Background and aim Oil pulling (OP) has been studied as an adjunct to routine oral hygiene, but pediatric evidence - especially for castor oil - is limited. The registered primary outcome broadly covered gingival health and bacterial flora at two weeks; no secondary outcome was registered. This report compared those components after virgin coconut oil (VCO), virgin castor oil, or distilled-water rinsing. Plaque Index and the direct VCO-versus-castor comparison were exploratory. Materials and methods This exploratory randomized controlled trial enrolled 70 children, of whom 51 met the eligibility criteria and were allocated equally to VCO, castor-oil, or distilled-water groups (n = 17 each). Participants performed the assigned intervention once daily for two weeks. The Turesky modification of the Quigley-Hein Plaque Index, the Löe-Silness Gingival Index, and salivary MacConkey-culturable bacterial counts were assessed at baseline (T0) and after two weeks (T2). Intergroup values were compared using one-way analysis of variance (ANOVA) followed by Tukey HSD tests (p < 0.05). Results Forty-five children (n = 15 per group) completed the study. Both oil-pulling groups showed statistically significant reductions in Plaque Index, Gingival Index, and bacterial load from baseline to the two-week follow-up (p < 0.05). At postoperative evaluation, Group B (castor oil) demonstrated the greatest reduction in Plaque Index (3.90 ± 0.46 to 2.50 ± 0.38) and Gingival Index (2.00 ± 0.32 to 0.90 ± 0.28). Bacterial counts decreased markedly in both Groups A and B (from approximately 6.75 and 6.65 to 5.70 and 5.65 × 10[3] CFU/mL, respectively), whereas Group C showed negligible change (6.55 to 6.50 × 10[3] CFU/mL). Intergroup comparisons confirmed significantly better outcomes in both intervention groups relative to the control. Conclusion Over two weeks, VCO and castor-OP were associated with lower mean plaque and gingival-index scores and lower salivary MacConkey-culturable bacterial counts than distilled-water rinsing. The small sample, baseline imbalances, short follow-up, and restricted microbiological method limited causal and clinical interpretation. Larger trials using baseline-adjusted analyses and oral-microbiome-specific methods are required before routine paediatric use can be recommended.

Two-dimensional (2D) monolayer cultures remain the workhorse of colorectal cancer (CRC) research and high-throughput screening (HTS), yet they poorly recapitulate tissue architecture, extracellular matrix (ECM) cues, metabolic gradients, and multicellular heterogeneity that govern drug response in vivo. Over the past decade, three-dimensional (3D) culture systems-including spheroids, patient-derived organoids (PDOs), tissue-originated spheroids (CTOS), and collagen-embedded models-have revealed striking 2D vs. 3D discordance in proliferation, signaling, epithelial-mesenchymal transition (EMT), plasticity, and sensitivity to standard chemo- and targeted therapies. This review synthesizes how 3D CRC models are used to decode mechanisms of primary and acquired resistance to anti-CRC therapies, from metabolic phenotypes and ECM remodeling to EMT, polarity loss, and rare invasive trajectories identified by imaging and quantification of morphologic features of CRC cells grown in 3D. Parallel advances in PDO-based functional precision oncology demonstrate that organoid drug screens can prospectively predict patient response in advanced CRC, while 3D combination screens uncover format-specific synergies that are invisible in 2D. Building on this foundation, we highlight recent technical innovations and discuss how integrating morphometric, metabolic, and trajectory-level readouts can move CRC drug discovery "beyond live/dead." Rather than propose a single best model, we outline practical guidelines for matching 3D platform choice (cell lines vs. PDO/CTOS, collagen vs. Matrigel, static vs. live-imaging HTS) to mechanistic questions and therapeutic goals in CRC. We propose an integrated roadmap-matrix-embedded 3D cultures, high-content morphological endpoints, trajectory analytics, and rigorous HTS validation-to decode resistance mechanisms and prioritize clinically actionable combinations in CRC.

The therapeutic goal in multiple myeloma (MM) is changing from long-term disease control to a potential cure. While anti-CD38-containing quadruplets have improved outcomes, relapses continue to occur in many patients. In recent years, advancements in immunotherapy have fundamentally changed this paradigm. Bispecific antibodies (BsAbs) and chimeric antigen receptor T-cells (CAR T-cells) have deepened responses and achieved sustained MRD negativity in an unprecedented way, raising the possibility of curing MM in a subset of patients. Recently, the International Myeloma Society (IMS) reached a consensus and discussed a framework to define cure. This review examines how novel immunotherapies are redefining frontline strategies in newly diagnosed MM (NDMM) and attempts to summarize how BsAbs, CAR T-cells, and cereblon E3 ligase modulators (CELMoDs) can be integrated into induction, consolidation, and maintenance phases. It also discusses the evolving role of autologous stem cell transplant (ASCT) in the immunotherapy era and MRD-guided treatment and discontinuation strategies. Together, this review attempts to provide a roadmap to fulfill the criteria for cure.

