New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

The anammox case-a new experimental manifesto for microbiological eco-physiology

The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional ‘cultivation based techniques’ alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers. This revised version was published online in August 2006 with corrections to the Cover Date.     

Pharmacogenomics - it''s not just pharmacogenetics

New technologies in both combinatorial chemistry and combinatorial biology promise to unlock new opportunities for drug discovery and lead optimisation. Using such genome-based technologies to measure the dynamic properties of pharmacological systems, pharmacogenomics can now provide an objective measure of a drug's biological efficacy, including its potential adverse effects.     

Pancreatic adenocarcinoma induces bone marrow mobilization of myeloid-derived suppressor cells which promote primary tumor growth

Purpose

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells that are upregulated in cancer. Little is known about the prevalence and importance of MDSC in pancreas adenocarcinoma (PA).

Experimental design

Peripheral blood, bone marrow, and tumor samples were collected from pancreatic cancer patients, analyzed for MDSC (CD15⁺CD11b⁺) by flow cytometry and compared to cancer-free controls. The suppressive capacity of MDSC (CD11b⁺Gr-1⁺) and the effectiveness of MDSC depletion were assessed in C57BL/6 mice inoculated with Pan02, a murine PA, and treated with placebo or zoledronic acid, a potent aminobisphosphonate previously shown to target MDSC. The tumor microenvironment was analyzed for MDSC (Gr1⁺CD11b⁺), effector T cells, and tumor cytokine levels.

Results

Patients with PA demonstrated increased frequency of MDSC in the bone marrow and peripheral circulation which correlated with disease stage. Normal pancreas tissue showed no MDSC infiltrate, while human tumors avidly recruited MDSC. Murine tumors similarly recruited MDSC that suppressed CD8⁺ T cells in vitro and accelerated tumor growth in vivo. Treatment with zoledronic acid impaired intratumoral MDSC accumulation resulting in delayed tumor growth rate, prolonged median survival, and increased recruitment of T cells to the tumor. This was associated with a more robust type 1 response with increased levels of IFN-γ and decreased levels of IL-10.

Conclusions

MDSC are important mediators of tumor-induced immunosuppression in pancreatic cancer. Inhibiting MDSC accumulation with zoledronic acid improves the host anti-tumor response in animal studies suggesting that efforts to block MDSC may represent a novel treatment strategy for pancreatic cancer.     

Plant Conservation in the Caribbean Island Biodiversity Hotspot

While the Caribbean is a recognized “biodiversity hotspot”, plant conservation has not received adequate attention; particularly, given the high levels of endemism in many plant groups. Besides establishing protected areas, there needs to be a sustained effort to study the taxonomy, systematics and ecology of the flora. Recent phylogenetic studies have shown high levels of endemism and conservation studies indicate a large propotion of the flora is threatened with extinction. Eight recommendations are given for plant conservation in the region.     

Expanding the Verrucomicrobial Methanotrophic World: Description of Three Novel Species of Methylacidimicrobium gen. nov.

Methanotrophic Verrucomicrobia have been found in geothermal environments characterized by high temperatures and low pH values. However, it has recently been hypothesized that methanotrophic Verrucomicrobia could be present under a broader range of environmental conditions. Here we describe the isolation and characterization of three new species of mesophilic acidophilic verrucomicrobial methanotrophs from a volcanic soil in Italy. The three new species showed 97% to 98% 16S rRNA gene identity to each other but were related only distantly (89% to 90% on the 16S rRNA level) to the thermophilic genus Methylacidiphilum. We propose the new genus Methylacidimicrobium, including the novel species Methylacidimicrobium fagopyrum, Methylacidimicrobium tartarophylax, and Methylacidimicrobium cyclopophantes. These mesophilic Methylacidimicrobium spp. were more acid tolerant than their thermophilic relatives; the most tolerant species, M. tartarophylax, still grew at pH 0.5. The variation in growth temperature optima (35 to 44°C) and maximum growth rates (µmax; 0.013 to 0.040 h⁻¹) suggested that all species were adapted to a specific niche within the geothermal environment. All three species grew autotrophically using the Calvin cycle. The cells of all species contained glycogen particles and electron-dense particles in their cytoplasm as visualized by electron microscopy. In addition, the cells of one of the species (M. fagopyrum) contained intracytoplasmic membrane stacks. The discovery of these three new species and their growth characteristics expands the known diversity of verrucomicrobial methanotrophs and shows that they are present in many more ecosystems than previously assumed.     

Peptide YY and neuropeptide Y induce villin expression, reduce adhesion, and enhance migration in small intestinal cells through the regulation of CD63, matrix metalloproteinase-3, and Cdc42 activity

Peptide YY (PYY) and neuropeptide Y (NPY) are regulatory peptides synthesized in the intestine and brain, respectively, that modify physiological functions affecting nutrient assimilation and feeding behavior. Because PYY and NPY also alter the expression of intestine-specific differentiation marker proteins and the tetraspanin CD63, which is involved in cell adhesion, we investigated whether intestinal cell differentiation could be linked to mucosal cell adhesion and migration through these peptides. PYY and NPY significantly decreased cell adhesion and increased cell migration in a dose-dependent manner prior to cell confluency in our model system, non-tumorigenic small intestinal hBRIE 380i cells. Both peptides reduced CD63 expression and CD63-dependent cell adhesion. CD63 overexpression increased and antisense CD63 cDNA decreased intestinal cell adhesion. In parallel, both PYY and NPY increased expression of matrix metalloproteinase-3 (MMP-3) to a level sufficient to induce cell migration by activating the Rho GTPase Cdc42. The effects of both peptides on cell migration were blocked in cells constitutively overexpressing dominant-negative Cdc42. PYY and NPY also significantly induced the expression of the differentiation marker villin, which could be eliminated by an MMP inhibitor at a concentration that inhibits cell migration. Increased MMP-3 activity, which enhanced cell migration, also induced villin mRNA levels. Therefore, these data indicate that the alteration of adhesion and migration by PYY and NPY occurs in part by synchronous modulation of three proteins that are involved in extracellular matrix-basolateral membrane interactions, CD63, MMP-3 and Cdc42, and that PYY/NPY regulation of expression of mucosal proteins such as villin is linked to the process of cell migration and adhesion.     

Novel phenylamino acetamide derivatives as potent and selective kappa opioid receptor agonists

A novel series of phenylamino acetamide derivatives was synthesized. These amides were shown to be potent and selective kappa opioid receptor agonists.