Identification of hypervirulent Klebsiella pneumoniae based on biomarkers and Galleria mellonella infection model.

BACKGROUND: Currently, clinical laboratories lack an effective method to differentiate between classical Klebsiella pneumoniae (cKP) and hypervirulent Klebsiella pneumoniae (hvKP) strains, leading to delays in diagnosing and treating hvKP infections. Previous studies have identified peg-344, iroB, iucA, prmpA, prmpA2, and siderophores (SP) yields greater than 30 µg/ml as reliable markers for distinguishing hvKP from cKp strains. However, these diagnostic tests were conducted on a relatively small study population and lacked sufficient clinical data support. In this study, hvKP strains were identified by biomarker analysis and the Galleria mellonella model. Combined with in vitro and in vivo experiments, the reliability of clinical identification method of hvKP was verified, which provided an experimental basis for timely diagnosis of hvKP infection. RESULTS: According to the clinical data, a total of 108 strains of hvKP were preliminary screened. Among them, 94 strains were further identified using PCR analysis of biomarkers and quantitative determination of SP. The high virulence of hvKP was subsequently confirmed through infection experiments on Galleria mellonella. Additionally, susceptibility testing revealed the identification of 58 carbapenem-resistant hvKP (CR-hvKP) strains and 36 carbapenem-sensitive hvKP (CS-hvKP) strains. By comparing molecular diagnostic indexes, molecular characteristics such as high SP production of CR-hvKP were found. CONCLUSION: The combination of clinical data and molecular diagnostic index analysis effectively enables the identification of hvKP, particularly CR-hvKP. This study provides a scientific basis for accurate clinical identification and timely treatment of hvKP.

The prognostic value of weight loss during radiotherapy among patients with nasopharyngeal carcinoma: a large-scale cohort study.

BACKGROUND: We aim to investigate the prognostic value of weight loss during radiotherapy (RT) among patients with nasopharyngeal carcinoma (NPC). METHODS: A total of 1149 NPC patients who received radical RT were retrospectively analyzed. Patients' weight were measured at initiation of RT (WPre-RT) and every week during RT (WRT1,2,3,4,5,6,7). Percentage of weight loss (PWL) at 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th week of RT (RT-PWL1,2,3,4,5,6,7) were calculated using the following equation: (WPre-RT -WRT1,2,3,4,5,6,7)/WPre-RT × 100%. The optimal threshold of RT-PWL7 was determined by recursive partitioning analyses (RPAs). Our endpoints included disease-free survival (DFS), overall survival (OS), distant metastasis-free survival (DMFS), and locoregional relapse-free survival (LRRFS). RESULTS: The median RT-PWLs were 0, 0, 1.5, 2.9, 4.1, 5.5, 6.6% at 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th week of RT, respectively. RT-PWL7 optimal threshold with respect to DFS was 5.3% based on RPAs. Therefore, a consistent threshold of 5% (<5% vs > ≥5%) was selected to classify NPC patients into low RT-PWL7 and high RT-PWL7 groups for survival analysis. Compared to high RT-PWL7 (≥5%), patients with low RT-PWL7 (< 5%) had significantly better ten-year DFS (61.2% vs 78.8%; P < 0.001), OS (70.1% vs 86.6%; P < 0.001), and DMFS (80.2% vs 88.5%; P = 0.007). However, no difference was observed between LRRFS groups (91.7% vs 94.3%; P = 0.173). In multivariate analysis, high RT-PWL7 was an independent risk factor for DFS (HR, 1.56; 95%CI, 1.19-2.03; P = 0.001), OS (HR, 1.54; 95%CI, 1.11-2.15; P = 0.011), and DMFS (HR, 1.47; 95%CI, 1.03-2.10; P = 0.033) in patients with NPC. In addition, treatment strategy, plasma Epstein-Barr virus DNA, and N stage were associated with weight loss. CONCLUSIONS: High RT-PWL7 was significantly associated with decreased DFS, OS, and DMFS for NPC patients. Clinicians should continuously inform patients on the health impact of minimizing RT-PWL7 under 5% during radiotherapy.

Critical amino acids in the TM2 of EAAT2 are essential for membrane-bound localization, substrate binding, transporter function and anion currents.

