International Journal of Molecular Sciences (IJMS)

Citation: Tandoh, K.Z.; Avalos-Padilla, Y.; Ameyaw, P.; Laryea-Akrong, E.K.; Awandare, G.A.; Wilson, M.D.; Quashie, N.B.; Fernàndez-Busquets, X.; Duah-Quashie, N.O. Extracellular Vesicle Abundance, but Not a High Aggregation-Prone Peptide Cargo, Is Associated with Dihydroartemisinin Exposure in Plasmodium falciparum. Int. J. Mol. Sci. 2025, 26, 3962. Abstract: Our understanding of the molecular mechanisms undergirding artemisinin (ART) resistance in Plasmodium falciparum is currently based on two organizing principles: reduced hemoglobin trafficking into the digestive food vacuole, resulting in lower levels of activated ART, and increased tolerance to ART-induced oxidative stress in the infected erythrocyte. We had previously proposed an extracellular vesicle (EV) export model of ART resistance in P. falciparum. This model predicts that EV abundance will be altered by ART exposure and that the peptide cargo of EVs from the ART-exposed condition will be enriched with aggregation-prone peptides. We tested the predictions of the EV export hypothesis in this study using in vitro culture assays of an ART-resistant transgenic line engineered on a 3D7 background (R561H) and a 3D7 knockout line (PfVps60KO) with deficient EV production phenotype. EV enrichment was obtained from in vitro parasite culture supernatants via a series of ultracentrifugation and filtration steps, followed by size exclusion chromatography. A quality check on EVs was performed using dynamic light scattering. Liquid chromatography with tandem mass spectrometry was used to determine the proteome cargo from extracted EVs, and parasite peptides were queried for aggregation-prone tendency using open-access software. We report that dihydroartemisinin (DHA) exposure was positively correlated with EV abundance (coefficient estimate = 1038.58, confidence interval of 194.86-1882.30, and p-value = 0.018) and suggests that EV biogenesis is part of the parasite's response to DHA/ART. Furthermore, our findings suggest the expression of a non-constitutive DHA-induced alternate EV biogenesis pathway as the PfVps60KO was observed to produce the highest number of EVs under DHA exposure. Finally, we show that EVs from both ART-susceptible and resistant parasites under DHA exposure carry a cargo of Chorein N-terminal domain-containing protein (PF3D7_1021700) with a high aggregation-prone index (prion-like domain [PrLD] score = 26.5) out of nine identified parasite peptides. The former of these findings is in concordance with the EV Int. J. Mol. Sci. 2025, 26, 3962 https://doi.org/10.3390/ijms26093962 Int. J. Mol. Sci. 2025, 26, 3962 2 of 14 export hypothesis, which posits that the removal of DHA/ART-induced aggregated and/or misfolded peptides is critical to the parasite's survival under DHA/ART exposure. This observation further implicates EVs in the development of the ART-resistant phenotype. However, the finding of one aggregation-prone peptide out of the nine parasite proteins in the EV cargo does not sufficiently support the EV export hypothesis. Future replicates of this study and further interrogations of the EV export hypothesis are needed.

Ischemic stroke (IS) is the leading cause of long-term disability and the second leading cause of death worldwide. It remains a significant clinical problem because only supportive therapies exist, such as thrombolytic agents and surgical thrombectomy, which do not restore function. Understanding the molecular pathogenesis of IS, including dysfunction in oxidative homeostasis, apoptosis, neuroinflammation and neuroprotection, is crucial to developing therapies. Non-coding RNAs (ncRNAs) are master regulators, and one ncRNA that stands out is miR-155, a pro-inflammatory micro-RNA elevated in stroke. This review addresses the biological mechanisms reported in the literature that support using miR-155 as a biomarker and therapeutic agent to treat IS in patients.

