Tumor-derived hypoxic small extracellular vesicles promote endothelial cell migration and tube formation via ALS2/Rab5/ß-catenin signaling.

Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the ß-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of ß-catenin. These events converged in the expression of ß-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.

Ipsilesional Hippocampal GABA Is Elevated and Correlates With Cognitive Impairment and Maladaptive Neurogenesis After Cortical Stroke in Mice.

BACKGROUND: Cognitive dysfunction is a frequent stroke sequela, but its pathogenesis and treatment remain unresolved. Involvement of aberrant hippocampal neurogenesis and maladaptive circuitry remodeling has been proposed, but their mechanisms are unknown. Our aim was to evaluate potential underlying molecular/cellular events implicated. METHODS: Stroke was induced by permanent occlusion of the middle cerebral artery occlusion in 2-month-old C57BL/6 male mice. Hippocampal metabolites/neurotransmitters were analyzed longitudinally by in vivo magnetic resonance spectroscopy. Cognitive function was evaluated with the contextual fear conditioning test. Microglia, astrocytes, neuroblasts, interneurons, γ-aminobutyric acid (GABA), and c-fos were analyzed by immunofluorescence. RESULTS: Approximately 50% of mice exhibited progressive post-middle cerebral artery occlusion cognitive impairment. Notably, immature hippocampal neurons in the impaired group displayed more severe aberrant phenotypes than those from the nonimpaired group. Using magnetic resonance spectroscopy, significant bilateral changes in hippocampal metabolites, such as myo-inositol or N-acetylaspartic acid, were found that correlated, respectively, with numbers of glia and immature neuroblasts in the ischemic group. Importantly, some metabolites were specifically altered in the ipsilateral hippocampus suggesting its involvement in aberrant hippocampal neurogenesis and remodeling processes. Specifically, middle cerebral artery occlusion animals with higher hippocampal GABA levels displayed worse cognitive outcome. Implication of GABA in this setting was supported by the amelioration of ischemia-induced memory deficits and aberrant hippocampal neurogenesis after blocking pharmacologically GABAergic neurotransmission, an intervention which was ineffective when neurogenesis was inhibited. These data suggest that GABA exerts its detrimental effect, at least partly, by affecting morphology and integration of newborn neurons into the hippocampal circuits. CONCLUSIONS: Hippocampal GABAergic neurotransmission could be considered a novel diagnostic and therapeutic target for poststroke cognitive impairment.

The salivary peptide histatin-1 enhances bone repair in vivo.

The salivary peptide histatin-1 was recently described as a novel osteogenic factor that stimulates cell adhesion, migration, and differentiation in bone-lineage cells. Since these cell responses collectively contribute to bone regeneration, we hypothesized that histatin-1 harbors the capacity to enhance bone tissue repair at the preclinical level. By using a model of monocortical bone defect, we explored the effects of histatin-1 in tibial mineralization and organic matrix formation in vivo. To this end, different amounts of histatin-1 were embedded in one-mm3 collagen sponges and then applied to tibial monocortical defects in C57bl/6 mice. After seven days, mice were euthanized, and samples were processed for subsequent analysis. Micro-computed tomography screening showed that histatin-1 increased intraosseous mineralization, and this phenomenon was accompanied by augmented collagen matrix deposition and closure of cortical defect edges, as determined by Hematoxylin-Eosin and Masson's Trichrome staining. Moreover, immunohistochemical analyses showed that histatin-1 increased the expression of the osteogenic marker alkaline phosphatase, which was accompanied by augmented blood vessel formation. Collectively, our findings show that histatin-1 itself promotes bone regeneration in an orthotopic model, proposing this molecule as a therapeutic candidate for use in bone regenerative medicine.

Epigenetic responses of hare barley (Hordeum murinum subsp. leporinum) to climate change: an experimental, trait-based approach.

The impact of reduced rainfall and increased temperatures forecasted by climate change models on plant communities will depend on the capacity of plant species to acclimate and adapt to new environmental conditions. The acclimation process is mainly driven by epigenetic regulation, including structural and chemical modifications on the genome that do not affect the nucleotide sequence. In plants, one of the best-known epigenetic mechanisms is cytosine-methylation. We evaluated the impact of 30% reduced rainfall (hereafter "drought" treatment; D), 3 °C increased air temperature ("warming"; W), and the combination of D and W (WD) on the phenotypic and epigenetic variability of Hordeum murinum subsp. leporinum L., a grass species of high relevance in Mediterranean agroforestry systems. A full factorial experiment was set up in a savannah-like ecosystem located in southwestern Spain. H. murinum exhibited a large phenotypic plasticity in response to climatic conditions. Plants subjected to warmer conditions (i.e., W and WD treatments) flowered earlier, and those subjected to combined stress (WD) showed a higher investment in leaf area per unit of leaf mass (i.e., higher SLA) and produced heavier seeds. Our results also indicated that both the level and patterns of methylation varied substantially with the climatic treatments, with the combination of D and W inducing a clearly different epigenetic response compared to that promoted by D and W separately. The main conclusion achieved in this work suggests a potential role of epigenetic regulation of gene expression for the maintenance of homoeostasis and functional stability under future climate change scenarios.

Wnt/ß-Catenin Signaling in Oral Carcinogenesis.

Oral carcinogenesis is a complex and multifactorial process that involves cumulative genetic and molecular alterations, leading to uncontrolled cell proliferation, impaired DNA repair and defective cell death. At the early stages, the onset of potentially malignant lesions in the oral mucosa, or oral dysplasia, is associated with higher rates of malignant progression towards carcinoma in situ and invasive carcinoma. Efforts have been made to get insights about signaling pathways that are deregulated in oral dysplasia, as these could be translated into novel markers and might represent promising therapeutic targets. In this context, recent evidence underscored the relevance of the Wnt/ß-catenin signaling pathway in oral dysplasia, as this pathway is progressively "switched on" through the different grades of dysplasia (mild, moderate and severe dysplasia), with the consequent nuclear translocation of ß-catenin and expression of target genes associated with the maintenance of representative traits of oral dysplasia, namely cell proliferation and viability. Intriguingly, recent studies provide an unanticipated connection between active ß-catenin signaling and deregulated endosome trafficking in oral dysplasia, highlighting the relevance of endocytic components in oral carcinogenesis. This review summarizes evidence about the role of the Wnt/ß-catenin signaling pathway and the underlying mechanisms that account for its aberrant activation in oral carcinogenesis.

Molecular Features and mRNA Expression of the Receptor for Activated C Kinase 1 from Symbiodinium microadriaticum ssp. microadriaticum During Growth and the Light/Dark cycle.

Two genes of the RACK1 homolog from the photosynthetic dinoflagellate Symbiodinium microadriaticum ssp. microadriaticum (SmicRACK1), termed SmicRACK1A and SmicRACK1B, were found tandemly arrayed and displayed a single synonymous substitution (T/C) encoding threonine. They included two exons of 942 bp each, encoding 313 amino acids with seven WD-40 repeats and two PKC-binding motifs. The protein theoretical mass and pI were 34,200 Da and 5.9, respectively. SmicRACK1 showed maximum identities with RACK1 homologs at the amino acid and nucleotide level, respectively, of 92 and 84% with S. minutum, and phylogenetic analysis revealed clustered related RACK1 sequences from the marine dinoflagellates S. minutum, Heterocapsa triquetra, Karenia brevis, and Alexandrium tamarense. Interestingly, light-dependent regulatory elements were found both within the 282 bp SmicRACK1A promotor sequence, and within an intergenic sequence of 359 nucleotides that separated both genes, which strongly suggest light-related functions. This was further supported by mRNA accumulation analysis, which fluctuated along the light and dark phases of the growth cycle showing maximum specific peaks under either condition. Finally, qRT-PCR analysis revealed differential SmicRACK1 mRNA accumulation with maxima at 6 and 20 d of culture. Our SmicRACK1 characterization suggests roles in active growth and proliferation, as well as light/dark cycle regulation in S. microadriaticum.

Ultrasound-Assisted Extraction Optimization of Phenolic Compounds from Citrus latifolia Waste for Chitosan Bioactive Nanoparticles Development.

