KRAS-driven non-small cell lung cancer (NSCLC) patients have no effective targeted treatment. In this study, we aimed to investigate targeting epidermal growth factor receptor (EGFR) as a therapeutic approach in KRAS-driven lung cancer cells. We show that ablation of EGFR significantly suppressed tumor growth in KRAS-dependent cells and induced significantly higher expression of CX chemokine receptor 7 (CXCR7) and activation of MAPK (ERK1/2). Conversely, rescue of EGFR led to CXCR7 downregulation in EGFR-/- cells. Dual EGFR and CXCR7 inhibition led to substantial reduction of MAPK (pERK) and synergistic inhibition of cell growth. Analysis of two additional EGFR knockout NSCLC cell lines using CRISPR/Cas9 revealed genotype dependency of CXCR7 expression. In addition, treatment of different cells with gefitinib increased CXCR7 expression in EGFRwt but decreased it in EGFRmut cells. CXCR7 protein expression was detected in all NSCLC patient samples, with higher levels in adenocarcinoma as compared to squamous cell lung carcinoma and healthy control cases. In conclusion, EGFR and CXCR7 have a crucial interaction in NSCLC, and dual inhibition may be a potential therapeutic option for NSCLC patients.

Ectopically expressed Ring1 and YY1 binding protein (RYBP) induces tumor cell apoptosis through promoting the formation of the death-inducing signaling complex (DISC) in the cytoplasm. However, transiently overexpressed as well as endogenous RYBP in tumor tissues were observed to be mainly located in the nucleus while that in adjacent non-tumor tissues distributed majorly in the cytoplasm. Currently, we do not know the nuclear localization signals and biological function of different subcellular location of RYBP. In this study, we employed bioinformatic analysis, deletion, point mutation, enhanced green fluorescence protein (EGFP) fusion and others, to investigate the elements responsible for RYBP nuclear import and to explore the anti-tumor activities of cytoplasm- and nuclear-located RYBP. Herein, we identified three functional monopartite nuclear localization signals (NLSs), all of which located at the N-terminus of RYBP. Through four basic amino acid replacements within the NLSs, we obtained a cytoplasm-located RYBP mutant (RYBPmut). Compared with wild-type counterpart, RYBPmut exhibited more potent abilities to bind to caspase 8, to prevent MDM2-mediated polyubiquitination and degradation of p53, thereby leading to its stabilization. Further investigation revealed that, in contrast to its wild type, RYBPmut showed more potentials to inhibit tumor cell proliferation and to induce apoptosis, in both p53-dependent and -independent manner. Collectively, our current study revealed the molecular mechanism responsible for RYBP nuclear translocation, and provided evidences to support that RYBPmut could be a more promising candidate agent for cancer treatment.

The aim of the current study was to find the epileptic focus and examine its causal relationship to other brain regions in children with new-onset benign childhood epilepsy with centrotemporal spikes (BECTS). Resting-state functional magnetic resonance imaging (fMRI) was performed in 66 children with BECTS and 37 matched control children. We compared the amplitude of low frequency fluctuation (ALFF) signals between the two groups to find the potential epileptogenic zone (EZ), then used Granger causality analysis (GCA) to explore the causal effects of EZ on the whole brain. Children with BECTS had significantly increased ALFF in the right Broca's area, and decreased ALFF in bilateral fusiform gyrus. The patients also showed increased driving effect from the EZ in Broca's area to the right prefrontal lobe, and decreased effects to the frontal lobe and posterior parts of the language network. The causal effect on left Wernicke's area negatively correlated with verbal IQ (VIQ) score. Our research on new-onset BECTS patients illustrates a possible compensatory mechanism in the language network at early stages of BECTS, and the negative correlation of GCA and VIQ suggest the disturbance of epileptiform activity on language. These findings shed light on the mechanisms of and language dysfunction in BECTS.

Receptor tyrosine kinases, such as VEGFR, PDGFR and EGFR, play important roles in renal cancer. In this study, we investigated EGFR knockout as a therapeutic approach in renal cell carcinoma (RCC). We showed that a renal cell carcinoma cell line (RC21) has higher expression of EGFR as compared to other frequently used cell lines such as HEK293, A549, Hela and DLD1. Ablation of EGFR by CRISPR/Cas9 significantly restrained tumor cell growth and activated the MAPK (pERK1/2) pathway. The VEGFR and PDGFR inhibitor, sunitinib, attenuated the expression of MAPK (pERK1/2) and pAKT induced by EGFR loss and further inhibited EGFR-/- cell proliferation. We showed that loss of EGFR eventually leads to resistance to SAHA and cisplatin. Furthermore, EGFR loss induced G2/M phase arrest and resulted in an increased resistance to TNF-related apoptosis-inducing ligand (TRAIL) in renal cell carcinoma. Thus, ablation of overexpressed EGFR by CRISPR/Cas9 alone or in combination with sunitinib may be a new treatment option for renal cell carcinoma.

