Pharmaceuticals

Non-small cell lung cancer (NSCLC), as the predominant subtype of lung cancer, presents a considerable clinical challenge due to its high rates of recurrence and the significant adverse effects associated with conventional therapeutic modalities. In response to these challenges, this study explored the new combined anticancer effects of epigallocatechin gallate (EGCG) together with thermal cycling-stimulation (TCS). The findings demonstrated that the combination of EGCG and TCS synergistically decreased the viability of A549 and NCI-H460 NSCLC cells, while exhibiting minimal cytotoxic effects on IMR-90 normal lung fibroblasts. Further investigation revealed that EGCG mitigated the TCS-induced upregulation of heat shock proteins HSP70 and HSP105 and concurrently diminished the expression levels of proteasome subunits. This combined effect disrupted proteostasis, resulting in pronounced endoplasmic reticulum (ER) stress. Subsequently, a positive feedback mechanism was established between inositol 1,4,5-trisphosphate receptor (IP3R)-mediated ER Ca2+ release and excessive reactive oxygen species (ROS) production, ultimately leading the cells to undergo mitochondrial apoptosis. This combined treatment reduces the necessary dosage of EGCG, thereby overcoming limitations related to its poor bioavailability and systemic toxicity, while also preventing the development of thermotolerance induced by TCS. Consequently, this method offers a new and potentially practical therapeutic strategy for treating NSCLC.

Temozolomide is the gold standard chemotherapeutic agent used in the treatment of glioblastoma multiforme. Yet its pharmacological use has been linked to the emergence of depressive- and/or anxiety-like behaviors, probably through the inhibition of hippocampal neurogenesis. Since prior studies reporting these negative effects were based on prolonged treatment paradigms (i.e., from 2 weeks to up to 6 months), and given the few reports that have included female rodents in their studies, our approach aimed at further characterizing the behavioral effects induced by temozolomide (25 mg/kg, 1 or 2 cycles, 5 days/cycle) in a mixed-sex cohort of adult rats. To do so, rats were scored across time through specific behavioral tests that capture diverse manifestations of affective-like responses (forced-swim, open field, novelty-suppressed feeding and sucrose preference) or cognitive performance (Barnes maze). At the neurochemical level, we ascertained the effects of 2 cycles of temozolomide on hippocampal neurogenesis (neural progenitors with NeuroD) and other potential neuroplasticity targets (i.e., FADD, BDNF). The main results showed that temozolomide induced unexpected antidepressant-like responses in a treatment-duration manner while decreased hippocampal FADD, a neuroplastic marker previously associated with the acute and repeated actions of most antidepressants. These results break the prior dogma linking increased hippocampal neurogenesis with antidepressant-like efficacy, and suggest that other mechanisms of action, such as the one described through the neuroplastic molecule FADD, might be responsible for the antidepressant-like actions of temozolomide, even in the presence of impaired neurogenesis. Our results, in conjunction with the prior data, suggested cycle- and/or length-dependent treatment effects in terms of temozolomides antidepressant- vs. depressant-like profile, while proposing a novel biomarker of its treatment response.

Migraine headaches affect over one billion people internationally and can be defined as episodes of acute severe pain wrapping around the head and are normally accompanied by nausea, blurry vision, and sensitivity to light and sound. While triggers that cause migraines may vary among patients, evidence shows they are involved with the trigeminovascular system (a network of blood vessels in the brain in conjunction with the trigeminal nerve). Activation of the trigeminal neurons triggers the release of vasoactive neuropeptides, such as calcitonin gene-related peptide (CGRP), leading to neurogenic inflammation and vasodilation of cranial blood vessels. The development of 5-HT1B serotonin agonist drugs, commonly known as triptans, have been an effective measure of migraine relief. The drugs created in this research were found to have improved docking scores within the 5-HT1B binding site compared to that of naratriptan. The two drugs proposed in this paper would need to undergo further investigation to determine the feasibility of laboratory synthesis and clinical trials.