Bioactive Phenols-loaded chitosan nanoparticles (PL-CNps) were developed by ionic gelation from Persian lemon (Citrus latifolia) waste (PLW) and chitosan nanoparticles. Response Surface Methodology (RSM) was used to determine the optimal Ultrasound-Assisted Extraction (UAE) conditions for the total phenolic compounds (TPC) recovery from PLW (58.13 mg GAE/g dw), evaluating the ethanol concentration, extraction time, amplitude, and solid/liquid ratio. Eight compounds expressed as mg/g dry weight (dw) were identified by ultra-performance liquid chromatography coupled photo diode array (UPLC-PDA) analysis: eriocitrin (20.71 ± 0.09), diosmin (18.59 ± 0.13), hesperidin (7.30 ± 0.04), sinapic acid (3.67 ± 0.04), catechin (2.92 ± 0.05), coumaric acid (2.86 ± 0.01), neohesperidin (1.63 ± 0.00), and naringenin (0.44 ± 0.00). The PL-CNps presented size of 232.7 nm, polydispersity index of 0.182, Z potential of -3.8 mV, and encapsulation efficiency of 81.16%. The results indicated that a synergic effect between phenolic compounds from PLW and chitosan nanoparticles was observed in antioxidant and antibacterial activity, according to Limpel's equation. Such results indicate that PLW in such bioprocesses shows excellent potential as substrates for the production of value-added compounds with a special application for the food industry.

Inhibition of PORCN Blocks Wnt Signaling to Attenuate Progression of Oral Carcinogenesis.

PURPOSE: Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/ß-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis. EXPERIMENTAL DESIGN: We performed a model of tobacco-induced oral cancer in vitro, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an in vivo model of oral carcinogenesis and ex vivo samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59. RESULTS: Both in vitro and ex vivo oral carcinogenesis approaches revealed decreased levels of nuclear ß-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 in vitro resulted in diminished cell migration, viability, and invasion. Finally, by using an in vivo model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions. CONCLUSIONS: The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.

The PsbO homolog from Symbiodinium kawagutii (Dinophyceae) characterized using biochemical and molecular methods.

A photosystem II component, the PsbO protein is essential for maximum rates of oxygen production during photosynthesis, and has been extensively characterized in plants and cyanobacteria but not in symbiotic dinoflagellates. Its close interaction with D1 protein has important environmental implications since D1 has been identified as the primary site of damage in endosymbiotic dinoflagellates after thermal stress. We identified and biochemically characterized the PsbO homolog from Symbiodinium kawagutii as a 28-kDa protein, and immunolocalized it to chloroplast membranes. Chloroplast association was further confirmed by western blot on photosynthetic membrane preparations. TX-114 phase partitioning, chromatography, and SDS-PAGE for single band separation and partial peptide sequencing yielded peptides identical or with high identity to PsbO from dinoflagellates. Analysis of a cDNA library revealed three genes differing by only one aminoacid residue in the in silico-translated ORFs despite greater differences at nucleotide level in the untranslated, putative regulatory sequences. The consensus full amino acid sequence displayed all the characteristic domains and features of PsbO from other sources, but changes in functionally critical, highly conserved motifs were detected. Our biochemical, molecular, and immunolocalization data led to the conclusion that the 28-kDa protein from S. kawagutii is the PsbO homolog, thereby named SkPsbO. We discuss the implications of critical amino acid substitutions for a putative regulatory role of this protein.

Biochemical and molecular characterization of the SBiP1 chaperone from Symbiodinium microadriaticum CassKB8 and light parameters that modulate its phosphorylation.

