ETHNOPHARMACOLOGICAL RELEVANCE:Conventional antidepressant therapy necessitates prolonged medication, which can result in a plethora of adverse effects, including osteoporosis. Eucommiae Folium (EF) has been demonstrated to alleviate depressive symptoms and osteoporosis. Gardeniae Fructus (GF) also has remarked antidepressant potential, which is often used together with EF as a herb pair in the clinics. This herb pair is highly valuable in synergistically and safely exerting antidepressant effects. AIM OF THE STUDY:PACAP is a novel antidepressant target, via enhancing neuroplasticity. Here, we aimed to examine the synergistic effects of EF and GF (EG) on depression, and test underlying mechanisms of PACAP and its downstream signaling. MATERIALS AND METHODS:The antidepressant effects of EG were confirmed in both CUMS and LPS depression models, with fluoxetine as a positive control. Network pharmacology analysis was utilized to reveal the associated mechanisms. The key signaling mechanisms were examined with immunoblotting and immunofluorescence, and further verified by knockdown of PACAP with virus-mediated RNAi. RESULTS:EG exhibited synergistic efficacy in antidepressant response. EG alleviated depressive and anxiety symptoms in both the CUMS and LPS models, with effects comparable to fluoxetine. Network pharmacology analysis revealed the important role of PI3K/AKT/mTOR signaling pathway, which was verified in the hippocampus following EG treatment. Moreover, the expression of the potential upstream activator, PACAP, was also upregulated, in addition to enhanced the expressions of the synaptic proteins PSD95 and Synapsin-1. EG reduced the number of Iba-1+ microglial cells and their cell areas, and increased the number of BDNF+ cells in the hippocampus. Importantly, hippocampal PACAP knockdown blunted the antidepressant effects of EG and the PI3K/AKT/mTOR signaling. CONCLUSIONS:The herb pair of Eucommiae Folium and Gardeniae Fructus synergistically elicited antidepressant effects, likely via activating hippocampal PACAP and its downstream PI3K/AKT/mTOR signaling.

E2F transcription factors are key regulators of the cell cycle and play an important role in tumorigenesis. N6-methyladenosine (m6A), the most prevalent internal RNA modification in eukaryotic cells, is also crucial for cancer progression. However, the interplay between E2F family members and m6A modification in glioblastoma multiforme (GBM) remains poorly understood. We applied weighted gene co-expression network analysis (WGCNA) to identify m6A-related gene modules co-expressed with E2F7, a gene that is significantly associated with adverse prognosis and tumor stemness. Through intersection analysis, eukaryotic translation initiation factor 3 subunit B (EIF3B) emerged as a hub gene, showing a strong positive correlation with E2F7 in GBM datasets. Functional validation was performed in GBM cells to assess the regulatory relationship between EIF3B and E2F7, including examination of mRNA and protein expression, mRNA stability, and m6A modification levels. GBM stemness was evaluated by tumor sphere formation assays and detection of stemness-associated markers. Functional validation revealed that EIF3B knockdown reduced E2F7 mRNA and protein expression, promoted E2F7 mRNA degradation, and decreased its m6A modification, suggesting an association between EIF3B and m6A-related regulation of E2F7 stability. Furthermore, EIF3B knockdown markedly suppressed GBM stemness, as demonstrated by impaired tumor sphere formation and downregulation of stemness-associated markers. Clinically, EIF3B expression increased with glioma grade and correlated with poor prognosis. Collectively, these findings suggest that the EIF3B-E2F7 axis is associated with m6A-related regulation in GBM and may warrant further investigation as a candidate for therapeutic targeting.

