Development of advanced image analysis techniques for the in situ characterization of multiphase dispersions occurring in bioreactors

Fermentation bioprocesses typically involve two liquid phases (i.e. water and organic compounds) and one gas phase (air), together with suspended solids (i.e. biomass), which are the components to be dispersed. Characterization of multiphase dispersions is required as it determines mass transfer efficiency and bioreactor homogeneity. It is also needed for the appropriate design of contacting equipment, helping in establishing optimum operational conditions. This work describes the development of image analysis based techniques with advantages (in terms of data acquisition and processing), for the characterization of oil drops and bubble diameters in complex simulated fermentation broths. The system consists of fully digital acquisition of in situ images obtained from the inside of a mixing tank using a CCD camera synchronized with a stroboscopic light source, which are processed with a versatile commercial software. To improve the automation of particle recognition and counting, the Hough transform (HT) was used, so bubbles and oil drops were automatically detected and the processing time was reduced by 55% without losing accuracy with respect to a fully manual analysis. The system has been used for the detailed characterization of a number of operational conditions, including oil content, biomass morphology, presence of surfactants (such as proteins) and viscosity of the aqueous phase.     

Beauveria Bassiana Pathogenicity to the Citrus Rust Mite Phyllocoptruta Oleivora

The pathogenicity of Beauveria bassiana to one of the major pests of citrus crops, Phyllocoptruta oleivora, was assessed by inoculating mites with different concentrations of conidia (1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷ and 1×10⁸). Treated mites were kept at controlled conditions (25 ± 0.5°C, 12 h photoperiod and 98% relative humidity) and mite survivorship was evaluated daily. Mortality was found to increase in time and was dependent on the conidia concentration, with values ranging from 24 to 91% for the lowest and highest conidia concentration, respectively. The calculated LC₅₀ on the fifth day was 4.23×10⁶ conidia/ml. Mean lethal time was 3.98, 9.79, 3.09 and 2.74 days for 5×10⁶, 1×10⁷, 5×10⁷ and 1×10⁸ conidia/ml, respectively. Conidia were found to adhere all over the mite body surface, especially at the anal region, where vegetative mycelium was found entering the mite body. We noticed the formation of small crystals inside the mite’s body that were produced during colonization of the body cavity by the fungus. This is the first report of B. bassiana pathogenicity for this species.     

Role of microtubules in estradiol-17beta-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity

Estradiol-17beta-D-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 micromol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 micromol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process.     

Mediterranean Diplodus annularis (Teleostei: Sparidae) and its brain parasite: unforeseen outcome

Patterns of parasite load and aggregation of the bird trematode Cardiocephaloides longicollis in its main intermediate host in the Mediterranean, the annular sea bream, Diplodus annularis, were studied in a large sample collected off Valencia (Spain) and are discussed within the context of the parasite induced host mortality hypothesis. The metacercariae were located within large composite cysts of host origin in the ventricles of the optic lobes of the cerebrum. A weak immunological response was detected in older fish, which was significantly associated with the total parasite load. Although the mean abundance of C. longicollis showed a tendency to increase with host size, the infection levels were generally homogeneous with a noticeable plateauing in the intermediate size classes. The distribution of the metacercariae was aggregated and agreed with the negative binomial distribution. There was a marked decline in parasite aggregation in the largest size-class, suggesting parasite-induced host mortality in the oldest fish possibly due to predation by large non-host fish predators. On the other hand, levelling off of abundance and decrease in heterogeneity of parasite distribution within the intermediate age cohort could indicate that these sizes are being rapidly and/or constantly removed from the host population due to by-catch fishing. The overall high infection levels and the continuous recruitment across age cohorts provides evidence that an enhanced parasite transmission is taking place in the Gulf of Valencia due to increased spatial overlap of the hosts involved in the life cycle. We suggest a human-induced facilitation of the digenean life cycle due to the fact that gulls in the area feed extensively on discards, thus indicating the possibility of an unforeseen effect of fishing practices in a marine littoral system.     

ATP released via gap junction hemichannels from the pigment epithelium regulates neural retinal progenitor proliferation

The retinal pigment epithelium (RPE) plays an essential role in the normal development of the underlying neural retina, but the mechanisms by which this regulation occurs are largely unknown. Ca2+ transients, induced by the neurotransmitter ATP acting on purinergic receptors, both increase proliferation and stimulate DNA synthesis in neural retinal progenitor cells. Here, we show that the RPE regulates proliferation in the underlying neural retina by the release of a soluble factor and identify that factor as ATP. Further, we show that this ATP is released by efflux through gap junction connexin 43 hemichannels, the opening of which is evoked by spontaneous elevations of Ca2+ in trigger cells in the RPE. This release mechanism is localized within the RPE cells to the membranes facing the neural retina, a location ideally positioned to influence neural retinal development. ATP released from RPE hemichannels speeds both cell division and proliferation in the neural retina.     