The coding and promoter region sequences from the BiP-like protein SBiP1 from Symbiodinium microadriaticum CassKB8 were obtained by PCR, sequenced and compared with annotated sequences. The nucleotides corresponding to the full sequence were correctly annotated and the main SBiP1 features determined at the nucleotide and amino acid level. The translated protein was organized into the typical domains of the BiP/HSP70 family including a signal peptide, a substrate- and a nucleotide-binding domain, and an ER localization sequence. Conserved motifs included a highly conserved Thr513 phosphorylation site and two ADP-ribosylation sites from eukaryotic BiP's. Molecular modeling showed the corresponding domain regions and main exposed post-translational target sites in its three-dimensional structure, which also closely matched Homo sapiens BiP further indicating that it indeed corresponds to a BiP/HSP70 family protein. The gene promoter region showed at least eight light regulation-related sequences consistent with the molecule being highly phosphorylated in Thr under dark conditions and dephosphorylated upon light stimuli. We tested light parameter variations that could modulate the light mediated phosphorylation effect and found that SBiP1 Thr dephosphorylation was only significantly detected after 15-30 min light stimulation. Such light-induced dephosphorylation was observed even when dichlorophenyl dimethyl urea, a photosynthesis inhibitor, was also present in the cells during the light stimulation. Dephosphorylation occurred indistinctly under red, yellow, blue or the full visible light spectra. In additon, it was observed at a light intensity of as low as 1 µmole photon m-2 s-1. Our results indicate that: a) SBiP1 is a chaperone belonging to the BiP/HSP70 family proteins; b) its light-modulated phosphorylation/dephosphorylation most likely functions as an activity switch for the chaperone; c) this light-induced modulation occurs relatively slow but is highly sensitive to the full spectrum of visible light; and d) the light induced Thr dephosphorylation is independent of photosynthetic activity in these cells.

Biomarkers in Oral Fluids as Diagnostic Tool for Psoriasis.

Psoriasis is a prevalent worldwide chronic immuno-inflammatory skin disease with various variants and atypical cases. The use of biomarkers for the diagnosis of psoriasis can favor timely treatment and thus improve the quality of life of those affected. In general, the search for biomarkers in oral fluids is recommended as it is a non-invasive and fast technique. This narrative review aimed to identify biomarkers in gingival crevicular fluid (GCF) and saliva to diagnose psoriasis. To achieve this goal, we selected the available literature using the following MESH terms: "psoriasis", "saliva" and "gingival crevicular fluid". The studies analyzed for this review cover original research articles available in English. We found three full articles available for psoriasis biomarkers in GCF and ten articles available for psoriasis biomarkers in saliva. Studies showed that in the saliva of healthy individuals and those with psoriasis, there were differences in the levels of inflammatory cytokines, immunoglobulin A, and antioxidant biomarkers. In GCF, individuals with psoriasis showed higher levels of S100A8, IL-18 and sE-selectin in comparison to healthy individuals, independent of periodontal status. Despite these findings, more studies are required to determine an adequate panel of biomarkers to use in saliva or GCF for psoriasis.

Light-stimulated dephosphorylation of the BiP-like protein, SmicHSP75 (SBiP1) from Symbiodinium microadriaticum is inhibited by elevated but not low temperature and suggests regulation of the chaperone function.

Specific phosphorylation/dephosphorylation processes are fundamental for the transduction of external stimuli into physiological responses. A few of these processes appear to be modulated by light in cultured Symbiodinium microadriaticum since the BiP-like protein SmicHSP75 undergoes threonine dephosphorylation upon light stimuli. Several isoforms of the protein are encoded in the S. microadriaticum genome and thus, we identified and heterologously expressed a specific sequence corresponding to the previously identified SmicHSP75 isoform to obtain a highly specific antibody. We then determined by western blot analysis, that the detected light-stimulated changes in SmicHSP75 threonine phosphorylation were not due to changes in the protein expression and explored further the effect of lower than normal and higher stressful temperature, on the phosphorylation levels of the protein. Upon long-term (12 h) exposure of the cells to the low temperature of 21ºC under darkness, the protein was found significantly phosphorylated; however, light exposure for 30 min caused a dephosphorylation effect like the 26ºC control treatment. On the other hand, in cells exposed to 32ºC for 12 h under darkness, the highly Thr-phosphorylated SmicHSP75 was converted to a low-level phosphorylated protein. Likewise, short term (30 min) exposure to 32ºC under dark conditions caused dephosphorylation of the protein, similar to what was observed upon long-term exposure to 32ºC and upon light stimulation of cells under the normal temperature of 26ºC. These data suggested activation/inactivation of the chaperone function of SmicHSP75 by regulation of its Thr phosphorylation levels under heat stress conditions in Symbiodinium microadriaticum, independent of changes in protein expression.

