Biomedicine & Pharmacotherapy

Antimicrobial resistance is an impactful One Health issue. One of its drivers is the extensive use of antibiotics in both human and animal production systems, and despite regulatory restrictions on antibiotic use in poultry farming, antimicrobial resistance remains a major challenge. Consequently, animals are at higher risk of harder-to-treat diseases and play a role as resistance reservoirs, highlighting the need for alternative antimicrobial strategies. Towards this end, antimicrobial peptides (AMPs) have emerged as promising candidates due to their broad-spectrum activity and lower propensity to induce resistance. However, the effectiveness of AMPs against poultry pathogens, and in particular multi drug-resistant strains, is largely unclear. To tackle this question, we evaluated the synthetic AMP SET-M33 against four species of clinically relevant pathogens in poultry, namely Escherichia coli, Salmonella enterica, Enterococcus faecalis and Enterococcus cecorum. Using a panel of 141 field isolates, we found that SET-M33 broadly inhibited bacterial growth at low micromolar concentrations (median MICs of 2.5 M and 5 M for Gram-negative and Gram-positive strains, respectively), including in multi drug-resistant isolates. To examine the potential impact of SET-M33 on the host, we established a new in vitro co-cultivation system using chicken intestinal organoids. We found that SET-M33 retains its antimicrobial activity in organoid-microbe co-cultures at concentrations that preserved host viability. These findings demonstrate the potential of SET-M33 as a new antimicrobial agent against pathogens in poultry.

Alzheimers disease (AD) is a multifactorial disease with mixed pathologies. Consequentially, drugs targeting multiple pathological processes may offer synergistic benefits. While histone deacetylase (HDAC) inhibitors have demonstrated efficacy in alleviating AD-related pathologies in animal models, the neuroprotective Wnt/{beta}-catenin signaling pathway remains compromised in AD brain. CI-994 is a class I HDAC inhibitor containing N-(2-aminophenyl)-benzamide. Our recent studies indicate that CI-994 is also an activator of Wnt/{beta}-catenin signaling by stabilizing Wnt co-receptor LRP6. We herein use CI-994 as a scaffold to develop novel potent dual modulators of class I HDACs and Wnt/{beta}-catenin signaling for AD therapy. Our lead compound, W2A-28, selectively inhibits class I HDAC1, 2 and 3 with IC50 values of 0.51 M, 0.68 M, and 0.22 M, respectively, and shows no inhibitory activities on other HDACs. Furthermore, W2A-28 potently activates Wnt reporter activity with an EC50 value of 1.61 M in Wnt-3A-expressing HEK293 cells. As expected, activation of Wnt/{beta}-catenin signaling by W2A-28 is associated with elevated LRP6 protein level. Importantly, W2A-28 displays excellent microsomal stability in both mouse and human liver microsomal stability assays, alongside high permeability and a lack of active efflux in MDR1-MDCKII models. Critically, W2A-28 treatment significantly enhances histone acetylation, activates Wnt/{beta}-catenin signaling, and suppresses tau phosphorylation in AD patient-specific cerebral organoids carrying APOE {varepsilon}4/{varepsilon}4 or APOE {varepsilon}3/{varepsilon}4 with PSEN1 M146V mutation. Our findings position W2A-28 as a promising multi-target drug candidate for AD therapy.

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype lacking well-defined molecular targets, leaving chemotherapy as the primary treatment despite drug resistance, systemic toxicity, and high recurrence rates. Therefore, the development of effective and less toxic therapeutic agents is essential. This study investigated the anti-cancer potential of gloriosine, a bioactive alkaloid with antiproliferative activity and low toxicity toward normal breast cells. MethodsPotential targets of gloriosine were predicted using SwissTargetPrediction, TargetNet, and PharmMapper, and overlapping genes related to TNBC and glutamine metabolism were selected. Protein-protein interaction networks, Gene Ontology, and KEGG pathway enrichment analyses were performed. Molecular docking evaluated binding affinity, followed by in vitro validation using cell viability, colony formation, and wound healing assays. ROS levels were measured by DCFDA and GSH assays, and ferroptosis was assessed by Western blot and FerroOrange staining in MDA{square}MB{square}231 cells. ResultsA total of 100 potential targets were identified, with 60 overlapping with TNBC and glutamine metabolism-related genes. Key targets included SRC, EGFR, mTOR, and HSP90AA1. Enrichment analyses indicated involvement in cancer progression, metabolic regulation, and resistance pathways, including central carbon metabolism, EGFR inhibitor resistance, and ErbB signaling. Gloriosine showed strong binding affinity toward hub targets. Experimental studies confirmed concentration-dependent inhibition of cell proliferation and migration. Mechanistically, gloriosine suppressed glutamine metabolism via GLS1 downregulation and induced ferroptosis, evidenced by increased ROS, glutathione depletion, GPX4 downregulation, and elevated intracellular iron levels. ConclusionsGloriosine exerts significant anti-cancer effects in TNBC through multi-target modulation and induction of ferroptosis, highlighting its potential as a promising therapeutic candidate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/725321v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@ce0ebcorg.highwire.dtl.DTLVardef@29603borg.highwire.dtl.DTLVardef@6d0025org.highwire.dtl.DTLVardef@249700_HPS_FORMAT_FIGEXP M_FIG C_FIG Flow chart of the network pharmacological and in vitro study of gloriosine