Ovarian and colorectal cancers have the highest incidence and mortality in the world, after breast cancer. Despite the initial response to Pt-drugs or 5-fluorouracil (5-FU), many cancer cells develop resistance to these drugs. For this reason, new therapeutic strategies represent an important medical need, in particular for drugs that are being studied in combination with methods that promote their entry into the cell. Among these strategies, electrochemotherapy (ECT), the combination of drugs with electroporation (EP), a physical method that uses high-frequency electrical pulses to create pores into which chemotherapy drugs can permeate, is gaining interest. In this study, we have evaluated the effect of ECT on the growth of both ovarian (A2780 and A2780/CP) and colorectal (HCT116) cancer cell lines using platinum derivatives (Cisplatin, Carboplatin and Oxaliplatin), as DNA alkylating agents, and human thymidylate synthase (hTS) inhibitors, both traditional (5FU) and novel TS destabilizers (compounds E3 and E7). To this aim, synergism quotient-like analysis to determine whether electroporation gives an advantage in terms of cytotoxicity was applied to the relative IC20 and IC50 concentrations of each drug. Results showed that two of the three Pt-drugs have greater efficacy when combined with EP. 5-FU and the new TS inhibitors E3 and E7 also take advantage of ECT because EP increases drug uptake into the cell, even in resistant cells. In conclusion, ECT appears to be a viable strategy to obviate the problem of resistance in ovarian and colorectal cancers, to deliver compounds inside cells overcoming uptake limits, especially for the low lipophilic compounds whose cytotoxic efficacy is hampered by the obstacle of biological membranes.

RationalePrimary Ciliary Dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, defective mucociliary clearance, and progressive lung disease. Pathogenic variants in the DNAI1 gene are a well-known cause of PCD. Currently, no approved therapies address the underlying genetic defect. RCT1100 is an inhaled mRNA therapy encoding DNAI1 currently under clinical development. This study evaluates the functional effects of RCT1100 using a fast three-dimensional explant spheroid (3DE-S) model consisting of apical-out undifferentiated nasal epithelial cells derived from patients with DNAI1 PCD. Methods3DE-S were generated from nasal brushings of five patients with confirmed biallelic DNAI1 variants. RCT1100 was administered from day 5 directly to culture wells three times weekly for two weeks. Spheroid motility was assessed throughout treatment by quantifying the proportion of moving spheroid rolling and their movement velocity. Following six doses, spheroids were harvested for high-speed video microscopy for assessment of ciliary beat frequency. ResultsEvaluable data were obtained from three of five patient samples; two samples were excluded due to contamination. After six doses of RCT1100, ciliary beat frequency increased from a baseline range of 2.8-3.5 Hz to 6.7-6.8 Hz post-harvesting. Mean spheroid movement velocity increased from 0.11 {micro}m/sec to 3.87 {micro}m/sec following dosing with 10 {micro}g/mL RCT1100, with more than 80% of spheroids exhibiting coordinated rolling motion pattern. ConclusionThe 3DE-S is a robust platform for evaluating targeted therapies. RCT1100 significantly restored ciliary function, supporting its therapeutic potential and highlighting the utility of spheroid-based systems for precision medicine approaches in DNAI1 PCD.

In some countries, melatonin is sold without a physician prescription and dosage is unregulated. Transdermal products have become popular including those marketed for children. We measured consumer assumptions about these products among adult residents of the United States, analyzed lot-to-lot variability, and compared the pharmacokinetics of melatonin administered in oral, lotion, and bath product forms. Survey respondents (n=199) believed oral melatonin was more effective than transdermal products and that all melatonin products were relatively safe. Melatonin lotion products analyzed by HPLC displayed lot-to-lot variability as well as changes in formulation and product claims. To determine pharmacokinetics, three different treatments (oral tablets, lotion, and bath immersion) were administered to twelve undergraduate participants in a randomized, crossover design. Five additional participants completed bath product treatment only. Participants collected saliva samples up to 48 hours after administration, which were analyzed for melatonin by enzyme-linked immunosorbent assay. Oral (n=11) and lotion formulations (n=12) caused maximum salivary melatonin levels within 30 minutes after administration, but bath immersion did not cause increases in saliva melatonin (n=17). The half-life of oral melatonin was 1.17 [0.69 -- 1.65] hours versus 5.72 [3.75 -- 7.68] hours for lotion treatment (p = 0.011, effect size r = 0.770). Melatonin lotion may pose a risk to consumers who assume it is safe and less effective than oral tablets, when in fact it may be very potent and remain at high physiological levels into the following day. This study is registered on clinicaltrials.gov (NCT06382610) and was funded by the Sleep Research Society.