PvRACK1 loss-of-function impairs cell expansion and morphogenesis in Phaseolus vulgaris L. root nodules.

Receptor for activated C kinase (RACK1) is a highly conserved, eukaryotic protein of the WD-40 repeat family. Its peculiar ß-propeller structure allows its interaction with multiple proteins in various plant signal-transduction pathways, including those arising from hormone responses, development, and environmental stress. During Phaseolus vulgaris root development, RACK1 (PvRACK1) mRNA expression was induced by auxins, abscissic acid, cytokinin, and gibberellic acid. In addition, during P. vulgaris nodule development, PvRACK1 mRNA was highly accumulated at 12 to 15 days postinoculation, suggesting an important role after nodule meristem initiation and Rhizobium nodule infection. PvRACK1 transcript accumulation was downregulated by a specific RNA interference construct which was expressed in transgenic roots of composite plants of P. vulgaris inoculated with Rhizobium tropici. PvRACK1 downregulated transcript levels were monitored by quantitative reverse-transcription polymerase chain reaction analysis in individual transgenic roots and nodules. We observed a clear phenotype in PvRACK1-knockdown nodules, in which nodule number and nodule cell expansion were impaired, resulting in altered nodule size. Microscopic analysis indicated that, in PvRACK1-knockdown nodules, infected and uninfected cells were considerably smaller (80 and 60%, respectively) than in control nodules. In addition, noninfected cells and symbiosomes in silenced nodules showed significant defects in membrane structure under electron microscopy analysis. These findings indicate that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development.

Screening a Spliced Leader-Based Symbiodinium microadriaticum cDNA Library Using the Yeast-Two Hybrid System Reveals a Hemerythrin-Like Protein as a Putative SmicRACK1 Ligand.

The dinoflagellate Symbiodiniaceae family plays a central role in the health of the coral reef ecosystem via the symbiosis that establishes with its inhabiting cnidarians and supports the host metabolism. In the last few decades, coral reefs have been threatened by pollution and rising temperatures which have led to coral loss. These events have raised interest in studying Symbiodiniaceae and their hosts; however, progress in understanding their metabolism, signal transduction pathways, and physiology in general, has been slow because dinoflagellates present peculiar characteristics. We took advantage of one of these peculiarities; namely, the post-transcriptional addition of a Dino Spliced Leader (Dino-SL) to the 5' end of the nuclear mRNAs, and used it to generate cDNA libraries from Symbiodinium microadriaticum. We compared sequences from two Yeast-Two Hybrid System cDNA Libraries, one based on the Dino-SL sequence, and the other based on the SMART technology (Switching Mechanism at 5' end of RNA Transcript) which exploits the template switching function of the reverse transcriptase. Upon comparison of the performance of both libraries, we obtained a significantly higher yield, number and length of sequences, number of transcripts, and better 5' representation from the Dino-SL based library than from the SMART library. In addition, we confirmed that the cDNAs from the Dino-SL library were adequately expressed in the yeast cells used for the Yeast-Two Hybrid System which resulted in successful screening for putative SmicRACK1 ligands, which yielded a putative hemerythrin-like protein.

Post-stroke Neurogenesis: Friend or Foe?

