Biomolecules doi: 10.3390/biom16091309

Authors: Zhiyuan Chen Fan Yu Yanhua Qiu Jun Lin Meilian Yu Jinwei Yan Xianzhi Liu

The liver has a remarkable regenerative capacity, but persistent injury caused by drugs, metabolic dysfunction, alcohol, and chronic inflammation can overwhelm this process and promote fibrosis and hepatocellular carcinoma. Interleukin-11 (IL-11), a cytokine that signals through glycoprotein 130 (gp130), was historically considered hepatoprotective; however, accumulating experimental evidence predominantly identifies endogenous IL-11 as an amplifier of liver injury and maladaptive repair. In hepatocytes, IL-11 promotes oxidative stress, mitochondrial dysfunction, and cell death while limiting effective regeneration. In hepatic stellate cells, it sustains myofibroblastic activation and extracellular matrix production, while crosstalk with hepatocytes, macrophages, and the matrix reinforces a fibroinflammatory niche. In hepatocellular carcinoma, experimental and associative clinical evidence implicates IL-11 in tumor growth, invasion, metastatic colonization, postoperative recurrence, stromal remodeling, and immune suppression. These findings have supported the development of therapeutic strategies targeting the IL-11/IL-11RA axis, with the potential to provide broad therapeutic benefits across a wide spectrum of liver diseases. This review summarizes IL-11 regulation and signaling in liver disease; discusses its roles in liver injury, fibrosis, and hepatocellular carcinoma; and evaluates the opportunities and challenges of IL-11-targeted therapy.

Biomolecules doi: 10.3390/biom16091308

Authors: Dong-Zhi Wei Hui Wang Sen-Lin Peng Yu Huang Ganglong Yang Weijie Dong Xiao-Dong Gao Ning Wang

Congenital disorders of glycosylation (CDGs) are a growing group of inborn errors caused by gene defects in glycan biosynthesis pathways. Genetic testing of two patients from the same family harboring ALG1-related CDG (ALG1-CDG) revealed heterozygous variations in the c.1129 A>C and c.1263+3 A>T. Although most CDGs with abnormal N-glycosylation can be detected by transferrin screening, Western blotting of serum transferrin from the two siblings did not reveal significant glycosylation deficiency. In this study, the biochemical phenotype and pathological molecular basis of the CDGs in the siblings were further explored. Analysis of the patients’ ALG1 cDNA indicated that the associated heterozygous variants were c.1129 A>C (p.Met377Leu) and c.1188 T>A (p.Cys396X). Single-molecule real-time (SMRT) sequencing indicated that the variant frequencies of M377L and C396X were 50% and 36%, respectively. In addition, glycoproteomics analysis suggested that the glycoforms of transferrin were altered in the patients and that glycosylation site occupancy was decreased. It was also confirmed that N-glycosylation was altered in the patients’ serum transferrin. Conserved amino acid variants of yeast Alg1 were recombinantly overexpressed, purified and subjected to an in vitro quantitative activity assay. The results showed that these variants caused a decrease in enzymatic activity, suggesting pathogenicity.

Biomolecules doi: 10.3390/biom16091307

Authors: Xiao Dong Xiaoyang Wu Zeyu You Kexin Lu Huan Cai Bo Zhang Yuehua Gong Qinchuan Wang Yuan Yuan Huakang Tu

Mucins are a class of highly glycosylated macromolecules that constitute a cornerstone of the gastrointestinal mucosal barrier and play multiple essential roles in maintaining tissue homeostasis. During the progression from normal gastric mucosa through precancerous lesions to gastric cancer and metastasis, the expression profiles, glycosylation patterns, and spatial distribution of mucins undergo systematic and programmatic alterations, a process referred to as “mucin reprogramming.” Recent studies have revealed that this reprogramming is by no means a passive bystander phenomenon accompanying tumorigenesis; rather, it is a central biological event that actively drives malignant transformation, shapes an immunosuppressive microenvironment, and influences therapeutic response. This article aims to systematically delineate the dynamic landscape of mucin expression changes during gastric cancer progression, to provide an in-depth analysis of the underlying molecular mechanisms, and to focus on its clinical value and translational potential in the early diagnosis, molecular classification, prognostic assessment, and targeted therapy of gastric cancer. In addition, this review also discusses recent applications of artificial intelligence in the precise identification of mucin phenotypic features. By integrating the latest research advances, this review seeks to provide new perspectives and a theoretical basis for a deeper understanding of the mechanisms of mucin reprogramming during gastric cancer progression and for the development of novel diagnostic and therapeutic strategies.

Biomolecules doi: 10.3390/biom16091306

Authors: Gülfem Özduygu İhsan Topaloğlu Çağrı Atasoy Eren Erdoğdu Güntuğ Batıhan Semih Çiftçi Volkan Gelen Kübra Kaya Adem Kara Ali Yeşildağ Elif Erbaş Deniz Ekinci

Background: Hypoxia triggers pulmonary inflammation and oxidative stress, leading to endothelial dysfunction, apoptosis and lung injury. Although the flavonoid orientin exhibits potent antioxidant and anti-inflammatory properties, its pharmacokinetic behavior remains incompletely characterized. Nanoparticle-based delivery systems may enhance efficacy. This study investigated whether Orientin delivered via chitosan nanoparticles (CNP–orientin) protects against hypoxia-induced pulmonary inflammation, oxidative stress, and tissue injury in rats. Methods: A total of 48 female Sprague–Dawley rats were randomized into four normoxic and four hypoxic groups (n = 6 per group). Rats in the hypoxic groups were exposed to intermittent hypoxia (7% O2, 8 h/day for 7 days). Hypoxic rats were allocated to experimental groups, including an untreated hypoxia group and groups receiving orientin alone, chitosan nanoparticles alone or orientin-loaded chitosan nanoparticles (CNP–orientin), while normoxic rats served as controls. Hypoxic exposure was conducted using a controlled glove box system. At the end of the protocol, blood samples were collected for serum inflammatory marker analysis, and lung tissues were harvested to assess oxidative stress parameters, hypoxia- and inflammation-related signaling pathways, apoptosis-related gene expression, and histopathological lung injury and fibrosis. Results: Intermittent hypoxia significantly increased pulmonary HIF-1α and iNOS expression, oxidative stress markers (increased malondialdehyde and decreased superoxide dismutase and glutathione), pro-inflammatory cytokines (TNF-α, IL-1β), apoptotic signaling and histopathological lung injury compared with normoxic controls (p < 0.05). Treatment with orientin or chitosan nanoparticles alone attenuated hypoxia-associated biochemical and molecular alterations compared to untreated hypoxic rats (p < 0.05). Notably, CNP–orientin treatment significantly attenuated multiple oxidative stress, inflammatory, and apoptosis-related alterations compared with untreated hypoxic rats (p < 0.05). These protective effects were observed despite the substantially lower orientin-equivalent dose in the CNP–orientin formulation compared with free orientin. NF-κB and TNF-α expression, caspase-3 levels, antioxidant markers, and Bcl-2 expression differed significantly among hypoxic groups (p < 0.05). Histopathological lung injury and Modified Ashcroft fibrosis scores were significantly lower in the CNP–orientin group than in the untreated hypoxic group (p < 0.01 and p < 0.001, respectively). Conclusions: Orientin-loaded chitosan nanoparticles effectively mitigate hypoxia-induced pulmonary inflammation, oxidative stress, apoptosis and fibrotic injury. These findings identify CNP–orientin as a promising nanotherapeutic strategy for hypoxia-associated lung diseases and support further translational investigations.

Biomolecules doi: 10.3390/biom16091305

Authors: Marta Jaskulak Iwona Rybakowska Magdalena Gregorczyk Klaudia Antoniak-Pietrynczak Patrycja Jabłońska Katarzyna Zorena

Background/Objectives: Circulating amino acids are not only markers of nutritional status; in experimental and interventional models they have been linked to hepatic lipogenesis, lipoprotein assembly, mitochondrial fatty acid oxidation, and bile acid conjugation, which makes them plausible candidate correlates of obesity-related lipid dysregulation. Despite this, most metabolomic studies of excess adiposity have focused either on a single lipid parameter—typically triglycerides—or only on branched-chain amino acids (BCAAs). This pilot study was designed to generate hypotheses about these associations across the full standard lipid panel using a targeted 19-amino acid liquid chromatography–tandem mass spectrometry (LC-MS/MS) panel in adults spanning the body mass index (BMI) spectrum, with an analytical framework oriented around lipid phenotype variance rather than body weight classification. The design is cross-sectional and the analysis exploratory; no causal or predictive claim is made. Methods: Targeted LC-MS/MS quantification of 19 plasma amino acids was performed in 50 adults grouped as normal weight (n = 20; BMI 22 ± 1.5 kg/m2), overweight (n = 20; BMI 28 ± 1.5 kg/m2), or obese (n = 10; BMI 34 ± 2.0 kg/m2). The analytical framework included: (i) one-way analysis of variance (ANOVA) with Bonferroni correction; (ii) Pearson correlation analysis; (iii) principal component analysis (PCA) performed on the lipid profile itself with amino acid projection vectors; (iv) K-means clustering based on lipid phenotype (K = 3); (v) Ward-linkage hierarchical clustering of the amino acid–lipid correlation matrix; (vi) Random Forest permutation importance for all four lipid outcomes; and (vii) composite lipid risk indices including atherogenic index (TG/HDL-C) and non-HDL cholesterol. Results: A distinct amino acid correlation pattern was observed for each of the four lipid fractions. Triglycerides (TG) correlated most strongly with glutamic acid (r = 0.58) and inversely with glutamine (r = −0.58). High-density lipoprotein cholesterol (HDL-C) correlated most strongly with glutamic acid (r = −0.61) and serine (r = 0.49). The strongest correlates of low-density lipoprotein cholesterol (LDL-C) were phenylalanine (r = 0.56) and leucine (r = 0.56), and those of total cholesterol (TC) were leucine (r = 0.63) and, inversely, glycine (r = −0.54). The atherogenic index (TG/HDL-C) increased 2.9-fold from normal to obese and was most strongly correlated with glutamic acid, isoleucine, and glycine. In the multivariable models the amino acid panel accounted for a modest share of the variance in TG (adjusted R2 = 0.43; F(19,30) = 2.97, p = 0.004) and HDL-C (adjusted R2 = 0.35; F(19,30) = 2.40, p = 0.016). For LDL-C and TC the adjusted R2 values were close to zero (0.06 for both) and the overall models were not statistically significant (both p > 0.33); no interpretable amino acid signal was therefore present for these two fractions, and no predictors are reported for them. Lipid-based K-means clustering identified three lipid-phenotype clusters (Favorable, Intermediate, Adverse lipid profiles) with differences in amino acid z-scores. Conclusions: In this exploratory pilot cohort, lipid dysregulation in abnormal BMI was associated with two partially separable amino acid axes: a glutamic acid–glutamine axis (TG and partially HDL-C), and a glycine–serine putatively protective pattern opposing all atherogenic lipid parameters. These findings extend the established BCAA–insulin-resistance paradigm and suggest that targeted amino acid profiling—particularly for glutamic acid, leucine, glycine, and serine—may serve as a way of discovering candidate biomarkers for further study for dyslipidemia in individuals with abnormal BMI.

Biomolecules doi: 10.3390/biom16091304

Authors: Matej Zrimsek Jakob Gubensek

Many therapeutic apheresis procedures that remove plasma or plasma components also lower coagulation factor levels, which can increase patients’ bleeding risk. In this narrative review, we sum up data on clotting factor removal and the development of dilutional coagulopathy mostly associated with therapeutic plasma exchange (TPE) treatment and consider available tests for coagulation assessment. It is common in clinical practice to check fibrinogen levels regularly during repetitive TPE treatments and use it as a marker of hemostasis because of its long half-life and central role in hemostasis. We discuss the background of current clinical practice regarding fibrinogen thresholds discuss different threshold levels and data on the associated risk of bleeding. We stress the importance of additional bleeding risk assessment when patients are exposed to concomitant invasive procedures. We also describe the possibilities for fibrinogen replacement during TPE treatments with either fresh frozen plasma or fibrinogen concentrate and the associated effect on factor XIII levels. Finally, we briefly discuss alternative apheresis procedures regarding their fibrinogen-sparing potential.

Biomolecules doi: 10.3390/biom16091303

Authors: Anastasia Hale Alexandra Nusawardhana Claudia M. Nicolae George-Lucian Moldovan

Bisphenol A (BPA) is a prevalent chemical used in the production of plastics. While adverse effects on the reproductive system have been documented, more recent studies also associated BPA exposure with carcinogenesis as well as genomic instability. However, these studies were generally performed using BPA concentrations much higher than those observed in the serum or urine of the general population, making their relevance unclear. To address this, we report here an unbiased genetic study to identify mechanisms responding to levels of BPA exposure relevant to plastic manufacturers. We performed genome-wide CRISPR knockout screens in HeLa and RPE1 cells upon continuous exposure to 0.5 μM BPA, a concentration similar to the mean BPA concentration found in the urine of plastics manufacturing workers, for 19 days. We found genome stability genes among the top common hits between the two cell lines, suggesting that BPA causes DNA damage at this environmentally relevant exposure dose. We validated the DNA repair gene RAD51C and the RNA helicase DDX21 as genes required for BPA resistance. Our study suggests that BPA exposure at environmentally relevant doses can cause DNA damage, highlighting the relevance of BPA for carcinogenesis.

Biomolecules doi: 10.3390/biom16091302

Authors: Chiara Rosa Maria Uras Martina Taglieri Linda Di Gregorio Alessandro Poggi

The immune response of our body, whether it is directed to infective agents, allergens, or tumors, should support our recovery but can also be dangerous if dysregulated. There are molecular systems that have the role of tuning the immune response, such as activating and inhibitory receptors and co-stimulatory molecules. Among this plethora of receptors, immune-checkpoint receptors (ICRs), as well as natural killer receptors (NKRs), expressed on NK and/or T cells can up- or downregulate the function of activating and inhibitory receptors during immune cell crosstalk. Among NKRs, NKRP1A/CD161 has shown to be a promising target molecule for immunotherapy. This receptor showed inhibiting or co-stimulatory effects depending on the cell type analyzed as well as the analytical procedures, reagents used, and the experimental context. The aim of the following review will be to explore the structural and functional features of this receptor on immune cells, its relevance in the tumor microenvironment and its targeting as an immunotherapeutic tool in cancers.

Biomolecules doi: 10.3390/biom16091301

Authors: Chiara Fossati Federica Armeli Alessandra D’Amico Giacomo Frati Giovanni Enrico Casciaro Elena Cavarretta Vittoria Testa Alessio Borrelli Luigi Sciarra Chiara Lodoli Marco Zagarella Beatrice Oana Paun Vittorio Picchio Antonio Gianfelici Fabio Pigozzi Stefano Corrado Fabrizio Perroni Cristina Nocella Roberto Carnevale

Background: Intense physical exercise induces oxidative stress and inflammation in adult professional athletes, but data on adolescent elite athletes remain limited. Methods: Twenty-four elite adolescent soccer players were recruited and evaluated three times: pre-season baseline (T0), after one month of pre-season training (T1), and at the end of the first half of the competitive season (T2, approximately 4 months after T0). Saliva samples were collected as non-invasive surrogate indicators reflecting oxidative stress- and endothelial-related signaling. In vitro studies were performed on endothelial cells exposed to H2O2 concentrations observed in athletes. Results: Athletes showed a significant increase in salivary oxidative stress markers (NOX2 levels and H2O2) at T1 and T2 versus T0. Endothelial impairment was evidenced by reduced salivary NOx concentration and elevated endothelin (ET-1) levels. In vitro, endothelial cells exposed to H2O2 (8 μM) showed increased sNOX2-dp and ET-1 levels, reduced eNOS phosphorylation, and impaired tube formation (mesh number and area) compared with untreated cells. NOX2ds-tat treatment reduced cell damage and restored angiogenic capacity. Conclusions: Our findings indicate that adolescent elite athletes experience significant salivary oxidative stress- and endothelial-related alterations, potentially reflecting cardiovascular stress. These results underscore the importance of cardiovascular monitoring and the potential benefits of antioxidant strategies, even in young athletes participating in high-intensity sports.

Biomolecules doi: 10.3390/biom16091300

Authors: Trevor W. Stone Felix I. L. Clanchy Richard O. Williams

In this narrative review we summarise some of the roles played by glutamate and its receptors in both epilepsy and schizophrenia, before presenting the kynurenine pathway of tryptophan metabolism as a generator of compounds acting at least partly by modulating those receptors. Increasing evidence indicates that epilepsy involves interactions between neuronal excitability, cellular metabolism and immune system signalling. The kynurenine pathway of tryptophan metabolism occupies a central position within this neuroimmune–metabolic network through its regulation of glutamatergic neurotransmission and both innate and adaptive arms of the immune response. The modulation of glutamate receptors is achieved by activating NMDA-sensitive sites by quinolinic acid and the blockade of all glutamate ionotropic receptors by kynurenic acid. In addition, emerging evidence is discussed which implicates the GRIA3-mediated AMPA receptor subunit GluA3 function in epilepsy and schizophrenia, and observations suggesting antagonistic actions of 3-hydroxykynurenine at kainate receptors. The evidence reviewed suggests that the modulation of the kynurenine pathway metabolism may be relevant to the development of agents which increase understanding of the mechanisms involved in epilepsy, while also addressing the psychiatric and systemic inflammatory disorders that frequently accompany it. A dysregulation of neuroimmune metabolism represents a framework for improved understanding of epilepsy and its associated neuropsychiatric comorbidities, while highlighting opportunities and challenges for therapies targeting glutamatergic and immunometabolic pathways.

Biomolecules doi: 10.3390/biom16091299

Authors: Mehmet Emin Ayağ Mehmet Cudi Tuncer Şamil Öztürk

Cervical cancer remains a major cause of cancer-related mortality worldwide, highlighting the need for strategies that may improve responses to conventional chemotherapeutics. Epigallocatechin gallate (EGCG), a green-tea polyphenol with diverse biological activities, has been investigated as a potential chemosensitizing agent. This study evaluated the interaction between EGCG and doxorubicin (DOX) in HeLa cervical cancer cells, with HaCaT keratinocytes included as a non-malignant comparator. Cell viability and drug interactions were assessed using the CCK-8 assay and Chou–Talalay combination index (CI) analysis. Complementary assays evaluated membrane integrity, wound closure, apoptosis, cell-cycle distribution, intracellular DCF-associated fluorescence with or without N-acetylcysteine (NAC) pretreatment, caspase-3 immunoreactivity, and EGFR, FOXP3, CASP3, and CASP7 mRNA expression. Network-based analyses were additionally used to identify candidate molecular associations and pathways. CI analysis demonstrated synergistic EGCG–DOX interactions in HeLa cells under the tested conditions, whereas additive or antagonistic interactions predominated in HaCaT cells. Combined treatment produced the greatest reduction in viable cells, increased apoptosis, altered cell-cycle distribution, and reduced wound closure. It also produced the highest viability-normalized DCF-associated fluorescence, which was attenuated by NAC pretreatment, indicating an antioxidant-sensitive change in intracellular oxidative status without establishing a causal role in cytotoxicity. Combined treatment was further associated with increased total caspase-3 immunoreactivity and altered EGFR, FOXP3, CASP3, and CASP7 transcript levels. Overall, EGCG and DOX exhibited synergistic interactions and multiple treatment-associated cellular and transcriptional responses in HeLa cells under the present in vitro conditions. These findings do not establish cancer-specific selectivity or a definitive molecular mechanism but provide a basis for validation in additional cervical cancer models and at clinically relevant exposures.