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant-fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.

Ring1 and YY1 Binding Protein (RYBP) induces tumor-specific cell apoptosis, but the underlying molecular mechanism has not been fully understood. Here we conducted a yeast two hybrid screen and identified FANK1 (Fibronectin type III and ankyrin repeat domains 1) as a novel RYBP-interacting protein. This interaction was confirmed by coimmunoprecipitation, GST pulldown and immunofluorescence assays. We mapped that the FNIII domain at the N-terminal of FANK1 binds to the Serine/Threonine-rich region at the C-terminal of RYBP. Further studies showed that overexpression of RYBP stabilized, whereas knockdown of RYBP by its specific shRNAs reduced, the expression of FANK1. Mechanistic studies revealed that RYBP inhibited the proteasome degradation of polyubiquitinated FANK1, thus prolonging the half-life of FANK1 protein. Functional studies indicated that RYBP activates FANK1-mediated activator protein 1 (AP-1) signaling pathway which contributes to tumor cell apoptosis. Taken together, our current study uncovered a new mechanism which RYBP utilizes to exert its pro-apoptotic activity in human tumor cells.

Serine/arginine-rich (SR) proteins are well known as splicing factors in humans, model animals and plants. However, they are largely unknown in regulating pre-mRNA splicing of filamentous fungi. Here we report that the SR protein MoSrp1 enhances and suppresses alternative splicing in a model fungal plant pathogen Magnaporthe oryzae. Deletion of MoSRP1 caused multiple defects, including reduced virulence and thousands of aberrant alternative splicing events in mycelia, most of which were suppressed or enhanced intron splicing. A GUAG consensus bound by MoSrp1 was identified in more than 94% of the intron or/and proximate exons having the aberrant splicing. The dual functions of regulating alternative splicing of MoSrp1 were exemplified in enhancing and suppressing the consensus-mediated efficient splicing of the introns in MoATF1 and MoMTP1, respectively, which both were important for mycelial growth, conidiation, and virulence. Interestingly, MoSrp1 had a conserved sumoylation site that was essential to nuclear localization and enhancing GUAG binding. Further, we showed that MoSrp1 interacted with a splicing factor and two components of the exon-joining complex via its N-terminal RNA recognition domain, which was required to regulate mycelial growth, development and virulence. In contrast, the C-terminus was important only for virulence and stress responses but not for mycelial growth and development. In addition, only orthologues from Pezizomycotina species could completely rescue defects of the deletion mutants. This study reveals that the fungal conserved SR protein Srp1 regulates alternative splicing in a unique manner.

Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site (Ki = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.

To explore the applicability of the epidemiology-based mortality score in status epilepticus (EMSE) and the status epilepticus severity score (STESS) in predicting hospital mortality in patients with status epilepticus (SE) in western China. Furthermore, we sought to compare the abilities of the two scales to predict mortality from convulsive status epilepticus (CSE) and non-convulsive status epilepticus (NCSE).

The homologous cytokines macrophage migration inhibitory factor (MIF) and d-dopachrome tautomerase (d-DT or MIF2) play key roles in cancers. Molecules binding to the MIF tautomerase active site interfere with its biological activity. In contrast, the lack of potent MIF2 inhibitors hinders the exploration of MIF2 as a drug target. In this work, screening of a focused compound collection enabled the identification of a MIF2 tautomerase inhibitor R110. Subsequent optimization provided inhibitor 5d with an IC50 of 1.0 μM for MIF2 tautomerase activity and a high selectivity over MIF. 5d suppressed the proliferation of non-small cell lung cancer cells in two-dimensional (2D) and three-dimensional (3D) cell cultures, which can be explained by the induction of cell cycle arrest via deactivation of the mitogen-activated protein kinase (MAPK) pathway. Thus, we discovered and characterized MIF2 inhibitors (5d) with improved antiproliferative activity in cellular models systems, which indicates the potential of targeting MIF2 in cancer treatment.

YY1-associated factor 2 (YAF2) was frequently reported to modulate target gene transcription through both epigenetic and non-epigenetic means. However, other mechanisms were also utilized by YAF2 to carry out its biological functions. Here, we demonstrated that YAF2 from human tumor and non-tumor cells were mainly expressed as Serine 167 phosphorylated form. Further studies showed that the phosphorylated YAF2 up-regulated while its knockdown by specific siRNAs reduced fibronectin type III and ankyrin repeat domains 1 (FANK1) protein level. Mechanistic exploration disclosed that phosphorylated YAF2 inhibit proteasomal degradation of polyubiquitinated FANK1, leading to its increased stability. We then validated their interaction, and displayed that the FN3 domain of FANK1 binds to amino-terminal of YAF2. Functional studies showed that phosphorylated YAF2 inhibits tumor cell apoptosis in a FANK1-dependent manner. Taken together, our current findings demonstrated that phosphorylated YAF2 exhibits anti-apoptotic activity through targeting FANK1 expression in human tumor cells.

