qNMR for Profiling the Production of Fungal Secondary Metabolites

Analysis of complex mixtures is a common challenge in natural products research. Quantitative nuclear magnetic resonance spectroscopy offers analysis of complex mixtures at early stages and with benefits that are orthogonal to more common methods of quantitation, including ultraviolet absorption spectroscopy and mass spectrometry. Several experiments were conducted to construct a methodology for use in analysis of extracts of fungal cultures. A broadly applicable method was sought for analysis of both pure and complex samples through use of an externally calibrated method. This method has the benefit of not contaminating valuable samples with the calibrant, and it passed scrutiny for line fitting and reproducibility. The method was implemented to measure the yield of griseofulvin and dechlorogriseofulvin from three fungal isolates. An isolate of Xylaria cubensis (coded MSX48662) was found to biosynthesize griseofulvin in the greatest yield, 149 ± 8 mg per fermentation, and was selected for further supply experiments

Exploring subtle land use and land cover changes: a framework for future landscape studies

UMR AMAP, équipe 3International audienceLand cover and land use changes can have a wide variety of ecological effects, including significant impacts on soils and water quality. In rural areas, even subtle changes in farming practices can affect landscape features and functions, and consequently the environment. Fine-scale analyses have to be performed to better understand the land cover change processes. At the same time, models of land cover change have to be developed in order to anticipate where changes are more likely to occur next. Such predictive information is essential to propose and implement sustainable and efficient environmental policies. Future landscape studies can provide a framework to forecast how land use and land cover changes is likely to react differently to subtle changes. This paper proposes a four step framework to forecast landscape futures at fine scales by coupling scenarios and landscape modelling approaches. This methodology has been tested on two contrasting agricultural landscapes located in the United States and France, to identify possible landscape changes based on forecasting and backcasting agriculture intensification scenarios. Both examples demonstrate that relatively subtle land cover and land use changes can have a large impact on future landscapes. Results highlight how such subtle changes have to be considered in term of quantity, location, and frequency of land use and land cover to appropriately assess environmental impacts on water pollution (France) and soil erosion (US). The results highlight opportunities for improvements in landscape modelling

Content and performance of the MiniMUGA genotyping array: A new tool to improve rigor and reproducibility in mouse research

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research

Peripuberty stress leads to abnormal aggression, altered amygdala and orbitofrontal reactivity and increased prefrontal MAOA gene expression.

Although adverse early life experiences have been found to increase lifetime risk to develop violent behaviors, the neurobiological mechanisms underlying these long-term effects remain unclear. We present a novel animal model for pathological aggression induced by peripubertal exposure to stress with face, construct and predictive validity. We show that male rats submitted to fear-induction experiences during the peripubertal period exhibit high and sustained rates of increased aggression at adulthood, even against unthreatening individuals, and increased testosterone/corticosterone ratio. They also exhibit hyperactivity in the amygdala under both basal conditions (evaluated by 2-deoxy-glucose autoradiography) and after a resident-intruder (RI) test (evaluated by c-Fos immunohistochemistry), and hypoactivation of the medial orbitofrontal (MO) cortex after the social challenge. Alterations in the connectivity between the orbitofrontal cortex and the amygdala were linked to the aggressive phenotype. Increased and sustained expression levels of the monoamine oxidase A (MAOA) gene were found in the prefrontal cortex but not in the amygdala of peripubertally stressed animals. They were accompanied by increased activatory acetylation of histone H3, but not H4, at the promoter of the MAOA gene. Treatment with an MAOA inhibitor during adulthood reversed the peripuberty stress-induced antisocial behaviors. Beyond the characterization and validation of the model, we present novel data highlighting changes in the serotonergic system in the prefrontal cortex-and pointing at epigenetic control of the MAOA gene-in the establishment of the link between peripubertal stress and later pathological aggression. Our data emphasize the impact of biological factors triggered by peripubertal adverse experiences on the emergence of violent behaviors

\u3csup\u3e1\u3c/sup\u3eH, \u3csup\u3e15\u3c/sup\u3eN, \u3csup\u3e13\u3c/sup\u3eC and \u3csup\u3e13\u3c/sup\u3eCO Assignments and Secondary Structure Determination of Basic Fibroblast Growth Factor Using 3D Heteronuclear NMR Spectroscopy

The assignments of the 1H, 15N, 13CO, and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation \u3e95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of Cα, Cβ, and Hα to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue cor-relation of Cα, Cβ, and Hα to the backbone amide 1H and 15N in the CBCANH and HNHA experi-ments. In addition, Cα and Cβ chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and Cα correlations from the HNCA experiment. Aliphatic side-chain spin sys-tems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, Hα, and Hβ protons as well as 3JHNHα coupling constants, amide exchange, and 13Cα and 13Cβ secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel β-sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125, and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as β-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the β-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin–FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined

Structural Relatedness of Distinct Determinants Recognized by Monoclonal Antibody TP25.99 on ß\u3csub\u3e2\u3c/sub\u3e-Microglobulin-Associated and ß\u3csub\u3e2\u3c/sub\u3e-Microglobulin-Free HLA Class I Heavy Chains

The association of HLA class I heavy chains with ß2-microglobulin (ß2m) changes their antigenic profile. As a result, Abs react with either ß2m-free or ß2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both ß2m-associated and ß2m-free HLA class I heavy chains. The reactivity with ß2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194–198 in the α3 domain. The reactivity with ß2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on \u3c50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239–242, 245, and 246 in the α3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs