Human urinary metabolomic profile of PPARalpha induced fatty acid beta-oxidation

Activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) is associated with increased fatty acid catabolism and is commonly targeted for the treatment of hyperlipidemia. To identify latent, endogenous biomarkers of PPARalpha activation and hence increased fatty acid beta-oxidation, healthy human volunteers were given fenofibrate orally for 2 weeks and their urine was profiled by UPLC-QTOFMS. Biomarkers identified by the machine learning algorithm random forests included significant depletion by day 14 of both pantothenic acid (>5-fold) and acetylcarnitine (>20-fold), observations that are consistent with known targets of PPARalpha including pantothenate kinase and genes encoding proteins involved in the transport and synthesis of acylcarnitines. It was also concluded that serum cholesterol (-12.7%), triglycerides (-25.6%), uric acid (-34.7%), together with urinary propylcarnitine (>10-fold), isobutyrylcarnitine (>2.5-fold), (S)-(+)-2-methylbutyrylcarnitine (5-fold), and isovalerylcarnitine (>5-fold) were all reduced by day 14. Specificity of these biomarkers as indicators of PPARalpha activation was demonstrated using the Ppara-null mouse. Urinary pantothenic acid and acylcarnitines may prove useful indicators of PPARalpha-induced fatty acid beta-oxidation in humans. This study illustrates the utility of a pharmacometabolomic approach to understand drug effects on lipid metabolism in both human populations and in inbred mouse models.

Memo has a novel role in S1P signaling and crucial for vascular development

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.

Fluorescence lifetime imaging of the ocular fundus in mice

PURPOSE Fundus autofluorescence (AF) is characterized not only by its intensity or excitation and emission spectra but also by the lifetimes of the fluorophores. Fluorescence lifetime is influenced by the fluorophore's microenvironment and may provide information about the metabolic tissue state. We report quantitative and qualitative autofluorescence lifetime imaging of the ocular fundus in mice. METHODS A fluorescence lifetime imaging ophthalmoscope (FLIO) was used to measure fluorescence lifetimes of endogenous fluorophores in the murine retina. FLIO imaging was performed in 1-month-old C57BL/6, BALB/c, and C3A.Cg-Pde6b(+)Prph2(Rd2)/J mice. Measurements were repeated at monthly intervals over the course of 6 months. For correlation with structural changes, an optical coherence tomogram was acquired. RESULTS Fundus autofluorescence lifetime images were readily obtained in all mice. In the short spectral channel (498-560 nm), mean ± SEM AF lifetimes were 956 ± 15 picoseconds (ps) in C57BL/6; 801 ± 35 ps in BALB/c mice; and 882 ± 37 ps in C3A.Cg-Pde6b(+)Prph2(Rd2)/J mice. In the long spectral channel (560-720 nm), mean ± SEM AF lifetimes were 298 ± 14 ps in C57BL/6 mice, 241 ± 10 ps in BALB/c mice, and 288 ± 8 ps in C3A.Cg-Pde6b(+)Prph2(Rd2)/J mice. There was a general decrease in mean AF lifetimes with age. CONCLUSIONS Although fluorescence lifetime values differ among mouse strains, we found little variance within the groups. Fundus autofluorescence lifetime imaging in mice may provide additional information for understanding retinal disease processes and may facilitate monitoring of therapeutic effects in preclinical studies.

Penetrability of the inner mucus layer: who is out there?

Large numbers of microorganisms colonise the skin and mucous membranes of animals, with their highest density in the lower gastrointestinal tract. The impact of these microbes on the host can be demonstrated by comparing animals (usually mice) housed under germ-free conditions, or colonised with different compositions of microbes. Inbreeding and embryo manipulation programs have generated a wide variety of mouse strains with a fixed germ-line (isogenic) and hygiene comparisons robustly show remarkably strong interactions between the microbiota and the host, which can be summarised in three axioms. (I) Live microbes are largely confined to their spaces at body surfaces, provided the animal is not suffering from an infection. (II) There is promiscuous molecular exchange throughout the host and its microbiota in both directions [1]. (III) Every host organ system is profoundly shaped by the presence of body surface microbes. It follows that one must draw a line between live microbial and host “spaces” (I) to understand the crosstalk (II and III) at this interesting interface of the host-microbial superorganism. Of course, since microbes can adapt to very different niches, there has to be more than one line. In this issue of EMBO Reports, Johansson and colleagues have studied mucus, which is the main physical frontier for most microbes in the intestinal tract: they report how different non-pathogenic microbiota compositions affect its permeability and the functional protection of the epithelial surface [2].

Impact of reactive oxygen species (ROS) on the control of parasite loads and inflammation in Leishmania amazonensis infection

Background: Reactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date. Methods: In this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91phox−/− ) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production. Results: In vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91phox−/− mice, indicating a larger inflammatory response in gp91phox−/− even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91phox−/− at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils. Conclusion: ROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions.

Mice deficient in interleukin-4 (IL-4) or IL-4 receptor alpha have higher resistance to sporozoite infection with Plasmodium berghei (ANKA) than do naive wild-type mice

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Effect of Population Heterogenization on the Reproducibility of Mouse Behavior: A Multi-Laboratory Study

In animal experiments, animals, husbandry and test procedures are traditionally standardized to maximize test sensitivity and minimize animal use, assuming that this will also guarantee reproducibility. However, by reducing within-experiment variation, standardization may limit inference to the specific experimental conditions. Indeed, we have recently shown in mice that standardization may generate spurious results in behavioral tests, accounting for poor reproducibility, and that this can be avoided by population heterogenization through systematic variation of experimental conditions. Here, we examined whether a simple form of heterogenization effectively improves reproducibility of test results in a multi-laboratory situation. Each of six laboratories independently ordered 64 female mice of two inbred strains (C57BL/6NCrl, DBA/2NCrl) and examined them for strain differences in five commonly used behavioral tests under two different experimental designs. In the standardized design, experimental conditions were standardized as much as possible in each laboratory, while they were systematically varied with respect to the animals' test age and cage enrichment in the heterogenized design. Although heterogenization tended to improve reproducibility by increasing within-experiment variation relative to between-experiment variation, the effect was too weak to account for the large variation between laboratories. However, our findings confirm the potential of systematic heterogenization for improving reproducibility of animal experiments and highlight the need for effective and practicable heterogenization strategies.

Variable phenotypic expressivity in inbred retinal degeneration mouse lines: A comparative study of C3H/HeOu and FVB/N rd1 mice

PURPOSE Recent advances in optogenetics and gene therapy have led to promising new treatment strategies for blindness caused by retinal photoreceptor loss. Preclinical studies often rely on the retinal degeneration 1 (rd1 or Pde6b(rd1)) retinitis pigmentosa (RP) mouse model. The rd1 founder mutation is present in more than 100 actively used mouse lines. Since secondary genetic traits are well-known to modify the phenotypic progression of photoreceptor degeneration in animal models and human patients with RP, negligence of the genetic background in the rd1 mouse model is unwarranted. Moreover, the success of various potential therapies, including optogenetic gene therapy and prosthetic implants, depends on the progress of retinal degeneration, which might differ between rd1 mice. To examine the prospect of phenotypic expressivity in the rd1 mouse model, we compared the progress of retinal degeneration in two common rd1 lines, C3H/HeOu and FVB/N. METHODS We followed retinal degeneration over 24 weeks in FVB/N, C3H/HeOu, and congenic Pde6b(+) seeing mouse lines, using a range of experimental techniques including extracellular recordings from retinal ganglion cells, PCR quantification of cone opsin and Pde6b transcripts, in vivo flash electroretinogram (ERG), and behavioral optokinetic reflex (OKR) recordings. RESULTS We demonstrated a substantial difference in the speed of retinal degeneration and accompanying loss of visual function between the two rd1 lines. Photoreceptor degeneration and loss of vision were faster with an earlier onset in the FVB/N mice compared to C3H/HeOu mice, whereas the performance of the Pde6b(+) mice did not differ significantly in any of the tests. By postnatal week 4, the FVB/N mice expressed significantly less cone opsin and Pde6b mRNA and had neither ERG nor OKR responses. At 12 weeks of age, the retinal ganglion cells of the FVB/N mice had lost all light responses. In contrast, 4-week-old C3H/HeOu mice still had ERG and OKR responses, and we still recorded light responses from C3H/HeOu retinal ganglion cells until the age of 24 weeks. These results show that genetic background plays an important role in the rd1 mouse pathology. CONCLUSIONS Analogous to human RP, the mouse genetic background strongly influences the rd1 phenotype. Thus, different rd1 mouse lines may follow different timelines of retinal degeneration, making exact knowledge of genetic background imperative in all studies that use rd1 models.

UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-β-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

An attenuated Salmonella enterica serovar Typhimurium strain lacking the ZnuABC transporter induces protection in a mouse intestinal model of Salmonella infection

Salmonella enterica serovar Typhimurium has long been recognised as a zoonotic pathogen of economic significance in animals and humans. Attempts to protect humans and livestock may be based on immunization with vaccines aimed to induce a protective response. We recently demonstrated that the oral administration of a Salmonella enterica serovar Typhimurium strain unable to synthesize the zinc transporter ZnuABC is able to protect mice against systemic salmonellosis induced by a virulent homologous challenge. This finding suggested that this mutant strain could represent an interesting candidate vaccine for mucosal delivery. In this study, the protective effect of this Salmonella strain was tested in a streptomycin-pretreated mouse model of salmonellosis that is distinguished by the capability of evoking typhlitis and colitis. The here reported results demonstrate that mice immunized with Salmonella enterica serovar Typhimurium (S. Typhimurium) SA186 survive to the intestinal challenge and, compared to control mice, show a reduced number of virulent bacteria in the gut, with milder signs of inflammation. This study demonstrates that the oral administration a of S. Typhimurium strain lacking ZnuABC is able to elicit an effective immune response which protects mice against intestinal S. Typhimurium infection. These results, collectively, suggest that the streptomycin-pretreated mouse model of S. typhimurium infection can represent a valuable tool to screen S. typhimurium attenuated mutant strains and potentially help to assess their protective efficacy as potential live vaccines.

Generation and analysis of a mouse intestinal metatranscriptome through Illumina based RNA-sequencing

With the advent of high through-put sequencing (HTS), the emerging science of metagenomics is transforming our understanding of the relationships of microbial communities with their environments. While metagenomics aims to catalogue the genes present in a sample through assessing which genes are actively expressed, metatranscriptomics can provide a mechanistic understanding of community inter-relationships. To achieve these goals, several challenges need to be addressed from sample preparation to sequence processing, statistical analysis and functional annotation. Here we use an inbred non-obese diabetic (NOD) mouse model in which germ-free animals were colonized with a defined mixture of eight commensal bacteria, to explore methods of RNA extraction and to develop a pipeline for the generation and analysis of metatranscriptomic data. Applying the Illumina HTS platform, we sequenced 12 NOD cecal samples prepared using multiple RNA-extraction protocols. The absence of a complete set of reference genomes necessitated a peptide-based search strategy. Up to 16% of sequence reads could be matched to a known bacterial gene. Phylogenetic analysis of the mapped ORFs revealed a distribution consistent with ribosomal RNA, the majority from Bacteroides or Clostridium species. To place these HTS data within a systems context, we mapped the relative abundance of corresponding Escherichia coli homologs onto metabolic and protein-protein interaction networks. These maps identified bacterial processes with components that were well-represented in the datasets. In summary this study highlights the potential of exploiting the economy of HTS platforms for metatranscriptomics.

Stable two-element control of dTph1 transposition in mutator strains of Petunia by an inactive ACT1 introgression from a wild species

The high copy dTph1 transposon system of Petunia (Solanaceae) is one of the most powerful insertion mutagens in plants, but its activity cannot be controlled in the commonly used mutator strains. We analysed the regulation of dTph1 activity by QTL analysis in recombinant inbred lines of the mutator strain W138 and a wild species (P. integrifolia spp. inflata). Two genetic factors were identified that control dTph1 transposition. One corresponded to the ACT1 locus on chromosome I. A second, previously undescribed locus ACT2 mapped on chromosome V. As a 6-cM introgression in W138, the P. i. inflata act1(S6) allele behaved as a single recessive locus that fully eliminated transposition of all dTph1 elements in all stages of plant development and in a heritable fashion. Weak dTph1 activity was restored in act1(S6)/ACT2(S6) double introgression lines, indicating that the P. i. inflata allele at ACT2 conferred a low level of transposition. Thus, the act1(S6) allele is useful for simple and predictable control of transposition of the entire dTph1 family when introgressed into an ultra-high copy W138 mutator strain. We demonstrate the use of the ACT1(W138)/act1(S6) allele pair in a two-element dTph1 transposition system by producing 10 000 unique and fixed dTph1 insertions in a population of 1250 co-isogenic lines. This Petunia system produces the highest per plant insertion number of any known two-element system, providing a powerful and logistically simple tool for transposon mutagenesis of qualitative as well as quantitative traits.

Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model.

Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.