New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.     

A Major Gene for Resting Metabolic Rate Unassociated with Body Composition: Results from the Québec Family Study

A major gene hypothesis for resting metabolic rate (RMR) was investigated using segregation analysis (POINTER) of data on families participating in Phase 2 of the Québec Family Study. Complete analyses were conducted on RMR adjusted for age, and also on RMR adjusted for age and other covariates, primarily fat mass (FM) and fat-free mass (FFM). Prior to adjustment for covariates, support for a major gene hypothesis was equivocal — i.e., there was evidence for either a major gene or a multifactorial component (i.e., polygenic and/or familial environment). The multifactorial model was preferred over the major gene model, although the latter did segregate according to Mendelian expectations. However, after the effects of FM and FFM were accounted for, a major gene effect was unambiguous and compelling. The putative locus accounted for 57% of the variance, affected 7% of the sample, and led to high values of RMR. The lack of a significant multifactorial effect suggested that the familial etiology of RMR adjusted for FM and FFM was likely to be entirely a function of the major locus. Comparing the RMR results from pre- and post-adjustment for FM and FFM suggests a plausible hypothesis. We know from earlier studies in this sample that there is a putative major gene for FM and a major non-Mendelian effect for FFM. The current study leads us to speculate that: (1) the gene(s) affecting body size and body composition also may have an effect on RMR, and further (2) removal of the effect of the major gene(s) for body size and composition allowed for detection of an additional major gene affecting only the RMR. Thus, RMR appears to be an oligogenic trait.     

The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules

Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gα_i/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis.     

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.     

Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.     

Phylogeography of mitochondrial haplogroup D1: An early spread of subhaplogroup D1j from Central Argentina

We analyzed the patterns of variation of haplogroup D1 in central Argentina, including new data and published information from other populations of South America. Almost 28% (107/388) of the individuals sampled in the region belong to haplogroup D1, whereas more than 52% of them correspond to the recently described subhaplogroup D1j (Bodner et al.: Genome Res 22 (2012) 811–820), defined by the presence of additional transitions at np T152C–C16242T–T16311C to the nodal D1 motif. This lineage was found at high frequencies across a wide territory with marked geographical–ecological differences. Additionally, 12 individuals present the mutation C16187T that defines the recently named subhaplogroup D1g (Bodner et al.: Genome Res 22 (2012) 811–820), previously described in populations of Patagonia and Tierra del Fuego. Based on our results and additional data already published, we postulate that the most likely origin of subhaplogroup D1j is the region of Sierras Pampeanas, which occupies the center and part of the northwestern portion of Argentina. The extensive yet restricted geographical distribution, the relatively large internal diversity, and the absence or low incidence of D1j in other regions of South America suggest the existence of an ancient metapopulation covering the Sierras Pampeanas, being this lineage its genetic signature. Further support for a scenario of local origin for D1j in the Sierras Pampeanas stems from the fact that early derivatives from a putative ancestral lineage carrying the transitions T16311C–T152C have only been found in this region, supporting the hypothesis that it might represent an ancestral motif previous to the appearance of D1j‐specific change C16242T. © 2012 Wiley Periodicals, Inc.     

Genotype Variability in Photosynthetic Characteristics in Finger Millet

High variability in leaf gas exchange and related traits were found in 30 genotypes of field grown finger millet. The variability in carbon exchange rate per unit leaf area (P _N) can be partly attributed to the differences in the stomatal conductance (g_s) and area leaf mass (ALM). The P _N was positively correlated with total dry matter (TDM). However, no relationship between P _N and seed yield was found. The leaf area showed a positive and significant correlation with total biomass. None of the other gas-exchange traits had significant relationship either with TDM or with seed yield. The ALM showed a strong positive association with P _N. However, it was not correlated with either total biomass or seed yield. As a result, the use of ALM as surrogate for P _N for identifying high biomass producing genotypes only had a limited value. Hence selection for high P _N would result in higher biomass producing types.     

Optimal mass selection policies for schemes with overlapping generations and restricted inbreeding

Optimum breeding schemes for maximising the rate of genetic progress with a restriction on the rate of inbreeding (per year or per generation) are investigated for populations with overlapping generations undergoing mass selection. The optimisation is for the numbers of males and females to be selected and for their distribution over age classes. Expected rates of genetic progress (ΔG) are combined with expected rates of inbreeding (ΔF) in a linear objective function (Φ = ΔG - λΔF) which is maximised. A simulated annealing algorithm is used to obtain the solutions. The restriction on inbreeding is achieved by increasing the number of parents and, in small schemes with severe restrictions, by increasing the generation interval. In the latter case the optimum strategy for obtaining the maximum genetic gain is far from truncation selection across age classes. In most situations, the optimum mating ratio is one but the differences in genetic gain obtained with different mating ratios are small. Optimisation of schemes when restricting the rate of inbreeding per generation leads to shorter generation intervals than optimisation when restricting the rate of inbreeding per year.     

The ABRF Metabolomics Research Group 2013 Study: Investigation of Spiked Compound Differences in a Human Plasma Matrix

Metabolomics is an emerging field that involves qualitative and quantitative measurements of small molecule metabolites in a biological system. These measurements can be useful for developing biomarkers for diagnosis, prognosis, or predicting response to therapy. Currently, a wide variety of metabolomics approaches, including nontargeted and targeted profiling, are used across laboratories on a routine basis. A diverse set of analytical platforms, such as NMR, gas chromatography-mass spectrometry, Orbitrap mass spectrometry, and time-of-flight-mass spectrometry, which use various chromatographic and ionization techniques, are used for resolution, detection, identification, and quantitation of metabolites from various biological matrices. However, few attempts have been made to standardize experimental methodologies or comparative analyses across different laboratories. The Metabolomics Research Group of the Association of Biomolecular Resource Facilities organized a “round-robin” experiment type of interlaboratory study, wherein human plasma samples were spiked with different amounts of metabolite standards in 2 groups of biologic samples (A and B). The goal was a study that resembles a typical metabolomics analysis. Here, we report our efforts and discuss challenges that create bottlenecks for the field. Finally, we discuss benchmarks that could be used by laboratories to compare their methodologies.