Intramacrophage survival of uropathogenic Escherichia coli : differences between diverse clinical isolates and between mouse and human macrophages

Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24 h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24 h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1+ vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival.

A high and low fear phenotype mouse model

Fear-related illnesses such as post-traumatic stress disorder (PTSD) impose a tremendous burden on individual quality of life, families, and the national economy. In the military population, 17-20% of services members returning from deployment are diagnosed with PTSD. While treatments have improved for PTSD and are helpful for some, many people continue to suffer despite therapy. The aim of this research is to examine fear memory behaviourally and at the cellular level in the amygdala by using a unique inter-cross strain of high and low fear phenotype mice. An extended outcross C57BL/6J x DBA/2J (F8) are selected for high or low Pavlovian fear memory to context and cue. On presentation of either the original learning context or the cue (tone) mice display high or low levels of freezing as a behavioural measure of fear. In order to identify key aspects of the cellular basis of this difference in fear memory behaviour we are making measurements of protein levels and neuron numbers of a known pathway involved in the consolidation of a long term fear memory (pMAPK). Ongoing studies aim to determine if high fear behaviour is associated with differential signalling in the lateral amygdala compared to low fear behaviour. Additionally, by blocking this pathway in the lateral amygdala (LA), we aim to reduce fear behaviour following Pavlovian fear conditioning. This research will help to unravel the cellular mechanisms underlying high fear behaviour and advance the field toward targeted treatment and improved outcomes, ultimately improving human quality of life.

Molecular analysis of asymptomatic bacteriuria escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.

Strain- and host species-specific inflammasome activation, IL-1β release, and cell death in macrophages infected with uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans.

Strain field measurement using digital image correlation

Digital Image Correlation and Tracking (DIC/DDIT) is an optical method that employs tracking & image registration techniques for accurate 2D and 3D measurements of changes in images. This is often used to measure deformation (engineering), displacement, and strain, but it is widely applied in many areas of science and engineering. One very common application is for measuring the motion of an optical mouse.

Cryopreservation of Kunming mouse oocytes using slow cooling, ultrarapid cooling and vitrification protocols

The cryopreservation of oocytes has been only marginally successful with any of the current protocols, including slow cooling, rapid cooling and vitrification. We wished to test the hypothesis that oocytes from a single mouse strain would freeze successfully by 1 of the 3 mentioned protocols. Unfertilized Kunming mouse oocytes obtained 14 h after PMSG/hCG administration were randomly assigned to be cryopreserved after slow cooling, ultra rapid cooling and vitrification. Oocytes were thawed by straws being placed into 37 degrees C water, and their morphological appearance and in vitro fertilization capability were compared with that of oocytes that had not undergone cryopreservation. Survival of oocytes was indicated by the absence of darkened ooplasm or by broken membranes or zona pellucida. Functional integrity was evaluated by the formation of a 2-cell embryo after IVF. Survival rate of slow cooled oocytes did not differ from that seen in vitrified oocytes (55.1 vs 65.9%) but was significantly lower in the rapidly cooled oocytes (24.2%; P<0.01). The results of NF of slow cooled and vitrified oocytes were similar to those of the control group (72 and 73 vs 77%; P>0.05). It appears that Kunming mouse oocytes can be successfully cryopreserved using the slow cooling method with 1,2-propanediol and vitrification, which contains both permeating and nonpermeating cryoprotectants. (C) 1997 by Elsevier Science Inc.

Mouse Toxicity of Anabaena flos-aquae from Lake Dianchi, China

Some species of the genera Anabaena can produce various kinds of cyanotoxins, which may pose risks to environment and human health. Anabaena has frequently been observed in eutrophic freshwater of China in recent years, but its toxicity has been reported only in a few studies. In the present study, the toxicity of an Anabaena flos-aquae strain isolated from Lake Dianchi was investigated. Acute toxicity testing was performed by mouse bioassay using crude extracts from the lyophilized cultures. The mice exposed to crude extracts showed visible symptoms of toxicity and died within 10-24 h of the injection. Serum biochemical parameters were evaluated by the use of commercial diagnostic kits. Significant alterations were found in the serum biochemical parameters: alkaline phosphatase (AKP), gamma-glutamyl transpepticlase (gamma-GT), aspartate amino transferase (AST), alanine amino transferase (ALT), AST/ALT ratio, total protein content, albumin content, albumin/globulin (A/G) ratio, blood urea nitrogen (BUN), serum creatinine (Ssr), and total antioxidative capacity (T-AOC). Histopathological observations were carried out with hematoxylin and eosin (HE) stain under light microscope. Severe lesions were seen in the livers, kidneys, and lungs of the mice injected with crude extracts. The alterations of biochemical parameters were in a dose-dependent manner, and the severities of histological lesions were in the same manner. Based on biochemical and histological studies, this research firstly shows the presence of toxin-producing Anabaena species in Lake Dianchi and the toxic effects of its crude extracts on mammals. (C) 2008 Wiley Periodicals, Inc. Environ Toxicol 24: 10-18, 2009.

Alleviation of pre-exposure of mouse brain with low-dose C-12(6+) ion or Co-60 gamma-ray on male reproductive endocrine damages induced by subsequent high-dose irradiation

Irradiation has been widely reported to damage organisms by attacking on proteins, nucleic acid and lipids in cells. However, radiation hormesis after low-dose irradiation has become the focus of research in radiobiology in recent years. To investigate the effects of pre-exposure of mouse brain with low-dose C-12(6+) ion or Co-60 gamma (gamma)-ray on male reproductive endocrine capacity induced by subsequent high-dose irradiation, the brains of the B6C3F(1) hybrid strain male mice were irradiated with 0.05 Gy of C-12(6+) ion or Co-60 gamma-ray as the pre-exposure dose, and were then irradiated with 2 Gy as challenging irradiation dose at 4 h after pre-exposure. Serum pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), testosterone, testis weight, sperm count and shape were measured on the 35th day after irradiation. The results showed that there was a significant reduction in the levels of serum FSH, LH, testosterone, testis weight and sperm count, and a significant increase in sperm abnormalities by irradiation of the mouse brain with 2 Gy of C-12(6+) ion or Co-60 gamma-ray. Moreover, the effects were more obvious in the group irradiated by C-12(6+) ion than in that irradiated by Co-60 gamma-ray. Pre-exposure with low-dose C-12(6+) ion or Co-60 gamma-ray significantly alleviated the harmful effects induced by a subsequent high-dose irradiation.

Chromosomal aberrations induced by C-12(6+) ions and Co-60 gamma-rays in mouse immature oocytes

The ovaries of Kun-Ming strain mice (3 weeks) were irradiated with different doses of C-12(6+) ion or Co-60 gamma-ray. Chromosomal aberrations were analyzed in metaphase II oocytes at 7 weeks after irradiation. The relative biological effectiveness (RBE) of C C-12(6+) ion was calculated with respect to Co-60 gamma-ray for the induction of chromosornal aberrations. The C-12(6+) ion and Co-60 gamma-ray dose-response relationships for chromosomal aberrations were plotted by linear quadratic models. The data showed that there was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values for C-12(6+) ions relative to (CO)-C-60 gamma-rays were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses of 0.5, 1.0, 2.07 4.0 or 6.0 Gy, respectively. Moreover, a different distribution of the various types of aberrations has been found for C-12(6+) ion and Co-60 gamma-ray irradiations. The dose-response relationships for C-12(6+) ion and (CO)-C-60 gamma-ray exhibited positive correlations. The results from the present study may be helpful for assessing genetic damage following exposure of immature oocytes to ionizing radiation.

Induction of cytogenetic adaptive response in spermatogonia and spermatocytes by pre-exposure of mouse testis to low-dose C-12(6+) ions

To investigate the effects of pre-exposure of mouse testis to low-dose C-12(6+) ions on cytogenetics of spermatogonia and spermatocytes induced by subsequent high-dose irradiation. the testes of outbred Kun-Ming strain mice were irradiated with 0.05 Gy of C-12(6+) ions as the pre-exposure dose, and then irradiated with 2 Gy as challenging dose at 4 h after per-exposure. Poly(ADP-ribose) polymerase (PARPs) activity and PARP-1 protein expression were respectively measured by using the enzymatic and Western blot assays at 4 h after irradiation; chromosomal aberrations in spermatogonia and spermatocytes were analyzed by the air-drying method at 8 h after irradiation. The results showed that there was a significant increase in the frequency of chromosomal aberrations and significant reductions of PARP activity and PARP-1 expression level in the mouse testes irradiated with 2 Gy of C-12(6+) ions. However, pre-exposure of mouse testes to a low dose of C-12(6+) ions significantly increased PARPs activity and PARP-1 expression and alleviated the harmful effects induced by a subsequent high-dose irradiation. PARP activity inhibitor 3-aminobenzamide (3-AB) treatment blocked the effects of PARP-1 on cytogenetic adaptive response induced by low-dose C-12(6+) ion irradiation. The data suggest that pre-exposure of testes to a low dose of heavy ions can induce cytogenetic adaptive response to subsequent high-dose irradiation. The increase of PARP-1 protein induced by the low-dose ionizing irradiation may be involved in the mechanism of these observations. (C) 2008 Elsevier B.V. All rights reserved.

Adaptive hormetic response of pre-exposure of mouse brain with low-dose C-12(6+) ion or Co-60 gamma-ray on growth hormone (GH) and body weight induced by subsequent high-dose irradiation

The brain of the Kun-Ming strain mice were irradiated with 0.05 Gy of C-12(6+) ion or Co-60 gamma-ray as the pre-exposure dose, and were then irradiated with 2 Gy of 12C6+ ion or Co-60 gamma-ray as challenging irradiation dose at 4 h after per-exposure. Body weight and serum growth hormone (GH) concentration were measured at 35th day after irradiation. The results showed that irradiation of mouse brain with 2 Gy of C-12(6+) ion or Co-60 gamma-ray significantly diminished mouse body weight and level of serum GH. The relative biological effectiveness values of a 2 Gy dose of C-12(6+) ion calculated with respect to Co-60 gamma-ray were 1.47 and 1.34 for body weight and serum GH concentration, respectively. Pre-exposure with a low-dose (0.05 Gy) of C-12(6+) ion or Co-60 gamma-ray significantly alleviated reductions of mouse body weight and level of serum GH induced by a subsequent high-dose (2 Gy) irradiation. The data suggested that low-dose ionizing irradiation can induce adaptive hormetic responses to the harmful effects of pituitary by subsequent high-dose exposure.

