Novel role for SOX2 in the development of the zebrafish epithalamus

The sex determining region Y-box 2 (sox2) gene is one of the most important transcription factors during development, particularly the development of the central nervous system (CNS). It is expressed in embryonic stem cells and later in neural stem cells, where it modulates their maintenance and differentiation. In humans, heterozygous mutations are associated with eye malformations, including anophthalmia and severe microphthalmia. Also, a subset of patients has extra-ocular phenotypes, such as hearing loss, seizures and pituitary hypoplasia. Although the roles of sox2 in embryonic stem cells and eye development are well studied, the function of sox2 in brain development and disease is still elusive. The aim of this project was to characterize a novel role for sox2 in the development of zebrafish epithalamus, which was identified from an in silico screen previously performed in our laboratory. The zebrafish epithalamus, located in the dorsal diencephalon, consists of three main structures: the pineal gland, the parapineal organ and the habenular nuclei. The pineal gland, also known as epiphysis, is a photoreceptive (in zebrafish) and neuroendocrine organ that detects light and rhythmically produces melatonin in order to regulate the circadian rhythms. The parapineal organ is located to the left side of the pineal gland and is important for the elaboration of the asymmetries observed between the left and right habenular nuclei. Finally, the bilateral habenulae are part of the dorsal diencephalic conduction system that links the forebrain with the mid- and hindbrain. The left and right habenulae show both molecular and neuroanatomical asymmetries, including differences in neuropil organization, in levels of gene expression and in the morphology and connectivity of their neurons’ projections. The relatively simple architecture of the pineal gland and the asymmetric character of the habenulae provide a useful tool for studying cell-fate determination, cell migration and establishment of brain asymmetries. In this study, we used zebrafish as a model to dissect the novel functions of sox2 in the development of the epithalamus. We showed that sox2 works synergistically with Notch pathway to negatively regulate neurogenesis within the pineal gland. The pineal gland consists of only two cell types: the photoreceptors and the projection neurons. Previous studies showed that the Notch and BMP pathways are important for the proper specification of these cells. Here, we show that sox2 normally inhibits the photoreceptor cell fate, whereas it has no effect on the number of projection neurons. Therefore, sox2 complements Notch and BMP pathways in cell-fate determination within the pineal gland. In addition, downregulation of sox2 results in abnormal parapineal organ development and disruption of the asymmetric architecture of the habenulae. A subset of sox2 morphant embryos develops right-sided parapineal organs, which is consistent with abnormal bilateral expression of the Nodal gene, pitx2 (paired-like homeodomain transcription factor 2). Also, timelapse experiments showed that migration of the parapineal cells is defective, resulting in scattered cells. The aberrant parapineal development leads to disorganization of the habenular nuclei, as shown by the abnormal neuropil arrangement and the expression of the asymmetric marker kctd12.1 (potassium channel tetramerisation domain containing 12.1)

Higher phagocytic activity of thioglycollate-elicited peritoneal macrophages is related to metabolic status of the cells

BACKGROUND: Peritoneal macrophages are widely used in immunological studies. The cells can be collected under non-elicited (resident) or elicited (e.g., with Brewer thioglycollate broth injection) conditions, and their phenotype and functions differ. Recent studies have shown that macrophage phenotype and function are related to their metabolic states, and metabolic reprogramming has been an emerging concept for controlling macrophage function. In this study, we examined the metabolic state of resident and elicited macrophages and investigated how their metabolic state may affect cell function, including phagocytosis. FINDINGS: Flow cytometry showed that elicited macrophages expressed higher levels of MHC-II, LFA-1 and CD64 but lower levels of F4/80 compared to naïve resident peritoneal macrophages, suggesting a more mature and active phenotype. Elicited macrophages had significantly higher levels of phagocytic activity compared to that of resident macrophages. Metabolic studies showed that the Extracellular Acidification Rates (ECAR) and Oxygen Consumption Rates (OCR) were both significantly higher in elicited macrophages than those in resident macrophages. The treatment of macrophages with 2-Deoxy-D-glucose suppressed glycolysis and reduced phagocytosis, whereas treatment with oligomycin enhanced glycolysis and increased phagocytosis in elicited macrophages. CONCLUSION: Naïve resident peritoneal macrophages are less metabolically active compared to elicited macrophages. Elicited macrophages had higher levels of glycolysis and oxidative phosphorylation, which may be related to their increased phagocytic capacity and higher levels of maturation and activation. Further understanding of the molecular links between metabolic pathways and cell function would be crucial to develop strategies to control macrophage function through metabolic reprogramming

Attenuating Diabetic Vascular and Neuronal Defects by Targeting P2rx7.

