Molecular characterization and immunohistochemical localization of tilapia piscidin 3 in response to Aeromonas hydrophila infection in Nile tilapia

The antimicrobial activity of tilapia piscidin 3 (TP3) was determined in vitro against a locally isolated Aeromonas hydrophila. A 388 bp fragment was amplified from the TP3 cDNA and sequenced. The coding sequence (CDS) of TP3 was estimated to be 231 bp codes for 76 amino acids long and stop codon. In silico analysis was performed to detect both the signal peptide and the prodomain cleavage sites to follow the amino acids number 22 and 70, respectively. Based on this, a peptide 23 amino acids long with a remarkably high computed antimicrobial probability was synthesized and used in the subsequent experiments. The antimicrobial activity of TP3 was determined with minimum inhibitory concentration (MIC) and minim um bactericidal concentration (MBC) methods. TP3 exhibited relatively weak antimicrobial activities against the tested bacteria. A challenge experiment was then performed in Nile tilapia with low and high doses of A. hydrophila, followed by timely recognition; after 3, 6, 24 h, and 7 days of the specific TP3 gene expression, immunohistochemical localization was also performed. Histopathological examination revealed provoked inflammatory responses and congestion in the same organs of TP3 expression. Immunohistochemical localization showed that A. hydrophila induced tilapia fish to express TP3 after 24 h within the gills, intestine, hepatopancreas, spleen, and posterior kidney. In quantitative real time (RT)‐polymerase chain reaction analysis, the high dose showed higher mRNA expression levels than the low dose, and its expression levels increased in the A. hydrophila‐infected fish. It was therefore concluded that TP3 plays an essential role in fish immunity.     

Évolution du trait de côte à l’Holocène supérieur dans la Sebkha El-Guettiate de Skhira (Golfe de Gabès, Tunisie) à travers sa faune d’ostracodes et de foraminifères

A 122 cm-long core was taken in the El-Guettiate Sebkha of Skhira (southeastern part of Tunisia) in order to investigate the recent palaeoenvironmental evolution of this region. The quantitative and qualitative analysis of ostracod and benthic foraminifera assemblages coupled with a correspondence analysis allows the reconstruction of palaeoenvironmental changes during the Holocene in this area. Four typical associations of ostracods (open marine, coastal marine, lagoonal and estuarine brackish) and two associations of benthic foraminifera (coastal and lagoonal) were distinguished. The onset of restricted lagoonal environments linked to the building-up of sand spit led to the onset of restricted lagoon and brackish environment at cal. 5408 years BP. The Shannon and equitability index of diversity were used to decipher the structural variations of the populations of ostracods and benthic foraminifera along the sampled core. We note a reduction in the Shannon index from the bottom to the top, which indicates a progressive isolation of the biotope. The open lagoonal episode is characterized by high values of diversity. During restricted lagoonal episodes the Shannon and equitability index are reduced. The correspondence analysis reveals an environmental gradient related to the marine influence. It shows an antagonism between the widely opened estuarian lagoonal species and those of restricted lagoon. The less opened estuarian lagoonal taxa occupy an intermediate position. Based on these evidences, microfauna carried out in the El-Guettiate Sebkha allows us to recognize four phases beginning with a widely opened estuarian lagoon (ca. cal. 7460 years PB), followed by a restricted lagoon (ca. cal. 5408 years BP) and finally a brackish lagoon evolving towards the present-day sebkha environment. The opened estuarine lagoon is characterized by high values of species richness and diversity indices.     

Maurocalcine interacts with the cardiac ryanodine receptor without inducing channel modification

We have previously shown that MCa (maurocalcine), a toxin from the venom of the scorpion Maurus palmatus, binds to RyR1 (type 1 ryanodine receptor) and induces strong modifications of its gating behaviour. In the present study, we investigated the ability of MCa to bind to and modify the gating process of cardiac RyR2. By performing pull-down experiments we show that MCa interacts directly with RyR2 with an apparent affinity of 150 nM. By expressing different domains of RyR2 in vitro, we show that MCa binds to two domains of RyR2, which are homologous with those previously identified on RyR1. The effect of MCa binding to RyR2 was then evaluated by three different approaches: (i) [(3)H]ryanodine binding experiments, showing a very weak effect of MCa (up to 1 muM), (ii) Ca(2+) release measurements from cardiac sarcoplasmic reticulum vesicles, showing that MCa up to 1 muM is unable to induce Ca(2+) release, and (iii) single-channel recordings, showing that MCa has no effect on the open probability or on the RyR2 channel conductance level. Long-lasting opening events of RyR2 were observed in the presence of MCa only when the ionic current direction was opposite to the physiological direction, i.e. from the cytoplasmic face of RyR2 to its luminal face. Therefore, despite the conserved MCa binding ability of RyR1 and RyR2, functional studies show that, in contrast with what is observed with RyR1, MCa does not affect the gating properties of RyR2. These results highlight a different role of the MCa-binding domains in the gating process of RyR1 and RyR2.     

