Nuclear Magnetic Resonance-Based Metabolomics and Risk of CKD.

RATIONALE & OBJECTIVE: Chronic kidney disease (CKD) leads to lipid and metabolic abnormalities, but a comprehensive investigation of lipids, lipoprotein particles, and circulating metabolites associated with the risk of CKD has been lacking. We examined the associations of nuclear magnetic resonance (NMR)-based metabolomics data with CKD risk in the UK Biobank study. STUDY DESIGN: Observational cohort study. SETTING & PARTICIPANTS: A total of 91,532 participants in the UK Biobank Study without CKD and not receiving lipid-lowering therapy. EXPOSURE: Levels of metabolites including lipid concentration and composition within 14 lipoprotein subclasses, as well as other metabolic biomarkers were quantified via NMR spectroscopy. OUTCOME: Incident CKD identified using ICD codes in any primary care data, hospital admission records, or death register records. ANALYTICAL APPROACH: Cox proportional hazards regression models were used to estimate hazard ratios and 95% confidence intervals. RESULTS: We identified 2,269 CKD cases over a median follow-up period of 13.1 years via linkage with the electronic health records. After adjusting for covariates and correcting for multiple testing, 90 of 142 biomarkers were significantly associated with incident CKD. In general, higher concentrations of very-low-density lipoprotein (VLDL) particles were associated with a higher risk of CKD whereas higher concentrations of high-density lipoprotein (HDL) particles were associated with a lower risk of CKD. Higher concentrations of cholesterol, phospholipids, and total lipids within VLDL were associated with a higher risk of CKD, whereas within HDL they were associated with a lower risk of CKD. Further, higher triglyceride levels within all lipoprotein subclasses, including all HDL particles, were associated with greater risk of CKD. We also identified that several amino acids, fatty acids, and inflammatory biomarkers were associated with risk of CKD. LIMITATIONS: Potential underreporting of CKD cases because of case identification via electronic health records. CONCLUSIONS: Our findings highlight multiple known and novel pathways linking circulating metabolites to the risk of CKD. PLAIN-LANGUAGE SUMMARY: The relationship between individual lipoprotein particle subclasses and lipid-related traits and risk of chronic kidney disease (CKD) in general population is unclear. Using data from 91,532 participants in the UK Biobank, we evaluated the associations of metabolites measured using nuclear magnetic resonance testing with the risk of CKD. We identified that 90 out of 142 lipid biomarkers were significantly associated with incident CKD. We found that very-low-density lipoproteins, high-density lipoproteins, the lipid concentration and composition within these lipoproteins, triglycerides within all the lipoprotein subclasses, fatty acids, amino acids, and inflammation biomarkers were associated with CKD risk. These findings advance our knowledge about mechanistic pathways that may contribute to the development of CKD.

RNA-binding protein-regulated fibronectin is essential for EGFR-activated metastasis of head and neck squamous cell carcinoma.

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the role of RNA-binding proteins (RBPs) in EGFR-associated metastasis of HNSCC remains unclear. In this study, we reveal that RBPs, specifically nucleolin (NCL) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), correlated with the mesenchymal phenotype of HNSCC. The depletion of RBPs significantly attenuated EGF-induced HNSCC metastasis. Intriguingly, the EGF-induced EMT markers, such as fibronectin, were regulated by RBPs through the ERK and NF-κB pathway, followed by the enhancement of mRNA stability of fibronectin through the 5' untranslated region (5'-UTR) of the gene. The upregulation of fibronectin triggered the integrin signaling activation to enhance tumor cells' attachment to endothelial cells and increase endothelial permeability. In addition, the concurrence of EGFR and RBPs or EGFR and fibronectin was associated with overall survival and disease-free survival of HNSCC. The in vivo study showed that depletion of NCL, hnRNPA2B1, and fibronectin significantly inhibited EGF-promoted extravasation of tumor cells into lung tissues. The depletion of fibronectin or treatment with integrin inhibitors dramatically attenuated EGF-induced HNSCC metastatic nodules in the lung. Our data suggest that the RBPs/fibronectin axis is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.

A Quadruplex Ultrasensitive Immunoassay for Simultaneous Assessment of Human Reproductive Hormone Proteins in Multiple Biofluid Samples.

