In vitro inhibition of Pseudomonas aeruginosa PAO1 biofilm formation by DZ2002 through regulation of extracellular DNA and alginate production.

Introduction: Pseudomonas aeruginosa (P. aeruginosa) is a common pathogen associated with biofilm infections, which can lead to persistent infections. Therefore, there is an urgent need to develop new anti-biofilm drugs. DZ2002 is a reversible inhibitor that targets S-adenosylhomocysteine hydrolase and possesses anti-inflammatory and immune-regulatory activities. However, its anti-biofilm activity has not been reported yet. Methods and results: Therefore, we investigated the effect of DZ2002 on P. aeruginosa PAO1 biofilm formation by crystal violet staining (CV), real-time quantitative polymerase chain reaction (RT-qPCR) and confocal laser scanning microscopy (CLSM). The results indicated that although DZ2002 didn't affect the growth of planktonic PAO1, it could significantly inhibit the formation of mature biofilms. During the inhibition of biofilm formation by DZ2002, there was a parallel decrease in the synthesis of alginate and the expression level of alginate genes, along with a weakening of swarming motility. However, these results were unrelated to the expression of lasI, lasR, rhII, rhIR. Additionally, we also found that after treatment with DZ2002, the biofilms and extracellular DNA content of PAO1 were significantly reduced. Molecular docking results further confirmed that DZ2002 had a strong binding affinity with the active site of S-adenosylhomocysteine hydrolase (SahH) of PAO1. Discussion: In summary, our results indicated that DZ2002 may interact with SahH in PAO1, inhibiting the formation of mature biofilms by downregulating alginate synthesis, extracellular DNA production and swarming motility. These findings demonstrate the potential value of DZ2002 in treating biofilm infections associated with P. aeruginosa.

The signature based on seven genomic instability-related genes could predict the prognosis of acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) is the most common acute blood malignancy in adults. The complicated and dynamic genomic instability (GI) is the most prominent feature of AML. Our study aimed to explore the prognostic value of GI-related genes in AML patients. METHODS: The mRNA data and mutation data were downloaded from the TCGA and GEO databases. Differential expression analyses were completed in limma package. GO and KEGG functional enrichment was conducted using clusterProfiler function of R. Univariate Cox and LASSO Cox regression analyses were performed to screen key genes for Risk score model construction. Nomogram was built with rms package. RESULTS: We identified 114 DEGs between high TMB patients and low TMB AML patients, which were significantly enriched in 429 GO terms and 13 KEGG pathways. Based on the univariate Cox and LASSO Cox regression analyses, seven optimal genes were finally applied for Risk score model construction, including SELE, LGALS1, ITGAX, TMEM200A, SLC25A21, S100A4 and CRIP1. The Risk score could reliably predict the prognosis of AML patients. Age and Risk score were both independent prognostic indicators for AML, and the Nomogram based on them could also reliably predict the OS of AML patients. CONCLUSIONS: A prognostic signature based on seven GI-related genes and a predictive Nomogram for AML patients are finally successfully constructed.

[Enterovirus 71 can induce autophagy and apoptosis of THP-1 macrophages].

