Primary cultures of insect midgut cells : a system to study membrane permeability to proteins and specific tools to enhance permeation

Recent studies have shown that Bombyx mori larval midgut can transport proteins unaltered following the transcellular pathway by transcytosis. In insects, the steps involved in this complex process are still unknown. Recently, this topic attracts increasing research efforts because of the demand of new and efficient strategies for the oral delivery of bioinsecticides targeting haemocoelic receptors. A promising tool to investigate these aspects is represented by midgut cells in culture. We developed a culture of B. mori midgut cells following the procedure of Sadrud-Din et al. (1994; 1996). We analysed the characteristics of the culture, showing that up to 60% of the stem cells isolated from the midgut, differentiated after three weeks in culture into columnar and goblet cells, the two predominant cell types in the epithelium. These cells presented in vitro the same shape, morphology and polarity recorded in vivo, even if their dimensions were slightly reduced. Moreover, cultured cell homogenates displayed aminopeptidase N and alkaline phosphatase activity, proving that these two enzymes, involved in vivo in the intermediate and final digestion of nutrients, are expressed also in vitro. We used this cell culture to assess the specific mechanism involved in the endocytosis and the sequence of intracellular events implicated in the movement of endocytic vesicles, of fluorescein isothiocyanate (FITC)-albumin, a protein absorbed by transcytosis in the midgut of B. mori larvae. We demonstrated that FITC-albumin uptake increased over time and was energy dependent, since it was strongly reduced by both low temperature and metabolic inhibitors. Labelled albumin uptake as a function of increasing protein concentration showed a saturation kinetics and was inhibited by extracellular unlabelled albumin in a concentration dependent manner. These data are compatible with the occurrence of a receptor-mediated endocytosis. FITC-albumin internalization was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalised in the intermicrovillar areas of the apical plasma membrane. RT-PCR analysis and colocalisation experiments with an anti-megalin primary antibody indicated that the receptor involved was a putative homologue of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein (LDL)-receptor family, responsible for the uptake of various molecules, albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca2+ for albumin internalisation. Albumin uptake was also inhibited by gentamycin, insulin and transferrin, proteins known to be megalin ligands. We demonstrated also that the integrity of actin and microtubule organisation was essential for the correct functioning of the endocytic machinery. Once internalised, albumin colocalized with early endosomes and lysosomes, suggesting that only a part of the protein is transcytosed, since a conspicuous amount is directed to the degradative pathway. Searching for enhancers of protein transport, we evaluated the ability of Cell-Penetrating Peptides (CPPs) to cross the intestinal cell membrane delivering a fused model protein. One of the best known and more often used CPPs is Tat, a peptide formed by 11 amino acid residues, that derives from the human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) peptide, Tat-(47-57). Tat-EGFP (Enhanced Green Fluorescent Protein) fusion protein, produced in the laboratory of Prof. Rosa Rao (University of Naples Federico II), was used in the experiments. Tat-EGFP was internalised by columnar cells in culture more efficiently than EGFP alone and its uptake increased over time. The internalisation process appeared to be energy-independent, since it was not reduced by either low temperature or metabolic inhibitors. The ability of Tat to translocate EGFP entering the cell across the brush border membrane was observed from experiments performed in the midgut isolated in a perfusion apparatus and incubated with the fusion protein added to the mucosal side of the epithelium. Densoviruses (DNVs) are insect parvoviruses which are lethal for several insects at larval stages, including agronomical pests and insects vector-borne diseases. During my permanence at the \u201cLaboratoire de Biologie Int\ue9grative et Virologie\u201d in Montpellier (France), under the supervision of Dr. Mylene Ogliastro, we studied the interaction between Junonia coenia Densovirus (JcDNV) and the permissive host Spodoptera frugiperda. Cultures of S. frugiperda midgut cells were prepared to detect the sites of penetration of JcDNV and to analyse the internalisation mechanism. The virus was unable to infect stem cells and differentiated goblet cells. In columnar cells with well developed microvilli, whether at an early phase of differentiation or fully differentiated, the virus was visible after 10 minutes on the cell surface at both the apical and the basolateral side, and after 30 minutes within the cytoplasm, but never in the nucleus. Virus particles were apparent as spots, suggesting a distribution in intracellular compartments. Virus internalisation appeared to be energy dependent, since it was strongly reduced by low temperature