The substantial clinical burden and disability after stroke injury urges the need to explore therapeutic solutions. Recent compelling evidence supports that neurogenesis persists in the adult mammalian brain and is amenable to regulation in both physiological and pathological situations. Its ability to generate new neurons implies a potential to contribute to recovery after brain injury. However, post-stroke neurogenic response may have different functional consequences. On the one hand, the capacity of newborn neurons to replenish the damaged tissue may be limited. In addition, aberrant forms of neurogenesis have been identified in several insult settings. All these data suggest that adult neurogenesis is at a crossroads between the physiological and the pathological regulation of the neurological function in the injured central nervous system (CNS). Given the complexity of the CNS together with its interaction with the periphery, we ultimately lack in-depth understanding of the key cell types, cell-cell interactions, and molecular pathways involved in the neurogenic response after brain damage and their positive or otherwise deleterious impact. Here we will review the evidence on the stroke-induced neurogenic response and on its potential repercussions on functional outcome. First, we will briefly describe subventricular zone (SVZ) neurogenesis after stroke beside the main evidence supporting its positive role on functional restoration after stroke. Then, we will focus on hippocampal subgranular zone (SGZ) neurogenesis due to the relevance of hippocampus in cognitive functions; we will outline compelling evidence that supports that, after stroke, SGZ neurogenesis may adopt a maladaptive plasticity response further contributing to the development of post-stroke cognitive impairment and dementia. Finally, we will discuss the therapeutic potential of specific steps in the neurogenic cascade that might ameliorate brain malfunctioning and the development of post-stroke cognitive impairment in the chronic phase.

Germination behavior, biochemical features and sequence analysis of the RACK1/arcA homolog from Phaseolus vulgaris.

Partial peptide sequence of a 36 kDa protein from common bean embryo axes showed 100% identity with a reported beta-subunit of a heterotrimeric G protein from soybean. Analysis of the full sequence showed 96.6% identity with the reported soybean G(beta)-subunit, 86% with RACK1B and C from Arabidopsis and 66% with human and mouse RACK1, at the amino acid level. In addition, it showed 85.5, 85 and 83% identities with arcA from Solanum lycopersicum, Arabidopsis (RACK1A) and Nicotiana tabacum, respectively. The amino acid sequence displayed seven WD40 domains and two sites for activated protein kinase C binding. The protein showed a constant expression level but the mRNA had a maximum at 32 h post-imbibition. Western immunoblotting showed the protein in vegetative plant tissues, and in both microsomal and soluble fractions from embryo axes. Synthetic auxin treatment during germination delayed the peak of RACK1 mRNA expression to 48 h but did not affect the protein expression level while the polar auxin transport inhibitor, naphtylphtalamic acid had no effect on either mRNA or protein expression levels. Southern blot and genomic DNA amplification revealed a small gene family with at least one member without introns in the genome. Thus, the RACK1/arcA homolog from common bean has the following features: (1) it is highly conserved; (2) it is both soluble and insoluble within the embryo axis; (3) it is encoded by a small gene family; (4) its mRNA has a peak of expression at the time point of germination stop and (5) its expression is only slightly affected by auxin but unaffected by an auxin transport blocker.

Phosphorylation/dephosphorylation response to light stimuli of Symbiodinium proteins: specific light-induced dephosphorylation of an HSP-like 75 kDa protein from S. microadriaticum.

BACKGROUND: Some genera of the family Symbiodiniaceae establish mutualistic endosymbioses with various marine invertebrates, with coral being the most important ecologically. Little is known about the biochemical communication of this association and the perception and translation of signals from the environment in the symbiont. However, specific phosphorylation/dephosphorylation processes are fundamental for the transmission of external signals to activate physiological responses. In this work, we searched phosphorylatable proteins in amino acids of Ser, Thr and Tyr from three species of the family Symbiodiniaceae, Symbiodinium kawagutii, Symbiodinium sp. Mf11 and Symbiodinium microadriaticum. METHODS: We used specific antibodies to the phosphorylated aminoacids pSer, pThr and pTyr to identify proteins harboring them in total extracts from three species of Symbiodinium in culture. Extractions were carried out on logarithmic phase growing cultures under a 12 h light/dark photoperiod. Various light/dark, nutritional and other stimuli were applied to the cultures prior to the extractions, and proteins were subjected to SDS-PAGE and western immunoblotting. Partial peptide sequencing was carried out by MALDI-TOF on specific protein spots separated by 2D electrophoresis. RESULTS: At 4 h of the light cycle, several Thr-phosphorylated proteins were consistently detected in the three species suggesting a genus-dependent expression; however, most Ser- and Tyr-phosphorylated proteins were species-specific. Analysis of protein extracts of S. microadriaticum cultures demonstrated that the level of phosphorylation of two Thr-phosphorylated proteins with molecular weights of 43 and 75 kDa, responded inversely to a light stimulus. The 43 kDa protein, originally weakly Thr-phosphorylated when the cells were previously adapted to their 12 h dark cycle, underwent an increase in Thr phosphorylation when stimulated for 30 min with light. On the other hand, the 75 kDa protein, which was significantly Thr-phosphorylated in the dark, underwent dephosphorylation in Thr after 30 min of the light stimulus. The phosphorylation response of the 43 kDa protein only occurred in S. microadriaticum, whereas the dephosphorylation of the 75 kDa protein occurred in the three species studied suggesting a general response. The 75 kDa protein was separated on 2D gels as two isoforms and the sequenced spots corresponded to a BiP-like protein of the HSP70 protein family. The presence of differential phosphorylations on these proteins after a light stimulus imply important light-regulated physiological processes in these organisms.