Biomolecules doi: 10.3390/biom16091298

Authors: Jun Liu Tingting Cheng Jiefeng Kou Lili Wang Lixin Pei Yan Yang Conglong Lian Jinxu Lan Fei Zhang Suiqing Chen

Trihelix is a class of transcription factors unique to plants that play a major role in abiotic and biotic stress responses, seed isolate development, floral organ morphogenesis, and plant photomorphogenesis. Nevertheless, the Trihelix transcription factor family in Gardenia jasminoides (G. jasminoides) has not been systematically characterized. In this study, 11 GjTrihelix genes were identified from the G. jasminoides genome, unevenly distributed across five chromosomes, and can be classified into four subfamilies: GT-1, GT-2, SIP and SH4. Gene structure and functional motif analyses revealed high conservation within the same subfamily. Cis-acting element analysis showed that these genes are closely related to hormone responses, stress responses, and growth and development processes. Intraspecific synteny analysis showed a segmental duplication between GjTrihelix-3 and GjTrihelix-10. Interspecific collinearity analysis revealed that G. jasminoides shared 21 collinear gene pairs with soybean, compared with five pairs with Arabidopsis and 13 with Populus, indicating greater syntenic block conservation between G. jasminoides and soybean. Transcriptome data analysis demonstrated distinct spatiotemporal expression specificity of this gene family. Several genes were constitutively expressed in fruits; GjTrihelix-1 and GjTrihelix-3 were predominantly expressed in green fruits, while GjTrihelix-11 was highly expressed in red fruits. Under melatonin treatment, five GjTrihelix genes showed significant up-regulation and obvious transcriptional suppression of another five genes. Following infection by Botryosphaeria dothidea, GjTrihelix-5 and GjTrihelix-7 were progressively induced and peaked at 72 h. qRT-PCR results indicated that most GjTrihelix genes were highly expressed in leaves, while GjTrihelix-11 was highly expressed in flowers. Most GjTrihelix genes were significantly down-regulated under NaCl, ABA, GA3 and IAA stresses. This study provides new insights into the potential association of the Trihelix transcription factor family in G. jasminoides growth, development, and stress adaptation, offering theoretical references for stress-resistant G. jasminoides breeding.

Biomolecules doi: 10.3390/biom16091297

Authors: Yanrui Yang Xinyue Zeng Yuqiao Hu Yufei Su Chengqi Li Fajin Lv Kehui Zhao Jing Hu

Bufalin is a naturally occurring bufadienolide with broad-spectrum anticancer activity. Unlike conventional cytotoxic agents, bufalin exerts pleiotropic antitumour effects by directly modulating oncogenic proteins and promoting their degradation. It also disrupts metabolic plasticity, induces multiple forms of regulated cell death, counteracts therapeutic resistance, and remodels the immunosuppressive tumour microenvironment. However, the further application of bufalin is hindered by poor aqueous solubility, rapid systemic clearance, a narrow therapeutic window, and dose-limiting toxicity. Nanodrug delivery systems (NDDSs) offer a promising strategy for translating these interconnected pharmacological effects into spatially and temporally controlled therapeutic responses. This review summarises the distinctive anticancer mechanisms of bufalin and systematically evaluates the organic, inorganic, biomimetic, and hybrid nanocarriers developed for its delivery. Particular attention is given to tumour-responsive drug release, the targeting of cancer stem cells, the induction of ferroptosis and pyroptosis, metabolic modulation, sensitisation to phototherapy, and the activation of antitumour immunity. Finally, the major translational challenges are critically examined to inform the further rational development of bufalin-based nanomedicines.

Biomolecules doi: 10.3390/biom16091296

Authors: Yasemin Dadas Enes Karaman Ergul Bayram Durmus Ayan

Uterine corpus endometrial carcinoma (UCEC) is the most common gynecologic malignancy, and aberrant canonical NF-κB signaling is implicated in uterine corpus endometrial carcinoma (UCEC), yet the expression, epigenetic, and prognostic profile of IκB kinase (IKK) complex subunits CHUK (IKKα), IKBKB (IKKβ), and IKBKG (NEMO) remains undefined. This study assessed expression, methylation, miRNA regulation, and clinical relevance of IKK-complex genes in UCEC using public datasets. RNA-seq expression (TCGA-UCEC; GEPIA2) showed significant downregulation of IKBKB in tumors versus normal endometrium (unpaired Wilcoxon), whereas CHUK and IKBKG showed non-significant increases; immunohistochemistry (Human Protein Atlas) illustrated heterogeneity and suggested possible mRNA–protein discordance for IKBKB. Promoter methylation analysis (UALCAN; Illumina 450K) identified CHUK hypomethylation and IKBKG hypermethylation in tumors, with no significant change for IKBKB. Stratification showed IKBKB suppression across clinicopathological strata and TP53 mutant/wild-type tumors; IKBKG decreased across subtypes and stages. Pan-cancer profiling (TIMER2.0) highlighted IKBKB as the broadly dysregulated IKK member across malignancies. STRING networks indicated connectivity with NF-κB mediators (e.g., RELA, NFKB1, TRAF6). miRNA predictions (miRDB/TargetScan) revealed shared regulation (CHUK–IKBKB: 14 miRNAs; CHUK–IKBKG: 2; IKBKB–IKBKG: 0). Kaplan–Meier analysis indicated that elevated IKBKG expression correlated with reduced overall survival (HR = 1.85, p = 0.0037), while CHUK expression did not correlate with survival, and IKBKB exhibited a non-significant trend. In a multivariable Cox regression analysis, high IKBKG expression continued to show a significant association with reduced overall survival, even after adjusting for age, histological type, histological grade, and TCGA molecular subtype (adjusted HR = 1.58, 95% CI = 1.00–2.47, p = 0.048). These findings collectively suggest the potential prognostic significance of IKBKG in UCEC, although independent validation in larger, comprehensively annotated cohorts is still required. Collectively, these findings suggest IKBKG as a candidate negative prognostic marker and indicator in UCEC, warranting experimental validation.

Biomolecules doi: 10.3390/biom16091295

Authors: Mubeen Fatima Ali Rafi Muhammad Shoaib Zafar Usman Aftab Khush Bakhat Kiran Muhammad Shahzad Safdar Hussain Hongbo Wang

Allergic asthma is characterized by Th2-driven airway inflammation. Indoleamine 2,3-dioxygenase (IDO) maintains immune tolerance, but its role in modulating the anti-inflammatory actions of Curcumin (CN) is unclear. This study investigated whether CN retains efficacy in a rat model of Ovalbumin (OVA)-induced airway inflammation under pharmacological IDO inhibition. Rats were sensitized and challenged with OVA and treated with methylprednisolone (MP, 15 mg/kg), CN (200 mg/kg), the IDO inhibitor 1-MT (70 mg/kg), or CN+1-MT. Assessments included systemic and pulmonary leukocyte profile, delayed-type hypersensitivity (DTH), OVA-specific IgE, bronchoalveolar lavage fluid (BALF) inflammatory cells and nitric oxide (NO) levels, lung wet/dry weight ratio, gene expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-4, IL-6, Cxcr2 and transcription factor Nfkb1 and lung histopathology. CN alone or combined with 1-MT normalized leukocyte counts, suppressed eosinophilic and neutrophilic infiltration, restored BALF NO levels, reduced DTH response and OVA-specific IgE, decreased pulmonary edema, and preserved pulmonary vascular integrity. CN significantly attenuated TNF-α, IL-4, IL-6, and Cxcr2 gene expression despite pharmacological IDO inhibition. Histopathology revealed reduced inflammatory infiltration and maintained alveolar structure. CN maintains anti-inflammatory, immunomodulatory and tissue-protective efficacy in allergic airway inflammation despite pharmacological IDO inhibition. These findings call for future research on CN as a potential adjunctive therapy method for inflammatory airway diseases. However, to elucidate the underlying processes, more research on IDO activity and downstream immunometabolic pathways is needed.

Biomolecules doi: 10.3390/biom16091294

Authors: Ivis Páez Edgar Uquiche Adalberto Pessoa Robinson Soto-Ramirez Paola Díaz-Navarrete

Microbial lipids produced by oleaginous yeasts are a sustainable alternative to conventional lipid sources because of their ability to generate nutritionally valuable fatty acids. This study evaluated the effects of carbon-to-nitrogen (C/N) ratio, modified through molasses concentration, and cultivation temperature (15 and 25 °C) on biomass production, lipid accumulation, fatty acid composition, and nutritional quality of lipids produced by the native yeast Rhodotorula mucilaginosa 6S. Biomass, lipids, sugar consumption, and growth kinetics were determined, while fatty acid composition was analyzed by gas chromatography and nutritional quality was assessed through lipid nutritional indices. Biomass production was mainly influenced by the C/N ratio, whereas temperature had the greatest effect on lipid accumulation and fatty acid composition. Cultivation at 25 °C enhanced lipid yield and productivity, while 15 °C promoted faster growth and higher accumulation of polyunsaturated fatty acids (PUFAs), resulting in lipids with superior nutritional quality. The combination of 15 °C and a C/N ratio of 12 produced the most favorable lipid profile, with higher unsaturation, improved PUFA/SFA and MUFA/SFA ratios, balanced ω-6/ω-3 and LA/ALA ratios, low atherogenicity and thrombogenicity indices, and a high hypocholesterolemic/hypercholesterolemic ratio. These findings demonstrate the potential of molasses as a sustainable substrate for producing high-quality microbial lipids.

Biomolecules doi: 10.3390/biom16091293

Authors: Ilaria Laurenzana Antonella Caivano Alessio Di Ciancia Oreste Villani Maddalena Maietti Angelo De Stradis Giovanni D’Arena Filomena Nozza Luciana De Luca Daniela Lamorte

Background/Objectives: Chronic lymphocytic leukemia (CLL) is characterized by marked clinical heterogeneity, underscoring the need for reliable biomarkers to improve diagnosis and risk stratification. Extracellular vesicles (EVs) represent promising liquid biopsy candidates because they reflect the molecular profile of their cells of origin. In CLL, CD200 is a well-established marker of tumor cells; its expression on EVs and its role as a CLL biomarker were poorly characterized. This study investigated the diagnostic and prognostic value of serum EVs in CLL. Methods: Serum EVs were isolated from 83 patients with CLL and 20 healthy subjects (HS). EV morphology, size, and concentration were assessed by transmission electron microscopy and nanoparticle tracking analysis. Flow cytometry was used to evaluate the expression of CD19, CD20, and CD200 on EVs, while digital PCR quantified EV-associated miR-93-5p, miR-125b-5p, miR-150-5p, and miR-484. Results: Compared with HS, CLL patients showed increased total EV counts and higher levels of CD19+, CD20+, CD200+, and CD19+/CD20+ EVs, together with enhanced CD20 and CD200 expression and reduced miR-93-5p, miR-125b-5p, and miR-484 levels. In contrast, EV-associated miR-150-5p levels were comparable between CLL patients and HS. EV features were correlated with clinical and biological characteristics, including disease stage, cytogenetic abnormalities, treatment, and time to treatment (TTT). Older age was associated with lower EV concentrations but higher CD20 and CD200 expression. Advanced-stage disease was characterized by smaller EVs with increased CD200 expression. Notably, elevated CD200+ EV levels and reduced CD19 expression were associated with shorter TTT and earlier treatment initiation. Conclusions: Multiparametric profiling identified distinct quantitative, phenotypic, and molecular features of CLL-derived EVs and revealed CD200 as a novel EV-associated biomarker. These findings support the clinical utility of serum EVs as a new valuable approach for CLL diagnosis, prognosis, and treatment prediction.

Biomolecules doi: 10.3390/biom16091292

Authors: Ji-Eun Park Narae Kim So-Ra Kim Soo-Ho Lee Yoon-Jin Lee Kwang Seok Kim

Current studies have elucidated the mechanisms of radiation-induced skin injury (RISI) and identified several medical countermeasures to reduce its severity. However, no treatment has yet proven effective in preventing or reversing radiation-induced skin fibrosis. Here, we show that radiation upregulates lipoprotein-associated phospholipase A2 (Lp-PLA2) expression and induces endothelial cell dysfunction, characterized by an increased DNA damage response and reduced tube-forming capacity and mitochondrial function. To define the role of Lp-PLA2 in the progression of RISI, we treated human dermal microvascular endothelial cells and the skin of SKH1 hairless mice with darapladib, a selective Lp-PLA2 inhibitor. In endothelial cells, darapladib attenuated radiation-induced cellular damage and suppressed endothelial-to-mesenchymal transition (EndoMT). In irradiated mouse skin, darapladib reduced radiation-induced inflammation, adipose tissue disruption, dermal thickness, and skin fibrosis. Notably, darapladib modulated macrophage polarization and inhibited radiation-induced macrophage infiltration in irradiated skin. These findings suggest that Lp-PLA2 inhibition may reveal potential targets for the treatment of RISI and other fibrotic skin diseases.

Biomolecules doi: 10.3390/biom16091291

Authors: Mengwan Jiang Mingke Yang Peixuan Du Zhongke Sun

The gut microbiome is a complex ecological system crucial to human physiology. Commensal microbes in the gut provide resistance against pathogenic colonization, mainly due to their metabolites. Though studies have revealed a few mechanisms of microbial metabolite-mediated colonization resistance, various commensal microbes in the gut produce versatile metabolites and confront different pathogens. To pave the way for microbial metabolite-based treatment, clarification of what microbes and derived metabolites contribute to colonization resistance is a central topic. This focused review addressed the nexus of the gut microbiome, microbial metabolites, and colonization resistance against three representative pathogens, namely Clostridioides difficile, Salmonella enterica subspecies enterica serovar Typhimurium, and vancomycin-resistant Enterococcus. Different microbes and microbial metabolites involved in colonization resistance against these pathogens are discussed. Microbial metabolites, mainly short-chain fatty acids, secondary bile acids, and bacteriocins, were included. The emerging role of signal molecules in combating pathogenic infections is also addressed. In contrast to others, we further discussed different strategies that can enhance microbial metabolite-mediated colonization resistance, such as precise microbial preparation, targeted proliferation of a protective metabolite-producing microbiome, regulation of dietary patterns to optimize metabolic homeostasis, enhancing the local concentration of metabolites in the gut, and host-matched intervention.

Biomolecules doi: 10.3390/biom16091290

Authors: Uzair Iqbal Khadija Khalid Yunus Yukselten Farooq Ahmad Haseeb Ahmad Abdur Rehman Khalid Mohamed Shaltout Richard E. Sutton

Hepatitis B virus (HBV) infection is a worldwide health concern that infects nearly 254 million people globally and causes more than 1 million deaths annually. The highest prevalence is seen in sub-Saharan Africa and the Western Pacific region. Cirrhosis, liver failure, and hepatocellular carcinoma (HCC) are reported as leading complications of chronic HBV. The route of transmission of this infection is mainly by exposure to infected blood and bodily fluids. Transmission from mother-to-child remains the predominant route in highly endemic areas. Vaccination has significantly reduced HBV seroprevalence and complications. However, incomplete vaccination of newborns continues to be a major obstacle to elimination of the disease. Current prevention strategies include universal vaccination, perinatal prophylaxis with hepatitis B immune globulin, and maternal antiviral therapy in pregnant women with high viral load. The management of chronic hepatitis B virus infection predominantly depends on nucleoside analogs, including entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide, as well as pegylated interferon alfa. These therapies effectively suppress viral replication and reduce the risks of cirrhosis, HCC, and liver-related mortality, but they rarely achieve functional cure characterized by hepatitis B surface antigen loss. The persistence of covalently closed circular DNA (cccDNA) remains a major hindrance in HBV eradication. Therefore, novel therapeutic strategies targeting different stages of the viral life cycle, including capsid assembly modulators, small interfering RNAs, nucleic acid polymers, and cccDNA-directed approaches, are under active investigation. This review summarizes the epidemiology, prevention, current therapies, clinical outcomes, and emerging therapeutic advances in HBV infection, highlighting ongoing efforts toward achieving a functional cure and global HBV elimination.

Biomolecules doi: 10.3390/biom16091289

Authors: Tianhui Wang Yujia Chu Xin Li Haokun Zheng Jiang Feng Hui Wang

Circadian rhythms regulate hepatic metabolism and physiological homeostasis, which are essential for organisms to adapt to environmental cycles. Bats are the only mammals capable of sustained powered flight, accompanied by extreme metabolic demands and a nocturnal lifestyle. However, the regulatory mechanisms underlying hepatic circadian rhythms in bats remain poorly understood. Herein, we employed data-independent acquisition (DIA)-based quantitative proteomics to characterize the diurnal dynamics of hepatic proteins in Vespertilio sinensis across four representative physiological states, and further conducted integrative multi-omics analysis combined with transcriptomic data. The results revealed that approximately 5% of hepatic proteins exhibited circadian rhythmicity in V. sinensis, a proportion markedly higher than that of rhythmic transcripts and metabolites. Prominent transcription-protein rhythm decoupling was observed, implying that post-transcriptional regulatory processes may play critical roles in shaping circadian rhythms. Core metabolic pathways displayed temporally dynamic enrichment. The diurnal functional switching of the liver was achieved via stable expression of core proteins and temporal turnover of specific proteins, which efficiently coordinated energy supply, oxidative defense and detoxification metabolism. Trend clustering and functional enrichment analyses suggested that the liver may undergo lysosome-mediated immune repair during the daytime, while exhibiting enriched energy metabolic profiles that potentially support the elevated energy expenditure required for flight at night. This study systematically elucidates the protein regulatory patterns underlying hepatic diurnal rhythms in V. sinensis, and provides molecular clues for exploring extreme energy adaptation and circadian evolutionary scenarios in nocturnal flying mammals.

Biomolecules doi: 10.3390/biom16091288

Authors: Nayoung Park Hyewon Lee Jaebum Kim

Single-cell RNA sequencing (scRNA-seq) studies are frequently organized around comparisons—disease versus control, treatment response, or genetic perturbation—yet biological interpretation still depends on integrating multiple independent downstream analyses for differential expression, functional enrichment, regulatory network inference, and cell–cell communication analysis. Applying these tools consistently across comparisons typically requires substantial custom scripting, and their heterogeneous outputs must be manually harmonized before the results can be compared or reported together. We present scFlowReport, a lightweight, configuration-driven workflow that propagates a single user-defined comparison across cell-level and sample-aware pseudobulk differential expression, over-representation and ranked functional enrichment, transcription-factor regulon export for SCENIC, and group-resolved LIANA cell–cell communication analysis, starting from an already annotated Seurat object. The workflow automatically compiles complementary downstream results into standardized figures, summary tables, and a self-contained static HTML report that can be readily inspected and shared without requiring a persistent server. Application of scFlowReport to a publicly available Atopic Dermatitis scRNA-seq dataset demonstrated its utility by enabling researchers to obtain complementary biological evidence from multiple established downstream analyses. By coordinating complementary downstream analyses under a shared comparison framework, scFlowReport provides a practical and reproducible workflow for systematic interpretation of comparative single-cell transcriptomic data.

Biomolecules doi: 10.3390/biom16091287

Authors: Chathurani Ekanayake Aftab Mollah Maelee Thompson Elizabeth Hout Chamali Thalagaha Mudiyanselage Sanjaya Abeysirigunawardena

5-Methylcytosine (m5C) is a widespread mRNA modification that regulates gene expression and is frequently dysregulated in cancer. Here, we used phage display to identify peptides that bind an NSUN2 consensus RNA sequence in either its unmodified (UN2-RNA) or m5C-modified (MN2-RNA) form. A single peptide, un2p1 (TDYSTHRLSHSL), was selectively enriched against both targets. Biophysical analyses demonstrated that un2p1 binds this RNA sequence with high affinity and that m5C incorporation reduces binding by approximately ninefold, indicating that cytidine methylation acts as a biochemical switch for peptide–RNA recognition. Sequence alignments mapped un2p1 to a conserved structural loop and catalytic domain region within NSUN2, suggesting that the peptide functions as a molecular mimic of the endogenous RNA-recognition interface. In A549 lung adenocarcinoma cells, un2p1 treatment reduced global m5C levels and downregulated the oncogenic chaperonin CCT5, consistent with disruption of NSUN2-dependent Wnt/β-catenin signaling. These effects were attenuated in non-malignant HEK293 cells, which exhibited a compensatory increase in m5C levels and preserved viability. Together, these findings identify un2p1 as a sequence-specific, methylation-sensitive RNA-binding peptide that modulates the m5C epitranscriptome and selectively impairs lung cancer cell viability, highlighting a substrate-centric strategy for targeting NSUN2-mediated oncogenic pathways.

Biomolecules doi: 10.3390/biom16091286

Authors: Katja Venko Eva Žerovnik

Over more than a decade of development, various data-driven computational approaches based on the physicochemical properties of proteins have been developed for estimating the aggregation potential of proteins. The currently available algorithms and models enable a sequence-based design strategy to predict regions prone to transform into liquid–liquid phase-separated (LLPS) condensed or solidified aggregated states; for the latter, one can distinguish amyloid and prion-like aggregates. In this study, we performed a comprehensive in silico experiment; more than 40 models were used to test amino acid sequences of various proteins known to aggregate in neurodegenerative diseases, certain progressive myoclonic epilepsies and mental illnesses. Altogether, 20 proteins were analyzed and their proneness to aggregate or condensate was discussed in view of their possible normal and/or toxic function. The reported large set of computational models enables highly accurate predictions of proteins that are prone to aggregation and/or condensation. Furthermore, such aggregation profiling can be performed for any protein sequence of interest.

Biomolecules doi: 10.3390/biom16091285

Authors: Alessandro D’Annibale Rosita Marabottini

Although natural biopolymers represent promising sustainable packaging alternatives, their weak mechanical and barrier properties limit industrial use. While previous reviews focus on descriptive aspects of enzymatic modification, this review fills a critical literature gap by systematically bridging molecular-level laccase-driven reactions with quantitative techno-economic and safety and regulatory frameworks. We evaluate the kinetic and topological differences between direct tyrosyl-coupled protein homopolymerisation and mediator-assisted ‘graft-then-link’ polysaccharide strategies. Crucially, we analyse how entrapment versus surface-immobilised architectures dictate mass-transfer regimes, establishing their specific functional fitness for active oxygen scavenging or intelligent time-temperature monitoring. Beyond physical performance, we critically assess the translational bottlenecks currently hindering industrial scaling. For the first time, we integrate a quantitative techno-economic analysis using the Technology Readiness Level (TRL) framework, demonstrating that active film fabrication costs (EUR 0.01–0.10/m2) are heavily offset by high-protein food waste savings (>EUR 2.00/kg). Finally, we navigate European and US regulatory landscapes for enzymatically active materials and evaluate safety risks via the Threshold of Toxicological Concern (TTC) model and deterministic migration modelling. This comprehensive analysis establishes a ‘Safe-by-Design’ paradigm, guiding the scalable development of intrinsically safe, high-performance biocatalytic packaging.

Biomolecules doi: 10.3390/biom16091284

Authors: Patrizia Nardini Sara Bertorello Luca Filippi Virginia Zizi Ida Cioffi Francesco Cei Simone Baldi Daniele Bani Maura Calvani Camilla Fazi Amedeo Amedei Alessandro Pini

Oxygen availability is a key regulator of organ maturation during the perinatal period. Disruption of physiological oxygen homeostasis contributes to prematurity-associated disorders, yet its effects on the coordinated maturation of the enteric nervous system (ENS) and gut microbiome remain poorly understood. Because β3-adrenergic receptor (β3-AR) signaling has emerged as a mediator of tissue adaptation to oxygen, we investigated whether activation of this pathway modulates hyperoxia-induced alterations in the developing colon. Newborn rats were exposed to normoxia or hyperoxia (85% O2) from birth to postnatal day 14 and treated with the β3-AR agonist BRL37344 (1 or 3 mg/kg). Enteric neuronal and glial populations were evaluated by quantitative immunofluorescence, whereas the colonic microbiome (CM) was characterized by 16S rRNA gene sequencing. Hyperoxia reduced neuronal density and altered neurochemical coding within the submucosal plexus, disrupted enteric glial organization in both the colonic submucosal plexus and mucosa, and remodeled the intestinal microbiome without affecting overall community diversity. BRL37344 treatment partially preserved submucosal neurochemical coding, modulated neuron–glia organization within the submucosal plexus, prevented the loss of mucosal enteric glial cells, and reshaped microbial composition. Collectively, these findings demonstrate that neonatal hyperoxia disrupts coordinated postnatal maturation of the ENS and CM and indicate that β3-AR signaling may contribute to postnatal intestinal adaptation to neonatal oxygen imbalance.

Biomolecules doi: 10.3390/biom16091283

Authors: Chun-Mei Gong Kun-Ze Liu Peng Wang Mu-Yan Wen Jie Wang Zhen-Ying Li Wei-Jingyi Lu Zi-Liang Wang Li-Fang Lu Ren-Jun Feng

Schizophrenia is a highly heterogeneous neuropsychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive impairment, with substantial long-term functional consequences. Although dopaminergic dysfunction remains central to current disease models and treatment, dopamine-centered frameworks alone cannot fully explain cognitive deficits, treatment resistance, or marked variability in therapeutic response. Emerging evidence supports a broader view in which genetic susceptibility and environmental exposures converge on multiple interacting biological systems, including dopaminergic and glutamatergic neurotransmission, neuroimmune and glial dysfunction, kynurenine pathway metabolism, large-scale brain network dysconnectivity, and gut–brain communication. In this review, we integrate these mechanisms within a systems-level framework and discuss how their interactions may contribute to symptom heterogeneity and disease progression. We further examine the limitations of conventional dopamine D2-based antipsychotics, emerging non-dopaminergic pharmacological strategies, and adjunctive interventions targeting cognition and functional recovery. Finally, we highlight major translational barriers, including treatment resistance, medication non-adherence, adverse-effect burden, and the lack of clinically actionable biomarkers. An integrated understanding of converging neurobiological mechanisms may provide a stronger foundation for mechanism-informed patient stratification and precision treatment in schizophrenia.

Biomolecules doi: 10.3390/biom16091282

Authors: Ibtihal Segmani Chiara D’Aprile Laura Cherchi Eleonora Pozzi Alessia Chiorazzi Annalisa Canta Paola Alberti Cristina Meregalli Lisa Fantoni Elisa Ballarini Virginia Rodriguez Menendez Elisabetta Donzelli Silvia Fermi Arianna Scuteri Houda Filali Guido Cavaletti Valentina Alda Carozzi

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a dose-limiting complication of paclitaxel (PTX) therapy, for which effective treatments are still lacking. This study evaluated the neuroprotective potential of Thymoquinone (TQ), a bioactive derivative of Nigella sativa, in mitigating chronic PTX-induced neurotoxicity without compromising antineoplastic activity. We first performed in vitro experiments using Sprague–Dawley rat embryonic (E15) Dorsal Root Ganglia (DRG) (Envigo Laboratory (Udine, Italy)) to assess neurotoxicity through neurite outgrowth evaluation. To investigate the molecular mechanisms underlying TQ’s putative neuroprotective mechanisms, SIRT1 protein expression was additionally evaluated by western blot, while MCF-7 and MDA-MB-231 breast cancer cells were used to monitor cytotoxicity via MTT assay. We then moved to in vivo experiments, in which chronic neuropathy was induced in rats using PTX (10 mg/kg, i.v., weekly for 4 weeks). TQ was co-administered orally (5–10 mg/kg/day). The effects of TQ on peripheral neuropathy were assessed through behavioral testing, neurophysiological assessments, and histological analysis of Intraepidermal Nerve Fibre density (IENF), DRG and peripheral nerves. In vitro, TQ (5 μM) significantly attenuated PTX-induced neurite shortening at 24 h; TQ co-treatment fully prevented PTX-induced SIRT1 downregulation in embryonic DRG neurons and did not compromise PTX cytotoxicity in MCF-7 cells, while significantly potentiating it in MDA-MB-231 triple-negative breast cancer cells. In vivo results demonstrated that PTX-treated animals exhibited mild erythroid myelosuppression at the end of treatment. Regarding efficacy, TQ consistently prevented PTX-induced mechanical allodynia throughout the treatment period, and TQ (10 mg/kg) transiently mitigated IENF depletion at mid-treatment; however, no improvement in neurophysiological parameters or peripheral nerve morphology was observed at either time point. Collectively, these findings suggest that, under conditions of chronic PTX exposure, TQ exerts a predominantly analgesic effect in vivo, without conferring meaningful structural neuroprotection against PTX-induced peripheral nerve degeneration.

Biomolecules doi: 10.3390/biom16091281

Authors: Apostolos Fasoulopoulos Michail Varras Fani-Niki Varra Viktoria-Konstantina Varra Anastassios Philippou Alexandros Gryparis Argyro Papadopetraki Petros Stellatos Athina Pesiridou Kleopatra Paparizou Anastasia Evangelia Konstantinidou

Background: Fetal growth restriction (FGR) is associated with placental dysfunction and adverse perinatal outcomes. Although insulin-like growth factor-1 (IGF-1) signaling is important for placental and fetal development, the mRNA expression, immunopositivity and potential biological significance of specific IGF-1 isoforms in FGR remain incompletely characterized. This study investigated placental IGF-1Eb mRNA expression and immunopositivity in FGR pregnancies compared with appropriate-for-gestational-age (AGA) pregnancies. Methods: A total of 62 third-trimester human placentas were analyzed, including 47 from pregnancies complicated by FGR and 15 from pregnancies with AGA fetal growth. The mRNA expression of the IGF-1Eb isoform was assessed by reverse-transcription quantitative PCR in a subset of 28 fresh placental samples. IGF-1Eb protein immunoreactivity was assessed in paraffin-embedded tissue sections. Histopathological lesions were classified according to the Amsterdam criteria, and associations with clinical, demographic, and pathological parameters were evaluated using appropriate statistical analyses. Results: IGF-1Eb mRNA expression did not differ significantly between the FGR and AGA groups. In contrast, significant differences in IGF-1Eb immunoreactivity were observed in selected placental compartments. Moderate immunoreactivity in the perivillous syncytiotrophoblast was more frequent in FGR placentas and was associated with histological features of maternal vascular malperfusion, as well as with gestational age, neonatal birth weight, placental weight, maternal body mass index, and fetal sex. Increased IGF-1Eb immunopositivity was also observed in the endothelium of maternal decidual and fetal villous vessels in FGR placentas. No significant differences were observed in IGF-1Eb immunoreactivity in the extravillous trophoblast. Conclusions: In this observational study, placental IGF-1Eb protein immunoreactivity, but not mRNA expression, differed between FGR and AGA pregnancies in selected placental compartments. These findings indicate compartment-specific differences in IGF-1Eb protein immunoreactivity, associated with FGR and placental pathological features. However, the observational and cross-sectional design does not establish causality or a functional role for IGF-1Eb in placental dysfunction. Larger prospective studies incorporating functional validation are required to clarify the biological significance of these findings.

Biomolecules doi: 10.3390/biom16091280

Authors: Kacper Górecki Jakub Polakowski Natalia Więckowska Renata Grzywa Justyna Frączyk Beata Kolesińska Agnieszka Wróbel-Tałałaj Danuta Drozdowska Łukasz Janczewski

The search for new compounds capable of inhibiting the activity of enzymes involved in the development of neurodegenerative diseases, including Alzheimer’s disease, has attracted continuing interest for many years. Natural isothiocyanates and their synthetic analogs, as well as compounds containing a 1,3,5-triazine core, constitute a group of molecules with complex mechanisms of action involving multiple molecular targets. Although literature data confirm the activity of both isothiocyanates and s-triazines, these two active fragments have not yet been combined, and it remains unclear whether a synergistic effect can be achieved. In this study, 11 novel isothiocyanate–triazine conjugates, consisting of a 1,3,5-triazine core substituted with aliphatic or cyclic secondary amines and a phosphorus analogue of isothiocyanate [6-(isothiocyanatohexyl)phosphonate)ethyl], were synthesized with yields of 46–80%. The pharmacokinetic profiles and parameters related to Lipinski’s rule of five were determined for all compounds. All compounds were evaluated as inhibitors of acetylcholinesterase (AChE) and β-secretase (BACE1). Compound 1i showed the highest inhibitory activity against AChE (IC50 = 0.182 ± 0.02 µM), while compound 1b exhibited the strongest inhibition of BACE1 (IC50 = 8.48 ± 2.18 µM). To explore the potential of these multifunctional derivatives, plausible enzyme-ligand binding models were proposed based on molecular docking simulations.

Biomolecules doi: 10.3390/biom16091279

Authors: Junyan Wang Kang Zhang Jingyan Zhang Zhiting Guo Guowei Xu Xiaowei Feng Xiaoliang Chen Shuqi Liu Zhengzhong Luo Lei Wang Jianxi Li

Acute lung injury (ALI) is a common clinical acute respiratory disorder driven primarily by a diffuse pulmonary inflammatory response. Cordycepin (COR) is a bioactive metabolite extracted from the fungus Cordyceps militaris, which possesses antioxidant and anti-inflammatory properties. However, its underlying molecular mechanism remains to be elucidated. This study evaluated the therapeutic effect of COR on ALI and the underlying molecular mechanisms. MH-S cells were primed with lipopolysaccharide (LPS) at 1 µg/mL for 24 h and then treated with varying doses of COR for an additional 24 h. WB and RT-qPCR analyses showed that COR inhibited the phosphorylation of TGF-β-activated kinase 1 (TAK1) as well as the key kinases in the MAPK and NF-κB pathways in LPS-induced MH-S cells, as evidenced by decreased TAK1, p38, JNK, IκB-α and P65 expression levels, as well as decreased TNF-α, IL-6, IL-1β, MAP3K7, MAPK8 and MAPK14 relative expression. Six-week-old BALB/c mice were intranasally instilled with LPS at 3 mg/kg, followed 24 h later by oral gavage administration of various concentrations of COR. The results showed that COR can effectively suppress the progression of pulmonary tissue injury; similarly, the expression levels of key proteins and inflammatory factors in the TAK1-MAPK and NF-κB signaling pathways were downregulated. In summary, COR exerts a therapeutic effect on ALI by directly targeting TAK1 to inhibit the activation of the MAPK and NF-κB signaling pathways and concurrently suppressing key inflammatory cytokines.

Biomolecules doi: 10.3390/biom16091278

Authors: Jaromír Vašíček Andrej Baláži Andrea Svoradová Jakub Vozaf Miroslav Bauer Marián Tomka Lucia Olexiková Peter Chrenek

The continual spermatogenesis throughout adulthood is ensured by a rare and unique cell group named spermatogonial stem cells (SSCs), which undergo self-renewal and/or differentiate into sperm cells. SSCs also become a promising genetic source for the protection of animal biodiversity. However, the isolation and culture of SSCs in vitro is still a big challenge and poorly explored in rabbits. The main objective of this study was to isolate, culture, and deeply characterize SSCs obtained from rabbit testes. Briefly, rabbit testicular tissue was mechanically and enzymatically dissociated, and obtained testicular somatic and germ cells were cultured for a short term in culture media supplemented with specific molecular factors maintaining SSC self-renewal and proliferation (GDNF, GFRα-1, FGF2, etc.). Immunofluorescent and PCR techniques were used for molecular profiling of cultured SSCs, while TEM analysis revealed their ultrastructure. After a few weeks, round and grape-like SSC colonies emerged, growing on the feeder cell layer. Rabbit SSCs showed positive staining for DBA, GFRA1, PLZF, RET, PGP9.5, DAZL, and DDX4. Increased expression of additional SSC markers was noticed using RT-qPCR and dd PCR (RET, PLZF, PGP9.5, DAZL, DDX4, CDH1, CD9, CD14, CD90, c-kit, ALDH, SSEA-4, SALL4, OCT4, and SOX2), while ultrastructure typical for primitive undifferentiated cells was observed under TEM. In conclusion, we successfully established a method for rabbit SSC isolation, culture, and phenotyping, which might facilitate their collection for further cryopreservation. However, the self-renewal, proliferative, and differentiation capacities of cultured SSCs still need to be confirmed through an in vivo SSC transplantation experiment.

Biomolecules doi: 10.3390/biom16091277

Authors: Ian Hall Carly A. Nowoj Lynne M. Dieckman

Eukaryotic genomes are organized into chromatin, a highly compact structure in which DNA is packaged into nucleosomes. Nucleosome formation, where DNA is wrapped around histone proteins, is essential for genome stability. This compaction protects DNA from damage and regulates accessibility of genes. Nucleosomes must be disassembled and reassembled during DNA replication and repair. These processes require precise regulation of histone folding, transfer, and deposition by a diverse network of histone chaperones. Chromatin assembly factor 1 (CAF-1) is a conserved histone chaperone that specifically deposits newly synthesized histones during replication-coupled and repair-coupled nucleosome assembly. The sliding clamp proliferating cell nuclear antigen (PCNA) serves as a regulatory scaffold during these processes by recruiting CAF-1 and many other proteins to sites of DNA replication and repair. Recent structural and biochemical studies have revealed increasingly complex mechanisms underlying PCNA-mediated CAF-1 recruitment, involving multiple protein interaction motifs, DNA-binding domains, and regulatory mechanisms that ensure efficient nucleosome assembly. This review summarizes current advances in understanding the molecular mechanisms by which human and yeast CAF-1 complexes are recruited to sites of DNA synthesis and how CAF-1 function is coordinated with other histone chaperones during replication and repair. These studies have provided important insights into how cells coordinate DNA metabolism with epigenome maintenance to preserve genome integrity.

Biomolecules doi: 10.3390/biom16091276

Authors: Miaomiao Wang Heng Xu Zhihui Hao Haiyang Jiang Jianzhong Shen Chongshan Dai

Due to limitations in the use of antibiotics as feed additives to promote growth, the development of safe, effective, and environmentally friendly antibiotic alternatives is a critical and urgent research priority in animal nutrition and veterinary medicine. Plants contain abundant active substances, which are important sources for developing new feed additives or discovering new active substances. Ageratina adenophora (Spreng.) R.M.King & H.Rob. (A. adenophora) is an herb native to Central America and a globally invasive species that has spread across Asia, Australia, Africa, and other continents. Studies indicated that it possesses a complex chemical composition, with over 100 constituents identified, including terpenoids, flavonoids, and phenolic acids, which exhibit diverse biological activities, including antibacterial, antioxidant, insecticidal, acaricidal, anti-inflammatory, neuroprotective, antidiabetic, antitumor, and gut health regulation effects. However, it also possesses distinct toxicity, primarily targeting organs such as the intestines, liver, and spleen, causing tissue damage by activating specific signaling pathways. This may limit its utilization as a feed additive or in medical drug development. Therefore, this review systematically summarizes the botanical characteristics, geographic distribution, phytochemical composition, biological activities, and toxicological effects of A. adenophora. We also discussed its potential application and current challenges. We hope this review can promote its development and utilization as potential feed additives or medicinal resources.

Biomolecules doi: 10.3390/biom16091275

Authors: Shunling Yuan Jiaxin Liu Lihan Lin Yiping Liu Shan Xu Liangwu Qiu

A high-fat diet (HFD) promotes lipid metabolic disorders, chronic low-grade inflammation and skeletal muscle dysfunction. Kukoamine B (KB), a Lycium-derived polyamine alkaloid with reported anti-inflammatory and antioxidant activities, may protect against HFD-associated muscle impairment. C57BL/6J mice were fed an HFD and orally administered KB for 12 weeks. Muscle function was evaluated using inverted grid, forelimb grip strength and treadmill exhaustion tests. Histological, transcriptomic, ELISA, immunofluorescence and Western blot analyses were used to assess muscle injury and inflammatory signalling. KB reduced intramuscular lipid deposition, collagen accumulation and myofibre damage, improved muscle strength and exercise endurance, and partially restored AKT/mTOR-associated protein metabolic signalling. Transcriptomic analysis showed that KB downregulated HFD-activated inflammatory responses, chemokine signalling, lipopolysaccharide responses and Toll-like receptor-related pathways. KB also reduced plasma and skeletal muscle LPS levels, inhibited TLR4/MyD88/NF-κB signalling, and decreased TNF-α and IL-1β expression. TAK-242 mimicked the anti-inflammatory and muscle function-improving effects of KB, whereas their combination produced no statistically detectable additional effect under the conditions tested. These findings suggest that KB attenuates HFD-induced skeletal muscle dysfunction, potentially involving reduced LPS burden and suppression of TLR4/NF-κB-mediated metabolic inflammation.