Hepatoblastoma (HB) is the most common pediatric primary liver tumor. About 20% of affected children have pulmonary metastasis at presentation. Survival rates for these children are dismal, not exceeding 25%. To study this subset of patients, we sequenced a metastatic HB cell line, HLM_2, and identified downregulation of the Liver X Receptor (LXR)/Retinoid X Receptor (RXR) pathway. LXR/RXRs function as transcriptional regulators that influence genes implicated in HB development, including the Wnt/β-catenin signaling pathway. We assessed the effects of a novel LXR/RXR agonist, UAB116, on metastatic HB, hypothesizing that this compound would affect genes governing the Wnt/β-catenin pathway, decreasing the metastatic phenotype of HLM_2 metastatic HB cells. We evaluated its effects on viability, proliferation, stemness, clonogenicity, and motility, and performed RNA sequencing to study differential gene regulation. Treatment with UAB116 for 72 h decreased HLM_2 proliferation, stemness, clonogenicity, and invasion. RNA sequencing identified an eight-fold increase in TRIM29, a gene known to inhibit β-catenin, in cells treated with UAB116. Administration of the LXR/RXR agonist, UAB116, reduces proliferation, stemness, and invasiveness of metastatic HB cells, potentially by upregulation of TRIM29, a known modulator of the Wnt/β-catenin pathway, providing support for further exploration of LXR/RXR agonism as a therapeutic strategy for metastatic HB.

Cancer has rapidly emerged as a leading global cause of premature mortality, with significant economic implications projected to reach USD 25.2 trillion from 2020 to 2050. Among the various types of cancer, primary bone cancers, though uncommon, are projected to see nearly 4000 new cases diagnosed in the United States in 2024. The complexity of treating bone cancer arises from its rarity, diversity, and the challenges associated with surgical interventions, metastatic spread, and post-operative complications. Advancements in bone tissue engineering (BTE) have introduced innovative therapeutic approaches to promote bone regeneration and address tumor recurrence. This interdisciplinary field integrates biomaterials, scaffolds, and gene therapy, utilizing technologies such as 3D bioprinting to create custom scaffolds that facilitate cellular activities essential for tissue regeneration. Recent developments in biodegradable, bioactive materials aim to enhance the biocompatibility and effectiveness of scaffolds, while nanotechnology presents promising avenues for targeted drug delivery and improved therapeutic outcomes. This review outlines the current landscape of BTE, highlighting scaffold fabrication techniques, the advantages of incorporating stem cell and gene therapies, and future directions, including the integration of artificial intelligence in scaffold design for personalized medicine in orthopedic oncology. This work underscores the necessity for ongoing research and innovation, aiming to improve therapeutic strategies specifically designed to address the unique challenges posed by bone sarcomas and metastatic cancers.

The dysregulation of the Hippo signaling pathway leads to the aberrant activation of oncogenic YAP and TAZ, driving tumor progression. In breast cancer, this disruption promotes proliferation and metastasis. This study investigates the effects of CA3, a selective YAP inhibitor, on the proteome of triple-negative breast cancer MDA-MB-231 and luminal-A-like MCF7 cells. Proteomic changes were analyzed via nano-LC-MS/MS, while cytotoxicity, apoptosis, and autophagy were assessed through WST-1 assays, flow cytometry, and Western blot analyses. Bioinformatics tools were employed to identify enriched pathways. MDA-MB-231 cells exhibited an increased expression of DNA repair proteins (p < 0.05), indicating a compensatory response to maintain genomic stability. In contrast, MCF7 cells showed a downregulation of DNA repair factors (p < 0.005). Additionally, metabolic reprogramming was apparent in MCF7 cells (p < 0.001). Apoptosis assays revealed a rise in cell death, while cell cycle analysis indicated pronounced G1-phase arrest in MDA-MB-231 cells (p < 0.01). Moreover, autophagic suppression was particularly evident in MCF7 cells. This study, for the first time, provides evidence that breast cancer subtypes exhibit distinct dependencies on YAP-driven pathways, revealing potential therapeutic vulnerabilities. Targeting Hippo signaling alongside DNA repair in triple-negative breast cancer or combining YAP inhibition with metabolic blockade in luminal breast cancer holds significant potential to enhance treatment efficacy.

Spatial omics integrates molecular profiling with spatial tissue context, enabling high-resolution analysis of gene expression, protein interactions, and epigenetic modifications. This approach provides critical insights into disease mechanisms and therapeutic responses, with applications in cancer, neurology, and immunology. Spatial omics technologies, including spatial transcriptomics, proteomics, and epigenomics, facilitate the study of cellular heterogeneity, tissue organization, and cell–cell interactions within their native environments. Despite challenges in data complexity and integration, advancements in multi-omics pipelines and computational tools are enhancing data accuracy and biological interpretation. This review provides a comprehensive overview of key spatial omics technologies, their analytical methods, validation strategies, and clinical applications. By integrating spatially resolved molecular data with traditional omics, spatial omics is transforming precision medicine, biomarker discovery, and personalized therapy. Future research should focus on improving standardization, reproducibility, and multimodal data integration to fully realize the potential of spatial omics in clinical and translational research.

Preeclampsia (PE), a pregnancy complication characterized by high blood pressure and organ damage, has been suggested to be associated with mitochondrial dysfunction, although evidence remains limited. This study aimed to investigate the activity of oxidative phosphorylation (OXPHOS) enzymes and the expression of related proteins in placental tissues from women diagnosed with early-onset preeclampsia (eoPE, <34 weeks of gestation), late-onset preeclampsia (loPE, ≥34 weeks of gestation), and normotensive controls. Placental samples were analyzed using immunohistochemistry, western blotting, and enzymatic activity assays to assess the activity and expression of OXPHOS complexes. Complex I activity was increased by 80% in eoPE and 56% in loPE, with positive correlations between normalized complex I expression, gestational age at delivery (r = 0.85, p = 0.01), and birth weight (r = 0.88, p = 0.004) in loPE. Relative complex II expression in loPE showed positive correlations with pregnancy duration (r = 0.76, p = 0.03) and birth weight (r = 0.77, p = 0.03), while in controls, complex II expression correlated with pregnancy duration (r = 0.64, p = 0.03). Additionally, complex IV enzyme activity in eoPE was negatively correlated with maternal age at birth (r = −0.69, p = 0.03). The observed correlations highlight mitochondrial metabolism as a promising biomarker for predicting disease progression and guiding therapeutic interventions in preeclampsia. Unraveling its precise role in PE pathogenesis is critical to advancing diagnostic precision and improving maternal-fetal outcomes.

Neurodegenerative diseases involve oxidative stress and enzyme dysregulation, necessitating novel neuroprotective agents. This study evaluates the neuroprotective and antioxidant potential of seven pyrrole-based compounds with predicted radical scavenging activity and inhibitory effects on monoamine oxidase B (MAO-B) and acetylcholinesterase (AChE). The compounds were tested in vitro using SH-SY5Y neuroblastoma cells and subcellular rat brain fractions, including synaptosomes, mitochondria, and microsomes. Neuroprotective and antioxidant effects were assessed in oxidative stress models, including H2O2-induced stress in SH-SY5Y cells, 6-hydroxydopamine toxicity in synaptosomes, tert-butyl hydroperoxide-induced stress in mitochondria, and non-enzyme lipid peroxidation in microsomes. In silico screening for lipophilicity, hydrogen bonding, total polar surface area (TPSA), and ionization properties, was performed to evaluate bioavailability. All compounds exhibited a weak neurotoxic effect on the subcellular fractions at a concentration of 100 µM. However, in oxidative stress models, they demonstrated significant neuroprotective and antioxidant effects at 100 µM. In SH-SY5Y cells, compounds 7, 9, 12, 14, and 15 exhibited low toxicity and strong protective effects at concentrations as low as 1 µM. In silico analysis prioritized compounds 1, 7, 9, 12, and 14 for further development based on their favorable bioavailability. The tested pyrrole-based compounds exhibit promising neuroprotective and antioxidant properties, with several candidates showing potential for further development based on both in vitro efficacy and predicted oral bioavailability.

Natural products play a crucial role in drug discovery because of their structural diversity and biological activity. However, identifying their molecular targets remains a challenge. Traditional target identification approaches such as affinity-based protein profiling and activity-based protein profiling are limited by the need for chemical modification or reactive groups in natural products. The emergence of label-free techniques offers a powerful alternative for studying drug–target engagement in a physiological context. In particular, the cellular thermal shift assay (CETSA) exploits ligand-induced protein stabilization—a phenomenon where ligand binding enhances a protein’s thermal stability by reducing conformational flexibility—to assess drug binding without requiring chemical modifications. CETSA’s integration with advanced mass spectrometry and high-throughput platforms has dramatically expanded proteome coverage and sensitivity, enabling the simultaneous quantification of thousands of proteins and the identification of low-abundance targets in native cellular environments. This review highlights the application of key CETSA-based methods to target identification in natural products including Western blot-based CETSA, isothermal dose–response CETSA, mass spectrometry-based CETSA, and high-throughput CETSA. Case studies are presented that demonstrate their effectiveness in uncovering the mechanisms of action of different drugs. The current limitations of CETSA-based strategies are also explored, and future improvements to optimize their potential for drug discovery are discussed. Integrating CETSA with complementary approaches can enhance the target identification accuracy and efficiency for natural products and ultimately advance development of therapeutic applications.

Cancer-associated cachexia (CAC), also known as wasting syndrome, is a systemic condition that affects multiple tissues and organs via a variety of metabolic pathways. Systemic inflammation, progressive weight loss, depletion of adipose tissue, and skeletal muscle impairment are some of the hallmark features of cachexia. Despite various studies on the clinical features of CAC, the complexity of the syndrome continues to pose significant challenges in clinical practice, leading to late diagnoses and the absence of a standardised treatment. Men and women respond differently to CAC, which may be prompted by the pre-existing physiologic sex differences. This review presents the sexual dimorphism associated with the hallmark pathways involved in CAC. A comprehensive understanding of sexual dimorphism in these pathways could drive research on cachexia to prioritise the inclusion of more females in related studies in order to achieve personalised sex-based therapeutic approaches and, consequently, enhance treatment efficacy and better patient outcomes.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation and systemic involvement. This study investigates the therapeutic potential of oral hyaluronan (HA) with different molecular weights (SHA: 0.99 MDa and VHA: 1.73 MDa) as monotherapy and in combination with methotrexate (MTX) in a preclinical adjuvant arthritis (AA) model in Lewis rats. The aim was to evaluate the impact of these treatments on biometric, inflammatory, and oxidative stress parameters. The preliminary study tested two doses of HA (0.5 mg/kg and 5 mg/kg), and the pivotal study focused on the combination of 0.5 mg/kg HA with 0.3 mg/kg MTX. Based on our experimental findings on combined therapy, the MTX + SHA combination demonstrated superior efficacy compared to MTX + VHA and MTX monotherapy. Specifically, the MTX + SHA regimen significantly promoted weight gain and reduced hind-paw volume in all monitored experimental days. This treatment markedly reduced plasmatic IL-17A levels (day 21) and GGT activity in both the spleen and joints (day 28), showing the most pronounced effects among all groups, including the MTX monotherapy group. The MTX + VHA combination showed a therapeutic response comparable to MTX alone, indicating no additional benefit. These findings suggest a superior efficacy of the MTX + SHA combination in comparison to other studied treatments. The overall efficacy can be ranked as: MTX ≈ MTX + VHA < MTX + SHA.

Diabetes mellitus (DM) is a chronic metabolic disorder that results in hyperglycemia, leading to multiple microvascular and macrovascular complications, including significant ocular damage resulting in the development of diabetic retinopathy (DR) and diabetic macular edema (DME). Many factors contribute to the pathogenesis of DR and DME, including hyperglycemia-mediated vascular and neuronal abnormalities and local and systemic inflammation. Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) have been implicated in the initiation and progression of DR and DME through a variety of mechanistic processes. In this review, we provide a comprehensive synopsis of the diverse roles and molecular pathways supporting IGF-1 in the pathogenesis of DR and DME, elucidating its range of effects from detrimental to protective, depending on the context and stage of disease. We further investigate the underlying inflammatory processes regulated by IGF-1 and examine how the interaction of IGF-1 with key signaling molecules influences these inflammatory mechanisms. Additionally, the potential of serum IGF-1 as a biomarker for the progression of DR and DME in clinical practice is discussed. Finally, we consider current therapeutic approaches for DR and DME in relation to IGF-1 and explore novel therapeutic targets and innovative delivery methods. By providing an in-depth understanding of IGF-1’s role in the pathogenesis and progression of DR and DME, this review underscores the diagnostic utility of serum IGF-1 and puts forth new treatment strategies to improve the management of DR and DME.

