PFKFB3 (6-phosphofructo-2-kinase) synthesizes fructose 2,6-bisphosphate (F2,6P2), which is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), the rate-limiting enzyme of glycolysis. Overexpression of the PFKFB3 enzyme leads to high glycolytic metabolism, which is required for cancer cells to survive in the harsh tumor microenvironment. The objective of this study was to investigate the antitumor activity of PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), a small molecule inhibitor of PFKFB3, against gastric cancer and to explore its potential mechanisms. The effects of PFK15 on proliferation, apoptosis and cell cycle progression in gastric cancer cells were evaluated by cytotoxicity and apoptosis assays, flow cytometry, and western blotting. In addition, the invasion inhibition effects of PFK15 were measured by transwell invasion assay and western blot analysis, and a xenograft tumor model was used to verify the therapeutic effect of PFK15 in vivo. Results showed that PFK15 inhibited the proliferation, caused cell cycle arrest in G0/G1 phase by blocking the Cyclin-CDKs/Rb/E2F signaling pathway, and induced apoptosis through mitochondria in gastric cancer cells. Tumor volume and weight were also significantly reduced upon intraperitoneal injection with PFK15 at 25 mg/kg. In addition, PFK15 inhibited the invasion of gastric cancer cells by downregulating focal adhesion kinase (FAK) expression and upregulating E-cadherin expression. Taken together, our findings indicate that PFK15 is a promising anticancer drug for treating gastric cancer.

PurposeThe application of metagenomic next-generation sequencing (mNGS) in the diagnosis of tuberculous meningitis (TBM) remains poorly characterized. Here, we retrospectively analyzed data from patients with TBM who had taken both mNGS and conventional tests including culture of Mycobacterium tuberculosis (MTB), polymerase chain reaction (PCR) and acid-fast bacillus (AFB) stain, and the sensitivity and specificity of these methods were compared.MethodsWe retrospectively recruited TBM patients admitted to the hospital between December 2015 and October 2018. The first collection of cerebrospinal fluid (CSF) samples underwent both mNGS and conventional tests. In addition, patients with bacterial/cryptococcal meningitis or viral meningoencephalitis were mNGS positive controls, and a patient with auto-immune encephalitis was an mNGS negative control.ResultsTwenty three TBM patients were classified as 12 definite and 11 clinical diagnoses, which were based on clinical manifestations, pathogen evidence, CSF parameters, brain imaging, and treatment response. The mNGS method identified sequences of Mycobacterium tuberculosis complex (MBTC) from 18 samples (18/23, 78.26%). In patients with definite TBM, the sensitivity of mNGS, AFB, PCR, and culture to detect MTB in the first CSF samples were 66.67, 33.33, 25, and 8.33%, respectively. The specificity of each method was 100%. Among the four negative mNGS cases (4/23, 17.39%), three turned out positive by repeated AFB stain. The agreement of mNGS with the total of conventional methods was 44.44% (8/18). Combination of mNGS and conventional methods increased the detection rate to 95.65%. One patient was diagnosed as complex of TBM and cryptococcal meningitis, in which AFB stain and cryptococcal antigen enzyme immunoassay were positive and the DNA of Cryptococcus neoformans was detected by mNGS.ConclusionOur study indicates that mNGS is an alternative method to detect the presence of mycobacterial DNA in CSF samples from patients with TBM and deserves to be applied as a front-line CSF test.

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Background and purpose: We retrospectively analyzed the clinical characteristics of children with autoimmune encephalitis (AE) in two Chinese tertiary pediatric neurology centers. We also compared anti-NMDAR encephalitis with and without co-positive MOG antibody, as well as specific autoantibody-positive AE and autoantibody-negative but probable AE. Methods: A retrospective study of children (0–18 years old) with AE in Peking University First Hospital and Children's Hospital Affiliated to Capital Institute of Pediatrics was carried out from May 2012 to January 2017. Demographics, clinical features, laboratory, and imaging findings, outcome, and co-positivity with MOG antibody were analyzed. Results: A total of 103 children had AE, 89 (86.4%) had anti-NMDAR encephalitis, 2 (1.9%) had anti-LGI1 encephalitis, 1 (0.9%) had anti-CASPR2 encephalitis, and 11 (10.7%) were diagnosed as autoantibody-negative but probable AE. Among the 89 children with anti-NMDAR encephalitis, 35 were males and 54 were females. The follow-up time was 1–3 years. A total of 15 cases (15/89, 16.9%) with anti-NMDAR encephalitis had co-positive MOG antibody (serum or cerebrospinal fluid or both). These patients were more likely to experience relapse later in life ( P = 0.014). We had two cases with anti-LGI1 encephalitis, that is, one with sleep disorder onset, and the other one with seizure onset, both of whom recovered after treatment. One case with anti-CASPR2 encephalitis was treated with an antiepileptic drug and fully recovered. There were 11 cases diagnosed as autoantibody-negative but probable AE who had relatively poorer outcome than those with autoantibody-positive AE (15.2%, 14/89). However, the difference was not significant ( P = 0.08). Only one 12-year-old girl with NMDAR-antibody AE had ovarian teratoma. Conclusion: Most subjects with AE in our Chinese cohort had anti-NMDAR AE, which had relatively good prognosis. Children with anti-LGI1 or anti-CASPR2 encephalitis were rare and showed good response on immunotherapy. Co-positive MOG antibody was relatively common in anti-NMDAR encephalitis, which was related to high relapse rate. In our study, the prognosis of autoantibody-negative but probable AE seemed worse than that of specific autoantibody-positive AE.