Biomolecules doi: 10.3390/biom16091274

Authors: Ian Hall Iain M. Davies Stephanie A. Limaye Ivy L. Williams Cael E. Carlson Trevor J. Snetsinger Andy E. Agbakpo James W. Checco Lynne M. Dieckman

Proliferating cell nuclear antigen (PCNA) is an essential sliding clamp that coordinates nearly all DNA-templated processes. It does so by recruiting a diverse array of factors to DNA through PCNA-interacting protein (PIP) motifs on PCNA-binding proteins. Chromatin assembly factor 1 (CAF-1) is a histone chaperone that deposits histones onto silent regions of the genome immediately following DNA replication. PCNA recruitment of CAF-1 to the replication fork is essential for nucleosome assembly and epigenetic inheritance. In yeast, CAF-1 recruitment to PCNA is assumed to be facilitated using one PIP motif. Here, we identify a second PIP motif in CAF-1, designated PIP1, located within the N-terminal intrinsically disordered region of the protein. Binding kinetics demonstrate that the PIP1 motif sequence binds PCNA with substantially lower affinity than the previously characterized PIP motif, designated PIP2. Structural and binding data reveal PIP1 as an extended PIP motif, in which C-terminal flanking residues make extensive, atypical contacts with PCNA that significantly increase its affinity. Neither PIP1 nor PIP2 alone is required for CAF-1–mediated gene silencing in vivo, but simultaneous disruption of both abolishes CAF-1 function. Together, these findings suggest CAF-1 engages PCNA multivalently and provide new insight into how PCNA selectively recognizes binding partners during nucleosome assembly.

Biomolecules doi: 10.3390/biom16091273

Authors: Junyi Gao Fei Wu Bing Li Yuhang Ji Hongxu Dong Dongfeng Wang Zixuan Liu

Silica nanoparticles (SiNPs) are common nanoparticles that are widely used in industrial and medical applications. Inhalation exposure to SiNPs is frequently unavoidable in occupational and everyday settings. While the adverse effects of SiNPs on lung injury have been extensively documented, the intrinsic mechanisms underlying SiNPs-induced pulmonary inflammation remain incompletely understood. This study demonstrates that the stimulator of interferon genes (STING) plays an essential role in SiNPs-triggered lung inflammation. In vitro, SiNPs induced death of bone marrow-derived macrophages (BMDMs) and activated the STING pathway. Knockout of STING expression alleviated SiNPs-induced inflammatory responses and subsequent cell death. Further mechanistic investigations revealed that STING activation promotes nuclear translocation of NF-κB p65 and subsequent activation of the NF-κB pathway, ultimately driving the secretion of inflammatory cytokines. In vivo, SiNPs-induced NF-κB activation and inflammatory cell infiltration were significantly attenuated in STING-deficient (STING−/−) mice. These findings reveal that targeting the STING signaling pathway may represent a potential therapeutic strategy for mitigating lung inflammation caused by silica particles.

Biomolecules doi: 10.3390/biom16091272

Authors: Elia Ranzato Simona Martinotti

Traditional wound therapies continue to be predominantly exogenous and address extracellular causes of the pathology without addressing the impaired function of cellular pathways that are trapped in the state of constant inflammation. The present review explores a novel putative nutriepigenetic framework, discussing how the honey matrix could act as a proposed modulator of the altered epigenetic landscape in non-healing ulcers. Honey contains a complex mixture of bioactive agents (polyphenols, flavonoids, and plant-derived xenomiRs) that are hypothesized to interact with multiple chromatin control points simultaneously. We discuss models wherein honey-induced aquaporin-mediated H2O2 influx and intracellular calcium transients may correlate with SIRT1/SIRT6 modulation and CRM1-mediated nuclear export of Class IIa HDACs. This review evaluates whether such multi-target signaling could foster chromatin relaxation to support gene expression required for cell migration and tissue remodeling.

Biomolecules doi: 10.3390/biom16091270

Authors: Fangyuan Qin Kaizhi Jiao Fei Zhi Yu Li Siping Deng Tianli Wu Dongneng Jiang

Spotted scat (Scatophagus argus) is an economically important fish species with an XX/XY sex determination (SD) system and the candidate sex-determining gene Dmrt1Y. Alternative splicing (AS) contributes to transcript diversity and post-transcriptional regulation, but its role in fish gonadal function remains unclear. In this study, we conducted a comprehensive analysis of AS events in adult ovaries (n = 3) and testes (n = 3) of S. argus using ONT-based full-length transcriptome sequencing. An average of 7.30 Gb of clean data per sample was obtained, with a mean N50 length of 1502 bp. A total of 3333 differentially expressed genes (DEGs) were identified, including 2064 ovary-upregulated and 1269 testis-upregulated genes. Additionally, 9417 and 17,216 AS events were detected in the ovary and testis, respectively. Notably, no AS events were detected in the Dmrt1Y gene. A total of 2253 differential alternative splicing (DAS) events involving 1300 genes were identified between ovaries and testes. Of these, only 213 genes were also differentially expressed. Sex-biased AS patterns were observed in genes potentially associated with gonadal function and germ-cell regulation, including Ncoa5, Tp53, and Fancl. Male-biased AS events in Tp53 and Fancl were predicted to alter coding regions corresponding to the DNA-binding and UBC-like domains, respectively. Furthermore, male-biased Ncoa5 isoforms lacking exons 3 and 4 (381 bp) were predicted to result in partial loss of the RNA recognition motif (RRM). Collectively, this study provides insights into AS regulation in adult gonads of S. argus and highlights the potential contribution of AS to reproduction.

Biomolecules doi: 10.3390/biom16091271

Authors: Nabarun Chakraborty Gregory P. Holmes-Hampton Lily S. Neff Vidya P. Kumar Swapna Kannan Amrita Cheema Chandan Guha Sanchita P. Ghosh Rasha Hammamieh

Introduction: Thrombopoietin mimetics (TPOm) are known to increase blood cell counts, thereby ameliorating various diseases, including cardiomyopathy—a leading cause of mortality because of delayed radiotoxic effects. Previous preclinical studies demonstrated significant survival enhancement following lethal radiation exposure with a single dose of JNJ-26366821 in two mouse strains, CD2F1 and C57BL/6. Materials and Methods: In this study, a single dose of JNJ-26366821 (1.0 mg/kg) or saline was administered 24 h post-8.0 Gy total body irradiation (TBI) to male C57BL/6 mice. Mice were euthanized on days 1, 7, 15, and 30 post-TBI, and serum and heart samples were collected. Unirradiated mice treated with saline served as baseline controls. Inflammatory serum cytokines/chemokines, growth factors and multi-omics analyses, including proteomics, metabolomics, and lipidomics, were evaluated to assess therapeutic impacts. Results: JNJ-26366821 demonstrated overall positive effects, restraining radiation-induced inflammatory surges by 7 days post-TBI. Functional analysis revealed that JNJ-26366821 treatment in unirradiated mice activated cellular homeostasis, which persisted after post-exposure intervention in irradiated mice. The drug immediately reduced cell death, but networks supporting cardiovascular health, such as angiogenesis and vasculogenesis, remained inhibited 2 h post-intervention. By 7 days post-TBI, JNJ-26366821 mobilized molecules in heart tissue to reconstitute cellular development and maintenance. Protein synthesis and metabolism were activated between 15 and 30 days post-TBI, potentially compensating for radiation-induced muscle wasting. Conclusions: JNJ-26366821 demonstrated promising mitigating effects on cellular homeostasis and cardiovascular health when administered as a single dose at 24 h post-exposure.

Biomolecules doi: 10.3390/biom16091268

Authors: Moon-Soo Kim Hae Sol Do Hye Yeon Jang Kang Eun Lee Sun Ju Lee

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system began as a tool for programmable genome editing. CRISPR/Cas technologies have evolved into a versatile platform for functional genomic screening, epigenome editing, therapeutic target discovery, and highly sensitive molecular diagnostics. New effectors and engineered variants continue to push these applications into personalized medicine and point-of-care testing. Here, this review highlights recent advances in therapeutic target discovery and therapeutic and molecular diagnostic development utilizing CRISPR/Cas technologies. We discuss how CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), base editing, and prime editing have improved our understanding of disease mechanisms, while creating new opportunities for therapeutic intervention. Current applications in cancer immunotherapy, infectious disease, and neurological disorders are also discussed. In diagnostics, CRISPR-based platforms enable sensitive detection of infectious pathogens, cancer biomarkers, and genetic disorders using programmable nuclease activity in both laboratory and point-of-care settings. Collectively, these advances continue to expand the role of CRISPR/Cas technology across biological discovery, disease diagnostics, and therapeutic development, driving progress in precision medicine.

Biomolecules doi: 10.3390/biom16091269

Authors: Klaus Groschner Ichiro Wakabayashi

Arterial hypertension is a key trigger for all major, life-threatening cardiovascular events such as arrhythmia, stroke, cardiac infarction and heart failure. Ethanol consumption has been identified as a critical risk factor, which promotes hypertension and thereby corrupts cardiovascular health and limits overall life expectancy. For heavy drinkers (≥72 g/day), reduction in half of the alcohol consumption reportedly results in considerable decreases in systolic and diastolic blood pressure levels amounting to 5.50 and 3.97 mmHg, respectively. Based on the complex network of ethanol effects on mammalian physiology, diverse mechanistic concepts have been postulated for a causal linkage between alcohol consumption and distorted blood pressure regulation. The impact of ethanol on the cardiovascular system involves a wide range of ethanol-sensitive cell types, ranging from neurons to vascular smooth muscle, endothelial and immune-cells, all of which express an interdependent array of potential ethanol target structures. Here, we provide an update on currently available evidence for a causal link between alcohol consumption and the etiology of arterial hypertension. The potential role of ethanol target structures with particular focus on membrane ion channels is discussed along with novel concepts of lipid-dependent modulation of signaling processes in the plasma membrane by ethanol.

Biomolecules doi: 10.3390/biom16091267

Authors: Fabian Zincke Dennis Kobelt Susan Kläger Fiona Pachl Gerrit Erdmann Mathias Dahlmann Wolfgang Walther Bernhard Küster Ulrike Stein

Understanding the mechanisms of metastasis is one of the most pressing issues in cancer therapy. Metastasis-associated in colon cancer 1 (MACC1) is an important biomarker and functional driver of tumor progression and metastasis. However, the molecular mechanisms underlying its activity remain incompletely understood. Here, we demonstrate that MACC1 acts as an important adaptor protein that promotes hyperactivation of receptor tyrosine kinase (RTK) signaling pathways in colorectal cancer (CRC) cells. Based on mass spectrometry-based interactomics, we identified key MACC1 interactors, including GRB2, SHP2, SHC1, and STAT5B, that preferentially associate with tyrosine-phosphorylated residues Y365, Y379, and Y789. Site-directed mutagenesis of Y379 and Y789 reduced MACC1-induced migration, proliferation, and ERK phosphorylation. Using digital Western blotting (DigiWest), we observed a broad MACC1-dependent hyperactivation of downstream signaling effectors, including MEK, ERK, β-catenin, SRC, FAK, CREB, and VASP. Targeting MACC1-induced signaling with clinically relevant inhibitors effectively reversed MACC1-driven clonogenicity. Our findings support a role for MACC1 in promoting hyperactivation of RTK-associated signaling and reveal pharmacological vulnerabilities of potential relevance to metastasis-prone cancers characterized by elevated MACC1 expression.

Biomolecules doi: 10.3390/biom16091266

Authors: Fruzsina Pilhál Bianka Szalainé Ágoston Imre Jákli Ernő Keszei András Perczel

Spontaneous deamidation and isomerization of asparagine (Asn) residues is a major pathway of nonenzymatic protein aging, where Asn-Gly-containing peptides (NG motifs) represent the most reactive sequence. In this context, the first step of isomerization is succinimide formation, which is initiated by a concerted proton-transfer and nucleophilic activation step. Although the overall mechanism is established, the detailed nature of the rate-determining succinimide formation step and the possible role of nuclear quantum effects remain unclear. Here, we combine density functional theory (DFT), intrinsic reaction coordinate (IRC) analysis, and quantitative NMR kinetics to investigate hydrogen/deuterium (H/D) substitution effects on NG isomerization in different model peptides, which represent both conformationally flexible and restricted systems. Deuteration preserves the reaction pathway and structural evolution, indicating an invariant classical reaction coordinate. However, kinetic isotope effects in the flexible peptide system reveal a mixed classical–tunneling mechanism in the rate-determining step with observable deviations from classical over-the-barrier behavior. In contrast, the conformationally restricted peptide shows suppressed tunneling contributions. These findings demonstrate that conformational accessibility modulates proton tunneling in spontaneous peptide rearrangements and extends nuclear quantum effects beyond enzymatic systems to nonenzymatic processes associated with protein aging.

Biomolecules doi: 10.3390/biom16091265

Authors: Hualian Mo Wenxing Chen Zhaoxi Yang Junjie Zhou Xinze Zhang Siyuan Yang Xiaoman She Wenyang Zhang Shijuan Yan Shaowen Wu

Aflatoxin B1 (AFB1) is a potent foodborne mycotoxin for which nanobodies offer compact, engineerable detection reagents, yet the molecular basis of their affinity differences remains poorly defined. Here, closely related nanobodies G8, NB28, and NB26 were compared. Fluorescence correlation spectroscopy showed G8 had the lowest dissociation constant for AFB1-BSA, followed by NB28 and NB26. To resolve this affinity separation, we performed triplicate 3 μs molecular dynamics simulations for each complex. While all complexes remained stable, G8 maintained broader, persistent aromatic and hydrophobic contacts extending into its elongated CDR3, whereas NB28 and NB26 relied on localized contacts centered on PHE47 and TRP101. Per-residue MM/GBSA decomposition confirmed favorable thermodynamic contributions from these persistent G8 residues. Ultimately, integrating contact occupancy, energy decomposition, and pairwise co-occurrence identified distinct cooperative contact networks (six in G8, versus two in NB28/NB26). This workflow demonstrates how mapping dynamic, cooperative binding networks rather than static proximity can systematically prioritize candidate residues for engineering high-affinity detection reagents in food-safety surveillance.

Biomolecules doi: 10.3390/biom16091264

Authors: Marie-Paule Lefranc

IMGT®, the international information system® (IMGT), was created in 1989 by Marie-Paule Lefranc (Université de Montpellier and CNRS) in Montpellier, France, to deal with and to manage the huge diversity of immunoglobulins (IG) or antibodies and T-cell receptors (TR), which are the antigen receptors (AR) of the adaptive immune response (AIR) of jawed vertebrates. The founding of IMGT® marked the advent of immunoinformatics, a new science which emerged at the interface between immunogenetics and bioinformatics. The biocuration of the IMGT data (IG and TR sequences, genes and structures) and the implementation of the IMGT system (7 databases, 17 tools, 25,000 Web resources pages) are based on the IMGT Scientific chart rules (keywords, labels, nomenclature, numbering…) generated from the IMGT-ONTOLOGY axioms and concepts. The IMGT nomenclature (IMGT-NC) and the IMGT unique numbering, the two pillars of immunoinformatics, have been used to define 335 engineered variants for effector properties and formats of therapeutic antibodies (including 12 chimerisotypes) and TR, fusion proteins for immune applications (FPIA) and composite proteins for clinical applications (CPCA). IMGT-NC engineered variant names from the World Health Organization (WHO) International Nonproprietary Name (INN) programme descriptions contribute to the common language for immunoinformatics and artificial intelligence (AI).

Biomolecules doi: 10.3390/biom16091263

Authors: Carlos A. Guzmán-Martín Yaneli Juárez-Vicuña Rafael Bojalil Evelyn Aranda-Cano Mario Peña-Peña Yamnia Q. Alvarez-Alvarez Fengyang Huang Javier González-Ramírez Laura Aline Martínez-Martínez Fausto Sánchez-Muñoz

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombotic and inflammatory manifestations whose molecular regulatory mechanisms remain incompletely understood. Long non-coding RNAs (lncRNAs) and N6-methyladenosine (m6A)-related regulation are increasingly recognized as components of immune gene regulation, but their involvement in APS remains poorly characterized. This study investigated BRD3OS (LINC00094) expression in APS and explored its molecular and predicted structural context in relation to m6A-associated regulation. An exploratory case–control study was conducted using an initial lncRNA PCR-array discovery cohort followed by targeted RT-qPCR validation in an independent cohort. Candidate prioritization incorporated multiple expression and technical features and was evaluated through sensitivity analyses. BRD3OS expression, selected m6A regulators (METTL3, METTL14, WTAP, and FTO), inflammatory mediators, and global m6A abundance in total peripheral blood mononuclear cell (PBMC) RNA were evaluated. Bioinformatic network analysis was used to contextualize BRD3OS within APS- and m6A-related molecular systems. RNAfold and RNAplfold were used to characterize the predicted structural context and accessibility of DRACH consensus motifs, with additional analyses evaluating fragment-boundary and composite-score robustness. BRD3OS was significantly downregulated in PBMCs from patients with APS in the independent validation cohort. METTL3, METTL14, and WTAP expression was also reduced, whereas global m6A levels in total PBMC RNA were increased. These observations indicate concurrent alterations in BRD3OS expression and the broader m6A-related molecular environment but do not establish transcript-specific methylation of BRD3OS. Bioinformatic network analysis placed BRD3OS within predicted RNA-centered regulatory relationships relevant to APS. DRACH motifs exhibited heterogeneous predicted structural accessibility, with unpaired structural environments showing greater RNAplfold-derived accessibility than paired regions. Quantitative accessibility estimates were highly concordant across overlapping transcript fragments, although sensitivity analyses indicated that the identity of individual highest-ranked candidates depended on the weighting scheme. BRD3OS downregulation represents a reproducible molecular finding in APS. Concurrent alterations in global m6A abundance and selected m6A regulators suggest broader epitranscriptomic dysregulation; however, these measurements cannot establish m6A modification of BRD3OS or a causal relationship between these observations. Structural and network analyses therefore provide a hypothesis-generating framework for prioritizing candidate regions and interactions for future transcript-specific methylation mapping and functional validation.

Biomolecules doi: 10.3390/biom16091261

Authors: Shuji Kitagawa Naoki Mimura Hironobu Kambara Masaaki Hori Erina Yamanishi Ayako Ogo Tatsushi Shiomi Kazuhiko Yoshimatsu Tomio Ueno Shuya Yano

Colorectal cancers (CRCs) include stroma-dominant tumors with desmoplasia and differentiated, gland-forming tumors with little stroma. We asked whether this difference reflects stromal abundance alone or also involves a distinct cancer cell state. HEST-1K sections were classified as M-type (stroma-dominant; five patient/tissue units) or D-type (differentiated and cancer cell-dominant; seven units). Prespecified EMT/pEMT, ECM/integrin, YAP/TAZ-TEAD, and DTP/persister gene sets were examined across spatial, bulk, and single-cell datasets and patient-derived malignant ascites cultures. M-type tumors contained broader CAF-rich compartments and higher activity of all four programs in EPCAM/KRT-high regions, including epithelial-dense tumor cores. Activity was greatest near CAF-rich areas. In GSE39582, EMT/pEMT, ECM/integrin, DTP/persister, and invasive epithelial programs were independently associated with recurrence. Ascites-derived cultures from stroma-dominant tumors showed higher ECM/integrin and survival programs. Independent institutional analyses supported the public data findings: focused real-time PCR showed higher MAPK/TGF-beta-related gene expression in M-type-derived than in D-type-derived ascites cells, and IHC showed stronger epithelial CD44v6 expression in M-type primary tumors and metastatic lymph node lesions. CAF co-culture increased VIM-promoter activity. These findings characterize stroma-dominant CRC as a spatially organized ecosystem in which a CAF-rich compartment is associated with stress-adapted cancer cell programs. Its poor prognosis may therefore involve not only stromal abundance but also a morphology-associated cancer cell state detectable after dissemination.

Biomolecules doi: 10.3390/biom16091262

Authors: Ye Wang Shiyun Sun Yuhan Zhang Guoqing Li Yunlong Xu Yingxue Shi Pedro A. Jose Zhiwei Yang Xing Liu Xiaoliang Jiang

Hypertension is an important risk factor for cardiovascular and renal diseases, yet the mechanisms linking ion channel dysfunction to hypertension remain poorly understood. The voltage-gated potassium channel Kv1.3 (encoded by Kcna3) regulates membrane potential, but its role in the pathogenesis of hypertension remains unclear. In this study, we employed Kcna3 knockout (KO) mice, transcriptomic profiling, and pharmacological inhibition to investigate the role of Kv1.3. Kcna3-deficient mice showed increased blood pressure and renal fibrosis. Transcriptomic profiling showed activation of the renin–angiotensin–aldosterone system (RAAS), with increased renin expression in both Kcna3-deficient mice and Kv1.3 inhibitor-treated cells. Mechanistically, loss of Kv1.3 increased intracellular Ca2+ accumulation, activating the phosphoinositide 3-kinase (PI3K)-AKT and protein kinase A (PKA) pathways, leading to cAMP response element binding protein (CREB) phosphorylation and renin upregulation. Pharmacological inhibition of Ca2+ signaling or PKA reduced CREB phosphorylation and renin expression, confirming a causal signaling cascade. Thus, Kv1.3 links membrane excitability to RAAS activation via a Ca2+-dependent AKT–PKA–CREB signaling axis and represents a potential therapeutic target for hypertension and associated renal injury.

Biomolecules doi: 10.3390/biom16091260

Authors: Alaa A. El Moghrabi Ali Al Khatib Israa Ahmad Cheikh Charbel Al Hage Dima Ismail Mariam Zhour Philip Mwesigwa Nadine Darwiche

Cannabinoids are terpenophenolic compounds derived from Cannabis sativa L. that exert a broad range of biological and pharmacological activities. Increasing evidence highlights their potential as modulators of cancer progression specifically through the suppression of tumor cell growth, angiogenesis, and metastasis across multiple tumor models. This review provides a comprehensive overview of the molecular mechanisms by which natural and synthetic cannabinoids induce regulated cancer cell death. Current evidence demonstrates that cannabinoids regulate multiple forms of cancer cell death, including apoptosis, autophagy-dependent cell death, necroptosis, ferroptosis, and parthanatos. These effects are mediated through complex and interconnected signaling pathways such as TRIB3/AKT/mTORC1, PI3K/AKT/mTOR, MAPK/ERK, NF-κB, ERK/JNK/p38-MAPK, and ceramide/Raf1/ERK/ROS. In addition to their direct antitumor effects, cannabinoids can enhance the efficacy of conventional anticancer therapies through the coordinated regulation of complementary cell death pathways. They also provide clinically relevant supportive benefits in palliative care, alleviating chemotherapy-induced nausea, cachexia, and mood or sleep disturbances. Collectively, these findings identify cannabinoids as promising anticancer agents and therapeutic adjuvants, predominantly in the preclinical setting. However, significant challenges remain regarding their safety, optimal dosing, formulation, and clinical efficacy. Further mechanistic studies, rigorous preclinical research, and well-designed clinical trials are required to establish the translation of cannabinoid-based therapies into precision oncology.

Biomolecules doi: 10.3390/biom16091259

Authors: Aiming Jiang Juntong Liao Yinyin Shi Wenyan Song Sisi Luo Longjiang Li Zhuoyuan Zhang

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) remains a challenging complication of bisphosphonate therapy because jaw extraction sockets exposed to bisphosphonates represent impaired wound environments rather than ordinary bone defects. This narrative review summarizes clinical, cellular, animal, and biomaterial evidence on the mechanisms that limit BRONJ repair and discusses how these pathological barriers can inform local material design. Current evidence suggests that BRONJ repair is constrained by impaired osteoclast-mediated remodeling, osteocyte and osteoblast dysfunction, oxidative stress, unresolved inflammation, angiogenic insufficiency, microbial challenge, mucosal instability, and changes in bone material properties. Biomaterial strategies investigated to date include local delivery of regenerative factors, restoration of remodeling activity, extracellular vesicles, nucleic acid nanostructures, platelet-derived matrices, antibacterial and ion-releasing hydrogels, angiogenic or lymphangiogenic systems, and mechanically adaptive scaffolds. Most studies remain preclinical and are based on rodent extraction or mandibular defect models, and few establish a direct causal link between a specific material property and durable BRONJ resolution. Future materials should be judged not only by their ability to enhance bone formation, but also by whether they can re-establish a sealed, vascularized, immune-balanced, and remodeling-competent socket capable of sustained jawbone repair.

Biomolecules doi: 10.3390/biom16091258

Authors: Christina Papadouli Sofia Vardali Theodoros Karatzinos Fotis Lykotrafitis Myrto Maniaki Panagiota Panagiotaki George Rigos Ioannis Nengas Morgane Henry Chrysanthi Nikoloudaki Dimitra Kogiannou Petros Chronopoulos Eleni Golomazou

As aquafeed formulations include more plant-based materials, mycotoxin contamination is becoming relevant for fish farming. Deoxynivalenol (DON) and fumonisin B1 (FB1) frequently occur in fish feeds and may impair fish performance and health. This study evaluated the impact of DON and FB1 on growth and health parameters in gilthead seabream (Sparus aurata). Fish were fed ad libitum for seven weeks with six diets assessed in triplicate: DON A (300 ppb), DON B (2000 ppb), DON C (5000 ppb), FB1 A (5 ppb), FB1 B (10 ppb), FB1 C (40 ppb). The highest contamination levels significantly reduced feed intake, body weight, total length, and biomass, while body weight was also reduced in the lowest FB1 treatment. The feed conversion ratio increased in exposed fish, whereas the specific growth rate decreased in DON B, DON C, FB1 A, and FB1 C. Both mycotoxins altered hematological profiles, immune-related enzymatic activities, and liver morphology. DNA damage increased with exposure level and was greatest at the highest DON concentration. Overall, this study shows that short-term dietary exposure can impair seabream performance and health, supporting stricter mycotoxin monitoring in fish feeds and research on chronic exposure, recovery, and species-specific safety thresholds.

Biomolecules doi: 10.3390/biom16091257

Authors: Yuchen Wu Ginam Cho Youngshin Lim

Inter-organelle membrane contact sites (MCSs) enable direct communication between organelles, and this communication is fundamental to cellular homeostasis, coordinated calcium (Ca2+) signaling, lipid metabolism, energy production, and stress responses. While MCSs are evolutionarily conserved, emerging evidence indicates that their organization and function are highly context dependent. In the brain, neurons and glial cells differ markedly in their physiological roles, morphologies, and metabolic demands, suggesting that inter-organelle contact networks might be specialized in a cell-type-dependent manner. Although much of the existing literature focuses on neurons, growing evidence indicates that these contact sites also play important roles in glial cells. Here, we review recent advances in our understanding of MCSs in the nervous system, focusing on cell-type-specific differences between neurons and glial cells. We highlight the spatial specialization of MCSs within neurons, emphasizing how subcellular localization shapes their functional output. Our analysis of the current literature suggests that neuronal MCSs are primarily optimized for rapid Ca2+ signaling and metabolic adaptation, whereas glial MCSs preferentially coordinate lipid metabolism, inflammatory signaling, and tissue homeostasis. We also review the context-dependent and disease-driven remodeling of MCSs in the brain, reflecting alterations in contact-site composition and function rather than simply increased or decreased organelle proximity. Furthermore, we discuss emerging therapeutic perspectives aimed at modulating inter-organelle communication in multiple neurological diseases and outline key unresolved questions and future directions necessary to elucidate how inter-organelle contact sites shape brain physiology and disease. Collectively, the evidence reviewed here indicates that MCSs serve as dynamic signaling platforms, with their specific physiological and pathological functions varying according to cell type, subcellular localization, and molecular composition.

Biomolecules doi: 10.3390/biom16091256

Authors: Mingyan Jin Chunliang Liu Song Lyu Aoxue Li Kesheng Dai Jun Wan

The catheter is among the most commonly used blood-contacting medical devices, but its use can induce surface-mediated coagulation activation, leading to catheter-related thrombosis (CRT). The occurrence of CRT causes venous thromboembolism and catheter malfunction, but current antithrombotic strategies have unsatisfactory efficacy and safety profiles. Here, we review recent advances in the understanding of the pathology of CRT, particularly the roles of the contact pathway factors, and promising novel therapeutic options. Recent studies using genetically modified animals, factor-deficient plasmas, specific inhibitors and purified systems demonstrated an important contribution of contact pathway factors XII and XI to catheter-related blood clotting. Accordingly, contact pathway inhibition has efficacy comparable to that of heparins in mitigating catheter-related coagulation or intraluminal occlusion in various in vitro and animal models, while having lower bleeding risk. Early human studies suggest potential thromboprotective effects of FXI inhibition in catheter placement and hemodialysis settings. However, inhibition of factors XII or XI may impair the defense against infection or disturb normal cardiac function, respectively. Larger human trials are needed to further confirm the efficacy and safety of these contact pathway inhibitors, and to explore whether low-dose combinations of contact pathway inhibitors with heparins are more effective for CRT protection.

Biomolecules doi: 10.3390/biom16091255

Authors: Xiaojia Guo Yanzhe Huang Yinghan Hu Lingyun Zhang Zongbao K. Zhao

The ubiquitous nicotinamide adenine dinucleotide (NAD) engages in diverse biological processes, leading to non-selective energy transfer toward target synthetic reactions. To achieve selective energy transfer in complex biological systems, we previously constructed artificial systems mediated by the non-natural cofactor nicotinamide cytosine dinucleotide (NCD), which can be specifically recognized by engineered enzymes with minimal cross-talk with natural cofactors. For enhanced energy transfer and higher product yields, efficient conversion of NCD to NCDH is required to deliver reducing power in NCD-mediated biosynthetic pathways. Here, we established a comprehensive strategy for selective reduction in intracellular NCD. First, coupled enzymatic colorimetric assays with high specificity were validated for quantifying NAD, NADP, and NCD. With phosphite as the energy source, we selectively elevated the intracellular NCDH/NCD ratio with minimal perturbation to NADH/NAD and NADPH/NADP ratios in E. coli. To overcome the limitation of phosphite transmembrane transport, cell-free systems were constructed to confirm that phosphite could drive near-complete NCD reduction. Finally, cells were treated with polymyxin B, which promoted phosphite uptake and thereby enabled maximal reduction in intracellular NCD. An NCDH/NCD ratio of 47 was achieved, demonstrating that 98% of the intracellular NCD pool existed in the reduced form. This work demonstrates that NCD can function as an independent redox cofactor for selective regulation, providing viable strategies for artificial cofactor-driven biosynthesis.

Biomolecules doi: 10.3390/biom16091254

Authors: Pere Domingo Paula Prieto

The spectrum of diseases in individuals with human immunodeficiency virus (HIV) receiving successful antiretroviral therapy has evolved over time. In the past, they developed opportunistic infections and malignancies, whereas today, cardiovascular disease is among the most common causes of illness and premature death. Traditional risk factors for atherosclerosis (hypertension, hyperlipidemia, smoking, diabetes, family history of heart disease) are more prevalent in people with HIV than in the general population. However, it is well established that HIV itself causes increased immune activation, chronic inflammation, vascular dysfunction, and a cluster of metabolic abnormalities that contribute to a faster-than-usual rate of biological aging and a higher risk of developing atherosclerosis, a risk not fully captured by current risk models. In the REPRIEVE study, treatment with pitavastatin was shown to reduce the rate of first cardiovascular events among individuals with HIV receiving antiretroviral therapy. Importantly, the beneficial effects of statins on atherosclerosis likely extend beyond lowering cholesterol to include effects on vascular function and on immune and metabolic systems altered by HIV. Even among individuals on statins, a considerable risk of cardiovascular disease remains. Here, We provide a narrative review of current evidence and an expert perspective on emerging approaches to residual cardiovascular risk after REPRIEVE. We review the current understanding of atherosclerosis pathogenesis in individuals with HIV, focusing on recent findings from the REPRIEVE trial. We outline current approaches to improving cardiovascular risk assessment across clinical, biological, and computational levels. We also examine a growing number of therapeutic options that address residual inflammation and atherogenic metabolic disturbance in individuals with HIV on long-term, effective antiretroviral therapy. Significantly, after REPRIEVE, we must move from prescribing statins to all individuals with HIV toward more individualized cardiovascular disease prevention strategies, integrating clinical information, a variety of biomarkers, imaging studies, and even molecular information to generate optimal individualized cardiovascular disease prevention regimens that reflect the complexity of this outcome in naturally diverse individuals.

Biomolecules doi: 10.3390/biom16091253

Authors: Keyu Guo Xiaofang Chen Chenyin Wang Yaru Liu Chao Liu Yexiang Wu Xiuyong Fan Yanni Xu Shuyi Si Yongxin Zhang Jing Zhang

Rising drug resistance undermines current anti-influenza virus therapies. Although targeting the hemagglutinin (HA)–sialic acid receptor interaction is a promising strategy, progress is impeded by the lack of subtype-independent screening models. Herein, we established a fluorescence-based cell high-throughput model using fluorescein isothiocyanate-conjugated Sambucus Nigra Lectin (FITC-SNA) as a stable HA surrogate and α-2,6-sialyltransferase (ST6GAL1)-overexpressing MDCK cells to mimic the HA–receptor interface. This platform was designed to serve as an efficient primary screening tool to rapidly filter large compound libraries for potential binders to the receptor-binding interface. Screening 10,000 compounds identified Obatoclax Mesylate and Ethylparaben as primary hits. Both exhibited broad-spectrum HA inhibition activity, validating the model’s capability to identify compounds interfering with viral attachment. Further cellular antiviral assays revealed cytotoxicity for both compounds, resulting in low selectivity indexes (SIs), indicating that while these molecules effectively target the interaction site, they require substantial structural optimization for therapeutic use. Molecular docking confirmed their binding to type A H1N1, H3N2, and B/Victoria HA proteins, while ADMET predictions highlighted specific structural optimization needs to mitigate toxicity. In conclusion, this subtype-independent, highly specific high-throughput screening (HTS) model provides an efficient and reliable platform for early-stage influenza drug discovery and lead compound development.

Biomolecules doi: 10.3390/biom16091252

Authors: Youyou Zheng Kent A. Lee Unimunkh Uriyanghai Christine Wai Mihaela Mocanu Anthony Z. Yang Huanjuan Su Lianxia Li Vinay A. Sudarsanam John S. Poulton Prabir Roy-Chaudhury Gang Xi

Uremic conditions are common in end-stage kidney disease (ESKD) patients. Accelerated vascular diseases in uremic patients lead to heart failure, stroke, and hypertension. To investigate the effects of uremia on porcine arterial smooth muscle cells (aSMCs), bulk RNA sequencing was used to identify uremia-induced alterations in signaling pathways of aSMCs that might explain the aggressive cardiovascular diseases seen in patients with chronic kidney disease (CKD) and ESKD. Bulk RNA sequencing was performed on porcine aSMCs cultured with serum from normal or uremic pigs. Differentially expressed gene (DEG) analysis revealed that 295 genes were upregulated and 138 genes were downregulated after uremic serum exposure. Gene Ontology molecular function analysis demonstrated that ATP-dependent activity, translation factor activity, and ATP-dependent protein folding chaperones were predicted to be negatively enriched after uremic serum exposure, while proton transmembrane transporter activity, antioxidant activity, and glutathione peroxidase activity were predicted to be positively enriched. Gene set enrichment analysis indicated that the cell cycle was predicted to be negatively enriched after uremic serum exposure in aSMCs. Overrepresentation analysis found that focal adhesion, protein processing in the endoplasmic reticulum (ER) and cell senescence were predicted to be negatively enriched, while lysosome, phagosome, apoptosis, and autophagy were predicted to be positively enriched after uremic serum exposure. This study suggests that the signaling pathways that regulate cellular redox homeostasis, the cellular waste disposal system, ER stress and autophagy are major signaling pathways involved in aSMCs’ responses to uremic serum exposure. These pathways may contribute to the severe arterial-specific clinical symptoms observed in CKD/ESKD patients, such as arterial stiffness, vascular calcification and cardiovascular disease.

Biomolecules doi: 10.3390/biom16091251

Authors: Noa Fine-Shamir Avital Beig Arik Dahan

Developing effective oral formulations for poorly soluble anticancer drugs remains a major pharmaceutical challenge due to the combined limitations of solubility, permeability, and efflux transporter activity. In this work, we investigated the influence of the nonionic surfactants Cremophor EL, Pluronic P-85, and Pluronic F-68 on the solubility and intestinal permeability of the anticancer drug etoposide. All surfactants significantly increased etoposide aqueous solubility. While in vitro permeability across an artificial membrane demonstrated the expected solubility-permeability tradeoff, in vivo SPIP studies in rats revealed a distinctive solubility-permeability interplay for Cremophor EL and Pluronic P-85, which simultaneously enhanced solubility and permeability, likely through P-gp inhibition. In contrast, Pluronic F-68 exhibited the classical solubility-permeability tradeoff, consistent with its reported negligible P-gp inhibitory activity. Mechanistic analysis indicated that surfactant hydrophobicity and molecular weight critically influence P-gp inhibition via ATPase modulation. Surfactants with higher hydrophobicity and moderate molecular weight can integrate into the phospholipid bilayer, enabling direct interaction with P-gp and disruption of its ATPase function. These findings provide strategic insights for the rational design of oral formulations capable of overcoming the solubility-permeability tradeoff, improving the bioavailability of challenging anticancer drugs, and may facilitate the transition from intravenous to oral chemotherapy.

Biomolecules doi: 10.3390/biom16091250

Authors: Baojian Yu Zekai Shao Zhuona Ni Yuxin Gao Ziyang Ding Weifeng Jiang Lin Li Lisheng Chu

The neurovascular unit (NVU) is composed of a diverse array of cells and an extracellular matrix (ECM). Neural cells and blood vessels are intricately interconnected, forming a cohesive whole. Specific cellular components and structures within the NVU play an indispensable role in maintaining homeostasis of the central nervous system (CNS). With the advancement and maturation of cell co-culture technology, various three-dimensional (3D) NVU models continue to emerge, offering a more objective and comprehensive perspective for in vitro studies of CNS diseases. Specifically, these 3D NVU models include Transwell Chamber models, gel-polydimethylsiloxane (PDMS)-based 3D models, self-assembled NVU models and microfluidic NVU models, which reconstruct the complex NVU architecture to varying degrees. This review systematically summarizes multiple 3D construction strategies for in vitro NVU to overcome the limitations of conventional cellular tests or animal experiments, highlights the critical roles of biomimetic gel in recapitulating native cell-gel crosstalk, comparatively analyzes four major 3D NVU technical routes in terms of cellular composition, vascular morphology, barrier performance, and reproducibility, categorizes application scenarios of 3D NVU platforms oriented to practical research demands, including oxygen-glucose deprivation/reoxygenation (OGD/R) injury modeling, blood-brain barrier (BBB) permeability assay, CNS drug penetration screening, neuroinflammation and neurotoxicity evaluation, proposes practical principles for model selection under different experimental purposes, and concludes with current bottlenecks, including imperfect vascular network maturation and lack of unified evaluation criteria, together with future perspectives for standardized 3D NVU in vitro. By comparing the advantages and limitations of these approaches, we aim to clarify their optimal applicability for investigating specific pathological mechanisms and screening potential therapeutics.