Analysis of tumoral RNA from bone marrow (BM) biopsy is essential for diagnosing childhood B-cell acute lymphoblastic leukemia (B-ALL), risk stratification, and monitoring, by detecting fusions and gene expression patterns. However, frequent BM biopsies are invasive and traumatic for patients. Small extracellular vesicles (sEVs) circulating in blood contain a variety of biomolecules, including RNA, that may contribute to cancer progression, offering a promising source of non-invasive biomarkers from liquid biopsies. While most EV studies have focused on small RNAs like microRNAs (miRNAs), the role of longer RNA species, including messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), remains underexplored despite their demonstrated potential for risk-based patient stratification when starting from BM biopsies. We used immuno-purification to isolate sEVs from peripheral blood at diagnosis in B-ALL patients and cell model-based conditioned culture medium (CCM) with ETV6::RUNX1 and TCF3::PBX1 fusions. Using whole-transcriptome sequencing targeting transcripts over 200 nt and a novel data analysis pipeline, we identified 102 RNA transcripts (67 mRNAs, 16 lncRNAs, 10 circRNAs, 4 pseudogenes, and 5 others) in patient-derived sEVs. These transcripts could serve as biomarkers for two distinct molecular subgroups of B-ALL, each with different risk profiles at diagnosis. This is the first study characterizing the long transcriptome in blood-derived sEVs for childhood B-ALL, highlighting the potential use of circulating RNAs for improved risk-based stratification.

Bladder cancer, especially muscle-invasive bladder cancer (MIBC), poses significant treatment challenges due to its aggressive nature and poor prognosis, often necessitating cisplatin-based chemotherapy. While cisplatin effectively reduces tumor burden, its nephrotoxic effects, specifically cisplatin-induced acute kidney injury (AKI), limit its clinical use. This study investigates SH3YL1 as a potential biomarker for bladder cancer progression and AKI. Plasma and urine SH3YL1 levels were measured in bladder cancer patients undergoing cisplatin treatment, showing elevated baseline levels compared to controls, suggesting a link with bladder cancer pathology rather than cisplatin-induced AKI. Functional network and Gene Ontology (GO) enrichment analyses identified SH3YL1’s interactions with NADPH oxidase pathways, particularly NOX family genes, and highlighted its roles in cell adhesion, migration, and cytoskeletal organization—processes critical for tumor invasiveness. Notably, SH3YL1 and NOX4 expression were significantly higher in MIBC than in non-muscle-invasive bladder cancer (NMIBC), with a strong correlation between SH3YL1 and NOX4 (r = 0.62) in MIBC, suggesting a subtype-specific interaction. Kaplan–Meier survival analysis using The Cancer Genome Atlas bladder cancer (TCGA-BLCA) data further demonstrated that low SH3YL1 expression is significantly associated with poor overall and disease-specific survival in MIBC patients, reinforcing its role as a prognostic biomarker. In conclusion, SH3YL1 is a promising biomarker for identifying the invasive characteristics of MIBC and predicting patient outcomes. These findings underscore the importance of SH3YL1–NOX4 pathways in MIBC and suggest the need for further research into targeted biomarkers for bladder cancer progression and cisplatin-induced AKI to improve patient outcomes in high-risk cases.

Persistent high-risk Human Papillomavirus infection is the primary factor in cervical carcinogenesis. However, other host-related features are believed to play a role as well. Recent research suggests that the vaginal microbiome and the immune microenvironment play a significant role in the acquisition and persistence of Human Papillomavirus infection, as well as in the regression or progression of cervical intraepithelial lesions. Studies in this emerging field describe factors associated with this interaction, though the precise nature remains incompletely understood. In this narrative review, we aim to summarize the current literature on the topic and propose hypotheses and recommendations for future research and treatment strategies.

Type 1 diabetes (T1D) is an autoimmune condition characterized by the destruction of insulin-producing pancreatic beta cells, leading to lifelong insulin dependence and significant complications. Early detection of T1D is essential to delay disease onset and improve outcomes. Recent advancements in artificial intelligence (AI) and machine learning (ML) have provided powerful tools for predicting and diagnosing T1D. This systematic review evaluates the current landscape of AI/ML-based approaches for early T1D detection. A comprehensive search across PubMed, EMBASE, Science Direct, and Scopus identified 1447 studies, of which 10 met the inclusion criteria for narrative synthesis after screening and full-text review. The studies utilized diverse ML models, including logistic regression, support vector machines, random forests, and artificial neural networks. The datasets encompassed clinical parameters, genetic risk markers, continuous glucose monitoring (CGM) data, and proteomic and metabolomic biomarkers. The included studies involved a total of 49,172 participants and employed case–control, retrospective cohort, and prospective cohort designs. Models integrating multimodal data achieved the highest predictive accuracy, with area under the curve (AUC) values reaching up to 0.993 in sex-specific models. CGM data and plasma biomarkers, such as CXCL10 and IL-1RA, also emerged as valuable tools for identifying at-risk individuals. While the results highlight the potential of AI/ML in revolutionizing T1D risk stratification and diagnosis, challenges remain. Data heterogeneity and limited model generalizability present barriers to widespread implementation. Future research should prioritize the development of universal frameworks and real-world validation to enhance the reliability and clinical integration of these tools. Ultimately, AI/ML technologies hold transformative potential for clinical practice by enabling earlier diagnosis, guiding targeted interventions, and improving long-term patient outcomes. These advancements could support clinicians in making more informed, timely decisions, thus reducing diagnostic delays and paving the way for personalized prevention strategies in both pediatric and adult populations.

Severe immune effector cell-associated neurotoxicity syndrome (ICANS) occurs in about 30% of all patients with large B-cell lymphoma (LBCL) who are treated with axicabtagene ciloleucel (axi-cel). There are currently limited treatment strategies other than the standard corticosteroids, and it is essential to find additional therapies to manage severe ICANS. We conducted a retrospective study of neurologic outcomes among patients who received axi-cel for LBCL from May 2015 to February 2019. We identified patients who developed severe ICANS and were treated with glucocorticoids followed by intravenous immunoglobulin (IVIG) (n = 9) or glucocorticoids alone (n = 10). There was no statistically significant difference in the time to resolution (TTR) of severe ICANS between groups; however, patients in the IVIG had more severe grades of ICANS with a lower performance status at baseline. The cumulative steroid days were 11.2 in the IVIG arm and 13.5 in the glucocorticoids-only arm. The use of IVIG for severe ICANS after axi-cel therapy was tolerable and safe and is generally recommended in the CAR-T setting in patients with hypogammaglobinemia. The use of IVIG as a potential therapeutic agent for severe ICANS can be further explored in future prospective studies.

Citation: Hernández, B.; Coïc, Y.-M.; Kruglik, S.G.; Sanchez-Cortes, S.; Ghomi, M. Influence of Side Chain-Backbone Interactions and Explicit Hydration on Characteristic Aromatic Raman Fingerprints as Analysed in Tripeptides Gly-Xxx-Gly (Xxx = Phe, Tyr, Trp). Int. J. Mol. Sci. 2025, 26, 3911. https://doi. Abstract: Because of the involvement of π-electron cyclic constituents in their side chains, the so-called aromatic residues give rise to a number of strong, narrow, and well-resolved lines spread over the middle wavenumber (1800-600 cm −1) region of the Raman spectra of peptides and proteins. The number of characteristic aromatic markers increases with the structural complexity (Phe → Tyr → Trp), herein referred to as (F i = 1,. . ., 6) in Phe, (Y i = 1,. . ., 7) in Tyr, and (W i = 1,. . ., 8) in Trp. Herein, we undertake an overview of these markers through the analysis of a representative data base gathered from the most structurally simple tripeptides, Gly-Xxx-Gly (where Xxx = Phe, Tyr, Trp). In this framework, off-resonance Raman spectra obtained from the aqueous samples of these tripeptides were jointly used with the structural and vibrational data collected from the density functional theory (DFT) calculations using the M062X hybrid functional and 6-311++G(d,p) atomic basis set. The conformation dependence of aromatic Raman markers was explored upon a representative set of 75 conformers, having five different backbone secondary structures (i.e., β-strand, polyproline-II, helix, classic, and inverse γ-turn), and plausible side chain ro-tamers. The hydration effects were considered upon using both implicit (polarizable solvent continuum) and explicit (minimal number of 5-7 water molecules) models. Raman spectra were calculated through a multiconformational approach based on the thermal (Boltzmann) average of the spectra arising from all calculated conformers. A subsequent discussion highlights the conformational landscape of conformers and the wavenumber dispersion of aromatic Raman markers. In particular, a new interpretation was proposed for the characteristic Raman doublets arising from Tyr (~850-830 cm −1) and Trp (~1360-1340 cm −1), definitely excluding the previously suggested Fermi-resonance-based assignment of these markers through the consideration of the interactions between the aromatic side chain and its adjacent peptide bonds.

3CL protease (3CLpro), a key enzyme of SARS-CoV-2 replication, is one of the most selective targets of antivirals, as no homologous protease has been recognized in the human body. As proteolysis-targeting chimeras (PROTACs) are superior to traditional inhibitors, based on the reported cereblon (CRBN) ligands thalidomide and lenalidomide, 3CLpro ligands of peptidomimetic inhibitors, and suitable linkers, we aimed to develop novel PROTACs that may trigger efficient intracellular 3CLpro degradation through a balance of hydrophilicity and lipophilicity. In brief, we designed and synthesized 5 PROTAC molecules. The 3CLpro degradation efficiency of the PROTACs was assayed in stable SARS-CoV-2 3CLpro expression HEK293 cell models and evaluated by Western blot. All compounds showed prominent 3CLpro degradation activity with tolerable HEK293 cytotoxicity. The most prominent PROTAC compounds, 15 and 16, have DC50 values of approximately 1 µM, and Dmax of 89.3% and 75% respectively, indicating good potential for further application.

Curcumin exhibits antioxidant, antibacterial, antitumor, and anti-inflammatory biological properties. Its dual functionality as both a food additive and a pH-sensitive colorant has led to extensive applications in meat products and other food systems, thereby garnering significant research interest. In recent years, curcumin-loaded active-intelligent food packaging films have emerged as a promising innovation due to their multifunctional capabilities: not only do they prevent microbial contamination and extend food shelf life, but they also enable real-time freshness monitoring through visual colorimetric responses. This paper first delineates the molecular structure and fundamental biological mechanisms of curcumin. Subsequently, it systematically reviews the strategies for curcumin incorporation (including encapsulation techniques and composite formulations) and advanced fabrication methodologies for developing active-intelligent biodegradable films. Finally, the current applications of curcumin in polymer-based smart packaging systems are critically analyzed, with prospective research directions proposed to address existing technological limitations.

MuRF1 [muscle RING (Really Interesting New Gene)-finger protein-1] is an ubiquitin-protein ligase (E3), which encode by TRIM63 (tripartite motif containing 63) gene, playing a crucial role in regulating cardiac muscle size and function through ubiquitylation. Among hypertrophic cardiomyopathy (HCM) patients, 24 TRIM63 variants have been identified, with 1 additional variant linked to restrictive cardiomyopathy. However, only three variants have been previously investigated for their functional effects. The structural impacts of the 25 variants remain unexplored. This study investigated the effects of 25 MuRF1 variants on ubiquitylation activity using in vitro ubiquitylation assays and structural predictions using computational approaches. The variants were generated using site-directed PCR (Polymerase Chain Reaction) mutagenesis and subsequently purified with amylose affinity chromatography. In vitro ubiquitylation assays demonstrated that all 25 variants compromised the ability of MuRF1 to monoubiquitylate a titin fragment (A168-A170), while 17 variants significantly impaired or completely abolished auto-monoubiquitylation. Structural modelling predicted that 10 MuRF1 variants disrupted zinc binding or key stabilising interactions, compromising structural integrity. In contrast, three variants were predicted to enhance the structural stability of MuRF1, while six others were predicted to have no discernible impact on the structure. This study underscores the importance of functional assays and structural predictions in evaluating MuRF1 variant pathogenicity and provides novel insights into mechanisms by which these variants contribute to HCM and related cardiomyopathies.

As an important zoonotic pathogen, Toxoplasma gondii relies on a unique organelle known as the apicoplast, which has garnered significant attention as a potential drug target for anti-Toxoplasma therapy. To better understand the structure and function of the apicoplast, we previously constructed a membrane protein database of the apicoplast. During this process, we identified the major facilitator superfamily (MFS) transporter protein HAP12, which partially colocalizes with the apicoplast. Evolutionary analysis revealed that HAP12 is highly conserved across the Apicomplexa family and model organisms. HAP12 depletion impaired T. gondii invasion and survival but did not affect the stability of several key organelles, including the apicoplast. Moreover, depletion of HAP12 resulted in a characteristic delayed-death phenotype in the apicoplast. Mouse virulence assays confirmed that HAP12 is an essential protein for parasite survival. This study provides new insights into potential drug and vaccine targets for combating Toxoplasma infections.

Although various factors contribute to the transition from acute kidney injury (AKI) to chronic kidney disease (CKD), no clinically effective pharmacological treatment has been established. We investigated whether chymase inhibition is effective in preventing renal fibrosis, a key process in the transition from AKI to CKD. Male BALB/c mice were subjected to unilateral ischemia-reperfusion (I/R) injury, and TY-51469, a chymase-specific inhibitor, was administered intraperitoneally at a dose of 10 mg/kg/day for 6 weeks. The 45 min ischemic period followed by 6 weeks of reperfusion resulted in severe renal atrophy. Renal fibrosis was particularly pronounced in the transition region between the cortex and medulla in placebo-treated mice. The expression of mouse mast cell protease 4 (MMCP-4, a mouse chymase) mRNA, the number of chymase-positive mast cells, and fibrosis-related factors, such as transforming growth factor (TGF)-β1 and collagen I, were all significantly increased in I/R-injured kidneys. However, treatment with TY-51469 significantly suppressed fibrosis formation, along with the inhibition of renal chymase and TGF-β1 expression. These findings suggest that chymase inhibition may be a potential therapeutic strategy for preventing the transition from AKI to CKD by reducing fibrosis.

Bis-indole-derived compounds including 1,1-bis(3′-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) analogs bind both orphan nuclear receptors 4A1 (NR4A1) and NR4A2, and DIM-3,5 compounds act as dual receptor inverse agonists and inhibit both NR4A1- and NR4A2-regulated responses. Chromatin immunoprecipitation assays show that β1-integrin and the methyltransferase gene G9a are regulated by both NR4A1 and NR4A2 acting as cofactors for Sp1- and Sp4-dependent gene expression. DIM-3,5 treatment results in the loss of one or more of these nuclear factors from the β1-integrin and G9a promoters. Single-cell and RNAseq analyses show that both receptors regulate common (<10%) and unique genes in SW480 colon cancer cells; however, functional enrichment analysis of the differentially expressed genes converges to several common pathways and gene ontology terms.

This study aimed to develop an ex vivo retinal model to examine inflammatory processes in diabetic retinopathy (DR) without animal testing. Porcine eyes were collected from a local abattoir, dissected, and cultivated for four days in five experimental groups: control group (Co), 25 mM and 50 mM mannitol groups (Man25, Man50) as osmotic controls, and 25 mM and 50 mM glucose groups (Glc25, Glc50) as diabetic groups. A TUNEL assay was used to determine relative cell death. Immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect inflammatory markers. An increase in the cell death rate in Man50 (30%), Glc25 (36%) and Glc50 (37%) compared to Co (12%) (p < 0.01, p < 0.001, p < 0.001, respectively) and between Glc25 and Man25 (21%) (p < 0.01) was found. Immunofluorescence staining and qRT-PCR analysis revealed a TNF-α increase in Glc25 compared to Man25 and Co. iNOS was increased in Glc25 vs. Man25 but not in Co vs. Glc25. iNOS gene expression was upregulated with Glc25 treatment compared to Co and Man25 groups. Expression levels of IL-6 and CD31 were significantly higher in Glc25 than in Co and Man25. Glucose treatment increased cell death and inflammation, prompting us to present a DR model for better understanding DR and testing new therapies.