Excitatory amino acid transporter 2 (EAAT2), the gene of which is known as solute carrier family 1 member 2 (SLC1A2), is an important membrane-bound transporter that mediates approximately 90% of the transport and clearance of l-glutamate at synapses in the central nervous system (CNS). Transmembrane domain 2 (TM2) of EAAT2 is close to hairpin loop 2 (HP2) and far away from HP1 in the inward-facing conformation. In the present study, 14 crucial amino acid residues of TM2 were identified via alanine-scanning mutations. Further analysis in EAAT2-transfected HeLa cells in vitro showed that alanine substitutions of these residues resulted in a decrease in the efficiency of trafficking/targeting to the plasma membrane and/or reduced functionality of membrane-bound, which resulted in impaired transporter activity. After additional mutations, the transporter activities of some alanine-substitution mutants recovered. Specifically, the P95A mutant decreased EAAT2-associated anion currents. The Michaelis constant (Km ) values of the mutant proteins L85A, L92A and L101A were increased significantly, whereas R87 and P95A were decreased significantly, indicating that the mutations L85A, L92A and L101A reduced the affinity of the transporter and the substrate, whereas R87A and P95A enhanced this affinity. The maximum velocity (Vmax) values of all 14 alanine mutant proteins were decreased significantly, indicating that all these mutations reduced the substrate transport rate. These results suggest that critical residues in TM2 affect not only the protein expression and membrane-bound localization of EAAT2, but also its interactions with substrates. Additionally, our findings elucidate that the P95A mutant decreased EAAT2-related anion currents. Our results indicate that the TM2 of EAAT2 plays a vital role in the transport process. The key residues in TM2 affect protein expression in the membrane, substrate transport and the anion currents of EAAT2.

Environmental enrichment prevents Aß oligomer-induced synaptic dysfunction through mirna-132 and hdac3 signaling pathways.

As the most common cause of progressive cognitive decline in humans, Alzheimer's disease (AD) has been intensively studied, but the mechanisms underlying its profound synaptic dysfunction remain unclear. Here we confirm that exposing wild-type mice to an enriched environment (EE) facilitates signaling in the hippocampus that promotes long-term potentiation (LTP). Exposing the hippocampus of mice kept in standard housing to soluble Aß oligomers impairs LTP, but EE can fully prevent this. Mechanistically, the key molecular features of the EE benefit are an upregulation of miRNA-132 and an inhibition of histone deacetylase (HDAC) signaling. Specifically, soluble Aß oligomers decreased miR-132 expression and increased HDAC3 levels in cultured primary neurons. Further, we provide evidence that HDAC3 is a direct target of miR-132. Overexpressing miR-132 or injecting an HDAC3 inhibitor into mice in standard housing mimics the benefits of EE in enhancing hippocampal LTP and preventing hippocampal impairment by Aß oligomers in vivo. We conclude that EE enhances hippocampal synaptic plasticity by upregulating miRNA-132 and reducing HDAC3 signaling in a way that counteracts the synaptotoxicity of human Aß oligomers. Our findings provide a rationale for prolonged exposure to cognitive novelty and/or epigenetic modulation to lessen the progressive effects of Aß accumulation during human brain aging.

PIWI-interacting RNA-36712 restrains breast cancer progression and chemoresistance by interaction with SEPW1 pseudogene SEPW1P RNA.

BACKGROUND: Breast cancer is one of the most common malignancies and the major cause of cancer-related death in women. Although the importance of PIWI-interacting RNAs (piRNAs) in cancer has been increasingly recognized, few studies have been explored the functional mechanism of piRNAs in breast cancer development and progression. METHODS: We examined the top 20 highly expressed piRNAs based on the analysis of TCGA breast cancer data in two patient cohorts to test the roles of piRNAs in breast cancer. The effects of piRNA-36,712 on the malignant phenotypes and chemosensitivity of breast cancer cells were detected in vitro and in vivo. MS2-RIP and reporter gene assays were conducted to identify the interaction and regulation among piRNA-36,712, miRNAs and SEPW1P. Kaplan-Meier estimate with log-rank test was used to compare patient survival by different piRNA-36,712 expression levels. RESULTS: We found piRNA-36,712 level was significantly lower in breast cancer than in normal breast tissues and low level was correlated with poor clinical outcome in patients. Functional studies demonstrated that piRNA-36,712 interacts with RNAs produced by SEPW1P, a retroprocessed pseudogene of SEPW1, and subsequently inhibits SEPW1 expression through competition of SEPW1 mRNA with SEPW1P RNA for microRNA-7 and microRNA-324. We also found that higher SEPW1 expression due to downregulation of piRNA-36,712 in breast cancer may suppress P53, leading to the upregulated Slug but decreased P21 and E-cadherin levels, thus promoting cancer cell proliferation, invasion and migration. Furthermore, we found that piRNA-36,712 had synergistic anticancer effects with the paclitaxel and doxorubicin, two chemotherapeutic agents for breast cancer. CONCLUSIONS: These findings suggest that piRNA-36,712 is a novel tumor suppressor and may serve as a potential predictor for the prognosis of breast cancer patients.

