Anti-tumor effects of brucine immuno-nanoparticles on hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma is difficult to diagnose early, and most patients are already in the late stages of the disease when they are admitted to hospital. The total 5-year survival rate is less than 5%. Recent studies have showed that brucine has a good anti-tumor effect, but high toxicity, poor water solubility, short half-life, narrow therapeutic window, and a toxic dose that is close to the therapeutic dose, which all limit its clinical application. This study evaluated the effects of brucine immuno-nanoparticles (BIN) on hepatocellular carcinoma. MATERIALS AND METHODS: Anionic polymerization, chemical modification technology, and phacoemulsification technology were used to prepare a carboxylated polyethylene glycol-polylactic acid copolymer carrier material. Chemical coupling technology was utilized to develop antihuman AFP McAb-polyethylene glycol-polylactic acid copolymer BIN. The size, shape, zeta potential, drug loading, encapsulation efficiency, and release of these immune-nanoparticles were studied in vitro. The targeting, and growth, invasion, and metastasis inhibitory effects of this treatment on liver cancer SMMC-7721 cells were tested. RESULTS: BIN were of uniform size with an average particle size of 249 ± 77 nm and zeta potential of -18.7 ± 4.19 mV. The encapsulation efficiency was 76.0% ± 2.3% and the drug load was 5.6% ± 0.2%. Complete uptake and even distribution around the liver cancer cell membrane were observed. CONCLUSION: BIN had even size distribution, was stable, and had a slow-releasing effect. BIN targeted the cell membrane of the liver cancer cell SMMC-7721 and significantly inhibited the growth, adhesion, invasion, and metastasis of SMMC-7721 cells. As a novel drug carrier system, BIN are a potentially promising targeting treatment for liver cancer

The anaphase promoting complex impacts repair choice by protecting ubiquitin signalling at DNA damage sites

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). While HDR can only occur in S/G2, NHEJ can happen in all cell cycle phases (except mitosis). How then is the repair choice made in S/G2 cells? Here we provide evidence demonstrating that APCCdh1 plays a critical role in choosing the repair pathways in S/G2 cells. Our results suggest that the default for all DSBs is to recruit 53BP1 and RIF1. BRCA1 is blocked from being recruited to broken ends because its recruitment signal, K63-linked poly-ubiquitin chains on histones, is actively destroyed by the deubiquitinating enzyme USP1. We show that the removal of USP1 depends on APCCdh1 and requires Chk1 activation known to be catalysed by ssDNA-RPA-ATR signalling at the ends designated for HDR, linking the status of end processing to RIF1 or BRCA1 recruitment.We thank S.-Y. Lin (MD Anderson Cancer Center) for cell lines; J. Rosen (Baylor College of Medicine) for reagents; H. Masai (Tokyo Metropolitan Institute of Medical Science) for U2OS-Fucci cell line; D. Durocher (University of Toronto) for HeLa-Fucci cell line; E. Citterio (Netherlands Cancer Institute) for GFP-USP3 construct; M.S.Y. Huen (The University of Hong Kong) for RNF168 antibody. This work was performed with facilities and instruments in the Imaging Core of National Center for Protein Science (Beijing), the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the NIH (P30 AI036211, P30 CA125123 and S10 RR024574), the Integrated Microscopy Core at Baylor College of Medicine with funding from the NIH (HD007495, DK56338 and CA125123), and the John S. Dunn Gulf Coast Consortium for Chemical Genomics. We also thank other members of the Zhang lab for helpful discussion and support. This work was supported in part by an international collaboration grant (# 2013DFB30210) and a 973 Project grant (# 2013CB910300) from Chinese Minister of Science and Technology, in part by a Chinese National Natural Science Foundation grant (# 81171920), in part by a grant from The Committee of Science and Technology of Beijing Municipality, China (# Z141100000214015), and in part by NIH grants CA116097 and CA122623 to P.Z. J.J. is supported by grants from National Institutes of Health (R01GM102529) and the Welch Foundation (AU-1711). S.H. is supported by grants (# 81272488 and 81472795) from Chinese National Natural Science Foundation. Y.Z. is supported by grants from the National Natural Scientific Foundation of China (No. 81430055), Programs for Changjiang Scholars and Innovative Research Team in University (No. IRT_15R13).S

Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

<p>Abstract</p> <p>Background</p> <p>Fumarase catalyzes the reversible hydration of fumarate to <smcaps>L</smcaps>-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of <smcaps>L</smcaps>-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications.</p> <p>Results</p> <p>A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from <it>Bacteroides </it>sp. 2_1_33B and <it>Parabacteroides distasonis </it>ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in <it>E. coli </it>BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form <smcaps>L</smcaps>-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg²⁺. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: <it>K</it>_m= 0.48 mM, <it>V</it>_max= 827 μM/min/mg, and <it>k</it>_cat/<it>K</it>_m= 1900 mM/s.</p> <p>Conclusions</p> <p>We isolated a novel fumarase gene, <it>fumF</it>, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of <smcaps>L</smcaps>-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.</p

Yiqi Huayu Jiedu Decoction Inhibits the Invasion and Metastasis of Gastric Cancer Cells through TGF- β

