Identification and Validation of a Prognostic Model Based on Three MVI-Related Genes in Hepatocellular Carcinoma

MVI has significant clinical value for treatment selection and prognosis evaluation in hepatocellular carcinoma (HCC). We aimed to construct a model based on MVI-Related Genes (MVIRGs) for risk assessment and prognosis prediction in patients with HCC. This study utilized various statistical analysis methods for prognostic model construction and validation in the Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts, respectively. In addition, immunohistochemistry and qRT-PCR were used to analyze and identify the value of the model in our cohort. After the analyses, 153 differentially expressed MVIRGs were identified, and three key genes were selected to construct a prognostic model. The high-risk group showed significantly lower overall survival (OS), and this trend was observed in all subgroups: different age groups, genders, stages, and grades. Risk score was a risk factor independent of age, gender, stage, and grade. Moreover, the ICGC cohort validated the prognostic value of the model corresponding to the TCGA. In our cohort, qRT-PCR and immunohistochemistry showed that all three genes had higher expression levels in HCC samples than in normal controls. High expression levels of genes and high-risk scores showed significantly lower recurrence-free survival (RFS) and OS, especially in MVI-positive HCC samples. Therefore, the prognostic model constructed by three MVIRGs can reliably predict the RFS and OS of patients with HCC and is valuable for guiding clinical treatment selection and prognostic assessment of HCC.     

Amphetamine Administration into the Ventral Striatum Facilitates Behavioral Interaction with Unconditioned Visual Signals in Rats

Background

Administration of psychomotor stimulants like amphetamine facilitates behavior in the presence of incentive distal stimuli, which have acquired the motivational properties of primary rewards through associative learning. This facilitation appears to be mediated by the mesolimbic dopamine system, which may also be involved in facilitating behavior in the presence of distal stimuli that have not been previously paired with primary rewards. However, it is unclear whether psychomotor stimulants facilitate behavioral interaction with unconditioned distal stimuli.

Principal Findings

We found that noncontingent administration of amphetamine into subregions of the rat ventral striatum, particularly in the vicinity of the medial olfactory tubercle, facilitates lever pressing followed by visual signals that had not been paired with primary rewards. Noncontingent administration of amphetamine failed to facilitate lever pressing when it was followed by either tones or delayed presentation or absence of visual signals, suggesting that visual signals are key for enhanced behavioral interaction. Systemic administration of amphetamine markedly increased locomotor activity, but did not necessarily increase lever pressing rewarded by visual signals, suggesting that lever pressing is not a byproduct of heightened locomotor activity. Lever pressing facilitated by amphetamine was reduced by co-administration of the dopamine receptor antagonists SCH 23390 (D1 selective) or sulpiride (D2 selective).

Conclusions

Our results suggest that amphetamine administration into the ventral striatum, particularly in the vicinity of the medial olfactory tubercle, activates dopaminergic mechanisms that strongly enhance behavioral interaction with unconditioned visual stimuli.     

Selection for High Oridonin Yield in the Chinese Medicinal Plant Isodon (Lamiaceae) Using a Combined Phylogenetics and Population Genetics Approach

Oridonin is a diterpenoid with anti-cancer activity that occurs in the Chinese medicinal plant Isodon rubescens and some related species. While the bioactivity of oridonin has been well studied, the extent of natural variation in the production of this compound is poorly known. This study characterizes natural variation in oridonin production in order to guide selection of populations of Isodon with highest oridonin yield. Different populations of I. rubescens and related species were collected in China, and their offspring were grown in a greenhouse. Samples were examined for oridonin content, genotyped using 11 microsatellites, and representatives were sequenced for three phylogenetic markers (ITS, rps16, trnL-trnF). Oridonin production was mapped on a molecular phylogeny of the genus Isodon using samples from each population as well as previously published Genbank sequences. Oridonin has been reported in 12 out of 74 species of Isodon examined for diterpenoids, and the phylogeny indicates that oridonin production has arisen at least three times in the genus. Oridonin production was surprisingly consistent between wild-collected parents and greenhouse-grown offspring, despite evidence of gene flow between oridonin-producing and non-producing populations of Isodon. Additionally, microsatellite genetic distance between individuals was significantly correlated with chemical distance in both parents and offspring. Neither heritability nor correlation with genetic distance were significant when the comparison was restricted to only populations of I. rubescens, but this result should be corroborated using additional samples. Based on these results, future screening of Isodon populations for oridonin yield should initially prioritize a broad survey of all species known to produce oridonin, rather than focusing on multiple populations of one species, such as I. rubescens. Of the samples examined here, I. rubescens or I. japonicus from Henan province would provide the best source of oridonin.     

Marine drugs--macrolactins

The increasing demands for new lead compounds in pharmaceutical and agrochemical industries have driven scientists to search for new bioactive natural products. Marine microorganisms are rich sources of novel, bioactive secondary metabolites, and have attracted much attention of chemists, pharmacologists, and molecular biologists. This mini-review mainly focuses on macrolactins, a group of 24-membered lactone marine natural products, aiming at giving an overview on their sources, structures, biological activities, as well as their potential medical applications.     

Rational redesign of the 4-chlorobenzoate binding site of 4-chlorobenzoate: coenzyme a ligase for expanded substrate range

Environmental aromatic acids are transformed to chemical energy in bacteria that possess the requisite secondary pathways. Some of these pathways rely on the activation of the aromatic acid by coenzyme A (CoA) thioesterification catalyzed by an aromatic acid: CoA ligase. Adaptation of such pathways to the bioremediation of man-made pollutants such as polychlorinated biphenyl (PCB) and dichlorodiphenyltrichloroethane (DDT) requires that the chlorinated benzoic acid byproduct that is formed be able to be eliminated by further degradation. To take advantage of natural benzoic acid degrading pathways requiring initial ring activation by thioesterification, the pathway aromatic acid:CoA ligase must be an effective catalyst with the chlorinated benzoic acid. This study, which focuses on the 4-chlorobenzoate:CoA ligase (CBL) of the 4-monochlorobiphenyl degrading bacterium Alcaligenes sp. strain ALP83, was carried out to determine if the 4-chlorobenzoate binding site of this enzyme can be transformed by rational design to recognize the chlorobenzoic acids formed in the course of breakdown of other environmental PCB congeners. The fundamental question addressed in this study is whether it is possible to add or subtract space from the substrate-binding pocket of this ligase (to complement the topology of the unnatural aromatic substrate) without causing disruption of the ligase catalytic machinery. Herein, we report the results of a substrate specificity analysis that, when interpreted within the context of the X-ray crystal structures, set the stage for the rational design of the ligase for thioesterification of two PCB-derived chlorobenzoic acids. The ligase was first optimized to catalyze CoA thioesterification of 3,4-dichlorobenzoic acid, a poor substrate, by truncating Ile303, a large hydrophobic residue that packs against the ring meta-C(H) group. The structural basis for the approximately 100-fold enhancement in the rate of 3,4-dichlorobenzoate thioesterification catalyzed by the I303A and I303G CBL mutants was validated by determination of the crystal structure of the 3,4-dichlorobenzoate-bound enzymes. Determinations of the structures of I303 mutant complexes of 3-chlorobenzoate, a very poor substrate, revealed nonproductive binding as a result of the inability of the substrate ring C(4)H group to fill the pocket that binds the C(4)Cl group of the native substrate. The C(4)Cl pocket of the CBL I303A mutant was then reduced in size by strategic amino acid replacement. A 54-fold improvement in catalytic efficiency was observed for the CBL F184W/I303A/V209T triple mutant. The results of this investigation are interpreted as evidence that the plasticity of the ligase catalytic scaffold is sufficient to allow expansion of substrate range by rational design. The combination of structural and kinetic analyses of the constructed mutants proved to be an effective approach to engineering the ligase for novel substrates.     