The success of Chimeric Antigen Receptor (CAR) T cell therapy is heavily dependent on the quality of the final cellular product. Current expansion protocols often rely on reagents that require removal from cell culture media, posing logistical challenges in manufacturing, and can also lead to terminal differentiation. Here, we evaluate the use of a soluble, bead-free T cell activator, T cell expansion protein (T-CEP), as a streamlined alternative for generating potent CAR-T cells. Human T cells were activated with T-CEP or known T cell activators (Dynabeads and TransAct) and transduced with either CD19 or interleukin-13 (IL-13) mutein (tetravariant-13; TV-13)-based CAR lentiviral vectors. Our results demonstrate that T-CEP supports robust CAR-T cell expansion and achieves transduction efficiencies comparable to commercial reagents for both types of CAR-T cells. Notably, T-CEP significantly favored the expansion of CD8+ T cells, yielding an enhanced CD27+ phenotype and a lower CD4:CD8 ratio compared to TransAct. Cytotoxicity assays confirmed that T-CEP-expanded CAR-T cells possess cytolytic function equivalent to commercial reagents for both CARs, while exhibiting lower levels of inflammatory cytokine secretion. In summary, T-CEP represents a competitive alternative to existing expansion agents, as it does not require its removal during CAR-T manufacturing and generates a CD8+ dominant, less-differentiated phenotype without compromising efficacy.

Glioblastoma is a very aggressive brain tumor with few therapeutics options. Type I and II Interferons (IFNs) co-formulation HeberFERON has been used in cancer treatment, with promising results in high grade brain tumors. High throughput techniques in easy-to-handle models have been important to interrogate biomolecules changes, describe mechanisms and find pharmacodynamic biomarkers. This study aims to elucidate the effect of HeberFERON over the cell proteome in comparison to its individual IFNs components. Proteomic changes with HeberFERON in the glioblastoma-derived cell line U-87MG, in comparison with individual IFN-2b and IFN-{gamma}, were studied using a nanoLC instrument EasyLC coupled to Velos Pro mass spectrometer; Maxquant and Perseus were also used. Several enrichment tools, networking analysis and canSAR for drug targets were employed. Translation, RNA processing, mitotic cell cycle, cytoskeleton and chromosome organization, apoptosis, autophagy, DNA repair are enriched to limit cellular growing together with changes in immune response components, supporting HeberFERON as a multitarget treatment. This co-formulation is distinguished at modulating RNA splicing with SMN complex, cytoskeleton organization and microtubule-based movement, nuclear envelope breakdown, DNA conformational changes, and oxidative phosphorylation, with a better drawing of effects over a variety of systems inside the tumoral cell. Together with previous microarray experiment, informative genes and proteins as pharmacodynamic biomarkers for antiproliferative effects showed up (ex. STAT1/2, CENPE, ATRIP, MAP1B, LIMA1, VCP, several ribosomal, spliceosome and proteasomal complexes proteins). This study complements transcriptomic and phosphoproteomic previous experiments in this model and underscore HeberFERON as a glioblastoma therapeutic.

Primary human bronchial epithelial cells (pHBECs) of the airways of smokers are chronically exposed to cigarette smoke, which may induce chronic obstructive pulmonary disease (COPD) ranked fourth among the most common global causes of death. Using an established protocol for differentiation of pHBECs to a pseudostratified epithelium at an air liquid interface (ALI), we analyzed functional expression of transient receptor potential vanilloid 4 (TRPV4) proteins after application of cigarette smoke extract (CSE), which upregulated seven smoke exposure regulated genes (SERGs). TRPV4 protein expression in the plasma membrane and localization next to the cilia of ciliated cells was reduced, while cell barrier function was not altered after chronic exposure to CSE for 28 days compared to untreated control cells. Accordingly, TRPV4-mediated Ca2+ influx was blocked in pHBECs after CSE exposure. Moreover, Os-9 protein, which after binding mediates protection from degradation of TRPV4 protein by polyubiquitination, was significantly less expressed in pHBECs upon CSE exposure. Most interestingly, overexpression of OS-9 in pHBECs rescued reduced TRPV4 protein levels induced by CSE. Our study identifies a novel molecular mechanism of toxicity by CSE interfering with TRPV4 and OS-9 expression in pHBECs, which may blaze the trail for new therapeutic options in COPD.

