Hyperoside, a natural dietary flavonoid widely found in a variety of fruits, vegetables, and medicinal plants, exhibits hypoglycemic as well as hepatorenal protective activities. However, its effects on type 2 diabetes mellitus (T2DM) and the underlying mechanisms remain poorly understood. In this study, T2DM mice were established after 8 weeks of high-fat diet (HFD) feeding and were subsequently treated with hyperoside for 6 weeks. Then, indices of glucose and lipid metabolism, energy expenditure, hepatic histopathology, brown adipose tissue (BAT) thermogenesis, inguinal white adipose tissue (iWAT) browning, macrophage polarization in iWAT, and gut microbiota composition were evaluated. Hyperoside significantly reduced HFD-induced body weight gain in T2DM mice without suppressing appetite. HFD-induced insulin resistance, dyslipidemia, hepatic steatosis, and fibrosis were markedly ameliorated by hyperoside. Hyperoside also increased oxygen consumption, carbon dioxide production, and energy expenditure, and decreased the respiratory exchange ratio. In addition, hyperoside treatment was accompanied by increased UCP1 and PGC-1α protein expression and upregulated mRNA expression of PGC-1α, UCP1, PRDM16, and Cidea in BAT and iWAT, consistent with enhanced BAT thermogenic activity and an iWAT browning-related phenotype. Flow cytometry analysis further revealed that hyperoside inhibited M1 macrophage polarization and reduced the M1/M2 ratio in iWAT. Moreover, hyperoside partially modulated gut microbial diversity and taxonomic composition in T2DM mice, increased the relative abundance of Bacteroidetes and the Bacteroidetes/Firmicutes ratio, and partially restored the relative abundance of Muribaculaceae and Lachnospiraceae_NK4A136_group. In conclusion, hyperoside ameliorated metabolic dysfunction in HFD-induced T2DM mice, accompanied by enhanced BAT thermogenic activity, iWAT browning-related changes, modulation of macrophage polarization in iWAT, and remodeling of the gut microbiota. These findings suggest that hyperoside may represent a potential natural product candidate with metabolic benefits, although further pharmacokinetic, safety, long-term, and clinical validation is required.

This study aims to identify indicator parameters for biofilms in drinking water distribution systems (DWDS). Biofilms grown on coupons of three pipe materials (cast iron, cement, and polyvinyl chloride) in two sampler types (pipe loops and annular reactors) installed at full scale DWDS were quantified by three analytical methods: heterotrophic plate count (HPC), total cell count by flow cytometry (FC), and in situ by confocal laser scanning microscopy (CLSM). Quantification by both biofilm HPC and CLSM biomass is recommended to avoid under-reporting of viable but non-culturable microbial populations. Multiple multivariable statistical methods were used to explore complex full-scale data sets. Principal component analysis (PCA) showed similar variability of select water parameters and biofilm quantity regardless of sampler type. Indicators of biofilm quantity were identified by taking the preponderance of results of correlation analysis, PCA, and multiple step linear regression. Indicators of biofilm quantity found here include total calcium (average range 55-108 ppm CaCO3), total alkalinity (49-173 ppm CaCO3), and pH (7.3-8.7). Total chlorine decay and HPC of planktonic cells were indicators of biofilms both within a single DWDS and in the aggregated data of all seven utilities sampled. Assimilable organic carbon and phosphorus appear to have secondary impacts on biofilm quantity. Oxygen reduction potential (439-644 mV measured at two utilities) may also be useful as a biofilm indicator worth further investigation.