Reproductive hormones play vital roles in reproductive health and can be used to assess a woman's ovarian function and diagnose diseases associated with reproductive endocrine disorders. As these hormones are important biomarkers for reproductive health monitoring and diagnosis, a rapid, high-throughput, and low-invasive detection and simultaneous assessment of the levels of multiple reproductive hormones has important clinical applications. In this work, a quadruplex ultrasensitive immunoassay was developed for simultaneous assessment of 4 human reproductive hormone proteins (follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and anti-Müllerian hormone (AMH)) in a variety of human biofluid samples. This assay takes advantage of single-molecule imaging of microwell arrays and capture antibody beads as a reaction interface to construct multiplex bead array immunoassays. The analyte-bound beads can easily be parsed to individual wells and detected via fluorophores, emitting distinct wavelengths associated to the beads. As a result, this proposed quadruplex immunoassay exhibits four good 4-parameter logistic calibration curves ranging from 2.7 to 2000, 1.6 to 1200, 1.8 to 1300, and 0.3 to 220 pg/mL with limits of detection of 0.32, 0.28, 0.14, and 0.02 pg/mL for FSH, LH, PRL, and AMH, respectively. Furthermore, the developed quadruplex immunoassay was used to test clinical venous serum samples where it showed remarkable consistency with clinical test results in methodological comparison and the diagnosis of polycystic ovary syndrome. In addition, we successfully applied the ultrasensitive capability of this assay to the simultaneous testing and evaluation of four proteins in fingertip blood as well as urine samples, in which the urinary AMH level (1.42-156 pg/mL) was measured and assessed quantitatively for the first time.

Glycoprotein α-Subunit of Glucosidase II (GIIα) is a novel prognostic biomarker correlated with unfavorable outcome of urothelial carcinoma.

BACKGROUND: Urothelial carcinoma (UC) is among the most prevalent malignancies. The muscle-invasive bladder cancer (MIBC) shows an invasive feature and has poor prognosis, while the non-muscle invasive bladder cancer (NMIBC) shows a better prognosis as compared with the MIBC. However, a significant proportion (10%-30%) of NMIBC cases progress to MIBC. Identification of efficient biomarkers for the prediction of the course of UC remains challenging nowadays. Recently, there is an emerging study showed that post-translational modifications (PTMs) by glycosylation is an important process correlated with tumor angiogenesis, invasion and metastasis. Herein, we reported a data-driven discovery and experimental validation of GANAB, a key regulator of glycosylation, as a novel prognostic marker in UC. METHODS: In the present study, we conducted immunohistochemistry (IHC) assay to evaluate the correlation between the expression levels of GANAB protein and the prognosis of UC in our cohort of 107 samples using whole slide image (WSI) analysis. In vitro experiments using RNAi were also conducted to investigate the biological functions of GANAB in UC cell lines. RESULTS: We observed that positive GANAB protein expression was significantly correlated with poor prognosis of UC in our cohort, with p-value of 0.0017 in Log-rank test. Notably, tumor cells at the invasive front of the tumor margin showed stronger GANAB expression than the tumor cells inside the tumor body in UCs. We further validated that the elevated expression levels of GANAB were significantly correlated with high grade tumors (p-values of 1.72 × 10-10), advanced stages (6.47 × 10-6), and elevated in luminal molecular subtypes. Moreover, knocking-down GANAB using RNAi in UM-UC-3 and T24 cells inhibited cell proliferation and migration in vitro. Knockdown of GANAB resulted in cell cycle arrest at G1 phase. We demonstrated that GANAB mediated HIF1A and ATF6 transcriptional activation in the ER stress signaling, and regulated the gene expression of cell cycle-related transcriptional factors E2F7 and FOXM1. CONCLUSIONS: The elevated expression of GANAB is a novel indicator of poorer prognosis of UC. Our data suggests that GANAB is not only a new and promising prognostic biomarker for UC, but also may provide important cues for the development of PTM-based therapeutics for UC treatment.

Effects of N6 -(4-hydroxybenzyl) adenine riboside in stress-induced insomnia in rodents.