The Na+/K+-ATPase (NKA) regulates ion balance in the kidney and influences cellular processes like proliferation and apoptosis through its signal transduction. The endogenous ligand 20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to inflammation and fibrosis in chronic kidney disease (CKD) and inhibits NKA activity in renal tubules. However, the molecular mechanism of this interaction remains unclear. In this study, we used in-silico approach to investigate the potential interaction between 20-HETE and NKA. Various ligands, including known NKA ligands such as cardiotonic steroids (CTS), 20-HETE, and negative controls, were docked using rigid and Induced Fit Docking to predict the affinity of the ligands toward NKA. Binding free energy calculations with the Prime Molecular mechanics with generalized Born and surface area (Prime MM/GBSA) tools were used to confirm the involvement of key amino acids in ligand-receptor interactions. The docking analyses revealed that 20-HETE exhibited a binding affinity comparable to negative control, with some differences between rigid and induced fit docking. Binding free energy data highlighted key amino acids in the 20-HETE and NKA interaction. Interaction fingerprint and mutations such as Ala330Gly and Val329Ala significantly reduced binding free energy, while Thr804Ala showed a notable decrease, underscoring the potential importance of these amino acids in ligand stabilization. These findings provide computational evidence supporting potential direct interaction between 20-HETE and NKA and identify candidate residues for future experimental validation.

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

Background and aimsTherapeutic outcomes for advanced hepatocellular carcinoma remain inadequate, despite recent advances using immunotherapy. Long-term effectiveness of systemic therapies, including second-line multi-tyrosine kinase inhibitor sorafenib, is limited by resistance mechanisms and adverse effects. Upregulated deubiquitinase UCH-L1 is frequently correlated with poor prognosis in cancers. Here, we investigated the therapeutic potential of combining pharmacological UCH-L1-inhibition with sorafenib in HCC. MethodsUCH-L1 expression was analysed in TCGA-LIHC data and patient-derived HCC tissues. Sorafenib and LDN57444 effects were evaluated in vitro in cytotoxicity and invasion assays. Gene and protein expression were examined by RT-qPCR, Western blotting and immunohistochemistry. In vivo efficacy of drug synergy was assessed in an orthotopic xenograft mouse HCC model. ResultsIn silico data-analysis revealed significantly higher UCH-L1 levels in patient HCC tumours versus non-tumour, associated with reduced overall survival. Low-dose sorafenib upregulated UCH-L1 in HCC cell line Hep3B. Paradoxically, this also promoted invasiveness and sustained MEK1/2-ERK1/2-pathway activation. Combining low-dose sorafenib with LDN57444 produced strong synergistic cytotoxicity in vitro, reverted MAPK-activation and suppressed invasion. Consistently, at low sorafenib dose co-treatment with LDN57444 completely inhibited tumour growth of Hep3B xenografts and enhanced sorafenib efficacy. ConclusionLDN57444 sensitises HCC cells to low-dose sorafenib by reverting drug-induced pro-oncogenic signalling and thereby strongly synergises with sorafenib to enhance anti-tumour efficacy in a HCC mouse model. This presents UCH-L1 as a player in treatment-induced adaptive response and supports further exploring UCH-L1-targeting in combination with sorafenib as therapeutic avenue for advanced HCC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/725527v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@176dc91org.highwire.dtl.DTLVardef@8acae8org.highwire.dtl.DTLVardef@f71bborg.highwire.dtl.DTLVardef@1f3c5aa_HPS_FORMAT_FIGEXP M_FIG C_FIG Lay summaryThis study explores a new treatment approach for hepatocellular carcinoma (HCC) by combining two drugs: LDN57444, which blocks the enzyme UCH-L1, and sorafenib, a FDA-approved multi-tyrosine kinase inhibitor. We evaluated the effect of this drug combination in vitro using a HCC cell line and in an mouse HCC-model. The drug combination displayed strong, synergy in lowering HCC cell viability, and greatly reduced invasiveness and in vivo tumour growth. LDN57444 sensitised HCC cells to low doses of sorafenib by preventing UCH-L1-mediated activation of pro-oncogenic signalling. These findings highlight the potential of this new drug combination for treating advanced HCC thereby potentially reducing side-effects and countering drug resistance. Impact and implicationsOur preclinical research introduces a novel combination strategy against advanced HCC that holds potential to improve existing therapies, particularly the second-line multi-tyrosine kinase inhibitor sorafenib. The proposed combination of sorafenib with an inhibitor of the deubiquitinase UCH-L1 not only enhances sorafenib efficacy but present promise to also counter resistance mechanisms. Moreover, because effective responses are achieved at lower drug doses, this may in addition reduce therapy-associated adverse effects further increasing potential impact. While sorafenib is FDA-approved, the UCH-L1 inhibitor LDN57444 needs further (clinical) development to bring our promising findings to full translational potential for HCC patients and physicians.