Simultaneous Learning of Fuzzy Sets

We extend a procedure based on support vector clustering and devoted to inferring the membership function of a fuzzy set to the case of a universe of discourse over which several fuzzy sets are defined. The extended approach learns simultaneously these sets without requiring as previous knowledge either their number or labels approximating membership values. This data-driven approach is completed via expert knowledge incorporation in the form of predefined shapes for the membership functions. The procedure is successfully tested on a benchmark

Liver X receptors, nervous system, and lipid metabolism

Lipids in the nervous system are represented by cholesterol and phospholipids as constituents of cell membranes and, in particular, of myelin. Therefore, lipids are finely regulated to guarantee physiological functions. In the central nervous system, cholesterol is locally synthesized due to the presence of the blood brain barrier. In the peripheral nervous system cholesterol is either up-taken by lipoproteins and/or produced by de novo biosynthesis. Defects in lipid homeostasis in these tissues lead to structural and functional changes that often result in different pathological conditions depending on the affected pathways (i.e. cholesterol biosynthesis, cholesterol efflux, fatty acid biosynthesis etc.). Alterations in cholesterol metabolism in the central nervous system are linked to several disorders such as Alzheimer's disease, Huntington disease, Parkinson disease, Multiple sclerosis, Smith-Lemli-Opitz syndrome, Niemann-Pick type C disease, and glioblastoma. In the peripheral nervous system changes in lipid metabolism are associated with the development of peripheral neuropathy that may be caused by metabolic disorders, injuries, therapeutics, and autoimmune diseases. Transcription factors, such as the Liver X receptors (LXR), regulate both cholesterol and fatty acid metabolism in several tissues including the nervous system. In the last few years several studies elucidated the biology of LXR in the nervous system due to the availability of knock-out mice and the development of synthetic ligands. Here, we review a survey of the literature focused on the central and peripheral nervous system and in physiological and pathological settings with particular attention to the roles played by LXR in both districts

HDAC3 is a molecular brake of the metabolic switch supporting white adipose tissue browning.

White adipose tissue (WAT) can undergo a phenotypic switch, known as browning, in response to environmental stimuli such as cold. Post-translational modifications of histones have been shown to regulate cellular energy metabolism, but their role in white adipose tissue physiology remains incompletely understood. Here we show that histone deacetylase 3 (HDAC3) regulates WAT metabolism and function. Selective ablation of Hdac3 in fat switches the metabolic signature of WAT by activating a futile cycle of de novo fatty acid synthesis and β-oxidation that potentiates WAT oxidative capacity and ultimately supports browning. Specific ablation of Hdac3 in adipose tissue increases acetylation of enhancers in Pparg and Ucp1 genes, and of putative regulatory regions of the Ppara gene. Our results unveil HDAC3 as a regulator of WAT physiology, which acts as a molecular brake that inhibits fatty acid metabolism and WAT browning.Histone deacetylases, such as HDAC3, have been shown to alter cellular metabolism in various tissues. Here the authors show that HDAC3 regulates WAT metabolism by activating a futile cycle of fatty acid synthesis and oxidation, which supports WAT browning

Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4+ T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals.

Low-grade systemic inflammation associated to obesity leads to cardiovascular complications, caused partly by infiltration of adipose and vascular tissue by effector T cells. The signals leading to T cell differentiation and tissue infiltration during obesity are poorly understood. We tested whether saturated fatty acid-induced metabolic stress affects differentiation and trafficking patterns of CD4+ T cells. Memory CD4+ T cells primed in high-fat diet-fed donors preferentially migrated to non-lymphoid, inflammatory sites, independent of the metabolic status of the hosts. This was due to biased CD4+ T cell differentiation into CD44hi-CCR7lo-CD62Llo-CXCR3+-LFA1+ effector memory-like T cells upon priming in high-fat diet-fed animals. Similar phenotype was observed in obese subjects in a cohort of free-living people. This developmental bias was independent of any crosstalk between CD4+ T cells and dendritic cells and was mediated via direct exposure of CD4+ T cells to palmitate, leading to increased activation of a PI3K p110δ-Akt-dependent pathway upon priming

Ve-ptp modulates vascular integrity by promoting adherens junction maturation

Background: Endothelial cell junctions control blood vessel permeability. Altered permeability can be associated with vascular fragility that leads to vessel weakness and haemorrhage formation. In vivo studies on the function of genes involved in the maintenance of vascular integrity are essential to better understand the molecular basis of diseases linked to permeability defects. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein present at endothelial adherens junctions (AJs). Methodology/Principal Findings: We investigated the role of Ve-ptp in AJ maturation/stability and in the modulation of endothelial permeability using zebrafish (Danio rerio). Whole-mount in situ hybridizations revealed zve-ptp expression exclusively in the developing vascular system. Generation of altered zve-ptp transcripts, induced separately by two different splicing morpholinos, resulted in permeability defects closely linked to vascular wall fragility. The ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in zve-ptp morphants but not in control embryos. Conclusions/Significance: Here we show the first in vivo evidence of a potentially critical role played by Ve-ptp in AJ maturation, an important event for permeability modulation and for the development of a functional vascular system