Leucine ureido derivatives as aminopeptidase N inhibitors using click chemistry. Part II

Aminopeptidase N (APN) has been proved to be deeply associated with cancer angiogenesis, metastasis and invasion. Therefore, APN gains increasing attention as a promising anti-tumor target. In the current study, we report the design, synthesis, biological evaluation and structure-activity relationship of one new series of leucine ureido derivatives containing the 1,2,3-triazole moiety. Among them, compound 31f was identified as the best APN inhibitor with IC₅₀ value being two orders of magnitude lower than that of the positive control bestatin. Compound 31f possessed selective cytotoxicity to several tumor cell lines over the normal cell line human umbilical vein endothelial cells (HUVECs). Notably, when combined with 5-fluorouracil (5-Fu), 31f exhibited synergistic anti-proliferation effect against several tumor cell lines. At the same concentration, 31f exhibited much better anti-angiogenesis activities than bestatin in the HUVECs capillary tube formation assay and the rat thoracic aorta rings test. In the in vitro anti-invasion assay, 31f also exhibited superior potency over bestatin. Moreover, considerable in vivo antitumor potencies of 31f alone or in combination with 5-Fu were observed without significant toxic signs in a mouse heptoma H22 tumor transplant model.     

Angiogenic synergism,vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2

The establishment of functional and stable vascular networks is essential for angiogenic therapy. Here we report that a combination of two angiogenic factors, platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-2, synergistically induces vascular networks, which remain stable for more than a year even after depletion of angiogenic factors. In both rat and rabbit ischemic hind limb models, PDGF-BB and FGF-2 together markedly stimulated collateral arteriogenesis after ligation of the femoral artery, with a significant increase in vascularization and improvement in paw blood flow. A possible mechanism of angiogenic synergism between PDGF-BB and FGF-2 involves upregulation of the expression of PDGF receptor (PDGFR)-alpha and PDGFR-beta by FGF-2 in newly formed blood vessels. Our data show that a specific combination of angiogenic factors establishes functional and stable vascular networks, and provides guidance for the ongoing clinical trials of angiogenic factors for the treatment of ischemic diseases.     

Selection of trkB-binding peptides from a phage-displayed random peptide library

Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurodegenerative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 μg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXΦXXΦXXC (X represents the random amino acids, Φ represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further development of small peptides mimicking BDNF activity.     

Involvement of ZEB1 and E-cadherin in the invasion of lung squamous cell carcinoma

This study intended to investigate the expression of the ZEB1 and E-cadherin proteins in lung squamous cell carcinoma (LSCC) tissues and to examine the clinicopathological correlation between protein levels and LSCC. RT-PCR and Western blot were used to examine the expression of ZEB1 and E-cadherin mRNAs and proteins in LSCC tissues as well as in adjacent normal tissues, and then analyze the relationship between the clinicopathological characteristics and the expression changes of ZEB1 and E-cadherin mRNAs in LSCC. In addition, RNAi was used to knockdown the expression of the ZEB1 gene in Human HCC827 cells; subsequently, changes in the invasive ability of the resultant cells were studied. The positive rates of ZEB1 and E-cadherin mRNAs in LSCC tissues were 69.2 and 38.5 %, respectively. They differed significantly from the corresponding positive rates in the adjacent normal lung tissues (15.4 and 80.8 %, p < 0.05). There was a negative correlation between the protein levels of ZEB1 and E-cadherin in LSCC tissues (r = -0.714, p < 0.001); in addition, it was found that ZEB1 protein expression in LSCC tissues was significantly higher than that in the neighboring normal lung tissues (p < 0.05), and its expression was also significantly higher in patients with lymph node metastases and distant metastases compared to those patients without metastatic disease (p < 0.05). On the contrary, E-cadherin expression was significantly lower in LSCC tissues than that in the neighboring normal tissue (p < 0.05). It was lower in patients with lymph node metastasis and distant metastasis compared to patients without metastatic disease (p < 0.05). However, the expression of ZEB1 and E-cadherin was independent of gender, age, tumor size, or tumor differentiation level (p > 0.05). Transfection of ZEB1 siRNA into HCC827 cells significantly reduced the ZEB1 protein level (p < 0.01) and significantly elevated E-cadherin levels (p < 0.01). Moreover, significantly less ZEB1 siRNA-transfected cells migrated through Transwell chambers in the LSCC tissue than that in the control groups (untransfected or transfected with control siRNA, p < 0.01). The expression of the ZEB1 gene in LSCC tissues is downregulated with the expression of E-cadherin. On the other hand, the expression of siRNA against ZEB1 promotes E-cadherin expression and suppresses the invasive ability conferred by E-cadherin. In conclusion, our data suggested that overexpression of the ZEB1 gene is possibly associated with the occurrence, development, invasion of LSCC.