Critical amino acid residues of maurocalcine involved in pharmacology, lipid interaction and cell penetration

Maurocalcine (MCa) is a 33-amino acid residue peptide that was initially identified in the Tunisian scorpion Scorpio maurus palmatus. This peptide triggers interest for three main reasons. First, it helps unravelling the mechanistic basis of Ca(2+) mobilization from the sarcoplasmic reticulum because of its sequence homology with a calcium channel domain involved in excitation-contraction coupling. Second, it shows potent pharmacological properties because of its ability to activate the ryanodine receptor. Finally, it is of technological value because of its ability to carry cell-impermeable compounds across the plasma membrane. Herein, we characterized the molecular determinants that underlie the pharmacological and cell-penetrating properties of maurocalcine. We identify several key amino acid residues of the peptide that will help the design of cell-penetrating analogues devoid of pharmacological activity and cell toxicity. Close examination of the determinants underlying cell penetration of maurocalcine reveals that basic amino acid residues are required for an interaction with negatively charged lipids of the plasma membrane. Maurocalcine analogues that penetrate better have also stronger interaction with negatively charged lipids. Conversely, less effective analogues present a diminished ability to interact with these lipids. These findings will also help the design of still more potent cell penetrating analogues of maurocalcine.     

The potential of <Emphasis Type="Italic">Rhizobium</Emphasis> strains for biological control of <Emphasis Type="Italic">Orobanche crenata</Emphasis>

Orobanche crenata Forsk is a chlorophyll lacking holoparasite that subsists on the roots of plants and causes significant damage to the culture of leguminous plants and, in particular, to peas (Pisum sativum L.). Here, we investigated the potential of Rhizobium strains for biological control of Orobanche crenata using a commercial pea cultivar (Douce de province) and different Rhizobium strains. Firstly, benefit of bacterial inoculation on plant growth and efficiency in N-incorporation were demonstrated with four isolates, P.SOM, P.1001, P.Mat.95 and P.1236. After five Rhizobium strains (three efficient: P.SOM, P.1236, P.Mat.95 and two not efficient: P.OM1.92, P.MleTem.92) were investigated for their ability to control Orobanche crenata using pot and Petri dish experiments. Inoculation of peas with two (P.SOM and P.1236) of the five strains induced a significant decrease in O. crenata seed germination and in the number of tubercles on pea roots. Furthermore, other symptoms, including the non-penetration of the germinated seeds into pea roots followed by radicle browning and death of the parasites, were observed in the presence of these inoculated pea plants. The hypothesis that roots secrete toxic compounds related to Rhizobium inoculation is discussed.     

Critical amino acid residues of maurocalcine involved in pharmacology, lipid interaction and cell penetration

Maurocalcine (MCa) is a 33-amino acid residue peptide that was initially identified in the Tunisian scorpion Scorpio maurus palmatus. This peptide triggers interest for three main reasons. First, it helps unravelling the mechanistic basis of Ca²⁺ mobilization from the sarcoplasmic reticulum because of its sequence homology with a calcium channel domain involved in excitation-contraction coupling. Second, it shows potent pharmacological properties because of its ability to activate the ryanodine receptor. Finally, it is of technological value because of its ability to carry cell-impermeable compounds across the plasma membrane. Herein, we characterized the molecular determinants that underlie the pharmacological and cell-penetrating properties of maurocalcine. We identify several key amino acid residues of the peptide that will help the design of cell-penetrating analogues devoid of pharmacological activity and cell toxicity. Close examination of the determinants underlying cell penetration of maurocalcine reveals that basic amino acid residues are required for an interaction with negatively charged lipids of the plasma membrane. Maurocalcine analogues that penetrate better have also stronger interaction with negatively charged lipids. Conversely, less effective analogues present a diminished ability to interact with these lipids. These findings will also help the design of still more potent cell penetrating analogues of maurocalcine.     