Bombesin and angiotensin II rapidly stimulate Src phosphorylation at Tyr-418 in fibroblasts and intestinal epithelial cells through a PP2-insensitive pathway

Src is activated in response to a variety of growth factors and hormones that bind G protein-coupled receptors (GPCRs), and its activity is regulated by phosphorylation at key sites, including the autophosphorylation site Tyr-418 and the inhibitory site Tyr-529. To better understand the mechanisms controlling Src activation, we examined Src phosphorylation in Swiss 3T3 fibroblasts stimulated with bombesin and in IEC-18 intestinal epithelial cells stimulated with angiotensin II (Ang II). Phosphorylation at Src Tyr-418, the activation loop site, was rapidly and markedly increased after GPCR agonist addition in both cell types. However, treatment of intact cells with the selective Src family kinase inhibitor PP2, at concentrations which abolished Src-mediated phosphorylation of focal adhesion kinase (FAK) at Tyr-577, unexpectedly led to increased phosphorylation at Src Tyr-418 and diminished phosphorylation at Tyr-529. In Swiss 3T3 cells, PP2 enhanced Tyr-418 phosphorylation after 1 min of bombesin stimulation, while in IEC-18 cells, PP2 increased Ang II-stimulated Tyr-418 phosphorylation at all times tested. These results imply that a distinct, non-Src family kinase may be responsible for phosphorylating Src at Tyr-418 in intact fibroblasts and epithelial cells stimulated by GPCR agonists.     

Expression cloning screening of a unique and full-length set of cDNA clones is an efficient method for identifying genes involved in Xenopus neurogenesis

Functional screens, where a large numbers of cDNA clones are assayed for certain biological activity, are a useful tool in elucidating gene function. In Xenopus, gain of function screens are performed by pool screening, whereby RNA transcribed in vitro from groups of cDNA clones, ranging from thousands to a hundred, are injected into early embryos. Once an activity is detected in a pool, the active clone is identified by sib-selection. Such screens are intrinsically biased towards potent genes, whose RNA is active at low quantities. To improve the sensitivity and efficiency of a gain of function screen we have bioinformatically processed an arrayed and EST sequenced set of 100,000 gastrula and neurula cDNA clones, to create a unique and full-length set of approximately 2500 clones. Reducing the redundancy and excluding truncated clones from the starting clone set reduced the total number of clones to be screened, in turn allowing us to reduce the pool size to just eight clones per pool. We report that the efficiency of screening this clone set is five-fold higher compared to a redundant set derived from the same libraries. We have screened 960 cDNA clones from this set, for genes that are involved in neurogenesis. We describe the overexpression phenotypes of 18 single clones, the majority of which show a previously uncharacterised phenotype and some of which are completely novel. In situ hybridisation analysis shows that a large number of these genes are specifically expressed in neural tissue. These results demonstrate the effectiveness of a unique full-length set of cDNA clones for uncovering players in a developmental pathway.     

ABC-type neutral amino acid permease N-I is required for optimal diazotrophic growth and is repressed in the heterocysts of Anabaena sp. strain PCC 7120

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N2 in differentiated cells called heterocysts. The products of Anabaena open reading frames (ORFs) all1046, all1047, all1284, alr1834 and all2912 were identified as putative elements of a neutral amino acid permease. Anabaena mutants of these ORFs were strongly affected (1-12% of the wild-type activity) in the transport of Pro, Phe, Leu and Gly and also impaired (17-30% of the wild-type activity) in the transport of Ala and Ser. These results identified those ORFs as the nat genes encoding the N-I neutral amino acid permease. According to amino acid sequence homologies, natA (all1046) and natE (all2912) encode ATPases, natC (all1047) and natD (all1284) encode transmembrane proteins, and natB (alr1834) encodes a periplasmic substrate-binding protein of an ABC-type uptake transporter. The natA, natC, natD and natE mutants showed defects in Gln and His uptake that were not observed in the natB mutant suggesting that NatB is not a binding protein for Gln or His. The nat mutants released hydrophobic amino acids to the medium, and amino acid release took place at higher levels in cultures incubated in the absence of combined N than in the presence of nitrate. Alanine was the amino acid released at highest levels, and its release was impaired in a mutant unable to develop heterocysts. The nat mutants were also impaired in diazotrophic growth, with natA, natC, natD and natE mutants showing more severe defects than the natB mutant. Expression of natA and natC, which constitute an operon, natCA, as well as of natB was studied and found to take place in vegetative cells but not in the heterocysts. These results indicate that the N-I permease is necessary for normal growth of Anabaena sp. strain PCC 7120 on N2, and that this permease has a role in the diazotrophic filament specifically in the vegetative cells.     

Synthesis and binding affinity of novel 3-aminoethyl-1-tetralones, potential atypical antipsychotics

A series of 3-aminoethyl-1-tetralones, conformationally constrained higher homologues of haloperidol (standard for typical antipsychotic profile), have been obtained by a four-step route from valerolactone. Their binding affinities at dopamine D(2) and serotonin 5-HT2A and 5-HT2C receptors were determined, showing in some cases an atypical antipsychotic profile.