Characterization of Ferroptosis-Related Molecular Subtypes with Immune Infiltrations in Neuropathic Pain.

Background: Neuropathic pain (NP) caused by a lesion or disease of the somatosensory nervous system is a common chronic pain condition that has a major impact on quality of life. However, NP pathogenesis remains unclear. The purpose of this study was to identify differentially expressed genes (DEGs) and specific and meaningful gene targets for the diagnosis and treatment of NP. Methods: Data from rat spinal nerve ligations and the sham group were downloaded from the Gene Expression Omnibus (GEO) database. Based on the single-sample gene set enrichment analysis (ssGSEA) method, 29 immune gene sets were identified in each sample, and these samples were correlated with the immune infiltration phenotype. LASSO regression modeling was used to screen key genes to identify diagnostic gene markers. According to GSEA and GSVA, NP is concentrated in a large number of immune-related pathways and genes. Additionally, we used the DGIdb database and correlation test to construct gene-drug and transcription factor interaction networks for differentially expressed genes relevant to NP-related ferroptosis. We used WGCNA to identify gene co-expression modules of NP, and explored the relationship between gene networks and phenotypes. Finally, we crossed core genes with diagnostic markers and analyzed gene correlation with molecular subtypes and immune cells. Results: We identified 224 DEGs, including 191 upregulated genes and 33 downregulated genes. APC co-stimulation, CCR, cytolytic activity, humid-promoting, neutrophils, NK cells, and RGS4, CXCL2, DRD4 and other 7 genes related to ferroptosis were involved in NP development. Key genes of RGS4 and HIF-1 signaling pathway were screened. Conclusion: This study contributes to our understanding of the neuroimmune mechanism of neuropathic pain, provides a reference for NP biomarkers and drug targets. Ferroptosis may be the next research direction to explore NP mechanism.

Systematic analysis of the potential off-target activities of osimertinib by computational target fishing.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor used to treat non-small cell lung cancer. However, its off-targets are obscure, and systematic analysis of off-target activities remains to be performed. Here, we identified the off-targets of osimertinib using PharmMapper and DRAR-CPI and analyzed the intersected targets using the GeneMANIA and DAVID servers. A drug-target-pathway network was constructed to visualize the associations. The results showed that osimertinib is associated with 31 off-targets, 40 Kyoto Encyclopedia of Genes and Genomes pathways, and 9 diseases. Network analysis revealed that the targets were involved in cancer and other physiological processes. In addition to EGFR, molecular docking analysis showed that seven proteins, namely Janus kinase 3, peroxisome proliferator-activated receptor alpha, renin, mitogen-activated protein kinases, lymphocyte-specific protein tyrosine kinase, cell division protein kinase 2 and proto-oncogene tyrosine-protein kinase Src, could also be potential targets of osimertinib. In conclusion, osimertinib is predicted to target multiple proteins and pathways, resulting in the formation of an action network via which it exerts systematic pharmacological effects.

Transforming growth factor-ß: An early predictor of a functional cure in chronic hepatitis B treated with interferon.

BACKGROUND: The relationship between the serum transforming growth factor (TGF)-ß level and HBsAg loss has not been clearly elaborated in patients with chronic hepatitis B (CHB). METHODS: Two cohorts of patients with CHB were studied. Cohort A: A total of 207 hepatitis B e antigen (HBeAg)-negative CHB patients who finished ≥1 year nucleos(t)ide analogue monotherapy and sequentially received PEGylated interferon treatment for less than 96 weeks were included. Cohort B: Forty HBeAg-positive patients who initially received entecavir therapy for at least 96 weeks were included. Their viral markers and serum TGF-ß levels were measured at different time points during therapy. RESULTS: The levels of serum TGF-ß and HBsAg (0-24 W) were significantly lower in the patients who had HBsAg< 0.05 IU/mL at 48 weeks than in patients who did not in cohort A. We got the same results when we further divided the patients into subgroups according to the initial HBsAg cut-off values (1000 IU/mL, 100 IU/mL, 50 IU/mL) in cohort A. However, HBeAg seroconversion did not lead to the downregulation of TGF-ß levels. The levels of serum TGF-ß were significantly correlated with HBsAg quantitation in cohort A (12-24 W) but not in cohort B (0-48 W). The levels of TGF-ß at week 12 could be used as an early index to predict a functional cure (AUC=0.818) as well as the levels of HBsAg itself (AUC=0.882) in HBeAg-negative chronic hepatitis B patients treated with PEGylated interferon. CONCLUSIONS: The levels of serum TGF-ß were significantly associated with HBsAg loss but not with HBeAg seroconversion and could be used as an early index to predict a functional cure in CHB patients treated with PEGylated interferon.

Distinct T-cell receptor profiles associated with hepatitis B e antigen seroconversion during entecavir treatment.

BACKGROUND & AIMS: T-cell receptor (TCR) repertoire is ambiguously changed in chronic hepatitis B (CHB) patients during antivirus therapy. We tried to assess TCR repertoire dynamics and its clinical significance upon HBeAg seroconversion in CHB patients. METHODS: Twenty CHB patients undergoing 1-year entecavir (ETV) treatment were enrolled, including 10 complete response (CR) vs 10 non-complete response (NCR) patients based on HBeAg seroconversion at week 48. The TCRß complementarity-determining region 3 (CDR3) of peripheral CD4+ and CD8+ T cells at weeks 0, 12 and 48 was analyzed by unbiased high-throughput sequencing. The TCR repertoire profiles and their correlations with serological parameters were analyzed. RESULTS: The diversity of TCRß repertoires was decreasing in CR patients but increasing in NCR patients. The distribution pattern of TCR repertoires stratified according to clonotype frequencies changed in the opposite direction between CR and NCR patients. Narrow amounts of newly appearing clonotypes in CR patients experienced a more intensive and robust expansion and this phenomenon could occur as early as week 12 for the CD4+ subset but later at week 48 for the CD8+ subset. There existed some CR-exclusive clonotypes with a relatively low but increasing frequency at week 48. The number of unique TCRß clonotypes was positively correlated with the ALT or HBV DNA level in CR patients but showed no or negative correlation in NCR patients. CONCLUSION: Distinct TCR profiles contribute to predicting HBeAg seroconversion in CHB patients during ETV treatment and certain TCRß CDR3 motif may be utilized for CHB immunotherapy in the future.

CACYBP Enhances Cytoplasmic Retention of P27Kip1 to Promote Hepatocellular Carcinoma Progression in the Absence of RNF41 Mediated Degradation.

Calcyclin-binding protein (CACYBP) is a multi-ligand protein implicated in the progression of various human cancers. However, its function in hepatocellular carcinoma (HCC) remains unknown. Methods: The expression of CACYBP and RNF41 (RING finger protein 41) in HCC cancer and adjacent non-tumor tissues was detected by immunohistochemistry. CCK-8 assays, colony formation assays, flow cytometry detection and xenograft models were used to evaluate the impact of CACYBP expression on HCC cell growth, apoptosis and cell cycle regulation. Immunoprecipitation and ubiquitination assays were performed to determine how RNF41 regulates CACYBP. The regulatory mechanism of RNF41-CACYBP signaling axis on P27Kip1 was investigated by western blotting and immunofluorescence. Results: CACYBP was highly expressed and associated with poor prognosis in HCC. CACYBP expression was required for HCC cell growth in vitro and in vivo. Moreover, we identified RNF41 as a specific binding partner of CACYBP at exogenous and endogenous levels. RNF41 recruited CACYBP by its C-terminal substrate binding domain, subsequently ubiquitinating CACYBP and promoting its degradation in both proteasome- and lysosome-dependent pathways. In HCC tissues, RNF41 expression was reduced and conferred a negative correlation with CACYBP expression. Mechanistically, CACYBP overexpression stimulated the Ser10, Thr157 and Thr198 phosphorylation of P27Kip1 and its cytoplasmic retention, and RNF41 co-expression attenuated this phenomenon. CACYBP depletion led to decreased levels of cyclin D1, cyclin A2, CDK2 and CDK4, causing a typical cell cycle arrest at G1/S phase and increasing apoptosis in HCC cells. P27Kip1-S10D but not P27Kip1-S10A reconstitution rescued partially the cell cycle function and apoptotic feature after CACYBP depletion. Conclusion: Our findings provide novel insights into the functional role and regulatory mechanism of CACYBP in HCC.