Glucocorticoids (GCs) remain the fastest-acting anti-inflammatory agents but are constrained by systemic exposure that suppresses the hypothalamic pituitary adrenal (HPA) axis, silences adaptive immunity, and drives chronic toxicities. Chronic inflammatory diseases are sustained by long-lived CD206+ macrophages containing immune-resistant pathogenic material not cleared physiologically. We developed 101-PGC-005 ('005), a macrophage-targeted type 1a dexamethasone prodrug engineered for low-affinity, recycling-compatible uptake via CD206, with intracellular release triggered by acidic endosomes. We evaluated '005 in mechanistic assays, pathogen-diverse preclinical models, three human pharmacokinetic (PK) studies, and an adaptive-design randomized Phase II/III trial in 309 hospitalized patients with moderate COVID-19. In two completed Phase I human studies, a first-in-human dose-escalation and repeated-dose study and a dedicated single/multiple-dose PK and safety study; '005 circulated as intact prodrug with rapid systemic clearance (Tmax ~0.5 h; terminal half-life ~1.9 h), with no measurable free dexamethasone after single dosing and only low, clinically non-significant free dexamethasone after repeated dosing, and intact prodrug recovered unchanged in urine. Morning cortisol and ACTH were preserved after 30 mg once daily for three consecutive days (1.5 times the intended therapeutic dose). A cerebrospinal fluid PK study is evaluating central-compartment penetration. In the Phase II/III trial, powered for non-inferiority, conducted across six sites in India under GCP with Ministry of Health approval and independent DSMB oversight; '005 (20 mg IV daily for 3 days) was superior to dexamethasone (6 mg IV daily for 3 -10 days) on the primary endpoint of time to > a 2-point improvement on the WHO ordinal scale (HR 2.31; 95% CI 1.83-2.93; p < 0.0001; median 3 vs. 4 days). '005 was also superior on viral clearance (HR 1.47; 95% CI 1.17-1.84; p = 0.0001), hospital discharge rate, SpO2; recovery, and fever resolution. Zero patients in the '005 arm received investigator-initiated corticosteroid supplementation despite protocol allowance. All 309 randomized patients completed the study (ITT = per-protocol). Safety profiles were equivalent (TEAEs 54.8% vs 54.5%; p = 0.958), with no Grade 3+ events, SAEs, deaths, or discontinuations in either arm. Mechanistically, '005 delivered dual benefit: acute debulking of inflammatory macrophages and selective depletion of chronically activated pathology-sustaining macrophages, while preserving CXCL10 antiviral signaling and physiologic HPA control. Critically, HPA preservation is not merely a safety feature, it is a core efficacy mechanism: by clearing the pathogenic macrophage burden that was overriding HPA regulation, '005 restores the conditions for endogenous cortisol to resume its pulsatile, demand-responsive anti-inflammatory role across all GR-expressing cells, lymphocytes, endothelial cells, neurons, and newly differentiated macrophages, that '005 itself cannot reach. These findings support regulatory-grade evidence for macrophage-targeted corticosteroid therapy and provide the foundation for further development across acute inflammatory indications (sepsis, viral pneumonia, cytokine-release syndromes) and chronic macrophage-driven diseases (atherosclerosis, metabolic steatohepatitis, neurodegeneration, tumor-associated macrophages).

Background: Chronic and non-healing wounds remain a major clinical challenge with limited therapeutic options. Angiogenesis and inflammation are central to tissue repair, and mesenchymal stem cells (MSC) contribute to these processes through their trophic and immunomodulatory secretome. Cannabidiol (CBD) exhibits antioxidant and immunomodulatory properties. However, whether CBD-rich Cannabis sativa extract stimulate MSC toward a pro-angiogenic secretome remains unclear. Purpose: This study aims to determine whether purified CBD or a phytochemically CBD-rich full spectrum extract stimulate umbilical cord-derived human MSC (UC-hMSC) to secrete pro-angiogenic factors and enhance endothelial responses relevant to wound healing. Methods: UC-hMSC were preconditioned with either purified CBD or a CBD-rich full-spectrum extract. Transcriptional changes were assessed by qPCR. The functional impact of the resulting secretome was evaluated in vitro using HUVEC-based proliferation and tube formation assays, and in vivo through the chick chorioallantoic membrane assay. To explore underlying mechanisms, we examined HIF-1 stabilization and VEGFA release in UC-hMSC, and VEGFR-2/ERK signaling in HUVEC. Results: Purified CBD and full-spectrum CBD extract preconditioned UC-hMSC secretomes, increased HUVEC proliferation, tube formation, and enhanced vascular branching in the CAM assay. Mechanistic analyses indicated activation of the HIF-1/VEGF axis in UC-hMSC, and ERK1/2 activation in HUVEC that was sensitive to VEGFR-2 blockade. Conclusion: Purified CBD and CBD-rich full-spectrum extract prime UC-hMSC toward a pro-angiogenic secretome that promotes endothelial activation and neovascularization. These findings suggest that cannabinoid-based preconditioning of UC-hMSC involves the HIF-1/VEGF axis and VEGFR-2/ERK signaling pathways in endothelial cells, supporting further investigation of this approach in wound healing and regenerative therapies.

Background/Objectives Dental anxiety remains a significant psychological barrier to oral healthcare, particularly for invasive surgical procedures such as tooth extraction. Aromatherapy using Lavandula angustifolia (lavender) has been proposed as a non-pharmacological adjunct to manage acute preoperative anxiety. This clinical trial evaluated the efficacy of inhaled lavender essential oil on anxiety severity in patients awaiting elective single-tooth extraction. Methods An assessor-blinded randomized controlled clinical trial with limited participant blinding (inherent to the recognizable scent of the intervention), registered with the Iranian Registry of Clinical Trials (IRCT20190920044824N1), was conducted among 60 patients requiring non-surgical tooth extraction. Participants were randomly assigned to an intervention group (n=30), receiving two drops of pure lavender essential oil on a sterile gauze for 20 minutes of inhalation, or a control group (n=30), receiving sweet almond oil as an olfactory placebo. The primary outcome was the severity of somatic anxiety symptoms, measured pre- and post-intervention using the Beck Anxiety Inventory (BAI). Because a significant baseline-by-treatment interaction was detected, a General Linear Model (GLM) retaining this interaction term was used to estimate adjusted group means at the grand-mean baseline, with bootstrap 95% confidence intervals (10,000 resamples). Two pre-specified checks of robustness were performed: (i) an unadjusted, baseline-naive Mann-Whitney U comparison of raw post-intervention scores, and (ii) a sensitivity analysis re-fitting the adjusted model after excluding two baseline outliers identified by inspection of model residuals. Results There were no significant baseline differences between the intervention and control groups regarding sex distribution (p=0.100), mean age (p=0.479), or pre-intervention BAI scores (Mann-Whitney p=0.215). A significant baseline-by-treatment interaction was detected (p<0.001). In the full sample (n=60), the baseline-adjusted GLM estimated a lower post-intervention BAI mean in the intervention group (22.38) than the control group (23.09), an adjusted difference of -0.71 (bootstrap 95% CI: -1.41 to -0.06; p=0.028). However, this signal was not robust: after excluding two patients identified as outliers in the model residuals (n=58), the adjusted difference attenuated to -0.51 and was no longer statistically significant (bootstrap 95% CI: -1.11 to 0.10; p=0.098). The unadjusted comparison of raw post-intervention scores was likewise not significant in the full sample (Mann-Whitney p=0.754). Conclusions A baseline-adjusted analysis of this trial produced an initial signal suggesting a small anxiolytic effect of inhaled lavender, but this signal was not stable under a pre-specified outlier-exclusion sensitivity analysis and was not supported by the unadjusted comparison of raw scores. Taken together, the totality of evidence from this trial is insufficient to conclude that inhaled lavender aromatherapy produces a reliable reduction in acute preoperative dental anxiety. Beyond its clinical findings, this trial offers a concrete, quantified illustration of how baseline imbalance and a small number of influential observations can generate a fragile, model-dependent treatment signal in a modestly sized randomized trial, a methodological caution directly relevant to the design and analysis of future small RCTs in dental and complementary medicine research.