Genetic transformation of cell-walled plant and algae cells: delivering DNA through the cell wall.

Transformation techniques are a fundamental tool for functional genomics studies. These techniques are routinely used in many prokaryotic and eukaryotic organisms, but in eukaryotes that are surrounded by a cell wall, these protocols have proven difficult to successfully deliver heterologous or homologous DNA within their cytoplasm and nucleus. Such cell-walled organisms represent a challenge that requires the development of genetic transformation techniques that are able to overcome their natural barrier, to achieve targeted gene expression. Here, we review the techniques that have been proven successful and applied to these cell-walled eukaryotic organisms. We focus, especially, on plant cells, microalgae, and the latest approaches to mediate DNA uptake by the photosynthetic dinoflagellate Symbiodinium.

Indomethacin reproducibly induces metamorphosis in Cassiopea xamachana scyphistomae.

Cassiopea xamachana jellyfish are an attractive model system to study metamorphosis and/or cnidarian-dinoflagellate symbiosis due to the ease of cultivation of their planula larvae and scyphistomae through their asexual cycle, in which the latter can bud new larvae and continue the cycle without differentiation into ephyrae. Then, a subsequent induction of metamorphosis and full differentiation into ephyrae is believed to occur when the symbionts are acquired by the scyphistomae. Although strobilation induction and differentiation into ephyrae can be accomplished in various ways, a controlled, reproducible metamorphosis induction has not been reported. Such controlled metamorphosis induction is necessary for an ensured synchronicity and reproducibility of biological, biochemical, and molecular analyses. For this purpose, we tested if differentiation could be pharmacologically stimulated as in Aurelia aurita, by the metamorphic inducers thyroxine, KI, NaI, Lugol's iodine, H2O2, indomethacin, or retinol. We found reproducibly induced strobilation by 50 µM indomethacin after six days of exposure, and 10-25 µM after 7 days. Strobilation under optimal conditions reached 80-100% with subsequent ephyrae release after exposure. Thyroxine yielded inconsistent results as it caused strobilation occasionally, while all other chemicals had no effect. Thus, indomethacin can be used as a convenient tool for assessment of biological phenomena through a controlled metamorphic process in C. xamachana scyphistomae.

Using phage display technology to obtain Crybodies active against non-target insects.

The insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) are increasingly important in the biological control of insect pests and vectors of human disease. Markets for Bt products and transgenic plants expressing their toxins are driven by their specificity, safety and the move away from chemical control agents. However, the high specificity of Cry toxins can also prove to be a limitation when there is no known Cry toxin active against a particular target. Novel activities can be discovered by screening natural Bt isolates or through modifications of the Cry proteins. Here we demonstrate the use of λ-phage displaying Cry1Aa13 toxin variants modified in domain II loop 2 (Crybodies) to select retargeted toxins. Through biopanning using gut tissue from larvae of the non-target insect Aedes aegypti, we isolated a number of phage for further testing. Two of the overexpressed Cry toxin variants showed significant activity against A. aegypti larvae while another induced mortality at the pupal stage. We present the first report of the use of phage display to identify novel activities toward insects from distant taxonomic Orders and establish this technology based on the use of Crybodies as a powerful tool for developing tailor-made insecticides against new target insects.