Biphasic influence of dexamethasone exposure on embryonic vertebrate skeleton development.

Dexamethasone (Dex) has anti-inflammatory and immunomodulatory properties against many conditions. There is a potential teratogenic risk, however, for pregnant women receiving Dex treatment. It has been claimed that Dex exposure during pregnancy could affect osteogenesis in the developing embryo, which still remains highly controversial. In this study, we employed chick embryos to investigate the effects of Dex exposure on skeletal development using combined in vivo and in vitro approach. First, we demonstrated that Dex (10(-8)-10(-6)µmol/egg) exposure resulted in a shortening of the developing long bones of chick embryos, and it accelerated the deposition of calcium salts. Secondly, histological analysis of chick embryo phalanxes exhibited Dex exposure inhibited the proliferation of chondrocytes, increased apoptosis of chondrocytes and osteocytes, and led to atypical arranged hypertrophic chondrocytes. The expression of genes related to skeletogenesis was also analyzed by semi-quantitative RT-PCR. The expression of ALP, Col1a2 and Col2a1 was decreased in the Dex treated phalanxes. A detectable increase was observed in Runx-2 and Mmp-13 expression. We next examined how Dex affected the different stages of skeletogenesis in vitro. Utilizing limb bud mesenchyme micromass cultures, we determined that Dex exposure exerted no effect on apoptosis but impaired chondrogenic cell proliferation. Interestingly, low dose of Dex moderately prompted nodule formation as revealed by alcian blue staining, but higher doses of Dex significantly inhibited similar chondrogenic differentiation. Dex exposure did not induce apoptosis when the chondrogenic precursors were still at the mesenchymal stage, however, cell viability was suppressed when the mesenchyme differentiated into chondrocytes. Alizarin red staining revealed that the capacity to form mineralized bone nodules was correspondingly enhanced as Dex concentrations increased. The mRNA level of Sox-9 was slightly increased in mesenchymal cell mass treated by low concentration of Dex. Mmp-13 expression was obviously up-regulated by Dex in both mesenchymal cells and primary chondrocyte cultures. And Col10a1 expression was also increased by Dex exposure in chondrocyte. In summary, we have revealed that different concentrations of Dex exposure during early gestation could exert a biphasic effect on vertebrate skeletal development.

Modulation of esophageal squamous cell carcinoma progression: the impact of CCR7 on JAK2/STAT3 signaling pathway.

BACKGROUND: Existing studies have already revealed the involvement of C-C chemokine receptor type 7 (CCR7) in diverse human cancers, including esophageal cell squamous carcinoma (ESCA). Our current study, aims to explore the relevant mechanisms implicated. METHODS: ESCA cell lines were collected for CCR7 expression quantification using western blot. Following the transfection, the viability, migration and invasion of ESCA cells were evaluated via cell counting kit-8 and Transwell assays. The specific molecular mechanisms underlying the effects of CCR7 in ESCA cells were explored via calculating the expressions of proteins related to metastasis and Janus kinase 2/signal transduction and transcription activation 3 (JAK2/STAT3) signaling pathway via western blot. The correlation between CCR7 and metastasis-related proteins was explored via Pearson's correlation test. RESULTS: CCR7 was high-expressed in ESCA cells and CCR7 knockdown repressed the viability, migration and invasion of ESCA cells, concurrent with the increased expression of E-cadherin (E-cad, which was also known as CDH1 and lowly expressed in ESCA cells) and the decreased expressions of vimentin (Vim, which was highly expressed in ESCA cells) and matrix metalloproteinase-9 (MMP-9, which was also highly expressed in ESCA cells). Meanwhile, CCR7 was positively correlated with Vim and MMP-9 yet negatively correlated with E-cad in ESCA cells, which indicated that CCR7 has a role in promoting tumor progression in ESCA cells. Besides, the phosphorylation of STAT3 and JAK2 in ESCA cells was elevated, which was diminished following CCR7 knockdown. CONCLUSION: This study proves the modulation of CCR7 on ESCA in vitro, which was achieved via JAK2/STAT3 signaling pathway. Our discovery will provide new therapeutic basis and insights for ESCA.

