Spatially resolved transcriptomics captures gene expression in tissue context, enabling direct mapping of defense pathways across organs and cell layers. This chapter describes a complete Stereo-seq workflow for Arabidopsis leaves treated with salicylic acid (SA), from plant growth and hormone application through cryosectioning, fixation, bright-field imaging and optional histology, on-chip permeabilization, in situ reverse transcription and library construction on STOmics V1.3 1 × 1 cm chips, sequencing on DNBSEQ platforms, and analysis using STOmics Analysis Workflow (SAW) with downstream spatial statistics and pathway enrichment. Practical parameters are given for section thickness, fixation, imaging, permeabilization optimization, sequencing configuration, and quality control. The bioinformatics analyses include binning strategies, alignment to TAIR10, normalization, clustering, spatial differential expression, pathway testing, and integration with single-cell RNA-seq references for cell-type deconvolution.

Salicylic acid (SA) and jasmonic acid (JA) coordinate plant immunity through partially antagonistic signaling pathways that differ across cell types and over time. Single-cell and single-nucleus RNA sequencing now resolve these hormone responses at cellular resolution, allowing clear separation of early primary signaling waves from secondary and downstream transcriptional programs. In the context of plant-pathogen interactions, these approaches are particularly powerful for uncovering how distinct cell populations perceive SA and JA, reprogram defense networks, and balance growth-immunity trade-offs. This chapter provides end-to-end wet-lab protocols for nuclei-based snRNA-seq tailored to SA and JA treatments, including sample preparation, nuclei isolation, and library construction. In parallel, a bioinformatics analysis workflow is described, covering quality control, ambient RNA correction, doublet removal, dataset integration, cell type mapping, differential expression analysis, and pathway and module scoring. Together, these experimental and computational pipelines enable researchers to dissect hormone-driven immune responses at single-cell resolution and to generate cell-type-resolved hypotheses that can guide downstream genetic and physiological studies.

Salicylic acid (SA) functions as a key signaling molecule in plant defense, orchestrating responses to diverse biotic and abiotic stresses. A clear understanding of SA-mediated immunity requires systematic analysis of SA-responsive transcriptional programs at both targeted and genome-wide levels. This chapter describes methodological strategies for examining SA-responsive gene expression through quantitative polymerase chain reaction (qPCR) as well as bulk transcriptome profiling using the Bio-Rad SEQuoia Complete Stranded RNA Kit. The workflow covers experimental design, sample preparation, data acquisition, and result interpretation within the framework of plant immune responses. Integrating targeted qPCR assays with SEQuoia-enabled transcriptome analysis enables robust characterization of regulatory networks underlying SA-induced immunity and supports reproducible comparisons across experiments. This combined approach provides insights into the complexity of plant defense signaling and its transcriptional control.

Multidrug-resistant Staphylococcus aureus (MRSA). This study investigated the antimicrobial efficacy of zinc oxide (ZnO) nanoparticles synthesised through a blue laser-enhanced hydrothermal method combined with the flavonoid Naringenin (NAR) against multidrug-resistant S. aureus isolates. Sixty S. aureus strains were identified using a combination of morphological, biochemical and molecular analyses (VITEK-2 and 16 S rRNA). All strains showed total resistance (100%) to oxacillin and cefoxitin and high resistance levels (60-90%) for vancomycin, erythromycin and fluoroquinolones. Characterization confirmed hexagonal ZNPs with a crystallite size of 38.78 nm. Disk diffusion assays demonstrated enhanced efficacy of ZNPs-NAR combination, achieved a 16.67 mm inhibition zone (p ≤ 0.0001). Notably, while ZNPs singly suppressed leukocidin toxin genes lukD and lukE by 90.3% and 94.9%, inhibition, and NAR reduced expression by 62.4% and 61.9%, the ZNPs-NAR combination showed lower gene suppression (59.3% and 52.8%) yet superior bactericidal activity. This discrepancy reveals that the enhanced bactericidal effect stems not from amplified transcriptional inhibition, but from complementary mechanisms: ZNPs likely disrupt bacterial membranes to facilitate NAR uptake, while NAR concurrently interferes with intracellular targets like DNA replication. The findings demonstrate that nanoparticle-phytochemical combinations can achieve superior antimicrobial effects through synergistic mechanisms, offering a promising strategy to combat multidrug-resistant infections and reduce reliance on conventional antibiotics.