Retinal vascular and neuronal degeneration are established pathological features of diabetic retinopathy. Data suggest that defects in the neuroglial network precede the clinically recognisable vascular lesions in the retina. Therefore, new treatments that target early-onset neurodegeneration would be expected to have great value in preventing the early stages of diabetic retinopathy. Here, we show that the nucleoside reverse transcriptase inhibitor lamivudine (3TC), a newly discovered P2rx7 inhibitor, can attenuate progression of both neuronal and vascular pathology in diabetic retinopathy. We found that the expression of P2rx7 was increased in the murine retina as early as one month following diabetes induction. Compared to non-diabetic controls, diabetic mice treated with 3TC were protected against the formation of acellular capillaries in the retina. This occurred concomitantly with a maintenance in neuroglial function, as shown by improved a- and b-wave amplitude, as well as oscillatory potentials. An improvement in the number of GABAergic amacrine cells and the synaptophysin-positive area was also observed in the inner retina of 3TC-treated diabetic mice. Our data suggest that 3TC has therapeutic potential since it can target both neuronal and vascular defects caused by diabetes

IL-33 deficiency causes persistent inflammation and severe neurodegeneration in retinal detachment.

BACKGROUND: Interleukin-33 (IL-33) belongs to the IL-1 cytokine family and resides in the nuclei of various cell types. In the neural retina, IL-33 is predominately expressed in Müller cells although its role in health and disease is ill-defined. Müller cell gliosis is a critical response during the acute phase of retinal detachment (RD), and in this study, we investigated if IL-33 was modulatory in the inflammatory and neurodegenerative pathology which is characteristic of this important clinical condition. METHODS: RD was induced by subretinal injection of sodium hyaluronate into C57BL/6 J (WT) and IL-33-/- mice and confirmed by fundus imaging and optical coherence tomography (OCT). The expression of inflammatory cytokines, complement components and growth factors was examined by RT-PCR. Retinal neurodegeneration, Müller cell activation and immune cell infiltration were assessed using immunohistochemistry. The expression of inflammatory cytokines in primary Müller cells and bone marrow-derived macrophages (BM-DMs) was assessed by RT-PCR and Cytometric Bead Array. RESULTS: RD persisted for at least 28 days after the injection of sodium hyaluronate, accompanied by significant cone photoreceptor degeneration. The mRNA levels of CCL2, C1ra, C1s, IL-18, IL-1β, TNFα, IL-33 and glial fibrillary acidic protein (GFAP) were significantly increased at day 1 post-RD, reduced gradually and, with the exception of GFAP and C1ra, returned to the basal levels by day 28 in WT mice. In IL-33-/- mice, RD induced an exacerbated inflammatory response with significantly higher levels of CCL2, IL-1β and GFAP when compared to WT. Sustained GFAP activation and immune cell infiltration was detected at day 28 post-RD in IL-33-/- mice. Electroretinography revealed a lower A-wave amplitude at day 28 post-RD in IL-33-/- mice compared to that in WT RD mice. IL-33-/- mice subjected to RD also had significantly more severe cone photoreceptor degeneration compared to WT counterparts. Surprisingly, Müller cells from IL-33-/- mice expressed significantly lower levels of CCL2 and IL-6 compared with those from WT mice, particularly under hypoxic conditions, whereas IL-33-/- bone marrow-derived macrophages expressed higher levels of inducible nitric oxide synthase, TNFα, IL-1β and CCL2 after LPS + IFNγ stimulation compared to WT macrophages. CONCLUSION: IL-33 deficiency enhanced retinal degeneration and gliosis following RD which was related to sustained subretinal inflammation from infiltrating macrophages. IL-33 may provide a previously unrecognised protective response by negatively regulating macrophage activation following retinal detachment

Effect of intermittent fasting on circulating inflammatory markers in obesity: A review of human trials

Obesity is associated with low-grade inflammation. Weight loss, by means of dietary restriction, has been shown to reduce systemic inflammation. Intermittent fasting has recently gained popularity as a weight loss diet, but its effects on inflammatory markers in individuals with obesity have yet to be summarized. Accordingly, this review examined how the two main forms of intermittent fasting, i.e., time restricted eating (TRE) and alternate day fasting (ADF), impact body weight and key circulating inflammatory markers (i.e., C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6)), in adults with obesity. Results from this review reveal that TRE with various eating window durations (4–10 h per day) has no effect on circulating levels of CRP, TNF-alpha or IL-6, with 1–5% weight loss. As for ADF, reductions in CRP concentrations were noted when >6% weight loss was achieved. However, ADF had no effect on TNF-alpha or IL-6 concentrations, with this degree of weight loss. Thus, intermittent fasting has little or no effect on key inflammatory markers, but more research is warranted to confirm these preliminary findings