Glioblastoma (GBM) is the most aggressive primary malignancy of the central nervous system, and acquired resistance to temozolomide (TMZ) is a major cause of tumor recurrence and poor clinical outcomes. However, the tumor-intrinsic drivers and metabolic adaptations underlying this process remain incompletely understood. In this study, we integrated transcriptomic data from established TMZ-sensitive and TMZ-resistant models with public bulk and single-nucleus sequencing datasets, and validated candidate genes using clinical specimens, cell-based drug-sensitivity assays, xenograft models, metabolic measurements, luciferase reporter assays, chromatin immunoprecipitation, and small-molecule binding assays. Immunoglobulin superfamily member 3 (IGSF3) was consistently upregulated in TMZ-resistant cells, resistant xenografts, and recurrent GBM samples, and was mainly enriched in malignant glioma cells. Functionally, IGSF3 overexpression promoted resistance to TMZ-related treatment, whereas IGSF3 knockdown restored drug sensitivity. Mechanistically, IGSF3 was detected in the nucleus, where it enhanced the activity of the asparagine synthetase (ASNS) promoter. In addition, IGSF3 was found to be enriched at the ASNS promoter, thereby increasing ASNS expression and asparagine production, reduced reactive oxygen species accumulation, preserved glutathione redox balance, and limited DNA damage induced by treatment. ASNS gain- and loss-of-function rescue experiments showed that ASNS was required for the pro-resistance effect of IGSF3. Finally, Tucatinib bound to IGSF3, suppressed the IGSF3-ASNS pathway, and enhanced TMZ efficacy in vitro and in vivo. These findings identify the IGSF3-ASNS axis as a tumor-intrinsic metabolic adaptation that drives acquired TMZ resistance in GBM and suggest a potential therapeutic strategy for recurrent GBM.

Temozolomide (TMZ) remains the first-line therapy for glioblastoma (GBM) patients. However, the mechanisms underlying the emergence of acquired TMZ resistance after treatment are unclear. Here, we reveal the critical role of macrophage phagocytosis in GBM recurrence and acquired TMZ resistance. Mechanistically, TMZ treatment sustained GSK3β activation and promoted its interaction with DNMT1. This led to the phosphorylation of DNMT1 at the previously unrecognized S977 site for its K981-dependent ubiquitination and destabilization. The downregulation of DNMT1 leads to hypomethylation of the CD47 promoter and increases the expression of CD47, a key inhibitor of macrophage phagocytosis. Elevated CD47 expression suppresses macrophage phagocytosis and promotes the survival of TMZ-treated GBM cells. The small molecule WIN 51708 disrupts the p-GSK3β-Y216-DNMT1 interaction, which stabilizes DNMT1, decreases CD47 expression, restores phagocytosis in vivo, and resensitizes tumors to TMZ. The GSK3β-DNMT1-CD47 axis was found to be conserved in a TMZ-resistant PDX model and in samples from patients with recurrent GBM, supporting its clinical translational value. Our findings underscore the potential of the combined administration of WIN 51708 and TMZ as a strategy to resensitize GBM tumors.

Major Depressive Disorder (MDD) is a common mental illness, for which current western pharmacotherapies often suffer from limited targets, delayed onset of action, and high relapse rates. Traditional chinese medicine (TCM) have potentials in multi-target regulation for improving the treatment outcome. A great number of classic TCM herbal formulas such as Yueju Pill and Chaihu Shugan San, as well as their active ingredients, can alleviate depressive symptoms via mechanisms such as neuroplasticity, inflammatory response, and tryptophan metabolism. The TCM "liver" organ is more functional and integrative, with "dispersing and discharging" characteristic. It is proposed that stagnation due to liver's dispersing and discharging dysfunction may lead to depression, and thus "soothing the liver and relieving stagnation" is critical for the treatment. Previous studies have shown the involvement of liver regulation of metabolism of tryptophan or bile acid, immunity in depression. An increasing number of evidence indicates the important role of epigenetics in depression, which is contingent on the metabolic products by liver. Here the research progress on liver-brain epigenetic axis and its connection with immune system for the development and treatment of depression is reviewed. Epigenetic mechanisms include DNA/RNA methylation, histone modification, and non-coding RNA regulation. The metabolic products by liver, including factors such as S-adenosylmethionine (SAM), provide the doner for epigenetic modification. Immune system is profoundly involved for the liver-brain epigenetic axis. Emerging studies of TCM treatment of depression start to investigate the mechanism. Further exploration of the liver-brain epigenetic axis and its interaction to immune systems in regulation of depression are discussed.

