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.     

Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide

Triazolopyrimidine sulfanilides are a class of highly active herbicides whose primary target is acetolactate synthase. Spontaneous mutants of tobacco (Nicotiana tabacum) (KS-43) and cotton (Gossypium hirsutum) (PS-3 and DO-2) resistant to triazolopyrimidine sulfonanilide were selected in tissue culture. Acetolactate synthase partially purified from the three mutants were 80- to 1000-fold less sensitive to inhibition by the compound compared with the corresponding wild-type enzyme. The mutants also varied in the cross-resistance pattern to other acetolactate synthase inhibiting herbicides in the sulfonylurea, imidazolinone, and pyrimidyl-oxy-benzoate chemical families. Thus, acetolactate synthase from KS-43, PS-3, and DO-2 cultures have different mutations. The affinities for pyruvate, thiamine pyrophosphate, as well as the activity of the mutant enzymes were found to be comparable to the corresponding wild-type enzymes. However, the enzyme from PS-3 was highly resistant to feedback inhibition by valine and leucine. In contrast, acetolactate synthase from KS-43 and DO-2 were inhibited by valine and leucine to nearly the same extent as the wild-type enzymes. Also, PS-3 cultures accumulated much higher levels of the branched chain amino acids compared to the wild-type cotton culture. The mutation in the PS-3 enzyme has therefore rendered it insensitive to feedback regulation by valine and leucine.     

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.     

Comparison of the evolutionary dynamics of symbiotic and housekeeping loci: a case for the genetic coherence of rhizobial lineages

In prokaryotes, lateral gene transfer across chromosomal lineages may be mediated by plasmids, phages, transposable elements, and other accessory DNA elements. However, the importance of such transfer and the evolutionary forces that may restrict gene exchange remain largely unexplored in native settings. In this study, tests of phylogenetic congruence are employed to explore the range of horizontal transfer of symbiotic (sym) loci among distinct chromosomal lineages of native rhizobia, the nitrogen-fixing symbiont of legumes. Rhizobial strains isolated from nodules of several host plant genera were sequenced at three loci: symbiotic nodulation genes (nodB and nodC), the chromosomal housekeeping locus glutamine synthetase II (GSII), and a portion of the 16S rRNA gene. Molecular phylogenetic analysis shows that each locus generally subdivides strains into the same major groups, which correspond to the genera Rhizobium, Sinorhizobium, and Mesorhizobium. This broad phylogenetic congruence indicates a lack of lateral transfer across major chromosomal subdivisions, and it contrasts with previous studies of agricultural populations showing broad transfer of sym loci across divergent chromosomal lineages. A general correspondence of the three rhizobial genera with major legume groups suggests that host plant associations may be important in the differentiation of rhizobial nod and chromosomal loci and may restrict lateral transfer among strains. The second major result is a significant incongruence of nod and GSII phylogenies within rhizobial subdivisions, which strongly suggests horizontal transfer of nod genes among congenerics. This combined evidence for lateral gene transfer within, but not between, genetic subdivisions supports the view that rhizobial genera are "reproductively isolated" and diverge independently. Differences across rhizobial genera in the specificity of host associations imply that the evolutionary dynamics of the symbiosis vary considerably across lineages in native settings.      

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.