Signaling Cascade Research Articles

Neuronatin Promotes the Progression of Non-small Cell Lung Cancer by Activating the NF-κB Signaling.

Understanding the regulatory mechanisms involving neuronatin (NNAT) in non-small cell lung cancer (NSCLC) is an ongoing challenge. This study aimed to elucidate the impact of NNAT knockdown on NSCLC by employing both in vitro and in vivo approaches. To investigate the role of NNAT, its expression was silenced in NSCLC cell lines A549 and H226. Subsequently, various parameters, including cell proliferation, invasion, migration, and apoptosis, were assessed. Additionally, cell-derived xenograft models were established to evaluate the effect of NNAT knockdown on tumor growth. The expression of key molecules, including cyclin D1, B-cell leukemia/lymphoma 2 (Bcl-2), p65, matrix metalloproteinase (MMP) 2, and nerve growth factor (NGF) were examined both in vitro and in vivo. Nerve fiber density within tumor tissues was analyzed using silver staining. Upon NNAT knockdown, a remarkable reduction in NSCLC cell proliferation, invasion, and migration was observed, accompanied by elevated levels of apoptosis. Furthermore, the expression of cyclin D1, Bcl-2, MMP2, and phosphorylated p65 (p-p65) showed significant downregulation. In vivo, NNAT knockdown led to substantial inhibition of tumor growth and a concurrent decrease in cyclinD1, Bcl-2, MMP2, and p-p65 expression within tumor tissues. Importantly, NNAT knockdown also led to a decrease in nerve fiber density and downregulation of NGF expression within the xenograft tumor tissues. Collectively, these findings suggest that neuronatin plays a pivotal role in driving NSCLC progression, potentially through the activation of the nuclear factor-kappa B signaling cascade. Additionally, neuronatin may contribute to the modulation of tumor microenvironment innervation in NSCLC. Targeting neuronatin inhibition emerges as a promising strategy for potential anti-NSCLC therapeutic intervention.

Norepinephrine triggers glutamatergic long-term potentiation in hypothalamic paraventricular nucleus magnocellular neuroendocrine cells through postsynaptic β1-AR/PKA signaling pathway in vitro in rats.

Norepinephrine (NE) modulates synaptic transmission and long-term plasticity through distinct subtype adrenergic receptor (AR)-mediated-intracellular signaling cascades. However, the role of NE modulates glutamatergic long-term potentiation (LTP) in the hypothalamic paraventricular nucleus (PVN) magnocellular neuroendocrine cells (MNCs) is unclear. We here investigate the effect of NE on high frequency stimulation (HFS)-induced glutamatergic LTP in rat hypothalamic PVN MNCs in vitro, by whole-cell patch-clamp recording, biocytin staining and pharmacological methods. Delivery of HFS induced glutamatergic LTP with a decrease in N2/N1 ratio in the PVN MNCs, which was enhanced by application of NE (100 nM). HFS-induced LTP was abolished by the blockade of N-methyl-D-aspartate receptors (NMDAR) with D-APV, but it was rescued by the application of NE. NE failed to rescue HFS-induced LTP of MNCs in the presence of a selective β1-AR antagonist, CGP 20712. However, application of β1-AR agonist, dobutamine HCl rescued HFS-induced LTP of MNCs in the absence of NMDAR activity. In the absence of NMDAR activity, NE failed to rescue HFS-induced MNC LTP when protein kinase A (PKA) was inhibited by extracellular applying KT5720 or intracellular administration of PKI. These results indicate that NE activates β1-AR and triggers HFS to induce a novel glutamatergic LTP of hypothalamic PVN NMCs via the postsynaptic PKA signaling pathway in vitro in rats.

