PAMAM-PEG-PAMAM: novel triblock copolymer as a biocompatible and efficient gene delivery carrier

A novel triblock copolymer, PAMAM-block-PEG-block-PAMAM was synthesized and applied as a gene carrier. PAMAM dendrimer is proven to be an efficient gene carrier itself, but it is associated with certain problems such as low water solubility and considerable cytotoxicity. Therefore, we introduced PEG to engineer a nontoxic and highly transfection efficient polymeric gene carrier because PEG is known to convey water-solubility and biocompatibility to the conjugated copolymer. This copolymer could achieve self-assembly with plasmid DNA, forming compact nanosized particles with a narrow size distribution. Fulfilling our expectations, the copolymer was found to form highly water-soluble polyplexes with plasmid DNA, showed little cytotoxicity despite its poor degradability, and finally achieved high transfection efficiency comparable to PEI in 293 cells. Consequently, these data show that an approach involving the introduction of PEG to create a tree-like cationic copolymer possesses a great potential for use in gene delivery systems.     

Optimization of free ammonia concentration for nitrite accumulation in shortcut biological nitrogen removal process

A shortcut biological nitrogen removal (SBNR) utilizes the concept of a direct conversion of ammonium to nitrite and then to nitrogen gas. A successful SBNR requires accumulation of nitrite in the system and inhibition of the activity of nitrite oxidizers. A high concentration of free ammonia (FA) inhibits nitrite oxidizers, but unfortunately decreases the ammonium removal rate as well. Therefore, the optimal range of FA concentration is necessary not only to stabilize nitrite accumulation but also to achieve maximum ammonium removal. In order to derive such optimal FA concentrations, the specific substrate utilization rates of ammonium and nitrite oxidizers were measured. The optimal FA concentration range appeared to be 5–10 mg/L for the adapted sludge. The simulated results from the modified inhibition model expressed by FA and ammonium/nitrite concentrations were shown very similar to the experimental results.     

A combinatorial approach to hybrid enzymes independent of DNA homology

We present a methodology, termed incremental truncation for the creation of hybrid enzymes (ITCHY), that creates combinatorial fusion libraries between genes in a manner that is independent of DNA homology. We compared the ability of ITCHY and DNA shuffling to create interspecies fusion libraries between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, which have only 50% identity on the DNA level. Sequencing of several randomly selected positives from each library illustrated that ITCHY identified a more diverse set of active fusion points including those in regions of nonhomology and those with crossover points that diverged from the sequence alignment. Furthermore, some of the hybrids found by ITCHY that were fused at nonhomologous locations had activities that were greater than or equal to the activity of the hybrids found by DNA shuffling.     

Licorice-derived dehydroglyasperin C increases MKP-1 expression and suppresses inflammation-mediated neurodegeneration

Recent studies have demonstrated that microglial hyperactivation-mediated neuroinflammation is involved in the pathogenesis of several neurodegenerative diseases. Thus, inhibiting microglial production of the neurotoxic mediator tumor necrosis factor-α (TNF-α) is considered a promising strategy to protect against neurodegeneration. Here, we investigated the inhibitory effect of licorice-derived dehydroglyasperin C (DGC) on lipopolysaccharide (LPS)-induced TNF-α production and inflammation-mediated neurodegeneration. We found that DGC pre-treatment attenuated TNF-α production in response to LPS stimulation of BV-2 microglia. DGC pre-treatment attenuated LPS-induced inhibitor of κB-α (IκB-α) and p65 phosphorylation and decreased the DNA binding activity of nuclear factor-κB (NF-κB). DGC pre-treatment also inhibited LPS-mediated phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinase (ERK). Interestingly, DGC treatment of BV-2 microglia significantly increased MAPK phosphatase 1 (MKP-1) mRNA and protein expression, which is a phosphatase of p38 MAPK and ERK, suggesting that the DGC-mediated increase in MKP-1 expression might inhibit LPS-induced MAPKs and NF-κB activation and further TNF-α production. We also found that LPS-mediated microglial neurotoxicity can be attenuated by DGC. The addition of conditioned media (CM) from DGC- and LPS-treated microglia to neurons helped maintain healthy cell body and neurite morphology and increased the number of microtubule-associated protein 2-positive cells and the level of synaptophysin compared to treatment with CM from LPS-treated microglia. Taken together, these data suggest that DGC isolated from licorice may inhibit microglia hyperactivation by increasing MKP-1 expression and acting as a potent anti-neurodegenerative agent.     

THE UTILITY OF PROTEOMICS IN ALGAL TAXONOMY: BOSTRYCHIA RADICANS/B. MORITZIANA (RHODOMELACEAE,RHODOPHYTA) AS A MODEL STUDY1

A comparison of the proteome of eight genetically well‐characterized isolates of the Bostrychia radicans (Mont.) Mont./B. moritziana (Sond. ex Kütz.) J. Agardh species complex was undertaken to establish if genetic relationships among them can be determined using proteome data. Genetic distances were calculated on the basis of common and distinct spots in two‐dimensional gel electrophoresis (2‐DE). Proteomes of the male and female plants of each population were compared to analyze the range of genetic difference within an isolate. Haploid male and female plants of the same species had 3.7%–7.1% sex‐specific proteins. The degree of similarity of the proteome was consistent with previous DNA sequence data and sexual compatibility studies between the isolates. Two sexually compatible isolates from Venezuela showed a pair‐wise distance ranging from 0.14 to 0.21. The isolates from Mexico and Venezuela, which were partially compatible, showed a maximum pair‐wise distance of 0.26. A high level of genetic difference was found among isolates that were sexually incompatible. The isolate from Brazil was reproductively isolated from the Mexico and Venezuela isolates and showed a maximum pair‐wise distance of 0.65 and 0.58, respectively. Comparative proteomics may be helpful for studying genetic distances among algal samples, if intraisolate variation (gene expression) can be minimized or tested.     

<Emphasis Type="Italic">Phellinus</Emphasis> extracts inhibit migration and matrix metalloproteinase secretion in porcine coronary artery endothelial cells

Phellinus linteus is a fungus which is found primarily in tropical regions of the Americas, Africa, and Asia.P. linteus has been used in traditional medical practice for the treatment of arthritis, liver damage and cancer. Angiogenesis is a process that involves migration, proliferation and cell differentiation, as well as the formation of new capillary structures. The anti-angiogenic activities evidenced by natural compounds may actually be a critical effect for the inhibition of angiogenesis-dependent disease by these agents via the blockage of vascular development. This study assessed the effects of water extracts fromP. linteus (Phellinus extracts) on primary cultured porcine coronary artery endothelial cells (PCAECs).Phellinus extracts induced no changes in DNA synthesis or cell numbers, but inhibited the migration of PCAECs.Phellinus extracts also induced a reduction in the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9. Our results show that, in endothelial cells,Phellinus extracts may inhibit angiogenesis by reducing levels of MMP-2 and MMP-9 secretion.     

An In Vivo C. elegans Model System for Screening EGFR-Inhibiting Anti-Cancer Drugs

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.     

Time-calibrated phylogenomics of the porcine epidemic diarrhea virus: genome-wide insights into the spatio-temporal dynamics

Porcine epidemic diarrhea virus (PEDV), the causative agent of porcine epidemic diarrhea (PED), has led to tremendous economic losses in the global swine industry. Although the phylogeny of PEDV has been investigated extensively at the molecular level, there was no time-calibrated phylogenomic study on the virus. To improve insight into this topic, we analyzed 138 published genome sequences using the Bayesian coalescent analyses as well as Bayesian inferences and maximum likelihood methods. All of the global PEDV isolates were divided into six groups, except for one unclassified isolate. Of the six groups, Groups 1–5 comprised pandemic viruses while the remaining Group 6 contained classical isolates. Interestingly, the two clades, both pandemic and classical, consisted of clade-specific amino acid sequences in five genes: ORF1a, ORF1b, S, ORF3, and N. Within the pandemic clade, Group 1 and Group 2 originated from North America, whereas Group 3–Group 5 were derived from Asia. In Group 2, there was a common origin of S INDEL isolates. Within each group, there was no apparent association between temporal or geographic origin and heterogeneity of PEDVs. Our findings also showed that the PEDV virus evolved at a rate of 3.38?×?10^?4 substitutions/site/year, and the most recent common ancestor of the virus emerged 75.9 years ago. Our Bayesian skyline plot analysis indicated that the PEDV had maintained constant effective population size excluding only a short period, around 2012, when a valley shaped decline in the effective number of infections occurred.     

Metformin and tenovin‐6 synergistically induces apoptosis through LKB1‐independent SIRT1 down‐regulation in non‐small cell lung cancer cells

Sirtuin 1 (SIRT1) is known to play a role in a variety of tumorigenesis processes by deacetylating histone and non‐histone proteins; however, antitumour effects by suppressing SIRT1 activity in non‐small cell lung cancer (NSCLC) remain unclear. This study was designed to scrutinize clinicopathological significance of SIRT1 in NSCLC and investigate effects of metformin on SIRT1 inhibition. This study also evaluated new possibilities of drug combination using a SIRT1 inhibitor, tenovin‐6, in NSCLC cell lines. It was found that SIRT1 was overexpressed in 300 (62%) of 485 formalin‐fixed paraffin‐embedded NSCLC tissues. Its overexpression was significantly associated with reduced overall survival and poor recurrence‐free survival after adjusted for histology and pathologic stage. Thus, suppression of SIRT1 expression may be a reasonable therapeutic strategy for NSCLC. Metformin in combination with tenovin‐6 was found to be more effective in inhibiting cell growth than either agent alone in NSCLC cell lines with different liver kinase B1 (LKB1) status. In addition, metformin and tenovin‐6 synergistically suppressed SIRT1 expression in NSCLC cells regardless of LKB1 status. The marked reduction in SIRT1 expression by combination of metformin and tenovin‐6 increased acetylation of p53 at lysine 382 and enhanced p53 stability in LKB1‐deficient A549 cells. The combination suppressed SIRT1 promoter activity more effectively than either agent alone by up‐regulating hypermethylation in cancer 1 (HIC1) binding at SIRT1 promoter. Also, suppressed SIRT1 expression by the combination synergistically induced caspase‐3‐dependent apoptosis. The study concluded that metformin with tenovin‐6 may enhance antitumour effects through LKB1‐independent SIRT1 down‐regulation in NSCLC cells.     

Signaling pathways implicated in alpha-melanocyte stimulating hormone-induced lipolysis in 3T3-L1 adipocytes

Melanocortins, besides their central roles, have also recently been reported to regulate adipocyte metabolism. In this study, we attempted to characterize the mechanism underlying alpha-melanocyte-stimulating hormone (MSH)-induced lipolysis, and compared it with that of the adrenocorticotrophin hormone (ACTH) in 3T3-L1 adipocytes. Similar to ACTH, MSH treatment resulted in the release of glycerol into the cell supernatant. The activity of hormone-sensitive lipase, a rate-limiting enzyme, which is involved in lipolysis, was significantly increased by MSH treatment. In addition, a variety of kinases, including protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) were also phosphorylated as the result of MSH treatment, and their specific inhibitors caused a reduction in MSH-induced glycerol release and HSL activity, indicating that MSH-induced lipolysis was mediated by these kinases. These results suggest that PKA and ERK constitute the principal signaling pathways implicated in the MSH-induced lipolytic process via the regulation of HSL in 3T3-L1 adipocytes.