Zebrafish Numb and Numblike Are Involved in Primitive Erythrocyte Differentiation

BACKGROUND:Notch signaling is an evolutionarily conserved regulatory circuitry implicated in cell fate determination in various developmental processes including hematopoietic stem cell self-renewal and differentiation of blood lineages. Known endogenous inhibitors of Notch activity are Numb-Nb and Numblike-Nbl, which play partially redundant functions in specifying and maintaining neuronal differentiation. Nb and Nbl are expressed in most tissues including embryonic and adult hematopoietic tissues in mice and humans, suggesting possible roles for these proteins in hematopoiesis. METHODOLOGY AND PRINCIPAL FINDINGS:We employed zebrafish to investigate the possible functional role of Numb and Numblike during hematopoiesis, as this system allows a detailed analysis even in embryos with severe defects that would be lethal in other organisms. Here we describe that nb/nbl knockdown results in severe reduction or absence of embryonic erythrocytes in zebrafish. Interestingly, nb/nbl knocked-down embryos present severe downregulation of the erythroid transcription factor gata1. This results in erythroblasts which fail to mature and undergo apoptosis. Our results indicate that Notch activity is increased in embryos injected with nb/nbl morpholino, and we show that inhibition of Notch activation can partially rescue the hematopoietic phenotype. CONCLUSIONS AND SIGNIFICANCE:Our results provide the first in vivo evidence of an involvement of Numb and Numblike in zebrafish erythroid differentiation during primitive hematopoiesis. Furthermore, we found that, at least in part, the nb/nbl morphant phenotype is due to enhanced Notch activation within hematopoietic districts, which in turn results in primitive erythroid differentiation defects

Zebrafish prox1b Mutants Develop a Lymphatic Vasculature, and prox1b Does Not Specifically Mark Lymphatic Endothelial Cells

Background: The expression of the Prospero homeodomain transcription factor (Prox1) in a subset of cardinal venous cells specifies the lymphatic lineage in mice. Prox1 is also indispensible for the maintenance of lymphatic cell fate, and is therefore considered a master control gene for lymphangiogenesis in mammals. In zebrafish, there are two prox1 paralogues, the previously described prox1 (also known as prox1a) and the newly identified prox1b. Principal Findings: To investigate the role of the prox1b gene in zebrafish lymphangiogenesis, we knocked-down prox1b and found that depletion of prox1b mRNA did not cause lymphatic defects. We also generated two different prox1b mutant alleles, and maternal-zygotic homozygous mutant embryos were viable and did not show any lymphatic defects. Furthermore, the expression of prox1b was not restricted to lymphatic vessels during zebrafish development. Conclusion: We conclude that Prox1b activity is not essential for embryonic lymphatic development in zebrafish

Lipids under stress - a lipidomic approach for the study of mood disorders

The emerging field of lipidomics has identified lipids as key players in disease physiology. Their physicochemical diversity allows precise control of cell structure and signaling events through modulation of membrane prop- erties and trafficking of proteins. As such, lipids are important regulators of brain function and have been implicated in neurodegenerative and mood disorders. Importantly, environmental chronic stress has been associated with anxiety and depression and its exposure in rodents has been extensively used as a model to study these diseases. With the accessibility to modern mass- spectrometry lipidomic platforms, it is now possible to snapshot the extensively interconnected lipid network. Here, we review the fundamentals of lipid biology and outline a framework for the interpretation of lipidomic studies as a new approach to study brain pathophysiology. Thus, lipid profiling provides an exciting avenue for the identification of disease signatures with important implications for diagnosis and treatment of mood disorders.We would like to thank Nuno Sousa for critical reading of the manuscript. André Miranda is funded by Fundação para a Ciência e Tecnologia (PD/BD/105915/2014). Tiago Gil Oliveira is funded by Fundação para a Ciência e Tecnologia (PTDC/ SAU-NMC/118971/2010)

Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to modulate endothelial cell number in angiogenic vessels

During embryonic development, new arteries, and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis