Lysostaphin (Lysn) is an antibacterial metalloendopeptidase that cleaves the pentaglycin bridges in the cell wall of Staphylococci. Although many studies have demonstrated its high activity in vitro, the medical application of Lysn has been hampered by its short half-life in vivo. In order to enhance its stability in vivo without significantly suppressing the enzymatic activity, we designed and tested eight single cysteine substitutions in Lysn for covalent attachment of polyethylene glycol chains (PEGylation). The purified mutants, fully reduced by Dithiothreitol (DTT), were treated with mPEG-MAL(20 kDa). The PEG modification efficiency was above 70% as determined by reverse-phase high-pressure liquid chromatography (HPLC) analysis. The PEG-Lysn proteins were further purified by cation exchange chromatography (MacroCap SP), reaching at least 95% purity. The activities of the PEG-Lysn proteins were determined by the turbidity and minimum inhibitory concentration (MIC) assays. We found that the PEGylated V240C and T244C mutants retained about 50% of the original antibacterial activity of Lysn. Overall, this study will help develop highly stable and active PEG-Lysn to treat systemic S. aureus infections.
Amino Acid Substitution ; Lysostaphin ; chemistry ; Polyethylene Glycols ; chemistry ; Protein Engineering ; Recombinant Proteins ; chemistry ; Staphylococcus aureus

Aim To investigate the apoptosis mechanism of human gastric cancer cell SGC-7901 induced by Omphalia lapidescens protein pPeOp.Methods CCK-8 and flow cytometry were used to detect the inhibitory effect of different concentrations of pPeOp(30, 60, 90 mg·L-1) on SGC-7901.The mRNA and protein expression of TNF-R1, Fas/FasL, Bcl-2, caspase-3 and caspase-8 were detected by qRT-PCR and Western blot.Results SGC-7901 cells were treated with different concentrations of pPeOp(30, 60, 90 mg·L-1) for 24 h.CCK-8 test showed that there was no significant difference between PVP group and the control group.The survival rate of the 5-Fu group was(53.71±7.34)% (P<0.05).The survival rates of pPeOp group(30, 60, 90 mg·L-1) were(80.95±6.25)%, (53.48±5.70)% and(44.61±6.50)%(r=0.984,P=0.016),respectively.Flow cytometry showed that the apoptosis rate of PVP group had no significant difference with control group, and the apoptosis rate of 5-Fu group was about(39.30±3.34)%(P<0.05).The apoptotic rates of pPeOp group(30, 60, 90 mg·L-1) were(10.90±1.25)%, (28.80±2.70)% and (32.00±3.50)%,respectively(P<0.05).The mRNA and protein expression levels of Bcl-2 were down-regulated,whereas the expression of TNF-R1, Fas/FasL, caspase-3 and caspase-8 were significantly up-regulated(P<0.05).Conclusions pPeOp can significantly inhibit the proliferation of gastric cancer cell line SGC-7901 and induce apoptosis in a dose-dependent manner.Death receptor pathway and mitochondrial pathway may be related to pPeOp-induced apoptosis of gastric cancer SGC-7901.

Natural product is an important source of new drug research and development (R&D). Traditional Chinese medicine (TCM) innovation is the key step for its modernization and internationalization. However, due to the complexity of TCM, there are many difficulties and confusions in this process. Target-based drug discovery is the mainstream model and method of R&D. TTD, short for therapeutic target database, is developed by National University of Singapore. Besides a large amount of information on drug targets, the database also contains considerable information related to natural products. This paper briefly introduces the TTD, analyzes the natural products derived drugs/compounds recorded in TTD, which we think might provide some inspiration for the innovation of TCM.

OBJECTIVETo investigate the prophylactic and therapeutic effect of oxymatrine on experimental liver fibrosis and to reveal its mechanism.

METHODSBy establishing D-galactosamine-induced rat liver fibrosis model, we observed the effect of oxymatrine on serum and tissue biochemical indexes, content of liver hydroxyline, expression of TGF?1 mRNA and changes of tissue pathology.

