Surface assembly of poly(I:C) on PEGylated microspheres to shield from adverse interactions with fibroblasts.

By expressing an array of pattern recognition receptors (PRRs), fibroblasts play an important role in stimulating and modulating the response of the innate immune system. The TLR3 ligand polyriboinosinic acid-polyribocytidylic acid, poly(I:C), a mimic of viral dsRNA, is a vaccine adjuvant candidate to activate professional antigen presenting cells (APCs). However, owing to its ligation with extracellular TLR3 on fibroblasts, subcutaneously administered poly(I:C) bears danger towards autoimmunity. It is thus in the interest of its clinical safety to deliver poly(I:C) in such a way that its activation of professional APCs is as efficacious as possible, whereas its interference with non-immune cells such as fibroblasts is controlled or even avoided. Complementary to our previous work with monocyte-derived dendritic cells (MoDCs), here we sought to control the delivery of poly(I:C) surface-assembled on microspheres to human foreskin fibroblasts (HFFs). Negatively charged polystyrene (PS) microspheres were equipped with a poly(ethylene glycol) (PEG) corona through electrostatically driven coatings with a series of polycationic poly(L-lysine)-graft-poly(ethylene glycol) copolymers, PLL-g-PEG, of varying grafting ratios g from 2.2 up to 22.7. Stable surface assembly of poly(I:C) was achieved by incubation of polymer-coated microspheres with aqueous poly(I:C) solutions. Notably, recognition of both surface-assembled and free poly(I:C) by extracellular TLR3 on HFFs halted their phagocytic activity. Ligation of surface-assembled poly(I:C) with extracellular TLR3 on HFFs could be controlled by tuning the grafting ratio g and thus the chain density of the PEG corona. When assembled on PLL-5.7-PEG-coated microspheres, poly(I:C) was blocked from triggering class I MHC molecule expression on HFFs. Secretion of interleukin (IL)-6 by HFFs after exposure to surface-assembled poly(I:C) was distinctly lower as compared to free poly(I:C). Overall, surface assembly of poly(I:C) may have potential to contribute to the clinical safety of this vaccine adjuvant candidate.

Intravitreous injection of PLGA microspheres encapsulating GDNF promotes the survival of photoreceptors in the rd1/rd1 mouse.

PURPOSE: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres. METHODS: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections. RESULTS: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres. CONCLUSIONS: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

De Novo Production of Metabolites by Fungal Co-culture of Trichophyton rubrum and Bionectria ochroleuca.

The co-cultivation of fungi has recently been described as a promising strategy to induce the production of novel metabolites through possible gene activation. A large screening of fungal co-cultures in solid media has identified an unusual long-distance growth inhibition between Trichophyton rubrum and Bionectria ochroleuca. To study metabolite induction in this particular fungal interaction, differential LC-MS-based metabolomics was performed on pure strain cultures and on their co-cultures. The comparison of the resulting fingerprints highlighted five de novo induced compounds, which were purified using software-oriented semipreparative HPLC-MS. One metabolite was successfully identified as 4″-hydroxysulfoxy-2,2″-dimethylthielavin P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated form, as well as three other related compounds, were found in the pure strain culture of B. ochroleuca.

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.

ACID-SENSING ION CHANNELS: functional and molecular characterization in putative nociceptors, and modulation by nerve injury and serine protease