Luteolin detoxifies DEHP and prevents liver injury by degrading Uroc1 protein in mice

AbstractDi-(2-ethylhexyl) phthalate (DEHP), an environmental pollutant, has been widely detected in both environmental and clinical samples, representing a serious threat to the homeostasis of the endocrine system. The accumulation of DEHP is notably pronounced in the liver and can lead to liver damage. The lack of effective high-throughput screening system retards the discovery of such drugs that can specifically target and eliminate the detrimental impact of DEHP. Here, by developing a Cy5-modified single-strand DNA-aptamer-based approach targeting DEHP, we have identified luteolin as a potential drug, which showcasing robust efficacy in detoxifying the DEHP by facilitating the expulsion of DEHP in both mouse primary hepatocytes and livers. Mechanistically, luteolin enhances the protein degradation of hepatic urocanate hydratase 1 (Uroc1) by targeting its Ala270 and Val272 sites. More importantly, trans-urocanic acid (trans-UCA), as the substrate of Uroc1, possesses properties similar to luteolin by regulating the lysosomal exocytosis through the inhibition of the ERK1/2 signal cascade. In summary, luteolin serves as a potent therapeutic agent in efficiently detoxifying DEHP in the liver by regulating the UCA/Uroc1 axis.

Calcium-Binding Protein and Polymorphism in Musa spp. Somaclones Resistant to Fusarium oxysporum

The fresh fruits of ‘Grande Naine’ (Cavendish AAA—Musa spp.) dominate the world market, especially in countries with a population in a situation of social vulnerability. However, Fusarium wilt, caused by the fungus Fusarium oxysporum f.sp. cubense race 4 Subtropical (Foc ST4), emerges as a serious threat to banana production, requiring the development of resistant cultivars based on biotechnological strategies, such as the induction of mutation in tissue culture. This study aimed to identify and characterize genetic variation in somaclones resistant to Fusarium oxysporum f.sp. cubense subtropical race 4 (Foc ST4), derived from ‘Grand Naine’ bananas, by molecular markers based on retrotransposons IRAP (Inter-retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism). Nine combinations of IRAP and six combinations of REMAP primers were used. The low number of polymorphic bands did not allow for genetic diversity studies; however, ten polymorphic bands between the somaclones and control were sequenced. Of these, three presented good base calling and were aligned, namely, 1AF, 2AF, and 3AF bands. Only the 1AF band presented function related to stress response with homology to a calcium-binding protein. These proteins act early in plant infection as secondary messengers activated by pathogen-associated molecular patterns (PAMPs), initiating the cascade of plant defense signals. The fact that this band is present in all somaclones reinforces previous assessments of their resistance to Foc ST4. The use of markers IRAP and REMAP produced polymorphic bands that can, through future primer design and field validations, accelerate the identification of resistant banana genotypes for use in banana genetic breeding programs.

Female Reproductive System and Thyroid Dysfunction: Findings from a 12-Year Follow-Up in the Tehran Thyroid Study.

Background: The impact of thyroid dysfunction (TD) on the female reproductive system has been extensively documented. While there is evidence suggesting that alteration in female reproductive status may affect thyroid function, conflicting results have prevented definitive conclusions. This study aimed to investigate the associations of parity, spontaneous abortion (mentioned as abortion throughout this study), and menopause status with the prevalence and incidence of TD. Methods: From the Tehran thyroid study population, 2711 participants were included in the cross-sectional analysis to explore associations between female reproductive status and TD. Overall, 2191 participants with euthyroid were included in the survival study and followed up in 3-year intervals. Multinomial logistic regression was adopted in cross-sectional analysis and multivariable Cox proportional hazard model was used to determine associations between the incidence of TD with parity, abortion, and menopause status, adjusting for age, smoking, body mass index, and thyroid peroxidase antibodies positivity. Results: At the baseline, multiple parities (≥4) were significantly associated with overt hypothyroidism (odds ratio [OR] = 1.12; confidence interval [CI] 1.0-1.26) and subclinical hyperthyroidism (OR = 1.11 [CI 1.03-1.21]). Furthermore, multiple abortions were associated with overt hyperthyroidism (OR = 2.09 [CI 1.02-4.26]). Over the course of the study, multiple parities were significantly associated with the incident subclinical and clinical hypothyroidism. Conversely, a history of abortion was associated with a reduced risk of incident overt hypothyroidism. We found no significant association between menopause status and the prevalence or incidence of either hypothyroidism or hyperthyroidism. Conclusions: Our results suggest that the female reproductive system may be associated with thyroid function. Parity and abortion are associated with the occurrence of TD. A deeper understanding of the underlying mechanisms of the cellular and molecular alterations in signaling cascades during pregnancy is necessary to fully elucidate these associations.

