Development and validation of a UPLC/MS method for a nutritional metabolomic study of human plasma

In order to study the effect of a diet on metabolites found in body fluids such as plasma, we have developed and validated a UPLC/MS method. While methods using NMR have been well established to analyse different biological tissues, recent studies have described robust untargeted UPLC-MS methods for plasma analysis. One major concern when profiling plasma is the presence of an important quantity of proteins which have to be precipitated without any loss of metabolites prior to LC/MS analysis. The utilization of untargeted approaches in nutritional metabolomics still suffers from the lack of identification of specific biomarkers. We therefore suggest an alternative method still using a global approach but focusing at the same time on metabolites previously described in human plasma in order to detect biomarkers of metabolic dysregulations. Thus, to fulfil our objectives, analytical parameters were tested (i) the anticoagulant type for sample collection, (ii) the protein precipitation method and (iii) UPLC/MS analytical conditions. Three protein precipitation methods and two anticoagulants were tested and compared. The method utilizing blood collection on heparin and methanol precipitation was chosen for giving the most reproducible results while keeping the complexity of the sample. Finally, a validation was proposed to evaluate the stability of this analytical method applied to a large batch of samples for nutritional metabolomic studies.     

Effect of the luteinizing hormone on embryo production in superovulated rabbit does

For most domestic animals, the responses to superovulation treatments are not controlled as a consequence of the lack of knowledge on exogenous gonadotrophins effects on the ovarian function. The role of luteinizing hormone (LH) on the number and quality of embryos produced was evaluated on rabbit does superovulated with porcine FSH (pFSH). Parameters of embryos recovery, in vitro and in vivo embryo development rates after freezing/thawing were compared. We used three experimental groups: (1) control group without superovulation treatment, (2) "pFSH+pLH" and (3) "pFSH" groups where females were treated with pFSH, respectively, with (20%) or without (0%) porcine LH supplementation. The number of corpora lutea and the number of embryos produced were significantly higher (p<0.001) in superovulated does than in control group (27.1, 26.7 versus 11.9 corpora lutea and 20.3, 21.2 versus 9.6 embryos produced for pFSH+pLH, pFSH and control group, respectively). However, both gonadotrophins administrations (groups 2 and 3) led to defaults of ovulation when compared with untreated does. No significant difference was observed between the number and quality of the embryos produced by does treated with pFSH+pLH or with pFSH alone. Moreover, we observed no significant difference between results of in vivo and in vitro viability assays after thawing. We concluded that pFSH alone seems to be sufficient to stimulate the follicles growth and that exogenous pLH administrated has no effect on the quantity and quality of embryos. Further studies are needed to evaluate the hormonal patterns before and after the gonadotrophins injections in the rabbit species.     

Our social genome?

Apocalyptic views on the natural order, chimeras and genetic engineering should not detract from the fact that medical research, similar to the promotion of health, is a public good. Genomics crosses all species, thereby requiring a global approach that respects human rights and public health priorities. Public trust and public participation in research demand clear stewardship as well as transparent and accountable oversight. Characterizing fundamental genomic data as a public resource might counterbalance the current overemphasis on individual rights but this will not be simple. It is only through an attachment to justice and solidarity that the dignity and well-being of persons, both as humans and as citizens, can truly be fostered.     

CD8 T cell help for innate antitumor immunity

Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential "help" to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis.     

A role for ATP-citrate lyase, malic enzyme, and pyruvate/citrate cycling in glucose-induced insulin secretion

In pancreatic beta-cells, metabolic coupling factors generated during glucose metabolism and pyruvate cycling through anaplerosis/cataplerosis processes contribute to the regulation of insulin secretion. Pyruvate/citrate cycling across the mitochondrial membrane leads to the production of malonyl-CoA and NADPH, two candidate coupling factors. To examine the implication of pyruvate/citrate cycling in glucose-induced insulin secretion (GIIS), different steps of the cycle were inhibited in INS 832/13 cells by pharmacological inhibitors and/or RNA interference (RNAi) technology: mitochondrial citrate export, ATP-citrate lyase (ACL), and cytosolic malic enzyme (ME1). The inhibitors of the di- and tri-carboxylate carriers, n-butylmalonate and 1,2,3-benzenetricarboxylate, respectively, reduced GIIS, indicating the importance of transmitochondrial transport of tri- and dicarboxylates in the action of glucose. To directly test the role of ACL and ME1 in GIIS, small hairpin RNA (shRNA) were used to selectively decrease ACL or ME1 expression in transfected INS 832/13 cells. shRNA-ACL reduced ACL protein levels by 67%, and this was accompanied by a reduction in GIIS. The amplification/K(ATP)-independent pathway of GIIS was affected by RNAi knockdown of ACL. The ACL inhibitor radicicol also curtailed GIIS. shRNA-ME1 reduced ME1 activity by 62% and decreased GIIS. RNAi suppression of either ACL or ME1 did not affect glucose oxidation. However, because ACL is required for malonyl-CoA formation, inhibition of ACL expression by shRNA-ACL decreased glucose incorporation into palmitate and increased fatty acid oxidation in INS 832/13 cells. Taken together, the results underscore the importance of pyruvate/citrate cycling in pancreatic beta-cell metabolic signaling and the regulation of GIIS.     

Anterior regions of monkey parietal cortex process visual 3D shape

The intraparietal cortex is involved in the control of visually guided actions, like reach-to-grasp movements, which require extracting the 3D shape and position of objects from 2D retinal images. Using fMRI in behaving monkeys, we investigated the role of the intraparietal cortex in processing stereoscopic information for recovering the depth structure and the position in depth of objects. We found that while several areas (CIP, LIP, and AIP on the lateral bank; PIP and MIP on the medial bank) are activated by stereoscopic stimuli, AIP and an adjoining portion of LIP are sensitive only to depth structure. Furthermore, only these two regions are sensitive to both the depth structure and the 2D shape of small objects. These results indicate that extracting 3D spatial information from stereo involves several intraparietal areas, among which AIP and anterior LIP are more specifically engaged in extracting the 3D shape of objects.     

Bayesian spatial modeling of moose count data: increasing estimator efficiency and exploring ecological hypotheses

Moose management throughout much of Alaska and Canada relies on aerial count data, which are commonly collected using the geospatial population estimator (GSPE) protocol. The GSPE uses a model-based analytical approach and finite-population block kriging to estimate abundance from a collection of sampled survey units. Widespread implementation and well-documented analytical software have resulted in reliable estimates of moose abundance, density, and composition across a large proportion of their range. Analysis is conducted almost exclusively using the GSPE software, which fits a fixed model structure to data collected within a single year. The downside of this approach to analysis is that the fixed model structure is inefficient for estimation, leading to more field effort than would otherwise be necessary to achieve a desired level of estimator precision. We developed a more easily modified and flexible Bayesian spatial general additive model approach (BSG) that accommodates spatial and temporal covariates (e.g., habitat characteristics, trend), multiple survey events, prior information, and incomplete detection. Using a series of 6 GSPE surveys conducted in Yukon-Charley Rivers National Preserve, Alaska, USA, from 2003–2019, we established the equivalence of the 2 approaches under similar model structures. We then extended the BSG to demonstrate how a more comprehensive approach to analysis can affect estimator precision and be used to assess ecological relationships. The precision of annual abundance estimators from the BSG were improved by an average of 43% over those based on the standard GSPE analysis, highlighting the very real costs of assuming a fixed (i.e., suboptimal) model structure. The population increased at a rate of 2.3%/year (95% CrI = 0.8–3.8%), and the increase was largely explained by a parallel increase in wildfire extent (i.e., high quality moose habitat). These results suggest that our approach could be used to increase estimator efficiency or decrease future survey costs without any modifications to the basic protocol. While modification of the GSPE software is possible, practitioners may find the BSG approach more convenient for quickly developing model structures for a particular application, thereby allowing them to extract more information from existing and future datasets.     

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre^+/−xRtn4/Nogo-A^flox/flox) and neuron-specific (Thy1-Cre^tg+xRtn4^flox/flox) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4^flox/flox animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.In the adult mammalian central nervous system (CNS), axons have a very limited capacity to regenerate after traumatic injury. This lack of axonal regeneration is thought to be mainly due to the presence of growth-inhibiting molecules in the injured CNS environment^{1, 2} and due to the low intrinsic growth capacity of mature neurons.³Nogo-A is a well-studied inhibitory protein for axonal growth, plasticity and regeneration after CNS injury.^{4, 5} Nogo-A is predominantly expressed in oligodendrocytes in the adult CNS, where it is thought to stabilize the neuronal circuits in healthy conditions and to inhibit neurite growth and plasticity after lesion.² Neutralizing Nogo-A by function-blocking antibodies or genetic knockout (KO) has been shown to improve axonal sprouting and regeneration in the injured spinal cord and brain.^{6, 7, 8, 9, 10, 11}In addition to oligodendrocytes and myelin, Nogo-A is expressed in growing and immature neurons, as well as in some adult neurons.^{12, 13} Neurons express Nogo-A receptors such as the Nogo-66 receptor 1 (NgR1)¹⁴ and the Nogo-A-Δ20-specific sphingosine 1-phosphate receptor 2 (S1PR2).¹⁵ They can co-express them along with Nogo-A,¹³ an observation that raises the possibility of cis-interactions between the ligand and its receptors within or at the cell surface of the same cell. This mechanism has previously been described for axonal guidance molecules such as Ephrins and Semaphorins, and could have a major role in the neuronal response to extracellular growth inhibitors during development.^{16, 17}In the adult CNS, the expression of neuronal Nogo-A remains elevated mainly in plastic regions such as in the hippocampus, olfactory bulb or neocortex, and in the dorsal root ganglia.¹² Nogo-A and NgR1 were shown to regulate synaptic plasticity, for example, long-term potentiation in the hippocampus and in the sensory-motor cortex,^{18, 19, 20, 21, 22} whereas the effects of neuronal Nogo-A after injury are not yet well understood. During development, neuronal Nogo-A influences neuronal migration,^{23, 24} survival,^{25, 26} cell spreading and neurite growth.^{27, 28} In injured adult retinal ganglion cells (RGCs), silencing neuronal Nogo-A resulted in a marked reduction of regenerative sprouting and decreased expression of growth-associated molecules.²⁹ Furthermore, in the optic nerve, axonal regeneration was not improved in conventional Nogo-A KO animals, in which both glial and neuronal Nogo-A were deleted.²⁹ The present study therefore aimed to investigate whether glial and neuronal Nogo-A differently influence axonal growth in vivo using cell type-specific Nogo-A KO mouse lines and adeno-associated virus (AAV)-mediated recombination of the Nogo-A gene in neurons. The results show that significantly more axons grew through the lesion site in the oligodendrocyte-specific Nogo-A KO mice. In contrast, neuron-specific ablation of Nogo-A in RGCs reduced the number of regenerating axons after optic nerve crush injury (ONC). In summary, we show that inactivating Nogo-A specifically in oligodendrocytes appears to be the most successful strategy to promote axonal regeneration in the adult optic nerve.     

Consequences of genetic erosion on fitness and phenotypic plasticity in European tree frog populations (Hyla arborea)

The detrimental effects of genetic erosion on small isolated populations are widely recognized contrary to their interactions with environmental changes. The ability of genotypes to plastically respond to variability is probably essential for the persistence of these populations. Genetic erosion impact may be exacerbated if inbreeding affects plastic responses or if their maintenance were at higher phenotypic costs. To understand the interplay 'genetic erosion-fitness-phenotypic plasticity', we experimentally compared, in different environments, the larval performances and plastic responses to predation of European tree frogs (Hyla arborea) from isolated and connected populations. Tadpoles from isolated populations were less performant, but the traits affected were environmental dependant. Heterosis observed in crosses between isolated populations allowed attributing their low fitness to inbreeding. Phenotypic plasticity can be maintained in the face of genetic erosion as inducible defences in response to predator were identical in all populations. However, the higher survival and developmental costs for isolated populations in harsh conditions may lead to an additional fitness loss for isolated populations.