OBJECTIVE: To investigate enterovirus 71 (EV71)-induced of autophagy, apoptosis and the related signaling pathways in THP-1 macrophages. METHODS: THP-1 macrophages were infected with EV71 at the multiplicity of infection (MOI) of 0.1 for 2, 8 or 16 h, and the cell proliferation and toxicity were analyzed using CCK-8 kit. The intracellular viral nucleic acid in THP-1 macrophages were detected by fluorescence quantitative PCR, and the ultrastructural changes of the cells were observed using transmission electron microscopy. Cell apoptosis induced by EV71 infection was detected using Hoechst 33342 staining and AnnexinV/PI double staining. Western blotting was performed for analysis of changes in autophagy and apoptosis of the cells and in the expressions of the related proteins. The effect of EV71 infection on apoptosis of THP-1 macrophages incubated with 3-MA and Ac-DEVD-CHO inhibitor for 2 h was assessed using Western blotting. RESULTS: EV71 infection significantly lowered the cell survival rate of THP-1 macrophages at 2, 8 h and 16 h after the infection (P < 0.05). The total copy number of viral nucleic acid in THP-1 macrophages incubated with EV71 increased significantly and progressively over time (P < 0.01). Intracellular autophagosomes and virions could be seen in EV71-infected THP-1 macrophages. The total apoptotic rate of the infected cell also increased significantly over time (P < 0.01). EV71 infection significantly increased LC3 conversion (LC3-Ⅱ/ LC3-I) and the expression of cleaved caspase 3 protein and decreased the protein expressions of p62, Bcl-2 and caspase-3 (P < 0.01) without causing obvious changes in cleaved caspase-8 (P>0.05). 3-MA significantly inhibited the EV71-induced autophagy of THP-1 macrophages and reduced LC3 conversion (LC3-Ⅱ/LC3-I) and p62 protein expression at 8 h after EV71 infection (P < 0.01). Compared with DMSO, Ac-DEVD-CHO significantly inhibited EV71-induced apoptosis of THP-1 macrophages (15.5% vs 7.7%, P < 0.01). CONCLUSIONS: EV71 not only can infect and replicate in THP-1 macrophages, but also induces autophagy and cell apoptosis possibly by activating LC3/p62 autophagy pathway and caspase apoptosis pathway.

Downregulation of glypican-3 expression increases migration, invasion, and tumorigenicity of human ovarian cancer cells.

Glypican-3 (GPC3) is a membrane of heparan sulfate proteoglycan family involved in cell proliferation, adhesion, migration, invasion, and differentiation during the development of the majority of mesodermal tissues and organs. GPC3 is explored as a potential biomarker for hepatocellular carcinoma screening. However, as a tumor-associated antigen, its role in ovarian cancer remains elusive. In this report, the expression levels of GPC3 in the various ovarian cancer cells were determined with quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and GPC3 expression in ovarian cancer UCI 101 and A2780 cells was knocked down by siRNA transfection, and the effects of GPC3 knockdown on in vitro cell proliferation, migration, and invasion were respectively analyzed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and Transwell migration assay. Additionally, the effect of GPC3 knockdown on in vivo tumorigenesis were investigated in athymic nude mice. The results indicated that GPC3 knockdown significantly promoted cell proliferation and increased cell migration and invasion by upregulation of matrix metalloproteinase (MMP)-2 and MMP-9 expression and downregulation of tissue inhibitor of metalloproteinase-1 expression. Additionally, GPC3 knockdown also increased in vivo tumorigenicity of UCI 101 and A2780 cells and final tumor weights and volumes after subcutaneous cell injection in the nude mice. The results of immunohistochemical staining and Western blotting both demonstrated a lower expression of GPC3 antigen in the tumors of GPC3 knockdown groups than that of negative control groups. Moreover, transforming growth factor-ß2 protein expression in the tumors of GPC3 knockdown groups was significantly increased, which at least contributed to tumor growth in the nude mice. Taken together, these findings suggest that GPC3 knockdown promotes the progression of human ovarian cancer cells by increasing their migration, invasion, and tumorigenicity, and suggest that GPC3 is a potential therapeutic target for ovarian cancer patients.

Polyphosphate kinase 1 is required for the pathogenesis process of meningitic Escherichia coli K1 (RS218).