Correction: Siu et al. Hexokinase 2 Regulates Ovarian Cancer Cell Migration, Invasion and Stemness via FAK/ERK1/2/MMP9/NANOG/SOX9 Signaling Cascades. Cancers 2019, 11, 813

In the original publication [...]

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p<0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

Transcriptional responses to direct and indirect TGFB1 stimulation in cancerous and noncancerous mammary epithelial cells

BackgroundTransforming growth factor beta (TGFβ) is important for the morphogenesis and secretory function of the mammary gland. It is one of the main activators of the epithelial–mesenchymal transition (EMT), a process important for tissue remodeling and regeneration. It also provides cells with the plasticity to form metastases during tumor progression. Noncancerous and cancer cells respond differently to TGFβ. However, knowledge of the cellular signaling cascades triggered by TGFβ in various cell types is still limited.MethodsMCF10A (noncancerous, originating from fibrotic breast tissue) and MCF7 (cancer, estrogen receptor-positive) breast epithelial cells were treated with TGFB1 directly or through conditioned media from stimulated cells. Transcriptional changes (via RNA-seq) were assessed in untreated cells and after 1–6 days of treatment. Differentially expressed genes were detected with DESeq2 and the hallmark collection was selected for gene set enrichment analysis.ResultsTGFB1 induces EMT in both the MCF10A and MCF7 cell lines but via slightly different mechanisms (signaling through SMAD3 is more active in MCF7 cells). Many EMT-related genes are expressed in MCF10A cells at baseline. Both cell lines respond to TGFB1 by decreasing the expression of genes involved in cell proliferation: through the repression of MYC (and the protein targets) in MCF10A cells and the activation of p63-dependent signaling in MCF7 cells (CDKN1A and CDKN2B, which are responsible for the inhibition of cyclin-dependent kinases, are upregulated). In addition, estrogen receptor signaling is inhibited and caspase-dependent cell death is induced only in MCF7 cells. Direct incubation with TGFB1 and treatment of cells with conditioned media similarly affected transcriptional profiles. However, TGFB1-induced protein secretion is more pronounced in MCF10A cells; therefore, the signaling is propagated through conditioned media (bystander effect) more effectively in MCF10A cells than in MCF7 cells.ConclusionsEstrogen receptor-positive breast cancer patients may benefit from high levels of TGFB1 expression due to the repression of estrogen receptor signaling, inhibition of proliferation, and induction of apoptosis in cancer cells. However, some TGFB1-stimulated cells may undergo EMT, which increases the risk of metastasis.

Acute myocardial infarction leads to distinct sex differences in the coding transcriptome of human monocytes with a potential impact in disease aetiopathology

Abstract Background Immune responses are intricately linked to human disease, not only during pathogenic infections but also in sterile injury events such as in cardiovascular disease. Atherosclerosis is the main driver leading to coronary artery disease (CAD) and subsequent development of acute myocardial infarction (AMI) and heart failure. The vital role of monocytes in the pathophysiology of atherosclerosis is well established. In the onset of AMI, monocytes act as first responders, both in the inflammatory and tissue repair phases. It is now apparent that sex-specific differences in the inflammatory responses leading to AMI impact the severity and outcome of patients. Yet, the immune responses during AMI have been under-investigated in women, often under-represented in clinical studies, and more research in this topic is required. Purpose Identification of sex-specific differences in the coding transcriptome of monocytes subpopulations in the onset of AMI compared to matched healthy subjects and CAD patients. Methods We isolated monocytes from male and female AMI patients, CAD patients and healthy controls. FACS sorting of monocytes (CD14 and CD16) resulted into classical, intermediate, and non-classical subpopulations. We performed ribosomal depletion and bulk Illumina RNA sequencing for each monocyte subpopulation. Differentially expressed genes (DEG) were calculated using specific R packages. Pathway enrichment analysis was performed with Gene set enrichment analysis (GSEA). Other computational tools were used to determine enrichment of genes linked to GWAS identified in cardiovascular diseases or to infer pathway responsive genes upon perturbation (Progeny). Network comparison and visualization were performed using Cytoscape platform. Results Whereas observing a clear overlap of most pathways such as homeostasis, regulation of signalling cascades and inflammatory response, we also identified other pathways contrasting strongly between both sexes. In fact, TGF beta/ SMAD signalling, wound healing and epithelial to mesenchymal transition are increased in female cells. On the contrast the more pro-inflammatory BMP/SMAD signalling is increased in male cells accompanied by decreased respiratory electron transport and translation. Additionally, when comparing our data to signatures identified in trauma patients, we observe clear mapping of our gene sets, which are upregulated in AMI, with the first 3 trauma associated signatures, whereas one of the signatures associated to homeostasis associates clearly with our CAD data as well as the healthy subjects. Conclusions Human monocytes display a specific coding transcriptome and certain pathways are differentially expressed in AMI and differ greatly between male and female cohorts. Interestingly, our DEG map closely to trauma associated signatures, highlighting potential similarities in disease aetiopathology between trauma and AMI patients.

Stretched Vascular Endothelial Cells Polarized Neutrophils to N2 Type via TRPC1-IL13-STAT3 Axis.

VECs play a crucial role in regulating the function of neutrophils, which is essential for immune responses and inflammation. As stretch-sensitive cells, VECs sense mechanical stretch through surface mechanoreceptors, converting external mechanical stimuli into biochemical signals. This study aimed to explore the molecular mechanisms underlying the regulation of neutrophil behavior by stretched VECs. The key cytokine-inducing neutrophil N2 polarization in the conditioned medium from stretched vascular endothelial cells (CM-stretch) was validated through multifactorial matrix and flow cytometry. Additionally, the molecular mechanism underlying the response of vascular endothelial cells to stretch was systematically verified through layer-by-layer analysis using WB. IL13, not IL4, was ultimately identified as a key cytokine-inducing neutrophil N2 polarization in CM-stretch. Inhibition of the transient receptor potential channel (TRPC1) and siRNA-mediated knockdown of TRPC1 both significantly decreased IL13 production. Furthermore, neutralizing IL13 in the CM-stretch or inhibiting STAT3 phosphorylation inhibited neutrophil N2 polarization, as evidenced by reduced CD206 and VEGFA expression. These results demonstrate that stretched VECs initiate a signaling cascade that induces neutrophil N2 polarization through the TRPC1-IL13-STAT3 axis, suggesting that mechanical stretching of VECs could shift neutrophil function from a pro-inflammatory to a more regulatory and healing role.

