Immunohistochemistry and Fluorescence Microscopy Mice were terminally anesthetized and subjected to whole body perfusion with 4% paraformaldehyde in phosphate-buffered saline (PBS). Whole eyes were enucleated and post-fixed in the same fixative overnight before embedded in Tissue-TeK Cryomold Figure 7. Schematic model integrating oxidative stress, autophagy and lysosomal function into the etiology of AMD. RPE cells are exposed to high levels of cellular stress and, when healthy, damaged proteins and organelles are removed promptly by autophagy. Under disease conditions, such as decreased antioxidant defense and lysosome inhibition, self-renewal by autophagy becomes less efficient. The resulted cellular waste products can be exported by exocytosis and contribute to sub-RPE deposit and drusen formation. NRF2 can be involved in regulating both the antioxidant responses and the autophagic activities.
The IfitmDel mouse lacks all five of the Ifitm genes via LoxP deletion. This animal breeds nor- mally with no obvious defect in development. The IfitmDel animals exhibit a steady and sig- nificantly enhanced weight gain relative to wild-type controls beginning about three months of age and under normal feeding conditions. The increased weight corresponds with elevat- ed fat mass, and in tolerance tests they are hyporesponsive to insulin but respond normally to glucose. Both young (4 mo) and older (12 mo) IfitmDel mice have enhanced levels of serum leptin suggesting a defect in leptin/leptin receptor signaling. Analysis of the gene ex- pression profiles in the hypothalamus of IfitmDel animals, compared to WT, demonstrated an altered ratio of Pomc and Npy neuropeptide expression, which likely impairs the satiation response of the IfitmDel animal leading to an increased eating behavior. Also elevated in hy- pothalamus of IfitmDel mice were pro-inflammatory cytokine expression and reduced IL-10. Anatomical analysis of the hypothalamus using immunohistochemistry revealed that micro- glia exhibit an abnormal morphology in IfitmDel animals and respond abnormally to Poly:IC challenge. These abnormalities extend the phenotype of the IfitmDel mouse beyond abnor- mal responses to viral challenge to include a metabolic phenotype and weight gain. Further, this novel phenotype for the IfitmDel mouse could be related to abnormal neuropeptide pro- duction, inflammatory status and microglia status in the hypothalamus.
found bonaﬁde necroinﬂammatory foci comprising histiocytes, side- rophages, hepatocyte necrosis and apoptotic bodies (Fig. 5A). Discrete features of hepatocyte apoptotic cell death included cytoplasmic shrinkage and nuclear chromatin condensation and fragmentation (Fig. 5A). The frequency of the necroin ﬂammatory foci increased with age (Fig. 5B), in parallel with the increase of TUNEL-positive hepato- cytes (Fig. 5C). Remarkably, the progression of these two features with age matched the increase in the number of iron-rich macrophages (Fig. 4D) and in the percentage of ﬁbrotic area ( Fig. 2B), indicating that reticuloendothelial iron loading and ﬁbrosis were triggered by iron- related necroinﬂammation (sideronecrosis). This assumption is sup- ported, at least in part, by the analysis of liver histology. Indeed, Hfe/ Nrf2 -/- mice displayed iron overload of non-hepatocyte cells located in
The generation of Pdyn KO mice has been described elsewhere . In short, the entire coding sequence of the Pdyn gene was replaced by a basally silent construct Cre-recombinase under Teton control. Mice were backcrossed onto the balb/c and C57Bl/6N backgrounds over eight and 10 generations, respectively. For breeding and maintenance mice were group-housed with free access to food and water. Temperature was maintained at 23uC with 60% humidity and a 12 h light-dark cycle (lights on 7 am to 7 pm). Mice were tested at 3 to 6 months of agein all experiments. Age- and testing experience-matched wild-type littermates were used as controls. Tests were performed in the fixed order with fixed time intervals for all animals: open-field, elevated plus maze, light-dark choice, forced swim, and tail suspension test. All procedures involving animals were approved by the Austrian Animal Experimentation Ethics Board in compliance with the European convention for the protection of vertebrate animals used for experimental and other scientific purposes ETS no. 123. (Licence number BMWF-66.011/0018-II/10b/2009) Every effort was taken to minimize the number of animals used.