Biomolecules doi: 10.3390/biom16091249

Authors: Yuwei Ge Qianqian Wang Yuying Fu Tingting Wang Huajun Liao Chongchong Yan

Potato (Solanum tuberosum L.) seedlings are highly sensitive to freezing temperatures, which severely impairs growth and causes substantial losses in tuber yield and quality. This study aimed to screen freezing-tolerant potato germplasm accessions, identify candidate genes involved in the freezing response, and provide elite parental materials and a theoretical reference for molecular breeding of freezing-tolerant cultivars. We evaluated seedling freezing tolerance of 73 potato accessions using the freezing damage index (FDI), and performed transcriptome sequencing on leaf samples from highly freezing-tolerant (HT) accession 15-1881 and highly freezing-susceptible (HS) accession B8 following freezing treatment. Substantial variation in freezing tolerance was observed across the germplasm panel, with 5 HT accessions and 12 HS accessions identified, showing marked phenotypic and physiological differences under freezing stress. Transcriptomic analysis detected 6560 differentially expressed genes (DEGs) in 15-1881 and 5161 DEGs in B8, with 3558 DEGs specific to 15-1881. The phenylpropanoid biosynthesis pathway exhibited noticeable expression divergence between 15-1881 and B8, harboring 17 15-1881-specific DEGs including three tandem phenylalanine ammonia-lyase (PAL) genes. The HT germplasm accessions obtained in this study provide breeding resources for freezing-tolerant potato improvement, and the PAL genes characterized here represent promising candidate genes associated with freezing response in potato.

Biomolecules doi: 10.3390/biom16091248

Authors: Amelia T. Y. Yam Yuek Ling Chai Saima Hilal Cai Yuan Vincent C. T. Mok Narayanaswamy Venketasubramanian Boon Yeow Tan Ming Ann Sim Mitchell K. P. Lai Christopher P. Chen Joyce R. Chong

Dysregulated insulin signaling in the brain has been linked to cognitive impairment and dementia. Insulin-like growth factor 1 (IGF-1) is a peptide growth hormone crucial for neurogenesis and neuroprotection. Findings regarding potential involvement of IGF-1 in dementia have been conflicting, and the status of IGF-1 in clinical cohorts with Alzheimer’s disease (AD) and concomitant cerebrovascular disease (CeVD) burden is unknown. A Singapore-based memory clinic cohort consisting of 46 non-cognitively impaired (NCI), 101 with cognitive impairment, no dementia (CIND) and 81 AD dementia subjects underwent plasma IGF-1 measurements and neuroimaging assessments for association analyses of peripheral IGF-1 with regional brain volumes, as well as with neuroimaging CeVD markers (lacunes, cerebral microbleeds, white matter hyperintensities). Plasma IGF-1 levels were significantly lower in AD compared to NCI and CIND participants (both p < 0.001). Plasma IGF-1 was significantly associated with smaller hippocampal (p = 0.035), amygdala (p = 0.024), parietal lobe (p = 0.029), and frontal lobe (p = 0.002) volumes. In contrast, plasma IGF-1 did not associate with CeVD markers after covariate adjustments. Our findings suggest that plasma IGF-1 may be a blood-based biomarker for reduced brain volumes, while having no direct role in CeVD pathophysiology.

Biomolecules doi: 10.3390/biom16091247

Authors: Dániel Vörösvácki Nikolett Szakállas Alexandra Kalmár István Takács Béla Molnár

Transposable elements (TEs) account for nearly half of the human genome and shape chromatin organization, gene regulation, and genome evolution. However, their contributions to human physiology and disease remain incompletely understood. The most active elements in humans, LINE-1 (L1), Alu, and SVA, retain some copies with the ability to evade epigenetic repression and mobilize via target-primed reverse transcription (TPRT), whereas copies become inactive through various fragmentations and mutations. TE activity contributes to genomic instability and has been implicated in aging, cancer, neurological disorders, chromatin organization, and epigenetic regulation. Studying TE is challenging due to their repetitive and polymorphic nature. Recent advances in sequencing technologies and short- and long-read sequencing platforms, combined with specialized bioinformatic pipelines, currently enable more comprehensive characterization of TE insertions, deletions, expression, and epigenetic status. Computational approaches vary in sensitivity, specificity, and resource requirements, and their performance is influenced by sequencing modality, coverage, and the reference genome used. Assembly-based and read-based methods, as well as integrating methylation data or single-cell data, provide complementary insights into TE biology. This review summarizes the biology of active human TE, surveys state-of-the-art short- and long-read pipelines for TE analysis, and highlights their applications in studies of aging, cancer, and other complex diseases. We also provide practical guidance for selecting appropriate sequencing strategies and tools for TE-focused projects, and discuss emerging approaches and open questions in the field.

Biomolecules doi: 10.3390/biom16091246

Authors: Ratan K. Chaudhuri Raja K. Sivamani

Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease characterized by epidermal barrier dysfunction, immune dysregulation, microbial imbalance, and severe pruritus. Emerging evidence establishes that barrier disruption is a central pathogenic driver capable of initiating inflammatory signaling, neuroimmune activation, and chronic disease instability. This understanding has shifted therapeutic paradigms toward barrier-directed strategies aimed at restoring epidermal resilience. This narrative review evaluates the mechanistic and clinical evidence surrounding isosorbide fatty acid diester molecules—specifically isosorbide dicaprylate (IDC) and isosorbide di-(linoleate/oleate) (IDL)—as a barrier-first approach for AD management. Early in vitro and ex vivo investigations demonstrated that IDC significantly improves epidermal hydration, transepidermal water loss, and the expression of barrier-associated genes linked to epidermal integrity. Subsequent studies showed that IDL expands these effects through coordinated regulation of keratinocyte differentiation, lipid homeostasis, and inflammatory stress pathways. Furthermore, recent mechanistic data highlight synergistic anti-inflammatory and pruritus-modulating effects involving TRPA1-, TRPV3-, and TSLP-associated pathways, while preserving tissue integrity under cytokine-induced stress. Clinically, these findings are supported by randomized studies in pediatric and adult cohorts demonstrating significant reductions in pruritus, favorable Eczema Area and Severity Index (EASI) responses, decreased topical corticosteroid dependence, and a reduction in the relative abundance of Staphylococcus aureus. Collectively, these findings support a barrier-first therapeutic framework in which restoration of epidermal resilience may beneficially influence multiple interconnected pathways involved in atopic dermatitis.

Biomolecules doi: 10.3390/biom16091245

Authors: Ayesha Jabeen Ilaria Barison Honoria Ocagli Bruna Fata Diego Perazzolo Cristina Basso Roberto Luisetto Silvia Pozzo Fabrizio Mancin Enrico Grisan Dario Gregori Annalisa Angelini Marny Fedrigo Chiara Castellani

Background: Heart failure remains a leading cause of morbidity and mortality, and current therapies rarely repair established myocardial damage. Nanoparticle-based interventions have been investigated across heterogeneous models of myocardial injury, remodeling, cardiomyopathy, and heart failure, but the distribution and translational maturity of this evidence remain unclear. Methods: A systematic search of PubMed, Embase, Scopus, and Web of Science was conducted from database inception to June 2024. Eligible reports were mapped according to disease model, experimental system, carrier-level nanoparticle platform, payload, route, comparator, outcomes, biodistribution, safety assessment, and translational characteristics. Reports of non-therapeutic nanoparticle exposure were retained in a separate contextual safety/toxicology stratum and were not included in the therapeutic evidence-density map. Risk of bias was evaluated using design-appropriate tools. Results: Of 2640 records screened, 157 independent studies met the criteria: 140 in the main therapeutic/platform evidence map and 17 in a separate contextual safety/toxicology stratum. Within the main corpus, polymeric systems were the largest platform class (n = 50), followed by inorganic/mineral (n = 35), biological/biomimetic (n = 24), lipid-based (n = 23), carbon-based (n = 5), and hybrid/multicomponent systems (n = 3). Evidence was concentrated in acute myocardial injury (n = 76), while direct same-agent comparisons, long-term safety assessment, repeated dosing, quantitative biodistribution, and clinically aligned heart-failure models remained limited. Conclusions: The field demonstrates substantial formulation diversity and biological activity, but translation is constrained by fragmented characterization, sparse comparative evidence, and incomplete assessment of biological fate and safety.

Biomolecules doi: 10.3390/biom16091244

Authors: Jessica Costa Andrea Atrei Juan José Valle-Delgado Monika Österberg Rebecca Pogni

Laccases are multicopper oxidases capable of oxidizing a wide range of substrates using molecular oxygen as the terminal electron acceptor, producing water as the sole by-product. High-redox potential fungal laccases, such as those from Trametes versicolor, are particularly attractive for industrial and environmental applications, although their use is often limited by sensitivity to operational conditions. Enzyme immobilization represents an effective strategy to enhance laccase stability and reusability. In this work, magnetic nanoparticles (MNPs) were investigated as support for laccase immobilization due to their high surface area, biocompatibility, and ease of magnetic recovery. Two modified co-precipitation synthetic routes were systematically evaluated, and the size, morphology, and chemical composition of the products were characterized by microscopy, light scattering, and spectroscopic methods, while both adsorption and covalent immobilization strategies were explored. The MNP surface was found to be highly reactive toward radical species generated during laccase-catalyzed reactions, especially in the presence of small Fe2+ excess. While this can enhance the enzyme catalytic activity, it challenges the inertness of the support and promotes, in some cases, strong interactions between reaction products and the nanoparticle surface. These findings highlight a previously unexplored role of magnetic supports in laccase-based biocatalytic systems.

Biomolecules doi: 10.3390/biom16091243

Authors: Rachid Anane Su Melser Elodie Renouf Rachid Ennamany Jean-Michel Mérillon

Hibiscus plant cell cultures were developed to produce a cosmetic ingredient with anti-ageing properties. UHPLC-DAD-MS analysis of Hibiscus plant cell cultures revealed a high content of polyphenols, particularly hydroxycinnamic acid derivatives, including caffeoyl and p-coumaroyl conjugates. The biological activity of a 50/50 mixture of extracts from Hibiscus syriacus and Hibiscus rosa-sinensis cells was investigated in a human keratinocyte/fibroblast co-culture model, which better reproduces the reciprocal epithelial–mesenchymal interactions between epidermal keratinocytes and dermal fibroblasts than monocultures, using quantitative data-independent acquisition (DIA) LC-MS/MS proteomics combined with functional enrichment and protein–protein interaction analyses. A total of 7062 proteins were identified, of which 280 were differentially expressed (107 upregulated and 171 downregulated) following hibiscus treatment. The proteomic profile suggested coordinated molecular reprogramming associated with extracellular matrix remodelling, tissue repair, hydration, and attenuation of inflammatory signalling. Functional enrichment analysis revealed coordinated modulation of extracellular matrix organization, glycosaminoglycan metabolism, lysosomal function, cell communication, and inflammatory signalling. Upregulation of extracellular matrix and adhesion proteins, including lumican, collagen VIII, fibulin-5, syndecans, and glypicans, suggested coordinated extracellular matrix remodelling that may promote skin firmness and elasticity, while the downregulation of inflammatory regulators, including CARD16 and S100 family proteins, suggested attenuation of innate inflammatory responses. Overall, these findings provide mechanistic insights into the biological activity of Hibiscus cell culture extracts and support their potential as cosmetic ingredients promoting skin homeostasis and healthy skin ageing.

Biomolecules doi: 10.3390/biom16091242

Authors: Gennady N. Chuev Timur V. Mamedov Dmitry O. Morozov

Hydration plays an essential role in protein–protein interactions. Coarse-grained modeling provides an efficient way to treat hydrated protein complexes without the use of extra-large computational resources. To enhance the capabilities of coarse-grained modeling, we developed an integral equation theory based on the solution of the Ornstein–Zerinke equation to evaluate the hydration structure of peptides and proteins within the framework of coarse-grained modeling. Our current version is based on the SPICA force field, which considers distance-dependent interaction potentials between solvent particles and amino acid segments. Our approach involves two key procedures: an accurate estimation of the structure factor of the uniform fluid and the specific construction of bridge functions obtained from MD simulations. The use of a special hybrid closure allows us to reproduce not only the details of the structure factor, but also the isothermal compressibility obtained from the simulations. The developed bridge functions include two components: an analytical repulsive contribution, which is primarily responsible for the thermodynamic properties, and an attractive contribution obtained from MD simulations. The main assumption in the construction is that the contribution of individual amino acids to the attractive bridge function is additive. By parameterizing the bridge functions, we reproduced details of the hydration structure and accurately calculated the hydration energy for various peptides and proteins. Our method is computationally inexpensive and appears to be suitable for the rapid processing of hydrated proteins of any size.

Biomolecules doi: 10.3390/biom16091241

Authors: Aodi Fan Xinwei Chen Ke Yang Xuefang Ma Haohao Gao Binyan Wang Guanwei Fan Lan Li

The heart–kidney axis has emerged as a central framework for understanding the bidirectional interactions that drive cardiorenal syndrome and broader cardiovascular–kidney–metabolic (CKM) disease. However, existing interpretations have often emphasized hemodynamic and neurohormonal mechanisms without fully integrating the growing evidence for endocrine, inflammatory, and metabolic mediators that coordinate injury across organs. This Review was therefore undertaken to provide an updated and clinically relevant synthesis of the physiological basis of heart–kidney communication, the mechanisms underlying its disruption, and the therapeutic implications of these insights. To achieve this aim, we performed a systematic narrative review of the literature using PubMed, Embase, Web of Science, and Scopus for studies, supplemented by manual screening of reference lists. Priority was given to original studies, large cohort analyses, randomized controlled trials, meta-analyses, and authoritative reviews. We integrated evidence spanning physiological regulation, maladaptive signaling pathways, emerging mediators, experimental models, and evolving treatment strategies. The reviewed evidence indicates that heart–kidney crosstalk is driven not only by altered perfusion and venous congestion, but also by sustained activation of the renin–angiotensin–aldosterone system (RAAS) and sympathetic nervous system (SNS), inflammation, oxidative stress, mitochondrial dysfunction, anemia, uremic toxins, and disordered mineral metabolism. Among novel mediators, fibroblast growth factor 23 (FGF23) emerges as a major bone-derived, chronic kidney disease (CKD)-associated endocrine mediator linking renal injury to cardiac hypertrophy, fibrosis, calcium mishandling, and diastolic dysfunction, whereas Klotho appears to exert counter-regulatory protective effects. Heart-derived natriuretic peptides, including atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), remain important modulators of renal blood flow, natriuresis, and volume homeostasis. We further highlight the translational relevance of newer biomarkers and therapies, including sodium–glucose cotransporter 2 inhibitors (SGLT2is), glucagon-like peptide-1 receptor agonists (GLP-1 receptor agonists), and mineralocorticoid receptor antagonists (MRAs), which may help address cardiac and renal dysfunction in parallel. Overall, this Review supports a revised conceptual model in which the heart–kidney axis is governed by multidirectional hemodynamic, neurohormonal, immune, and endocrine signaling networks. A more integrated understanding of these mechanisms may improve biomarker discovery, refine risk stratification, and promote therapies that target both organs simultaneously.

Biomolecules doi: 10.3390/biom16091240

Authors: Bence György Gombás Erika Gábor Viktor Honti Orsolya Németh-Szatmári Ferenc Jankovics Zoltán Villányi

Assemblysomes are ribosome-nascent chain condensates that regulate co-translational processes through liquid–liquid phase separation, yet the sequence determinants underlying their formation remain incompletely understood. Previous studies identified the DNA helicase Sgs1 as an assemblysome-associated protein; however, whether its N-terminal region possesses intrinsic phase separation propensity has not been experimentally examined. Here, we investigated the first 135 amino acids of Sgs1 using a light-inducible optoDroplet assay. A mCherry–Cry2–Sgs11–135 fusion construct was compared with the established positive control FUS–mCherry–Cry2 and the negative control mCherry–Cry2 in live HEK293T cells. Following blue-light activation, Sgs11–135 reproducibly formed reversible condensates, indicating intrinsic phase separation propensity. Quantitative image analysis revealed light-dependent increases in condensate number, average condensate area, and integrated condensate fluorescence intensity. Compared with FUS, Sgs11–135 formed slightly fewer and smaller condensates but displayed reproducible light-dependent condensate formation. These findings indicate that the Sgs1 N-terminal region exhibits intrinsic phase separation propensity in a validated optogenetic assay. Although this proof-of-principle study does not establish the molecular mechanism of assemblysome formation, the results are consistent with the hypothesis that the Sgs1 N-terminus may contribute to the multivalent interactions underlying assemblysome organization.

Biomolecules doi: 10.3390/biom16091239

Authors: Qiaohua Li Shaoxuan Liu Jingxing Gao Jie Li Hong Shi

Primary human epidermal melanocytes are valuable models for pigmentation research, but maintaining prolonged expansion together with stable melanocytic characteristics can be challenging. Here, primary melanocytes were transduced with an hTERT-overexpression lentiviral vector to generate a mixed population (MIX) and three single-cell-derived clones (SC1, SC3, and SC4). RNA sequencing was performed on passage-6 primary melanocytes, MIX cells, SC clones, and three melanoma cell lines (A375, WM-115, and SK-MEL-1). hTERT overexpression extended melanocyte culture lifespan by more than 10 passages. TERT expression increased in MIX cells and was highest in SC clones. Principal component and gene-expression analyses showed that MIX and SC cells remained globally closer to primary melanocytes than to melanoma cell lines, without an evident melanoma-like transcriptomic shift. However, lifespan extension and clonal selection were accompanied by marked morphological changes and broad transcriptional remodeling involving cell-cycle regulation, telomere-associated processes, antiviral responses, extracellular matrix organization, adhesion, dendrite-related programs, and pigmentation. Melanocyte differentiation and pigmentation programs were generally reduced, particularly in clonal cultures. These findings support hTERT-extended melanocytes as expandable in vitro models, while highlighting that hTERT expression and single-cell cloning can induce functional model drift.

Biomolecules doi: 10.3390/biom16091238

Authors: Shuying Fu Yuhuan Meng Keying Liang Meiying Feng

Skeletal muscle regeneration depends on coordinated transitions of muscle stem cells (MuSCs), also known as satellite cells, from quiescence through activation and proliferative expansion to differentiation and fusion, while self-renewal replenishes the quiescent MuSC pool within a dynamically remodeled niche. Fibroblast growth factor (FGF) signaling regulates these transitions, but its effects vary as MuSCs and their niche change across regenerative stages. FGF output is shaped by ligand availability and extracellular presentation, fibroblast growth factor receptor (FGFR) isoform expression and coreceptor availability, receptor trafficking, intracellular feedback, and the state of the responding cell. Following acute injury, FGF inputs can support MuSC activation and expansion; signaling is subsequently reconfigured during differentiation, fusion, self-renewal, and return to quiescence. Aging-associated regenerative decline, chronic injury and dystrophic remodeling, denervation, and metabolic dysfunction disrupt this coordination and can uncouple FGF activity from productive repair. Rhabdomyosarcoma provides a distinct malignant context in which the FGF network is rewired to sustain oncogenic myogenic cell states. Here, we integrate molecular, cellular, and niche-level evidence across these settings to explain why FGF signaling produces divergent outcomes and to clarify how cellular context and timing should inform therapeutic modulation.

Biomolecules doi: 10.3390/biom16091236

Authors: Omer Unal Nilufer Akgun-Unal

Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia, and the diaphragm—the primary respiratory muscle—is adversely affected by this diabetic process. The aim of this study is to investigate the effects of zinc sulfate (ZnSO4) treatment on diaphragm muscle contractile dynamics, calcium homeostasis, apoptosis, and fibrosis in an 18-month-old female Type 2 diabetic rat model. Thirty-two 18-month-old female Wistar rats were randomly divided into four groups: Control (CON), CON + ZnSO4, Diabetes Mellitus (DM), and DM + ZnSO4. The DM model was induced by a high-fat diet and administration of 30 mg/kg streptozotocin (STZ); after the disease was confirmed, ZnSO4 was administered intraperitoneally at a daily dose of 10 mg/kg to the treatment groups. The mechanical functions of the diaphragm muscle were evaluated using a post-rest potentiation protocol in an isolated organ bath; qPCR analyses (Caspase-3, TGF-β1, SERCA2a) were performed to investigate cellular apoptosis, fibrosis, and calcium regulation. Compared with the CON group, the DM group exhibited a severe ~90% reduction in diaphragmatic contraction force (CF) and a ~97% decline in maximal contraction/relaxation velocities (±dF/dtmax) (p < 0.0001), which strongly correlated with a 30% suppression of SERCA2a gene expression (p < 0.01). Concomitantly, apoptotic Caspase-3 (~2.6-fold) and profibrotic TGF-β1 (~3.1-fold) mRNA levels were significantly elevated (p < 0.0001). In the DM + ZnSO4 group, daily zinc treatment (10 mg/kg/day, i.p. for 6 weeks, initiated 4 weeks after diabetes confirmation) did not reverse the elevated Caspase-3 and TGF-β1 expressions (p > 0.05). However, SERCA2a expression was fully preserved back to control levels (p < 0.05 vs. DM), leading to a substantial ~3-fold improvement in CF and velocities (p < 0.05 to p < 0.0001 vs. DM). On the other hand, the healthy CON + ZnSO4 group exhibited a physiological slowing of contractility (~53% decrease in CF), without histological damage, likely due to a competitive antagonism between excess divalent zinc (Zn2+) and calcium (Ca2+) on myofilaments. Although zinc cannot reverse the structural apoptotic and fibrotic remodeling in the aged diabetic diaphragm, it successfully rescues functional contractility by preserving SERCA2a transcriptional expression.