Anti-Diabetic Effects of Oleuropein

Background/Objectives: Oleuropein, a secoiridoid polyphenol found in olive oil as well as the fruit and leaves of the olive tree, has been reported to have antioxidant, cardioprotective, anti-inflammatory, anti-cancer, and anti-diabetic properties. Type 2 diabetes mellitus (TD2M) is a chronic metabolic disease characterized by impaired insulin action, termed insulin resistance. The development of T2DM is closely associated with obesity and chronic low-grade inflammation. In recent years, a rise in sedentary lifestyles and diets rich in refined carbohydrates and saturated fats has contributed to an increase in the prevalence of obesity and TD2M. Currently, the strategies for treating T2DM and its prevention lack efficacy and are associated with adverse side effects. Hence, there is an urgent need for novel treatment strategies, including naturally occurring compounds possessing hypoglycemic and insulin-sensitizing properties. Methods: This review summarizes the evidence of the anti-inflammatory and anti-diabetic properties of oleuropein from in vitro and in vivo animal studies, as well as the available clinical trials. Results: The existing evidence indicates that oleuropein may exert its anti-inflammatory effects by downregulating the levels of pro-inflammatory cytokines in hepatic and adipose tissue. Additionally, the evidence suggests that oleuropein targets skeletal muscle and enhances glucose uptake and its related protein signalling cascades, improving glucose tolerance and insulin sensitivity. Conclusions: Despite the evidence of oleuropein’s anti-inflammatory and anti-diabetic potential, more animal and clinical studies are needed to proceed towards its clinical/therapeutic use for metabolic diseases confidently.

Development of a Desorption Electrospray Ionization-Multiple-Reaction-Monitoring Mass Spectrometry (DESI-MRM) Workflow for Spatially Mapping Oxylipins in Pulmonary Tissue.

Oxylipins are a class of low-abundance lipids formed via oxygenation of fatty acids. These compounds include potent signaling molecules (e.g., octadecanoids, eicosanoids) that can exert essential functions in the pathophysiology of inflammatory diseases including asthma. While some oxylipin signaling cascades have been unraveled using LC-MS/MS-based methods, measurements in homogenate samples do not represent the spatial heterogeneity of lipid metabolism. Mass spectrometry imaging (MSI) directly detects analytes from a surface, which enables spatial mapping of oxylipin biosynthesis and migration within the tissue. MSI has lacked the sensitivity to routinely detect low-abundance oxylipins; however, new multiple-reaction-monitoring (MRM)-based MSI technologies show increased sensitivity. In this study, we developed a workflow to apply desorption electrospray ionization coupled to a triple quadrupole mass spectrometer (DESI-MRM) to spatially map oxylipins in pulmonary tissue. The targeted MSI workflow screened guinea pig lung extracts using LC-MS/MS to filter oxylipin targets based on their detectability by DESI-MRM. A panel of 5 oxylipins was then selected for DESI-MRM imaging derived from arachidonic acid (TXB2, 11-HETE, 12-HETE), linoleic acid (12,13-DiHOME), and α-linolenic acid (16-HOTrE). To parse this new data type, a custom-built R package (quantMSImageR) was developed with functionality to label regions of interest as well as quantify and analyze lipid distributions. The spatial distributions quantified by DESI-MRM were supported by LC-MS/MS analysis, with both indicating that 16-HOTrE and 12-HETE were associated with airways, while 12,13-DiHOME and arachidonic acid were mapped to parenchyma. This study realizes the potential of targeted MSI to routinely map low-abundance oxylipins with high specificity at scale.

The Neglected Suppressor of Cytokine Signalling (SOCS): SOCS4-7.

SOCS proteins are essential for the regulation of oncogenic, anti-pathogenic, and proinflammatory signalling cascades, including the JAK/STAT and NF-kB pathways, where they act as negative feedback regulators. Given their powerful role in a broad spectrum of biological processes, it is surprising that the functions of many SOCS proteins have not been widely explored. While the mechanisms of action of CIS, SOCS1-3 are well-documented, information regarding SOCS4-7 remains limited. However, recent studies have begun to elucidate the regulatory functions of these proteins during infection and disease, such as influenza infection, cancer and diabetes. Therefore, this review aims to describe and discuss studies detailing our current understanding of SOCS4-7, painting a clearer picture of the biological processes these regulatory proteins maintain. Indeed, our review highlights important evidence proving that all SOCS play a role in biological processes that are essential for normal immunological homeostasis, clearance of infection and avoidance of disease. Understanding how SOCS proteins interact with other proteins or how they are dysregulated in disease is likely to provide valuable insights for advancing therapeutic approaches.