Overexpression of miR-132-3p contributes to neuronal protection in in vitro and in vivo models of Alzheimer's disease.

One of the neuropathological hallmarks of Alzheimer's disease (AD) is accumulation and deposition of amyloid-beta (Aß1-42) plaques in the hippocampus. Recently, microRNAs (miRNAs), have been demonstrated to play an essential role in AD. We have previously demonstrated that miR-132-3p exerts neuroprotection via regulating histone deacetylase 3 (HDAC3) in a mouse model of AD. In the present study, we further unveiled neuroprotective roles of miR-132-3p in transgenic amyloid precursor protein/presenilin 1 (APP/PS1) mice compared with those in age-matched wild-type C57BL/6 mice. Lentiviral-mediated inhibition or overexpression of miR-132-3p in the hippocampus of APP/PS1 mice was used to explore the contributions of hippocampal miR-132-3p in spatial memory, amyloid burden, apoptosis, and the number of hippocampal cells in a mouse model of AD. Overexpression of hippocampal miR-132-3p ameliorated spatial memory deficits in the Morris water maze, reduced both Aß1-42 accumulation and apoptosis, and promoted the numbers of hippocampal cells in the brains of APP/PS1 mice. Furthermore, trichostatin A (TSA) promoted the expression of miR-132-3p in Aß1-42-burdened neurons while increasing the expression levels of synaptic proteins. Taken together, our results suggest that miR-132-3p may represent a promising therapeutic target for the treatment of AD.

Functional investigation of SLC1A2 variants associated with epilepsy.

Epilepsy is a common neurological disorder and glutamate excitotoxicity plays a key role in epileptic pathogenesis. Astrocytic glutamate transporter GLT-1 is responsible for preventing excitotoxicity via clearing extracellular accumulated glutamate. Previously, three variants (G82R, L85P, and P289R) in SLC1A2 (encoding GLT-1) have been clinically reported to be associated with epilepsy. However, the functional validation and underlying mechanism of these GLT-1 variants in epilepsy remain undetermined. In this study, we reported that these disease-linked mutants significantly decrease glutamate uptake, cell membrane expression of the glutamate transporter, and glutamate-elicited current. Additionally, we found that these variants may disturbed stromal-interacting molecule 1 (STIM1)/Orai1-mediated store-operated Ca2+ entry (SOCE) machinery in the endoplasmic reticulum (ER), in which GLT-1 may be a new partner of SOCE. Furthermore, knock-in mice with disease-associated variants showed a hyperactive phenotype accompanied by reduced glutamate transporter expression. Therefore, GLT-1 is a promising and reliable therapeutic target for epilepsy interventions.

Intraoperative low-dose dopamine is associated with worse survival in patients with hepatocellular carcinoma: A propensity score matching analysis.

Background: Dopamine is widely used in patients during surgery. We evaluated the association between intraoperative low-dose dopamine administration and recurrence-free survival (RFS) and overall survival (OS) in patients with hepatocellular carcinoma (HCC). Methods: Consecutive patients with nonmetastatic HCC who underwent radical hepatectomy were enrolled between 2008 and 2010. Univariate and multivariate logistic regression analyses were used to evaluate the prognostic factors for RFS and OS. Survival outcomes were evaluated using Kaplan-Meier analyses with the log-rank test. A one-to-one propensity score matching (PSM) analysis was performed to reduce confounding bias. Results: A total of 805 HCC patients, including 699 patients who did not receive dopamine consumption and 106 patients who received low-dose dopamine during the operation, were retrospectively analyzed. The patients who were assigned low-dose dopamine had worse RFS (p = 0.009) and OS (p = 0.041) than those who did not receive dopamine. Multivariate regression analysis showed that the intraoperative administration of low-dose dopamine was an independent unfavorable predictor for RFS (p = 0.004) but not for OS (p = 0.059). After PSM, the low-dose dopamine-treated group still had significantly poorer RFS (p = 0.003) and OS (p = 0.002). When stratified by time of recurrence, patients with low-dose dopamine use had a significantly greater chance of recurrence within 2 years (p = 0.007) but not after 2 years (p = 0.186). Conclusions: Intraoperative low-dose dopamine use has a negative impact on RFS and OS in HCC patients who have undergone radical hepatectomy. Further prospective studies are required to assess the effects of low-dose dopamine on surgical outcomes in HCC patients.