There are few studies in which biopsies have been performed in patients with AIP. In one large population study , 16 patients were found with renal impairment with no other cause than AIP. Nine of the 10 patients that underwent renal biopsy had hypertension. The biopsies showed varying degrees of nephro- sclerosis, moderate tubular atrophy and interstitial fibrosis and vessel wall thickening. Other few authors reported nephrosclerosis and shrunken kidney more in accordance to hypertension damage [6,7,8]. Finally, renal biopsy data from patients without hyper- tension or glomerular lesions but with features of tubulointerstitial disease suggest an enhanced susceptibility to the nephrotoxic effects of porphyrin precursors and porphyrins [9,10]. Both types of damages directly or indirectly may point to active AIP, with or without frequent hemin administration , as an important factor causing renal disease. Of interest, a selective accumulation and excretion of PBG has been found in most porphyric patients with renal failure [9,11] and an increased PBG/ALA ratio was proposed as a warning sign associated to changes in the renal filtration.
Groups of 5–6 week old mice either lacking receptors for IFN-α/β (A129) or from the wild- type strain (129Sv/Ev) were subcutaneously inoculated with 10 6 pfu ZIKV in the lower leg. Sur- vival analysis was compared between the two ZIKV-infected mouse strains and mock-infected animals which received PBS only, and demonstrated that all A129 animals met humane clinical endpoints 6 days after challenge with ZIKV (Fig 1A). Wild-type 129Sv/Ev mice all survived the 14 day length of the study, as did the control animals. When weights were compared between groups, it could be seen that the ZIKV-challenged A129 mice started to lose weight rapidly after day 3 post-challenge, whereas the other groups all demonstrated a gradual increase over the course of the study indicating that animals were healthy (Fig 1B). Similarly, the tempera- tures of the A129 mice showed differences compared to the other groups, with a gradual increase until day 4 post-challenge and then a rapid decrease (Fig 1C). The A129 ZIKV-chal- lenged mice were the only ones which exhibited signs of disease post-challenge, with signs first being noted on day 5 and increasing until humane endpoints were met on day 6 post-challenge (Fig 1D). None of the other groups had any clinical signs recorded for the duration of the study.
Genotyping and immunoblot analyses. All mouse procedures were performed in accordance with McGill University guidelines, which are set by the Canadian Council on Animal Care. Genomic DNA was isolated from tail biopsies and analyzed by Southern blotting and genomic PCR analysis. The DNA fragment utilized as the probe for the Southern blotting analysis was ampliﬁed with the following two oligonucleotides (59-AAG CCT TTA CTG GTT GTG T-39) and (59- CTT GAA ACG CAC CGT AGG CT-39). The wild-type sam68 allele was identiﬁed by genomic PCR using the following oligonucleotides 59-AAA TCC TAA CCC TCC TCA GTC AG-39 and 59-GAT ATG ATG GAT GAT ATC TGT CAG-39. The sam68-targeted allele was identiﬁed by genomic PCR using the following oligonucleotides 59-CTT GGG TGG AGA GGC TAT TCG-39 and 59-GTC GGG CAT GCG CGC CTT GAG C-39.
