Analysis of Polycomb Epigenetic Marks in HeLa Spheroids.

Biochemical composition of the nucleus affects both its physical properties and its morphology. In recent years, several studies demonstrated the formation of f-actin in the nuclei. These filaments intermingle with the chromatin fibers underlying the crucial role of the mechanical force in chromatin remodeling, being thus involved in transcription, differentiation, replication, and DNA repair. Given the suggested role of Ezh2 in the cross-talk between f-actin and chromatin, we describe here how to obtain HeLa cell spheroids and a method to perform immunofluorescence analysis of nuclear epigenetic marks in a 3D cell culture system.

A Perspective Around Cephalopods and Their Parasites, and Suggestions on How to Increase Knowledge in the Field.

Although interest in several areas of cephalopod research has emerged over the last decades (e.g., neurobiology, aquaculture, genetics, and welfare), especially following their 2010 inclusion in the EU Directive on the use of animals for experimental purposes, knowledge regarding the parasites of cephalopods is lacking. Cephalopods can be intermediate, paratenic, or definitive hosts to a range of parasites with a wide variety of life cycle strategies. Here, we briefly review the current knowledge in cephalopod parasitological research, summarizing the main parasite groups that affect these animals. We also emphasize some topics that, in our view, should be addressed in future research, including: (i) better understanding of life cycles and transmission pathways of common cephalopod parasites; (ii) improve knowledge of all phases of the life cycle (i.e., paralarvae, juveniles, adults and senescent animals) and on species from polar deep sea regions; (iii) exploration of the potential of using cephalopod-parasite specificity to assess population boundaries of both, hosts and parasites; (iv) risk evaluation of the potential of standard aquacultural practices to result in parasite outbreaks; (v) evaluation and description of the physiological and behavioral effects of parasites on their cephalopod hosts; (vi) standardization of the methods for accurate parasite sampling and identification; (vii) implementation of the latest molecular methods to facilitate and enable research in above mentioned areas; (viii) sharing of information and samples among researchers and aquaculturists. In our view, addressing these topics would allow us to better understand complex host-parasite interactions, yield insights into cephalopod life history, and help improve the rearing and welfare of these animals in captivity.

The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants.

Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM) that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules.

Cephalopods in neuroscience: regulations, research and the 3Rs.

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.

Nervous control of reproduction in Octopus vulgaris: a new model.

The classic study of Wells and Wells on the control of reproduction in Octopus demonstrated that the activity of the subpedunculate lobe of the brain and environmental illumination both inhibit the release of an unknown gonadotropin from the optic gland. This inhibitory control may be exerted by the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide). It was later demonstrated that the olfactory lobe is also likely to be involved in the control of optic gland activity. The presence of gonadotropin-releasing hormone in the olfactory lobe suggested that it might exert an excitatory action on optic gland activity. Other neuropeptides have now been localised in the olfactory lobe: neuropeptide Y, galanin, corticotropin-releasing factor, Ala-Pro-Gly-Trp-NH2 (APGWamide), as well as steroidogenic enzymes and an oestrogen receptor orthologue. This supports the hypothesis that this lobe may also play a part in the control of reproduction in Octopus. The olfactory lobe receives distant chemical stimuli and also appears to be an integrative centre containing a variety of neuropeptides involved in controlling the onset of sexual maturation of Octopus, via the optic gland hormone. This review attempts to summarise current knowledge about the role of the olfactory lobe and optic gland in the control of sexual maturation in Octopus, in the light of new findings and in the context of molluscan comparative physiology.

Cephalopod genomics: A plan of strategies and organization.

The Cephalopod Sequencing Consortium (CephSeq Consortium) was established at a NESCent Catalysis Group Meeting, "Paths to Cephalopod Genomics- Strategies, Choices, Organization," held in Durham, North Carolina, USA on May 24-27, 2012. Twenty-eight participants representing nine countries (Austria, Australia, China, Denmark, France, Italy, Japan, Spain and the USA) met to address the pressing need for genome sequencing of cephalopod mollusks. This group, drawn from cephalopod biologists, neuroscientists, developmental and evolutionary biologists, materials scientists, bioinformaticians and researchers active in sequencing, assembling and annotating genomes, agreed on a set of cephalopod species of particular importance for initial sequencing and developed strategies and an organization (CephSeq Consortium) to promote this sequencing. The conclusions and recommendations of this meeting are described in this white paper.

