Adaptive laboratory evolution of microbial co‐cultures for improved metabolite secretion

Adaptive laboratory evolution has proven highly effective for obtaining microorganisms with enhanced capabilities. Yet, this method is inherently restricted to the traits that are positively linked to cell fitness, such as nutrient utilization. Here, we introduce coevolution of obligatory mutualistic communities for improving secretion of fitness‐costly metabolites through natural selection. In this strategy, metabolic cross‐feeding connects secretion of the target metabolite, despite its cost to the secretor, to the survival and proliferation of the entire community. We thus co‐evolved wild‐type lactic acid bacteria and engineered auxotrophic Saccharomyces cerevisiae in a synthetic growth medium leading to bacterial isolates with enhanced secretion of two B‐group vitamins, viz., riboflavin and folate. The increased production was specific to the targeted vitamin, and evident also in milk, a more complex nutrient environment that naturally contains vitamins. Genomic, proteomic and metabolomic analyses of the evolved lactic acid bacteria, in combination with flux balance analysis, showed altered metabolic regulation towards increased supply of the vitamin precursors. Together, our findings demonstrate how microbial metabolism adapts to mutualistic lifestyle through enhanced metabolite exchange.     

Structure of the ribosome associating GTPase HflX

The HflX‐family is a widely distributed but poorly characterized family of translation factor‐related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0‐Å resolution in apo‐ and GDP‐bound forms. The enzyme displays a two‐domain architecture with a novel “HflX domain” at the N‐terminus, and a classical G‐domain at the C‐terminus. The HflX domain is composed of a four‐stranded parallel β‐sheet flanked by two α‐helices on either side, and an anti‐parallel coiled coil of two long α‐helices that lead to the G‐domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX‐domain upon GTP‐binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24‐fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX‐domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.     

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.     

Elevated CO2 Levels do not Affect the Shell Structure of the Bivalve Arctica islandica from the Western Baltic

Shells of the bivalve Arctica islandica are used to reconstruct paleo-environmental conditions (e.g. temperature) via biogeochemical proxies, i.e. biogenic components that are related closely to environmental parameters at the time of shell formation. Several studies have shown that proxies like element and isotope-ratios can be affected by shell growth and microstructure. Thus it is essential to evaluate the impact of changing environmental parameters such as high pCO₂ and consequent changes in carbonate chemistry on shell properties to validate these biogeochemical proxies for a wider range of environmental conditions. Growth experiments with Arctica islandica from the Western Baltic Sea kept under different pCO₂ levels (from 380 to 1120 µatm) indicate no affect of elevated pCO₂ on shell growth or crystal microstructure, indicating that A. islandica shows an adaptation to a wider range of pCO₂ levels than reported for other species. Accordingly, proxy information derived from A. islandica shells of this region contains no pCO₂ related bias.     

Consequences of Cathodal Stimulation for Behavior: When Does It Help and When Does It Hurt Performance?

Cathodal Transcranial Direct Current Stimulation (C-tDCS) has been reported, across different studies, to facilitate or hinder performance, or simply to have no tangible effect on behavior. This discrepancy is most prominent when C-tDCS is used to alter a cognitive function, questioning the assumption that cathodal stimulation always compromises performance. In this study, we aimed to study the effect of two variables on performance in a simple cognitive task (letter Flanker), when C-tDCS was applied to the left prefrontal cortex (PFC): (1) the time of testing relative to stimulation (during or after), and (2) the nature of the cognitive activity during stimulation in case of post-tDCS testing. In three experiments, we had participants either perform the Flanker task during C-tDCS (Experiment 1), or after C-tDCS. When the Flanker task was administered after C-tDCS, we varied whether during stimulation subjects were engaged in activities that posed low (Experiment 2) or high (Experiment 3) demands on the PFC. Our findings show that the nature of the task during C-tDCS has a systematic influence on the outcome, while timing per se does not.     

S/T Phosphorylation of DLL1 Is Required for Full Ligand Activity In Vitro but Dispensable for DLL1 Function In Vivo during Embryonic Patterning and Marginal Zone B Cell Development

Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.     

The Himalayas: Barrier and conduit for gene flow

The Himalayan mountain range is strategically located at the crossroads of the major cultural centers in Asia, the Middle East and Europe. Although previous Y‐chromosome studies indicate that the Himalayas served as a natural barrier for gene flow from the south to the Tibetan plateau, this region is believed to have played an important role as a corridor for human migrations between East and West Eurasia along the ancient Silk Road. To evaluate the effects of the Himalayan mountain range in shaping the maternal lineages of populations residing on either side of the cordillera, we analyzed mitochondrial DNA variation in 344 samples from three Nepalese collections (Newar, Kathmandu and Tamang) and a general population of Tibet. Our results revealed a predominantly East Asian‐specific component in Tibet and Tamang, whereas Newar and Kathmandu are both characterized by a combination of East and South Central Asian lineages. Interestingly, Newar and Kathmandu harbor several deep‐rooted Indian lineages, including M2, R5, and U2, whose coalescent times from this study (U2, >40 kya) and previous reports (M2 and R5, >50 kya) suggest that Nepal was inhabited during the initial peopling of South Central Asia. Comparisons with our previous Y‐chromosome data indicate sex‐biased migrations in Tamang and a founder effect and/or genetic drift in Tamang and Newar. Altogether, our results confirm that while the Himalayas acted as a geographic barrier for human movement from the Indian subcontinent to the Tibetan highland, it also served as a conduit for gene flow between Central and East Asia. Am J Phys Anthropol 151:169–182, 2013. © 2013 Wiley Periodicals, Inc.     

Mitochondrial Dysfunction—A Pharmacological Target in Alzheimer's Disease

Increasing evidences suggest that mitochondrial dysfunction plays an important role in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). Alterations of mitochondrial efficiency and function are mainly related to alterations in mitochondrial content, amount of respiratory enzymes, or changes in enzyme activities leading to oxidative stress, mitochondrial permeability transition pore opening, and enhanced apoptosis. More recently, structural changes of the network are related to bioenergetic function, and its consequences are a matter of intensive research. Several mitochondria-targeting compounds with potential efficacy in AD including dimebon, methylene blue, piracetam, simvastatin, Ginkgo biloba, curcumin, and omega-3 polyunsaturated fatty acids have been identified. The majority of preclinical data indicate beneficial effects, whereas most controlled clinical trials did not meet the expectations. Since mitochondrial dysfunction represents an early event in disease progression, one reason for the disappointing clinical results could be that pharmacological interventions might came too late. Thus, more studies are needed that focus on therapeutic strategies starting before severe disease progress.