Adenosine exhibits a somnogenic effect; however, there is no adenosinergic hypnotic because of cardiovascular effects. This study investigated whether N6-(4-hydroxybenzyl) adenine riboside (T1-11), extracted from Gastrodia elata, produces somnogenic effects in rodents. We determined the involvement of adenosine 2A receptors (A2ARs) in GABAergic neurons of the ventrolateral preoptic area (VLPO) and the cardiovascular effects. Change of cage bedding is employed as a stressor to induce insomnia in rodents, and electroencephalograms and electromyograms were used to acquire and analyse sleep-wake activity. We found that intracerebroventricular administration of T1-11 before a dark period increased non-rapid eye movement (NREM) and rapid eye movement (REM) sleep during a dark period, and T1-11-induced sleep increases were blocked by the A2AR antagonist, SCH58261, in naïve rats. Oral administration of T1-11 increased NREM sleep during both dark and light periods. Microinjection of the A2AR antagonist, SCH58261, into the VLPO blocked sleep effects of T1-11. In addition to the somnogenic effect in naïve mice, T1-11 suppressed the stress-induced insomnia and this suppressive effect was blocked by SCH58261. C-fos expression in GABAergic neurons of VLPO was increased after administration of T1-11 in Gad2-Cre::Ai14 mice, suggesting the activation of GABAergic neurons in the VLPO. T1-11 exhibited no effects on heart rate and the low frequency/high frequency ratio of heart rate variability. We concluded that T1-11 elicited somnogenic effects and effectively ameliorated acute stress-induced insomnia. The somnogenic effect is mediated by A2ARs to activate GABAergic neurons in the VLPO. This adenosine analogue could be a potential hypnotic because of no sympathetic and parasympathetic effects on the cardiovascular system.

Screening of tumor-associated antigens based on Oncomine database and evaluation of diagnostic value of autoantibodies in lung cancer.

OBJECTIVES: The purpose of this study is to discover novel tumor-associated antigens (TAAs) to improve the diagnosis of lung cancer (LC). MATERIALS AND METHODS: Oncomine database was used to discover potential TAAs from LC tissues, enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of autoantibodies against TAAs in two independent sets (identification set, n = 368; validation set, n = 1011). RESULTS: Analyses of sera from identification set showed that the sensitivity of autoantibodies against five TAAs (HMGB3, ZWINT, GREM1, NUSAP1 and MMP12) reached 57.1%, 42.4%, 38.0%, 36.4% and 20.7%, with area under ROC curve (AUC) of 0.85, 0.75, 0.71, 0.73 and 0.70, respectively. It also validated the diagnostic performances of these autoantibodies with AUC of 0.72, 0.65, 0.61, 0.64 and 0.64, respectively. Autoantibody against HMGB3 exhibited significantly increased frequency in early LC (53.3%) compared to advanced LC (29.3%) (P < .05). The positive rates of autoantibody against HMGB3 and NUSAP1 in serum of LC patients without distant metastasis were significantly higher than that of distant metastatic LC (P < .05). When each of the three protein biomarkers (CEA, CA125 and CYFRA21-1) was combined with anti-HMGB3 autoantibody, the sensitivity of early LC increased to 72.7%, 63.3% and 75.9% from 36.4%, 13.3% and 27.6%, respectively. CONCLUSION: Autoantibodies against 5 TAAs (HMGB3, ZWINT, GREM1, NUSAP1 and MMP12) might have favorable diagnostic values in LC detection, and autoantibody against HMGB3 has the potential to serve as a serological biomarker in early-stage LC. The combination of protein biomarkers and anti-HMGB3 might contribute to detection of early-stage LC.

Strategy and validation of a structure-based method for the discovery of selective inhibitors of PAK isoforms and the evaluation of their anti-cancer activity.

p21 activated kinase 4 (PAK4), which belongs to the serine/threonine (Ser/Thr) protein kinase family, is a representative member of the PAK family and plays a significant role in multiple processes associated with cancer development. In this study, structure-based virtual screening was performed to discover novel and selective small molecule scaffolds, and a 6-hydroxy-2-mercapto-3-phenylpyrimidin-4(3H)-one-based compound (SPU-106, 14#) was identified as an effective PAK4 inhibitor. By combining both a molecular docking study and molecular dynamics (MD) simulation strategies, the binding mode was determined in the PAK4 site. The SPU-106 compound could efficiently and selectively bind to the PAK4 kinase domain at an IC50 of 21.36 µM according to the kinase analysis. The designed molecular probe demonstrated that SPU-106 binds to the kinase domain in the C-terminus of PAK4. Further investigation revealed that the SPU-106 had a strong inhibitory effect on the invasion of SGC7901 cells but without any cytotoxicity. The western blot analysis indicated that the compound potently inhibited the PAK4/LIMK1/cofilin and PAK4/SCG10 signaling pathways. Thus, our work shows the successful application of computational strategies for the discovery of selective hits, and SPU-106 may be an effective PAK4 inhibitor for further development as an antitumor agent.

Old drug new tricks: Chlorhexidine acts as a potential allosteric inhibitor toward PAK1.

This paper describes the identification of chlorhexidine, an agent commonly used in clinical as a novel potential allosteric inhibitor of PAK1. In cellular assays, chlorhexidine showed a good inhibitory profile, and its inhibitory profile was even better than IPA-3, a well-known allosteric inhibitor. In pharmacology experiments, chlorhexidine successfully inhibited the relief of PAK1 dimer and inhibited the activation of PAK1. Our findings offer an insight for the new drug development of PAK1 inhibitor. We also provide a possible explanation for the phenomenon that the application of the chlorhexidine in peritoneal lavage inhibited the development of tumor.

Interleukin-1 receptor (IL-1R) mediates epilepsy-induced sleep disruption.

BACKGROUND: Sleep disruptions are common in epilepsy patients. Our previous study demonstrates that homeostatic factors and circadian rhythm may mediate epilepsy-induced sleep disturbances when epilepsy occurs at different zeitgeber hours. The proinflammatory cytokine, interleukin-1 (IL-1), is a somnogenic cytokine and may also be involved in epileptogenesis; however, few studies emphasize the effect of IL-1 in epilepsy-induced sleep disruption. We herein hypothesized that IL-1 receptor type 1 (IL-1R1) mediates the pathogenesis of epilepsy and epilepsy-induced sleep disturbances. We determined the role of IL-1R1 by using IL-1R1 knockout (IL-1R1 -/- KO) mice. RESULTS: Our results elucidated the decrease of non-rapid eye movement (NREM) sleep during the light period in IL-1R -/- mice and confirmed the somnogenic role of IL-1R1. Rapid electrical amygdala kindling was performed to induce epilepsy at the particular zeitgeber time (ZT) point, ZT13. Our results demonstrated that seizure thresholds induced by kindling stimuli, such as the after-discharge threshold and successful kindling rates, were not altered in IL-1R -/- mice when compared to those obtained from the wildtype mice (IL-1R +/+ mice). This result suggests that IL-1R1 is not involved in kindling-induced epileptogenesis. During sleep, ZT13 kindling stimulation significantly enhanced NREM sleep during the subsequent 6 h (ZT13-18) in wildtype mice, and sleep returned to the baseline the following day. However, the kindling-induced sleep alteration was absent in the IL-1R -/- KO mice. CONCLUSIONS: These results indicate that the IL-1 signal mediates epilepsy-induced sleep disturbance, but dose not participate in kindling-induced epileptogenesis.

Low-frequency electroacupuncture suppresses focal epilepsy and improves epilepsy-induced sleep disruptions.

BACKGROUND: The positive effects of acupuncture at Feng-Chi acupoints on treating epilepsy and insomnia have been well-documented in ancient Chinese literature. However, there is a lack of scientific evidence to elucidate the underlying mechanisms behind these effects. Our previous study demonstrated that high-frequency (100 Hz) electroacupuncture (EA) at Feng-Chi acupoints deteriorates both pilocarpine-induced focal epilepsy and sleep disruptions. This study investigated the effects of low-frequency (10 Hz) EA on epileptic activities and epilepsy-induced sleep disruptions. RESULTS: In rats, the Feng-Chi acupoint is located 3 mm away from the center of a line between the two ears. Rats received 30 min of 10 Hz EA stimuli per day before each day's dark period for three consecutive days. Our results indicated that administration of pilocarpine into the left CeA at the beginning of the dark period induced focal epilepsy and decreased both rapid eye movement (REM) sleep and non-REM (NREM) sleep during the consequent light period. Low-frequency (10 Hz) EA at Feng-Chi acupoints suppressed pilocarpine-induced epileptiform EEGs, and this effect was in turn blocked by naloxone (a broad-spectrum opioid receptor antagonist), but not by naloxonazine (a µ-receptor antagonist), naltrindole (a δ-receptor antagonist) and nor-binaltorphimine (a κ-receptor antagonist). Ten Hz EA enhanced NREM sleep during the dark period, and this enhancement was blocked by all of the opioid receptor antagonists. On the other hand, 10 Hz EA reversed pilocarpine-induced NREM suppression during the light period, and the EA's effect on the sleep disruption was only blocked by naloxonazine. CONCLUSIONS: These results indicate that low-frequency EA stimulation of Feng-Chi acupoints is beneficial in improving epilepsy and epilepsy-induced sleep disruptions, and that opioid receptors in the CeA mediate EA's therapeutic effects.

Transcranial Direct Current Stimulation (tDCS) Ameliorates Stress-Induced Sleep Disruption via Activating Infralimbic-Ventrolateral Preoptic Projections.

Transcranial direct current stimulation (tDCS) is acknowledged for its non-invasive modulation of neuronal activity in psychiatric disorders. However, its application in insomnia research yields varied outcomes depending on different tDCS types and patient conditions. Our primary objective is to elucidate its efficiency and uncover the underlying mechanisms in insomnia treatment. We hypothesized that anodal prefrontal cortex stimulation activates glutamatergic projections from the infralimbic cortex (IL) to the ventrolateral preoptic area (VLPO) to promote sleep. After administering 0.06 mA of electrical currents for 8 min, our results indicate significant non-rapid eye movement (NREM) enhancement in naïve mice within the initial 3 h post-stimulation, persisting up to 16-24 h. In the insomnia group, tDCS enhanced NREM sleep bout numbers during acute stress response and improved NREM and REM sleep duration in subsequent acute insomnia. Sleep quality, assessed through NREM delta powers, remains unaffected. Interference of the IL-VLPO pathway, utilizing designer receptors exclusively activated by designer drugs (DREADDs) with the cre-DIO system, partially blocked tDCS's sleep improvement in stress-induced insomnia. This study elucidated that the activation of the IL-VLPO pathway mediates tDCS's effect on stress-induced insomnia. These findings support the understanding of tDCS effects on sleep disturbances, providing valuable insights for future research and clinical applications in sleep therapy.

Porous nickel-cobalt phosphate with oxygen-rich vacancies in situ grown on dopamine-modified cellulose textiles as self-supporting high mass loadings supercapacitor electrode.

Transition-metal phosphates/phosphides showcase significant promise for energy-related applications because of their high theoretical electrochemical characteristics. However, sluggish electro/ion transfer rates and kinetically unfavorable reaction sites hinder their application at high mass loading. Herein, a self-supporting electrode based on transition-metal phosphates was successfully fabricated via a one-step electrodeposition process. The nanosheet structure of transition-metal phosphates, formed by interconnecting nanoparticles, effectively mitigates the impact of stress and achieves a high mass-loading (21 mg cm-2) of the electrode. Additionally, the oxygen vacancy-rich and porous nanostructure of transition-metal phosphates endows the as-prepared electrodes with a significantly increased conductivity and fast ion migration rate for enhancing electrochemical kinetics. Consequently, the as-fabricated transition-metal phosphate electrode displays the highest areal specific capacity of 39.2F cm-2. Furthermore, the asymmetric supercapacitor achieves a maximum energy density of 0.79 mWh cm-2 and a high capacity retention of 93.0 % for 10000 cycles under 60 mA cm-2. This work provides an ideal strategy for fabricating flexible electrodes with high mass loading and synthesizing transition-metal phosphate electrodes rich in oxygen vacancies.

NAD+ metabolism enzyme NNMT in cancer-associated fibroblasts drives tumor progression and resistance to immunotherapy by modulating macrophages in urothelial bladder cancer.

BACKGROUND: This study comprehensively investigates the association between the expression of nicotinamide N-methyltransferase (NNMT) and clinical outcomes of urothelial bladder cancer (UBC), as well as the molecular mechanisms by which NNMT in cancer-associated fibroblast (CAF) modulates tumor progression and immunotherapy resistance in UBC. METHODS: Single-cell transcriptomic analyses, immunohistochemical and immunofluorescence assays were performed on bladder cancer samples to validate the relationship between NNMT expression and clinical outcomes. A series of experiments, including chromatin immunoprecipitation assay, liquid chromatography tandem mass spectrometry assay, and CRISPR‒Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) knockout, together with in vivo models, have been established to determine the molecular functions of NNMT in CAFs in UBC. RESULTS: We demonstrated that elevated expression of the nicotinamide adenine dinucleotide (NAD+) metabolism enzyme NNMT in CAFs (NNMT+ CAFs) was significantly associated with non-response to programmed death-ligand 1 (PD-L1) blockade immunotherapy in patients with UBC and predicted the unfavorable prognosis of UBC in two independent large cohorts. Targeting NNMT using the inhibitor 5-Amino-1-methylquinolinium iodide significantly reduced tumor growth and enhanced the apoptotic effects of the anti-PD-L1 antibody in UBC mouse models. Mechanistically, NNMT+ CAFs recruit tumor-associated macrophages via epigenetic reprogramming of serum amyloid A (SAA) to drive tumor cell proliferation and confer resistance to programmed death-1/PD-L1 blockade immunotherapy. CONCLUSIONS: NNMT+ CAFs were significantly associated with non-response to PD-L1 blockade immunotherapy in patients with UBC. Elevated NNMT, specifically in CAFs, upregulates SAA expression and enhances the recruitment and differentiation of macrophages in the tumor microenvironment, thereby directly or indirectly promoting tumor progression and conferring resistance to immunotherapies in bladder cancer.