Ion channels are pore-forming transmembrane proteins that allow ions to move down an electrochemical gradient and across the channel pore and regulate many cell functions. Among them, are the G-protein-gated inwardly-rectifying K+ channels 1 (GIRK1) that are ubiquitously expressed with major functions in the brain and heart. Interestingly, significantly higher GIRK1 expression has been found in estrogen receptor positive (ER+) breast cancer patients compared to patients with HER2+ tumors or normal patients, and that was statistically correlated with shorter survival times and metastatic potential. Herein, we report the preparation of a ∼4 nm GAT1508-coated poly(ethylene glycol) gold nanoparticle (PEGylated AuNP) biomarker for ER+ breast cancer cell screening through an optical microscope. A urea-based small molecule, GAT1508, with an N-methylpyrazole benzyl group on its head and a bromo-thiophene on its tail, has been shown to predominantly bind GIRK1 subunits and specifically activate GIRK1/2 channels. Two derivatives of GAT1508 were synthesized and characterized: an ethylamine derivative (GAT1508-EA) with a chain extension from the benzyl ring, and a propylamine derivative (GAT1508-PA) with a chain extension from the pyrazole ring. Electrophysiology (TEVC and whole-cell patch-clump) experiments as well as fluorescence studies (Thallium assay) showed that only GAT1508-PA inhibited GIRK1/2-mediated K+ currents in transfected HEK293GIRK1 cells. Docking studies showed strong binding for the propylamine GAT1508 derivative, both in the amine form (GAT1508-PA) as well as in the amide form (GAT1508-PA-EG2; coupled with PEG as in the AuNPs). GAT1508-PEG-AuNPs (GAT1508-NPs) were synthesized subsequently with ∼65 wt% metal loading. UV-Vis studies revealed the presence of the conjugated ligand at 260 nm. Flow cytometry studies showed binding of Alexa 594-labeled GAT1508-NPs in ER+ MCF-7 breast cancer cells with a strong interaction, while incubation of fixed MCF-7 cells with a GAT1508-NP solution led to optical detection of ER+ breast cancer cells, without the need of fluorescent dyes and additional amplification steps. Detection was not feasible in MDA-MB-231 cells, a triple (-) breast cell line that does not express GIRK1. This, to our knowledge, is the first study that couples nanotechnology with small molecule drug design and electrophysiology to develop ion channel-tracing molecular probes for the detection/screening of ER+ breast cancer.

Lysine-specific demethylase 1 (LSD1, KDM1A) drives oncogenic progression in acute myeloid leukemia (AML) through both catalytic histone demethylation and non-catalytic scaffolding interactions with oncogenic transcription factors. Conventional LSD1 inhibitors either suffer from severe off-target toxicity or fail to block LSD1's non-enzymatic oncogenic functions. In recent years, emerging targeted protein degradation technologies have enabled the elimination of target proteins, providing a promising alternative strategy for drug development. Herein, we report for the first time a panel of intracellular self-assembled LSD1 degraders rationally designed via hydrophobic tagging (Hyt) strategy. Upon covalent engagement with FAD cofactor within LSD1 active pocket, the parent precursor releases NEW-Hyts that continuously mediate LSD1 degradation. Systematic linkers and Hyt warheads investigation afforded compound 18 as the optimal candidate. Western blot verified that 18 induced LSD1 degradation up to 70.86% at 10 μM in MV4-11 AML cells, as a time- and dose-dependent manner. ELISA assay further confirmed the degradation activity of 18 against LSD1, with a degradation rate of 83.46% at 10 μM. Besides, CCK-8 proliferation assay demonstrated that 18 exerted potent anti-AML activity with an IC₅₀ value of 1.12 μM, far superior than parent LSD1 inhibitors (TCP and ORY-1001). Moreover, flow cytometry analysis further confirmed that 18 concentration-dependently induced AML cell apoptosis and arrested cell cycle progression at G0/G1 phase. Collectively, this work provides a novel low-molecular-weight (MW = 564.7), intracellular assembly LSD1 degrader, representing a promising strategy for targeted AML therapy.