DOPA decarboxylase (DDC) is a key enzyme that connects melanin production and innate immunity in insects, yet its regulatory network in major lepidopteran pests remains poorly understood. We combined CRISPR/Cas9 gene editing with multi-omics analysis to explore DDC's role in immune responses in the invasive pest Spodoptera frugiperda. Knocking out DDC caused complete albinism, severe developmental delays, pronounced developmental defects, and increased susceptibility to bacterial challenge. Following the E. coli challenge, DDC-mutant larvae showed substantially lower final survival than infected wild-type larvae (6.7% vs. 66.7%) and failed to pupate, despite pronounced cuticular darkening. Transcriptomic and metabolomic analyses showed that DDC loss disrupted the tyrosine-dopamine pathway and was associated with altered expression of genes related to the prophenoloxidase cascade. Notably, laccase2 was transcriptionally upregulated after challenge; however, its contribution to infection-associated cuticular darkening remains to be determined. This response did not restore dopamine-dependent immune signaling or whole-body metabolism, as evidenced by disruptions in glycine/serine pathways and reduced immune gene activity. These results separate pigmentation from immune functions within the melanin pathway, highlighting DDC as a crucial immune-metabolic hub. Given its roles in development and immunity and the high conservation of the dopamine pathway in invertebrates, DDC is a promising target for species-specific, resistance-proof RNAi pest control methods.

Monoamine neurotransmitters are central regulators of social behavior, yet it is unknown whether genomic imprinting of genes within their signaling pathways modulate these behaviors. The monoamine synthesis genes tyrosine hydroxylase (Th) and dopa decarboxylase (Ddc) exhibit tissue-specific, noncanonical imprinting in mice, with preferential maternal allele expression in the brain and paternal biased Ddc expression in peripheral tissues. Prior work indicated that imprinting of these genes causes parent-of-origin dependent allelic influences on mood-related and foraging behaviors. Here, we test whether alleles in this pathway also shape social behavior in a parent-of-origin-dependent manner. We identify strong maternal allele-biased expression of Th and Ddc in the anteroventral and preoptic periventricular (AVPV/PVpo) area of the hypothalamus using reciprocal CAST/EiJ x C57Bl/6J F1 hybrids and pyrosequencing-based allelic quantification. This region contains a sexually dimorphic dopaminergic cell population that is implicated in regulating social behaviors with prominent sex differences, though we did not detect sex differences in allelic bias in either tissue for Th or Ddc. To determine whether the parent-of-origin of Th and Ddc alleles influences social behavior we use reciprocal heterozygous null-allele mice. In males, maternally inherited Th null alleles reduce sociability, whereas paternally inherited Ddc null alleles increase aggression and impair social recognition. These behavioral outcomes align with the tissue-specific maternal Th and paternal Ddc expression biases. Together, these findings provide the first evidence that noncanonical imprinting of monoamine synthesis genes modulates social behavior in a mammalian organism. Parent-of-origin effects in the monoamine pathway may therefore contribute to natural variation in social behavior.

Background: Triple-negative breast cancer (TNBC) exhibits aggressive biology and heterogeneous immune infiltration. Lysine-specific demethylase 1 (LSD1/KDM1A) is an epigenetic regulator implicated in oncogenic transcriptional programs, but its role in immune exclusion remains incompletely defined. Methods: LSD1 expression was evaluated by immunohistochemistry in TNBC specimens and correlated with survival. Functional effects of LSD1 depletion or pharmacological inhibition were assessed in TNBC cell lines and patient-derived xenograft organoids (PDXOs). Transcriptomic, ChIP-seq, multiplex immunofluorescence, and GeoMX digital spatial profiling analyses were integrated to characterize LSD1-associated transcriptional, chromatin, and immune programs. PBMC-PDXO co-cultures evaluated the effects of LSD1 inhibition on tumor growth and immune cell infiltration. Results: High LSD1 expression was associated with inferior overall survival, hypoxia-associated programs and suppressed immune pathways. LSD1-high tumors exhibited reduced infiltration of T cells, B cells, NKT-like cells, and macrophages. Spatial profiling revealed suppressed innate immune responses and type I interferon signaling within B-cell and T-cell compartments, respectively. ChIP-seq demonstrated preferential LSD1 occupancy at AP-1 motif-enriched regions, while co-immunoprecipitation supported association with AP-1 complexes. Integration of LSD1 chromatin occupancy sites with transcriptomics data identified SPPL2A and NCOA3 as candidate LSD1-regulated genes associated with interferon-γ response pathways. LSD1 depletion increased SPPL2A, NCOA3, MHC-I and PD-L1 expression. LSD1 inhibition reduced PDXO growth and increased immune cell infiltration. Conclusions: LSD1 is associated with a poor clinical outcome and immune exclusion in TNBC. These findings support a role for LSD1 in AP-1-associated chromatin regulation and interferon-γ-related transcriptional response; they warrant further investigation into LSD1 inhibition as a therapeutic strategy for immune-cold TNBCs.

