Medical bioremediation of age-related diseases

Catabolic insufficiency in humans leads to the gradual accumulation of a number of pathogenic compounds associated with age-related diseases, including atherosclerosis, Alzheimer's disease, and macular degeneration. Removal of these compounds is a widely researched therapeutic option, but the use of antibodies and endogenous human enzymes has failed to produce effective treatments, and may pose risks to cellular homeostasis. Another alternative is "medical bioremediation," the use of microbial enzymes to augment missing catabolic functions. The microbial genetic diversity in most natural environments provides a resource that can be mined for enzymes capable of degrading just about any energy-rich organic compound. This review discusses targets for biodegradation, the identification of candidate microbial enzymes, and enzyme-delivery methods.     

Microbial production host selection for converting second-generation feedstocks into bioproducts

Background  

Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates are complex mixtures of different fermentable sugars, but also inhibitors and salts that affect the performance of the microbial production host. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum), two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) were compared for their (i) ability to utilize monosaccharides present in lignocellulosic hydrolysates, (ii) resistance against inhibitors present in lignocellulosic hydrolysates, (iii) their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). The feedstock hydrolysates were generated in two manners: (i) thermal pretreatment under mild acid conditions followed by enzymatic hydrolysis and (ii) a non-enzymatic method in which the lignocellulosic biomass is pretreated and hydrolyzed by concentrated sulfuric acid. Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated.     

Radial oxygen loss by the cushion plant Eriocaulon schimperi prevents methane emissions from an East‐African mountain mire

• Groundwater‐fed fens are known sources of methane (CH₄) emissions to the atmosphere, and these are known to be mediated by the vegetation. In a fen located in the Bale Mountains, Ethiopia, we assessed the effects of a cushion plant (Eriocaulon schimperi) and a sedge (Carex monostachya) on rhizosphere biogeochemistry.
• Methane and CO₂ concentrations and pH were measured in pore‐water at different depths in the profile. Redox potentials and NaCl‐extractable element concentrations were analysed in soil samples from sites dominated by either E. schimperii or C. monostachya. Nutrient and element concentration were analysed in plant tissues.
• At Carex‐dominated sites, CH₄ concentrations increased from 70 μmol·l^?1 at a depth of 10 cm to 130 μmol·l^?1 at a depth of 100 cm. CH₄ concentrations at Eriocaulon‐dominated sites were almost zero (<1 μmol·l^?1) to a depth of 100 cm. Simultaneously, soil redox potentials and CO₂ concentrations were higher at Eriocaulon‐dominated sites, indicating a low potential for CH₄ production and a high potential for CH₄ oxidation.
• Eriocaulon schimperi displayed a root investment strategy to cope with the harsh environment, similar to the cushion plant Astelia pumila in Patagonian bogs. This strategy is characterised by high root/shoot ratios, high root porosity and density under high redox conditions. Both cushion plant species create an aerobic rhizosphere through radial oxygen loss from deep roots, which strongly reduce CH₄ fluxes to the atmosphere.

     

Affinity cleavage and targeted catalysis of proteins using the avidin-biotin system

The avidin-biotin system was used in order to target enzymes to their substrates in complex mixtures of proteins in solution. The approach described here thus mimics natural systems in which enzymes usually act in selective fashion, due, perhaps, to proximity effects. For affinity cleavage studies, biotinyl transferrin was used as a model target substrate. Avidin or streptavidin was then employed to bridge between the biotinylated target protein and a biotinyl protease. Bovine serum albumin was included in the reaction mixtures to assess the level of nonspecific cleavage. In the case of an unbiotinylated target protein, avidin could be used to inhibit the hydrolytic action of the biotinyl protease. In some systems, a biotinyl antibody could be used to direct the avidin-bridged biotinyl protease to an unbiotinylated target antigen. The data support the contention that preferential cleavage reflects two separate phenomena: (i) avidin confers a conformational alteration of the biotinylated target protein, and (ii) the biotinyl protease is targeted (via the avidin bridge) to the proximity of the biotinylated target protein, thereby promoting cleavage of the conformationally altered molecule. This is the first report in which a proteolytic enzyme could be selectively targeted to specifically hydrolyze a defined protein substrate in solutions containing a complex mixture of other proteins. The approach appears to be a general phenomenon for "targeted catalysis", appropriate for other applications, particularly for affinity cleavage and targeted catalysis of cell-based macromolecules.     