In a randomised controlled trial (RCT), we compared the effects of treatment with furosemide + small volumes of hypertonic saline solution (HSS) with those of furosemide alone in patients with decompensated heart failure (HF), and their effects on inflammatory and remodelling markers and epigenetic signatures.

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subtype of breast cancer, with limited treatment options due to the absence of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2) expression. This characteristic renders TNBC resistant to hormone-based and HER2-targeted therapies, leaving cytotoxic chemotherapy as the predominant strategy and highlighting the urgency for novel interventions. In this study, we investigated the mechanism of action of GL24, a potent 4-(phenylsulfonyl)morpholine-based small molecule with selective tumor suppression effects on metastatic TNBC cells, while being ineffective against TNBC cells derived from the primary tumor site, using gene co-expression analysis. By considering the distinct phenotypic responses induced by GL24, we tailored our co-expression analysis approach, selecting gene pairs that exhibited differential co-expression in effective cells while excluding gene pairs that also showed differential patterns in non-effective cells. Constructing a co-expression network from these differential pairs, followed by enrichment analysis and functional annotation, revealed specific gene interactions and molecular pathways associated with GL24-mediated TNBC inhibition. These insights supported the previously established findings that showed convergence on apoptosis based on differentially expressed genes, while also providing complementary information by highlighting pathways involved in metabolic alterations, proliferation, and migration or invasion. This expanded understanding advances the knowledge of the mechanisms of GL24 in combating TNBC.

Exposure to immune stress or lipopolysaccharide (LPS) during critical developmental stages like puberty may lead to gut microbiome dysbiosis and epigenetic dysregulation in mammary glands, affecting gene expression and potentially elevating breast cancer susceptibility in adulthood. Although LPS's adverse impacts on intestinal and brain functions are well-documented, its effects on mammary glands remain underexplored. Using an immunocompetent BALB/c mouse model, we administered an acute LPS dose (1.5 mg/kg body weight) during puberty. The study evaluated the long-term consequences of LPS exposure alone and combined with AHCC (Lentinula edodes cultured extract, 2 g/kg body weight/day) on DNA methylation patterns, cytokine profiles, and microRNA expression in mammary glands at 9 weeks of age. Analyses included DNA methylation sequencing, multiplex immunoassays, quantitative PCR, and image processing. Pubertal LPS exposure produced persistent molecular dysregulation in mammary glands, including differential DNA methylation (> 5% change vs. control; FDR-adjusted p < 0.05), elevated inflammatory mediators, and altered microRNA expression. Differentially methylated regions were enriched in regulatory features, with decreased methylation at transcription start sites, promoters, and 5' UTRs of genes implicated in mammary development and oncogenic signaling (including Vav3, Pdgfa, Pdgfc, Jag2, Hras, Ksr1, Il2rb, Il17b, and Il17rb) in the LPS group, whereas the AHCC + LPS group exhibited a shift toward hypermethylation at these loci (approximately 5%-10% decrease). Inflammatory profiling showed increased IL-17A/F (∼2-fold vs. control; p < 0.05), while microRNA analyses indicated reduced let-7a/c (∼30% vs. control; p < 0.05). Notably, miR-130a and miR-34a increased ∼1.5-fold across all treatment groups relative to control. Pubertal LPS exposure induces enduring epigenetic and inflammatory changes in mammary glands that may heighten breast cancer risk. AHCC's mitigating role indicates potential for dietary interventions to counteract these effects.