Connecting the Edges: A Universal, Mobile-Centric, and Opportunistic Communications Architecture

The Internet has crossed new frontiers with access to it getting faster and cheaper. Considering that the architectural foundations of today's Internet were laid more than three decades ago, the Internet has done remarkably well until today coping with the growing demand. However, the future Internet architecture is expected to support not only the ever growing number of users and devices, but also a diverse set of new applications and services. Departing from the traditional host-centric access paradigm, where access to a desired content is mapped to its location, an information-centric model enables the association of access to a desired content with the content itself, irrespective of the location where it is being held. UMOBILE tailors the information-centric communication model to meet the requirements of opportunistic communications, integrating those connectivity approaches into a single architecture. By pushing services near the edge of the network, such an architecture can pervasively operate in any networking environment and allows for the development of innovative applications, providing access to data independent of the level of end-to-end connectivity availability

Discovery and assessment of conserved Pax6 target genes and enhancers

The characterization of transcriptional networks (TNs) is essential for understanding complex biological phenomena such as development, disease, and evolution. In this study, we have designed and implemented a procedure that combines in silico target screens with zebrafish and mouse validation, in order to identify cis-elements and genes directly regulated by Pax6. We chose Pax6 as the paradigm because of its crucial roles in organogenesis and human disease. We identified over 600 putative Pax6 binding sites and more than 200 predicted direct target genes, conserved in evolution from zebrafish to human and to mouse. This was accomplished using hidden Markov models (HMMs) generated from experimentally validated Pax6 binding sites. A small sample of genes, expressed in the neural lineage, was chosen from the predictions for RNA in situ validation using zebrafish and mouse models. Validation of DNA binding to some predicted cis-elements was also carried out using chromatin immunoprecipitation (ChIP) and zebrafish reporter transgenic studies. The results show that this combined procedure is a highly efficient tool to investigate the architecture of TNs and constitutes a useful complementary resource to ChIP and expression data sets because of its inherent spatiotemporal independence. We have identified several novel direct targets, including some putative disease genes, among them Foxp2; these will allow further dissection of Pax6 function in development and disease

Pleiotropic Effects of Sox2 during the Development of the Zebrafish Epithalamus

The zebrafish epithalamus is part of the diencephalon and encompasses three major components: the pineal, the parapineal and the habenular nuclei. Using sox2 knockdown, we show here that this key transcriptional regulator has pleiotropic effects during the development of these structures. Sox2 negatively regulates pineal neurogenesis. Also, Sox2 is identified as the unknown factor responsible for pineal photoreceptor prepatterning and performs this function independently of the BMP signaling. The correct levels of sox2 are critical for the functionally important asymmetrical positioning of the parapineal organ and for the migration of parapineal cells as a coherent structure. Deviations from this strict control result in defects associated with abnormal habenular laterality, which we have documented and quantified in sox2 morphants

Sustained high glucose exposure sensitizes macrophage responses to cytokine stimuli but reduces their phagocytic activity

Abstract Background Macrophages are tissue resident immune cells important for host defence and homeostasis. During diabetes, macrophages and other innate immune cells are known to have a pro-inflammatory phenotype, which is believed to contribute to the pathogenesis of various diabetic complications. However, diabetic patients are highly susceptible to bacterial infections, and often have impaired wound healing. The molecular mechanism underlying the paradox of macrophage function in diabetes is not fully understood. Recent evidence suggests that macrophage functions are governed by metabolic reprograming. Diabetes is a disorder that affects glucose metabolism; dysregulated macrophage function in diabetes may be related to alterations in their metabolic pathways. In this study, we seek to understand the effect of high glucose exposure on macrophage phenotype and functions. Results Bone marrow cells were cultured in short or long term high glucose and normal glucose medium; the number and phenotype of bone marrow derived macrophages were not affected by long-term high glucose treatment. Short-term high glucose increased the expression of IL-1β. Long-term high glucose increased the expression of IL-1β and TNFα but reduced the expression of IL-12p40 and nitric oxide production in M1 macrophage. The treatment also increased Arg-1 and IL-10 expression in M2 macrophages. Phagocytosis and bactericidal activity was reduced in long-term high glucose treated macrophages and peritoneal macrophages from diabetic mice. Long-term high glucose treatment reduced macrophage glycolytic capacity and glycolytic reserve without affecting mitochondrial ATP production and oxidative respiration. Conclusion Long-term high glucose sensitizes macrophages to cytokine stimulation and reduces phagocytosis and nitric oxide production, which may be related to impaired glycolytic capacity