Hypericin-photodynamic therapy leads to interleukin-6 secretion by HepG2 cells and their apoptosis via recruitment of BH3 interacting-domain death agonist and caspases

Photodynamic therapy (PDT) has emerged as a capable therapeutic modality for the treatment of cancer. PDT is a targeted cancer therapy that reportedly leads to tumor cell apoptosis and/or necrosis by facilitating the secretion of certain pro-inflammatory cytokines and expression of multiple apoptotic mediators in the tumor microenvironment. In addition, PDT also triggers oxidative stress that directs tumor cell killing and activation of inflammatory responses. However, the cellular and molecular mechanisms underlying the role of PDT in facilitating tumor cell apoptosis remain ambiguous. Here, we investigated the ability of PDT in association with hypericin (HY) to induce tumor cell apoptosis by facilitating the induction of reactive oxygen species (ROS) and secretion of Th1/Th2/Th17 cytokines in human hepatocellular liver carcinoma cell line (HepG2) cells. To discover if any apoptotic mediators were implicated in the enhancement of cell death of HY-PDT-treated tumor cells, selected gene profiling in response to HY-PDT treatment was implemented. Experimental results showed that interleukin (IL)-6 was significantly increased in all HY-PDT-treated cells, especially in 1 μg/ml HY-PDT, resulting in cell death. In addition, quantitative real-time PCR analysis revealed that the expression of apoptotic genes, such as BH3-interacting-domain death agonist (BID), cytochrome complex (CYT-C) and caspases (CASP3, 6, 7, 8 and 9) was remarkably higher in HY-PDT-treated HepG2 cells than the untreated HepG2 cells, entailing that tumor destruction of immune-mediated cell death occurs only in PDT-treated tumor cells. Hence, we showed that HY-PDT treatment induces apoptosis in HepG2 cells by facilitating cytotoxic ROS, and potentially recruits IL-6 and apoptosis mediators, providing additional hints for the existence of alternative mechanisms of anti-tumor immunity in hepatocellular carcinoma, which contribute to long-term suppression of tumor growth following PDT.     

Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples

High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-peak with reliable retention time (Rt.). However, in the current study, we have found that under the same LC-MS conditions, the Rt. and shape of LC-peaks of bile acids in urine samples from animals fed dissimilar diets differed significantly among each other. To verify this matrix effect, 17 authentic bile acid standards were dissolved in pure methanol or in methanol containing extracts of urine from pigs consuming either breast milk or infant formula and analyzed by LC-MS/MS. The matrix components in urine from piglets fed formula significantly reduced the LC-peak Rt. and areas of bile acids. This is the first characterization of this matrix effect on Rt. in the literature. Moreover, the matrix effect resulted in an unexpected LC behavior: one single compound yielded two LC-peaks, which broke the rule of one LC-peak for one compound. The three bile acid standards which exhibited this unconventional LC behavior were chenodeoxycholic acid, deoxycholic acid, and glycocholic acid. One possible explanation for this effect is that some matrix components may have loosely bonded to analytes, which changed the time analytes were retained on a chromatography column and interfered with the ionization of analytes in the MS ion source to alter the peak area. This study indicates that a comprehensive understanding of matrix effects is needed towards improving the use of HPLC and LC-MS/MS techniques for qualitative and quantitative analyses of analytes in pharmacokinetics, proteomics/metabolomics, drug development, and sports drug testing, especially when LC-MS/MS data are analyzed by automation software where identification of an analyte is based on its exact molecular weight and Rt.     

Compensatory evolution for a gene deletion is not limited to its immediate functional network

Background  

Genetic disruption of an important phenotype should favor compensatory mutations that restore the phenotype. If the genetic basis of the phenotype is modular, with a network of interacting genes whose functions are specific to that phenotype, compensatory mutations are expected among the genes of the affected network. This perspective was tested in the bacteriophage T3 using a genome deleted of its DNA ligase gene, disrupting DNA metabolism.