Dopamine internalization via Uptake2 and stimulation of intracellular D5-receptor-dependent calcium mobilization and CDP-diacylglycerol signaling

Dopamine stimulates CDP-diacylglycerol biosynthesis through D1-like receptors, particularly the D5 subtype most of which is intracellularly localized. CDP-diacylglycerol regulates phosphatidylinositol-4,5-bisphosphate-dependent signaling cascades by serving as obligatory substrate for phosphatidylinositol biosynthesis. Here, we used acute and organotypic brain tissues and cultured cells to explore the mechanism by which extracellular dopamine acts to modulate intracellular CDP-diacylglycerol. Dopamine stimulated CDP-diacylglycerol in organotypic and neural cells lacking the presynaptic dopamine transporter, and this action was selectively mimicked by D1-like receptor agonists SKF38393 and SKF83959. Dopaminergic CDP-diacylglycerol stimulation was blocked by decynium-22 which blocks Uptake2-like transporters and by anti-microtubule disrupters of cytoskeletal transport, suggesting transmembrane uptake and guided transport of the ligands to intracellular sites of CDP-diacylglycerol regulation. Fluorescent or radiolabeled dopamine was saturably transported into primary neurons or B35 neuroblastoma cells expressing the plasmamembrane monoamine transporter, PMAT. Microinjection of 10-nM final concentration of dopamine into human D5-receptor-transfected U2-OS cells rapidly and transiently increased cytosolic calcium concentrations by 316%, whereas non-D5-receptor-expressing U2-OS cells showed no response. Given that U2-OS cells natively express PMAT, bath application of 10 μM dopamine slowly increased cytosolic calcium in D5-expressing cells. These observations indicate that dopamine is actively transported by a PMAT-implicated Uptake2-like mechanism into postsynaptic-type dopaminoceptive cells where the monoamine stimulates its intracellular D5-type receptors to mobilize cytosolic calcium and promote CDP-diacylglycerol biosynthesis. This is probably the first demonstration of functional intracellular dopamine receptor coupling in neural tissue, thus challenging the conventional paradigm that postsynaptic dopamine uptake serves merely as a mechanism for deactivating spent or excessive synaptic transmitter.

GmWRKY33A positively regulates disease resistance in soybean (Glycine max)

The WRKY transcription factor gene family is a plant-specific transcription factor that plays important roles defense responses. Studies in model plant Arabidopsis demonstrated that WRKYs function downstream of mitogen activated-protein kinase (MAPK) signaling cascade and participate in defense responses through activating the expression of defense-related genes. However, the roles of WRKYs in defense responses have not been previously investigated in paleopolyploidy soybean. Bioinfomatic analysis revealed that there are three pair of GmWRKY33 genes in the soybean genome. The identity of first two pair of GmWRKY33 genes is greater than 84% (named as GmWRKY33A). The identity of genes within the same pair is greater than 95%. A 300 bp fragment highly homologous to these four GmWRKY33A was chosen to clone into bean pod mosaic virus (BPMV)-based silencing vector (BPMV-VIGS) to achieve the goal of silencing four GmWRKY33A genes simultaneously. In this study, we simultaneously silenced four homologous genes of GmWRKY33A using a bean pod mottle virus (BPMV) vector carrying a single fragment of GmWRKY33A. Comparing the silenced plants with the vector control plants, no evident morphological phenotypes were observed. However, the GmWRKY33A-silenced plants exhibited significantly reduced resistance to Pseudomonas syringae pv. glycinea (Psg), Xanthomonas axonopodis pv. glycine (Xag), as well as to soybean mosaic virus (SMV). Furthermore, we demonstrated that silencing these GmWRKY33A genes significantly inhibited the activation of GmMPK3/GmMPK6 induced by Psg infection. Collectively, our results suggest that GmWRKY33As are involved in soybean immunity through regulating the transcription of GmMPK3/6 genes or activating the kinase activities of GmMPK3/6. Taken together, our results demonstrated that GmWRKY33As are positive regulators of soybean immune responses.

Design and development of dual targeting CAR protein for the development of CAR T-cell therapy against KRAS mutated pancreatic ductal adenocarcinoma using computational approaches

Mutant KRAS promotes the proliferation, metastasis, and aggressiveness of various cancers including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal adenocarcinoma (CRC) respectively. Mutant KRAS therapeutics are limited, while Sotorasib and Adagrasib were the only FDA-approved drugs for the treatment of KRASG12C mutated NSCLC. Chimeric antigen receptor (CAR) T-cell therapy has been emerged as an effective strategy against hematological malignancies and being extended towards solid cancers including PDAC. mesothelin (MSLN) and Carcinoembryonic Antigen (CEA) were reported to be highly overexpressed in KRAS-mutated PDAC. Meanwhile, in clinical trials, several CAR T-cell therapy studies are mainly focused towards these two cancer antigens in PDAC, however, the dual targeting of these two neoantigens is not reported. In the present study, we have designed and developed a novel dual-targeting CAR protein by employing various bioinformatics approaches such as functional analysis (antigenicity, allergenicity, antigen binding sites & signalling cascades), qualitative analysis (physicochemical, prediction, refinement & validation of 2D and 3D structures), molecular docking, and in silico cloning. Our results revealed that the designed CAR protein specifically binds with both MSLN & CEA with significant binding affinities, and was predicted to be stable & non-allergenic. Additionally, the protein–protein interaction network reveals the T-cell mediated antitumor responses of each domain in the designed CAR. Conclusively, we have designed and developed a dual targeting (MSLN & CEA) CAR protein towards KRAS-mutated PDAC using computational approaches. Alongside, we further recommend to engineer this designed CAR in T-cells and evaluating their therapeutic efficiency in in vitro and in vivo studies in the near future.

Integrating network pharmacology, molecular docking, and experimental validation to reveal the mechanism of Radix Rehmanniae in psoriasis.

Radix Rehmanniae (RR) plays an important role in treating psoriasis. However, the active compounds of RR and potential mechanisms are unclear. The current study was designed to investigate the potential active ingredients, targets, and mechanisms of RR in treating psoriasis through network pharmacology, molecular docking, and vitro experiments. Initially, the TCMSP database and literature retrieval were used to access the active ingredients of RR. The psoriasis target proteins were obtained from Therapeutic Target Database, OMIM, GeneCards, and DrugBank databases. The target proteins were then converted into target genes using Uniprot. Secondly, overlapping genes were obtained through Venn online tool. Then, protein-protein interactions network diagram is finished by STRING database. Next, Cytoscape software was used to acquire the top 10 hub proteins; gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis were then used to predict possible mechanisms. Afterwards, molecular docking validation of the active ingredients with the main targets was performed by AutoDock software. Finally, lipopolysaccharides induced RAW264.7, to assess the effects and molecular mechanisms by MTT, RT-qPCR, and Western blot assays. Overall, there are 20 effective compounds and 33 targets involved in biological processes including apoptosis, intracellular signaling, vasodilation, and mitogen-activated protein kinase (MAPK) signaling cascade. The docking results showed strong binding capacity between the active ingredients and targets. We verified aucubin as the key active ingredient, tumor necrosis factor α, and IL6 as the core targets, and focused on the p38MAPK protein pathway. Cellular experiments showed that aucubin down-regulated the phosphorylated p38MAP protein and reduced the expression of tumor necrosis factor α mRNA, IL6 mRNA, and IL1βmRNA. In summary, RR is featured with multicomponent, multi-target, and multi-pathway in treating psoriasis; the preliminary mechanism may be associated with the down-regulation of p38MAPK phosphorylation and curbing the expression of inflammatory factor by aucubin. This paper provides the scientific basis for Traditional Chinese medicine treating psoriasis.