The gut microbiome plays a central role in host metabolism, immune function, and overall health, with disruptions in microbial composition (dysbiosis) being associated with a range of metabolic, inflammatory, and infectious conditions [1,2]. Consequently, strategies aiming to modulate the microbiome require selective activity that preserves beneficial commensals while limiting pathogenic organisms [3]. In this context, ThymoQuin(R)--a cold-pressed, standardized black cumin (Nigella sativa) seed oil developed by TriNutra Ltd. and defined by [≥]3% thymoquinone (TQ), controlled p-cymene levels, and low free fatty acids ([≤]1.25%)--was evaluated for its microbiome-relevant activity. In vitro minimum bactericidal concentration (MBC) assays across three independent batches demonstrated a biphasic, dose-dependent response. At intermediate concentrations (0.25-0.5%), Streptococcus thermophilus was strongly stimulated (up to 53-fold) and Lactiplantibacillus plantarum fully preserved, while Klebsiella pneumoniae was effectively reduced (>94%). Akkermansia muciniphila exhibited stable viability at concentrations below 1%, with reductions only observed at 1%. This is notable given its role as a mucin-degrading commensal that has been linked to metabolic health, but whose abundance may vary across physiological and disease contexts [4,5]. At concentrations [≥]1%, selective effects diminished, resulting in broader antimicrobial activity and reduced specificity. These findings indicate a defined concentration range in which selective microbiome modulation is maintained, whereas higher thymoquinone levels may increase the risk of non-selective detrimental effect on microbes.

Innovative therapeutic approaches providing clinically effective medication for Alzheimers disease (AD) patients are urgently needed. In recent years, monoclonal antibodies against Amyloid-{beta} (A{beta}) oligomers were approved as the first disease modifying AD therapies. However, their effects on cognitive decline of AD patients are still limited - most likely because neuroinflammatory processes downstream of A{beta} plaques remain activated, even when plaques are depleted. Accordingly, anti-inflammatory drugs are currently considered as valuable (co-)treatments to target A{beta} associated neuro-inflammation. The anti-inflammatory sphingosine-1-phosphate receptor (S1PR) modulator fingolimod (FTY720) has been shown to alleviate synaptic and cognitive deficits in numerous mouse models of AD. Whether other S1PR modulators exert similar beneficial effects is largely unknown. Here we used a transgenic APP/PS1 AD mouse model to investigate whether the S1PR modulator Ozanimod (RPC1063) can rescue AD pathology and synaptic dysfunctions even when treatment is initiated at 16-17 months of age, which is 10 months after onset of cognitive deficits. We performed quantitative dendritic spine analysis in hippocampal CA1 pyramidal neurons and immunohistochemical labelling of Iba1-positive activated microglia, GFAP-positive reactive astrocytes, and A{beta} plaques in hippocampus and neocortex. Our results reveal that 6 weeks of Ozanimod treatment via drinking water rescues synaptic spine deficits, counteracts A{beta} pathology and reduces neuro-inflammation in the hippocampus and neocortex of APP/PS1 mice. Therefore, it might hold promise to examine a potential disease-modifying effect of S1PR modulators in clinical trials with AD patients.

