Mode Of Action Research Articles

Optimizing Nav1.7-Targeted Analgesics: Revealing Off-Target Effects of Spider Venom-Derived Peptide Toxins and Engineering Strategies for Improvement.

The inhibition of Nav1.7 is a promising strategy for the development of analgesic treatments. Spider venom-derived peptide toxins are recognized as significant sources of Nav1.7 inhibitors. However, their development has been impeded by limited selectivity. In this study, eight peptide toxins from three distinct spider venom Nav channel families demonstrated robust inhibition of hNav1.7, rKv4.2, and rKv4.3 (rKv4.2/4.3) currents, exhibiting a similar mode of action. The analysis of structure and function relationship revealed a significant overlap in the pharmacophore responsible for inhibiting hNav1.7 and rKv4.2 by HNTX-III, although Lys25 seems to play a more pivotal role in the inhibition of rKv4.2/4.3. Pharmacophore-guided rational design is employed for the development of an mGpTx1 analogue, mGpTx1-SA, which retains its inhibition of hNav1.7 while significantly reducing its inhibition of rKv4.2/4.3 and eliminating cardiotoxicity. Moreover, mGpTx1-SA demonstrates potent analgesic effects in both inflammatory and neuropathic pain models, accompanied by an improved in vivo safety profile. The results suggest that off-target inhibition of rKv4.2/4.3 by specific spider peptide toxins targeting hNav1.7 may arise from a conserved binding motif. This insight promises to facilitate the design of hNav1.7-specific analgesics, aimed at minimizing rKv4.2/4.3 inhibition and associated toxicity, thereby enhancing their suitability for therapeutic applications.

Alternatives of mesenchymal stem cell-derived exosomes as potential therapeutic platforms

With outstanding therapeutic potential in the tissue regeneration and anti-inflammation, mesenchymal stem cell-derived exosomes (MSC-EXOs) have emerged as a prominent therapeutic in recent. However, poor production yield and reproducibility have remained as significant challenges of their practical applications. To surmount these challenges, various alternative materials with stem cell-like functions, have been recently investigated, however, there has been no comprehensive analysis in these alternatives so far. Here, we discuss the recent progress of alternatives of MSC-EXOs, including exosomes and exosome-like nanovesicles from various biological sources such as plants, milk, microbes, and body fluids. Moreover, we extensively compare each alternative by summarizing their unique functions and mode of actions to suggest the expected therapeutic target and future directions for developing alternatives for MSC-EXOs.

Fused Thiazolo[2,3‐b]Quinazolinone–Chromone Hybrids: Synthesis, Characterization, In Vitro Antibacterial Activity and In Silico Screening

ABSTRACTAntimicrobial resistance is one of the biggest threats to public health across the globe. Bacteria, fungi, viruses, and protozoans have been exhibiting resistance against antimicrobial drugs making them ineffective. Hence, the development of new antibiotics with a different mode of action is highly desirable. In this study, 10 new chromone‐incorporated fused thiazolo[2,3‐b]quinazolinone derivatives, 8a‐j, have been prepared via Biginelli reaction involving aromatic aldehydes, 1‐tetralone, and thiourea followed by a reaction with 2‐chloro‐N‐phenylacetamide, and Knoevenagel condensation with 3‐formylchromone. All the structures of the compounds were characterized by NMR, FTIR, and mass spectrometry. The in vitro antibacterial activities of all the synthesized compounds against the four different microbial strains were evaluated. Among them, few compounds demonstrated prominent activity against Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. No appreciable activity of any compound against Klebsiella pneumoniae was observed. Molecular docking studies were employed to reveal the interactions responsible for the potent compounds' activities against S. aureus, S. pyogenes, and P. aeruginosa. Both in vitro and in silico studies have been carried out by using standard agar well diffusion protocol and Auto Dock Vina in PyRx. The results indicated that compound 8c was the potential compound as it showed good affinity toward the receptors of all three organisms. Molecular dynamics simulation of the 8c‐1JIJ complex for 100 ns further confirmed the potentiality of 8c. The pharmacokinetic properties of the compounds indicate that the studied molecules have exhibited a favorable profile.

Lemongrass (Cymbopogon citratus) essential oil: an eco-friendly and fast-acting acaricide against the grain pest mite Aleuroglyphus ovatus

ABSTRACT This study was designed to evaluate the acaricidal effect and the mode of action of lemongrass essential oil (LEO) against Aleuroglyphus ovatus. Contact and fumigant mortality indicated that LEO had higher acaricidal activities at a low concentration, and the acaricidal action of LEO was significantly more effective by contact effects than by fumigant bioassay. Repellent rate against A. ovatus at different concentrations of LEO increased with application time. Observation on the toxic symptoms revealed that almost all mites were in immobilized type (IM type) in 30 min, but the symptom of knock-down type (KD type) appeared later. Cuticles of mites killed by LEO exhibited obvious colour change and deformation. Detoxification enzyme activity assay showed that glutathione S-transferase (GST) activity increased in mites treated by LEO compared to the controls. LEO increased catalase (CAT) activity of A. ovatus but inhibited superoxide dismutase (SOD) after 20 h. LEO also increased nitric oxide synthetase (NOS) and inhibited acetylcholine esterase (AChE) activities. Moreover, LEO acted as a fast-acting neurotoxin on A. ovatus. Our results suggest that LEO has an acaricidal potential and is a promising agent in control of A. ovatus.

Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation.

Deubiquitylases (DUBs) play a pivotal role in cell signalling and are often regulated by homo- or hetero-interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by selectively cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). BRCC36 is a Zn2+-dependent JAMM/MPN DUB, a challenging ubiquitin protease class for the design of selective inhibitors. We identified first-in-class DUB inhibitors that act as BRISC molecular glues (BLUEs). BLUEs inhibit DUB activity by stabilising a BRISC dimer consisting of 16 subunits. The BLUE-stabilised BRISC dimer is an autoinhibited conformation, whereby the active sites and interactions with the recruiting subunit SHMT2 are blocked. This unique mode of action leads to highly selective inhibitors for BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. Structure-guided inhibitor resistant mutants confirm BLUEs on-target activity in cells, and BLUE treatment results in reduced interferon-stimulated gene (ISG) expression in human peripheral blood mononuclear cells from Scleroderma patients, a disease linked with aberrant IFNAR1 activation. BLUEs represent a new class of molecules with potential utility in Type I interferon-mediated diseases and a template for designing selective inhibitors of large protein complexes by promoting protein-protein interactions instead of blocking them.

Sodium Houttuyniae attenuates ferroptosis by regulating TRAF6-c-Myc signaling pathways in lipopolysaccharide-induced acute lung injury (ALI)

The impact of Sodium Houttuyniae (SH) on lipopolysaccharide (LPS)-induced ALI has been investigated extensively. However, it remains ambiguous whether ferroptosis participates in this process. This study aimed to find out the impacts and probable mechanisms of SH on LPS-induced ferroptosis. A rat ALI model and type II alveolar epithelial (ATII) cell injury model were treated with LPS. Enzyme-linked immunosorbent assay (ELISA), hematoxylin-eosin (HE) staining, and Giemsa staining were executed to ascertain the effects of SH on LPS-induced ALI. Moreover, Transmission electron microscopy, Cell Counting Kit-8 (CCK8), ferrous iron colorimetric assay kit, Immunohistochemistry, Immunofluorescence, Reactive oxygen species assay kit, western blotting (Wb), and qRT-PCR examined the impacts of SH on LPS-induced ferroptosis and ferroptosis-related pathways. Theresults found that by using SH treatment, there was a remarkable attenuation of ALI by suppressing LPS-induced ferroptosis. Ferroptosis was demonstrated by a decline in the levels of glutathione peroxidase 4 (GPX4), FTH1, and glutathione (GSH) and a surge in the accumulation of malondialdehyde (MDA), reactive oxygen species (ROS), NOX1, NCOA4, and Fe2+, and disruption of mitochondrial structure, which were reversed by SH treatment. SH suppressed ferroptosis by regulating TRAF6-c-Myc in ALI rats and rat ATII cells. The results suggested that SH treatment attenuated LPS-induced ALI by repressing ferroptosis, and the mode of action can be linked to regulating the TRAF6-c-Myc signaling pathway in vivo and in vitro.

Dendritic cell effector mechanisms and tumor immune microenvironment infiltration define TLR8 modulation and PD-1 blockade.

The potent immunostimulatory effects of toll-like receptor 8 (TLR8) agonism in combination with PD-1 blockade have resulted in various preclinical investigations, yet the mechanism of action in humans remains unknown. To decipher the combinatory mode of action of TLR8 agonism and PD-1 blockade, we employed a unique, open-label, phase 1b pre-operative window of opportunity clinical trial ( NCT03906526 ) in head and neck squamous cell carcinoma (HNSCC) patients. Matched pre- and post-treatment tumor biopsies from the same lesion were obtained. We used single-cell RNA sequencing and custom multiplex staining to leverage the unique advantage of same-lesion longitudinal sampling. Patients receiving dual TLR8 agonism and anti-PD-1 blockade exhibited marked upregulation of innate immune effector genes and cytokines, highlighted by increased CLEC9A+ dendritic cell and CLEC7A/SYK expression. This was revealed via comparison with a previous cohort from an anti-PD-1 blockade monotherapy single-cell RNA sequencing study. Furthermore, in dual therapy patients, post-treatment mature dendritic cells increased in adjacency to CD8 + T-cells. Increased tumoral cytotoxic T-lymphocyte densities and expanded CXCL13 + CD8 + T- cell populations were observed in responders, with increased tertiary lymphoid structures (TLSs) across all three patients. This study provides key insights into the mode of action of TLR8 agonism and anti-PD-1 blockade immune targeting in HNSCC patients.

Corilagin alleviated intestinal ischemia-reperfusion injury by modulating endoplasmic reticulum stress via bonding with Bip

BackgroundIntestinal ischemia-reperfusion (II/R) injury is a common clinical emergency with high morbidity and mortality. Given the absence of efficacious prophylactic and therapeutic interventions and specific drugs, sustained efforts are essential to develop new targeted drugs. Corilagin, a naturally polyphenolic tannic acid widespread in longan, rambutan and many other edible economic crops with medicinal properties in China, is of interest due to its multiple bioactivities, including the potential to mitigate II/R injuries. Nevertheless, a clear understanding of its molecular targets and the intricate mechanisms against II/R injury remains obscure and requires further elucidation. ObjectiveThis study aimed to investigate corilagin's pharmacological impact and molecular mechanism for II/R injury. MethodsAn animal II/R model was established by clamping superior mesenteric artery (SMA), and the therapeutic efficacy of corilagin against II/R was evaluated by biochemical and pathological analysis. Next, integrated transcriptomic and proteomic analyses was performed to identify key targets. Moreover, endoplasmic reticulum stress (ERS) damage was respectively observed by transmission electron microscope (TEM), immunohistochemistry, TUNEL, flow cytometry and western blotting (WB). Finally, molecular docking, molecular dynamics (MD) simulation, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) assays were utilized to assess the interaction between corilagin and binding immunoglobulin protein (Bip, Grp78 or Hspa5), and co-IP assay was conducted to investigate the interaction between Bip and its substrate proteins. ResultsCorilagin exhibited robust protection against II/R injuries, effectively alleviating intestinal tissue damage and oxidative stress induced by II/R. The modulation of ERS as a potential regulatory mechanism was investigated through an integrated transcriptomic and proteomic analysis, identifying Bip as a key target contributing to corilagin's protective effects. Further experimental evidence using molecular docking, MD simulation, CETSA, and DARTS assays confirmed the potentially direct interaction of corilagin with Bip. This interaction promoted the ubiquitin-dependent degradation of the Bip-substrate complex, thereby suppressing ERS-related signalling pathways, including the IRE1 branch, PERK branch, and ATF6 branch, to alleviate tissue damage. ConclusionThis study confirmed that corilagin could selectively bind to Bip, facilitating its ubiquitin-dependent recognition and degradation, thereby inhibiting severe endoplasmic reticulum stress signalling and alleviating II/R injury. A detailed mechanistic insight into the action mode of corilagin had been proposed, supporting its potential usage as an ERS inhibitor.

Deciphering mechanisms of UV filter (benzophenone-3)- and high temperature-induced adverse effects in the coral Acropora tenuis, using ecotoxicogenomics.

Coral reefs are at risk of bleaching due to various environmental and anthropogenic stressors such as global warming and chemical pollutants. However, there is little understanding of stressor-specific mechanisms that cause coral bleaching. Therefore, conducting accurate ecotoxicological risk assessments and deciphering modes of action of potentially deleterious ultraviolet (UV) filters (sunscreen compounds) are crucial issues. In this study, we evaluated the toxicity and bleaching effect of benzophenone-3 (BP-3), which is widely used in sunscreen products, on the reef-building coral Acropora tenuis. Furthermore, to understand differences in UV filter- and temperature-induced adverse effects, a comparative ecotoxicogenomic approach using RNA-seq was integrated into a toxicity test to clarify differences in gene expression changes induced by BP-3 and heat stress (31 °C). The lethal concentration 50 % (LC50) was calculated as 3.9 mg/L, indicating that the aquatic environmental risk on corals posed by BP-3 was low based on the risk assessment in this study. Differentially expressed genes related to oxidative stress and extracellular matrix organization were involved in coral responses to both BP-3 and heat stress, but their patterns differed. Whereas immune and heat-shock responses were activated in response to heat stress, activation of a drug metabolism pathway and several signal transduction pathways were identified in BP-3 treatment groups. Our study enhances understanding of stress responses in corals induced by UV filters and thermal stress. Using potential gene markers identified in this study for eco-epidemiological surveys of stressed corals, we urgently need to develop effective countermeasures.

Enhanced exploration of the mode of action of a five-layer foam dressing: critical properties to support wound healing.

The aim of this in vitro experimental series was to explore the mode of action of a hydrocellular polyurethane foam dressing (HPFD) and how its advanced features support beneficial interactions with the wound bed to address common barriers to wound healing, thus supporting improved clinical outcomes. Multiple in vitro microbiological tests were performed, assessing prevention of bacterial ingress, surface removal of bacteria, bacterial sequestration and retention into the dressing in a clinically relevant environment. Odour molecule concentrations were measured using gas chromatography and further assays explored matrix metalloproteinase (MMP)-9 retention in the dressing using enzyme linked immunosorbent assay. The HPFD demonstrated marked reductions in bioburden levels across multiple tests. These included prevention of bacterial ingress for seven days, removal of surface bacteria and absorption into the dressing. Further tests identified that most bacteria were sequestered into the hyperabsorbent layer (90.5% for Pseudomonas aeruginosa and 89.6% for meticillin-resistant Staphylococcus aureus). Moreover, the majority of bacteria (99.99% for both test organisms) were retained within the dressing, even upon compression. Additional tests demonstrated a marked reduction of odour molecules following incubation with HPFD and total retention of protease MMP-9 within the dressing. Proactive management of the wound environment with an appropriate advanced wound dressing, such as the HPFD examined in these in vitro investigations, can not only help to minimise the barriers to healing, as observed across this test series by direct interaction with the wound bed, but may, as a result, provide an ideal environment for wound progression with minimal disturbance.

Patterns of herbicide resistance in Raphanus raphanistrum revealed by comprehensive testing and statistical analysis.

Raphanus raphanistrum causes $40 million total revenue losses annually in Western Australia partly due to its historically-documented ability to evolve herbicide resistance to multiple modes of action. In this study, 376 field-sampled populations of R. raphanistrum were tested for resistance to 21 herbicides applied at the recommended label rate. Eight treatments were herbicide mixtures with two, three or four modes of action. A total of 7199 individual resistance tests were conducted across 4 years by screening approximately 104 000 individual seeds and seedlings. The mean survival of individuals within a population for all standalone herbicides was 9%, whereas survival was significantly decreased to 3.5% with a herbicide mixture. Some herbicides such as triasulfuron (herbicide Group 2), 2,4-D (Group 4) or diflufenican (Group 12) were highly impacted by resistance, with frequencies of resistant populations being > 50%. Conversely, there was negligible resistance to glyphosate (Group 9) or protoporphyrinogen oxidase (PPO) inhibitors (tiafenacil, saflufenacil + trifludimoxazin, fomesafen: Group 14), and pre-emergence herbicides (i.e., atrazine or mesotrione: Groups 5 and 27, respectively) remained largely effective. Binary, ternary or quaternary mixtures of Groups 4, 6, 12 and 27 herbicides reduced the frequency of high-level resistant populations to 7.1%, 3.8% or 0%, respectively. The cost-effective control of R. raphanistrum remains a challenge due to herbicide resistance. Raphanus raphanistrum management relies heavily on herbicide uses not yet compromised by resistance, such as pre-emergence herbicides (atrazine, fomesafen, mesotrione), glyphosate, and mixtures of two, three or four modes of action including bromoxynil, diflufenican, MCPA, picolinafen, pyrasulfotole and topramezone. Strategies that integrate effective herbicide use patterns, novel modes of action and efficiently-mechanized non-chemical weed control options (i.e., seed destructors) can completely constrain the selection of herbicide resistance in this highly adaptable species. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of diammonium hydrogen phosphate coated with silica on flame retardancy of epoxy resin.

Epoxy resin has become one of the most widely used polymers owing to their excellent comprehensive properties. However, the inherent inflammability of epoxy resin (EP) has seriously limited its application in a range of fields with high fire safety requirements. Herein, a novel shell-core hierarchy architecture (DAP@SiO2) was prepared, composed of diammonium hydrogen phosphate (DAP) and in situ grown silica, and its structure and morphology were characterized by electron microscopy and infrared spectroscopy. The results indicated that silica particles were uniformly coated onto the surface of DAP. The modified DAP was used to reinforce the epoxy resin. The thermal stability of the EP blends was studied with the use of thermogravimetric analysis. The diammonium phosphate in DAP@SiO2 flame retardant produces pyrolysis gases such as NH3 and N2 during decomposition, diluting the concentration of oxygen and flammable volatile products around the flame. Secondly, silica migrates to the surface of epoxy resin to form a shielding layer, forming compounds containing Si-O-Si and O-Si-C structures, which can be cross-linked with other condensed phase products, greatly improving the thermal stability of the carbon layer. Fire behavior was evaluated using the limiting oxygen index (LOI), vertical burning test, and the cone calorimetry, and the flame retardancy mode of action was explained. With 12% of DAP@SiO2 involved, the EP blend passes UL-94 V-0 level, and its limiting oxygen index (LOI) reaches 33.2%. The incorporation of DAP@SiO2 in an EP matrix showed a slight reduction in the heat release and smoke production. The flame retardant mode of the EP polymer shows that its flame retardant and smoke suppression characteristics are related to the interaction of flame retardants in the gas phase and condensed phase. The mechanical properties test results illustrated that the tensile strength, elastic modulus and impact strength of EP/3%DAP@SiO2 are improved compared with pure EP. This is due to the crosslinking reaction between a large number of amino groups on the surface of DAP@SiO2 and the epoxy group on the epoxy resin, which significantly enhances the interfacial compatibility between DAP@SiO2 and epoxy resin, making the combination of DAP@SiO2 and epoxy resin closer.

Limited role of fungal diversity in maintaining soil processes in grassland soil under concurrent fungicide stress

