Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.     

Parasitic helminth cystatin inhibits DSS-induced intestinal inflammation via IL-10(+)F4/80(+) macrophage recruitment

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-γ and TNF-α in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-α in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10(+)F4/80(+) macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.     

Drosophila as a model system for studying lifespan and neuroprotective activities of plant-derived compounds

The fruit fly, Drosophila melanogaster, has been intensively used as a genetic model system for basic and applied research on human neurological diseases because of advantages over mammalian model systems such as ease of laboratory maintenance and genetic manipulations. Disease-associated gene mutations, whether endogenous or transgenically-inserted, often cause phenotypes in vivo that are similar to the clinical features of the human disorder. The Drosophila genome is simpler than that of mammals, in terms of gene and chromosome number, but nonetheless demonstrates extraordinary phylogenetic conservation of gene structure and function, especially notable among the genes whose mutations cause neurodevelopmental, neuropsychiatric, or neurodegenerative disorders. In addition, its well-established neuroanatomical, developmental, and molecular genetic research techniques allow many laboratories worldwide to study complex biological and genetic processes. Based on these merits of the Drosophila model system, it has been used for screening lifespan expansion and neuroprotective activities of plant extracts or their secondary metabolites to counteract pathological events such as mitochondrial damage by oxidative stress, which may cause sporadic neurodegenerative diseases. In this review, we have summarized that the fruit fly can be used for early-stage drug discovery and development to identify novel plant-derived compounds to protect against neurodegeneration in Alzheimer's disease and Parkinson's disease, and other neurological disorders caused by oxidative stress. Thus, the Drosophila system can directly or indirectly contribute to translational research for new therapeutic strategies to prevent or ameliorate neurodegenerative diseases.     

Establishment and maintenance of an axenic culture of <Emphasis Type="Italic">Ettlia</Emphasis> sp. using a species-specific approach

The establishment of an axenic culture of microalgae is essential step in understanding its physiology, genetics, and ecology. However, culturing of microalgae is usually accompanied by complex and variable associated prokaryotic and eukaryotic microorganisms. Conventional approaches used for obtaining axenic cultures of microalgae are time-consuming and often involve difficulties in maintaining and preserving axenicity. In this study, we developed a procedure for establishing an axenic culture of Ettlia sp. YC001 and demonstrate that we maintained the axenic culture through subculture in the long term. Three sequential treatments, an antibiotic cocktail, serial dilution, and plate spreading, were applied to strain YC001 and we confirmed axenicity using molecular and physiological methods. The bacterial community associated with strain YC001 was investigated to select antibiotics for their specific elimination. The xenic culture (1 × 10⁶ cells/mL) was treated with the antibiotic cocktail-5 (AC-5), carbendazim, chloramphenicol, imipenem, rifampicin, and tetracycline for 3 days, followed by serial dilution up to 1 × 102 cells and spreading on agar plates. The pure colonies were analyzed using denaturing gradient gel electrophoresis (DGGE), fluorescence-activated cell sorting (FACS), and scanning electron microscopy (SEM). The procedure we developed can be applied to other strains of microalgae for the establishment of axenic cultures.     

Cryopreservation induces macrophage colony stimulating factor from human periodontal ligament cells in vitro

Cryopreservation is used to protect vital periodontal ligaments during the transplantation of teeth. We investigated which gene products implicated in root resorption are upregulated in human periodontal ligament cells by cryopreservation, and whether cryopreservation affects the expression of macrophage-colony stimulating factor (M-CSF) in human periodontal ligament cells. We used customized microarrays to compare gene expression in human periodontal ligament cells cultured from teeth immediately after extraction and from cryopreserved teeth. Based on the result of these assays, we examined M-CSF expression in periodontal ligament cells from the immediately extracted tooth and cryopreserved teeth by real-time PCR, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence. We also investigated whether human bone marrow cells differentiate into tartrate-resistant acid phosphatase (TRAP) positive osteoclasts when stimulated with RANKL (Receptor Activator for Nuclear Factor κ B Ligand) together with any secreted M-CSF present in the supernatants of the periodontal ligament cells cultured from the various groups of teeth. M-CSF was twofold higher in the periodontal ligament cells from the rapid freezing teeth than in those from the immediately extracted group (p < 0.05). Cryopreservation increased M-CSF expression in the periodontal ligament cells when analyzed by real time PCR, ELISA, Western blotting, and immunofluorescence (p < 0.05). TRAP positive osteoclasts were formed in response to RANKL and the secreted M-CSF present in the supernatants of all the experimental groups except negative control. These results demonstrate that cryopreservation promotes the production of M-CSF, which plays an important role in root resorption by periodontal ligament cells.     

Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.     

Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Oral microbiome profiles: 16S rRNA pyrosequencing and microarray assay comparison

Objectives

The human oral microbiome is potentially related to diverse health conditions and high-throughput technology provides the possibility of surveying microbial community structure at high resolution. We compared two oral microbiome survey methods: broad-based microbiome identification by 16S rRNA gene sequencing and targeted characterization of microbes by custom DNA microarray.

Methods

Oral wash samples were collected from 20 individuals at Memorial Sloan-Kettering Cancer Center. 16S rRNA gene survey was performed by 454 pyrosequencing of the V3–V5 region (450 bp). Targeted identification by DNA microarray was carried out with the Human Oral Microbe Identification Microarray (HOMIM). Correlations and relative abundance were compared at phylum and genus level, between 16S rRNA sequence read ratio and HOMIM hybridization intensity.

Results

The major phyla, Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were identified with high correlation by the two methods (r = 0.70∼0.86). 16S rRNA gene pyrosequencing identified 77 genera and HOMIM identified 49, with 37 genera detected by both methods; more than 98% of classified bacteria were assigned in these 37 genera. Concordance by the two assays (presence/absence) and correlations were high for common genera (Streptococcus, Veillonella, Leptotrichia, Prevotella, and Haemophilus; Correlation = 0.70–0.84).

Conclusion

Microbiome community profiles assessed by 16S rRNA pyrosequencing and HOMIM were highly correlated at the phylum level and, when comparing the more commonly detected taxa, also at the genus level. Both methods are currently suitable for high-throughput epidemiologic investigations relating identified and more common oral microbial taxa to disease risk; yet, pyrosequencing may provide a broader spectrum of taxa identification, a distinct sequence-read record, and greater detection sensitivity.     

Adjuvant effect of liposome-encapsulated natural phosphodiester CpG-DNA

Immunostimulatory CpG-DNA targeting TLR9 is one of the most extensively evaluated vaccine adjuvants. Previously, we found that a particular form of natural phosphodiester bond CpG-DNA (PO-ODN) encapsulated in a phosphatidyl-Β-oleoyl- γ-palmitoyl ethanolamine (DOPE) : cholesterol hemisuccinate (CHEMS) (1 : 1 ratio) complex (Lipoplex(O)) is a potent adjuvant. Complexes containing peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Here, we showed that IL-12 production was increased in bone marrow derived dendritic cells in a CpG sequence-dependent manner when PO-ODN was encapsulated in Lipoplex(O), DOTAP or lipofectamine. However, the effects of Lipoplex(O) surpassed those of PO-ODN encapsulated in DOTAP or lipofectamine and also other various forms of liposome-encapsulated CpG-DNA in terms of potency for protein antigen-specific IgG production and Th1- associated IgG2a production. Therefore, Lipoplex(O) may have a unique potent immunoadjuvant activity which can be useful for various applications involving protein antigens as well as peptides.