Selenium-containing dihydropyrimidinones (DHPMs) have emerged as promising scaffolds for anticancer drug development owing to their structural versatility and ability to modulate biologically relevant targets. Here, we investigated the antileukemic activity of two previously reported selenium-containing monastrol analogs, 4b1 and 4d1, and explored their interaction with kinesin Eg5 using complementary cellular, biophysical, and computational approaches. Cytotoxicity was evaluated by MTT assay in tumor and non-tumor cell lines, followed by selectivity index determination. Cell-cycle distribution and apoptosis were analyzed by flow cytometry, morphological alterations by panoptic staining, and protein-ligand interactions by molecular docking and differential scanning fluorimetry (DSF). Both compounds displayed preferential cytotoxic activity toward AML cells within the tested concentration range, with MOLM-13 being the most sensitive cell line. 4b1 and 4d1 exhibited IC₅₀ values of 9.35 μM and 6.96 μM, respectively, while showing limited toxicity toward non-tumoral cells. Their antiproliferative effects were time-dependent and associated with distinct cellular responses: 4b1 induced G₂/M arrest accompanied by pronounced apoptosis, whereas 4d1 promoted G₀/G₁ accumulation with a more moderate apoptotic response. These findings were supported by morphological features consistent with apoptotic cell death. Molecular docking revealed that both analogs adopted predicted binding modes that were broadly consistent with the canonical binding orientation of monastrol within the Eg5 allosteric pocket, while DSF demonstrated ligand-induced thermal destabilization, providing experimental evidence consistent with ligand engagement of Eg5. Collectively, this study integrates cellular, biophysical, and computational evidence to demonstrate that selenium-containing monastrol analogs exhibit preferential cytotoxic activity toward AML cells and distinct biological responses, with findings consistent with engagement of the kinesin Eg5 allosteric site, supporting their potential as lead compounds for the development of novel therapies against acute myeloid leukemia.

Eosinophilic chronic rhinosinusitis (ECRS) is a refractory subtype characterized by dominant Th2 inflammation and progressive tissue remodeling, with high postoperative recurrence and limited efficacy of current biological agents. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) represent a promising cell-free nanotherapeutic approach, yet their efficacy in ECRS remains poorly studied. This study investigated the therapeutic efficacy of intranasal MSC-sEV against Th2/Th17 immune dysregulation and mucosal remodeling in a papain-induced ECRS model. Intranasal administration of MSC-sEV during the challenge phase significantly alleviated clinical symptoms and attenuated key pathological features, including epithelial hyperplasia, goblet cell hyperplasia, excessive mucus secretion and mucosal eosinophil infiltration, the latter confirmed by direct eosinophil quantification on H&E-stained sections. Immunohistochemistry showed that MSC-sEV attenuated airway remodeling by downregulating markers such as FXIII-A and CLCA1 while partially restoring E-cadherin levels, indicating attenuation of EMT-like changes. Flow cytometry of splenic lymphocytes revealed that MSC-sEV partially restored the disturbed systemic Th1/Th2/Th17 balance, with a more evident effect on the Th17-associated response, and immunohistochemistry for GATA3, IL-17 A and TNF-α confirmed concordant changes in the nasal mucosa. Transcriptomic analysis showed that these therapeutic effects were associated with downregulation of eosinophil-recruitment chemokines (Ccl11, Ccl24) and with reduced enrichment of IL-17, TNF and JAK-STAT signaling-corroborated at the protein level by decreased phosphorylation of STAT3 and NF-κB p65 and by RT-qPCR confirmation of nine key transcripts. Collectively, this study identifies MSC-sEV as an effective cell-free strategy that attenuates the development of ECRS by partially restoring immune homeostasis and attenuating epithelial remodeling.

Thyroid eye disease (TED) is an autoimmune orbital disorder in which interleukin-6 (IL-6) may reflect inflammatory activity. We evaluated tear IL-6 concentrations across disease activity groups, their association with the Clinical Activity Score (CAS), and paired tear-serum differences. This prospective cross-sectional study included 42 adults: 15 with active TED (CAS ≥3), 14 with inactive TED (CAS 0-2), and 13 healthy controls. IL-6 was measured in diluted tear samples collected by micro-volume wash and paired serum using cytometric bead array with flow cytometry. Analyses comprised one-way analysis of variance, paired Student t-tests, and Pearson correlation. Mean tear IL-6 concentrations were 22.33, 8.49, and 3.82 pg/mL in active TED, inactive TED, and controls, respectively, with a significant overall difference across groups (p = 0.002). Tear IL-6 concentrations exceeded serum concentrations in active and inactive TED (p = 0.001 and p = 0.005, respectively), with no significant difference in controls (p = 0.309). In active TED, tear IL-6 correlated positively with CAS (r = 0.55; 95% confidence interval, 0.05-0.83; p = 0.032). These exploratory findings support further investigation of tear IL-6 as a correlate of clinical activity. Unmeasured ocular-surface status, unquantified tear dilution, and extrapolation below the assay's lowest calibrator limit interpretation; larger longitudinal studies incorporating objective ocular-surface assessment and assays validated for tear fluid are needed before clinical application.

Multiple myeloma is a hematologic cancer caused by abnormal plasma cell proliferation in the bone marrow and is associated with significant immune dysfunction. Neutrophils, essential components of the innate immune response, may function poorly in patients with multiple myeloma, increasing the risk of infection and affecting disease progression. This study aimed to systematically evaluate the extent of neutrophil dysfunction in patients with newly diagnosed and relapsed multiple myeloma. The study employed a cross-sectional analytical design and recruited 72 participants: 30 multiple myeloma patients and 42 age- and sex-matched healthy controls from the University Teaching Hospital and the Cancer Diseases Hospital of Lusaka, Zambia. 4 mL of venous blood was collected from all participants in ethylenediaminetetraacetic acid vacutainers for laboratory analysis. Neutrophils were isolated from whole blood using Polymorphprep™, and functionality was assessed using in vitro assays, including a phagocytosis assay using fluorescently labeled bacterial particles and a reactive oxygen species assay using the fluorescent probe 2', 7'-Dichlorodihydrofluorescein diacetate, measured by flow cytometry. Statistical analysis was performed using STATA. Neutrophils from multiple myeloma patients demonstrated significantly lower phagocytic capacity (45.0% vs 75.9%, P < 0.001) and reactive oxygen species production (44.7% vs 74.9%, P < 0.001) compared to controls. The logistic regression analysis demonstrated strong associations between multiple myeloma and both reactive oxygen species (OR=5097.6, P < 0.001) and phagocytic dysfunction (OR=7272.7, P < 0.001). By contrast, age, sex, white blood cell count, and absolute neutrophil count were not significantly associated with these outcomes. The study found that patients with multiple myeloma exhibit qualitative impairment of neutrophil function, despite normal quantitative neutrophil counts, underscoring the importance of functional immune monitoring in patient management.

Capsular polysaccharides are key virulence determinants in extraintestinal pathogenic Escherichia coli, protecting bacteria from host immune defences while modulating interactions with host tissues. In group 2 capsule gene clusters, transcription of region 1 genes required for capsule export is controlled by a complex regulatory region (PR1) containing three tandem promoters (PR1-1, PR1-2 and PR1-3).In this study, targeted mutations were introduced into the -10 elements of each PR1 promoter in the uropathogenic strain UTI89. Transcriptional output from the PR1 region was assessed using a chromosomal PR1-lacZ fusion, and capsule expression was evaluated by K1 bacteriophage sensitivity assays, flow cytometry and immunofluorescence microscopy. Growth in Lysogeny Broth medium demonstrated that all three promoters contributed to capsule expression in vitro, with expression from both PR1-2 and PR1-3 being dependent on a functional upstream PR1-1.In contrast, infection of PD07i bladder epithelial cells revealed a distinct regulatory pattern in which, following invasion, intracellular capsule expression was restored in the absence of PR1-1 and PR1-2, indicating that PR1-3 was sufficient for capsule induction within bladder cells. The presence of a predicted binding site for the oxygen-responsive regulator fumarate and nitrate reduction regulatory protein near PR1-3 suggests a possible mechanism for intracellular activation of PR1-3 under microaerobic host conditions in the absence of a functional PR1-1 promoter.Together, these findings reveal a niche-dependent, multi-promoter regulatory architecture that enables adaptive control of capsule expression during infection.

Here, we present an in vivo approach to study virus-vector-host interactions during Toscana virus (TOSV) infection in mouse skin. We describe preparation of viral stocks and subcutaneous TOSV inoculation into dorsal upper foot skin, either alone, with sand-fly salivary gland extract (SGE), or following sand-fly biting. We then detail tissue collection and generation of single-cell suspensions. Finally, we describe flow cytometric detection of infected cells, magnetic isolation of dermal fibroblasts, and RNA extraction for transcriptomic analysis. For complete details on the use and execution of this protocol, please refer to Keskek Turk et al.1.