RESULTSThere was a decline of liver hydroxyline and serum AST and ALT in oxymatrine group compared to those of the D-GalN group. The hydroxyline content in oxymatrine pretreatment group was (0.50 0.11)mug/mg compared with (0.99 0.14)mug/mg in D-GalN group (t=8.366, P<0.01). The content in oxymatrine treatment group was (0.44 0.04)mug/mg compared with 0.70 0.06 in D-GalN group (t=9.839, P<0.01). The SOD activity was (149.81 15.28) NU/mg in oxymatrine pretreatment group and (95.22 16.33) NU/mg in the model group (t=7.309, P<0.01); (157.68 19.54) NU/mg in the treatment group compared with (119.88 14.94) NU/mg in the model group (t=4.348, P<0.01). MDA in the pretreatment group was (2.06 0.17) nmol/mg, lower than (4.57 0.37) nmol/mg in the model group (t=17.529, P<0.01). In the treatment group, it was (1.76 0.24)nmol/mg, lower than (3.10 0.17) nmol/mg in the model group (t=12.697, P<0.01). TGF?1 mRNA reduced in the pretreatment and treatment groups as compared with that in the model group (0.21 0.01 vs 0.50 0.01, t=48.665, P<0.01; 0.18 0.02 vs 0.38 0.01, t=22.464, P<0.01). Electron microscopy showed that oxymatrine group had milder hepatocyte degeneration and less fibrosis accumulation than did the model group. Microscopy revealed wide septa expansion from the portal area to the central venous, piecemeal and confluent necrosis and pseudo-nodular formation in part of the lobular in the model group. While in oxymatrine group these lesions were much improved.

CONCLUSIONSOxymatrine shows prophylactic and therapeutic effect in D-galactosamine induced rat liver fibrosis. This is partly by protecting hepatocyte and suppressing fibrosis accumulation through anti-lipoperoxidation.

Alkaloids ; therapeutic use ; Animals ; Anti-Arrhythmia Agents ; therapeutic use ; Calcium Hydroxide ; metabolism ; Chemoprevention ; Disease Models, Animal ; Galactosamine ; Liver Cirrhosis ; chemically induced ; drug therapy ; metabolism ; pathology ; prevention & control ; Liver Function Tests ; Male ; Quinolizines ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Superoxide Dismutase ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism

Objective:To explore the diagnostic efficacy of nomogram based on multi-parameter MRI for assessment of bowel fibrosis in patients with Crohn disease（CD).Methods:The clinical and imaging data of CD patients diagnosed by surgical histopathology in the First Affiliated Hospital of Sun Yat-sen University from June 2015 to March 2018 were prospectively collected. All the patients underwent conventional MRI and diffusion kurtosis imaging（DKI) within 2 weeks before surgery. Patients who underwent surgery between June 2015 and September 2017 were included in the model building group, and those who underwent surgery between October 2017 and March 2018 were included in the model validation group. We measured the apparent diffusion coefficient（ADC) from monoexponential model of diffusion-weighted imaging（DWI), apparent diffusional kurtosis（K app), and apparent diffusion for non-Gaussian distribution（D app) from non-Gaussian DKI model, and observed T 2WI signal intensity and enhancement pattern of the same segment. One to three intestinal specimens per patient were stained with Masson′s trichrome for the histological grading of fibrosis. Correlations between qualitative/quantitative MRI indexes and histological grades were evaluated using the Spearman rank test. Multivariate logistic regression analysis was performed to identify independent factors to be included into the nomogram for predicting the degree of bowel fibrosis and its diagnostic performance was assessed by internal and external validation. Results:A total of 40 CD patients were included, including 31 in the model construction group and 9 in the model verification group. A total of 81 intestinal specimens from 31 patients were graded as none-to-mild bowel fibrosis（ n=32) and moderate-to-severe bowel fibrosis（ n=49) according to a scoring system of fibrosis. In the training cohort, the K app value of moderate-to-severely fibrotic bowel walls was significantly higher than that of none-to-mildly fibrotic bowel walls, and the D appand ADC values of moderate-to-severely fibrotic bowel walls were significantly lower than those of none-to-mildly fibrotic bowel walls（ Z=-5.999, -4.521 and -3.893; P<0.001). There was no significant difference in T 2WI signal intensity or enhancement pattern between these two groups（χ2=1.571 and 0.103; P>0.05). Moderate and mild correlations of histological fibrosis grades with K appand D app( r=0.721 and -0.483; P<0.001), and a mild correlation with ADC（ r=-0.445, P<0.001) were found. Independent factors derived from multivariate logistic regression analysis to predict the degree of bowel fibrosis were K app and D app. Internal and external validation revealed good performance of the nomogram with concordance index of 0.901（95% confidence interval, 0.824-0.978) and 1.000, respectively, for differentiating none-to-mild from moderate-to-severe fibrosis. Conclusion:The DKI-based nomogram can be used to evaluate the bowel fibrosis in CD patients and provides a visual and simple prediction method for clinic.