The oxidative burst is likely a pivotal early event in immune activation in strawberry, a non-model plant, yet the underlying mechanisms remain largely unknown. Here we optimized a detached-leaf system for independent application of ROS inducers and pharmacological inhibitors to investigate the roles of NADPH oxidases, SOD, and calcium in ROS accumulation induced by the avirulent fungus Acremonium strictum SS71. Our results show that the dynamics of SS71-induced ROS accumulation in the detached-leaf system was consistent with previous whole-plant strawberry studies, reaching maximum levels at 6 hpi. The efficient scavenging of paraquat-induced ROS by vascular application of ascorbic acid further supports the validity of this model compared with the other delivery methods assayed. The pharmacological inhibitors used were DDC (a Cu,Zn-SOD inhibitor), DPI (a NADPH oxidase inhibitor), and EGTA (a calcium chelator). Concentrations lacking phytotoxic effects were selected based on cellular damage assessments following petiolar uptake, and the functional efficacy of these treatments was validated by SOD activity assays (DDC) and stomatal closure assays (DPI and EGTA). Finally, the effects of pharmacological inhibitors on SS71-induced ROS accumulation were evaluated. Our findings suggest that NADPH oxidases contribute to SS71-induced superoxide accumulation, although additional sources may also involve. SS71-induced hydrogen peroxide accumulation appears to be mainly dependent on Cu,Zn-SOD activity. Extracellular calcium influx does not appear to be required for SS71-induced ROS accumulation under the experimental conditions tested. These findings provide new insights into strawberry defense signaling and establish a useful detached-leaf system for studying cellular mechanisms in non-model species.

Genome-wide cell-free DNA (cfDNA) methylation profiling of 491 pre-diagnosis breast and prostate cancer plasma samples, some collected up to nine years before diagnosis, identifies early cancer-associated epigenetic alterations and predictive signatures. Differentially methylated regions were enriched in regulatory and repetitive elements and reflected cancer-tissue- and immune-cell-associated DNA methylation patterns. Logistic regression models trained on discovery set samples showed that cfDNA silencer-region methylation robustly predicted future prostate cancer, with high-risk individuals exhibiting a 3.55-fold increased hazard of developing prostate cancer. Enhancer hypermethylation modestly predicted incident breast cancer but strongly distinguished established late-stage breast cancers, with high-risk individuals showing a 2.3-fold increased hazard. These results highlight cfDNA methylation as a promising biomarker for early cancer risk prediction and surveillance.

Malignant transformation is driven by disruption of pathways regulating proliferation and cell fate, but these same disruptions can create a collateral vulnerability: loss of transcriptional and epigenetic control over transposable elements and other normally silenced genomic regions. Consequently, emerging cancer cells can accumulate transposable element-derived and other endogenous immunogenic nucleic acids capable of triggering antiviral responses, a process termed viral mimicry. Increasing evidence indicates that viral mimicry can eliminate precancerous cells and shape tumour evolution, positioning it as an intrinsic tumour-suppressive mechanism. Here we highlight how cancer-associated changes in DNA methylation, histone modifications, splicing and RNA processing can lead to the presence of immunogenic nucleic acids that can activate viral mimicry pathways. We outline how cancer cells suppress viral mimicry, including compensatory epigenetic repression, RNA editing, nucleic acid decay and dampening of interferon signalling to enable cancer cell growth. Finally, we highlight the evidence suggesting that escaping viral mimicry is a fundamental process for cancer initiation and progression, and suggest that viral mimicry escape is necessary for cancer transformation and a therapeutic target in combination with immunotherapies. By framing viral mimicry escape as a necessary part of cancer transformation, this Review provides a unifying conceptual model for its translational exploitation.