Résumé Les canaux ioniques ASICs (acid-sensing ion channels) appartiennent à la famille des canaux ENaC/Degenerin. Pour l'instant, quatre gènes (1 à 4) ont été clonés dont certains présentent des variants d'épissage. Leur activation par une acidification rapide du milieu extracellulaire génère un courant entrant transitoire essentiellement sodique accompagné pour certains types d'ASICs d'une phase soutenue. Les ASICs sont exprimés dans le système nerveux, central (SNC) et périphérique (SNP). On leur attribue un rôle dans l'apprentissage, la mémoire et l'ischémie cérébrale au niveau central ainsi que dans la nociception (douleur aiguë et inflammatoire) et la méchanotransduction au niveau périphérique. Toutefois, les données sont parfois contradictoires. Certaines études suggèrent qu'ils sont des senseurs primordiaux impliqués dans la détection de l'acidification et la douleur. D'autres études suggèrent plutôt qu'ils ont un rôle modulateur inhibiteur dans la douleur. De plus, le fait que leur activation génère majoritairement un courant transitoire alors que les fibres nerveuses impliquées dans la douleur répondent à un stimulus nocif avec une adaptation lente suggère que leurs propriétés doivent être modulés par des molécules endogènes. Dans une première partie de ma thèse, nous avons abordé la question de l'expression fonctionnelle des ASICs dans les neurones sensoriels primaires afférents du rat adulte pour clarifier le rôle des ASICs dans les neurones sensoriels. Nous avons caractérisé leurs propriétés biophysiques et pharmacologiques par la technique du patch-clamp en configuration « whole-cell ». Nous avons pu démontrer que près de 60% des neurones sensoriels de petit diamètre expriment des courants ASICs. Nous avons mis en évidence trois types de courant ASIC dans ces neurones. Les types 1 et 3 ont des propriétés compatibles avec un rôle de senseur du pH alors que le type 2 est majoritairement activé par des pH inférieurs à pH6. Le type 1 est médié par des homomers de la sous-unité ASIC1 a qui sont perméables aux Ca2+. Nous avons étudié leur co-expression avec des marqueurs des nocicepteurs ainsi que la possibilité d'induire une activité neuronale suite à une acidification qui soit dépendante des ASICs. Le but était d'associer un type de courant ASIC avec une fonction potentielle dans les neurones sensoriels. Une majorité des neurones exprimant les courants ASIC co-expriment des marqueurs des nocicepteurs. Toutefois, une plus grande proportion des neurones exprimant le type 1 n'est pas associée à la nociception par rapport aux types 2 et 3. Nous avons montré qu'il est possible d'induire des potentiels d'actions suite à une acidification. La probabilité d'induction est proportionnelle à la densité des courants ASIC et à l'acidité de la stimulation. Puis, nous avons utilisé cette classification comme un outil pour appréhender les potentielles modulations fonctionnelles des ASICs dans un model de neuropathie (spared nerve injury). Cette approche fut complétée par des expériences de «quantitative RT-PCR ». En situation de neuropathie, les courants ASIC sont dramatiquement changés au niveau de leur expression fonctionnelle et transcriptionnelle dans les neurones lésés ainsi que non-lésés. Dans une deuxième partie de ma thèse, suite au test de différentes substances sécrétées lors de l'inflammation et l'ischémie sur les propriétés des ASICs, nous avons caractérisé en détail la modulation des propriétés des courants ASICs notamment ASIC1 par les sérines protéases dans des systèmes d'expression recombinants ainsi que dans des neurones d'hippocampe. Nous avons montré que l'exposition aux sérine-protéases décale la dépendance au pH de l'activation ainsi que la « steady-state inactivation »des ASICs -1a et -1b vers des valeurs plus acidiques. Ainsi, l'exposition aux serine protéases conduit à une diminution du courant quand l'acidification a lieu à partir d'un pH7.4 et conduit à une augmentation du courant quand l'acidification alleu à partir d'un pH7. Nous avons aussi montré que cette régulation a lieu des les neurones d'hippocampe. Nos résultats dans les neurones sensoriels suggèrent que certains courants ASICs sont impliqués dans la transduction de l'acidification et de la douleur ainsi que dans une des phases du processus conduisant à la neuropathie. Une partie des courants de type 1 perméables au Ca 2+ peuvent être impliqués dans la neurosécrétion. La modulation par les sérines protéases pourrait expliquer qu'en situation d'acidose les canaux ASICs soient toujours activables. Résumé grand publique Les neurones sont les principales cellules du système nerveux. Le système nerveux est formé par le système nerveux central - principalement le cerveau, le cervelet et la moelle épinière - et le système nerveux périphérique -principalement les nerfs et les neurones sensoriels. Grâce à leur nombreux "bras" (les neurites), les neurones sont connectés entre eux, formant un véritable réseau de communication qui s'étend dans tout le corps. L'information se propage sous forme d'un phénomène électrique, l'influx nerveux (ou potentiels d'actions). A la base des phénomènes électriques dans les neurones il y a ce que l'on appelle les canaux ioniques. Un canal ionique est une sorte de tunnel qui traverse l'enveloppe qui entoure les cellules (la membrane) et par lequel passent les ions. La plupart de ces canaux sont normalement fermés et nécessitent d'être activés pour s'ouvrire et générer un influx nerveux. Les canaux ASICs sont activés par l'acidification et sont exprimés dans tout le système nerveux. Cette acidification a lieu notamment lors d'une attaque cérébrale (ischémie cérébrale) ou lors de l'inflammation. Les expériences sur les animaux ont montré que les canaux ASICs avaient entre autre un rôle dans la mort des neurones lors d'une attaque cérébrale et dans la douleur inflammatoire. Lors de ma thèse je me suis intéressé au rôle des ASICs dans la douleur et à l'influence des substances produites pendant l'inflammation sur leur activation par l'acidification. J'ai ainsi pu montrer chez le rat que la majorité des neurones sensoriels impliqués dans la douleur ont des canaux ASICs et que l'activation de ces canaux induit des potentiels d'action. Nous avons opéré des rats pour qu'ils présentent les symptômes d'une maladie chronique appelée neuropathie. La neuropathie se caractérise par une plus grande sensibilité à la douleur. Les rats neuropathiques présentent des changements de leurs canaux ASICs suggérant que ces canaux ont une peut-être un rôle dans la genèse ou les symptômes de cette maladie. J'ai aussi montré in vitro qu'un type d'enryme produit lors de l'inflammation et l'ischémie change les propriétés des ASICs. Ces résultats confirment un rôle des ASICs dans la douleur suggérant notamment un rôle jusque là encore non étudié dans la douleur neuropathique. De plus, ces résultats mettent en évidence une régulation des ASICs qui pourrait être importante si elle se confirmait in vivo de part les différents rôles des ASICs. Abstract Acid-sensing ion channels (ASICs) are members of the ENaC/Degenerin superfamily of ion channels. Their activation by a rapid extracellular acidification generates a transient and for some ASIC types also a sustained current mainly mediated by Na+. ASICs are expressed in the central (CNS) and in the peripheral (PNS) nervous system. In the CNS, ASICs have a putative role in learning, memory and in neuronal death after cerebral ischemia. In the PNS, ASICs have a putative role in nociception (acute and inflammatory pain) and in mechanotransduction. However, studies on ASIC function are somewhat controversial. Some studies suggest a crucial role of ASICs in transduction of acidification and in pain whereas other studies suggest rather a modulatory inhibitory role of ASICs in pain. Moreover, the basic property of ASICs, that they are activated only transiently is irreconcilable with the well-known property of nociception that the firing of nociceptive fibers demonstrated very little adaptation. Endogenous molecules may exist that can modulate ASIC properties. In a first part of my thesis, we addressed the question of the functional expression of ASICs in adult rat dorsal root ganglion (DRG) neurons. Our goal was to elucidate ASIC roles in DRG neurons. We characterized biophysical and pharmacological properties of ASIC currents using the patch-clamp technique in the whole-cell configuration. We observed that around 60% of small-diameter sensory neurons express ASICs currents. We described in these neurons three ASIC current types. Types 1 and 3 have properties compatible with a role of pH-sensor whereas type 2 is mainly activated by pH lower than pH6. Type 1 is mediated by ASIC1a homomultimers which are permeable to Ca 2+. We studied ASIC co-expression with nociceptor markers. The goal was to associate an ASIC current type with a potential function in sensory neurons. Most neurons expressing ASIC currents co-expressed nociceptor markers. However, a higher proportion of the neurons expressing type 1 was not associated with nociception compared to type 2 and -3. We completed this approach with current-clamp measurements of acidification-induced action potentials (APs). We showed that activation of ASICs in small-diameter neurons can induce APs. The probability of AP induction is positively correlated with the ASIC current density and the acidity of stimulation. Then, we used this classification as a tool to characterize the potential functional modulation of ASICs in the spared nerve injury model of neuropathy. This approach was completed by quantitative RT-PCR experiments. ASICs current expression was dramatically changed at the functional and transcriptional level in injured and non-injured small-diameter DRG neurons. In a second part of my thesis, following an initial screening of the effect of various substances secreted during inflammation and ischemia on ASIC current properties, we characterized in detail the modulation of ASICs, in particular of ASIC1 by serine proteases in a recombinant expression system as well as in hippocampal neurons. We showed that protease exposure shifts the pH dependence of ASIC1 activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in the current response if ASIC1 is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provided evidence that this bi-directional regulation of ASIC1a function also occurs in hippocampal neurons. Our results in DRG neurons suggest that some ASIC currents are involved in the transduction of peripheral acidification and pain. Furthermore, ASICs may participate to the processes leading to neuropathy. Some Ca 2+-permeable type 1 currents may be involved in neurosecretion. ASIC modulation by serine proteases may be physiologically relevant, allowing ASIC activation under sustained slightly acidic conditions.

Use of recovered frying oils in chicken and rabbit feeds: effect on the fatty acid and tocol composition and on the oxidation levels of meat, liver and plasma

The addition of some fat co- and by-products to feeds is usual nowadays; however, the regulations of their use are not always clear and vary between countries. For instance, the use of recycled cooking oils is not allowed in the European Union, but they are used in other countries. However, oils recovered from industrial frying processes could show satisfactory quality for this purpose. Here we studied the effects of including oils recovered from the frying industry in rabbit and chicken feeds (at 30 and 60 g/kg, respectively) on the fatty acid (FA) and tocol (tocopherol + tocotrienol) compositon of meat, liver and plasma, and on their oxidative stability. Three dietary treatments (replicated eight times) were compared: fresh non-used oil (LOX); oil discarded from the frying industry, having a high content of secondary oxidation compounds (HOX); and an intermediate level (MOX) obtained by mixing 50 : 50 of LOX and HOX. The FA composition of oil diets and tissues was assessed by GC, their tocol content by HPLC, the thiobarbituric acid value was used to assess tissue oxidation status, and the ferrous oxidation-xylenol orange method was used to assess the susceptibility of tissues to oxidation. Our results indicate that FA composition of rabbit and chicken meat, liver and plasma was scarcely altered by the addition of recovered frying oils to feed. Differences were encountered in the FA composition between species, which might be attributed mainly to differences in the FA digestion, absorption and metabolism between species, and to some physiological dietary factors (i.e. coprophagy in rabbits that involves fermentation with FA structure modification). The α-tocopherol (αT) content of tissues was reduced in response to the lower αT content in the recovered frying oil. Differences in the content of other tocols were encountered between chickens and rabbits, which might be attributable to the different tocol composition of their feeds, as well as to species differences in the digestion and metabolism of tocols. Tissue oxidation and susceptibility to oxidation were in general low and were not greatly affected by the degree of oxidation of the oil added to the feeds. The relative content of polyunsaturated fatty acids/αT in these types of samples would explain the differences observed between species in the susceptibility of each tissue to oxidation. According to our results, oils recovered from the frying industry could be useful for feed uses.

Use of recovered frying oils in chicken and rabbit feeds: effect on the fatty acid and tocol composition and on the oxidation levels of meat, liver and plasma

The addition of some fat co- and by-products to feeds is usual nowadays; however, the regulations of their use are not always clear and vary between countries. For instance, the use of recycled cooking oils is not allowed in the European Union, but they are used in other countries. However, oils recovered from industrial frying processes could show satisfactory quality for this purpose. Here we studied the effects of including oils recovered from the frying industry in rabbit and chicken feeds (at 30 and 60 g/kg, respectively) on the fatty acid (FA) and tocol (tocopherol1tocotrienol) compositon of meat, liver and plasma, and on their oxidative stability. Three dietary treatments (replicated eight times) were compared: fresh non-used oil (LOX); oil discarded from the frying industry, having a high content of secondary oxidation compounds (HOX); and an intermediate level (MOX) obtained by mixing 50 : 50 of LOX and HOX. The FA composition of oil diets and tissues was assessed by GC, their tocol content by HPLC, the thiobarbituric acid value was used to assess tissue oxidation status, and the ferrous oxidation-xylenol orange method was used to assess the susceptibility of tissues to oxidation. Our results indicate that FA composition of rabbit and chicken meat, liver and plasma was scarcely altered by the addition of recovered frying oils to feed. Differences were encountered in the FA composition between species, which might be attributed mainly to differences in the FA digestion, absorption and metabolism between species, and to some physiological dietary factors (i.e. coprophagy in rabbits that involves fermentation with FA structure modification). The a-tocopherol (aT) content of tissues was reduced in response to the lower aT content in the recovered frying oil. Differences in the content of other tocols were encountered between chickens and rabbits, which might be attributable to the different tocol composition of their feeds, as well as to species differences in the digestion and metabolism of tocols. Tissue oxidation and susceptibility to oxidation were in general low and were not greatly affected by the degree of oxidation of the oil added to the feeds. The relative content of polyunsaturated fatty acids/aT in these types of samples would explain the differences observed between species in the susceptibility of each tissue to oxidation. According to our results, oils recovered from the frying industry could be useful for feed uses.

Gene transfer of the Ly-3 chain gene of the mouse CD8 molecular complex: co-transfer with the Ly-2 polypeptide gene results in detectable cell surface expression of the Ly-3 antigenic determinants.

The CD8 molecule is a glycoprotein expressed on a subset of mature T lymphocytes. It has been postulated to be a receptor for class I major histocompatibility complex molecules. In the mouse, CD8 is a heterodimer composed of Ly-2 and Ly-3 chains. We have isolated and analyzed cDNA and cosmid clones corresponding to the Ly-3 subunit. One of the isolated, cosmid clones was subsequently transfected, alone or in combination with the Ly-2 gene, into mouse Ltk- cells. Analysis of the Ly-2,3 molecules expressed at the surface of the double transfectants indicated that they are serologically and biochemically indistinguishable from their normal counterparts expressed on lymphoid cells. Ltk- cells transfected with the Ly-2 gene alone were shown to react with a subset of anti-CD8 monoclonal antibodies whereas Ly-3 transfectants did not stain with any of the anti-Ly-3 antibodies employed in this study. Since at least one of these antibodies (53-5.8) has been previously shown to recognize an epitope which is retained on the Ly-3 subunit after dissociation of the heterodimeric Ly-2,3 complex, these observations suggest that the expression of the Ly-2 polypeptide is required to permit the detectable cell surface expression of the antigenic determinants carried by the Ly-3 subunit.

Esterification of oleic acid catalayzed by an Aspergillus niger strain. Influence of water activity and alcohol concentration

Effects of water activity and 1-propanol concentration on synthesis of propyl oleate from oleic acid using Aspergillus niger cell-bound lipases in isooctane are described. A. niger produces lipases (EC 3.1.1.3) which partly bind to the mycelium during growth. Ester production was monitored for 72 hours at different 1-propanol concentrations and water activities. Aliquots were sequentially withdrawn and propyl esters were quantified using GC and methyl palmitate as an internal standard. In all assayed conditions A. niger cell-bound lipases catalysed propyl oleate synthesis, but at different degrees.

Nolz1 promotes striatal neurogenesis through the regulation of retinoic acid signaling

Background: Nolz1 is a zinc finger transcription factor whose expression is enriched in the lateral ganglionic eminence (LGE), although its function is still unknown. Results: Here we analyze the role of Nolz1 during LGE development. We show that Nolz1 expression is high in proliferating neural progenitor cells (NPCs) of the LGE subventricular zone. In addition, low levels of Nolz1 are detected in the mantle zone, as well as in the adult striatum. Similarly, Nolz1 is highly expressed in proliferating LGE-derived NPC cultures, but its levels rapidly decrease upon cell differentiation, pointing to a role of Nolz1 in the control of NPC proliferation and/or differentiation. In agreement with this hypothesis, we find that Nolz1 over-expression promotes cell cycle exit of NPCs in neurosphere cultures and negatively regulates proliferation in telencephalic organotypic cultures. Within LGE primary cultures, Nolz1 over-expression promotes the acquisition of a neuronal phenotype, since it increases the number of β-III tubulin (Tuj1)- and microtubule-associated protein (MAP)2-positive neurons, and inhibits astrocyte generation and/or differentiation. Retinoic acid (RA) is one of the most important morphogens involved in striatal neurogenesis, and regulates Nolz1 expression in different systems. Here we show that Nolz1 also responds to this morphogen in E12.5 LGE-derived cell cultures. However, Nolz1 expression is not regulated by RA in E14.5 LGE-derived cell cultures, nor is it affected during LGE development in mouse models that present decreased RA levels. Interestingly, we find that Gsx2, which is necessary for normal RA signaling during LGE development, is also required for Nolz1 expression, which is lost in Gsx2 knockout mice. These findings suggest that Nolz1 might act downstream of Gsx2 to regulate RA-induced neurogenesis. Keeping with this hypothesis, we show that Nolz1 induces the selective expression of the RA receptor (RAR)β without altering RARα or RARγ. In addition, Nozl1 over-expression increases RA signaling since it stimulates the RA response element. This RA signaling is essential for Nolz1-induced neurogenesis, which is impaired in a RA-free environment or in the presence of a RAR inverse agonist. It has been proposed that Drosophila Gsx2 and Nolz1 homologues could cooperate with the transcriptional co-repressors Groucho-TLE to regulate cell proliferation. In agreement with this view, we show that Nolz1 could act in collaboration with TLE-4, as they are expressed at the same time in NPC cultures and during mouse development. Conclusions: Nolz1 promotes RA signaling in the LGE, contributing to the striatal neurogenesis during development.

Nolz1 promotes striatal neurogenesis through the regulation of retinoic acid signaling

Background: Nolz1 is a zinc finger transcription factor whose expression is enriched in the lateral ganglionic eminence (LGE), although its function is still unknown. Results: Here we analyze the role of Nolz1 during LGE development. We show that Nolz1 expression is high in proliferating neural progenitor cells (NPCs) of the LGE subventricular zone. In addition, low levels of Nolz1 are detected in the mantle zone, as well as in the adult striatum. Similarly, Nolz1 is highly expressed in proliferating LGE-derived NPC cultures, but its levels rapidly decrease upon cell differentiation, pointing to a role of Nolz1 in the control of NPC proliferation and/or differentiation. In agreement with this hypothesis, we find that Nolz1 over-expression promotes cell cycle exit of NPCs in neurosphere cultures and negatively regulates proliferation in telencephalic organotypic cultures. Within LGE primary cultures, Nolz1 over-expression promotes the acquisition of a neuronal phenotype, since it increases the number of β-III tubulin (Tuj1)- and microtubule-associated protein (MAP)2-positive neurons, and inhibits astrocyte generation and/or differentiation. Retinoic acid (RA) is one of the most important morphogens involved in striatal neurogenesis, and regulates Nolz1 expression in different systems. Here we show that Nolz1 also responds to this morphogen in E12.5 LGE-derived cell cultures. However, Nolz1 expression is not regulated by RA in E14.5 LGE-derived cell cultures, nor is it affected during LGE development in mouse models that present decreased RA levels. Interestingly, we find that Gsx2, which is necessary for normal RA signaling during LGE development, is also required for Nolz1 expression, which is lost in Gsx2 knockout mice. These findings suggest that Nolz1 might act downstream of Gsx2 to regulate RA-induced neurogenesis. Keeping with this hypothesis, we show that Nolz1 induces the selective expression of the RA receptor (RAR)β without altering RARα or RARγ. In addition, Nozl1 over-expression increases RA signaling since it stimulates the RA response element. This RA signaling is essential for Nolz1-induced neurogenesis, which is impaired in a RA-free environment or in the presence of a RAR inverse agonist. It has been proposed that Drosophila Gsx2 and Nolz1 homologues could cooperate with the transcriptional co-repressors Groucho-TLE to regulate cell proliferation. In agreement with this view, we show that Nolz1 could act in collaboration with TLE-4, as they are expressed at the same time in NPC cultures and during mouse development. Conclusions: Nolz1 promotes RA signaling in the LGE, contributing to the striatal neurogenesis during development.

Assessment of the levels of degradation in fat co- and by-products for feed uses and their relationships with some lipid composition parameters

This paper discusses the levels of degradation of some co- and byproducts of the food chain intended for feed uses. As the first part of a research project, 'Feeding Fats Safety', financed by the sixth Framework Programme-EC, a total of 123 samples were collected from 10 European countries, corresponding to fat co- and byproducts such as animal fats, fish oils, acid oils from refining, recycled cooking oils, and other. Several composition and degradation parameters (moisture, acid value, diacylglycerols and monoacylglycerols, peroxides, secondary oxidation products, polymers of triacylglycerols, fatty acid composition, tocopherols, and tocotrienols) were evaluated. These findings led to the conclusion that some fat by- and coproducts, such as fish oils, lecithins, and acid oils, show poor, nonstandardized quality and that production processes need to be greatly improved. Conclusions are also put forward about the applicability and utility of each analytical parameter for characterization and quality control.

Jasmonic acid and the THO/TREX complex participate in phosphate starvation response

All plants are typically confronted to simultaneous biotic and abiotic stress throughout their life cycle. Low inorganic phosphate (Pi) is the most common nutrient deficiency limiting plant growth in natural and agricultural ecosystems while insect herbivory accounts for major losses in plant productivity and impacts on ecological and evolutionary changes in plant populations. Here we report that plants experiencing Pi deficiency induce the jasmonic acid (JA) pathway and enhance their defence against insect herbivory. The phol mutant is impaired in the translocation of Pi from roots to shoots and shows the typical symptoms associated with Pi deficiency, including high anthocyanin and poor shoot growth. These phol shoot phenotypes were significantly attenuated by blocking the JA biosynthesis or signalling pathways. Wounded phol leaves hyper-accumulated JA in comparison to wild type, leading to increased resistance against the generalist herbivore Spodoptera littoralis. Pi deficiency also triggered enhanced resistance to herbivory in wild-type Arabidopsis as well as tomato and tobacco, revealing that the link between Pi deficiency and JA-mediated herbivory resistance is conserved in a diversity of plants, including crops. We performed a phol suppressor screen to identify new components involved in the adaptation of plants to Pi deficiency. We report that the THO RNA TRANSCRIPTION AND EXPORT (THO/TREX) complex is a crucial component involved in modulating the Pi- deficiency response. Knockout mutants of at least three members of the THO/TREX complex, including TEX1, HPR1, and TH06, can suppress the phol shoot phenotype. Grafting experiments showed that loss of function of TEX1 only in the root was sufficient to suppress the reduced shoot growth phenotype of phol while maintaining low Pi contents. This indicates that TEX1 is involved in a long distance root-to-shoot signalling component of the Pi-deficiency response. We identified a small MYB-like transcription factor, RAD LIKE 3 (RL3), as a potential downstream target of the THO/TREX complex. RL3 expression is induced in phol mutants but attenuated in phol-7 texl-4 double mutants. Identical to knockout mutants of the THO/TREX complex, rl3 mutants can suppress the phol shoot phenotypes. Interestingly, RL3 is induced during Pi deficiency and is described in the literature as likely being mobile. It is therefore a promising new candidate involved in the root-to-shoot Pi-deficiency signalling response. Finally, we report that PHOl and its homologue PH01:H3 are involved in the co-regulation of Pi and zinc (Zn) homeostasis. PH01;H3 is up-regulated in response to Zn deficiency and, like PHOl, is expressed in the root vascular cylinder and localizes to the Golgi when expressed transiently in tobacco cells. The phol;h3 mutant accumulates more Pi as compared to wild-type when grown in Zn-deficient medium, but this increase is abolished in the phol phol;h3 double mutant. These results suggest that PH01;H3 restricts the PHOl-mediated root-to-shoot Pi transfer in responsé to Zn deficiency. Résumé Au cours de leur cycle de vie, toutes les plantes sont généralement confrontées à divers stress biotiques et abiotiques. La carence nutritionnelle la plus fréquente, limitant la croissance des plantes dans les écosystèmes naturels et agricoles, est la faible teneur en phosphate inorganique (Pi). Au niveau des stress biotiques, les insectes herbivores sont responsables de pertes majeures de rendement et ont un impact considérable sur les changements écologiques et évolutifs dans les populations des plantes. Au cours de ce travail, nous avons mis en évidence que les plantes en situation de carence en Pi induisent la voie de l'acide jasmonique (JA) et augmentent leur défense contre les insectes herbivores. Le mutant phol est déficient dans le transport du phosphate des racines aux feuilles et démontre les symptômes typiques associés à la carence, tels que la forte concentration en anthocyane et une faible croissance foliaire. Ces phénotypes du mutant phol sont significativement atténués lors d'un blocage de la voie de la biosynthèse ou des voies de signalisation du JA. La blessure des feuilles induit une hyper-accumulation de JA chez phol, résultant en une augmentation de la résistance contre l'herbivore généraliste Spodoptera littoralis. Outre Arabidopsis, la carence en Pi induit une résistance accrue aux insectes herbivores aussi chez la tomate et le tabac. Cette découverte révèle que le lien entre la carence en Pi et la résistance aux insectes herbivores via le JA est conservé dans différentes espèces végétales, y compris les plantes de grandes cultures. Nous avons effectué un crible du suppresseur de phol afin d'identifier de nouveaux acteurs impliqués dans l'adaptation de la plante à la carence en Pi. Nous rapportons que le complexe nommé THO RNA TRANSCRIPTION AND EXPORT (THO/TREX) est un élément crucial participant à la réponse des feuilles à la carence en Pi. Les mutations d'au moins trois des membres que composent le complexe THO/TREX, incluant TEX1, HPR1 et 77/06, peuvent supprimer le phénotype de phol. Des expériences de greffes ont montré que la perte de fonction de TEX1, seulement dans la racine, est suffisante pour supprimer le phénotype de la croissance réduite des parties aériennes observé chez le mutant phol, tout en maintenant de faibles teneurs en Pi foliaire. Ceci indique que TEX1 est impliqué dans la signalisation longue distance entre les racines et les parties aériennes. Nous avons identifié un petit facteur de transcription proche de la famille des MYB, RAD LIKE 3 (RL3), comme une cible potentielle en aval du complexe THO / TREX. L'expression du gène RL3 est induite dans le mutant phol mais atténuée dans le double mutant phol-7 texl-4. Exactement comme les plantes mutées d'un des membres du complexe THO/TREX, le mutant rl3 peut supprimer le phénotype foliaire de phol. RL3 est induit au cours d'une carence en Pi et est décrit dans la littérature comme étant potentiellement mobile. Par conséquent, il serait un nouveau candidat potentiellement impliqué dans la réponse longue distance entre les racines et les parties aériennes lors d'un déficit en Pi. Enfin, nous reportons que PHOl et son homologue PHOl: H3 sont impliqués dans la co- régulation de l'homéostasie du Pi et du zinc (Zn). PHOl; H3 est sur-exprimé en réponse au déficit en Zn et, comme PHOl, est exprimé dans les tissus vasculaires des racines et se localise dans l'appareil de Golgi lorsqu'il est exprimé de manière transitoire dans des cellules de tabac. Le mutant phol; h3 accumule plus de Pi par rapport aux plantes sauvages lorsqu'il est cultivé sur un milieu déficient en Zn, mais cette augmentation en Pi est abolie dans le double mutant phol phol; h3. Ces résultats suggèrent qu'en réponse à une carence en Zn, PHOl; H3 limite l'action de PHOl et diminue le transfert du Pi des racines aux parties aériennes.

Consequences of lineage-specific gene loss on functional evolution of surviving paralogs: ALDH1A and retinoic acid signaling in vertebrate genomes

Genome duplications increase genetic diversity and may facilitate the evolution of gene subfunctions. Little attention, however, has focused on the evolutionary impact of lineage-specific gene loss. Here, we show that identifying lineage-specific gene loss after genome duplication is important for understanding the evolution of gene subfunctions in surviving paralogs and for improving functional connectivity among human and model organism genomes. We examine the general principles of gene loss following duplication, coupled with expression analysis of the retinaldehyde dehydrogenase Aldh1a gene family during retinoic acid signaling in eye development as a case study. Humans have three ALDH1A genes, but teleosts have just one or two. We used comparative genomics and conserved syntenies to identify loss of ohnologs (paralogs derived from genome duplication) and to clarify uncertain phylogenies. Analysis showed that Aldh1a1 and Aldh1a2 form a clade that is sister to Aldh1a3-related genes. Genome comparisons showed secondarily loss of aldh1a1 in teleosts, revealing that Aldh1a1 is not a tetrapod innovation and that aldh1a3 was recently lost in medaka, making it the first known vertebrate with a single aldh1a gene. Interestingly, results revealed asymmetric distribution of surviving ohnologs between co-orthologous teleost chromosome segments, suggesting that local genome architecture can influence ohnolog survival. We propose a model that reconstructs the chromosomal history of the Aldh1a family in the ancestral vertebrate genome, coupled with the evolution of gene functions in surviving Aldh1a ohnologs after R1, R2, and R3 genome duplications. Results provide evidence for early subfunctionalization and late subfunction-partitioning and suggest a mechanistic model based on altered regulation leading to heterochronic gene expression to explain the acquisition or modification of subfunctions by surviving ohnologs that preserve unaltered ancestral developmental programs in the face of gene loss.

Estratégias de cultivo para produção dos plásticos biodegradáveis poli(3-Hidroxibutirato) e poli(3-hidroxibutirato-co-3-hidroxivalerato) por bactérias

Polyhydroxyalkanoates (PHAs) are carbon and energy storage materials that are accumulated as intracellular granules in a variety of microorganisms during unbalanced growth. PHAs have drawn attention due to their properties similar to conventional plastics and complete biodegradability. They can be used for food and cosmetics packaging, and in medicine and agriculture. However, their applicability is reduced because of their high production cost compared to conventional plastics. An overview on production strategies of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) aiming at reducing the production costs is presented.