Amygdala opioid receptors mediate the electroacupuncture-induced deterioration of sleep disruptions in epilepsy rats.

BACKGROUND: Clinical and experimental evidence demonstrates that sleep and epilepsy reciprocally affect each other. Previous studies indicated that epilepsy alters sleep homeostasis; in contrast, sleep disturbance deteriorates epilepsy. If a therapy possesses both epilepsy suppression and sleep improvement, it would be the priority choice for seizure control. Effects of acupuncture of Feng-Chi (GB20) acupoints on epilepsy suppression and insomnia treatment have been documented in the ancient Chinese literature, Lingshu Jing (Classic of the Miraculous Pivot). Therefore, this study was designed to investigate the effect of electroacupuncture (EA) stimulation of bilateral Feng-Chi acupoints on sleep disruptions in rats with focal epilepsy. RESULTS: Our result indicates that administration of pilocarpine into the left central nucleus of amygdala (CeA) induced focal epilepsy and decreased both rapid eye movement (REM) sleep and non-REM (NREM) sleep. High-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints, in which a 30-min EA stimulation was performed before the dark period of the light:dark cycle in three consecutive days, further deteriorated pilocarpine-induced sleep disruptions. The EA-induced exacerbation of sleep disruption was blocked by microinjection of naloxone, µ- (naloxonazine), κ- (nor-binaltorphimine) or δ-receptor antagonists (natrindole) into the CeA, suggesting the involvement of amygdaloid opioid receptors. CONCLUSION: The present study suggests that high-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints exhibits no benefit in improving pilocarpine-induced sleep disruptions; in contrast, EA further deteriorated sleep disturbances. Opioid receptors in the CeA mediated EA-induced exacerbation of sleep disruptions in epileptic rats.

Activation of amygdala opioid receptors by electroacupuncture of Feng-Chi (GB20) acupoints exacerbates focal epilepsy.

BACKGROUND: The effect of seizure suppression by acupuncture of Feng-Chi (GB20) acupoints has been documented in the ancient Chinese literature, Lingshu Jing (Classic of the Miraculous Pivot), however, there is a lack of scientific evidence to prove it. This current study was designed to elucidate the effect of electroacupuncture (EA) stimulation of bilateral Feng-Chi (GB20) acupoints on the epileptic activity by employing an animal model of focal epilepsy. METHODS: Administration of pilocarpine into the left central nucleus of amygdala (CeA) induced the focal epilepsy in rats. Rats received a 30-min 100 Hz EA stimulation of bilateral Feng-Chi acupoints per day, beginning at 30 minutes before the dark period and performing in three consecutive days. The broad-spectrum opioid receptor antagonist (naloxone), µ-receptor antagonist (naloxonazine), δ-receptor antagonist (naltrindole) and κ-receptor antagonist (nor-binaltorphimine) were administered directly into the CeA to elucidate the involvement of CeA opioid receptors in the EA effect. RESULTS: High-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints did not suppress the pilocarpine-induced epileptiform electroencephalograms (EEGs), whereas it further increased the duration of epileptiform EEGs. We also observed that epilepsy occurred while 100 Hz EA stimulation of Feng-Chi acupoints was delivered into naïve rats. EA-induced augmentation of epileptic activity was blocked by microinjection of naloxone, µ- (naloxonazine), κ- (nor-binaltorphimine) or δ-receptor antagonists (natrindole) into the CeA, suggesting that activation of opioid receptors in the CeA mediates EA-exacerbated epilepsy. CONCLUSIONS: The present study suggests that high-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints has no effect to protect against pilocarpine-induced focal epilepsy; in contrast, EA further exacerbated focal epilepsy induced by pilocarpine. Opioid receptors in the CeA mediated EA-induced exacerbation of focal epilepsy.

Uncovering the ceRNA network and DNA methylation associated with gene expression in nasopharyngeal carcinoma.

