Genome-Wide Microarray Expression and Genomic Alterations by Array-CGH Analysis in Neuroblastoma Stem-Like Cells

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.     

Calibration of the Normal Cutoff Values of Systolic Dyssynchrony of the Left Ventricular Synchronicity in Normal Subjects Using Real-Time 3-Dimensional Echocardiography and the Effects of Age and Heart Rate

The normal data of left ventricular (LV) synchronicity by real-time 3-dimensional echocardiography (RT3DE) are lacking. We assessed the normal range/cutoff values of LV dyssynchrony parameters by RT3DE. For this purpose, RT3DE was performed in 130 healthy subjects, aged 53 ± 12 years. Time to the point of minimal regional systolic volume (Tmsv) was measured from time–volume curves in each segment. Standard deviation (SD) and maximal difference (Dif) of Tmsv were calculated from 16 (6 basal/6 mid/4 apical), 12 (6 basal/6 mid), and 6 (basal) LV segments together with the corresponding parameters adjusted by R–R interval. The data show non-significant difference between Tmsv-16-SD (9.24 ± 3.54 ms) and Tmsv-12-SD (8.80 ± 3.82 ms); with a correlation between two by both unadjusted (ms; r = 0.87) and adjusted (%R–R; r = 0.84) methods (P < 0.001). Heart rate correlated negatively with Tmsv (r = ?0.13 to ?0.34, P < 0.05–0.001) but had no effect on parameters adjusted for %R–R. Age and gender did not affect any of these parameters. Inter-observer variability was 3.3–4.6 % for 16, 4.8–9.1 % for 12, and 14.4–19.7 % for 6 segments. Thus, RT3DE is a reliable technique for detecting LV systolic dyssynchrony whereas the heart rate, but not age and gender, affects Tmsv parameters. Dyssynchrony parameters by 16 or 12 segments are superior to 6 segments in yielding comprehensive information and lower variability.     

Role of angiotensin II and oxidative stress on renal aquaporins expression in hypernatremic rats

The aim of this study was to assess whether endogenous Ang II and oxidative stress produced by acute hypertonic sodium overload may regulate the expression of aquaporin-1 (AQP-1) and aquaporin-2 (AQP-2) in the kidney. Groups of anesthetized male Sprague–Dawley rats were infused with isotonic saline solution (control) or with hypertonic saline solution (Na group, 1 M NaCl), either alone or with losartan (10 mg kg^?1) or tempol (0.5 mg min^?1 kg^?1) during 2 h. Renal function parameters were measured. Groups of unanesthetized animals were injected intraperitoneally with hypertonic saline solution, with or without free access to water intake, Na+W, and Na?W, respectively. The expression of AQP-1, AQP-2, Ang II, eNOS, and NF-kB were evaluated in the kidney by Western blot and immunohistochemistry. AQP-2 distribution was assessed by immunofluorescence. Na group showed increased natriuresis and diuresis, and Ang II and NF-kB expression, but decreased eNOS expression. Losartan or tempol enhanced further the diuresis, and AQP-2 and eNOS expression, as well as decreased Ang II and NF-kB expression. Confocal immunofluorescence imaging revealed labeling of AQP-2 in the apical plasma membrane with less labeling in the intracellular vesicles than the apical membrane in kidney medullary collecting duct principal cells both in C and Na groups. Importantly, our data also show that losartan and tempol induces a predominantly accumulation of AQP-2 in intracellular vesicles. In unanesthetized rats, Na+W group presented increased diuresis, natriuresis, and AQP-2 expression (112?±?25 vs 64?±?16; *p?<?0.05). Water deprivation increased plasma sodium and diuresis but decreased AQP-2 (46?±?22 vs 112?±?25; §p?<?0.05) and eNOS expression in the kidney. This study is a novel demonstration that renal endogenous Ang II–oxidative stress, induced in vivo in hypernatremic rats by an acute sodium overload, regulates AQP-2 expression.     

Regional and segmental differences in the embryonic expression of a putative leech Hox gene,Lox2, by central neurons immunoreactive to FMRFamide-like neuropeptides

We performed immunofluorescence experiments using a rat polyclonal antibody on formaldehyde-fixed whole-mount embryos to characterize the expression of a putative leech Hox gene, Lox2, during embryonic development. The main goal was to determine whether the differentiation of subsets of FMRFamide-like immunoreactive (FLI) neurons coincide with the expression domain of Lox2. The earliest expression of Lox2 was detected in relatively large, prominent nuclei in the posterior region at embryonic day 4, a very early stage. Lox2 expression was also detected in subsets of central neurons (neurons located in the CNS) located in midbody ganglia 6 (M6)–M21. In addition, Lox2 was expressed by a number of segment-specific and segmentally repeated central FLI neurons. Lox2-positive FLI neurons of interest included some of those previously identified: the rostral most ventral (RMV) neurons, the circular ventral (CV) neurons, and cell 261. The paired RMVs, which are located in all midbody ganglia, expressed Lox2 only in M7–M19. The CV neurons, specialized motor neurons that innervate the circular ventral muscles of the body wall, expressed Lox2 in M7–M19. The putative cell 261 expressed Lox2 in M7–M12, where Lox1 is also expressed. FMRFamide staining in putative segmental homologs of cell 261 was not detected in other segmental ganglia. Our results suggest a role for Lox2 in very early embryonic development (before the formation of the CNS), and in the differentiation of segmentally repeated and region-specific FLI neurons.     

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX₉C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX₉C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.     

Decreased astrocytic thrombospondin‐1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin‐1 (TSP‐1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH₄Cl, 0.5–2.5 mM) for 1–10 days, and TSP‐1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra‐ and extracellular TSP‐1 levels. Exposure of cultured neurons to conditioned media from ammonia‐treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP‐1 over‐expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP‐1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP‐1 synthesis in other cell types, also reversed the ammonia‐induced TSP‐1 reduction. Likewise, we found a significant decline in TSP‐1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP‐1 may represent an important therapeutic target for CHE.

     

Lizard scales in an adaptive radiation: variation in scale number follows climatic and structural habitat diversity in Anolis lizards

Lizard scales vary in size, shape and texture among and within species. The overall function of scales in squamates is attributed to protection against abrasion, solar radiation and water loss. We quantified scale number of Anolis lizards across a large sample of species (142 species) and examined whether this variation was related either to structural or to climatic habitat diversity. We found that species in dry environments have fewer, larger scales than species in humid environments. This is consistent with the hypothesis that scales reduce evaporative water loss through the skin. In addition, scale number varied among groups of ecomorphs and was correlated with aspects of the structural microhabitat (i.e. perch height and perch diameter). This was unexpected because ecomorph groups are based on morphological features related to locomotion in different structural microhabitats. Body scales are not likely to play an important role in locomotion in Anolis lizards. The observed variation may relate to other features of the ecomorph niche and more work is needed to understand the putative adaptive basis of these patterns. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 570–579.     

Topography and Land Cover of Watersheds Predicts the Distribution of the Environmental Pathogen Mycobacterium ulcerans in Aquatic Insects

Background

An understanding of the factors driving the distribution of pathogens is useful in preventing disease. Often we achieve this understanding at a local microhabitat scale; however the larger scale processes are often neglected. This can result in misleading inferences about the distribution of the pathogen, inhibiting our ability to manage the disease. One such disease is Buruli ulcer, an emerging neglected tropical disease afflicting many thousands in Africa, caused by the environmental pathogen Mycobacterium ulcerans. Herein, we aim to describe the larger scale landscape process describing the distribution of M. ulcerans.

Methodology

Following extensive sampling of the community of aquatic macroinvertebrates in Cameroon, we select the 5 dominant insect Orders, and conduct an ecological niche model to describe how the distribution of M. ulcerans positive insects changes according to land cover and topography. We then explore the generalizability of the results by testing them against an independent dataset collected in a second endemic region, French Guiana.

Principal Findings

We find that the distribution of the bacterium in Cameroon is accurately described by the land cover and topography of the watershed, that there are notable seasonal differences in distribution, and that the Cameroon model does not predict the distribution of M. ulcerans in French Guiana.

Conclusions/Significance

Future studies of M. ulcerans would benefit from consideration of local structure of the local stream network in future sampling, and further work is needed on the reasons for notable differences in the distribution of this species from one region to another. This work represents a first step in the identification of large-scale environmental drivers of this species, for the purposes of disease risk mapping.     

Clinical and Parasitological Protection in a Leishmania infantum-Macaque Model Vaccinated with Adenovirus and the Recombinant A2 Antigen

Background

Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs.

Methodology/Principal Findings

Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol.

Conclusions/Significance

The remarkable clinical protection induced by A2 in an animal model that is evolutionary close to humans qualifies this antigen as a suitable vaccine candidate against human VL.