Chronic hepatitis B virus (HBV) infection contributes to hepatocellular carcinoma by disrupting host transcription, cell-cycle control, and apoptotic signaling. Isochlorogenic acid A (ICAA), a natural compound with antiviral and hepatoprotective properties, was previously shown to inhibit HBV replication by interfering with multiple steps of the viral life cycle. Because chronic HBV often reflects an imbalance between proliferation and cell death, we investigated how ICAA affects gene expression related to these processes in the presence or absence of HBV. We performed transcriptome analysis using RNA sequencing (RNA-seq) in HepAD38 cells (a HepG2-derived stable HBV-expressing line) and HepG2 control cells (HBV-negative) treated with ICAA or DMSO. HBV caused major differences in gene expression in HepAD38 cells compared with HBV-negative HepG2 cells. Principal component analysis showed that ICAA significantly altered HBV-dependent expression patterns, resulting in 189 differentially expressed genes (DEGs) that were regulated in opposite directions by both HBV and ICAA. Functional enrichment analysis highlighted pathways in viral carcinogenesis, apoptosis, MAPK signaling, and p53 signaling. Annexin V/propidium iodide assays showed apoptotic cells in both treated and untreated HepAD38 cultures, with only minor pattern changes. Mechanistically, in untreated HBV-positive cells caspase-9 cleavage failed to activate PARP, suggesting that induction of intrinsic apoptosis is followed by blocked execution. In contrast, ICAA inhibits caspase-9 cleavage in a dose-dependent manner, while activating PARP. Consistent with this, ICAA treatment increased apoptotic DNA fragmentation in HepAD38, reflecting the proapoptotic potential of ICAA under these conditions facilitating the elimination of HBV-positive cells by apoptosis. These findings highlight the potential therapeutic relevance of this compound in processes associated with HBV pathogenesis, together with its antiviral effect. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/733975v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@38d107org.highwire.dtl.DTLVardef@235a13org.highwire.dtl.DTLVardef@ee988aorg.highwire.dtl.DTLVardef@60cb13_HPS_FORMAT_FIGEXP M_FIG C_FIG

Polo-like kinase 1 (PLK1), a key regulatory protein controlling entry into and passage through mitosis, has been targeted through its kinase domain (KD) with mixed clinical success. Inhibition through the Polo-box domain (PBD) is a viable alternative through targeting the sub-cellular localization and kinase activity of PLK1. Novel non-peptidic compounds, termed abbapolins, were discovered through the REPLACE strategy and have been lead optimized through structure-based strategies and screening analogs in the NCI-60 tumor cell panel. Proteomic analysis revealed a correlation between abbapolin activity and PLK1 protein levels in the cell lines part of the NCI-60. Prostate cell lines were identified as among the most sensitive and led to further detailed studies of their activity in prostate cancer models. Compounds were evaluated for their pharmacokinetic properties, and in vivo efficacy, and results showed significant antitumor xenograft activity with no observable gross toxicity. Treated tumors were analyzed for loss of PLK1, which was previously shown to be induced by abbapolin binding. Results obtained showed a significant degradation of PLK1 in abbapolin-treated vs untreated tumors, thereby confirming on-target action in vivo and revealing PLK1 levels as a potential pharmacodynamic marker. Lead compounds were shown to sensitize PC tumors resistant to androgen deprivation therapy paving the way for future combination studies in vivo. These data provide an alternative pathway for effective PLK1 therapeutics that avoid the reported problems of molecules targeting the KD, in vivo proof-of-concept for the REPLACE strategy and validation for targeting the PBD as an anti-tumor drug development strategy.

Selective inhibition of phosphodiesterase 4B (PDE4B) remains a promising strategy for preserving the anti-inflammatory benefit of PDE4 inhibition in chronic obstructive pulmonary disease while reducing PDE4D-associated tolerability liabilities. This study integrated SHAP-interpretable machine learning, natural product virtual screening, hierarchical docking, post-docking MM-GBSA, isoform cross-docking, binding-pocket comparison, ADMET prediction, and 100 ns molecular dynamics simulations to identify PDE4B-selective inhibitors from the LOTUS natural product database. A Random Forest classifier trained on curated ChEMBL PDE4B bioactivity data achieved an external performance with AUC-ROC = 0.955, accuracy = 0.893, F1-score = 0.896, MCC = 0.785, and prioritized 119,698 predicted actives from 276,518 LOTUS compounds. SHAP analysis identified BertzCT and TPSA as major contributors to predicted activity. Sequential Lipinski, PAINS, and QED filtering retained 14,210 candidates for structure-based evaluation. Extra precision docking identified four leads with PDE4B docking scores of -9.123 to -12.080 kcal/mol, all outperforming roflumilast (-7.658 kcal/mol). Cross-docking and post-docking MM-GBSA supported preferential PDE4B binding for three candidates. The top lead, LTS0048837, maintained a stable PDE4B-bound pose during simulation, with comparatively stronger interaction persistence than its PDE4D complex and the roflumilast reference. These findings nominate LTS0048837 as a computationally prioritized PDE4B-selective natural product lead requiring experimental enzyme, cellular, and pharmacokinetic validation.

3:(-)-trans-Cannabidiol ((-)-CBD) is a principal phytocannabinoid from Cannabis sativa. (-)-CBD has complex pharmacology but is a relatively weak inhibitor of CB1 and CB2 receptor signalling. Cannabidiol has two chiral centres and thus four stereoisomers. (+)-trans-CBD ((+)-CBD) has a higher affinity than (-)-CBD at CB1 and CB2, but its pharmacodynamic effects at these receptors are incompletely described. We examined the activity of (+)-CBD at human CB1 and CB2 receptors using a fluorescence-based assay of membrane potential in AtT20 cells stably expressing CB1 or CB2 receptors. (+)-CBD produced a rapid, concentration-dependent hyperpolarization in CB2-expressing cells (pEC50 6.63 {+/-} 0.08) with a maximal effect [~]90% of the response to CP55940. The CB2 response was blocked by pertussis toxin pretreatment and competitively inhibited by the CB2 antagonist AM630 (Schild slope 1.1 {+/-} 0.1). (+)-CBD was a low-efficacy, low-potency CB1 agonist and inhibited somatostatin-receptor effects at high concentrations (10-30 {micro}M). It had no effect on the membrane potential of AtT20 wild-type cells. In silico modelling of ligand interactions with CB2 indicated that (+)-CBD but not (-)-CBD formed an H-bond with Ser285, a residue crucial for agonist activation of CB2. Our data suggests (+)-CBD acted as a CB2 agonist via the orthosteric binding site on the receptor. Synthetic CBD, including (+)-CBD, has previously been administered in clinical trials, presumably without consideration of its potential CB2 agonist activity. Given the relative safety of (-)-CBD in people, (+)-CBD may be a useful drug to explore CB2-sensitive disease states, should it prove similarly safe.

Evidence suggests that insomnia disorder is associated with pathophysiological alterations that may contribute to long-term physical, mental and inflammatory-related health risks. Cognitive behavioral therapy for insomnia (CBTi) is the first-line treatment for insomnia disorder, yet its effects on physiological outcomes remain unclear. This randomized-controlled trial examined the effects of CBTi on cardiovascular and immunological biomarkers. Sixty-two participants with insomnia disorder were randomized to group-CBTi (N = 33, 75.8% female, Mage = 48.8 + 17.1 years) or Waitlist (WL) control (N = 29, 75.9% female, Mage = 52.2 + 15.6 years). Cardiovascular parameters included systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), and nocturnal heart rate variability (HRV). Inflammatory markers from blood samples included C-reactive protein (CRP), tumor necrosis factor-alpha (TNF- ), interleukin-6 (IL-6) and brain-derived neurotrophic factor (BDNF). All outcomes were assessed at baseline (T1), post-treatment assessment (T2, following completion of CBTi or WL period), and 6-months for the WL group (T3, after CBTi for the WL participants). No significant Group-by-time effects were observed for SBP, DBP, HR, HRV and any inflammatory markers (ps > .05) from T1 to T2. When pooling treatment effects following CBTi exposure across both groups (T1 to T2 in CBTi group and T1 to T3 in WL group), no significant biomarker changes were observed. Overall, results indicate that CBTi did not produce detectable changes in cardiovascular or inflammatory markers among healthy individuals with insomnia disorder. These findings suggest physiological responses to CBTi are complex and may reflect dynamic and context-dependent processes (https://www.isrctn.com/ISRCTN13983243).