Functions and mechanisms of CDPKs in plant responses to abiotic stress

Calcium-dependent protein kinases (CDPKs/CPKs) are members of the Ca2+-sensitive Ser/Thr protein kinase family and play a crucial role in plant growth and development and responses to abiotic stress. CDPKs are capable of rapidly sensing changes in intracellular Ca2+ signals and recognizing and phosphorylating specific substrates, thereby transmitting and amplifying Ca2+ signal cascades downstream. They are involved in plant responses to stress conditions such as drought, saline-alkali stress, and injuries and regulate plant growth and development, gene expression, ion channel activity, and stomatal movement. The autophosphorylation of CDPKs can affect their activities and substrate specificity. CDPKs have the ability to bind to and phosphorylate multiple substrates. In addition to participating in respiratory burst oxidase homolog (RBOH), mitogen-activated protein kinase (MAPK), and plant hormone signaling pathways, CDPKs can also bind to 14-3-3 proteins, which enables the regulation of plant responses to stress and promotes plant growth and development. This paper summarized the research findings on the discovery, structure, classification, and roles of CDPKs in plant responses to stress and proposed the future research directions, aiming to provide the genetic resources and a theoretical basis for improving the stress tolerance of crops.

Identifying Herbal Candidates and Active Compounds for Psoriasis Through Multiscale Network Analysis

Psoriasis is a chronic inflammatory skin disorder characterized by the hyperproliferation of keratinocytes and immune system dysregulation, with significant needs due to the limitations and adverse effects of current treatments. In this study, we sought to discover novel herbal candidates and their active compounds for psoriasis by leveraging a multiscale network analysis. We conducted a comprehensive analysis of data from 348 medicinal herbs and their active compounds, identifying Piperis longi fructus, Pini koraiensis semen, Schisandrae fructus, and Cnidi fructus as top candidates without reported evidence. Key active compounds, such as piperine, piperlongumine, α-humulene, schizandrin A, schizandrin II, and torilin, were prioritized for their ability to target psoriasis-associated proteins, including STAT3, TNF, IL-6, and NF-κB. These compounds are involved in the modulation of critical inflammatory pathways, notably the MAPK signaling cascade, which plays a central role in psoriasis pathogenesis. Our findings suggest that these herbal compounds may not only mitigate inflammation but also regulate keratinocyte hyperproliferation, addressing fundamental mechanisms underlying the disease. This approach highlights the utility of multiscale network analysis in identifying promising natural therapies, offering new insights and potential avenues for safer and more effective psoriasis management.

Functional analysis of TkWRKY33: A key regulator in drought-induced natural rubber synthesis in Taraxacum kok-saghyz

WRKY proteins, which form a transcription factor superfamily that responds to jasmonic acid (JA) signals, regulate various developmental processes and stress responses in plants, including Taraxacum kok-saghyz (TKS). TKS serves as an ideal model plant for studying rubber production and lays the foundation for a comprehensive understanding of JA-mediated regulation of natural rubber synthesis. In the present study, we screened and identified a valuable transcription factor, TkWRKY33, based on transcriptome data from TKS in response to JA. We investigated its role in the regulation of natural rubber synthesis within the JA signaling pathway and its function in response to drought stress. Through protein-protein interactions and transcriptional regulation analysis, we found that TkWRKY33 may regulate natural rubber synthesis through the JA-TkMPK3-TkWRKY33-(TkGGPS5/TkACAT8) cascade pathway, possibly by participating in JA-activated mitogen-activated protein kinase (MAPK) signaling. Overexpression of TkWRKY33 in tobacco, along with functional analysis of drought resistance and comparative analysis of natural rubber content after drought stress, revealed that TkWRKY33 not only enhances plant drought resistance by regulating the expression of genes related to reactive oxygen species (ROS) scavenging through the JA signaling pathway, but also has a close relationship with the signal transduction pathway mediated by the JA hormone in regulating natural rubber synthesis.The TkWRKY33 is recognized as a valuable transcription factor, which likely plays a role in regulating natural rubber biosynthesis through the JA-activated MAPK cascade signaling pathway JA-TkMPK3-TkWRKY33-(TkGGPS5/TkACAT8).