Sepsis induced myocardial injury (SIMI) remains a life threatening complication with mortality rates exceeding 40% to 50%, yet effective therapies are lacking. This study investigates the cardioprotective effects of eupatilin (EUP), a bioactive flavonoid derived from Artemisia argyi, and reveals a previously unrecognized mechanism involving selective modulation of KAT5 mediated histone H3K27 lactylation (H3K27la). Using an LPS induced murine model and TNF-α stimulated human AC16 cardiomyocytes, we evaluated cardiac function, inflammatory responses, and apoptotic pathways through dose response analyses. Multi omics approaches including RNA seq, metabolomics, ChIP seq, and molecular docking were integrated to dissect the pharmacodynamic profile of EUP. EUP conferred concentration dependent cardioprotection with optimal effects at 25 μM. Compared with conventional glucocorticoid therapy, EUP showed enhanced target selectivity, markedly reducing pro inflammatory cytokines such as TNF-α, IL-1β, and IL-6 while improving cardiac function parameters including ejection fraction and fractional shortening. Mechanistically, EUP bound KAT5 with high affinity, suppressed its lactylation activity, and reduced H3K27la enrichment at the promoters of inflammatory genes. Metabolic flux analysis further indicated that EUP inhibited glycolytic lactate production and restored oxidative phosphorylation. Together, these findings identify EUP as a natural modulator of the KAT5-H3K27la axis, addressing both metabolic dysregulation and epigenetic reprogramming in SIMI. With a favorable pharmacokinetic profile and superior target specificity relative to standard immunosuppressive regimens, EUP holds promise for clinical translation in sepsis associated cardiac dysfunction.

Mitochondria are central to cellular homeostasis and play a critical role in aging and age-related disorders, making them promising therapeutical targets. Here, we identify terbinafine and miglustat as novel mitochondrial stress inducers that extend lifespan and improve healthspan in Caenorhabditis elegans. Through a two-step screening, we found that both compounds activate the mitochondrial stress response (MSR) and exhibit distinct mechanisms of action. Terbinafine and miglustat robustly activated the mitochondrial unfolded protein response (UPRmt) mediator ATFS-1, upregulated MSR pathways, and modulated mitochondrial function across species, similarly to doxycycline. Interestingly, both compounds also engaged the insulin/IGF-1 signaling (IIS) pathway in C. elegans, revealing an integrated stress response involving coordinated action of ATFS-1 and the FOXO transcription factor DAF-16, distinct from canonical IIS activation. Experiments in human HEK293T cells confirmed the translational potential, with both compounds inducing mitochondrial stress and modulating mitochondrial function in mammalian systems. This study highlights the potential of harnessing the MSR to promote longevity and mitigate age-related functional decline. The identification of terbinafine and miglustat as mitochondrial stressors paves the way for novel anti-aging therapies.

Triple-negative breast cancer (TNBC) is known for its more aggressive clinical behavior, poor prognosis, and distinctive patterns of metastasis. Neoadjuvant chemotherapy (NAC) can influence both tumor cells and the tumor microenvironment. Emerging evidence highlights the critical role of actin cytoskeletal dynamics in cancer progression.

Heart failure (HF) represents the terminal stage of various cardiovascular diseases, and its prevalence continues to rise while current therapeutic approaches remain insufficient to reverse disease progression. Epigenetic regulation, with a particular focus on histone methylation, has gained increasing attention for its involvement in the initiation and advancement of HF. Histone methylation is a reversible post-translational modification controlled by histone methyltransferases and demethylases, and it participates in essential biological processes such as gene expression regulation, cell cycle, apoptosis, and metabolic reprogramming. This review systematically summarizes the multifaceted roles of histone methylation in HF, including the specific functions in cardiac regeneration, hypertrophy, fibrosis, apoptosis, metabolic remodeling, and inflammation. The review also highlights the promising effects of inhibitors that target histone methylation enzymes in animal studies, including anti-hypertrophic, anti-fibrotic, and cardioprotective properties, suggesting significant potential for clinical translation.