BackgroundDiuretic agents are widely used on the treatment of water retention related diseases, among which acetazolamide (AZA) acts originally as a carbonic anhydrase (CA) inhibitor. Aquaporin-1 (AQP1) being located in renal proximal tubules is required for urine concentration. Previously our lab has reported AZA putatively modulated AQP1. Aim of this study is to testify our hypothesis that regulating AQP1 may mediate diuretic effect of AZA.Methodology/Principal FindingsFor in vivo study, we utilized Sprague Dawley rats, as well as AQP1 knock-out (AQP1−/−) mice to examine urine volume, and human kidney-2 (HK-2) cell line was used for in vitro mechanism study. In our present study we found that AZA decreased CAs activity initially but the activity gradually recovered. Contrarily, diuretic effect was consistently significant. AQP1 protein expression was significantly decreased on day 7 and 14. By utilizing AQP1−/− mice, we found diuretic effect of AZA was cancelled on day 14, while urine volume continuously increased in wild-type mice. Surface plasmon resonance (SPR) results indicated AQP1 was physiologically bound by myosin heavy chain (MHC), immunoprecipitation and immunofluorescence results confirmed this protein interaction. In vitro study results proved AZA facilitated AQP1 translocation onto cell membrane by promoting interaction with MHC, dependent on ERK/ myosin light chain kinase (MLCK) pathway activation. MHC inhibitor BDM and ERK inhibitor U0126 both abolished above effect of AZA. Eventually AZA induced AQP1 ubiquitination, while proteasome inhibitor MG132 reversed AZA's down-regulating effect upon AQP1.Conclusions/SignificanceOur results identified AZA exerted diuretic effect through an innovative mechanism by regulating AQP1 and verified its inhibitory mechanism was via promoting MHC-dependent translocation onto cell membrane and then ubiquitin mediated degradation, implicating a novel mechanism and target for diuretic agent discovering.

Background/Aims: Acute kidney injury (AKI) is a major complication of kidney transplantation, resulting in early graft dysfunction. Since diuretic acetazolamide (AZA) has been shown to improve contrast induced AKI, we hypothesized that AZA also protected against ischemia-reperfusion (I/R) caused AKI. Methods: An in vivo mouse renal I/R injury model and an in vitro H₂O₂ stimulated HK-2 cell injury model were utilized to examine the renoprotective effect of AZA. Renal injury and blood flow were measured. Nitric oxide synthase (eNOS)/Nitric oxide (NO), cell apoptosis and hypoxia-inducible factor-1α (HIF-1α) changes were analyzed. Results: AZA reduced kidney injury scores and improved renal function by decreasing serum creatinine and BUN levels after I/R. Impaired renal blood flow was restored by increasing eNOS activities and NO production, as indicated by Laser Doppler imaging. TUNEL staining presented that AZA reduced apoptotic cells due to attenuated caspase activation and increased Bcl-2/Bax ratio. Furthermore, HIF-1α induction by AZA was demonstrated. AZA also enhanced in vitro NO production, reduced cell apoptosis and increased HIF-1α expression. Knockdown of HIF-1α by RNAi confirmed that AZA exerted its protective role depending on HIF-1α. AZA's effects were significantly reduced by Akt inhibitor LY294002. Conclusions: The present study demonstrated that AZA exerted a renoprotective role against I/R induced AKI through activating HIF-1α and downstream pathways.

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The purposes of this study are to investigate the antitumor activities of NSK-01105, a novel sorafenib derivative, in in vitro and in vivo models, and explore the potential mechanisms. The effects of NSK-01105 on proliferation and apoptosis of prostate cancer cells were established by cytotoxicity assays, apoptosis analysis, flow cytometry analysis, and Western blot analysis. Two xenograft tumor models were used to verify the therapeutic effect of NSK-01105 in vivo. NSK-01105 exhibited broad-spectrum antitumor activity, particularly in prostate cancer cells. Characterization of apoptosis morphology was observed, and the percentage of apoptosis-positive cells significantly increased after NSK-01105 treatment for 24 h. Furthermore, a significant increase of the "sub-G1" population in LNCaP and PC-3 cells after NSK-01105 treatment was determined by cell cycle analysis. Tumor growth was significantly suppressed by once daily oral 30 mg/kg dose of NSK-01105 with the inhibition rates of 63.82% in LNCaP models and 64.29% in PC-3 models, respectively. The activation of Raf-1 kinase and epidermal growth factor receptor was downregulated by NSK-01105 at 10 μmol/L. Consequently, the dual inhibitions of Raf/MEK/ERK and PI3K/Akt/mTOR signal pathways were observed by Western blot analysis. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors by inhibiting cell proliferation and inducing apoptosis. NSK-01105 appears to be a promising orally active anticancer drug and deserves further investigation.