ETHNOPHARMACOLOGICAL RELEVANCE:Insomnia and anxiety are highly comorbid, severely compromising quality of life. Efficacy of current pharmacological interventions for this dual condition remains limited. Zhi-Gan Formula (ZG), consisting of Zhi-Zi-Chi Decoction and Ganmai Dazao Decoction, two classic Traditional Chinese Medicine (TCM) formulae clinically widely used for insomnia or anxiety, holds promise as a therapeutic option for insomnia-anxiety comorbidity. AIM OF THE STUDY:This study aimed to assess ZG's sleep-promoting and anxiolytic efficacy, and investigate the novel mechanism through which pituitary adenylate cyclase-activating polypeptide (PACAP) in the medial prefrontal cortex (mPFC) modulates comorbid sleep and anxiety conditions. MATERIALS AND METHODS:Mice received 4-chloro-DL-phenylalanine (PCPA) injections and were subsequently administered ZG or diazepam. Behaviors were assessed using the pentobarbital-induced sleep test, open-field test (OFT), and elevated plus-maze test (EPM). Key pathways were identified via network pharmacology analysis and validated using long-term potentiation (LTP) recordings and protein quantification. Viral-mediated PACAP knockdown vectors were transfected into the mPFC. RESULTS:PCPA administration induced insomnia and anxiety-like behaviors. ZG administered for 3 days significantly shortened sleep latency, prolonged sleep duration, and alleviated anxiety-like behaviors, whereas diazepam only partially improved anxiety-like behaviors. Network pharmacology analysis suggested ZG's engagement in neuropeptide-receptor interactions and synaptic transmission pathways. Assessments of synaptic plasticity showed that ZG improved mPFC LTP and the expression of synaptic proteins (PSD95, synapsin-1, BDNF) impaired in the model mice. Moreover, the expression of the neuropeptide PACAP and downstream eEF2 signaling for synaptic protein synthesis were all improved by ZG. Crucially, perfusion of a PACAP agonist in the mPFC brain slices from sleep-deprived mice rescued LTP deficits. Finally, mPFC PACAP knockdown abolished the therapeutic effects and the enhanced expressions of the synaptic proteins by ZG. CONCLUSIONS:ZG alleviated insomnia-anxiety comorbidity by restoring synaptic plasticity in the mPFC via the PACAP-eEF2-BDNF pathway, which may also shed light on the development of a novel therapeutic approach for the treatment of sleep-anxiety comorbidity.

Background To explore the pharmacological mechanism of the antidepressant-like effects of Ganmaidazao-Shanzha Decoction (GMS) through network pharmacological analysis and experimental verification. Methods The active ingredient and the target of action related to depression were clarified by searching the herb group identification, GeneCards and Drugbank, Pharmgkb, CTD, Disgenet databases, and the protein interaction relationship was obtained by using the String database, and the protein interaction network map was constructed by using Cytoscape software. We also performed gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of key targets of the antidepressant-like effects of GMS and predicted the core targets and pathways. Furthermore, BDNF pathway which involved in antidepressant-like effect of GMS was validated by mice model to validate the results of partial network pharmacology analysis. Results A total of 276 active ingredients and 1142 related targets of action were retrieved from GMS, and a total of 2164 common targets were obtained by mapping the obtained depression disease targets and drug targets. Protein interaction network analysis indicated that targets including AKT1, SRC, HSP90AA1, PRKACA, PIK3R1, PIK3CA and other targets may be the core targets. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the treatment of depression by GMS might include phosphorylation, synaptic plasticity and growth factor stimulus processes, and the signaling pathways might include AGE-RAGE signaling pathway in diabetic complications, Neurotrophin signaling pathway, and PI3K-Akt signaling pathway. The results of the in vivo tests showed that GMS could reverse the depressive behaviors induced by corticosterone in mice. The activity of GMS against depression was found to be mediated through neurotrophin signaling pathway by targeting the Brain-derived neurotropic factor (BDNF) signaling proteins and downstream synaptic proteins, promoting the c-fos positive cell restoration and finally leading to hippocampal neurogenesis in age difference. Conclusions Through the application of network pharmacology and in vivo validation experiments, this study revealed the active components and the potential targets of GMS for the treatment of depression, providing a theoretical basis for the research and clinical application of GMS.