Malignant melanoma is the most aggressive and lethal type of skin cancer associated with increased mortality rates. Moreover, beyond the genetic background, the altered epigenetic landscape (e.g., abnormal patterns of DNA methylation, aberrant histone modifications and de-regulated expression levels of ncRNAs) further contributes to the pathophysiology of the disease. In addition, despite the improvement of current anti-melanoma strategies and the development of new therapeutic approaches, the 5-year survival among melanoma patients is still high, mainly due to acquired-drug resistance. On the other hand, phytochemicals have been associated with various health-promoting properties through pleiotropic mechanisms including acting as potent epigenetic regulators restoring back a normal phenotype in various experimental cancer models. In this review article, we discuss the general characteristics of malignant melanoma and current therapeutic approaches while we report the epigenetic basis of the disease along with the main compounds capable of restoring a normal epigenetic landscape. Finally, we describe the role of various phytochemicals in targeting the epigenome of malignant melanoma thereby potentially acting as an alternative therapeutic approach.

Nanoparticles offer promising applications in agriculture due to their unique physicochemical properties. This study investigated selenium nanoparticles (SeNPs) as a rooting agent to improve rice tolerance to low phosphate stress. IR64 rice was grown in media with varying SeNP concentrations (0-25 ppm), and plants were analyzed at 2 and 6 weeks for phenotypic traits, histology, biochemistry, and gene expression. At 2 weeks, 25 ppm SeNPs led to a threefold increase in root length versus untreated controls. Histological analysis revealed increased root perimeter and diameter, along with reduced aerenchyma perimeter. After 6 weeks under phosphate starvation, SeNP-treated plants showed higher phosphate and selenium content in roots and shoots, increased shoot length and weight, and reduced root length, weight, and phenolic content. Gene expression analysis showed that SeNPs upregulated key phosphate deficiency response genes, including OsPAP21, OsPT9, OsSPX, and OsPHR2. The most dramatic change was observed in OsSPX expression, which increased nearly 250-fold in shoots under full phosphate conditions. This is the first study to demonstrate that SeNPs promote root growth, enhance phosphate uptake, and regulate gene expression, suggesting SeNPs may serve as a sustainable strategy to boost phosphate use efficiency in rice cultivation.

Constitutive defense profiles underlie potato resistance to PVX ROTH1 and P. infestans. Treatment with aqueous rosemary extract (ARE) primes susceptible cultivars, enhancing their defense and offering a sustainable strategy to boosts resilience against these major foliar pathogens. Potato (Solanum tuberosum L.) is a major global food crop increasingly threatened by viral and oomycete pathogens, such as potato virus X (PVX) and Phytophthora infestans, whose impact is exacerbated under changing environmental conditions. Although priming strategies offer a sustainable approach to enhance disease resistance, it remains unclear how cultivar-specific metabolic states influence priming efficiency. Infection responses to PVX (strain ROTH1) and a P. infestans isolate were evaluated in four commercial cultivars. Cultivar-dependent differences were observed: Innovator exhibited higher resistance, Kennebec and Spunta greater susceptibility, and Frital-INTA was resistant to PVX ROTH1 but susceptible to P. infestans. 1H NMR metabolomics combined with defense gene expression analysis under non-inoculated conditions revealed distinct cultivar-specific signatures. Resistant and susceptible cultivars segregated into separate clusters, identifying metabolites associated with constitutive defense states. These results indicate that basal metabolic configuration contributes to differential pathogen susceptibility and may influence responsiveness to priming. Spunta, due to its broad cultivation and susceptibility, was selected to assess aqueous rosemary extract (ARE)-mediated priming. ARE treatment reduced PVX ROTH1 accumulation by ~ 70% and decreased P. infestans lesion size by ~ 30%, demonstrating that it enhances potato immunity by integrating both constitutive and inducible defense mechanisms.