AIM: Polyphosphate kinase 1 (PPK1), encoded by the ppk1 gene, is one of the major enzymes to reversibly catalyze the synthesis of polyphosphate (poly P) from the terminal phosphate of ATP. Poly P confers resistance to stress in a number of bacterial species but its role in the virulence of meningitic bacterial pathogens is unknown. The aim of this study was to determine the role of PPK1 in the pathogenesis of Escherichia coli meningitis. MATERIALS & METHODS: An isogenic in-frame ppk1 deletion mutant (PD44) of E. coli K1 strain E44 was constructed and characterized. Human brain microvascular endothelial cells and neonatal rats were used as the in vitro and in vivo models, respectively, to evaluate bacterial adhesion/invasion and the abilities of bacteria crossing the blood-brain barrier (BBB) to cause meningitis. The survival of PD44 and E44 under osmotic and acid stress conditions were also examined. RESULTS: Poly P levels in E44 were clearly higher than those in PD44, especially at the stationary phase (SP). The ppk1 deletion mutant PD44 also showed poor survival rates during osmotic shock and acidic challenge, which the bacteria would face during pathogenesis. In vitro and in vivo assays revealed that PD44 was defective in bacterial adhesion and translocation across the BBB. By using the Evans blue method, we found that E44-induced permeability of the BBB in neonatal rats was significantly higher than that of the animals infected with PD44. Cytokine ELISA results showed that the TNF-α and IL-1ß levels in the serum and brain tissues of the neonatal rats infected with PD44 were lower than that of the E44 group. A more obvious meningeal inflammation could be observed in the brain tissues of the rats infected with E44 when compared with that of the PD44 group by histopathological examination. Furthermore, the mRNA expression of IbeR, which is an RpoS-like regulator contributing to the SP regulation in E44, was found to be decreased in PD44 when compared with the parent strain. PD44 was also deficient in mRNA expression of the invasin IbeA, the adhesin FimH and the outer member protein A, which contributes to E44 penetration across BBB and resistance to the stimulations of low pH and high osmolarity. CONCLUSION: These results indicate that ppk1 plays an important role in stress adaption and virulence in meningitic E. coli K1 strain E44, and controls the relevant phenotypes by modulating the expression of the SP regulatory gene ibeR and the virulence genes ibeA, fimH and ompA.

Autologous nucleus pulposus transplantation to lumbar 5 dorsal root ganglion after epineurium dissection in rats: a modified model of non-compressive lumbar herniated intervertebral disc.

BACKGROUND: Nucleus pulposus of intervertebral discs has proinflammatory characteristics that play a key role in neuropathic pain in lumbar herniated intervertebral disc. One of the most commonly used animal models (the traditional model) of non-compressive lumbar herniated intervertebral disc is created by L4-L5 hemilaminectomy and the application of autologous nucleus pulposus to cover the left L4 and L5 nerve roots in rats. However, such procedures have the disadvantages of excessive trauma and low success rate. We proposed a modified model of non-compressive lumbar herniated intervertebral disc in which only the left L5 dorsal root ganglion is exposed and transplanted with autologous nucleus pulposus following incision of epineurium. We aimed to compare the modified model with the traditional one with regard to trauma and success rate. METHODS: Thirty Sprague-Dawley male rats were randomized into three groups: sham operation group (n = 6), traditional group (n = 12), and modified group (n = 12). The amount of blood loss and operative time for each group were analyzed. The paw withdrawal threshold of the left hind limb to mechanical stimuli and paw withdrawal latency to heat stimuli were examined from the day before surgery to day 35 after surgery. RESULTS: Compared with the traditional group, the modified group had shorter operative time, smaller amount of blood loss, and higher success rate (91.7% versus 58.3%, P < 0.05). There was no decrease in paw withdrawal latency in any group. The sham operation group had no decrease in postoperative paw withdrawal threshold, whereas the modified and traditional groups had significant reduction in paw withdrawal threshold after surgery (mechanical hyperalgesia). CONCLUSIONS: Transplantation of nucleus pulposus onto the L5 dorsal root ganglion following incision of epineurium in rats established an improved animal model of non-compressive lumbar herniated intervertebral disc with less trauma and more stable pain ethology.

[Studies on phloroglucinol derivatives of Dryopteris fragrans L].

OBJECTIVE: To study the phloroglucinol derivatives of Dryopteris fragrans. METHODS: Isolation and purification were carried out on repeated silica gel, Sephadex LH-20 column chromatography and prepare HPLC. The structures of the compounds were determined by physicochemical properties and spectral analysis. RESULTS: Four compounds were isolated and identified as aspidin PB (I), dryofragin (II), aspidinol (III), aspidin BB (IV). CONCLUSION: Compounds IV is isolated from this plant for the first time.

[Study on terpene of Dryopteris fragrans L].

OBJECTIVE: To study the terpene of Dryopteris fragrans. METHODS: Isolation and purification were carried out on repeated silica gel, Sephadex LH-20 column chromatography and prepare HPLC. The structures of the compounds were determined by physicochemical properties and spectral analysis. RESULTS: Four compounds were isolated and identified as 10-hydroxyl-15-oxo-alpha-cadinol (I), albicany acetate (II), alpha-cadinene (III), albicanol (IV). CONCLUSION: Compounds I is isolated from this plant for the first time.