Molecular Mechanisms of Glutamate Toxicity in Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disease, the pathological features of which include the presence of Lewy bodies and the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta. However, until recently, research on the pathogenesis and treatment of PD have progressed slowly. Glutamate and dopamine are both important central neurotransmitters in mammals. A lack of enzymatic decomposition of extracellular glutamate results in glutamate accumulating at synapses, which is mainly absorbed by excitatory amino acid transporters (EAATs). Glutamate exerts its physiological effects by binding to and activating ligand-gated ion channels [ionotropic glutamate receptors (iGluRs)] and a class of G-protein-coupled receptors [metabotropic glutamate receptors (mGluRs)]. Timely clearance of glutamate from the synaptic cleft is necessary because high levels of extracellular glutamate overactivate glutamate receptors, resulting in excitotoxic effects in the central nervous system. Additionally, increased concentrations of extracellular glutamate inhibit cystine uptake, leading to glutathione depletion and oxidative glutamate toxicity. Studies have shown that oxidative glutamate toxicity in neurons lacking functional N-methyl-D-aspartate (NMDA) receptors may represent a component of the cellular death pathway induced by excitotoxicity. The association between inflammation and excitotoxicity (i.e., immunoexcitotoxicity) has received increased attention in recent years. Glial activation induces neuroinflammation and can stimulate excessive release of glutamate, which can induce excitotoxicity and, additionally, further exacerbate neuroinflammation. Glutamate, as an important central neurotransmitter, is closely related to the occurrence and development of PD. In this review, we discuss recent progress on elucidating glutamate as a relevant neurotransmitter in PD. Additionally, we summarize the relationship and commonality among glutamate excitotoxicity, oxidative toxicity, and immunoexcitotoxicity in order to posit a holistic view and molecular mechanism of glutamate toxicity in PD.

Serum piRNA-54265 is a New Biomarker for early detection and clinical surveillance of Human Colorectal Cancer.

Background: Our previous study has demonstrated an oncogenic role of PIWI-interacting RNA-54265 (piR-54265) in colorectal cancer (CRC). Here, we investigate whether it can be a blood biomarker for population screening and clinical applications. Methods: Serum piR-54265 levels were determined by a digital PCR method in 209 cancer-free healthy controls, 725 patients with CRC, 1303 patients with other types of digestive cancer and 192 patients with benign colorectal tumors. A prospective case-control analysis was conducted to assess the predictive value of serum piR-54265 for future CRC diagnosis. Receiver operating characteristic (ROC) curve was constructed to quantify the diagnostic performance of serum piR-54265 levels by assessing its sensitivity, specificity and respective areas under curve (AUC). The odds ratios (ORs) were computed using multivariate logistic regression models. Results: Serum piR-54265 levels were significantly elevated only in patients with CRC compared with controls and patients with other cancer types. The AUC for recognizing CRC was 0.896 (95% CI, 0.874-0.914), with a sensitivity and specificity being 85.7% and 65.1% at 1500 copies/µL as a cut-off value. The serum piR-54265 levels in patients declined substantially after surgery but increased significantly again when tumor relapses. The prediagnostic serum piR-54265 levels were significantly associated with future CRC diagnosis, with the ORs of 7.23, 2.80, 2.45, and 1.24 for those whose CRC was diagnosed within 1, 2, 3 and >3 years. Serum piR-54265 test is more sensitive than other blood CRC markers. Conclusion: Serum piR-54265 may serve as a valuable biomarker for CRC screening, early detection and clinical surveillance.