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that integrates into the host cell's DNA as a provirus. Transcription from the provirus is regulated in large part by cellular proteins and epigenetic factors. These may be repressive or permissive to productive infection. The host factors that regulate this balance are therefore attractive targets for HIV-1 therapeutics. Indeed, proviral chromatin is the focus of two of the current HIV-1 cure strategies. "Shock and Kill" uses latency reversal agents to open the provirus's chromatin, promoting high levels of gene expression that induce the killing of infected cells. "Block and Lock" uses latency promoting agents to induce heterochromatin, blocking transcription and forcing HIV-1 into a state of deep latency. Here, the compounds investigated in both strategies are reviewed, including their chemical structures, mechanisms of action, and clinical results. Finally, the use of CRISPR-Cas therapeutics and the impact of chromatin architecture on its efficacy are discussed.

Background/Objectives: There is a clear need for more options to control the progression of breast cancer and prevent the occurrence of breast cancer in minority populations that have a higher rate of mortality due to triple-negative breast cancer (TNBC) subtypes. Prevalent nutraceuticals such as Ashwagandha (also known as the Indian Winter Cherry) have anti-inflammatory and apoptotic capabilities, as well as the ability to inhibit cancer growth. The purpose of this study is to analyze the novel combination of withaferin A (derived from the Indian Winter Cherry and known to have histone deacetylase inhibition capabilities) and sodium butyrate (a short-chain fatty acid produced from the gut microbiome and known to have DNA methyltransferase inhibition capabilities) treatment on breast cancer-derived cell lines. There is a scientific gap of possible causality of decreasing breast cancer progression when treated with sodium butyrate and withaferin A. Methods: Two in vitro cell viability assays were utilized consisting of [MTT (4,5 Dimethylthiazol-2-yl)] and the neutral red assay to analyze the impact of treatment of compounds alone and in combination on breast cancer cells for 72 h. The Highest Single Agent (HSA) combination analysis was utilized to derive combination indexes for our breast cancer cell types. Protein and gene expression was investigated for Class 1 histone deacetylases, de novo DNA methyltransferase, the p65 subunit of NF-κB, and NFκB1. Lastly, DNA methyltransferase enzymatic activity was analyzed via the Epigentek DNMT Activity/Inhibition ELISA Easy Kit. Results: Through the cell viability assay [MTT (4,5 Dimethylthiazol-2-yl)], MCF-7, MDA-MB-231, and MDA-MB-157 cells were found to have a decrease in cell viability due to combinatorial treatment with withaferin A and sodium butyrate. Western blot results depicted a decrease in protein expression levels for DNA methyltransferases due to the administration of 2.5 mM sodium butyrate and 0.2 µM withaferin A alone and in combination for breast cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-157. Additionally, the combination of these two components have successfully inhibited the progression of the NFκB1 gene within analysis through the quantitative polymerase chain reaction (qPCR). Conclusions: The novel combination of withaferin A and sodium butyrate have markedly reduced the progression of breast cancer-derived cell lines for cell viability, epigenetic DNMT gene expression, as well as inhibiting NFκB1 signaling on the gene expression level.

Environmental exposure to heavy metals, particularly cadmium (Cd), has been increasingly associated with obesity, metabolic dysfunction, chronic inflammation, and related disorders such as type 2 diabetes and cardiovascular diseases. Adipose tissue (AT), a paracrine and endocrine organ central to systemic energy and inflammatory homeostasis, is a major site of heavy metal accumulation and a key target of Cd toxicity. However, the mechanisms by which Cd disrupts adipocyte function, especially through epigenetic pathways, remain poorly understood. In this study, we investigated the effects of Cd on epigenetic regulators, antioxidant enzyme activity, inflammatory mediators, and adipogenic programming in human adipose-derived stromal/stem cells (hASCs) and differentiated adipocytes. Cd exposure altered histone modifiers associated with lysine 27 of histone 3 (H3K27), disrupted redox balance in a concentration-dependent manner, impaired adipogenic differentiation and lipid accumulation, and modulated inflammatory and adipokine responses according to differentiation stage and Cd concentration. Our findings suggest that Cd compromises adipose cell homeostasis through mechanisms involving epigenetic dysregulation, oxidative stress imbalance, and altered adipogenic and inflammatory signalling. These observations point to possible long-term metabolic consequences of environmental Cd exposure due to its accumulation in adipose tissue.

Triple-negative breast cancer (TNBC) cells with intact homologous recombination (HR) repair mechanism can survive treatment with Olaparib, which further limits the clinical application of PARP1/2 inhibitors. Previous studies have demonstrated that inhibition of indoleamine 2,3-dioxygenase (IDO) can enhance the sensitivity of human tumor cells to PARP1/2 inhibitors. However, the mechanisms underlying their synergistic effects in the treatment of TNBC remain unclear. Herein, we demonstrate that the combination of Olaparib and Epacadostat significantly reduces the proliferation of BRCA-proficient MDA-MB-231 and MDA-MB-468 cells compared to either monotherapy. Mechanistically, Epacadostat reduces intracellular kynurenine and NAD+ levels, thereby sensitizing TNBCs to PARP1/2 inhibition and significantly amplifying Olaparib-induced DNA damage. Furthermore, Epacadostat and Olaparib synergistically increase cellular reactive oxygen species (ROS), leading to DNA oxidative damage and apoptosis. In vivo, Epacadostat and Olaparib significantly suppressed MDA-MB-468 tumor growth compared to the monotherapy groups, while promoting an increase in phosphorylated H2AX. Notably, the dual inhibition of IDO1 and PARP1/2 specifically reduced the expression of HR core genes and proteins, such as BRCA1 and RAD51, which may contribute to impaired DNA-damage repair and increased sensitivity to Olaparib. In summary, targeting both IDO1 and PARP1/2 represents a promising combination therapy for BRCA-proficient TNBC.

S-phase kinase-associated protein 2 (SKP2) is an oncogene and cell cycle regulator that mediates the ubiquitination of cell cycle regulators. Curcumol, a sesquiterpene natural product, has been reported to regulate SKP2-mediated ubiquitination degradation to overcome drug resistance in cancer cells. However, whether the cell cycle arrest effect of curcumol is related to SKP2's function in cancer cells and its mechanisms are still unclear.

Biochar, a carbon-rich material traditionally used to improve soil health and as a feed additive, has recently attracted attention for its potential biological activity. This study examined the effects of an aqueous biochar extract (BC-AE) on human intestinal epithelial cells (Caco-2), focusing on its influence on cell viability and apoptosis. The metabolic activity of Caco-2 cells exposed to BC-AE was first evaluated using an MTS assay. A concentration of 3 mg/mL, which promoted Caco-2 metabolic activity, was selected for further testing at 24 and 72 h. The effect of BC-AE on cell viability was assessed by epifluorescence microscopy (morphology) and flow cytometry (apoptosis profiling). The transcriptional response of cell viability-related genes (BAX, BAD, BCL-2, BCL-xL, MCL-1, P21, and P53) and microRNAs (miR-15b, miR-19, miR-21, miR-33a, miR-155, and miR-486) was analyzed by RT-qPCR. In parallel, selected proteins (BAD, BAX, BCL-2, and MCL-1) were examined by Western blotting. We showed that BC-AE decreased cell viability after 24 h via late apoptosis, while 72 h exposure increased necrosis without further viability loss. Both BAX and MCL-1 protein levels increased in Caco-2 cells after 72 h of BC-AE treatment, and miR-15b and miR-21 were upregulated, suggesting the involvement of a regulatory mechanism controlling cell survival. The obtained findings highlight the importance of considering both concentration and exposure duration when assessing biochar bioactivity and represent an additional contribution to the ongoing effort to better understand the biological role.