Background/ObjectivesNeuroinflammation-driven iron dysregulation and neurotoxic astrocyte polarization are increasingly recognized as interconnected pathological mechanisms in neurodegenerative diseases. Systemic inflammation triggered by strenuous exercise or infection can engage the central nervous system and astrocytic inflammatory responses and perturb iron homeostasis; however, targeted nutritional strategies to counteract these processes remain limited. Inflamate(R) is a multi-component botanical supplement comprising boswellic acids, astilbin, xanthohumol, and cinnamaldehyde, each with documented anti-inflammatory properties. However, whether this combined formulation can modulate the inflammatory-iron metabolic axis and astrocyte phenotypic polarization remains unexplored. This study aimed to investigate the effects of Inflamate(R) on LPS-induced pro-inflammatory gene expression, iron metabolism-related gene regulation, and A1/A2 astrocyte phenotypic polarization in mouse astrocytes. MethodsMouse astrocytes (AWT) were pre-treated with Inflamate(R) (0.0375 g/mL) or DMSO vehicle for 24 h, followed by lipopolysaccharide (LPS; 1 g/mL) stimulation for an additional 24 h. The non-cytotoxic working concentration was determined by morphological assessment, CCK-8 cell viability, and LDH cytotoxicity assays. Expression of 14 target genes spanning pro-inflammatory mediators (NOS2, IL6, C3, COX2, PLA2g15, SOCS3), iron metabolism regulators (FTH1, Hepcidin, TFRC, SLC40A1, RGMa, RGMb), and astrocyte polarization markers (S100A10, GFAP) was quantified by qRT-PCR. ResultsUnder normal culture conditions, Inflamate(R) did not significantly alter the expression of any target gene except S100A10, confirming the absence of baseline cytotoxicity or transcriptional homeostatic perturbation. Upon LPS stimulation, Inflamate(R) selectively suppressed NOS2 (approximately 64% reduction, p < 0.0001), IL6 (approximately 37% reduction, p < 0.0001), and C3 (approximately 47% reduction, p < 0.0001), while COX2, PLA2g15, and SOCS3 remained unaffected. Concurrently, Inflamate(R) significantly reduced LPS-induced Hepcidin expression to approximately 17% of the control level (p < 0.05) and attenuated FTH1 upregulation (p < 0.01), without altering the expression of iron transporters (TFRC, SLC40A1) or BMP-SMAD pathway components (RGMa, RGMb). Furthermore, Inflamate(R) upregulated the neuroprotective A2 marker S100A10 under both basal (p < 0.05) and LPS-stimulated conditions (p < 0.01), while the general reactivity marker GFAP remained unchanged. ConclusionsInflamate(R) exerts a selective, multi-target modulatory effect at the transcriptional level in LPS-stimulated astrocytes, encompassing suppression of the iNOS-NO and IL-6 signaling axes, attenuation of inflammation-driven hepcidin-ferritin iron dysregulation via the IL-6-STAT3 pathway, and promotion of a phenotypic shift from neurotoxic A1 toward neuroprotective A2 astrocyte polarization. Given that the IL-6-JAK-STAT3-hepcidin axis is also activated during exercise-induced systemic inflammation, these findings suggest that Inflamate(R) may represent a targeted nutritional strategy for preserving CNS iron homeostasis and supporting neuroprotective astrocyte function in both neurodegenerative and exercise-related neuroinflammatory contexts. Further validation in in vivo neurodegenerative and exercise models, including protein-level analyses, is warranted to confirm these transcriptional findings.

Fv-HSP72 is a rapid cell-penetrating human heat shock protein for the treatment of traumatic organ injuries. We have shown this re-engineered protein (HSP72) is capable of crossing the blood brain barrier (BBB) of rats suffering a controlled cortical impact (CCI) and remains in brain tissue for up to 12 hours; long after clearance from the cortex of uninjured rats. Peptide sequences unique to Fv-HSP72 allow for its differential detection from endogenous HSP72. Male Sprague-Dawley rats were divided into 10 groups of n=10 with those animals receiving a CCI subjected to a unilateral cortical contusion simulating a moderate to severe brain injury using an electronically controlled pneumatic impact device. Control groups were either uninjured (Sham), injured (TBI Only), or injured and given buffer (TBI+Vehicle). Rats treated with one of three Fv-HSP72 variants were dosed at 10 or 30mg/kg 15m post-impact, then sacrificed 48 hours later. Cortical tissues were extracted from the ipsilateral and contralateral hemispheres for biomarker analysis. Here we report results of our drug inhibiting neurodegeneration based on five biomarkers (NF-L, pNF-H, pTau [T181, T231, S396]). These results were statistically significant, especially for one of the Fv-HSP72 variants, when comparing differences both between treatment groups and within groups (i.e. when comparing ipsi-vs. contralateral hemispheres). Significant inhibition of oxidative stress (3-NT) and inflammatory (IL-6) biomarkers were also observed (both p<0.0001). With similar results obtained for a blast injury model being published elsewhere, the analyses suggest Fv-HSP72 is neuroprotective following a direct impact brain injury. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly inhibiting neuronal tissue damage in the brain when administered after a direct cortical impact.