SDS-digested freeze-fracture replica labeling (SDS-FRL) SDS-FRL can be used to observe the two dimensional structure of cell membranes and the distribution of membrane proteins [31– 33]. Excitatory synapses were identified by the intramembrane particle (IMP) cluster on its exoplasmic face . In this experiment, mice were given intraperitoneal injections of either saline or cocaine (20 mg/kg). After 3 days of saline injections, mice were divided into groups that received seven daily injections of either saline or cocaine with no measurement of locomotor activity. Following 14 days of withdrawal, mice were decapitated and their brains were removed. We measured the density of AMPA receptors on excitatory synapses in the NAc by SDS-FRL. Mice were anesthetized by pentobarbital (40 mg/kg, i.p.) and fixed with 2% paraformaldehyde by perfusion fixation. Coronal slices (150 m m thickness) were prepared by a micro slicer (Linearslicer PRO7, Dosaka, Kyoto, Japan) and the NAc core was trimmed. Trimmed sections were submerged in 0.1 M phosphate buffer with 30% glycerol at 4 uC overnight, and the sections were then frozen quickly by a high-pressure freezing machine (HPM010, BAL-TEC, Balzers, Liechtenstein). The frozen slices were then freeze-fractured and replicated with carbon (5 nm), shadowed by platinum (2 nm), and replicated again with carbon (15–20 nm) in a freeze fracture machine (JFD-II, JEOL, Tokyo, Japan). After thawing, tissue debris attached to the replicas were digested with gentle stirring at 80 uC for 16 hours in a solution of 15 mM Tris-HCl, 20% sucrose, and 2.5% sodium lauryl sulfate. We used three different primary antibodies and performed single labeling on each separated replica. These antibodies were the pan-AMPA antibody raised in rabbit, which binds to GluR1, GluR2, GluR3 and GluR4 receptors (the specificity of this antibody was described in ref. ), a GluR1 antibody (rabbit polyclonal antibody; the specificity of this antibody was described in ref
Age-related cataract results from increasing opacification of the ocular lens eventually leading to visual loss and is a problem found in many people throughout the world as they age. Surgery with intraocular lens implantation is currently the only effective procedure. In low income countries with poor access to cataract surgery, cataract is the main cause of vision impairment and blindness . In well-resourced countries cataract surgery is one of the highest health care expenditures . There is evidence of a genetic component to age-related cataract. Earlier studies found that family history was a risk factor for cataract [3–6] while the strongest evidence came from twin studies demonstrating a heritability of 48% for nuclear cataract  and 59% for cortical cataract . Most work on cataract genetics has focused on inherited congenital cataract and there has been limited success in
relative impact of B cells in the absence of immunoglobulin in the protective immune response to TB. We found that B cells can be directly detrimental to control of Mtb in the spleen but that excess IL-10 is directly responsible for the increased susceptibility seen inmice that have B cells but lack circulating immunoglobulin. Delivery of exogenous immunoglobulin alone failed to restore the capacity of the mutant mice to control bacterial growth in the lung or spleen. We found that macrophages are the main cellular source of IL-10 during infection and that the macrophages in these mice produce arginase as well as iNOS and are likely less efficient at controlling bacterial growth. We also show that there is no apparent role for immunoglobulin in the initiation and expression of type 1 immune responses specifically the generation of antigen- specific Th1 cells.
Our serial histological analysis of mouse embryos detected ridges as extension of the sinus horn mesenchyme and the associated PPMs around E11.5. During the next two days, these mesenchymal ridges expanded towards the lung hilus and narrowed the lumen of the initially broader pleuropericardial communication to a thin canal. Around E13.5, the mesothelial linings of the ridges and the lung bud fused, completely sealing the pleural from the pericardial cavity (schematized in Figure 8). The existence of such mesenchymal ridges that are crucial for PPC closure was noted in human embryos as well. By careful analysis of serially sectioned human embryos Salzer reported on such a ‘‘Mesenchymfalte’’ at the border of the Ductus Cuvieri and the PPM, and suggested that closure of the PPC occurs by an active mechanism rather than by mere passive growth of the neighboring structures in the region of the lung hilus. He argued that the final step of PPC closure represents a real fusion process between neighboring membranes that were juxtaposed by the previous expansion of the ridges and the overlying lung hilus . This study may have remained unnoticed in the field due to its German language.
