibitors/antagonists were used to investigate the underlying mechanisms. The results showed that ZEA decreased granulosa cell viability and significantly inhibited the expression of the gap junction proteins Connexin 43 (Cx43) and Connexin 37 (Cx37) in a concentration-dependent manner. ZEA treatment also significantly upregulated the expression of the NOD-like receptor familypyrindomain containing 3 (NLRP3) inflammasome-related proteins (NLRP3, ASC, Cleaved Caspase-1, and the downstream pro-inflammatory cytokine IL-1β) in a concentration-dependent manner. Pretreatment with the NLRP3-specific inhibitor MCC950 significantly reversed ZEA-induced downregulation of Cx43 and Cx37 and effectively blocked NLRP3 inflammasome activation, indicating that NLRP3 is a key target in ZEA-induced gap junction injury. Further experiments confirmed that ZEA treatment significantly increased oxidative stress levels in granulosa cells; pretreatment with the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) restored the ZEA-induced downregulation of Cx43 and Cx37 and suppressed NLRP3 inflammasome activation, suggesting that ROS acts as an upstream regulator of NLRP3 inflammasome activation. Moreover, ZEA treatment altered GPR30 expression levels, and pretreatment with the GPR30 antagonist G15 effectively inhibited ZEA-induced ROS production, NLRP3 inflammasome activation, and downregulation of Cx43/Cx37, indicating that ZEA exerts its effects through functional activation of GPR30. Collectively, ZEA activates the GPR30 receptor, induces ROS accumulation in granulosa cells, and subsequently triggers NLRP3 inflammasome activation, ultimately leading to downregulation of Cx43 and Cx37 and gap junction dysfunction. This study reveals a previously unrecognized molecular mechanism by which ZEA induces gap junction injury in ovarian GCs, providing potential therapeutic targets and a theoretical basis for preventing ZEA-induced ovarian dysfunction and improving animal reproductive health.