Antitumor Mechanisms of Curcumae Rhizoma Based on Network Pharmacology.

Curcumae Rhizoma, a traditional Chinese medication, is commonly used in both traditional treatment and modern clinical care. Its anticancer effects have attracted a great deal of attention, but the mechanisms of action remain obscure. In this study, we screened for the active compounds of Curcumae Rhizoma using a drug-likeness approach. Candidate protein targets with functions related to cancer were predicted by reverse docking and then checked by manual search of the PubMed database. Potential target genes were uploaded to the GeneMANIA server and DAVID 6.8 database for analysis. Finally, compound-target, target-pathway, and compound-target-pathway networks were constructed using Cytoscape 3.3. The results revealed that the anticancer activity of Curcumae Rhizoma potentially involves 13 active compounds, 33 potential targets, and 31 signaling pathways, thus constituting a "multiple compounds, multiple targets, and multiple pathways" network corresponding to the concept of systematic actions in TCM. These findings provide an overview of the anticancer action of Curcumae Rhizoma from a network perspective, as well as setting an example for future studies of other materials used in TCM.

Systemic pro-inflammatory response facilitates the development of cerebral edema during short hypoxia.

BACKGROUND: High-altitude cerebral edema (HACE) is the severe type of acute mountain sickness (AMS) and life threatening. A subclinical inflammation has been speculated, but the exact mechanisms underlying the HACE are not fully understood. METHODS: Human volunteers ascended to high altitude (3860 m, 2 days), and rats were exposed to hypoxia in a hypobaric chamber (5000 m, 2 days). Human acute mountain sickness was evaluated by the Lake Louise Score (LLS), and plasma corticotrophin-releasing hormone (CRH) and cytokines TNF-α, IL-1ß, and IL-6 were measured in rats and humans. Subsequently, rats were pre-treated with lipopolysaccharide (LPS, intraperitoneal (ip) 4 mg/kg, 11 h) to induce inflammation prior to 1 h hypoxia (7000 m elevation). TNF-α, IL-1ß, IL-6, nitric oxide (NO), CRH, and aquaporin-4 (AQP4) and their gene expression, Evans blue, Na(+)-K(+)-ATPase activity, p65 translocation, and cell swelling were measured in brain by ELISA, Western blotting, Q-PCR, RT-PCR, immunohistochemistry, and transmission electron micrography. MAPKs, NF-κB pathway, and water permeability of primary astrocytes were demonstrated. All measurements were performed with or without LPS challenge. The release of NO, TNF-α, and IL-6 in cultured primary microglia by CRH stimulation with or without PDTC (NF-κB inhibitor) or CP154,526 (CRHR1 antagonist) were measured. RESULTS: Hypobaric hypoxia enhanced plasma TNF-α, IL-1ß, and IL-6 and CRH levels in human and rats, which positively correlated with AMS. A single LPS injection (ip, 4 mg/kg, 12 h) into rats increased TNF-α and IL-1ß levels in the serum and cortex, and AQP4 and AQP4 mRNA expression in cortex and astrocytes, and astrocyte water permeability but did not cause brain edema. However, LPS treatment 11 h prior to 1 h hypoxia (elevation, 7000 m) challenge caused cerebral edema, which was associated with activation of NF-κB and MAPKs, hypoxia-reduced Na(+)-K(+)-ATPase activity and blood-brain barrier (BBB) disruption. Both LPS and CRH stimulated TNF-α, IL-6, and NO release in cultured rat microglia via NF-κB and cAMP/PKA. CONCLUSIONS: Preexisting systemic inflammation plus a short severe hypoxia elicits cerebral edema through upregulated AQP4 and water permeability by TLR4 and CRH/CRHR1 signaling. This study revealed that both infection and hypoxia can cause inflammatory response in the brain. Systemic inflammation can facilitate onset of hypoxic cerebral edema through interaction of astrocyte and microglia by activation of TLR4 and CRH/CRHR1 signaling. Anti-inflammatory agents and CRHR1 antagonist may be useful for prevention and treatment of AMS and HACE.

In vivo measurements of changes in pH triggered by oxalic acid in leaf tissue of transgenic oilseed rape.

Oxalic acid (OA), a non-host-specific toxin secreted by Sclerotinia sclerotiorum during pathogenesis, has been demonstrated to be a major phytotoxic and pathogenic factor. Oxalate oxidase (OXO) is an enzyme associated with the detoxification of OA, and hence the introduction of an OXO gene into oilseed rape (Brassica napus L.) to break down OA may be an alternative way of increasing the resistance of the plant to Sclerotinia sclerotiorum. In order to investigate the activation of OXO in transgenic oilseed rape, a convenient and accessible method was used to monitor changes in pH in response to stress induced by OA. The pH sensor, a platinum microcylinder electrode modified using polyaniline film, exhibited a linear response within the pH range from 3 to 7, with a Nernst response slope of 70 mV/pH at room temperature. The linear correlation coefficient was 0.9979. Changes induced by OA in the pH values of leaf tissue of different oilseed rape species from Brassica napus L. were monitored in real time in vivo using this electrode. The results clearly showed that the transgenic oilseed rape was more resistant to OA than non-transgenic oilseed rape.

The interface behavior and biocatalytic activity of superoxide dismutase at carbon nanotube.

The interface behavior and biocatalytic activity of superoxide dismutase (SOD) were studied at carbon nanotube (CNT) surface with cyclic voltammetry (CV). The results show that SOD participates in a rapid exchange with CNT and the process is a single-electron-single-proton process. The electron-transfer coefficient was calculated to be 0.52, the electron-transfer rate constant was 1.4s(-1) and the average surface coverage was measured to be 6.93 x 10(-11)+/-4.2 x 10(-12) mol cm(-2). The bioactive measurements show that SOD keeps its bioactivity at the CNT surface. SOD's remarkable ability to catalyze the dismutation of the superoxide anion (O (2)(-)) has been demonstrated.

Interactions of cytochrome c with DNA at glassy carbon surface.

Cyclic voltammetry (CV) was used to investigate the interactions of Cytochrome c (Cyt c) with deoxyribonucleic acid (DNA) at glassy carbon (GC) electrodes. The results indicate that there are strong interactions between Cyt c and DNA. The binding constant (k(A)) and binding free energy (Delta(r)G) of Cyt c with dsDNA are (1.69+/-0.38) x 10(5) L.mol(-1) and -(29.76+/-0.56) kJ.mol(-1), respectively; and those of Cyt c with ssDNA are (3.35+/-0.50) x 10(5) L.mol(-1) and -(31.49+/-0.37) kJ.mol(-1), respectively. The binding sites are achieved to be 3.3 bp per Cyt c molecule with dsDNA and 4.0 nucleotides (ssDNA) binding one Cyt c molecule. This experiment affords a valid method for investigating the interactions between DNA and proteins by electrochemical techniques.

The interface behavior of hemoglobin at carbon nanotube and the detection for H(2)O(2).

Direct electrochemistry of hemoglobin (Hb) is observed at carbon nanotube (CNT) interface. The adsorbing Hb can transfer electron directly at CNT interface compared with common carbon material. The heterogeneous electron transfer rate constant k of Hb can be calculated as 0.062s(-1), the transfer coefficient alpha is 0.21 and the average surface coverage of Hb on CNT surface is 3.58 x 10(-9) +/- 2.7 x 10(-10)mol/cm(2). It is found that the adsorbing Hb still keeps its catalytic activity to H(2)O(2). This sensor was used to detect H(2)O(2). The apparent Michaelis-Menten constant is calculated as 6.75 x 10(-4)molL(-1).