Although there are many studies which have assessed the microbial communities associated with both healthy and diseased corals, little is known of the bacterial associates of the coral Coe- lastrea aspera, the subject of the present investigation. However, the environmental physiology of this species has been extensively studied [11–15]. Most recently, this physiologically robust species has shown an unexpected decline at sites around Phuket following a severe bleaching event observed in 2010 . One proposed explanation for this demise, was that of senescence . Interestingly, colonial animals such as corals have historically and theoretically been described as non-ageing organisms . However, recent studies on certain species of reef coral appear to show declines in physiological performance with age [18–20]. One aspect which has been well documented to be strongly dependent on agein numerous higher organ- isms, is the relationship hosts share with their associated ‘natural’ microbiota throughout their life cycle [21,22]. For example, microbial communities associated with the avian cloaca have been shown to significantly alter with the age of the host . Furthermore, humans often exhibit marked shifts in their microbial communities, a result again often associated with aging. Interestingly this has been documented to occur both externally  and internally (i.e. within the human gut) [21,24,25]. We therefore considered that investigation into the micro- bial communities of different sizes of corals (using size as a proxy for age), might provide some insight into whether corals show similar age-related patterns as those described in higher organisms. In this preliminary study we examined the diversity of microbial communities asso- ciated with different size-classes of the reef coral C. aspera, at a site on Phuket Island, Thailand; where the size and age structure of this particular species are well known .
Although diabetic retinopathy is recognized as the leading cause of blindness in working age adults, we have yet to define the cellular mechanisms responsible for diabetes-induced loss of retinal neurons. Several lines of evidence suggest a link between decreased sympathetic innervation and diabetes. For example, hyperglyce- mia has been shown to cause dysfunctional neurotransmitter release from the sympathetic ganglia projection to the retina . In our own studies, we have previously shown that removal of the superior cervical ganglion or knockout of dopamine beta hydroxylase (a key enzyme in the conversion of dopamine to norepinephrine in sympathetic neurons) results in a retinal phenotype that is similar to that seen in diabetic animals [2,3]. Likewise, we showed that treatment with adrenergic receptor antagonists, in particular b-adrenergic receptor antagonists, caused a similar diabetic phenotype in retina [4,5]. These results led us to hypothesize that restoration of b-adrenergic signaling in diabetic retina might prevent or reduce retinal damage due to diabetes. To test this hypothesis, we treated streptozotocin-induced diabetic rats with a general b-adrenergic receptor agonist. As predicted, the treatment prevented retinal damage in this model system. [6,7].
We here provide evidence for a novel role of the complement control protein CD55 in granulocyte homeostasis and anti- bacterial host defense. CD55-deficientmice display increased numbers of circulating granulocytes. On average, we found twice as many granulocytes in the blood stream, the marginated pool, and the spleen of Cd55 -/- compared to wild-type mice. This mild granulocytosis clearly differs from the marked changes inmice that lack leukocyte adhesion molecules . A comparable phenotype has recently been reported in two independently generated CD97- deficientmice [26,27]. The Kelly laboratory and we found that mice lacking CD97 have about two-fold increased granulocyte numbers in blood and spleen in the steady state. This phenotype was not the result of enhanced bone marrow emigration to the blood neither of reduced clearance of circulating cells. Cd97 -/- mice showed a greater response to daily G-CSF treatment, which induces granulopoiesis in the bone marrow . In line with this observation, we here demonstrate increased numbers of Gr-1- positive cells in cell cycle in the bone marrow of both CD55- deficientmice and CD97-deficientmice, indicating a higher granulopoietic activity in both strains.