Background. Yiqi Huayu Jiedu Decoction (YHJD) can obviously improve the quality of life of those patients with gastric cancer and prolong their survival. Methods. In vitro experiments, we observe YHJD’s effect on the cells’ proliferation by MTT assay. Cell adhesion assay, wound-healing assay, and Transwell invasion assay serve to detect its influence on cells’ adhesion, migration, and invasion, respectively. Inhibitor (10 μM/L of SB431542) and activator (10 ng/mL of TGF-β) of TGF-β/Smad pathway were used to estimate whether YHJD’s impact on the biological behavior of gastric cancer cells was related to TGF-β/Smad pathway. In in vivo studies, YHJD was administered to the nude mice transplanted with gastric cancer to observe its effect on the tumor. Western blotting and immunohistochemical assay were used to test relevant cytokines of TGF-β/Smad pathway and epithelial-mesenchymal transition (EMT) in MGC-803 cells and the tumor bearing nude mice. Results. YHJD inhibited proliferation, adhesion, migration, and invasion of MGC-803 gastric cancer cells in vitro. In in vivo studies, YHJD reduced the volume of the transplanted tumors. It also enhanced the expression of E-cadherin and decreased the levels of N-cadherin, TGF-β, Snail, and Slug in both MGC-803 cells and the transplanted tumor by western blot assay. The immunohistochemical assay revealed that YHJD raised E-cadherin in the tumors of the mice; on the contrary, the expression of N-cadherin, Twist, vimentin, TGF-βR I, p-Smad2, p-Smad3, Snail, and Slug reduced. Conclusion. YHJD can effectively inhibit the invasion and metastasis of gastric cancer cells. The mechanism may be related to TGF-β/Smad pathway

Homotopic Connectivity in Early Pontine Infarction Predicts Late Motor Recovery

Connectivity-based methods are essential to explore brain reorganization after a stroke and to provide meaningful predictors for late motor recovery. We aim to investigate the homotopic connectivity alterations during a 180-day follow-up of patients with pontine infarction to find an early biomarker for late motor recovery prediction. In our study, resting-state functional MRI was performed in 15 patients (11 males, 4 females, age: 57.87 ± 6.50) with unilateral pontine infarction and impaired motor function during a period of 6 months (7, 14, 30, 90, and 180 days after stroke onset). Clinical neurological assessments were performed using the Fugl–Meyer scale (FM).15 matched healthy volunteers were also recruited. Whole-brain functional homotopy in each individual scan was measured by voxel-mirrored homotopic connectivity (VMHC) values. Group-level analysis was performed between stroke patients and normal controls. A Pearson correlation was performed to evaluate correlations between early VMHC and the subsequent 4 visits for behavioral measures during day 14 to day 180. We found in early stroke (within 7 days after onset), decreased VMHC was detected in the bilateral precentral and postcentral gyrus and precuneus/posterior cingulate cortex (PCC), while increased VMHC was found in the hippocampus/amygdala and frontal pole (P &lt; 0.01). During follow-up, VMHC in the precentral and postcentral gyrus increased to the normal level from day 90, while VMHC in the precuneus/PCC presented decreased intensity during all time points (P &lt; 0.05). The hippocampus/amygdala and frontal pole presented a higher level of VMHC during all time points (P &lt; 0.05). Negative correlation was found between early VMHC in the hippocampus/amygdala with FM on day 14 (r = −0.59, p = 0.021), day 30 (r = −0.643, p = 0.01), day 90 (r = −0.693, p = 0.004), and day 180 (r = −0.668, p = 0.007). Furthermore, early VMHC in the frontal pole was negatively correlated with FM scores on day 30 (r = −0.662, p = 0.013), day 90 (r = −0.606, p = 0.017), and day 180 (r = −0.552, p = 0.033). Our study demonstrated the potential utility of early homotopic connectivity for prediction of late motor recovery in pontine infarction

Molecular and Phylogenetic Analyses Suggest an Additional Hepatitis B Virus Genotype “I”

A novel hepatitis B virus (HBV) strain (W29) was isolated from serum samples in the northwest of China. Phylogenetic and distance analyses indicate that this strain is grouped with a series of distinct strains discovered in Vietnam and Laos that have been proposed to be a new genotype I. TreeOrderScan and GroupScan methods were used to study the intergenotype recombination of this special group. Recombination plots and tree maps of W29 and these putative genotype I strains exhibit distinct characteristics that are unexpected in typical genotype C strains of HBV. The amino acids of P gene, S gene, X gene, and C gene of all genotypes (including subtypes) were compared, and eight unique sites were found in genotype I. In vitro and in vivo experiments were also conducted to determine phenotypic characteristics between W29 and other representative strains of different genotypes obtained from China. Secretion of HBsAg in Huh7 cells is uniformly abundant among genotypes A, B, C, and I (W29), but not genotype D. HBeAg secretion is low in genotype I (W29), whose level is close to genotype A and much lower than genotypes B, C, and D. Results from the acute hydrodynamic injection mouse model also exhibit a similar pattern. From an overview of the results, the viral markers of W29 (I1) in Huh7 cells and mice had a more similar level to genotype A than genotype C, although the latter was closer to W29 in distance analysis. All evidence suggests that W29, together with other related strains found in Vietnam and Laos, should be classified into a new genotype

Search for Quasi-Periodical Oscillations in Precursors of Short and Long Gamma Ray Bursts

The precursors of short and long Gamma Ray Bursts (SGRBs and LGRBs) can serve as probes of their progenitors, as well as shedding light on the physical processes of mergers or core-collapse supernovae. Some models predict the possible existence of Quasi-Periodically Oscillations (QPO) in the precursors of SGRBs. Although many previous studies have performed QPO search in the main emission of SGRBs and LGRBs, so far there was no systematic QPO search in their precursors. In this work, we perform a detailed QPO search in the precursors of SGRBs and LGRBs detected by Fermi/GBM from 2008 to 2019 using the power density spectrum (PDS) in frequency domain and Gaussian processes (GP) in time domain. We do not find any convinced QPO signal with significance above 3 $\sigma$, possibly due to the low fluxes of precursors. Finally, the PDS continuum properties of both the precursors and main emissions are also studied for the first time, and no significant difference is found in the distributions of the PDS slope for precursors and main emissions in both SGRBs and LGRBs.Comment: submitte