BackgroundEpidemiological studies consistently report inverse associations between caffeinated coffee consumption and dementia risk. However, the molecular mechanisms linking coffee-derived phytochemicals to neuroprotection remain only partially understood. ObjectiveTo evaluate, through integrated in silico pharmacology, the relative contribution of adenosine receptor modulation versus direct amyloidogenic enzyme and kinase inhibition in mediating the putative neuroprotective effects of major coffee constituents. MethodsMolecular docking analyses were conducted for caffeine, paraxanthine, chlorogenic acid, trigonelline, cafestol, and kahweol against adenosine A2A and A1 receptors (A2AR, A1R), {beta}-secretase 1 (BACE1), glycogen synthase kinase-3{beta} (GSK-3{beta}), and NLRP3 inflammasome components. Docking was performed using the CB-Dock2 platform. Binding affinities, interaction patterns, and ligand efficiency metrics were assessed. Blood-brain barrier permeability and ADMET properties were predicted using pkCSM. ResultsCaffeine and paraxanthine demonstrated structurally coherent binding within the orthosteric pockets of A2AR and A1R, supported by favorable predicted blood-brain barrier penetration and high unbound fractions. Ligand efficiency analysis identified adenosine receptors as the most pharmacologically plausible targets for small xanthine derivatives. Although larger phytochemicals exhibited stronger absolute docking scores at BACE1, GSK-3{beta}, and NLRP3, predicted pharmacokinetic constraints suggest a small biological effect due to a limited central exposure. ConclusionsThese findings support an adenosine receptor-centered mechanism as the dominant molecular axis linking caffeinated coffee consumption to reduced dementia risk, favoring neuroinflammatory and signaling modulation over direct enzymatic inhibition. Experimental validation is warranted to confirm translational relevance. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/723029v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a02629org.highwire.dtl.DTLVardef@129890dorg.highwire.dtl.DTLVardef@1e4c05corg.highwire.dtl.DTLVardef@110ec7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Temporal lobe epilepsy (TLE) has an unmet need for precision treatments targeting the seizure focus while avoiding effects on other body parts to minimise side effects. Photopharmacology could enable precision treatment by combining systemic administration of a photoswitchable drug with implantation of an optic fibre in the epileptic focus to induce light-dependent drug conversion from an inactive to an active configuration that interacts with its target receptor to suppress seizures. The photoswitchable {Delta}9-tetrahydrocannabinol ({Delta}9-THC) derivative, azo-THC-3, transitions from an inactive trans to an active cis configuration upon UV irradiation. We demonstrate that local or systemic administration of azo-THC-3 and local UV irradiation in the hippocampus supresses difficult-to-treat seizures in the intrahippocampal kainic acid mouse model of TLE. Furthermore, our findings illustrate that the photoswitch strategy avoids hypolocomotion, a common side effect of systemic {Delta}9-THC administration. As such, we provide the first demonstration of seizure suppression with the systemic administration of a photoswitchable compound and its local photoactivation in the seizure focus. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/720358v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1e42794org.highwire.dtl.DTLVardef@1e26891org.highwire.dtl.DTLVardef@13f2b6forg.highwire.dtl.DTLVardef@3c8e48_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundLauric acid (LA) is a medium-chain saturated fatty acid found in several foods, including vegetable oils and seeds. Previous studies have demonstrated that LA exhibits neuroprotective, antioxidant, and anti-inflammatory properties in experimental models of neuropsychiatric disorders. Therefore, the present study aimed to investigate the behavioral and neurochemical effects of LA in a corticosterone-induced murine model of depression. MethodsMale Swiss mice received corticosterone (CORT; 20 mg/kg, subcutaneously) for 23 consecutive days, while the control group received vehicle only. During the last nine days of the experimental protocol, the animals received the respective treatments by oral gavage: LA (10 or 20 mg/kg), fluvoxamine (FLUV; 50 mg/kg), or vehicle, administered 1 hour after CORT injection. One hour after treatment administration, the animals were subjected to the behavioral tests: Forced Swimming Test (FST), Tail Suspension Test (TST), and Open Field Test (OFT). At the end of the experimental protocol, the animals were euthanized, and the prefrontal cortex (PFC), hippocampus (HPC), and striatum (STR) were collected for neurochemical analyses. ResultsChronic CORT treatment significantly increased immobility time in the FST and TST, characterizing depressive-like behavior. Treatment with LA reversed these behavioral alterations, showing an effect similar to that observed in the FLUV-treated group. In the OFT, LA did not promote significant changes in locomotor activity, suggesting the absence of psychostimulant effects. Regarding neurochemical analyses, LA treatment did not reduce malondialdehyde (MDA) or nitrite/nitrate (NO2-/NO3-) levels, nor did it alter reduced glutathione (GSH) levels in the evaluated brain regions. ConclusionThe results demonstrated that LA treatment was able to reverse corticosterone-induced behavioral alterations in mice, indicating a potential antidepressant-like effect. Furthermore, the observed effects were not associated with nonspecific locomotor alterations. Although LA did not promote significant changes in the evaluated neurochemical markers, these findings reinforce its potential as a therapeutic agent for depressive disorders and highlight the need for further studies to elucidate its mechanisms of action and possible clinical applicability.