The Characteristics and Survival Outcomes in Patients Aged 70 Years and Older with Nasopharyngeal Carcinoma in the Intensity-Modulated Radiotherapy Era.

PURPOSE: We aim to examine nasopharyngeal carcinoma (NPC) characteristics and survival outcomes in patients aged 70 years and older in the intensity-modulated radiotherapy (IMRT) era. METHODS AND MATERIALS: From 2006 to 2013, 126 non-metastatic NPC patients aged ≥ 70 years who were treated with IMRT +/‒ chemotherapy were included. Adult Comorbidity Evaluation 27 (ACE-27) was used to measure patient comorbidities. The overall survival (OS) and cancer-specific survival (CSS)were calculatedwith the Kaplan-Meier method, and differenceswere compared using the log-rank test. The Cox proportional hazards model was used to carry out multivariate analyses. RESULTS: For the entire group, only two patients (1.6%) presented stage I disease, and up to 84.1% patients had stage III-IVB disease. All patients had a comorbidity score of 0 in 24 (19.0%), 1 in 45 (35.7%), 2 in 42 (33.3%), and 3 in 15 (11.9%) patients. The main acute grade during radiotherapy was 3-4 adverse events consisting of mucositis (25.4%), bone marrow suppression (16.7%), and dermatitis (8.7%). After treatment, four patients (3.2%) developed temporal lobe injury. Five-year CSS and OS rates were 67.3% (95% confidence interval [CI], 58.6% to 77.4%) and 54.0% (95% CI, 45.6% to 63.9%), respectively. Five-year OS was significantly higher for ACE-27 score 0-1 than ACE-27 score 2-3 (72.9% and 39.9%, respectively; p < 0.001). Multivariate analyses showed ACE-27 score 0-1 was significantly associated with superior OS (hazard ratio [HR], 3.02; 95% CI, 1.64 to 5.55; p < 0.001). In addition, the rate of OS was higher for stage I-III than that of stage IV, with borderline significance (HR, 1.67; 95% CI, 0.99 to 2.82; p=0.053). But no significant advantage was observed in OS when chemotherapy was used (p > 0.05). CONCLUSION: Our findings suggest IMRT +/- chemotherapy has a manageable toxicity and provides an acceptable survival in patients aged ≥ 70 years with NPC. ACE-27 score was significantly associated with survival outcomes in this group population.

Combining pretreatment plasma Epstein-Barr virus DNA level and cervical node necrosis improves prognostic stratification in patients with nasopharyngeal carcinoma: A cohort study.

This study aimed to evaluate the prognostic value of combining pretreatment Epstein-Barr virus (EBV) DNA level and cervical node necrosis (CNN) for patients with nasopharyngeal carcinoma (NPC) receiving intensity-modulated radiotherapy (IMRT). A total of 607 incident nonmetastatic NPC patients treated with IMRT ± chemotherapy were reviewed. Patients were divided into four groups based on EBV DNA level and CNN status. The primary endpoint was progression-free survival (PFS). Kaplan-Meier curves with log-rank test were applied to compare survival outcomes and the Cox proportional model was used to identify independent prognostic factors. Pretreatment EBV DNA level and CNN status were independent prognostic factors. Patients in the low-level EBV DNA group or non-CNN group had significantly better 5-year PFS. Multivariate analyses demonstrated that CNN was an independent prognostic factor for overall survival (OS) (HR = 1.927, 95% CI: 1.129-3.290, P = .016), PFS (HR = 1.492, 95% CI: 1.005-2.214, P = .047), distant metastasis-free survival (DMFS) (HR = 1.661, 95% CI: 1.044-2.644, P = .032), but not locoregional relapse-free survival. EBV DNA levels correlated significantly with CNN with a correlation coefficient of .324 (P < .001). Compared with low-level EBV DNA and non-CNN grouping, high-level EBV DNA and CNN grouping had poor PFS. The combined classification was an independent prognostic factor for OS (P < .001), PFS (P = .001), and DMFS (P = .018). Pretreatment plasma EBV DNA level and CNN status both closely correlated with prognosis of NPC patients in the IMRT era. Combined EBV DNA level and CNN status improves risk stratification and prognostic value.

Excessive miR-25-3p maturation via N6-methyladenosine stimulated by cigarette smoke promotes pancreatic cancer progression.