Biomolecules doi: 10.3390/biom16091237

Authors: Shijin Lan Ying Wang Yutong Fu Shixing Yang Quan Shen Xiaochun Wang Wen Zhang Deqiang Wang Likai Ji

Tripartite motif-containing protein 21 (TRIM21/Ro52) is a pivotal E3 ubiquitin ligase and cytoplasmic fragment crystallizable receptor (FcR). It plays a crucial role in viral infections, autoimmune disorders, and cancers by regulating multiple cell signaling axes, including the NF-κB, RIG-I-like receptor (RLR), cGAS-STING, and Toll-like receptor (TLR) pathways. Type I interferon (IFN-I), a pleiotropic cytokine, is produced via these immune signaling pathways, which are often triggered by viral components. TRIM21 both activates IFN-I signaling and mediates its negative feedback through post-translational modification of key immune signaling proteins, thereby maintaining immune homeostasis. In recent years, TRIM21 has been found to dually regulate autophagy and IFN-I in the context of virus–host interplay. Herein, we systematically summarize the functional roles of TRIM21 in virus-triggered intracellular immunity, aiming to provide insights for researchers and inspire further investigation in this area.

Biomolecules doi: 10.3390/biom16091235

Authors: Diana Culej Bošnjak Doris Janjić Petra Korać Mariastefania Antica Maja Matulić

The urokinase plasminogen activator or urokinase is a highly specific extracellular protease involved in numerous physiological and pathological processes. Its activity is a consequence of its interplay with its inhibitor, PAI1, and receptor, uPAR, and is finely regulated at several levels. The aim of the work was to investigate whether endoplasmic reticulum stress can modulate urokinase activity. Two tumor cell lines grown in cell culture were treated with Thapsigargin and sodium salicylate, inducers of ER stress response. Urokinase activity was determined in the conditioned media, and expression of uPA system molecules and molecules involved in response to ER stress in cell lysates was measured. ER stress influenced urokinase activity: while in the glioblastoma line its activity was increased, in breast cancer cells it was decreased. Differences in activity were a consequence of urokinase and PAI1 expression at the protein and RNA level. However, ER stress decreased cell migration, invasion, and proliferation regardless of the changes in urokinase activity. Gene expression analysis indicated that cell specific activation of some transcription factors and pathways could be responsible for different urokinase activity regulation.

Biomolecules doi: 10.3390/biom16091234

Authors: Mariarosaria De Rosa Theresa M. Heidenreich Libby Childs Benura Azeroglu Sneh M. Toprani Nader Aryamanesh Pablo Galaviz Hilda A. Pickett Eros Lazzerini Denchi Zachary D. Nagel Patricia L. Opresko

Telomeres are highly susceptible to oxidative DNA damage, particularly 8-oxoguanine (8-oxoG), which is processed by glycosylase-initiated base excision repair (BER). OGG1 removes 8-oxoG opposite C, and MUTYH removes A misinserted opposite 8-oxoG to prevent mutations. While OGG1 has an established role in telomere protection, the contribution of MUTYH to telomere stability in cancer cells after oxidative DNA damage remains poorly understood. Using a chemoptogenetic system to induce targeted 8-oxoG lesions specifically at telomeres in HeLa cancer cells, we demonstrate that MUTYH is required to prevent telomere shortening, telomere loss, and genomic instability after chronic damage. Yet, telomere damage in MUTYH-deficient cells does not cause sustained DNA damage signaling or reduced cellular proliferation. Whole-genome sequencing further reveals enrichment of G to T transversions within telomeric repeats in MUTYH-deficient cells, consistent with increased mutagenesis due to unrepaired 8-oxoG:A mispairs. Combined loss of MUTYH and OGG1 rescues damage-induced telomere aberrations and genomic instability, implicating BER-generated single-strand break (SSB) intermediates as major contributors to telomere instability. In agreement, exo-FISH and S1-END-seq analyses reveal that repair-proficient cells rapidly accumulate SSB intermediates after damage, which are later resolved, whereas glycosylase-deficient cells exhibit SSBs at later time points. Together, these findings identify MUTYH as a critical guardian of telomere integrity during chronic oxidative stress and provide insight into how defective BER at telomeres contributes to genomic instability in cancer cells, with implications for cancers associated with MUTYH deficiency and mutations.

Biomolecules doi: 10.3390/biom16091233

Authors: Yanxin Wang Zhenhao Zhao Tongyu Li Guoqi Liu Jiale Wang Zhoukun Li

β-1,6-Glucan is a functionally crucial polysaccharide of the fungal cell wall, although typically less abundant than β-1,3-glucan and chitin, its content, chain length, and branching vary considerably among species. Structurally, it serves as a covalent cross-linker tethering the external mannoprotein layer to the internal β-1,3-glucan-chitin network, contributing to cell wall integrity and plasticity. Biosynthetically, unlike chitin and β-1,3-glucan, which are synthesized by the plasma membrane-associated synthases, β-1,6-glucan biosynthesis depends on a multi-protein cooperative network spanning the endoplasmic reticulum (ER), Golgi, and cell surface, whose core catalytic machinery remains incompletely defined. Genetic and in vitro reconstitution studies have begun to delineate the contributions of ER-resident proteins (Kre5, Big1, Cwh41/Gls1, Rot2/Gls2, and Cne1), Golgi-localized Kre6/Skn1 family members, and cell-surface components (Kre9, Knh1, Kre1, and Kre11). Functionally, its biological roles are established by two complementary lines of evidence, namely enzymatic digestion by endogenous or exogenous β-1,6-glucanases and inactivation of the biosynthetic machinery. Collectively, these studies show that β-1,6-glucan is essential for cell wall architecture, GPI-anchored protein localization, fungal growth, morphogenesis, and virulence, and acts as a potent immunomodulatory molecule at the fungus–host interface. Elucidating its structure, biosynthesis, and function will advance fungal cell wall biology.

Biomolecules doi: 10.3390/biom16091232

Authors: Ching-Chieh Lin Yi-Chou Hou Po-Jen Hsiao Kuo-Cheng Lu

Bone remodeling is rhythmically regulated, yet the contribution of the circadian–melatonin axis to osteoporosis remains incompletely defined, in part because mechanistic findings obtained at high experimental concentrations are frequently extrapolated to physiological signaling. This narrative review examines that inference. PubMed/MEDLINE, Embase, Scopus, and Web of Science were searched from inception to July 2026 for English-language studies of melatonin, circadian clock genes, and bone; molecular, preclinical, epidemiological, and clinical evidence was appraised with attention to receptor dependence, exposure concentration, and study architecture. In osteoblast-lineage cells, melatonin promotes osteogenic differentiation through MT2-linked Wnt/β-catenin and MEK1/2–MEK5 signaling, post-translational stabilization of SP7, and modulation of the OPG/RANKL axis. By contrast, direct antiosteoclastic and antioxidant effects are usually reported at micromolar concentrations, four to six orders of magnitude above nocturnal plasma levels, and are increasingly attributable to receptor-independent chemistry converging on the ROS–KEAP1–NRF2 node shared with structurally unrelated antioxidant compounds. This exposure mismatch suggests that conventional oral doses engage receptor-mediated osteoblast pathways rather than reproduce high-dose antiresorptive effects; sustained exposure at or above 1 µM is not attainable by conventional oral administration, and the chronic safety of the doses that would be required has not been characterized. In humans, bone resorption has an intrinsic circadian rhythm, and night-shift work is associated with adverse skeletal outcomes, although causality remains unresolved. The five available randomized trials are small and heterogeneous; none was powered for fracture prevention, and none compared administration times for a skeletal endpoint. Melatonin therefore cannot currently be recommended for the treatment of osteoporosis. Human bone and marrow pharmacokinetics, receptor-specific in vivo dose–response experiments, and adequately powered monotherapy trials in established primary osteoporosis are the studies that would change this assessment.

Biomolecules doi: 10.3390/biom16091231

Authors: Eunjeong Lee Blaine Hunter Gordon Jasmina S. Redzic Anthony J. Saviola Sean P. Maroney Steven Shaw Mila Cordero Shaun Bevers Angelo D’Alessandro Kirk C. Hansen Sarah E. Clark Elan Eisenmesser

Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.

Biomolecules doi: 10.3390/biom16091230

Authors: Agnieszka Pałucha-Poniewiera

The use of animal models to study mental illnesses, such as depression, requires proper standardization and extensive expertise. Achieving good construct, face, and predictive validity in depression models is quite challenging. Currently, only a few environmental models, mostly based on chronic stress, and a limited number of genetic models fulfill these criteria. In the quest for new antidepressants, initial screening tests are employed as a preliminary step in research. While these tests do not always meet the requirements of a disease model, they are useful for the early identification of substances that may have antidepressant potential, paving the way for further studies based on established models. This approach to discovering antidepressants was originally designed for traditional medications, which typically act by modulating serotonergic, noradrenergic, and dopaminergic systems, and require multi-week administration to produce a therapeutic effect. In contrast, the new antidepressant ketamine offers rapid therapeutic effects following a single dose and exhibits distinctive behavioral outcomes in both screening tests and animal depression models. These effects have inspired a new model for the search for rapid-acting ketamine-like antidepressants. This review presents the behavioral effects of ketamine and discusses the methodologies used in the search for novel rapid-acting antidepressant drugs (RAADs).

Biomolecules doi: 10.3390/biom16091229

Authors: Assia Azouaghe Florence Back Walid Daoudi Abdelmalik El Aatiaoui Céline Spack Diana Potes Vecini David Hoogewijs

Photodynamic therapy (PDT) exploits photoactivated materials that generate reactive oxygen species (ROS) to induce selective cancer cell death. Nitrogen-doped carbon dots (N-CDs) have emerged as promising photosensitizers owing to their favorable optical properties, while hybridization with titanium dioxide (TiO2) may further enhance photoinduced ROS generation through improved charge separation. Here, we synthesized a series of N-CD/TiO2 hybrid composites with varying TiO2 content using a hydrothermal approach and systematically investigated the relationship between their physicochemical characteristics and biological activity. The hybrid materials were characterized by Fourier-transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, dynamic light scattering, and UV–visible spectroscopy. Among the formulations investigated, the composite containing 90% N-CDs and 10% TiO2 (N-CDs10T) exhibited the smallest hydrodynamic diameter, a relatively narrow particle size distribution, favorable optical properties, and the strongest irradiation-dependent biological responses. Biological activity was evaluated in A549 lung adenocarcinoma and Kelly neuroblastoma cells. Under dark conditions, all formulations displayed relatively low intrinsic cytotoxicity. Following irradiation with 365 nm UVA light, however, N-CDs10T induced a marked increase in intracellular ROS production, activation of antioxidant response element (ARE)-dependent signaling, disruption of cell-cycle progression, apoptosis-associated cell death, and inhibition of cell proliferation and migration. Kelly cells exhibited greater sensitivity than A549 cells, with IC50 values decreasing from 0.98 mg/mL under dark conditions to 0.52 mg/mL following irradiation. Collectively, these findings demonstrate that N-CD/TiO2 hybrid composites function as photoresponsive materials that enhance ROS-mediated cytotoxicity upon light activation. Beyond demonstrating phototoxicity, this study systematically links hybrid composition with oxidative stress signaling and multiple cellular responses, providing a comprehensive biological evaluation of N-CD/TiO2 hybrid materials. While additional studies are required to identify the predominant ROS, evaluate selectivity in non-malignant cells, and optimize activation at clinically relevant wavelengths, the present work establishes a proof of concept for the development of N-CD/TiO2 hybrid composites for photodynamic applications.

Biomolecules doi: 10.3390/biom16091228

Authors: Joanna M. Shepherd Lucy R. Gibb Hew D. T. Torrance Joanna Manson Daniel J. Pennington Paul Vulliamy Karim Brohi

The cellular immune response underlying post-injury multiple organ dysfunction syndrome (MODS) remains incompletely described. We hypothesized that early perturbations in innate lymphocyte behavior are critical to the development of MODS in trauma patients. To address this, we examined lymphocyte subsets in a prospective cohort of major trauma patients recruited at a single major trauma hospital. Circulating lymphocytes were profiled with flow cytometry in serial samples drawn within the hyperacute (≤2 h) and acute (24 h, 72 h) post-injury periods. Plasma levels of specific mediators derived from innate lymphocytes were also measured in a larger cohort. We observed a marked hyperacute increase in circulating NK (particularly the CD56dim subset) and Vδ1 cells that were associated with MODS or early mortality. Absolute counts of NK activation markers CD69 and NKG2D were also higher in patients with adverse outcomes, although the proportion of NK cells expressing NKG2D was reduced. Exploratory cluster analyses of NK activating and inhibiting receptors identified patient groups with differing injury characteristics and outcomes. In plasma, patients who developed MODS had significantly higher levels of NK-associated cytotoxic mediators and cytokines. Collectively, these data indicate a specific pattern of hyperacute NK cell activation after major trauma that is characterized by a pattern consistent with cytotoxic lymphocyte activation and is associated with clinical outcome.

Biomolecules doi: 10.3390/biom16091227

Authors: Yue-Lin Fang Yu-Jou Hsu Chao-Hsien Sung Chia-Chi Kung Shiuan-Ruei Shiu Chih-Yu Hung Mei-Jung Chen Der-Chen Chang I-Chia Liang Chi-Feng Hung

Background: Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss and is strongly driven by oxidative stress and inflammation. This study investigated the protective effects of cynarin against sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) injury, focusing on the MAPK and NF-κB signaling pathways. Materials and Methods: Human ARPE-19 cells were exposed to NaIO3, and cell viability was assessed by the MTT assay. Protein expression of MAPK components (p38, JNK, ERK) and the NF-κB pathway was analyzed by Western blotting, and pro-inflammatory cytokine (IL-1β, IL-6, TNF-α) mRNA expression was measured by RT-qPCR. In vivo, NaIO3-induced retinal degeneration in C57BL/6 mice was treated with cynarin (3 or 10 mg/kg) for seven days, and retinal changes were evaluated by fundus photography, fluorescein angiography, and OCT. Results: Cynarin preserved ARPE-19 cell viability without cytotoxicity. It significantly attenuated NaIO3-induced p38 and JNK phosphorylation, IκB degradation, and NF-κB activation while downregulating IL-1β, IL-6, and TNF-α expression. In vivo, cynarin reduced drusen-like lesions, hyperfluorescent abnormalities, and retinal thinning, and dose-dependently suppressed ocular pro-inflammatory cytokines. Conclusions: Cynarin protects against oxidative stress-induced retinal degeneration by suppressing MAPK and NF-κB inflammatory signaling, representing a promising therapeutic candidate for preventing or delaying NaIO3-induced dry AMD-like retinal injury.

Biomolecules doi: 10.3390/biom16091226

Authors: Zhishan Xu Dingbo Song Fangqi Hu Qiuhan Liang Mengyao Zhang Chao Lei Jinyuan Li Haiyi Guo Zhongbin Deng Zishan Yang

Inflammatory bowel disease (IBD) and colorectal cancer (CRC) are heterogeneous intestinal disorders that pose significant threats to human health and share common pathological features, including intestinal mucosal barrier disruption and gut microbiota dysbiosis, in which fucosylation acts as a critical regulatory mediator. Fucosylation is a highly conserved post-translational glycosylation modification involving the enzymatic transfer of fucose residues to glycoproteins and glycolipids. This tightly regulated process plays essential roles in maintaining intestinal homeostasis, mediating host–microbiota interactions and regulating immune responses. This review adopts a physiology-to-pathology framework, delineating fucosylation’s operational principles in healthy intestines and its dysregulation in IBD and CRC. It summarizes the spatial distribution of fucosylation, its regulatory mechanisms, and its roles in disease pathogenesis, and also discusses its potential as a diagnostic biomarker and therapeutic target. Finally, this review highlights future research directions to bridge mechanistic insights with clinical translation, emphasizing the promise of fucosylation in the precision diagnosis and treatment of intestinal disorders.

Biomolecules doi: 10.3390/biom16091225

Authors: Zhen-Zhen Dong Wanying Zheng Shuojie Lv Ling Peng Yihan Wang Qingjing Wang

Depression (major depressive disorder, MDD) is a globally prevalent, highly disabling, and complex mental disorder whose pathogenesis has not been fully elucidated. In recent years, the role of the gut microbiota in depression via the “microbiota–gut–brain axis” (MGB axis) has attracted increasing attention. A large body of evidence indicates that the gut microbiota and its metabolites can engage in bidirectional communication with the central nervous system through three core pathways—neural, immune, and metabolic—thereby profoundly influencing the onset and progression of depression. This article reviews the specific mechanisms by which the gut microbiota affects depression through the aforementioned pathways, including regulating the balance of neurotransmitters (e.g., GABA and 5-HT), mediating neuroinflammatory responses, and adjusting the levels of metabolites such as short-chain fatty acids. This study aims to provide a theoretical basis for an in-depth understanding of the pathophysiological mechanisms of depression and the development of novel microbiota-based intervention therapeutic strategies.

Biomolecules doi: 10.3390/biom16091224

Authors: Thanawat Pattananandecha Sutasinee Apichai Chalermpong Saenjum Young-Joon Surh

Bone remodeling is a dynamic and tightly regulated process that maintains skeletal homeostasis through a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Disruption of this balance contributes to the development of bone-related disorders, particularly osteopenia, osteoporosis and osteogenesis imperfecta, which weaken, deform, or cause fractures. Increasing evidence indicates that oxidative stress and chronic inflammation impair osteoblast functions while promoting osteoclast differentiation and activity. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with cytoprotective, antioxidant, and anti-inflammatory properties. Besides its primary role in cellular defense against oxidative stress and inflammatory damage, HO-1 has been shown to be involved in both osteoblast differentiation and osteoclastogenesis. Through its interaction with key regulatory systems, including the receptor activator of nuclear factor κB (RANK)–receptor activator of nuclear factor κB ligand (RANKL)–osteoprotegerin axis and redox-sensitive signaling pathways, HO-1 contributes to maintenance of optimal bone remodeling. The enzyme also plays a role in modulating metabolic processes in the bone. This review highlights the role of HO-1 in bone formation, bone resorption, and related pathophysiologic conditions. Furthermore, the therapeutic potential of HO-1 as a target for bone disorders is discussed.

Biomolecules doi: 10.3390/biom16091223

Authors: He Zang Xinrui Chen Xiang Li Xue Yang Qingwei Tan Dafu Chen Rui Guo Jianfeng Qiu

Chalkbrood disease caused by Ascosphaera apis threatens honey bee brood, yet the transcriptional mechanisms that coordinate antifungal immune responses in Apis cerana remain unclear. Here, RNA interference (RNAi) was used to examine two Dorsal homologs and selected dorsal1 transcript isoforms during larval infection. A. apis inoculation increased dorsal1, dorsal2, and five antimicrobial peptide (AMP) transcripts in larval midguts, consistent with an infection-associated humoral response. Gene-level RNAi produced selective transcript responses: dorsal1 knockdown was accompanied by reduced apidaecin and defensin1 expression, whereas dorsal2 knockdown was accompanied principally by reduced defensin1 expression. abaecin, defensin2, and hymenoptaecin transcripts were not significantly altered after dorsal knockdown under the tested conditions. Isoform-targeted RNAi of RNA9886, RNA9888, and RNA9890 was likewise associated with distinct AMP transcript responses, with RNA9888 and RNA9890 more closely associated with defensin1. These transcript-level data support selective and partially overlapping Dorsal-associated regulation during the A. cerana larval response to A. apis, while direct differences in Dorsal protein abundance or activity remain to be established.