In resting conditions, FTO deficientmice were characterized by higher heart rate values than wild-type mice, both during the active (dark) and inactive (light) phase of the daily cycle. Likewise, we found signs of elevated body temperature inmice lacking the FTO gene. Clearly, differences in heart rate and body temperature may have been determined by different levels of somatomotor activity, which indeed resulted significantly higher in knockout mice during the active phase of the daily cycle. However, given that heart rate was consistently higher in FTO deficientmice even when somatomotor activity levels were not greater, we believe that autonomic mechanisms concurred to determine higher heart rate in these animals. Supporting this view, HRV analysis revealed that knockout mice were characterized by a lower vagal modulation of heart rate (RMSSD and HF indexes) than wild-type counterparts. In addition, the fact that FTO deficientmice showed higher LF to HF ratio (index of sympatho-vagal balance) is suggestive of a larger contribution of the sympathetic modulation of heart rate inmice lacking the FTO gene. Signs that link FTO deficiency to increased cardiac sympathetic drive were evident during stress conditions. Following the injection of saline and during Figure 3. Susceptibility to cardiac tachyarrhythmias. Panel A shows an example of ECG traces belonging to a representative Fto 2/2 mouse
Changes relevant to endothelial injury were assessed in this study by determination of circulating vasoconstrictor peptides and plasma nitrite levels. Circulating endothelin isoform profiles are shown in Figure 5A–H. In general, a similarity in the patterns of profiles is noticeable for ET-1, ET-2, ET-3 isoforms both under acute and chronic exposure scenarios. When mice were acutely exposed to radiation, ET-3 isoform exhibited significant increase in plasma at the highest dose (2 Gy) of exposure compared to the control and 0.5 Gy exposure groups (one-way ANOVA, p = 0.010). Overall analysis of radiation exposures on circulating ET isoforms (2-way ANOVA with Dose [0, 2 Gy] and Time [acute, chronic] as factors) exhibited significant increases in plasma ET-1 (p = 0.016) and ET-3 (p = 0.020) with radiation exposure, and increased plasma ET-1/ET-3 ratio (p = 0.032) with time (Figure 5I–J). Circulating nitrite levels suggested an increasing trend with acute exposure but no observable changes were seen with chronic radiation exposure (Figure 6A–B). Inmice acutely exposed to 0.5 Gy radiation, serum cholesterol levels were significantly increased (one-way ANOVA, p = 0.011) compared to the corresponding controls (Figure 7A). Also, serum cholesterol Figure 1. Marker of lipid oxidation. Plasma levels of 8–isoprostane in Apo-E2/2 mice following acute (A) and chronic (B) radiation exposure. Mean 6 SEM. Acute, 0 (n = 3), 0.5 Gy (n = 4), 2 Gy (n = 4). Chronic, 0 (n = 3), 2 Gy (n = 4).
Sprouty proteins are known negative regulators of the ERK pathway , a pathway in- volved in cell division, survival and transformation , we anticipated that ERK would play a critical role in the development of ICC hyperplasia. ICC represent less than 1% of the total cell population of the highly heterogeneous gut wall. Modifications in gene or protein expression occurring in ICC might thus vanish in the ambient ‘noise’ when using techniques which re- quire tissue homogenization, e.g. qPCR or Western blot. Therefore, immunoreactivity was used to study signaling pathways in situ with resolution of the different cell types. Surprisingly, pERK was undetectable in the ICC of Spry4 KO mice. In order to exclude compensatory upre- gulation of other Sprouty family members, we performed qPCR of the antrum for Spry1, Spry2, and Spred1. None of these genes were upregulated in Spry4 KO antrum. No commercially available antibodies for SPRY1 and SPRED1 were fond suitable for IF on our material while SPRY2-ir showed no change between the different genotypes. Nevertheless, despite these limi- tations, our data suggest that no compensation by other SPRYs appears to occur. Recently, sev- eral papers indicated that SPRY4 can play a role in other signaling pathways [33,34,44,45]. Hence, pAKT and pp70S6 IF were performed in order to investigate the PI3K/AKT/mTOR and pSTAT5a/b for the JAK/STAT pathways, but no detectable differences in immunoreactivi- ty could be seen between the different genotypes.
Dystrophin is a 427-kDa protein expressed in striated mus- cles and the central nervous system (CNS). It is localized at the cytoplasmic face of the plasma membrane linking the intra- cellular cytoskeleton with the extracellular matrix (Ervasti, 2007; Pilgram et al., 2010). Three full-length dystrophin (Dp427) isoforms, which are transcribed from distinct promoters of the DMD gene, are expressed in muscles (Dp427M), throughout the brain (Dp427B), and in cerebellar Purkinje cells (Dp427P). Four other shorter isoforms (Dp260, Dp140, Dp116, and Dp71), regulated by internal promoters, are expressed in the CNS and other tissues (Blake and Kroger, 2000; Perronnet and Vaillend, 2010). The mutant mdx mouse is a well-studied model of DMD that lacks the expression of full-length dystro- phin in both the muscles and brain (Sicinski et al., 1989). At the cell membrane, dystrophin is associated with a complex of glycoproteins (DGC) comprised of dystroglycans, syntrophins, dystrobrevins, sarcoglycans, and sarcospan (Blake et al., 2002; Ervasti, 2007). In striated muscles, the lack of dystrophin disrupts the macromolecular complex and damages the plasma membrane, resulting in muscle degeneration and necrosis (Petrof, 2002). At the neuromuscular synapse, dystrophin and some components of the DGC are necessary for the maturation of post-junctional folds and the regulation of nicotinic acetylcholine receptors (nAChRs) (Ghedini et al., 2008; Grady et al., 2000; Huh and Fuhrer, 2002). The protein complex also plays a role in Ca 2þ homeostasis and cell
Expression of the Vi capsular polysaccharide reduces comple- ment activation by the alternative pathway [12,16], as indicated by diminished fixation of complement fragment C3b on the surface of the capsulated S. Typhi wild-type strain compared to a non- capsulated S. Typhi mutant (DtviB-vexE mutant) (Fig. 2E and 2F). To further investigate whether chemotactic responses were dependent on C3, we performed single-cell experiments with serum and/or neutrophils from mice with C3-deficiency . In serum from C3-deficientmice (bred on a C57BL/6 background), neutrophils from C3-deficientmice did not exhibit a chemotactic response toward the non-capsulated S. Typhi DtviB-vexE mutant (Fig. 3A, Video S9). In contrast, neutrophils from C3-deficientmice extended chemotactic pseudopodia toward the non-capsu- Figure 2. The Vi capsular polysaccharide inhibits complement-dependent chemotaxis in vitro . (A and D) Neutrophil migration into the bottom compartment of a Boyden chamber containing the indicated attractants was measured in the presence or absence of the complement inhibitor Futhan. Bars represent averages 6 standard error from at least 3 different donors. Vehicle; HBSS vehicle control, fMLP; N-formyl-L-methionyl- L-leucyl-L-phenylalanine; wt, wild type. (B) Schematic drawing of Vi capsular biosynthesis genes (black arrows) of S. Typhi. (C) Expression of the Vi capsular polysaccharide (Y-axis) was detected in cultures of the S. Typhi wild type (wt, left panel) and a non-capsulated S. Typhi strain (DtviB-vexE mutant, right panel) by flow cytometry using rabbit anti-Vi serum. (E) Fixation of C3b (Y-axis) on the surface of the S. Typhi wild type (wt, left panel) or a non-capsulated S. Typhi strain (DtviB-vexE mutant, right panel) that had been incubated for 30 minutes in 1% human serum was detected by flow cytometry using FITC-conjugated goat anti-human C3b antibody. (F) Quantification of C3b fixation detected by flow cytometry. A gate was set using a control in which bacteria were not stained with goat anti-human C3b antibody. Bars represent averages 6 standard error from three independent experiments for each strain.
ARE contain- ing endogenous target expression (mRNA levels of GCLC, NQO1) was also not observed. It is understood that 46 NQO1- ARE reporter plasmid is engineered with only Nrf2 binding elements in which case the activity of reporter plasmid is solely dependent on Nrf2. However, in the endogenous cellular setting, target gene expression could be dependent on the enhancers and suppressors other than Nrf2 which could bind to non-ARE DNA regions. Therefore, the luciferase based transcriptional activity measurement of NQO1-ARE construct would be very sensitive to Nrf2 levels as opposed to endogenous transcriptional activity measurement (mRNA levels by real time) and thus, connecting these two different measurement strategies based on exact magnitude of changes would be practically difficult. Nevertheless, our findings demonstrate that in neuronal SH-SY5Y cells, miR144/153/27a/142-5p induced Nrf2 downregulation affects the nucleo-cytoplasmic concentration of Nrf2 which is reflected in its inefficient transactivating ability.