OBJECTIVE: This study aimed to uncover abnormally expressed genes regulated by competitive endogenous RNA (ceRNA) and DNA methylation nasopharyngeal carcinoma and to validate the role of lncRNAs in the ceRNA network on nasopharyngeal carcinoma progression. METHODS: Based on the GSE64634 (mRNA), GSE32960 (miRNA), GSE95166 (lncRNA), and GSE126683 (lncRNA) datasets, we screened differentially expressed mRNAs, miRNAs and lncRNAs in nasopharyngeal carcinoma. A ceRNA network was subsequently constructed. Differentially methylated genes were screened using the GSE62336 dataset. The abnormally expressed genes regulated by both the ceRNA network and DNA methylation were identified. In the ceRNA network, the expression of RP11-545G3.1 lncRNA was validated in nasopharyngeal carcinoma tissues and cells by RT-qPCR. After a knockdown of RP11-545G3.1, the viability, migration, and invasion of CNE-2 and NP69 cells was assessed by CCK-8, wound healing and Transwell assays. RESULTS: This study identified abnormally expressed mRNAs, miRNAs and lncRNAs in nasopharyngeal carcinoma tissues. A ceRNA network was constructed, which contained three lncRNAs, 15 miRNAs and 129 mRNAs. Among the nodes in the PPI network based on the mRNAs in the ceRNA network, HMGA1 was assessed in relation to the overall and disease-free survival of nasopharyngeal carcinoma. We screened two up-regulated genes regulated by the ceRNA network and hypomethylation and 26 down-regulated genes regulated by the ceRNA network and hypermethylation. RP11-545G3.1 was highly expressed in the nasopharyngeal carcinoma tissues and cells. Moreover, the knockdown of RP11-545G3.1 reduced the viability, migration, and invasion of CNE-2 and NP69 cells. CONCLUSION: Our findings uncovered the epigenetic regulation in nasopharyngeal carcinoma and identified the implications of RP11-545G3.1 on the progression of nasopharyngeal carcinoma.

Hypocretin role in posttraumatic stress disorder-like behaviors induced by a novel stress protocol in mice.

Introduction: Posttraumatic stress disorder (PTSD) is a psychiatric disorder developed in individuals who expose to traumatic events. These patients may experience symptoms, such as recurrent unwanted memory of the traumatic event, avoidance of reminders of the trauma, increased arousal, and cognitive difficulty. The hypocretinergic system originates from the lateral hypothalamic area (LHA) and projects diffusely to the whole brain, and hypocretin may be involved in the features of stress-related disorder, PTSD. Methods: Our study aimed to investigate the role of basolateral amygdala (BLA) hypocretin signals in the pathophysiology of PTSD-like symptoms induced by the modified multiple-prolonged stress (MPS) protocol. The BLA, a brain region involved in fear-related behaviors, receives the hypocretin projections. In this study, TCS1102, a dual hypocretin receptor antagonist, was used to block the hypocretin signal in BLA. Results: Our data indicated that the MPS protocol is a potential PTSD-like paradigm in mice. Meanwhile, the blockade of hypocretin signaling in the BLA relieved the MPS-induced fear response, and partially reduced PTSD-like anxiety behaviors performed by the open field test (OFT) and elevated plus maze (EPM) task. Discussion: Our findings suggest that the hypocretinergic system is a potential therapeutic approach for PTSD treatment. With further research, the hypocretin-based medication can be a candidate for human PTSD treatment.

CD4+ T cells related to disease severity in elderly and frailty community-acquired pneumonia patients: A retrospective cohort study.

BACKGROUNDS: Elderly and frailty individuals show a more senescent immune system, which may relate to worse outcome in community-acquired pneumonia (CAP). This study aimed to explore prognostic factors related to immune. METHODS: Sixty of elderly (≥65 years) and frailty (clinical frailty scale ≥5 scores) nonsevere CAP patients and 60 severe CAP (SCAP) patients were recruited at our center. Clinical and laboratory data, and several assessment scores were collected. RESULTS: Compared with nonsevere CAP group, the elderly and frailty SCAP patients showed higher level of BMI, PaCO2 and lactate in arterial blood-gas, CURB-65 score, ICU admission, mechanical ventilation, shock accidence, and longer hospital stay using two-tailed t test. The SCAP group also showed increased CRP, IL-6, and PCT, and decreased CD3+ T cells, CD4+ T cells, and CD8+ T cells. Logistic regression analysis showed that CD4+ T cells, IL-6 and PCT were independent prognostic factors for SCAP. The area under the receiver operating characteristic (ROC) curve for CD4+ T cells combined with PCT was 0.771 (95% CI 0.683-0.859), and the sensitivity and specificity were both 76.7%. Paired t test analysis showed that low CD4+ T cells in SCAP patients increased after treatment. CONCLUSIONS: CD4+ T cells decreased in elderly and frailty SCAP patients, and CD4+ T cells combined with PCT were relatively accurate in the prediction of elderly and frailty SCAP.

The APC/C E3 ligase subunit ANAPC11 mediates FOXO3 protein degradation to promote cell proliferation and lymph node metastasis in urothelial bladder cancer.