Ectopic expression of the Ets transcription factor ER81 in transgenic mouse mammary gland enhances both urokinase plasminogen activator and stromelysin-1 transcription

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.

Resolving candidate genes of mouse skeletal muscle QTL via RNA-Seq and expression network analyses

BACKGROUND:

We have recently identified a number of Quantitative Trait Loci (QTL) contributing to the 2-fold muscle weight difference between the LG/J and SM/J mouse strains and refined their confidence intervals. To facilitate nomination of the candidate genes responsible for these differences we examined the transcriptome of the tibialis anterior (TA) muscle of each strain by RNA-Seq.

13,726 genes were expressed in mouse skeletal muscle. Intersection of a set of 1061 differentially expressed transcripts with a mouse muscle Bayesian Network identified a coherent set of differentially expressed genes that we term the LG/J and SM/J Regulatory Network (LSRN). The integration of the QTL, transcriptome and the network analyses identified eight key drivers of the LSRN (Kdr, Plbd1, Mgp, Fah, Prss23, 2310014F06Rik, Grtp1, Stk10) residing within five QTL regions, which were either polymorphic or differentially expressed between the two strains and are strong candidates for quantitative trait genes (QTGs) underlying muscle mass. The insight gained from network analysis including the ability to make testable predictions is illustrated by annotating the LSRN with knowledge-based signatures and showing that the SM/J state of the network corresponds to a more oxidative state. We validated this prediction by NADH tetrazolium reductase staining in the TA muscle revealing higher oxidative potential of the SM/J compared to the LG/J strain (p<0.03).

Thus, integration of fine resolution QTL mapping, RNA-Seq transcriptome information and mouse muscle Bayesian Network analysis provides a novel and unbiased strategy for nomination of muscle QTGs.

Differences in Mucosal Gene Expression in the Colon of Two Inbred Mouse Strains after Colonization with Commensal Gut Bacteria

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.

The activity of mouse Cerberus like 2 during cardiogenesis - genetic and morphogenetic studies

Cardiogenesis is a delicate and complex process that requires the coordination of an intricate network of pathways and the different cell types. Therefore, understanding heart development at the morphogenetic level is an essential requirement to uncover the causes of congenital heart disease and to provide insight for disease therapies. Mouse Cerberus like 2 (Cerl2) has been defined as a Nodal antagonist in the node with an important role in the Left-Right (L/R) axis establishment, at the early embryonic development. As expected, Cerl2 knockout mice (Cerl2-/-) showed multiple laterality defects with associated cardiac failure. In order to identify the endogenous role of Cerl2 during heart formation independent of its described functions in the node, we accurately analyzed animals where laterality defects were not present. We thereby unravel the consequences of Cerl2 lossof- function in the heart, namely increased left ventricular thickness due to hyperplasia of cardiomyocytes and de-regulated expression of cardiac genes. Furthermore, the Cerl2 mutant neonates present impaired cardiac function. Once that the cardiac expression of Cerl2 is mostly observed in the left ventricle until around midgestration, this result suggest a specific regulatory role of Cerl2 during the formation of the left ventricular myoarchitecture. Here, we present two possible molecular mechanisms underlying the cardiac Cerl2 function, the regulation of Cerl2 antagonist in activation of the TGFßs/Nodal/Activin/Smad2 signaling identified by increased Smad2 phosphorilation in Cerl2-/- hearts and the negative feedback between Cerl2 and Wnt/ß-catenin signaling in heart formation. In this work and since embryonic stem cells derived from 129 mice strain is extensively used to produce targeted mutants, we also present echocardiographic reference values to progressive use of juveniles and young adult 129/Sv strain in cardiac studies. In addition, we investigate the cardiac physiology of the surviving Cerl2 mutants in 129/Sv background over time through a follow-up study using echocardiographic analysis. Our results revealed that Cerl2-/- mice are able to improve and maintain the diastolic and most of systolic cardiac physiologic parameters as analyzed until young adult age. Since Cerl2 is no longer expressed in the postnatal heart, we suggest that an intrinsic and compensatory mechanism of adaptation may be active for recovering the decreased cardiac function found in Cerl2 mutant neonates. Altogether, these data highlight the role of Cerl2 during embryonic heart development in mice. Furthermore, we also suggest that Cerl2-/- may be an interesting model to uncover the molecular, cellular and physiological mechanisms behind the improvement of the cardiac function, contributing to the development of therapeutic approaches to treat heart failures.