Radiotherapy is widely used in the management of brain tumors; however, it is often associated with delayed adverse effects, including cognitive decline and depression-like behavior. These effects are thought to arise, in part, from suppressed hippocampal neurogenesis, altered neuronal architecture, and microglial dysfunction. Despite this, the precise mechanisms underlying irradiation-induced cognitive deficits, as well as effective therapeutic interventions, remain poorly understood. In the present study, six-month-old male mice were subjected to a single 9 Gy dose of cranial irradiation, followed by behavioral assessments several weeks post-exposure. We observed that cranial irradiation significantly impaired hippocampal-, prefrontal cortex-, and cortical-dependent memory functions. Notably, treatment with dehydrozingerone (DH), a curcumin analog (50 mg/kg, oral administration for two weeks), markedly prevented these cognitive deficits. At the molecular level, irradiation disrupted the activity of key enzymes involved in the tricarboxylic acid (TCA) cycle and the glutamate-glutamine/GABA cycle, both of which were restored following DH treatment. Furthermore, irradiation induced dysregulation of genes and proteins associated with glycolysis (Atp2b1, mt-Nd2, mt-Atp6), mitochondrial energetics (mt-Atp8, mt-Cytb), glucose transport (Slc4a5), insulin resistance (Etnppl), lipid metabolism (Pla2g3, Plin4), and inflammation (Ighg2c), all of which were significantly normalized by DH. Importantly, DH also prevented irradiation-induced loss of cell-type-specific glucose transporter expression, including GLUT3 in neurons and GLUT5 in microglia. In conclusion, our findings suggest that DH is a promising therapeutic candidate for mitigating irradiation-induced energy deficits and cognitive impairments, likely through modulation of metabolic and mitochondrial pathways.

Atopic dermatitis (AD) is a highly prevalent, relapse-remitting, inflammatory skin disease, the hallmark symptom of which is chronic itch. Mechanisms underlying AD itch are multifactorial, involving various cells, receptors, and mediators. Developing a physiologically relevant, human model system for AD itch research and drug development is crucial. To this end, human induced pluripotent stem cell-derived sensory neurons (iPSCSNs) were cultured with human primary keratinocytes to form deconstructed skin models. Using Ca2+-imaging in a direct contact, 2.5D co-culturing format, which mimics natural skin innervation and permits both paracrine exchange and juxtacrine signaling, iPSCSNs exhibited functional TRPA1 responses not seen in monotypic iPSCSN cultures or in iPSCSNs conditioned with keratinocyte medium. Different AD-associated cytokines were used to stimulate the co-culture systems to mimic an inflamed lesional skin environment, whereby TNF was found to increase iPSCSN chemosensitivity. Finally, both TRPA1 and JAK1/2 inhibition reduced iPSCSN responses to pruritogens (TSLP, IL-31), thus supporting TRPA1 as a therapeutic target for AD itch in humans. This study demonstrates that human deconstructed skin models can be a useful tool in AD and broader pruritus research. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/724000v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@11c0c9borg.highwire.dtl.DTLVardef@7fa518org.highwire.dtl.DTLVardef@2fe7a2org.highwire.dtl.DTLVardef@1105fa7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pioneering research is adapting chimeric antigen receptors (CARs) from oncology to Alzheimers disease (AD) by targeting amyloid beta (A{beta}). Newer synthetic receptor systems can go beyond, transforming cells into targeted biological drug factories that can couple A{beta} detection to synthesis and secretion of genetically encoded therapeutics. Among candidate systems, T cells Redirected for Universal Cytokine Killing (TRUCK), synthetic Notch (synNotch), and Synthetic Intramembrane Proteolysis Receptors (SNIPR) have shown promise in oncology. Here, we adapt these platforms to AD using a shared A{beta}-targeting binding domain derived from Aducanumab (Aduhelm), coupled to inducible expression cassettes driving identical transgenes: secreted Metridia luciferase (MetLuc) and a Lecanemab (Leqembi)-based chimeric human-mouse antibody (chLecanemab). To validate these systems in vitro, Jurkat clones expressing each receptor were treated with oligomer-enriched A{beta} (A{beta}O) to model AD, and receptor output was quantified by media MetLuc levels and chLecanemab colocalization with A{beta} aggregates. For TRUCK systems, we show the A{beta}-targeting CAR successfully activated Jurkat cells by flow cytometry. We also show that six Nuclear Factor of Activated T-cells (NFAT) tandem repeat response elements (6xNFAT) paired with either minimal interleukin-2, synthetic TATA box, or minimal cytomegalovirus promoters resulted in functional regulatory regions. Despite this, all TRUCK variants failed to significantly upregulate MetLuc in response to A{beta}O. In contrast, both synNotch and SNIPR responded robustly to A{beta}O, with SNIPR outperforming synNotch in both MetLuc and chLecanemab production. These findings establish SNIPR and synNotch as promising platforms for future research on cell-based targeted therapeutic delivery in AD.

BackgroundDichapetalin M (Dic M), an active compound extracted from medicinal plants in the Dichapetalum genus, has been previously shown to possess anti-proliferative activity against cancer cell lines. However, the specific mechanism through which it exerts its anticancer effects remains unknown. PurposeThis study focused on elucidating the mechanism of action of dichapetalin M to further explore its potential as a therapeutic agent for resistant and metastatic breast cancer. MethodWe confirmed the Estrogen Receptor (ER) as a target of Dic M, using an in vitro approach. Furthermore, we examined both the apoptotic and migrastatic effects of dichapetalin M by assessing its impact on the expression of key apoptosis-related and cancer cell migration genes. Finally, we evaluated the compounds effect on Multi-drug Resistance Gene MDR1 expression, a gene linked to cancer drug resistance. ResultsOur target validation experiments demonstrated that Dic M exhibited considerably higher cytotoxicity in ER-positive breast cell lines compared to ER-negative cell lines. Furthermore, treatment of MCF-7 cells (which are ER-positive) with Dic M led to a dose-dependent increase in AREG (amphiregulin), a downstream effector of the Estrogen Receptor. Additionally, Dic M inhibited actin polymerization and significantly downregulated genes involved in the turnover of actin monomers. Scratch-wound assay results further demonstrate that Dic M reduces the rate of cell migration, although its impact on EMT-related gene expression was only observed at high doses. Additionally, Dic M treatment in MCF-7 cells resulted in a significant decrease in the expression of pro-apoptotic genes and MDR1 expression. ConclusionsThese findings indicate that Dic M likely interacts with the Estrogen Receptor and employs the apoptotic pathway to exert its cytotoxic and anti-proliferative effects. Dic M exhibits promising potential, such as anti-migrastatic properties and downregulation of a key breast cancer resistance gene, warranting further investigation.

Background and AimsSteatotic liver disease (SLD) is characterized by excessive lipid accumulation in hepatocytes, and alcohol consumption may modify the disease course, but the evidence is inclusive. This systematic review and meta-analysis aimed to holistically evaluate the impact of mild, moderate, and high levels of alcohol consumption on hepatic and extrahepatic outcomes in SLD. MethodsWe systematically searched EMBASE, MEDLINE, Web of Science, and the Cochrane Central Register of Controlled Trials for relevant studies. The study outcomes included liver related events, malignancy, mortality and cardiovascular disease among adults with SLD who consumed alcohol. ResultsOf 2228 records identified, twenty-six studies comprising 466611 adults with SLD were included. High alcohol consumption was associated with an increased risk of liver-related events compared with abstinence (2.97, 95% CI 1.61-5.50; p<0.001), and a similar association was observed among alcohol drinkers overall (HR 1.93, 95% CI 1.60-2.33; p<0.001). Moderate alcohol consumption was associated with a higher incidence of malignancy (HR 1.41, 95% CI 1.13-1.78; p=0.677). In contrast, mild alcohol consumption was associated with lower all-cause mortality compared with abstinence (HR 0.88, 95% CI 0.78-0.98; p=0.001). No association was observed between alcohol consumption and cardiovascular disease incidence or hepatocellular carcinoma ConclusionsAlcohol intake may increase the risk of liver-related complications and cancer risk in individuals with SLD. Mild alcohol consumption was associated with lower all-cause mortality, and alcohol intake showed no association with cardiovascular disease incidence. Further studies are needed to clarify the dose-dependent effects of alcohol on hepatic and extrahepatic outcomes in SLD.

BackgroundThe discontinuation of Fasiglifam (TAK-875), a GPR40/FFAR1 full agonist, during Phase 3 clinical trials due to hepatotoxicity led to widespread abandonment of GPR40 as a viable therapeutic target for type 2 diabetes mellitus (T2DM). However, mechanistic evidence suggests that Fasiglifams hepatotoxicity arises from mitochondrial liability driven by high lipophilicity (aLogP = 5.31), rather than from on-target GPR40 signaling. We hypothesized that target-level failure was incorrectly inferred from compound-level safety concerns, and that superior candidates exist within publicly available databases. MethodsWe queried ChEMBL Release 36 (28 GB SQLite, 74 tables) for all compounds with documented GPR40/FFAR1 activity (UniProt: O14842). Compounds were filtered by EC50 [&le;] 10 nM in nM units with standard relation "=". Drug-likeness was assessed using Lipinskis Rule of Five (Ro5), aLogP, molecular weight (MW), hydrogen bond donors/acceptors (HBD/HBA), and polar surface area (PSA). A parallel analysis of Therapeutic Target Database (TTD v10.1.01, 4,298 targets) provided clinical context. A real-world evidence (RWE) patient stratification framework was constructed using EMR data from tens of millions of patients with >10 years of longitudinal follow-up. ResultsOf 2,637 GPR40-active compounds in ChEMBL 36, 526 (19.9%) demonstrated EC50 < 100 nM and 102 (3.9%) demonstrated EC50 < 10 nM. Eight compounds met stringent drug-likeness criteria (Ro5 violations = 0, aLogP < 5.0, EC50 [&le;] 1 nM). The lead compound (CHEMBL4859651) exhibited EC50 = 0.04 nM (8.75-fold more potent than Fasiglifam), MW = 297 Da (43% lower), and aLogP = 4.30 (19% lower), with zero Ro5 violations. Mean MW of the eight candidates was 317 {+/-} 28 Da versus 524 Da for Fasiglifam. A parallel GCK analysis identified a protein-protein interaction target (CHEMBL3885579, GCK-GKRP interface) harboring 40 exclusive compounds as an orthogonal strategy for partial GCK activation. ConclusionsSystematic cheminformatic analysis reveals that compounds with substantially superior activity and drug-likeness profiles relative to Fasiglifam exist within ChEMBL 36. Fasiglifams hepatotoxicity is attributable to compound-specific physicochemical properties, not GPR40-mediated toxicity. RWE patient stratification may further mitigate hepatotoxicity risk for next-generation GPR40 agonists. These findings argue for systematic reappraisal of GPR40 as a viable therapeutic target for T2DM.

Liver sinusoidal endothelial cells (LSEC) rapidly dedifferentiate in 2D-monoculture, losing their high endocytic activity and characteristic morphology, limiting their use in mechanistic studies. We established and validated culture conditions that preserve LSEC endocytic capacity for at least 10 days, enabling efficient in vitro siRNA-mediated gene silencing. Mouse LSEC were cultured in 5% oxygen, growth media partially exchanged daily and assessed for cell viability, endocytic capacity, morphology and ultrastructure. Despite typical culture-induced defenestration, the cells showed high viability and efficient endocytosis via scavenger-receptors. This allowed for siRNA-mediated mannose receptor knockdown exemplified by 96% and 76% reduction in Mrc1 mRNA and protein expression at 72h (validated by qPCR and Western blot), with functional assays confirming decreased mannose-receptor-mediated endocytosis. Extended maintenance of LSEC viability and functions, previously restricted to complex co-culture systems, provide a practical platform for investigating LSEC-specific molecular mechanisms and hepatic sinusoid physiology.