Urothelial bladder cancer (UBC) is one of the most prevalent malignancies worldwide, with striking tumor heterogeneity. Elucidating the molecular mechanisms that can be exploited for the treatment of aggressive UBC is a particularly relevant goal. Protein ubiquitination is a critical post-translational modification (PTM) that mediates the degradation of target protein via the proteasome. However, the roles of aberrant protein ubiquitination in UBC development and the underlying mechanisms by which it drives tumor progression remain unclear. In this study, taking advantage of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9 technology, we identified the ubiquitin E3 ligase ANAPC11, a critical subunit of the anaphase-promoting complex/cyclosome (APC/C), as a potential oncogenic molecule in UBC cells. Our clinical analysis showed that elevated expression of ANAPC11 was significantly correlated with high T stage, positive lymph node (LN) metastasis, and poor outcomes in UBC patients. By employing a series of in vitro experiments, we demonstrated that ANAPC11 enhanced the proliferation and invasiveness of UBC cells, while knockout of ANAPC11 inhibited the growth and LN metastasis of UBC cells in vivo. By conducting immunoprecipitation coupled with mass spectrometry, we confirmed that ANAPC11 increased the ubiquitination level of the Forkhead transcription factor FOXO3. The resulting decrease in FOXO3 protein stability led to the downregulation of the cell cycle regulator p21 and decreased expression of GULP1, a downstream effector of androgen receptor signaling. Taken together, these findings indicated that ANAPC11 plays an oncogenic role in UBC by modulating FOXO3 protein degradation. The ANAPC11-FOXO3 regulatory axis might serve as a novel therapeutic target for UBC.

LncRNA MIR4435-2HG drives cancer progression by modulating cell cycle regulators and mTOR signaling in stroma-enriched subtypes of urothelial carcinoma of the bladder.

BACKGROUND: The risk for recurrence and metastasis after treatment for urothelial carcinoma of the bladder (UCB) is high. Therefore, identifying efficient prognostic markers and novel therapeutic targets is urgently needed. Several long noncoding RNAs (lncRNAs) have been reported to be correlated with UCB progression. In this study, we found that the subtype-specific lncRNA MIR4435-2 host gene (MIR4435-2HG) plays a novel oncogenic role in UCB. METHODS: RNA-Seq data of TCGA/BLCA were analyzed. The expression of MIR4435-2HG was measured by qRT-PCR in 16 pairs of bladder cancer tissues and adjacent normal tissues. The clinical relecance of MIR4435-2HG was validated via in situ hybridization performed on an in-house cohort of 116 UCB patient samples. RNA pull-down followed by mass spectrometry was performed to identify MIR4435-2HG-binding proteins. To identify signaling pathways involved in MIR4435-2HG activity, comprehensive in vitro and in vivo studies and RNA-Seq assays were performed using UCB cells in which MIR4435-2HG expression was knocked down or exogenously overexpressed. In addition, we performed RNA immunoprecipitation and Western blot analyses to validate the identified MIR4435-2HG-binding proteins and to determine the molecular mechanisms by which MIR4435-2HG promotes UCB progression. RESULTS: We found that MIR4435-2HG was significantly upregulated in the stromal-enriched subtype of UCB. Increased MIR4435-2HG expression was positively correlated with a high histological grade, advanced T stages, larger tumors, lymph node metastasis and a poor prognosis. In vitro experiments revealed that MIR4435-2HG expression silencing suppressed cell proliferation and induced apoptosis. Inhibition of MIR4434-2HG delayed xenograft tumor growth, while MIR4435-2HG overexpression reversed the MIR4435-2HG silencing-induced inhibition of UCB tumor phenotype acquisition. Mechanistically, we found that MIR4435-2HG positively regulated the expression of a variety of cell cycle regulators, including BRCA2 and CCND1. Knocking down MIR4435-2HG increased the sensitivity of tumor cells to the VEGFR inhibitor cediranib. Furthermore, we found that MIR4435-2HG regulated mTOR signaling and epithelial-mesenchymal transition (EMT) signaling pathways by modulating the phosphorylation of mTOR, 70S6K and 4EBP1. Finally, we confirmed that MIR4435-2HG enhances tumor metastasis through regulation of the EMT pathway. CONCLUSIONS: Our data indicate that upregulated MIR4435-2HG expression levels are significantly correlated with a poor prognosis of UCB patients. MIR4435-2HG promotes bladder cancer progression, mediates cell cycle (de)regulation and modulates mTOR signaling. MIR4435-2HG is an oncogenic lncRNA in UCB that may serve as a diagnostic and therapeutic target.