Direct analysis of microbial populations in soil and freshwater aquifers using nucleic acid based techniques

The first DNA-based methods for direct quantification of soil protozoa, and a DNA-based quantification method to describe the spread of phenanthrene-degrading bacteria in soil and freshwater aquifers, have recently been developed at the BIOPRO Research Centre at the Geological Survey of Denmark and Greenland (GEUS). Well-known genes for phenoxyalcanoic acid degradation have been used to monitor the in situ degradation of phenoxyalcanoic acid pesticides. Studies have been initiated on the short-lived mRNA molecules that are expected to provide a shortcut to the understanding of low, yet important, microbial activity in geological samples. This article reviews recent developments in techniques based on analysis of nucleic acids from soils and aquifers. Analytical work has been carried out mainly on soil samples from a former asphalt production plant at Ringe (Fig. 1). The Ringe plant constitutes one of the most polluted industrial sites in Denmark, and is a priority site of studies by the BIOPRO Research Centre. Although rich in carbon, the Ringe subsoil is an oligotrophic environment due to the high content of polycyclic aromatic hydrocarbons (PAH). This is an environment where the supply of nutrients to microorganisms is low, leading to slow growth, low total numbers of microorganisms and small cells. To study microbial communities of oligotrophic environments, analytical methods with low detection limits are needed. Until recently, microorganisms of natural environments were mainly studied by cultivation-dependent methods. However, microorganisms that can be cultured on agar plates are now known to represent only a small fraction of the total microbial community. Modern methods, therefore, need to be based on the detection of biomolecules in the microorganisms rather than being dependent on growth of the microorganisms. The best available techniques are based on DNA and RNA molecules (Fig. 2), which due to their high level of resolution allow closely related organisms or functional genes to be distinguished. In the following review, examples are given of applications of these nucleic acid based methods

A Highly Selective Cc Chemokine Receptor (Ccr)8 Antagonist Encoded by the Poxvirus Molluscum Contagiosum

The MC148 CC chemokine from the human poxvirus molluscum contagiosum (MCV) was probed in parallel with viral macrophage inflammatory protein (vMIP)-II encoded by human herpesvirus 8 (HHV8) in 16 classified human chemokine receptors. In competition binding using radiolabeled endogenous chemokines as well as radiolabeled MC148, MC148 bound with high affinity only to CCR8. In calcium mobilization assays, MC148 had no effect on its own on any of the chemokine receptors, but in a dose-dependent manner blocked the stimulatory effect of the endogenous I-309 chemokine on CCR8 without affecting chemokine-induced signaling of any other receptor. In contrast, vMIP-II acted as an antagonist on 10 of the 16 chemokine receptors, covering all four classes: XCR, CCR, CXCR, and CX3CR. In chemotaxis assays, MC148 specifically blocked the I-309–induced response but, for example, not stromal cell–derived factor 1α, monocyte chemoattractant protein 1, or interleukin 8–induced chemotaxis. We thus concluded that the two viruses choose two different ways to block the chemokine system: HHV8 encodes the broad-spectrum chemokine antagonist vMIP-II, whereas MCV encodes a highly selective CCR8 antagonist, MC148, conceivably to interfere with monocyte invasion and dendritic cell function. Because of its pharmacological selectivity, the MC148 protein could be a useful tool in the delineation of the role played by CCR8 and its endogenous ligand, I-309

A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain

Maladaptive plasticity involving increased expression of AMPA‐type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell‐permeable, high‐affinity (~2 nM) peptide inhibitor, Tat‐P$_4$‐(C5)$_2$, of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat‐P$_4$‐(C5)$_2$ disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA‐receptor surface expression in vivo. Moreover, Tat‐P$_4$‐(C5)$_2$ administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat‐P$_4$‐(C5)$_2$ as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non‐tandem protein–protein interaction domains

miRNA-mRNA Integrated Analysis Reveals Roles for miRNAs in Primary Breast Tumors

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