Disulfiram (DSF), long used as an aversive agent in alcohol dependence therapy, has recently regained attention as a promising candidate for oncological drug repurposing. After administration, DSF is rapidly reduced to diethyldithiocarbamate (DDC), which, in the presence of Cu[2+], forms the complex Cu(DDC)2. This compound acts as a strong inducer of oxidative stress, an inhibitor of the ubiquitin-proteasome system, and a suppressor of endogenous hydrogen sulfide (H2S) synthesis. DSF also modifies protein and non-protein thiol groups, disrupting cancer cell metabolism and promoting apoptosis. Despite robust preclinical evidence, clinical translation remains limited. Key obstacles include DSF's rapid metabolism, insufficient availability of free copper ions in humans, and the lack of predictive biomarkers capable of identifying responsive patients. Another challenge is DSF's low oral bioavailability, which prevents the drug from reaching tumor tissue at therapeutically effective concentrations. Consequently, current research focuses on advanced nanocarrier systems designed to protect DSF from premature degradation and ensure its controlled release within the tumor microenvironment. This review summarizes the multifaceted anticancer mechanisms of DSF and discusses biological and pharmacological factors underlying the discrepancies between experimental findings and clinical outcomes.

The avian trachea is a critical component of respiration and vocalization, as well as pathogen defense and immunity. Tracheal neuroimmune pathways involving serotonin and catecholamines are well established in mammals. Although earlier work identified an endocrine system in the avian tracheal epithelium involved in dopamine metabolism, little remains known regarding the neurophysiological systems of the trachea in poultry species. As such, the present study sought to characterize the pathways involved in the synthesis, transport, and breakdown of indolamines and catecholamines in the trachea of the chicken and turkey. Trachea was collected from chickens and turkeys at 3 weeks of age. Following tissue protein extraction, protein lysates were analyzed using the Jess immunoassay system for the identification and quantification of target proteins. Targeted proteins included catechol-O-methyltransferase, monoamine oxidases A and B, serotonin and norepinephrine transporters, dopamine decarboxylase (DDC), dopamine beta hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase-1 (TPH-1). All enzymes and transporters were consistently detected in the trachea of both the chicken and turkey. These findings establish that neurochemical metabolic pathways are present in the trachea of poultry species. As serotonin and catecholamines are known to drive pathogen response, affect cilial movement, as well as regulate inflammation, the results presented herein are expected to inform future applications that seek to target serotonergic and catecholaminergic pathways within the respiratory tract and gut-respiratory axis across poultry species.

High stocking density in recirculating aquaculture systems (RAS) can promote the accumulation of endogenous bioactive metabolites, yet the environmental factors controlling their formation remain unclear. L-3,4-dihydroxyphenylalanine (L-DOPA) has recently linked to growth inhabitation in crowded turbot (Scophthalmus maximus) culture. Here, we used a 2 × 2 factorial design with two dissolved oxygen (DO) levels (HDO, 10 mg/L; NDO, 7 mg/L) and two stocking densities (HD, 9.39 kg/m[2]; LD, 4.70 kg/m[2]) to examine whether oxygen availability regulates waterborne L-DOPA accumulation and associated biological effects in turbot RAS. After 60 days, fish under normoxic conditions and high density (NDO-HD) showed marked growth inhibition, whereas HDO restored growth to levels comparable with low-density treatments. Waterborne L-DOPA in the NDO-HD group increased to approximately twice that in the other groups, while HDO prevented this density-associated accumulation. Elevated L-DOPA coincided with increased blood methemoglobin, lactate and cortisol, suggesting impaired oxygen transport, enhanced anaerobic metabolism and systemic stress. It was also associated with sustained activation of HIF-1 signaling pathway, including upregulating of hypoxia-inducible factor-1α (hif-1α), erythropoietin (epoa) and EGL nine homolog 1(egln1a), and with disrupted L-DOPA metabolism, characterized by increased tyrosine hydroxylase (th) and reduced phenylalanine hydroxylase (pah) and dopa decarboxylase (ddc) expression. Transcriptomic analysis further indicated that oxygen supplementation reduced stress-related transcriptional responses and supported pathways related to energy metabolism and anabolic growth. These findings identify DO as a key environmental regulator of endogenous L-DOPA accumulation and suggest that oxygen management can reduce endogenous bioactive metabolite buildup in intensive RAS.