Distance-based analysis of variance for brain connectivity

The field of neuroimaging dedicated to mapping connections in the brain is increasingly being recognized as key for understanding neurodevelopment and pathology. Networks of these connections are quantitatively represented using complex structures, including matrices, functions, and graphs, which require specialized statistical techniques for estimation and inference about developmental and disorder-related changes. Unfortunately, classical statistical testing procedures are not well suited to high-dimensional testing problems. In the context of global or regional tests for differences in neuroimaging data, traditional analysis of variance (ANOVA) is not directly applicable without first summarizing the data into univariate or low-dimensional features, a process that might mask the salient features of high-dimensional distributions. In this work, we consider a general framework for two-sample testing of complex structures by studying generalized within-group and between-group variances based on distances between complex and potentially high-dimensional observations. We derive an asymptotic approximation to the null distribution of the ANOVA test statistic, and conduct simulation studies with scalar and graph outcomes to study finite sample properties of the test. Finally, we apply our test to our motivating study of structural connectivity in autism spectrum disorder.     

Influence of hydrological connectivity of riverine wetlands on nitrogen removal via denitrification

Wetland ecosystems in agricultural areas often become progressively more isolated from main water bodies. Stagnation favors the accumulation of organic matter as the supply of electron acceptors with water renewal is limited. In this context it is expected that nitrogen recycling prevails over nitrogen dissipation. To test this hypothesis, denitrification rates, fluxes of dissolved oxygen (SOD), inorganic carbon (DIC) and nitrogen and sediment features were measured in winter and summer 2007 on 22 shallow riverine wetlands in the Po River Plain (Northern Italy). Fluxes were determined from incubations of intact cores by measurement of concentration changes or isotope pairing in the case of denitrification. Sampled sites were eutrophic to hypertrophic; 10 were connected and 12 were isolated from the adjacent rivers, resulting in large differences in nitrate concentrations in the water column (from <5 to 1,133 μM). Benthic metabolism and denitrification rates were investigated by two overarching factors: season and hydrological connectivity. SOD and DIC fluxes resulted in respiratory quotients greater than one at most sampling sites. Sediment respiration was coupled to both ammonium efflux, which increased from winter to summer, and nitrate consumption, with higher rates in river-connected wetlands. Denitrification rates measured in river-connected wetlands (35–1,888 μmol N m^?2 h^?1) were up to two orders of magnitude higher than rates measured in isolated wetlands (2–231 μmol N m^?2 h^?1), suggesting a strong regulation of the process by nitrate availability. These rates were also significantly higher in summer (9–1,888 μmol N m^?2 h^?1) than in winter (2–365 μmol N m^?2 h^?1). Denitrification supported by water column nitrate (D_W) accounted for 60–100% of total denitrification (Dtot); denitrification coupled to nitrification (D_N) was probably controlled by limited oxygen availability within sediments. Denitrification efficiency, calculated as the ratio between N removal via denitrification and N regeneration, and the relative role of denitrification for organic matter oxidation, were high in connected wetlands but not in isolated sites. This study confirms the importance of restoring hydraulic connectivity of riverine wetlands for the maintenance of important biogeochemical functions such as nitrogen removal via denitrification.     

Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Sulfolobus solfataricus via chemical rescue of an inactive mutant

We have recently reported that a functional alpha-L-fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622-14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-alpha-L-fucoside (pNp-Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the alpha-L-fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 10(3)-fold activity reduction on pNp-Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp-Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 alpha-L-fucosidases. This is the first time that this approach is used for alpha-L-glycosidases, widening the applicability of this method.     

Biosynthesis of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 using liquid acid protein residue of soybean as culture medium

Vitamin B12 deficiency still persists, mainly caused by low intake of animal food products affecting vegetarians, vegans, and populations of underdeveloped countries. In this study, we investigate the biosynthesis of vitamin B12 by potential probiotic bacterium using an agroindustry residue, the liquid acid protein residue of soybean (LAPRS), as a low-cost, animal derivate-free alternative culture medium. Cultures of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 growing in LAPRS for vitamin B12 biosynthesis were studied using the Plackett–Burman experimental approach, followed by a central composite design 2² to optimize the concentration of significant variables. We also performed a proteolytic treatment of LAPRS and evaluated the optimized–hydrolyzed medium influence on the microbial growth and metabolism in shaker flask and bioreactor experiments. In this all-plant source medium, P. freudenreichii subsp. shermanii produced high concentrations of cells and high amounts of vitamin B12 (0.6 mg/g cells) after process optimization. These results suggest the possibility of producing vitamin B12 by a potential probiotic bacterium in a very cheap, animal derivate-free medium to address the needs of specific population groups, at the same time reducing the production costs of this essential vitamin.     

Discovery of a novel styrene monooxygenase originating from the metagenome

Oxygenases form an interesting class of biocatalysts, as they typically perform oxygenations with exquisite chemo-, regio-, and/or enantioselectivity. It has been observed that, once heterologously expressed in Escherichia coli, some oxygenases are able to form the blue pigment indigo. We have exploited this characteristic to screen a metagenomic library derived from loam soil and identified a novel oxygenase. This oxygenase shows 50% sequence identity with styrene monooxygenases from pseudomonads (StyA). Only a limited number of homologs can be found in the genome sequence database, indicating that this biocatalyst is a member of a relatively small family of bacterial monooxygenases. The newly identified monooxygenase catalyzes the epoxidation of styrene and styrene derivatives and forms the corresponding (S)-epoxides with excellent enantiomeric excess [e.g., (S)-styrene oxide is formed with >99% enantiomeric excess, ee] and therefore is named styrene monooxgenase subunit A (SmoA). SmoA shows high enantioselectivity towards aromatic sulfides [e.g., (R)-ethyl phenyl sulfoxide is formed with 92% ee]. This excellent enantioselectivity in combination with the moderate sequence identity forms a clear indication that SmoA from a metagenomic origin represents a new enzyme within the small family of styrene monooxygenases.     

INCREASED RESIDUAL VARIANCE AT DEVELOPMENTAL SWITCH POINTS: STATISTICAL ARTIFACT OR INDICATOR OF EXPOSED GENOTYPIC INFLUENCE?

Discussing the organization of developmental switches, West-Eberhard (2003) proposed the use of regression residual plots to locate the neutral point of the switch, which is characterized by maximum genotypic influence on the resulting phenotype. However, statistical artifacts due to measurement error in nonlinear models might account for a substantial proportion of the increase of residuals variance at the switch point. Simulations based on field data show that increases in residual variance occur as artifacts when normal amounts of measurement error are present, even in absence of any genotypic variance. The results suggest that interpretation of nonlinear variation in thresold traits is problematic and requires considering this statistical effect. A method to estimate the weight of genotypic contribution to residual variance is proposed, and its assumptions and limitations are discussed.     

Synthesis and stability in biological media of 1H-imidazole-1-carboxylates of ROS203, an antagonist of the histamine H3 receptor

A series of carbamate derivatives of the H(3) antagonist ROS203 (1) were prepared, and their lipophilicity and steric hindrance were modulated by introducing linear or branched alkyl chains of various lengths. In vitro stability studies were conducted to evaluate how structural modulations affect the intrinsic reactivity of the carbamoyl moiety and its recognition by metabolic enzymes. Linear alkyl carbamates were the most susceptible to enzymatic hydrolysis, with bioconversion rates being higher in rat liver and plasma. Chain ramification significantly enhanced the enzymatic stability of the set, with two derivatives (1g and 1h) being more stable by a factor of 8-40 than the ethyl carbamate 1a. Incubation with bovine serum albumin (BSA) showed a protective role of proteins on chemical and porcine-liver esterase (PLE)-catalyzed hydrolysis. Ex vivo binding data after i.v. administration of 1h revealed prolonged displacement of the labeled ligand [(3)H]-(R)-alpha-methylhistamine ([(3)H]RAMHA) from rat-brain cortical membranes, when compared to 1. However, the high rates of bioconversion in liver, as well as the chemical instability of 1h, suggest that further work is needed to optimize the enzymatic and chemical stability of these compounds.     

Bacterial colonization of a fumigated alkaline saline soil

After chloroform fumigating an arable soil, the relative abundance of phylotypes belonging to only two phyla (Actinobacteria and Firmicutes) and two orders [Actinomycetales and Bacillales (mostly Bacillus)] increased in a subsequent aerobic incubation, while it decreased for a wide range of bacterial groups. It remained to be seen if similar bacterial groups were affected when an extreme alkaline saline soil was fumigated. Soil with electrolytic conductivity between 139 and 157 dS m^?1, and pH 10.0 and 10.3 was fumigated and the bacterial community structure determined after 0, 1, 5 and 10 days by analysis of the 16S rRNA gene, while an unfumigated soil served as control. The relative abundance of the Firmicutes increased in the fumigated soil (52.8 %) compared to the unfumigated soil (34.2 %), while that of the Bacteroidetes decreased from 16.2 % in the unfumigated soil to 8.8 % in the fumigated soil. Fumigation increased the relative abundance of the genus Bacillus from 14.7 % in the unfumigated soil to 25.7 %. It was found that phylotypes belonging to the Firmicutes, mostly of the genus Bacillus, were dominant in colonizing the fumigated alkaline saline as found in the arable soil, while the relative abundance of a wide range of bacterial groups decreased.     

A Quantitative and Dynamic Model of the Arabidopsis Flowering Time Gene Regulatory Network

Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.     

Trophic relationships between the larvae of two freshwater mussels and their fish hosts

Freshwater mussels of the order Unionoida have life cycles that include larval attachment to and later metamorphosis on suitable host fishes. Information on the trophic relationship between unionoid larvae and their host fishes is scarce. We investigated the trophic interaction between fish hosts and encysted larvae of two species of freshwater mussels, Margaritifera margaritifera and Unio crassus, using stable isotope analyses of larvae and juvenile mussels as well as of host fish gill and muscle tissues before and after infestation. Due to different life histories and durations of host‐encystment, mass and size increase in M. margaritifera during the host‐dependent phase were greater than those of U. crassus. δ¹³C and δ¹⁵N signatures of juvenile mussels approached isotopic signatures of fish tissues, indicating a parasitic relationship between mussels and their hosts. Shifts were more pronounced for M. margaritifera, which had a five‐fold longer host‐dependent phase than U. crassus. The results of this study suggest that stable isotope analyses are a valuable tool for characterizing trophic relationships and life history strategies in host–parasite systems. In the case of unionoid mussels, stable isotopic shifts of the larvae are indicative of the nutritional versus phoretic importance of the host.