BackgroundFungicides are an effective tool for protecting crops and maintaining a steady food supply. However, as pathogens continue to evolve, it is crucial to prolong the effectiveness of fungicides by delaying resistance development. A key strategy to achieving this is to combine or rotate fungicides with different modes of action. As fungicides lack specificity, they inevitably affect both pathogenic and non-pathogenic fungi when surrounding environments are unintentionally contaminated. Our study aims to investigate the effects of recommended application methods to prevent resistance development, specifically repeated-single fungicide, simultaneous mixture, and sequential applications on non-target soil fungi, and the subsequent impacts on important soil processes. We used fungicides with different modes of action on soil microcosms inoculated with fungi at varying levels of diversity (3, 5, and 8 species) isolated from a protected grassland.ResultsWe found that repeated treatments of individual isopyrazam and prothioconazole differentially inhibited fungal activity. Although mixture applications are considered more protectant against crop pathogen resistance than repeated application, our study revealed stronger negative effects of simultaneous application on saprobic fungi and consequently on soil processes. However, contrary to expectations, higher fungal diversity did not translate to improved soil function under these conditions.ConclusionsThe simultaneous application of fungicides with different modes of action (MoA) has more pronounced non-target effects on soil compared to the individual or sequential application of fungicides. These non-target effects extend beyond the intended control of pathogenic fungi, impacting saprobic and beneficial soil microbes and the critical processes they drive. When fungicides are applied concurrently, microbial activities in the soil are significantly altered, even in soils with high microbial diversity. Our study emphasizes the importance of carefully considering the unintended consequences of fungicide use in agriculture. As we strive for a secure food supply, it is crucial to investigate the broader environmental impacts of these chemical interventions, including their effects on non-pathogenic fungi and overall soil health.

Navigating the fungal battlefield: cysteine-rich antifungal proteins and peptides from Eurotiales

Fungi are ubiquitous in the environment and play a key role in the decomposition and recycling of nutrients. On the one hand, their special properties are a great asset for the agricultural and industrial sector, as they are used as source of nutrients, producers of enzymes, pigments, flavorings, and biocontrol agents, and in food processing, bio-remediation and plant growth promotion. On the other hand, they pose a serious challenge to our lives and the environment, as they are responsible for fungal infections in plants, animals and humans. Although host immunity opposes invading pathogens, certain factors favor the manifestation of fungal diseases. The prevalence of fungal infections is on the rise, and there is an alarming increase in the resistance of fungal pathogens to approved drugs. The limited number of antimycotics, the obstacles encountered in the development of new drugs due to the poor tolerability of antifungal agents in patients, the limited number of unique antifungal targets, and the low species specificity contribute to the gradual depletion of the antifungal pipeline and newly discovered antifungal drugs are rare. Promising candidates as next-generation therapeutics are antimicrobial proteins and peptides (AMPs) produced by numerous prokaryotic and eukaryotic organisms belonging to all kingdom classes. Importantly, filamentous fungi from the order Eurotiales have been shown to be a rich source of AMPs with specific antifungal activity. A growing number of published studies reflects the efforts made in the search for new antifungal proteins and peptides (AFPs), their efficacy, species specificity and applicability. In this review, we discuss important aspects related to fungi, their impact on our life and issues involved in treating fungal infections in plants, animals and humans. We specifically highlight the potential of AFPs from Eurotiales as promising alternative antifungal therapeutics. This article provides insight into the structural features, mode of action, and progress made toward their potential application in a clinical and agricultural setting. It also identifies the challenges that must be overcome in order to develop AFPs into therapeutics.

Targeting RNA with small molecules, from RNA structures to precision medicines: IUPHAR review: 40.

RNA plays important roles in regulating both health and disease biology in all kingdoms of life. Notably, RNA can form intricate three-dimensional structures, and their biological functions are dependent on these structures. Targeting the structured regions of RNA with small molecules has gained increasing attention over the past decade, because it provides both chemical probes to study fundamental biology processes and lead medicines for diseases with unmet medical needs. Recent advances in RNA structure prediction and determination and RNA biology have accelerated the rational design and development of RNA-targeted small molecules to modulate disease pathology. However, challenges remain in advancing RNA-targeted small molecules towards clinical applications. This review summarizes strategies to study RNA structures, to identify small molecules recognizing these structures, and to augment the functionality of RNA-binding small molecules. We focus on recent advances in developing RNA-targeted small molecules as potential therapeutics in a variety of diseases, encompassing different modes of actions and targeting strategies. Furthermore, we present the current gaps between early-stage discovery of RNA-binding small molecules and their clinical applications, as well as a roadmap to overcome these challenges in the near future.