Amphetamine stimulates movement through thalamocortical glutamate release

The ventrolateral thalamus (VL) is a primary relay point between the basal ganglia and the primary motor cortex (M1). Using dual probe microdialysis and locomotor behavior monitoring, we investigated the contribution of VL input into M1 during amphetamine (AMPH)‐stimulated monoamine release and hyperlocomotion in rats. Tetrodotoxin (10 μM) perfusion into the VL significantly lowered hyperactivity induced by AMPH (1 mg/kg i.p.). This behavioral response corresponded to reduced cortical glutamate and monoamine release. To determine which glutamate receptors the thalamocortical projections acted upon, we perfused either the α‐amino‐3‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl)propanoic acid (AMPA)/kainate receptor antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo[f]quinoxaline‐2,3‐dione (NBQX) (10 μM) or the N‐methyl‐D‐aspartic acid (NMDA) receptor antagonist (MK‐801) intracortically followed by systemic AMPH. The results show that AMPA/kainate, and to a lesser extent NMDA receptors, mediated the observed effects. As glutamate–monoamine interactions could possibly occur through local or circuit‐based mechanisms, we isolated and perfused M1 tissue ex vivo to determine the extent of local glutamate–dopamine interactions. Taken together, these results demonstrate that AMPH generates hyperlocomotive states via thalamocortical signaling and that cortical AMPA receptors are an important mediator of these effects.

     

Thyreophagus corticalis as a vector of hypovirulence in Cryphonectria parasitica in chestnut stands

The natural spread of hypovirulence in Cryphonectria parasitica (Murr.) Barr. occurs in chestnut (Castanea sativa Mill) stands and orchards in Italy and other European countries, leading to spontaneous recovery of the diseased trees. Little is known about how hypovirulence spreads in chestnut stands but various corticolous mite species frequently detected on chestnut cankers could be one of the many factors playing a role in the spread. Artificial virulent cankers created in inoculation field tests and treated with Thyreophagus corticalis (Acari, Sarcoptiformes, Acaridae) raised on hypovirulent cultures showed similar growth to those treated with mycelia of the hypovirulent strain over 18 months of inoculation. Cultures re-isolated from virulent cankers treated with mites were found to contain hypovirus like those derived from pairings of virulent and hypovirulent strains. Viral dsRNA could be carried externally and/or ingested by mites from the hypovirulent mycelia and then transmitted to the mycelia of virulent strains, causing their conversion. In a laboratory study, all fecal pellets collected from mites reared on hypovirulent and virulent strains grown on semi-selective media gave rise to colonies of C. parasitica with similar morphological characters and virulence to the original cultures. Field inoculation of stump sprouts with the resulting colonies revealed that mite digestive tract passage did not alter the virulence of the studied strains. These results are of interest for the biological control of chestnut blight.     

Pharmacological and Behavioral Characterization of D-473, an Orally Active Triple Reuptake Inhibitor Targeting Dopamine,Serotonin and Norepinephrine Transporters

Major depressive disorder (MDD) is a debilitating disease affecting a wide cross section of people around the world. The current therapy for depression is less than adequate and there is a considerable unmet need for more efficacious treatment. Dopamine has been shown to play a significant role in depression including production of anhedonia which has been one of the untreated symptoms in MDD. It has been hypothesized that drugs acting at all three monoamine transporters including dopamine transporter should provide more efficacious antidepressants activity. This has led to the development of triple reuptake inhibitor D-473 which is a novel pyran based molecule and interacts with all three monoamine transporters. The monoamine uptake inhibition activity in the cloned human transporters expressed in HEK-293 cells (70.4, 9.18 and 39.7 for DAT, SERT and NET, respectively) indicates a serotonin preferring triple reuptake inhibition profile for this drug. The drug D-473 exhibited good brain penetration and produced efficacious activity in rat forced swim test under oral administration. The optimal efficacy dose did not produce any locomotor activation. Microdialysis experiment demonstrated that systemic administration of D-473 elevated extracellular level of the three monoamines DA, 5-HT, and NE efficaciously in the dorsal lateral striatum (DLS) and the medial prefrontal cortex (mPFC) area, indicating in vivo blockade of all three monoamine transporters by D-473. Thus, the current biological data from D-473 indicate potent antidepressant activity of the molecule.     

Image Derived Input Function for [18F]-FEPPA: Application to Quantify Translocator Protein (18 kDa) in the Human Brain

In [¹⁸F]-FEPPA positron emission topography (PET) imaging, automatic blood sampling system (ABSS) is currently the gold standard to obtain the blood time activity curve (TAC) required to extract the input function (IF). Here, we compare the performance of two image-based methods of IF extraction to the ABSS gold standard method for the quantification of translocator protein (TSPO) in the human brain. The IFs were obtained from a direct delineation of the internal carotid signal (CS) and a new concept of independent component analysis (ICA). PET scans were obtained from 18 healthy volunteers. The estimated total distribution volume (V_T) by CS-IF and ICA-IF were compared to the reference V_T obtained by ABSS-IF in the frontal and temporal cortex, cerebellum, striatum and thalamus regions. The V_T values estimated using ICA-IF were more reliable than CS-IF for all brain regions. Specifically, the slope regression in the frontal cortex with ICA-IF was r² = 0.91 (p<0.05), and r² = 0.71 (p<0.05) using CS-IF.