Biomolecules doi: 10.3390/biom16091222

Authors: Paul M. Harrison

In proteins, tracts compositionally biased by a subset of amino acids are often linked to intrinsic disorder. Such compositionally biased regions (CBRs) are sometimes analyzed for ‘sequence complexity’ (‘information entropy’) despite being an unlikely substrate for natural selection per se, and therefore not functionally implicated. Here, an algorithmic strategy applying compositional bias detection was designed to capture the wide diversity of CBRs in structured protein domains (termed ‘sCBRs’), ranging from trihomopeptides to >200 residues, with conservation and partner binding as functional lenses. sCBRs are common, with about 1/4th of domains harbouring them, but domains dominated by sCBRs over >50% of their lengths are rare (~1 in 200). Despite general assumptions, very short sCBRs are highly significantly sequence-conserved, and associated with ligand binding, even when common nucleotide/phosphate-binding or glycine-rich cases are disregarded. However, regardless of length, ~50% of cases are evolutionarily dynamic, undergoing clade-specific expansion/contraction. Protein-binding associations include aversions for short (≤16 residues) valine-rich regions in protein interfaces, and enrichments of longer alanine-rich cases (>16 residues). Only ~5% of cases are (at least partly) in intrinsically disordered loops, and are significantly shorter than sCBRs generally. Functional implications of sCBRs are discussed with many examples. The sCBR data might help with hypothesis generation and protein design/engineering.

Biomolecules doi: 10.3390/biom16091221

Authors: Rashmi Kumariya Carole A. Bewley

Human immunodeficiency virus (HIV) is an enveloped virus with a remarkable capacity for genetic diversification, enabling rapid escape from host immune responses and therapeutic interventions. Despite extensive global efforts, the development of an effective vaccine has remained elusive owing to the virus’s high genetic variability and antigenic diversity. Consequently, considerable effort has been directed toward the development of therapeutic agents targeting viral entry, reverse transcriptase, integrase, protease, and more recently, capsid. Although antiretroviral therapy (ART) remains the cornerstone of HIV treatment, it is associated with challenges including drug resistance, adverse side effects, and limitations in access and affordability. Targeting viral entry offers distinct advantages by blocking infection at the earliest stage of the viral life cycle and enabling the neutralization of free virions, as well as Fc-mediated elimination of HIV-infected cells in some cases. This review highlights promising protein-based HIV entry inhibitors that have demonstrated efficacy in preclinical studies, and discusses ongoing efforts to optimize their valency, avidity, specificity, serum half-life, effector functions, and production platforms to improve their therapeutic potential and economic feasibility.

Biomolecules doi: 10.3390/biom16081220

Authors: Sergeja Adamič Zamljen Sara Godena Nikola Major Smiljana Goreta Ban Tvrtko Karlo Kovačević Marija Polić Pasković Igor Pasković

Olive leaves represent a metabolically active tissue that plays an important role in plant responses to biotic stress. The present study comprised two independent experiments investigating biochemical responses of olive leaves to bacterial and fungal challenge under controlled conditions. Changes in primary metabolites (sugars, organic acids and free amino acids), phenolic compounds and lipid peroxidation were analyzed using chromatographic and spectrophotometric methods. In the bacterial experiment, pronounced differences were observed in primary metabolism. Tryptophan concentrations ranged from approximately 50 mg kg−1 DW to more than 360 mg kg−1 DW in ‘Istarska bjelica’, while sucrose concentrations reached up to 87 g kg−1 DW, demonstrating cultivar-dependent differences in carbohydrate metabolism. Phenolic profiling showed that secoiridoids were the dominant phenolic class, with oleuropein concentrations exceeding 27 g kg−1 DW across bacterial treatments. In the fungal experiment, amino acids showed greater variability than sugars and phenolic compounds, whereas MDA concentrations ranged from approximately 190 to 300 nmol g−1 DW but did not differ significantly among pathogen treatments. Overall, the two experiments showed distinct patterns of metabolite variation associated with bacterial and fungal challenge. These findings contribute to a better understanding of cultivar-dependent metabolic responses and provide a basis for future studies of olive–microbe interactions.

Biomolecules doi: 10.3390/biom16081219

Authors: Yakov Rabinovich Yoav Vardizer Shirley Pincovich Marva Wolowelsky Sofia Kulyamzin Miriam Ehrenberg Shiri Zayit-Soudry Inbal Man Peles Rina Leibu Nitza Goldenberg-Cohen Tamar Ben-Yosef

Microphthalmia, anophthalmia and ocular coloboma (MAC) are rare developmental eye disorders. Although over 100 causative genes have been identified, the molecular spectrum and genotype–phenotype correlations remain incompletely understood, particularly in genetically diverse populations. We set out to molecularly characterize MAC in the Israeli population. Forty-seven MAC-affected individuals from 43 unrelated families were enrolled. DNA of all probands was subjected to whole exome sequencing. The most common phenotype was microphthalmia (64% of patients). Definite or possible molecular diagnoses were achieved in 13/43 probands (30%) and involved 10 different genes (MFRP, SMO, GJA8, SOX2, RARB, TSPAN12, SHH, PTPN11, BEST1, and TP63). An in vitro splicing assay was used to explore the pathogenicity of a variant in the SMO gene. Following stringent filtering of exome data, 226 rare possibly pathogenic variants were identified in 218 genes not previously associated with MAC. The rate of molecular diagnosis achieved in this Israeli MAC cohort is similar to the reported range in other studies. The results further demonstrate the genetic heterogeneity of MAC, while supporting the involvement of complex inheritance and/or environmental factors in many of the cases. Further studies are required to reveal these underlying etiological factors, and to support the novel genotype–phenotype associations suggested here.

Biomolecules doi: 10.3390/biom16081217

Authors: Vladislava Martyshova Sergey Sedykh

Methyl-dependent restriction endonucleases are promising tools for analyzing eukaryotic DNA methylation patterns. However, quantitative assessment of their substrate specificity requires the determination of the kinetic parameters of enzymatic reactions. Here, we present a method for determining initial reaction rates based on fluorescent probes and real-time monitoring of changes in fluorescence intensity. Initial rates of methyl-dependent GlaI and BlsI restriction endonucleases were determined as the slope of the linear part of the kinetic curves, after which the Michaelis–Menten constants (KM) and reaction rates (Vmax) were calculated using nonlinear regression. For both enzymes, KM values were determined for the first time, indicating a high affinity of the methyl-dependent restriction endonucleases for methylated sites. KM values for fully methylated duplexes were in the range of (4.4–7.0)·102 nM for GlaI and 2.4–55 nM for BlsI. KM values were significantly lower for the hemimethylated duplexes: (1.9–8.0)·102 nM for GlaI and (0.7–15.0)·102 nM for BlsI; and even lower for unmethylated duplexes: (27–46)·102 nM for GlaI and (0.28–23)·102 nM for BlsI. Maximum reaction rates varied within relatively narrow limits: Vmax values were in range (4.5–19.5)·10−4 nM/s for GlaI and (1.4–18)·10−4 nM/s for BlsI, respectively. Vmax values were depended weakly on the degree of methylation compared to the KM. The proposed fluorescence method was applied to determine the kinetic parameters of methyl-dependent restriction endonucleases for the first time. It may serve as a simpler and more environmentally friendly alternative to traditional electrophoretic approaches that use radioactive labels.

Biomolecules doi: 10.3390/biom16081218

Authors: Jada Sangha David A. Hood

Skeletal muscle mitochondria possess the ability to autoregulate their health and functioning by the orchestration of mitochondrial quality control (MQC) pathways. This plasticity allows them to adapt to various stimuli, such as exercise. However, under pathological conditions, mitochondria can become dysfunctional, generating damage-associated molecular patterns (DAMPs), such as reactive oxygen species (ROS) and oxidized mitochondrial DNA (mtDNA). These DAMPs can launch an innate immune response, with consequences of widespread inflammation and atrophy. Integral to this is the NLRP3 inflammasome complex. Activation of the NLRP3 inflammasome results in maturation of caspase-1, which processes pro-inflammatory cytokines IL-1β and IL-18, as well as GSDMD. Consequently, the pore-forming GSDMD-N fragment induces pyroptosis, releasing mature IL-1β and IL-18. Exercise training is widely accepted as a potent mechanism to promote skeletal muscle health, particularly by remodeling the mitochondrial network and reducing the production of DAMPs. It has also been shown promote an anti-inflammatory milieu with the release of various myokines. Indeed, the potential of exercise to mitigate NLRP3 inflammasome-mediated inflammation and atrophy is promising. This review will examine the mechanisms underpinning inflammasome priming and activation, as well the effects of exercise, with an emphasis on the skeletal muscle.

Biomolecules doi: 10.3390/biom16081216

Authors: Büşra Başar Gökcen Büşra Atabilen Pınar Menşure Nur Çelik Zeynep Büşra Aksoy Bence Raposa Duygu Ağagündüz

Mesenchymal stem/stromal cells (MSCs) are promising candidates in regenerative medicine, but their effectiveness is significantly influenced by the surrounding metabolic and nutritional conditions. Increasing evidence suggests that lipids act not only as energy sources but also as regulators of MSC fate. This review explores how lipid metabolism influences the balance among stemness, immunomodulation, and differentiation into adipogenic or osteogenic lineages. It does so through mechanisms such as fatty acid uptake, β-oxidation, de novo lipogenesis, and membrane remodeling, all orchestrated by CD36, carnitine palmitoyltransferase 1A, PPARγ, AMP-activated protein kinase, and the PI3K/AKT/mTOR pathway. We then examine how diet reshapes the MSC lipidome: obesity and high-fat diets promote adipogenesis and senescence, while omega-3 fatty acids, caloric restriction, micronutrients, and a balanced microbiota help preserve regenerative capacity. Lastly, we discuss how combining lipidomics with multi-omics could uncover lipid-metabolic signatures and regulatory nodes that connect diet to MSC function. Overall, the diet–lipid–MSC axis emerges as a modifiable determinant of MSC function.

Biomolecules doi: 10.3390/biom16081215

Authors: Itzhak Fischer

The microtubule-associated protein tau, encoded by the MAPT gene, serves as a major component of the neuronal cytoskeleton, facilitating the assembly, stabilization, and spatial organization of microtubules. Much of the work on tau has focused on the low-molecular-weight (LMW) isoforms abundantly expressed in the central nervous system (CNS) and their pathological aggregation in tauopathies. However, a different variant known as “Big tau”, present in the peripheral nervous system (PNS) and selective CNS regions has distinct structural and functional properties and offers a unique perspective on protein evolution. Big tau is characterized by the inclusion of a large, alternatively spliced insert termed exon 4a, which expands the protein’s projection domain by approximately 250 amino acids and increases the molecular weight to 90–110 kDa. The evolutionary trajectory of exon 4a presents a fascinating enigma that challenges conventional models of protein conservation. Across the vertebrate phylogeny, spanning from fishes, amphibians and birds to mammals, the primary amino acid sequence of exon 4a exhibits extreme divergence, often reaching background levels of identity when comparing distant classes. In contrast, the physical length of this domain remains remarkably stable, hovering around the 250-amino acid mark regardless of the species. This pattern suggests that the selective pressure acting on Big tau is not directed toward specific sequence motifs or functional domains, but rather toward the biophysical properties and physical dimensions of the domain. Here, we posit that exon 4a evolved as an essential molecular spacer optimized for the structural demands of long-projection neurons and high-caliber axons as well as a protective structure for the pathologic aggregation of tau. The paper examines the genomic architecture and biophysical identity underlying the stable-size and low sequence identity of exon 4a, presenting two evolutionary mechanisms as working hypotheses: a Prototype Model of neutral drift of an ancient insert, and an Independent Exonization of convergent recruitment of non-coding DNA by transposable elements or intron retention. Finally, we emphasize the need for additional experimental work in vitro and in vivo to resolve unanswered questions about the structure of the 4a exon, the physiological role Big tau and its potential insight into tauopathies therapeutics.

Biomolecules doi: 10.3390/biom16081214

Authors: Ulan Sarsenbayeva Didier Gasparutto Nicolas Geacintov Alexander A. Ishchenko Gulzhan Zhamanbayeva Kamalidin O. Sharipov Carlos H. Trasviña-Arenas Sheila S. David Dmitry O. Zharkov Bakhyt T. Matkarimov Murat Saparbaev Sabira Taipakova

The Escherichia coli adenine-DNA glycosylase (MutY) and its human homologue, MUTYH, protect cells against oxygen-free radical-induced mutagenesis by excising regular adenine impaired with 8-oxo-7,8-dihydro-guanine (8oxoG) in the base excision repair (BER) pathway. However, removal of adenine by MutY and MUTYH from an A·8oxoG pair generated via misincorporation of an oxidized nucleotide during DNA synthesis might induce A·T→C·G transversions. Here, to examine MutY and MUTYH in vitro activities, we used short synthetic DNA duplexes in which the target adenine residue was positioned opposite a variety of DNA base modifications. MUTYH does not excise mismatched adenine in non-canonical DNA substrates, whereas MutY excises adenine mispaired with 1,3-d(GpNpG) cisplatin intra-strand crosslink. In addition, we characterized four MUTYH variants associated with cancer risk, which exhibit the following order of DNA glycosylase deficiency: WT ≥ G169D > G202E ≈ Y165C >> D222N. Human adenine-DNA glycosylase MUTYH and its mutant variants, contrary to bacterial MutY, are not prone to aberrant removal of regular adenine residues opposite modified residues in DNA duplexes. We hypothesize that E. coli MutY is prone to aberrant repair under certain conditions and that this may prevent incorporation of adenine opposite blocking lesions in the template strand.

Biomolecules doi: 10.3390/biom16081213

Authors: Bianka Nicolle Pena Marcelino Kiersten Girard Lauren Letourneau Andrew Lewin David Lewin Anna Presicci Luke Reistrom Tyler Williams H. John Sharifi

Sulforaphane (SFN), a natural compound found in cruciferous vegetables, mobilizes the transcription factor NRF2 to protect macrophages from HIV-1. SFN/NRF2 exerts this protective effect by promoting the reduced phosphorylation of the antiviral protein SAMHD1. Phosphorylation at threonine 592 (T592) potently inhibits the capacity of SAMHD1 to restrict HIV-1. How SFN, and other NRF2 mobilizers reduce SAMHD1 T592 phosphorylation is unclear. p21 (CDKN1A) is an NRF2-responsive protein that accumulates in primary macrophages after SFN treatment. p21 blocks SAMHD1 T592 phosphorylation through the inhibition of several cyclin-dependent kinases. We therefore hypothesized that SFN acts through p21 to reduce SAMHD1 T592 phosphorylation in macrophages. Here, we use RNAi, CRISPR-Cas9, and pharmacological inhibition to deplete or delete p21 in macrophages and demonstrate that p21 is necessary for SFN to efficiently reduce SAMHD1 T592 phosphorylation and restrict HIV-1 transduction.

Biomolecules doi: 10.3390/biom16081212

Authors: Isabell Schütt Jonathan Teuffel Ben H. Hlawatschke Philip Einwohlt Bernd Kreikemeyer Rebecca C. Wade Tomas Fiedler

Streptococcus cristatus (S. cristatus) belongs to the viridans group of streptococci and is a commensal of the human upper respiratory tract. With the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, GapN, and the oxidative part of the pentose phosphate pathway (oxPPP), S. cristatus can use two different metabolic pathways to provide reduced nicotinamide adenine dinucleotide phosphate (NADPH), an essential cofactor of anabolic reactions such as fatty acid and amino acid biosynthesis. Regarding their NADP-reducing capacity, streptococci can be categorized into three groups: those that have only GapN, those that use only the oxPPP, and those that use both pathways. Here, we report on the experimental and computational characterization of the catalytic properties of the three NADP-reducing enzymes: GapN, glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH) of S. cristatus. Kinetic analyses showed moderate substrate and cofactor affinities, with GapN displaying the tightest substrate binding, followed by 6PGDH and G6PDH, in agreement with structural and computational predictions. All three enzymes preferentially utilized NADP+, with only G6PDH exhibiting limited NAD+ promiscuity. Growth-phase-dependent activity patterns suggest dynamic adjustment of NADPH-generating pathways, with reduced GapN contribution and sustained oxPPP activity in the stationary phase. Regulatory screening indicated limited allosteric control, though feedback inhibition by NADPH and the ATP sensitivity of G6PDH point to conserved redox regulatory mechanisms. Comparative analysis across streptococci supports the concept that the coexistence of GapN and the oxidative pentose phosphate pathway in S. cristatus may provide metabolic flexibility by offering alternative routes for NADPH generation.

Biomolecules doi: 10.3390/biom16081211

Authors: Bryony Davies Faheem Seedat Lina Bergman Catherine Cluver Angga Wiratama Lokeswara Michelle Ma Morganne Wilbourne Shuhan Jiang Antonio Galvez Adam Handel Andrew Fower Carlos Escudero Wei Zhang Manu Vatish

Circulating small extracellular vesicles (sEVs) released from the placenta carry bioactive compounds. Placental sEVs (psEVs) have been implicated as drivers of pathology in preeclampsia, a common disorder of pregnancy. This study investigates the hypothesis that psEVs are detectable in cerebrospinal fluid (CSF) during pregnancy, and are present at higher concentrations in preeclamptic and eclamptic pregnancies. Two techniques were used to search for psEVs. Firstly, the ExoCounter assay was performed on neat CSF from normotensive, preeclamptic and eclamptic pregnancies, and non-pregnant controls (n = 11, 12, 10 and 4, respectively). Quantitative PCR was used to search for psEV-associated microRNAs in the CSF of pregnant women. Neither assay found evidence of psEVs in the CSF of pregnant women, regardless of whether they had preeclampsia or eclampsia. This study suggests that psEVs do not reside in CSF during pregnancy and may be more likely to impact central nervous tissues through peripheral changes or interaction with the blood–brain barrier without crossing.

Biomolecules doi: 10.3390/biom16081210

Authors: Rui Pu Guo-Pan Gong Wen-Li Song Yue Yin Zi-Yang Chen Pan Jin

Skeletal disorders, including osteoporosis, osteoarthritis, rheumatoid arthritis, and osteonecrosis of the femoral head, are common chronic conditions that substantially affect health and quality of life. Ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, has increasingly been implicated in abnormal bone remodeling, cartilage degeneration, synovial pathology, and impaired skeletal homeostasis. Aerobic exercise is an important non-pharmacological approach for maintaining skeletal health, but the role of ferroptosis in its protective effects remains incompletely understood. Previous reviews have mainly discussed ferroptosis in skeletal disorders or the beneficial effects of exercise on skeletal health as separate topics. In contrast, this review places aerobic exercise, ferroptosis, and skeletal disorders within a unified framework and summarizes current evidence across osteoporosis, osteoarthritis, rheumatoid arthritis, and osteonecrosis of the femoral head. We further discuss how aerobic exercise may influence ferroptosis through the regulation of iron homeostasis, lipid peroxidation, antioxidant defense, and inflammatory responses, with attention to recently emerging molecular evidence and to the distinction between direct findings from bone- and joint-related tissues and supportive evidence from non-skeletal systems. Current direct evidence is concentrated mainly in osteoblast-related bone loss and osteoarthritis and is derived predominantly from animal and cellular studies, whereas direct clinical evidence in humans remains limited. Overall, available evidence supports ferroptosis as a potential mechanistic link between aerobic exercise and skeletal protection, but its role in mediating exercise-induced benefits in humans has yet to be established. Further clinical validation of this relationship may help clarify the biological basis of aerobic exercise interventions and support the development of more targeted exercise strategies for the prevention and management of skeletal disorders.