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine and essential signaling protein associated with inflammation and cancers. One of the newly described roles of MIF is binding to apoptosis-inducing factor (AIF) that "brings" cells to death in pathological conditions. The interaction between MIF and AIF and their nuclear translocation stands as a central event in parthanatos. However, classical competitive MIF tautomerase inhibitors do not interfere with MIF functions in parthanatos. In this study, we employed a pharmacophore-switch to provide allosteric MIF tautomerase inhibitors that interfere with the MIF/AIF co-localization. Synthesis and screening of a focused compound collection around the 1,2,3-triazole core enabled identification of the allosteric tautomerase MIF inhibitor 6y with low micromolar potency (IC50 = 1.7 ± 0.1 μM). This inhibitor prevented MIF/AIF nuclear translocation and protects cells from parthanatos. These findings indicate that alternative modes to target MIF hold promise to investigate MIF function in parthanatos-mediated diseases.

Long noncoding RNAs (lncRNAs) play an important role in many neurological diseases. This study aimed to investigate differentially expressed lncRNAs and messenger RNAs (mRNAs) in the susceptibility gaining process of primed DBA/1 mice, a sudden unexpected death in epilepsy (SUDEP) model, to illustrate the potential role of lncRNAs in SUDEP.

A considerable proportion of epilepsy patients who achieved long-term seizure freedom with standardized treatment of antiseizure medication will attempt to withdraw medications. Epilepsy is currently considered as a network disease, however, the characteristics of brain function and neural network before and after medication withdrawal remain to be discovered.

Spontaneous low-frequency oscillations in different frequency bands have diverse physiological meanings. The amplitude of low-frequency fluctuation (ALFF) and functional connectivity (FC) in different frequency bands in Benign Childhood Epilepsy with Centrotemporal Spikes (BECTS) are unknown and worth exploring.

ETS1 (E26 transformation specific-1) is the founding member of ETS transcriptional factor family. It transcriptionally modulates numerous gene expressions, and is involved in cellular differentiation, tissue remodeling, angiogenesis, drug resistance and tumorigenesis. ETS1 is usually regarded as an oncogene. However, its apoptosis-inducing activity was also frequently reported. Here, we identified RYBP (Ring1 and YY1 binding protein), a critical epigenetic regulator and apoptosis enhancer, as a novel transcriptional target of ETS1. Specifically, we found that overexpression of ETS1 up-regulates the promoter activity of RYBP in HEK293T and tumor cells from different tissue origins, indicating a universal transcriptional regulatory effect. Subsequently, both overexpression and RNA interfering experiments demonstrated that ETS1 positively modulates RYBP expression from both mRNA and protein levels. Bioinformatics analysis combined with site-directed mutagenesis suggested that there probably exist a multiple of ETS1 binding sites in RYBP promoter region, and chromatin immunoprecipitation assay validates the physical association between ETS1 protein and RYBP promoter region. Functional studies showed that ectopic expression of ETS1 significantly suppresses tumor cell proliferation. However, this inhibitory effect was partially compromised when RYBP was concomitantly knocked down by its specific short hairpin RNA. Meanwhile, we provide evidence to demonstrate that cyclin-dependent kinase inhibitor p21 is possibly involved in this regulatory loop. Taken together, our current study identified RYBP as a new transcriptional target which is utilized by ETS1 to carry out its tumor cell growth inhibitory effect.

To evaluate the predictors of seizure reduction outcome after vagus nerve stimulation (VNS) in patients with drug-resistant epilepsy (DRE).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as a promising anti-cancer therapeutic. However, many cancers have been found to be or to become inherently resistant to TRAIL. A combination of epigenetic modifiers, such as histone deacetylase inhibitors (HDACi's), with TRAIL was effective to overcome TRAIL resistance in some cancers. Broad spectrum HDACi's, however, show considerable toxicity constraining clinical use. Since overexpression of class I histone deacetylase (HDAC) has been found in colon tumors relative to normal mucosa, we have focused on small spectrum HDACi's. We have now tested agonistic receptor-specific TRAIL variants rhTRAIL 4C7 and DHER in combination with several class I specific HDACi's on TRAIL-resistant colon cancer cells DLD-1 and WiDr. Our data show that TRAIL-mediated apoptosis is largely improved in WiDr cells by pre-incubation with Entinostat-a HDAC1, 2, and 3 inhibitor- and in DLD-1 cells by RGFP966-a HDAC3-specific inhibitor- or PCI34051-a HDAC8-specific inhibitor. We are the first to report that using RGFP966 or PCI34051 in combination with rhTRAIL 4C7 or DHER represents an effective cancer therapy. The intricate relation of HDACs and TRAIL-induced apoptosis was confirmed in cells by knockdown of HDAC1, 2, or 3 gene expression, which showed more early apoptotic cells upon adding rhTRAIL 4C7 or DHER. We observed that RGFP966 and PCI34051 increased DR4 expression after incubation on DLD-1 cells, while RGFP966 induced more DR5 expression on WiDr cells, indicating a different role for DR4 or DR5 in these combinations. At last, we show that combined treatment of RGFP966 with TRAIL variants (rhTRAIL 4C7/DHER) increases apoptosis on 3D tumor spheroid models.

Dysregulated epidermal growth factor receptor (EGFR) is an oncogenic driver of many human cancers, promoting aberrant cell proliferation, migration, and survival. Pharmacological targeting of EGFR is often challenged by acquired mechanisms of resistance. Ligand-dependent mechanisms in EGFR wild-type cells rely on ligand or receptor overexpression, allowing cells to outcompete inhibitors and perpetuate signaling in an autocrine manner. Importantly, EGFR ligands are synthesized as membrane-bound precursors that must be solubilized to enable receptor-ligand interactions. The A disintegrin and metalloproteinase 17 (ADAM17) is considered the main sheddase of several EGFR ligands, and a potential pharmacological target. However, its broad substrate range and ubiquitous expression complicate its therapeutic targeting. Here, we present a novel bispecific fusion protein construct consisting of the inhibitory prodomain of ADAM17 (TPD), fused to an EGFR-targeting designed ankyrin repeat protein (DARPin). TPD is a natural inhibitor of ADAM17, maintaining the protease in a zymogen-like form. Meanwhile, the high affinity anti-EGFR DARPin E01 binds to EGFR and inhibits ligand binding. The resulting fusion protein E01-GS-TPD retained binding ability to both molecular targets EGFR and ADAM17. The large difference in affinity for each target resulted in enrichment of the fusion protein in EGFR-positive cells compared to EGFR-negative cells, suggesting a possible application in autocrine signaling inhibition. Accordingly, E01-GS-TPD decreased migration and proliferation of EGFR-dependent cell lines with no significant increase in apoptotic cell death. Finally, inhibition of proliferation was observed through EGFR ligand-dependent mechanisms as growth inhibition was not observed in EGFR mutant or KRAS mutant cell lines. The use of bispecific proteins targeting the EGFR/ADAM17 axis could be an innovative strategy for the treatment of EGFR-dependent cancers.

Background and Objectives: The research on the therapeutic effect of preoperative radiotherapy (PRRT) for patients with early non-small cell lung cancer (NSCLC) is still insufficient, and the impact of postoperative radiotherapy (PORT) on the prognosis of patients with early NSCLC remains controversial. We conducted this study to investigate the effect of PORT and PRRT on prognosis for these patients. Materials and Methods: In total, 3640 patients with stage II NSCLC who underwent a lobectomy or pneumonectomy were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. Multivariate regression was adopted to identify the independent influence of PORT or PRRT on patients' prognosis. Subgroup analysis of survival was performed in patients with different combinations of key clinical features. We also used Kaplan-Meier analysis and competitive risk analysis to explore to which extent PORT or PRRT impacted the overall survival and cumulative mortality. Results: PORT was an independent risk factor of NSCLC-specific death among patients with N0 stage (HR, 1.648; 95% CI, 1.309-2.075, p < 0.001) and in N1 stage with <3 positive lymph nodes (HR, 2.698; 95% CI, 1.910-3.812, p < 0.001) in multivariate analysis. Findings from subgroup analysis for the risk of NSCLC-specific death, competitive risk analysis of NSCLC-specific cumulative mortality, and overall survival analysis also demonstrated PORT was detrimental to patients in these two subgroups above (p < 0.05). However, in patients with N1 stage with ≥3 positive lymph nodes, PORT may help prolong median survival. PRRT was an independent risk factor for NSCLC-specific death in multivariate analysis of patients with N0 stage (HR, 1.790; 95% CI, 1.201-2.668, p = 0.004), and significantly decreased overall survival in these patients (p < 0.001). Conclusion: PORT is associated with worse survival outcome and better cumulative mortality of stage II patients of NSCLC with N0 disease or N1 disease (<3 nodes), while PRRT is associated with reduced prognosis in patients with N0 stage. On the other hand, PORT may help to improve the prognosis of patients with N1 stage who have three or more lymph node metastases. Hence, PORT and PRRT should not be recommended for patients with N0 stage. However, in patients with "high volume" N1 stage, PORT might improve oncological outcomes.