Microbial bile salt hydrolase (BSH) plays a central role in shaping bile acid composition and gut-liver metabolic signaling, yet its therapeutic potential in metabolic dysfunction-associated steatohepatitis (MASH) remains incompletely defined. Here, we evaluated the efficacy of the non-absorbable BSH inhibitor GR-7 in a diet induced mouse model of steatohepatitis using early and late intervention strategies with different dosing regimens. GR-7 reduced food intake and exerted stage- and dose-dependent therapeutic effects, with early intervention robustly suppressing hepatic fibrosis even at low dose, whereas late-stage administration of high-dose GR-7 markedly reduced hepatic steatosis and inflammation, as evidenced by decreased liver weight, hepatic triglyceride and cholesterol levels, and plasma ALT. Although late intervention did not result in statistically significant histological reversal of fibrosis, a trend toward improvement was observed, together with suppression of fibrogenic gene expression, suggesting that prolonged treatment may further enhance antifibrotic efficacy. Mechanistically, GR-7 effectively inhibited microbial BSH activity in vivo, leading to reduced cecal unconjugated primary and secondary bile acids--including deoxycholic acid and lithocholic acid, which was associated with improved gut barrier integrity and reduced hepatic inflammation. In parallel, BSH inhibition reprogrammed hepatic bile acid metabolism toward activation of the alternative CYP27A1-mediated synthesis pathway, accompanied by reduced food intake, thereby contributing to improved hepatic lipid accumulation. Furthermore, late-stage high-dose treatment selectively remodeled the hepatic immune landscape rather than fully restoring homeostasis, highlighting immune recalibration as a key component of therapeutic response. Together, these findings identify microbial BSH inhibition as a promising microbiome-targeted therapeutic strategy for MASH. HighlightsO_LIThe non-absorbable BSH inhibitor GR-7 improves steatosis, inflammation, and fibrosis in of Western diet-induced steatohepatitis model in mice in a dose-dependent manner. C_LIO_LIGR-7 reduces food intake and body weight gain. C_LIO_LIGR-7 reduces cytotoxic secondary bile acids, including DCA and LCA. C_LIO_LIGR-7 reprograms hepatic bile acid metabolism and immune responses. C_LI

Excessive alcohol consumption remains a major public health challenge with limited therapeutic options. Both glucagon-like peptide-1 (GLP-1) and fibroblast growth factor-21 (FGF21) independently regulate alcohol intake through complementary metabolic and reward pathways, but their combined potential has not been explored. Here, we report that a long-acting dual agonist, GLP1-ELP-FGF21 modulates behavioural, neurophysiological, and cognitive components of alcohol seeking in mice. A single GLP1-ELP-FGF21 dose reversibly reduces voluntary alcohol intake for at least 72 hours in male mice, has sustained effects in female mice, and markedly blunts nucleus accumbens dopamine transients aligned to the initiation and termination of lick bouts during alcohol consumption. To assess its effects on decision-making, we used a novel two-choice (alcohol versus food) decision task modelled with evidence-accumulation frameworks. Alcohol choice behaviour conformed to evidence accumulation decision models: Linear Ballistic Accumulator (LBM) and Racing diffusion models (RDM). Critically, GLP1-ELP-FGF21 selectively reduces choices for alcohol and slows the latent accumulation rate for alcohol options, without affecting food-directed choice or non-decision processes. Sensory-specific satiety devaluation confirms that reductions in reward value are explained by reductions in accumulation rates. Together, these results highlight GLP1-ELP-FGF21 as a therapeutic strategy for alcohol use disorder via modulation of central reward pathways and decision-making when confronted with alcohol rewards.

The search for antimicrobials with a low propensity to select resistance has intensified in response to the global antimicrobial resistance crisis. Norway spruce resin (Picea abies) has long been used in Northern European wound care traditions and has shown broad antimicrobial activity in earlier microbiological studies. In the present study, we evaluated whether prolonged exposure to medical-grade spruce resin promotes reduced susceptibility in clinically relevant bacterial species. A 20-day serial-passage experiment was performed with Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis using sub-inhibitory resin concentrations and broth microdilution readouts at baseline, day 10, and day 20. Resistance development was predefined as a [&ge;]4-fold increase in inhibitory concentration. Baseline inhibitory concentrations were 1.25% for S. aureus, 5.0% for P. aeruginosa, and 2.5% for E. faecalis. After 20 days, inhibitory concentrations were 2.5%, 10.0%, and 2.5%, respectively, corresponding to at most 2-fold changes and remaining below the predefined threshold for resistance development. Validation and vehicle-control arms indicated that these shifts were not attributable to medium transfer or solvent-related bias. These findings suggest that medical-grade Norway spruce resin has a low short-term tendency to select for reduced susceptibility under serial-passage conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723837v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@160479forg.highwire.dtl.DTLVardef@1fe1e95org.highwire.dtl.DTLVardef@89dec3org.highwire.dtl.DTLVardef@17ff134_HPS_FORMAT_FIGEXP M_FIG C_FIG