Cancer-related fatigue (CRF) is a common symptom in cancer patients, affecting their quality of life. Prunella vulgaris and its seed oil (PVSO) are edible-medicinal foods with bioactive compounds that may alleviate CRF. The present study aimed to test the effects of PVSO on CRF and underlying mechanisms. We found PVSO improved fatigue-like behaviors in both LPS-induced and tumor-bearing models. In the breast cancer tumor-bearing animals, PVSO reduced tumor growth, and suppressed serum and hypothalamic inflammatory cytokines (TNF-α, IL-1β, and IL-6) and NF-κB signaling. Furthermore, the hypothalamic corticotropin-releasing hormone (CRH) expression and serum CRH/cortisol levels dampened in tumor-bearing animals were normalized by PVSO. Our results suggest that PVSO exhibits potent anti-CRF effects likely via attenuation of cancer-induced hypothalamic inflammation and subsequent normalization of the dysregulated HPA axis. PVSO may be developed as a functional food for breast cancer-related fatigue.

Glioblastoma (GBM) is an aggressive malignant brain tumor, characterized by a poor prognosis and a limited response to chemoradiotherapy and immunotherapy. Increasing evidence indicates that the extracellular matrix (ECM), particularly collagen proteins, contributes to tumor progression and immune evasion. In this study, we identified COL1A2, COL6A2, COL8A1, and COL8A2 as survival-related genes that were overexpressed in GBM and significantly upregulated in short-term survivors. Subsequently, COL6A2 was verified to be associated with chemotherapy and immunosuppression. Functional assays demonstrated that COL6A2 promotes GBM cell proliferation, invasion, and chemoresistance. Cytometry by time-of-flight (CyTOF) and Tumor Immune Estimation Resource (TIMER) analysis revealed that high COL6A2 expression correlates with immunosuppressive features in the tumor microenvironment, particularly the accumulation of immature dendritic cells (DCs) and impaired cytotoxic T-cell activity. Mechanistically, COL6A2 silencing restored DCs' activation and enhanced the infiltration and function of effector immune cells. Our findings highlight COL6A2 as a key oncogenic and immunomodulatory ECM component in GBM and suggest that targeting collagen-mediated immune suppression may improve therapeutic outcomes in GBM patients.

Depression, marked by persistent low mood and anhedonia, poses significant global health challenges, whereas mainstream antidepressants like SSRIs often have delayed onset and limited efficacy. Yueju Pill, a traditional Chinese herbal medicine formulated 800 years ago to treat “stagnation syndrome”, which overlaps with depression. Yueju pill has been shown to have the rapid onset and sustained antidepressant potential since 2013. It has been revealed to have similarities to the prototype rapid antidepressant ketamine in neuroplasticity mechanisms, including instant stimulation of protein synthesis signaling in the hippocampus and prefrontal cortex, subsequently enhancing expressions of BDNF and synaptic proteins. More recently, some targets and compound substrates that were not known before have been revealed from Yueju pill, and some clinical evidence has been provided. This review will focus on the advances in the discovery of a novel target, the neuropeptide PACAP in the hippocampus for the onset of depression treatment, the study paradigm employed to identify the synergism of the composing compounds in Yueju pill targeting PACAP, the mechanisms of neuroinflammation and gut-brain axis, and clinical trials showing fast alleviation of depression symptoms by adjunct or monotherapy with Yueju pill, in relationship to the improvement in serum BDNF levels. The perspective for a broader use of Yueju pill as a therapeutic avenue for depression and further mechanistic and clinical research directions is also provided.

Real-time quantitative PCR (qPCR), droplet digital PCR (ddPCR), and CRISPR/Cas diagnostics typically rely on expensive, target-specific fluorescence probes or CRISPR RNA (crRNA) for precise nucleic acid detection, and their multiplexing capability is limited by the scarcity of fluorescent colors in fluorometric thermal cyclers. To overcome these limitations, we introduced Engineered Hairpin Cleavage Amplification (EHCA), a novel technique that is compatible with various platforms such as qPCR, ddPCR, CRISPR/Cas12a and melting curve analysis (MCA), utilizing universal fluorescence probes/crRNA. EHCA employs a unique mechanism in which Taq polymerase cleaves engineered hairpins to release secondary primers, thereby extending helper targets or fluorescence probes. EHCA, efficiently developed with the aid of computational tools, exhibited comparable detection sensitivity and precision to specific probe assays. Furthermore, EHCA-MCA demonstrated multiplexed detection capabilities by generating fluorescent double strands of different lengths (Tm) and colors. Detection of nucleic acids with high sensitivity was achieved using EHCA-MCA at annealing temperatures between 46 and 66 degrees C, highlighting the remarkable temperature-robustness. In evaluating 213 clinical samples for high-risk HPV genotyping, the 14-plex EHCA-MCA yielded a sensitivity of 92.2 %, specificity of 98.1 % and detection accuracy of 96.7 %. With its versatility, cost-effectiveness, simplicity, high sensitivity, and multiplexing capabilities, the EHCA strategy is anticipated to be widely utilized.