The natural compound periplogenin suppresses the growth of prostate carcinoma cells by directly targeting ATP1A1

Natural compounds constitute a major resource for the development of medicines for multiple diseases. While many natural compounds show strong biological activity, the mechanisms that confer clinical benefits are often elusive and have been attributed to multiple pathways. Periplogenin (PPG), a natural compound isolated from Cortex periplocae, exhibits strong anti-tumor activities in several human cancer cell lines. However, its molecular mode of action remained unclear. In this study, we leveraged a forward genetic screening approach in DU145 prostate cancer cells to uncover the molecular target of PPG using chemical mutagenesis. Next generation sequencing revealed that a single amino acid substitution at amino acid 804 in ATP1A1 (ATPase Na + /K + Transporting Subunit Alpha 1) confers resistance to the cytotoxic activity of PPG. Mechanistically, ATP1A1 T804 forms a hydrogen bond with PPG which is abolished by the T804A substitution in ATP1A1, resulting in resistance to PPG treatment in vitro. Importantly, in vivo, PPG strongly suppressed tumor development in a DU145 xenograft model whereas DU145 xenograft tumors carrying a ATP1A1-T804A mutation were largely unaffected by the treatment. These findings demonstrate that PPG suppresses the growth of DU145 prostate cancer cells in vitro and in vivo by directly binding to ATP1A1 and highlight the power of our unbiased forward genetic screening approach to uncover direct drug target structures at single amino acid resolution.

Selection of reference genes for expression profiling in biostimulation research of soybean

BackgroundPlant biostimulants constitute a promising environmentally friendly alternative for increasing crop yield and tolerance to unfavorable conditions. Among various types of such formulations, botanical extracts are gaining more recognition as products supporting plant performance. Moreover, novel tools such as cold-plasma or low-pressure microwave plasma discharge are being proposed as techniques that might improve their efficacy. Elucidation of the biostimulant’s mode of action requires complex research at a molecular level. Transcriptional changes occurring after biostimulant spraying might be investigated using RT-qPCR. However, this technique requires data normalization against stable endogenous controls.ResultsHere, we tested the expression stability of ten candidate genes in soybean plants exposed to various biostimulants treatment. Selection of the best-performing reference genes was conducted using four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method). According to the obtained results, Bic-C2 (RNA-binding protein Bicaudal-C) and CYP (cyclophilin type peptidyl-prolyl cis–trans isomerase) showed highest expression stability, while expression of EF1B (elongation factor 1-beta) fluctuated the most among a tested set of candidate genes.ConclusionsOverall, we recommend using Bic-C2 together with CYP for the RT-qPCR data normalization in soybean biostimulation experiments. To our best knowledge, this is the first comprehensive study of reference genes stability in plants subjected to biostimulant treatment. The results of this study will aid in further biostimulant research in crop plants, facilitating analyses performed on the transcriptional level.Graphical

DNA methylation and histone modifications associated with antipsychotic treatment: a systematic review.

Antipsychotic medications are essential when treating schizophrenia spectrum and other psychotic disorders, but the efficacy and tolerability of these medications vary from person to person. This interindividual variation is likely mediated, at least in part, by epigenomic processes that have yet to be fully elucidated. Herein, we systematically identified and evaluated 65 studies that examine the influence of antipsychotic drugs on epigenomic changes, including global methylation (9 studies), genome-wide methylation (22 studies), candidate gene methylation (16 studies), and histone modification (18 studies). Our evaluation revealed that haloperidol was consistently associated with increased global hypermethylation, which corroborates with genome-wide analyses, mostly performed by methylation arrays. In contrast, clozapine seems to promote hypomethylation across the epigenome. Candidate-gene methylation studies reveal varying effects post-antipsychotic therapy. Some genes like Glra1 and Drd2 are frequently found to undergo hypermethylation, whereas other genes such as SLC6A4, DUSP6, and DTNBP1 are more likely to exhibit hypomethylation in promoter regions. In examining histone modifications, the literature suggests that clozapine changes histone methylation patterns in the prefrontal cortex, particularly elevating H3K4me3 at the Gad1 gene and affecting the transcription of genes like mGlu2 by modifying histone acetylation and interacting with HDAC2 enzymes. Risperidone and quetiapine, however, exhibit distinct impacts on histone marks across different brain regions and cell types, with risperidone reducing H3K27ac in the striatum and quetiapine modifying global H3K9me2 levels in the prefrontal cortex, suggesting antipsychotics demonstrate selective influence on histone modifications, which demonstrates a complex and targeted mode of action. While this review summarizes current knowledge, the intricate dynamics between antipsychotics and epigenetics clearly warrant more exhaustive exploration with the potential to redefine our understanding and treatment of psychiatric conditions. By deciphering the epigenetic changes associated with drug treatment and therapeutic outcomes, we can move closer to personalized medicine in psychiatry.

In Vitro Studies of Genistein Lipophilic Derivatives as Potential UV Radiation Protectors

The major environmental factor responsible for skin cancer is ultraviolet (UV) radiation, present in sunlight. UV radiation is directly linked to the production of reactive oxygen species (ROS), which accumulate in exposed cells and cause serious damage. The antioxidant systems present in cells cannot always sufficiently neutralize the ROS. Therefore, supplementation with exogenous antioxidants has been proposed. The antioxidant properties of some isoflavones, such as genistein, have already been well-proven. Genistein has limited bioavailability. However, its derivatives, with increased lipophilicity, could facilitate its transfer into cells, where they can expose its antioxidative potential. This study aims to investigate three genistein derivatives, with greater lipophilicity than the native compound, regarding their cytotoxicity, antioxidative properties, and effect on the cell cycle in normal human dermal fibroblasts (NHDF) and a melanoma cancer cell line (Me45). Results showed that lipophilic modification of the genistein molecule changes the biological response of NHDF and Me45 cell lines to UV-C radiation, but the lipophilicity cannot be directly linked with the activity of the compounds. A comparison of the effects of the genistein derivatives on healthy and cancerous cells suggests that their mode of action strongly depends on the type of cell involved.

Polarized benzene rings can promote the interaction between CaM and the CaMBD region of nNOS.

The neuronal nitric oxide synthase (nNOS) subtype of nitric oxide synthase (NOS) is an enzyme required for learning and memory. Overactivation of nNOS can lead to oxidative/nitrite stress, which is complicit in the pathophysiology of various neurological and psychiatric disorders. Previous studies have shown that calmodulin (CaM) forms complexes with Ca2+ and binds to the calmodulin-binding domain (CaMBD) of nNOS, thereby upregulating its catalytic activity in hippocampal neurons. To date, there has been no explanation for the non-covalent interactions in the CaMBD-CaM binding structure model of nNOS. In this study, we aimed to investigate the intrinsic factors involved in the binding of CaM to NOS-CaMBD and designed interfering peptides based on the N0 peptide structure of the original nNOS-CaMBD sequence: N1 (obtained from the L734F mutation), N2 (obtained from the F731Y and F740Y mutations), and N3 (obtained from the F731L, V738L, and F740L mutations). We employed homology modeling to construct six CaM-peptide complex models, aiming to elucidate the roles of key amino acid residues within the N0 peptide in its interaction with CaM by means of molecular dynamics simulations. The effect of the peptides on the activation and release of NO by nNOS in neurons was assessed using murine primary neuronal cells. When measuring neuronal NO content, it was found that adding N2 and N3 to cultivated neurons significantly increased nNOS activity, leading to the increased NO production. We found that interfering peptides could stably bind to CaM. Among them, N2 and CaM exhibited the strongest binding ability, indicating that the polarized benzene ring significantly enhanced the binding between nNOS-CaMBD and CaM. Conversely, the binding ability between N0 and CaM was the weakest, as they exhibited the worst polar contact, weakest hydrogen bonding, and the lowest binding free energy. The simulation results also highlighted several important amino acid residues: The K76 of CaM plays an important role in polar contact and hydrogen bonding formation, the L734 residue suppressed model flexibility to a certain extent and had an adverse effect on the overall binding free energy of the model. These results, compared with the results of cellular NO content, a preliminary verification of the antagonistic competitive mechanism between CaM allosteric activation of nNOS and SUMOylation hyperactivation was performed. In summary, this study explored the ability and mode of action of key residues in nNOS-CaMBD on the binding of interfering peptides to CaM, thereby providing new structural perspectives for the activation of nNOS by CaM and recommendations for drug design targeting the specific inhibition of nNOS.