N6-methyladenosine (m6A) modification is an important mechanism in miRNA processing and maturation, but the role of its aberrant regulation in human diseases remained unclear. Here, we demonstrate that oncogenic primary microRNA-25 (miR-25) in pancreatic duct epithelial cells can be excessively maturated by cigarette smoke condensate (CSC) via enhanced m6A modification that is mediated by NF-κB associated protein (NKAP). This modification is catalyzed by overexpressed methyltransferase-like 3 (METTL3) due to hypomethylation of the METTL3 promoter also caused by CSC. Mature miR-25, miR-25-3p, suppresses PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2), resulting in the activation of oncogenic AKT-p70S6K signaling, which provokes malignant phenotypes of pancreatic cancer cells. High levels of miR-25-3p are detected in smokers and in pancreatic cancers tissues that are correlated with poor prognosis of pancreatic cancer patients. These results collectively indicate that cigarette smoke-induced miR-25-3p excessive maturation via m6A modification promotes the development and progression of pancreatic cancer.

Simvastatin Inhibits Activation of NADPH Oxidase/p38 MAPK Pathway and Enhances Expression of Antioxidant Protein in Parkinson Disease Models.

Evidence suggests that oxidative stress is involved in the pathogenesis of Parkinson disease (PD). Simvastatin has been suggested to protect against oxidative stress in several diseases. However, the molecular mechanisms by which simvastatin protects against neuropathology and oxidative damage in PD are poorly elucidated. In this study, we aimed to investigate the potential neuroprotective effects of simvastatin owing to its anti-oxidative properties in 6-hydroxydopamine (6-OHDA)-treated SH-SY5Y cells and mice. The results of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence and CCK-8 assay demonstrated that simvastatin reduced intracellular reactive oxygen species (ROS) levels and reversed apoptosis in 6-OHDA-treated SH-SY5Y cells. Mechanistic studies revealed that 6-OHDA-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/p38 mitogen-activated protein kinase (MAPK) pathway was inhibited and nuclear factor-κB (NF-κB) nuclear transcription decreased in SH-SY5Y cells after simvastatin treatment. Enhanced expression levels of superoxide dismutase (SOD), heme oxygenase-1 (HO-1), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and glutamate-cysteine ligase modifier subunit (GCLM) were observed after simvastatin treatment in 6-OHDA-treated SH-SY5Y cells. In vivo studies revealed that administration of simvastatin by gavage decreased limb-use asymmetry and apomorphine-induced rotations in 6-OHDA-lesioned mice. Simvastatin increased dopaminergic neurons and reduced protein tyrosine nitration and gliosis in the midbrain of PD mice. An inhibitory effect on activation of the NADPH oxidase/p38 MAPK was observed, and increased antioxidant protein expression in the midbrain were seen in the simvastatin plus 6-OHDA group compared with the 6-OHDA-lesioned group. Taken together, these results demonstrate that simvastatin might inhibit the activation of NADPH oxidase/p38 MAPK pathway, enhance antioxidant protein expression and protect against oxidative stress, thereby providing a novel antioxidant mechanism that has therapeutic validity.

PIWI-interacting RNA-54265 is oncogenic and a potential therapeutic target in colorectal adenocarcinoma.

Although PIWI-interacting RNAs (piRNAs) have recently been linked to human diseases, their roles and functions in malignancies remain unclear. This study aimed to investigate the significance of some piRNAs in colorectal cancer (CRC). Methods: We first analyzed the expression profile of piRNAs in CRC using the TCGA and GEO databases. The top 20 highly expressed piRNAs were selected and tested in our CRC tumor and non-tumor tissue samples. We then examined the relevance of the significantly differentially expressed piRNA to the CRC outcomes in 218 patients receiving postoperative chemotherapy and 317 patients receiving neoadjuvant chemotherapy. A series of biochemical and molecular biological assays were conducted to elucidate the functional mechanism of a piRNA of interest in CRC. Furthermore, experiments with mice xenografts were performed to evaluate the therapeutic effect of an inhibitor specific to the piRNA. Results: We found that among the examined 20 piRNAs, only piRNA-54265 was overexpressed in CRC compared with non-tumor tissues and higher levels in tumor or in serum were significantly associated with poor survival in patients. Functional assays demonstrated that piRNA-54265 binds PIWIL2 protein and this is necessary for the formation of PIWIL2/STAT3/phosphorylated-SRC (p-SRC) complex, which activates STAT3 signaling and promotes proliferation, metastasis and chemoresistance of CRC cells. Treatment with a piRNA-54265 inhibitor significantly suppressed the growth and metastasis of implanted tumors in mice. Conclusion: These results indicate that piRNA-54265 is an oncogenic RNA in CRC and thus might be a therapeutic target.