Ethnopharmacological relevance: Geniposide (GP) and shanzhiside methyl ester (SM) are the two important bioactive compounds in the classical traditional Chinese herbal medicine Yueju Pill, which is currently used as an over-the-counter (OTC) medicine in China. Yueju has been demonstrated with antidepressant-like effects with the prescriptional dose. As GP and SM both have antidepressant potential, the synergism of them could be crucial to the function of Yueju. Objectives: The neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) has been implicated in the onset of antidepressant-like response. Here we investigated the synergism of the chronic treatment with GP and SM, at proportional doses to Yueju, on antidepressant-like effects, and underlying mechanism of PACAPrelated signaling in a neuroinflammation-based depression model. Materials and methods: Depression-related behaviors were tested in the lipopolysaccharide (LPS)-induced depression model. The molecular signaling of neuroinflammation and neuroplasticity was investigated using Western blot analysis, immunofluorescence and pharmacological inhibition of mTOR signaling. Results: Chronic treatment of GP and SM (GS) at the dose which is proportional to the prescriptional dose of Yueju synergistically elicited antidepressant-like effects. Chronic treatment of the GS or the conventional antidepressant fluoxetine (FLX) showed antidepressant-like effects in LPS-injected mice. In vitro analysis indicated the synergism of GS on PACAP expression. In the hippocampus of LPS-injected mice, both GS and FLX enhanced PACAP expression, downregulated the inflammatory signaling of Iba-1/NF-& kcy;B/IL-1(3 and NLRP3, and upregulated the neuroplasticity signaling of mTOR-BDNF/PSD95. Additionally, both treatments reduced microglia activation indicated by Iba-1 immunofluorescent staining. Rapamycin, an mTOR inhibitor, blunted the antidepressant-like effects and the upregulation of BDNF expression induced by chronic GS. Conclusion: The antidepressant-like effects elicited by chronic fluoxetine or by synergistic doses of GS were involved in the upregulation of hippocampal PACAP levels, in association with ameliorated neuroinflammation and neuroplasticity signaling in LPS-injected mice. GS synergism may play a key part in the antidepressant-like effects of the prescriptional dose of Yueju.

BackgroundChemoresistance and recurrence following treatment are the greatest impediments to the prognosis of glioblastoma (GBM). Increasing evidence indicates that cancer-associated fibroblasts (CAFs) play a significant role in the progression of glioblastoma. Nevertheless, the role and source of CAFs in recurrent and chemotherapy-resistant GBMs still remain ambiguous.MethodsSpatial transcriptome (ST) sequencing was conducted on the tissue microarray encompassing primary and recurrent glioma samples in order to characterize the cellular composition. Subsequently, the infiltration of CAFs in our formerly established in vivo temozolomide (TMZ)-resistant model was inspected through immunohistochemical staining. Additionally, we carried out RNA-seq and label-free quantitation (LFQ) proteomics on HCMECs co-cultured with TMZ-sensitive (TMZ-S) or TMZ-resistant (TMZ-R) cells to explore the mechanism.ResultsThis investigation revealed that CAFs and astrocytes are enriched in recurrent GBM, and this phenotype is associated with the expression of extracellular matrix (ECM) proteins associated with COL1A1 and FN1 deposition. Further investigations revealed that tenascin-C (TNC) and filamin C (FLNC), which potentially mediate endothelial-to-mesenchymal transition (EndMT), are the predominant factors that induce the deposition of ECM proteins in the resistance-promoting microenvironment. Additionally, the natural product punicalin (PNC) was found to downregulate EndMT-related proteins, multidrug resistance-associated membrane proteins, and collagen-related proteins by targeting TNC and FLNC, thereby increasing the susceptibility of temozolomide (TMZ)-resistant cells to chemotherapeutic agents both in vitro and in vivo.ConclusionThese discoveries indicate that TNC and FLNC induced EndMT was a key resource of CAFs and targeting TNC and FLNC to inhibit EndMT and the collagen pathway is a promising tactic for reversing drug resistance in tumours. The development of combined chemotherapeutic strategies based on the features of tumour microenvironment endothelial cells and ECM deposition has high potential clinical value in increasing the efficacy of tumour treatment.

The detection of mutations in circulating tumor DNA (ctDNA) is challenging due to the significant fragmentation of ctDNA and the high prevalence of the wild-type template. Additionally, variant detection through qPCR is typically dependent on target-specific fluorescence probes, and no more than five targets can be identified in a single reaction due to the limited fluorescence colors in thermal cyclers. To address these limitations, we introduce the Dual-Role Mediator Blocker Amplification (DMBA) strategy, enabling sensitive and multiplex mutation detection without reliance on specific fluorescence probes. This strategy is applicable in both qPCR and melting curve analysis (MCA) platforms. The mediator blockers in DMBA play dual roles: enhancing discrimination between wild-type and mutant DNA and releasing mediator primers. These mediator primers extend the helper target and cleave universal fluorescence probes in qPCR, enabling the detection of mutations at variant allele fractions (VAFs) as low as 0.01%. The DMBA MCA method can identify multiple mutations, overcoming limitations in fluorescence channels by using mediator primers to extend universal fluorescence probes, producing fluorescent double strands with different T m's and colors. Multiplexed DMBA-MCA was developed to detect seven variants at 0.1-0.5% VAF in one tube. Our innovative method offers advantages including exceptional sensitivity, elimination of the requirement for specific fluorescence probes, shorter amplicons, and high multiplexing capacity, potentially revolutionizing clinical practice and precision medicine.

Fructus Aurantii, a Chinese herbal medicine, has been indicated to have antidepressant effects in our previous study. However, the main component and specific mechanisms of the antidepressant effects of Fructus Aurantii still need to be further revealed. This study aimed to explore the main antidepressant component of Fructus Aurantii and the underlying mechanisms of its antidepressant effects in the hippocampus. The results showed that the component of meranzin hydrate (MH) was enrichment in Fructus Aurantii. MH could alleviate depressive phenotypes in LPS-induced mice after a single administration 1 day later. High genetic and proteinic levels of caspase4 in the hippocampus in LPS-induced mice were reversed by MH after a single administration 1 day later. Moreover, MH was capable of relieving inflammatory factors (TNF-a and IL-1β) and LPS in the serum in LPS-induced mice. Subsequently, activation of hippocampal caspase4 blocked MH's antidepressant effects and its effects on suppression of microglia and improvement of astrocyte in the hippocampus. Furthermore, MH could increase long-term potential (LTP) in the hippocampal dentate gyrus (DG) and activation of hippocampal caspase4 blocked MH's enhancement on neuronal activities and synaptic plasticity in the hippocampal DG. To sum up, the antidepressant effects of a rich component MH in Fructus Aurantii suppressed the activation of caspase4 by maintaining glial cells function to promote neuronal activities and synaptic plasticity in the hippocampus.

Acetylation is critically required for p53 activation, though it remains poorly understood how p53 acetylation is regulated in glioblastoma (GBM). This study reveals that p53 acetylation is a favorable prognostic marker for GBM, regardless of p53 status, and that Smad1, a key negative regulator of p53 acetylation, is involved in this process. Smad1 forms a complex with p53 and p300, inhibiting p300's interaction with p53 and leading to reduced p53 acetylation and increased Smad1 acetylation in GBM. This results in enhanced tumor growth and resistance to chemotherapy, particularly in tumors with missense mutant p53. Acetylation of K373 is found to be essential for Smad1's oncogenic function but does not confer chemoresistance in the absence of p53. Through molecular docking, it is discovered that Smad1 and p53 both interact with the acetyltransferase domain of p300, but at different amino acid sites. Disturbing the interface of Smad1 through amino acid mutations abolishes the Smad1-p300 complex and promotes p53 acetylation. Therefore, a small molecule is identified through virtual screening that specifically disrupts the Smad1-p300 interaction, offering a promising strategy for inhibiting GBM and increasing chemosensitivity by inhibiting Smad1 acetylation and restoring p53 acetylation.

