We describe here the implementation of the statistical total correlation spectroscopy (STOCSY) analysis method for aiding the identification of potential biomarker molecules in metabonomic studies based on NMR spectroscopic data. STOCSY takes advantage of the multicollinearity of the intensity variables in a set of spectra (in this case 1H NMR spectra) to generate a pseudo-two-dimensional NMR spectrum that displays the correlation among the intensities of the various peaks across the whole sample. This method is not limited to the usual connectivities that are deducible from more standard two-dimensional NMR spectroscopic methods, such as TOCSY. Moreover, two or more molecules involved in the same pathway can also present high intermolecular correlations because of biological covariance or can even be anticorrelated. This combination of STOCSY with supervised pattern recognition and particularly orthogonal projection on latent structure-discriminant analysis (O-PLS-DA) offers a new powerful framework for analysis of metabonomic data. In a first step O-PLS-DA extracts the part of NMR spectra related to discrimination. This information is then cross-combined with the STOCSY results to help identify the molecules responsible for the metabolic variation. To illustrate the applicability of the method, it has been applied to 1H NMR spectra of urine from a metabonomic study of a model of insulin resistance based on the administration of a carbohydrate diet to three different mice strains (C57BL/6Oxjr, BALB/cOxjr, and 129S6/SvEvOxjr) in which a series of metabolites of biological importance can be conclusively assigned and identified by use of the STOCSY approach.
The pathogenesis of infection in tilapia has not been fully described. To understand this, we investigated the clinic-pathological features of acute experimental septicemia in tilapia ( ) after receiving an intra-peritoneal injection with THN-1901GFP. Immunohistochemistry and sections of pathological tissues were used to estimate the level of damage in the head-kidney, liver, spleen and trunk-kidney. The expression of FasL was analyzed by western blotting in these samples based on their damage levels. Leucocytes were isolated from the head-kidney and incubated with THN-1901GFP. Then, phagocytosis, programmed cell death and the expression of FasL were analyzed. The infected tissues showed varying degrees of necrosis and histolysis. The serous membrane of the intestine was dissolved by THN-1901GFP. Antigens of THN-1901GFP accumulated in different parts of the infected organs. In the head-kidney and spleen, the expression of FasL was up-regulated in parallel with increased tissue damage. After being incubated with THN-1901GFP, the phagocytic capacity and ability were both very high and the expression of FasL remained high in leucocytes. THN-1901GFP was able to survive for a long period of time after being engulfed by phagocytic cells. These findings offer insight into the pathogenesis of infection in tilapia.
Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2′,5′)pA(3′,5′)p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTING adopts a “closed” conformation upon binding c[G(2′,5′)pA(3′,5′)p] and its linkage isomer c[G(2′,5′)pA(2′,5′)p], as does mouse mSting on binding c[G(2′,5′)pA(3′,5′)p], c[G(3′,5′)pA(3′,5′)p] and the antiviral agent DMXAA, leading to similar “closed” conformations. Comparing hSTING to mSting, 2′,5′-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3′,5′-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING. 2′,5′-linkage-containing cGAMP second messengers bind to human and mouse STING with stronger affinity than 3′,5′ isomers to trigger interferon signaling. A point mutation in human STING may explain the lack of sensitivity to DMXAA, a drug with potent antiviral and antitumorigenic effects in mice.