Background/Aims: This study aimed to explore the effect of gut microbiota-regulated Kupffer cells (KCs) on colorectal cancer (CRC) liver metastasis. Methods: A series of in vivo and in vitro researches were showed to demonstrate the gut microbiota and its possible mechanism in CRC liver metastasis. Results: Fewer liver metastases were identified in the ampicillin-streptomycin-colistin and colistin groups. Increased proportions of Parabacteroides goldsteinii, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Bacteroides uniforms were observed in the colistin group. The significant expansion of KCs was identified in the ampicillin-streptomycin-colistin and colistin groups. B.vulgatus levels were positively correlated with KC levels. More liver metastases were observed in the vancomycin group. An increased abundance of Parabacteroides distasonis and Proteus mirabilis and an obvious reduction of KCs were noted in the vancomycin group. P. mirabilis levels were negatively related to KC levels. The number of liver metastatic nodules was increased in the P. mirabilis group and decreased in the B. vulgatus group. The number of KCs decreased in the P. mirabilis group and increased in the B. vulgatus group. In vitro, as P. mirabilis or B. vulgatus doses increased, there was an opposite effect on KC proliferation in dose- and time-dependent manners. P. mirabilis induced CT26 cell migration by controlling KC proliferation, whereas B. vulgatus prevented this migration. Conclusions An increased abundance of P. mirabilis and decreased amount of B. vulgatus play key roles in CRC liver metastasis, which might be related to KC reductions in the liver.

The mammalian target of rapamycin (mTOR) pathway is abnormally activated in lung cancer. However, the anti-lung cancer effect of mTOR inhibitors as monotherapy is modest. Here, we identified that ginsenoside Rh2, an active component of Panax ginseng C. A. Mey., enhanced the anti-cancer effect of the mTOR inhibitor everolimus both in vitro and in vivo. Moreover, ginsenoside Rh2 alleviated the hepatic fat accumulation caused by everolimus in xenograft nude mice models. The combination of everolimus and ginsenoside Rh2 (labeled Eve-Rh2) induced caspase-independent cell death and cytoplasmic vacuolation in lung cancer cells, indicating that Eve-Rh2 prevented tumor progression by triggering paraptosis. Eve-Rh2 up-regulated the expression of c-MYC in cancer cells as well as tumor tissues. The increased c-MYC mediated the accumulation of tribbles homolog 3 (TRIB3)/P62⁺ aggresomes and consequently triggered paraptosis, bypassing the classical c-MYC/MAX pathway. Our study offers a potential effective and safe strategy for the treatment of lung cancer. Moreover, we have identified a new mechanism of TRIB3/P62⁺ aggresomes-triggered paraptosis and revealed a unique function of c-MYC.