The neddylation pathway has been recently identified as an attractive anticancer target, and MLN4924, a specific NEDD8-activating enzyme inhibitor, has been developed as a first-in-class anticancer agent. However, neither the status of the neddylation pathway in glioblastoma (GBM) nor the effect of MLN4924 against GBM has been systematically investigated yet. To measure the activation state of the neddylation pathway in GBM, expression of the NEDD8-activating enzyme (E1), NEDD8-conjugating enzyme (E2), and global protein neddylation in GBM tumor tissues versus adjacent tissues were examined by immunoblotting analysis and immunohistochemistry staining. To assess the therapeutic efficacy of neddylation inhibition in GBM, cell proliferation in vitro and tumor growth in vivo were determined upon neddylation inhibition by MLN4924, an investigational NEDD8-activating enzyme inhibitor. The neddylation pathway was overactivated in a majority of GBM tumor tissues when compared with adjacent normal tissues. The upregulation of this pathway in GBM tissues was positively correlated with high-grade disease and postoperative recurrence but was negatively associated with patient overall survival. MLN4924 treatment inhibited cullin neddylation, inactivated cullin-RING E3 ligase, and led to the accumulation of tumor-suppressive cullin-RING E3 ligase substrates to trigger cell-cycle arrest and senescence or apoptosis in a cell-line dependent manner. Moreover, inhibition of neddylation by MLN4924 significantly suppressed tumor growth in an orthotopic xenograft model of human GBM. Our study indicates that an overactivated neddylation pathway may be involved in GBM progression and that inhibition of this oncogenic pathway is a potentially new therapeutic approach for GBM.

Aim: To identify intestinal microbiota compositions in elderly functional constipation (FC) patients. Materials & methods: Fecal samples from 61 FC patients and 48 healthy age-matched volunteers we...

Background: Shanghai, in east China, has one of the world’s highest burdens ofHelicobacter pyloriinfection. While multidrug regimens can effectively eradicateH. pylori, the increasing prevalence of antibiotic resistance (AR) inH. pylorihas been recognized by the WHO as ‘high priority’ for urgent need of new therapies. Moreover, the genetic characteristics ofH. pyloriAR in Shanghai is under-reported. The purpose of this study was to determine the resistance prevalence, re-substantiate resistance-conferring mutations, and investigate novel genetic elements associated withH. pyloriAR.Results: We performed whole genome sequencing and antimicrobial susceptibility testing of 112H. pyloristrains isolated from gastric biopsy specimens from Shanghai patients with different gastric diseases. No strains were resistant to amoxicillin. Levofloxacin, metronidazole and clarithromycin resistance was observed in 39 (34.8%), 73 (65.2%) and 18 (16.1%) strains, respectively. There was no association between gastroscopy diagnosis and resistance phenotypes. We reported the presence or absence of several subsystem protein coding genes includinghopE,hofF,spaB,cagYandpflA, and a combination of CRISPRs, which were potentially correlated with resistance phenotypes. TheH. pyloristrains were also annotated for 80 genome-wide AR genes (ARGs). A genome-wide ARG analysis was performed for the three antibiotics by correlating the phenotypes with the genetic variants, which identified the well-known intrinsic mutations conferring resistance to levofloxacin (N87T/I and/or D91G/Y mutations ingyrA), metronidazole (I38V mutation infdxB), and clarithromycin (A2143G and/or A2142G mutations in 23S rRNA), and added 174 novel variations, including 23 non-synonymous SNPs and 48 frameshift Indels that were significantly enriched in either the antibiotic-resistant or antibiotic-susceptible bacterial populations. The variant-level linkage disequilibrium analysis highlighted variations in a protease Lon with strong co-occurring correlation with a series of resistance-associated variants.Conclusion: Our study revealed multidrug antibiotic resistance inH. pyloristrains from Shanghai, which was characterized by high metronidazole and moderate levofloxacin resistance, and identified specific genomic characteristics in relation toH. pyloriAR. Continued surveillance ofH. pyloriAR in Shanghai is warranted in order to establish appropriate eradication treatment regimens for this population.

Objectives: The present study aimed to reveal expression status of the neddylation enzymes in HNSCC and to elucidate the anticancer efficacy and the underlying mechanisms of inhibiting neddylation pathway.Materials and methods: The expression levels of neddylation enzymes were estimated by Western blotting in human HNSCC specimens and bioinformatics analysis of the cancer genome atlas (TCGA) database. Cell apoptosis was evaluated by Annexin V fluorescein isothiocyanate/propidium iodide (Annexin V‐FITC/PI) stain and fluorescence‐activated cell sorting (FACS). Small interfering RNA (siRNA) and the CRISPR‐Cas9 system were used to elucidate the underlying molecular mechanism of MLN4924‐induced HNSCC apoptosis.Results: Expression levels of NAE1 and UBC12 were prominently higher in HNSCC tissues than that in normal tissues. Inactivation of the neddylation pathway significantly inhibited malignant phenotypes of HNSCC cells. Mechanistic studies revealed that MLN4924 induced the accumulation of CRL ligase substrate c‐Myc that transcriptionally activated pro‐apoptotic protein Noxa, which triggered apoptosis in HNSCC.Conclusions: These findings determined the over‐expression levels of neddylation enzymes in HNSCC and revealed novel mechanisms underlying neddylation inhibition induced growth suppression in HNSCC cells, which provided preclinical evidence for further clinical evaluation of neddylation inhibitors (eg, MLN4924) for the treatment of HNSCC.

Background: Urinary tract infections (UTIs) are one the most common infections. The rapid and accurate identification of uropathogens, and the determination of antimicrobial susceptibility, are essential aspects of the management of UTIs. However, existing detection methods are associated with certain limitations. In this study, a new urinary tract infection high-throughput multiplex genetic detection system (UTI-HMGS) was developed for the semi-quantitative detection of 18 pathogens and the simultaneously screening of nine resistance genes directly from the clinical urine sample within 4 hours. Methods: We designed and optimized a multiplex polymerase chain reaction (PCR) involving fluorescent dye-labeled specific primers to detect 18 pathogens and nine resistance genes. The specificity of the UTI-HMGS was tested using standard strains or plasmids for each gene target. The sensitivity of the UTI-HMGS assay was tested by the detection of serial tenfold dilutions of plasmids or simulated positive urine samples. We also collected clinical urine samples and used these to perform urine culture and antimicrobial susceptibility testing (AST). Finally, all urine samples were detected by UTI-HMGS and the results were compared with both urine culture and Sanger sequencing. Results: UTI-HMGS showed high levels of sensitivity and specificity for the detection of uropathogens when compared with culture and sequencing. In addition, ten species of bacteria and three species of fungi were detected semi-quantitatively to allow accurate discrimination of significant bacteriuria and candiduria. The sensitivity of the UTI-HMGS for the all the target genes could reach 50 copies per reaction. In total, 531 urine samples were collected and analyzed by UTI-HMGS, which exhibited high levels of sensitivity and specificity for the detection of uropathogens and resistance genes when compared with Sanger sequencing. The results from UTI-HMGS showed that the detection rates of 15 pathogens were significantly higher (P<0.05) than that of the culture method. In addition, there were 41(7.72%, 41/531) urine samples were positive for difficult-to-culture pathogens, which were missed detected by routine culture method. Conclusions: UTI-HMGS proved to be an efficient method for the direct semi-quantitative detection of 18 uropathogens and the simultaneously screening of nine antibiotic resistance genes in urine samples. The UTI-HMGS could represent an alternative method for the clinical detection and monitoring of antibiotic resistance.