Effect of Prolonged Radiotherapy Treatment Time on Survival Outcomes after Intensity-Modulated Radiation Therapy in Nasopharyngeal Carcinoma.

PURPOSE: To estimate the influence of prolonged radiation treatment time (RTT) on survival outcomes in nasopharyngeal carcinoma after continuous intensity-modulated radiation therapy. METHODS AND MATERIALS: Retrospectively review 321 patients with NPC treated between October 2009 and December 2010 and all of them underwent simultaneous accelerated intensity-modulated radiation therapy. The fractionated dose was 2-2.47 Gy/F (median 2.27 Gy), and the total dose for nasopharyngeal region was 64-74 Gy/ 28-33 fractions. The association of prolonged RTT and treatment interruption with PFS, LRFS and DFFS were assessed by univariate analysis and multivariate analysis. Survival analyses were carried out using Kaplan-Meier methodology and the log-rank test was used to assess the difference. The Cox regression proportional hazard model was used for multivariate analyses and evaluating the prognostic parameters for PFS, LRFS and DFFS. RESULTS: Univariate analysis revealed no significant associations between prolonged RTT and PFS, LRFS, DFFS when dichotomized using various cut-off values (all P>0.05). In multivariate analysis, RTT (range, 36-63 days) as a continuous variable, had no influence on any survival outcome as well (P>0.05). T and N classification were independent prognostic factors for PFS, LRFS and DFFS (all P<0.05, except T classification for LRFS, P = 0.057). Age was an independent prognostic factor for PFS (hazard ratio [HR], 1.033; P = 0.008) and DFFS (HR, 1.032; P = 0.043). CONCLUSION: We conclude that no such association between survival outcomes and radiation treatment duration (range: 36-63 days) can be found in the present retrospective study, however, we have to remind that prolongation in treatment should be limited in clinical application and interruptions caused by any reason should be minimized as much as possible.

Plasma Epstein-Barr Viral Deoxyribonucleic Acid Predicts Worse Outcomes in Pediatric Nonmetastatic Nasopharyngeal Carcinoma Patients: An Observational Study of 89 Cases in an Endemic Area.

To evaluate the clinical significance of pretreatment levels of plasma Epstein-Barr virus DNA (pEBV DNA) on prognoses in pediatric nasopharyngeal carcinoma (NPC) patients. Eighty-nine patients aged 21 years old or younger with nonmetastatic NPC were evaluated to determine the effect of pEBV DNA levels on progression-free survival (PFS), distant metastasis-free survival (DMFS), and overall survival (OS). Survival probabilities in patient groups that were segregated by clinical stage or pEBV DNA load (low or high) were compared. The median pretreatment concentrations of pEBV DNA were 3440 copies/mL in 35 patients with stage III disease and 14,900 copies/mL in 50 patients with stage IV disease (P = 0.059). The median concentration of pEBV DNA was 34,500 copies/mL in 17 patients with relapse, which was higher than the concentration in 72 patients without relapse, who had a median level of 4985 copies/mL (P = 0.057). Further study showed that pretreatment pEBV DNA load was an independent prognostic indicator in pediatric NPC patients. High pEBV DNA was associated with adverse clinical outcomes, including PFS [3-year PFS rate = 80.5% versus 95.8%, hazard ratio (HR) = 5.00, 95% confidence interval (CI) = 1.00-25.00; P = 0.050], DMFS (3-year DMFS rate = 80.5% versus 95.8%, HR = 5.20, 95% CI = 1.04-26.00; P = 0.045), and OS (3-year OS rate = 82.9% versus 95.8%, HR = 5.41, 95% CI = 1.08-27.22; P = 0.040). Pretreatment pEBV DNA load was an independent prognostic indicator for PFS, DMFS, and OS in pediatric patients with NPC. Prospective studies, however, are needed to validate these results.