Background Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer, and chemoresistance poses a significant challenge to the survival and prognosis of GBM. Although numerous regulatory mechanisms that contribute to chemoresistance have been identified, many questions remain unanswered. This study aims to identify the mechanism of temozolomide (TMZ) resistance in GBM. Methods Bioinformatics and antibody-based protein detection were used to examine the expression of E2F7 in gliomas and its correlation with prognosis. Additionally, IC 50 , cell viability, colony formation, apoptosis, doxorubicin (Dox) uptake, and intracranial transplantation were used to confirm the role of E2F7 in TMZ resistance, using our established TMZ-resistance (TMZ-R) model. Western blot and ChIP experiments provided confirmation of p53-driven regulation of E2F7. Results Elevated levels of E2F7 were detected in GBM tissue and were correlated with a poor prognosis for patients. E2F7 was found to be upregulated in TMZ-R tumors, and its high levels were linked to increased chemotherapy resistance by limiting drug uptake and decreasing DNA damage. The expression of E2F7 was also found to be regulated by the activation of p53. Conclusions The high expression of E2F7, regulated by activated p53, confers chemoresistance to GBM cells by inhibiting drug uptake and DNA damage. These findings highlight the significant connection between sustained p53 activation and GBM chemoresistance, offering the potential for new strategies to overcome this resistance.

Background:The development of ketamine-like rapid antidepressants holds promise for enhancing the therapeutic efficacy of depression,but the underlying cellular and molecular mechanisms remain unclear.Implicated in depression regulation,the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is investigated here to examine its role in mediating the rapid antidepressant response.Methods:The onset of antidepressant response was assessed through depression-related behavioral paradigms.The signaling mechanism of PACAP in the hippocampal dentate gyrus (DG) was evaluated by utilizing site-directed gene knockdown,pharmacological interventions,or optogenetic manipulations.Overall,446 mice were used for behavioral and molecular signaling testing.Mice were divided into control or experimental groups randomly in each experiment,and the experimental manipulations included:chronic paroxetine treatments (4 d,9 d,14 d) or a single treatment of ketamine;social defeat or lipopolysaccharides-injection induced depression models;different doses of PACAP(0.4 ng/site,2 ng/site,4 ng/site;microinjected into the hippocampal DG);pharmacological intra-DG interventions(CALM and PACAP6-38);intra-DG viral-mediated PACAP RNAi;and opotogenetics using channelrhodopsins 2 (ChR2)or endoplasmic natronomonas halorhodopsine 3.0 (eNpHR3.0).Behavioral paradigms included novelty suppressed feeding test,tail suspension test,forced swimming test,and sucrose preference test.Western blotting,ELISA,or quantitative real-time PCR (RT-PCR) analysis were used to detect the expressions of proteins/peptides or genes in the hippocampus.Results:Chronic administration of the slow-onset antidepressant paroxetine resulted in an increase in hippocampal PACAP expression,and intra-DG blockade of PACAP attenuated the onset of the antidepressant response.The levels of hippocampal PACAP expression were reduced in both two distinct depression animal models and intra-DG knockdown of PACAP induced depression-like behaviors.Conversely,a single infusion of PACAP into the DG region produced a rapid and sustained antidepressant response in both normal and chronically stressed mice.Optogenetic intra-DG excitation of PACAP-expressing neurons instantly elicited antidepressant responses,while optogenetic inhibition induced depression-like behaviors.The longer optogenetic excitation/inhibition elicited the more sustained antidepressant/depression-like responses.Intra-DG PACAP infusion immediately facilitated the signaling for rapid antidepressant response by inhibiting calcium/calmodulin-dependent protein kinaseⅡ(CaM KⅡ)-eukaryotic elongation factor 2 (eEF2) and activating the mammalian target of rapamycin (mTOR).Pre-activation of CaMKⅡsignaling within the DG blunted PACAP-induced rapid antidepressant response as well as eEF2-mTOR-brain-derived neurotrophic factor (BDNF) signaling.Finally,acute ketamine treatment upregulated hippocampal PACAP expression,whereas intraDG blockade of PACAP signaling attenuated ketamine’s rapid antidepressant response.Conclusions:Activation of hippocampal PACAP signaling induces a rapid antidepressant response through the regulation of CaMKⅡinhibition-governed eEF2-mTOR-BDNF signaling.