Research investigating interactions between aboveground (AG) and belowground (BG) herbivores has been central to characterizing AG-BG linkages in terrestrial ecosystems, with many of these interactions forming the basis of complex food webs spanning the two subsystems. Despite the growing literature on the effects of AG and BG herbivores on each other, underlying patterns have been difficult to identify due to a high degree of context dependency. In this study, we present the first quantitative meta-analysis of AG and BG herbivore interactions. Previous global predictions, specifically that BG herbivores normally promoted AG herbivore performance and AG herbivores normally reduced BG herbivore performance, were not supported. Instead, the meta-analysis identified four factors that determined the outcome of AG-BG interactions. (1) Sequence of herbivore arrival on host plants was important, with BG herbivores promoting AG herbivore performance only when introduced to the plant simultaneously, whereas AG herbivores had negative effects on BG herbivores only when introduced first. (2) AG herbivores negatively affected BG herbivore survival but tended to increase population growth rates. (3) AG herbivores negatively affected BG herbivore performance on annual plants, but not on perennials, and these effects were observed more consistently in laboratory than field studies. (4) The type of herbivore was also important, with BG insect herbivores belonging to the order Diptera (i.e., true flies) having the strongest negative effects on AG herbivores. Coleoptera (i.e., beetles) species were the most widely investigated BG herbivores and had positive impacts on AG Homoptera (e.g., aphids), but negative effects on AG Hymenoptera (e.g., sawflies). The strongest negative outcomes for BG herbivores were seen when the AG herbivore was a Coleoptera species. We found no evidence for publication bias in AG-BG herbivore interaction literature and conclude that several biological and experimental factors are important for predicting the outcome of AG-BG herbivore interactions. The sequence of herbivore arrival on the host plant was among the most influential.
Dose-response analysis can be carried out using multi-purpose commercial statistical software, but except for a few special cases the analysis easily becomes cumbersome as relevant, non-standard output requires manual programming. The extension package drc for the statistical environment R provides a flexible and versatile infrastructure for dose-response analyses in general. The present version of the package, reflecting extensions and modifications over the last decade, provides a user-friendly interface to specify the model assumptions about the dose-response relationship and comes with a number of extractors for summarizing fitted models and carrying out inference on derived parameters. The aim of the present paper is to provide an overview of state-of-the-art dose-response analysis, both in terms of general concepts that have evolved and matured over the years and by means of concrete examples.
Pesticides residues in aquatic ecosystems are an environmental concern which requires efficient analytical methods. In this study, we proposed a generic method for the quantification of 13 pesticides (azoxystrobin, clomazone, diflufenican, dimethachlor, carbendazim, iprodion, isoproturon, mesosulfuron-methyl, metazachlor, napropamid, quizalofop and thifensulfuron-methyl) in three environmental matrices. Pesticides from water were extracted using a solid phase extraction system and a single solid–liquid extraction method was optimized for sediment and fish muscle, followed by a unique analysis by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Limits of quantification were below 5 ng L for water (except for fluroxypyr and iprodion) and ranged between 0.1 ng g and 57.7 ng g for sediments and regarding fish, were below 1 ng g for 8 molecules and were determined between 5 and 49 ng g for the 5 other compounds. This method was finally used as a new routine practice for environmental research.
Kinetic studies showed that [Asp(3), Dhb(7)]MC-RR reacted with mercaptoethanol hundreds of times more slowly than MC-RR and a range of other [Mdha(7)]-containing microcystin congeners. The difference in reaction rate was sufficiently large that derivatization of microcystin-containing samples with mercaptoethanol, followed by LC-MS analysis, clearly discriminated between microcystins containing the isobaric [Dhb(7)]- and [Mdha(7)]-groups. Application of this approach, using LC-MS with both ion trap and triple-quadrupole mass spectrometers, to water samples and Planktothrix cultures from Lake Steinfjorden, Norway, demonstrated the presence of [Asp(3), Dhb(7)]MC-RR (5), [Asp(3)]MC-RY (14), and [Asp(3)]MC-LY (16), as well as analogues tentatively identified as [Asp(3)]MC-RR (4), [Asp(3), DMAdda(5), Dhb(7)]MC-LR (6), [Asp(3), Dhb(7)]MC-HtyR (8), [Asp(3)]MC-HtyR (9), [Asp(3), Dhb(7)]MC-LR (10), [Asp(3)]MC-LR (11), [Asp(3), Dhb(7)]MC-RY (15), and [Asp(3), Dhb(7)]MC-LY (17), together with low levels of several other analogues. This is the first use of this thiol-based LC-MS approach to identify Dhb-containing microcystins, and allowed identification of LC-MS peaks in a mixture of [Mdha(7)]- and [Dhb(7)]-congeners of [Asp(3)]MC-RR (4, 5), -RY (14, 15), and -LY (16, 17) in the samples from L. Steinsfjorden. This is also the first report of MC-RY-congeners outside of Africa, or in Planktothrix spp. Analysis of European crayfish (Astacus astacus) taken from L. Steinsfjorden revealed the presence of only trace levels of microcystins in the edible parts.
Bovine tuberculosis (bTB) was first detected in 2005 in cattle in northwestern Minnesota (MN) through slaughter surveillance. By the end of 2008, 12 cattle herds were infected with bTB, and the main cause for infection was determined to be the movement of infected animals between herds. Bovine tuberculosis was contained in a smaller area in northwestern Minnesota classified as modified accredited (MA), corresponding to a prevalence inferior to 0.1% in cattle. From January 2008 to 2011, all cattle movements within the bTB MA were recorded electronically. The primary objectives of this study were to characterize cattle movements within this region and identify cattle herds with higher risk of bTB introduction based on network parameters and known risk factors from the published literature. During the period that data was collected, 57,460 cattle were moved in 3762 movements corresponding to permits issued to 682 premises, mostly representing private farms, sale yards, slaughter facilities and county or state fairs. Although sale yards represented less than 2% of the premises (nodes), 60% of the movements were to or from a sale yard. The network showed an overall of 0.4%, a of 14.6% and a of 12.7%, reflecting the low connectivity of this cattle network. The degree distribution showed that 20% of nodes performed 90% of the movements. Farms were ranked based on the total risk score and divided into high, medium, and low risk groups based on the score and its variability. The higher risk group included 14% ( = 50) of the farms, corresponding to 80% of the cumulative risk for the farms in the bTB area. This analysis provides a baseline description about the contact structure of cattle movements in an area previously infected with bTB and develops a framework for a targeted surveillance approach for bTB to support future surveillance decisions.
Pointillistic based super‐resolution techniques, such as photoactivated localization microscopy (PALM), involve multiple cycles of sequential activation, imaging, and precise localization of single fluorescent molecules. A super‐resolution image, having nanoscopic structural information, is then constructed by compiling all the image sequences. Because the final image resolution is determined by the localization precision of detected single molecules and their density, accurate image reconstruction requires imaging of biological structures labeled with fluorescent molecules at high density. In such image datasets, stochastic variations in photon emission and intervening dark states lead to uncertainties in identification of single molecules. This, in turn, prevents the proper utilization of the wealth of information on molecular distribution and quantity. A recent strategy for overcoming this problem is pair‐correlation analysis applied to PALM. Using rigorous statistical algorithms to estimate the number of detected proteins, this approach allows the spatial organization of molecules to be quantitatively described. Inphotoactivated localization microscopy (PALM) photoactivable fluorescent proteins (PA‐FPs) are stochastically activated, imaged, their localization determined and then bleached. By repeating this cycle with different subsets of PA‐FPs and combining the data, a super‐resolution image is obtained. An overview of latest developments in pair‐correlation analysis is given here.
Dietary composition and rearing regime largely determine the trace elemental composition of pigs, and consequently their concentration in animal products. The present study evaluates thirteen macro- and trace element concentrations in pork from organic and conventional farms. Conventional pigs were given a commercial feed with added minerals; organic pigs were given a feed based on organic feedstuffs. The content of macro-elements (Na, K, Mg and Ca) and some trace elements (Ni, Fe, Zn and Sr) in organic and conventional meat samples showed no significant differences (P > 0.05). Several trace element concentrations in organic pork were significantly higher (P < 0.05) compared to conventional pork: Cr (808 and 500 μg/kg in organic and conventional pork, respectively), Mn (695 and 473 μg/kg) and Cu (1.80 and 1.49 mg/kg). The results showed considerable differences in mineral content between samples from pigs reared in organic and conventional systems. Our results also indicate that authentication of organic pork can be realized by applying multivariate chemometric methods such as discriminant analysis to this multi-element data.