Ethnopharmacological relevance Previous study has demonstrated lancao decoction (LC), a traditional Chinese medicine (TCM) fomula and recorded in “Huangdineijing”, has a therapeutic effect on cognitive impairment (early clinical manifestations of alzheimer's disease (AD), which suggests that LC may have potential therapeutic advantages for AD. Whether LC has the therapeutic effect on AD and its potential mechanisms were still further indicated. Aim of the study In this study, we aimed to uncover the potential advantage and neuronal mechanisms of LC in the treatment of AD in APP/PS1 mice in the hippocampus. Methods and materials We chose APP/PS1 mice to combing with behavioral tests including morris water maze (MWM) or y-maze to determine the role of LC in the therapeutic actions of AD. Network pharmacology was used to screen potential targets and pathways involving in LC's treatments of AD. Western blot was used to detect the phosphorylated expressions of proteins in hippocampus in APP/PS1 mice in the hippocampus. Pharmacological interventions were used to elucidate the relationship between the role of LC in the treatment of AD and the pathway, as well as the upstream and downstream interactions with neuronal activities. Results According to our previous LC effective dose (2.5 g/kg), the dose was also able to significantly reduce the latency to the platform, and significantly increase the number of crossing times and time spend in the target quadrant in APP/PS1 mice in MWM, which was consistent with donepezil (DON) after 14 days chronic treatments. Network pharmacology showed that PI3K/AKT and MAPK pathways were closely associated with LC's treatments of AD, and protein autophosphorylation played a role in this process. The phosphorylated expressions of PI3K and AKT were obviously reduced in APP/PS1 mice in the hippocampus, which were both reversed by LC or DON. The phosphorylated expressions of MAPK including P38, JNK and ERK were also significantly reduced in APP/PS1 mice hippocampus, but only the phosphorylated expression of ERK was reversed by LC or DON. Inhibiting the activities of PI3K/AKT pathway by LY294002 blocked LC's improvement of behavioral deficits in APP/PS1 mice, including reducing latency to platform and increasing the number of crossings time in MWM in APP/PS1 mice, which also blunted LC's up-regulated phosphorylated expressions of PI3K, AKT and ERK in the hippocampus. Moreover, suppressing the activities of ERK by PD98059 also blocked LC's improvement of AD-related behavioral deficits including decreasing latency to new arm and increasing time in new arm in y-maze test, which also inhibited LC's enhancement of synaptic proteins (PSD95 and synapsin1) in the hippocampus and the number of EGR1-positive cells in the hippocampal dentate gyrus (DG). Conclusions Take together, our study revealed that LC had the therapeutic effects on AD by activating the PI3K/AKT pathway to enhance ERK activity and further strengthened neuronal activities in the hippocampus.

Highly sensitive detection of gene variants holds immense significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a substantial challenge when identifying low-frequency mutant circulating tumor DNA (ctDNA) in cancer cases. Here, a novel technique called hairpin amplicon cleavage PCR (HAC-PCR) for detecting low-level DNA mutations with exceptional sensitivity was presented. What sets HAC-PCR apart is its ability to achieve high sensitivity independently of strand-displacement, base modification, or the use of additional reagents. The discrimination between wild-type targets (WT) and mutant targets (MT) is significantly improved by introducing uniquely designed hairpin structures. These hairpins hinder the amplification of WT and are cleaved during the elongation of MT. For the first time, the unique mechanism of hairpin amplicon cleavage by Taq polymerase was exploited for mutation identification. The optimized HAC-PCR consistently demonstrates the capability to discriminate mutant DNA at levels as low as 0.01% variant allele fraction (VAF) for five mutation types. Furthermore, this impressive sensitivity of 0.01% VAF has also been achieved in a multiplex assay. When applied to the testing of tissue DNA from 24 patients with thyroid cancers and plasma ctDNA from 12 patients with non-small cell lung cancers (NSCLCs), HAC-PCR exhibited a 100% concordance rate with the existing commercial kit and droplet digital PCR (ddPCR). Therefore, HAC-PCR stands as a cost-effective and highly effective tool for variant identification in clinical testing.