Steroidogenesis in the brain of Sepia officinalis and Octopus vulgaris.

The presence of vertebrate-like steroids, steroidogenic enzymes and steroid receptors has been reported exclusively in cephalopods gonads. The role played by these steroids has been also recently investigated. We here give evidence of steroidogenic activity in the brain of cephalopods. The activity of two key steroidogenic enzymes: 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD is present in the lobes of the nervous system of both Sepia and Octopus. Such enzymes convert pregnenolone to progesterone and androstenedione to testosterone respectively. Binding experiments seem to assign a functional role to the androgens in the brain of cephalopods. According to the present results, the absence of any progesterone binding moiety supports the hypothesis that progesterone may be a metabolite product along the steroidogenic chain leading to androgens. The presence of steroidogenic enzymes in specific lobes of the central nervous system is discussed in terms of the possible role that steroids can play in the sexual differentiation of the brain and in influencing some coded behaviours of cephalopods, such as learning processes.

GABA(A)- and AMPA-like receptors modulate the activity of an identified neuron within the central pattern generator of the pond snail Lymnaea stagnalis.

To examine the neurochemistry underlying the firing of the RPeD1 neuron in the respiratory central pattern generator of the pond snail, Lymnaea stagnalis, we examined electrophysiologically and pharmacologically either "active" or "silent" preparations by intracellular recording and pharmacology. GABA inhibited electrical firing by hyperpolarizing RPeD1, while picrotoxin, an antagonist of GABA(A) receptors, excited silent cells and reversed GABA-induced inhibition. Action potential activity was terminated by 1 mM glutamate (Glu) while silent cells were depolarized by the GluR agonists, AMPA, and NMDA. Kainate exerted a complex triphasic effect on membrane potential. However, only bath application of AMPA desensitized the firing. These data indicate that GABA inhibits RPeD1 via activation of GABA(A) receptors, while Glu stimulates the neuron by activating AMPA-sensitive GluRs.

Control of GnRH expression in the olfactory lobe of Octopus vulgaris.

In the cephalopod mollusk Octopus vulgaris, the gonadotropic hormone released by the optic gland controls sexual maturity. Several lobes of the central nervous system control the activity of this gland. In one of these lobes, the olfactory lobe, a gonadotropin releasing hormone (GnRH) neuronal system has been described. We assume that several inputs converge on the olfactory lobes in order to activate GnRH neurons and that a glutamatergic system mediates the integration of stimuli on these neuropeptidergic neurons. The presence of N-methyl-d-aspartate (NMDA) receptor immunoreactivity in the neuropil of olfactory lobes and in the fibers of the optic gland nerve, along with the GnRH nerve endings strongly supports this hypothesis. A distinctive role in the control of GnRH secretion has also been attributed, in vertebrates, to nitric oxide (NO). The lobes and nerves involved in the nervous control of reproduction in Octopus contain nitric oxide synthase (NOS). Using a set of experiments aimed at manipulate a putative l-glutamate/NMDA/NO signal transduction pathway, we have demonstrated, by quantitative real-time PCR, that NMDA enhances the expression of GnRH mRNA in a dose-response manner. The reverting effect of a selective antagonist of NMDA receptors (NMDARs), 2-amino-5-phosphopentanoic acid (D-APV), confirms that such an enhancing action is a NMDA receptor-mediated response. Nitric oxide and calcium also play a positive role on GnRH mRNA expression. The results suggest that in Octopusl-glutamate could be a key molecule in the nervous control of sexual maturation.

GnRH in the brain and ovary of Sepia officinalis.

We have cloned from brain, ovary and eggs of the cephalopod Sepia officinalis a 269-bp PCR product, which shares 100% sequence identity with the open reading frame of GnRH isoform isolated from Octopus vulgaris. Similar to Octopus, this sequence encodes a peptide that is organized as a preprohormone from which, after enzymatic cleavage, a dodecapeptide is released. Apart from its length, this peptide shares all the common features of vertebrate GnRHs. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses followed by sequencing have confirmed that the same peptide transcript is also present in the ovary, as well as in eggs released in the mantle cavity. The use of an antibody made specifically against the oct-GnRH has revealed that the peptide is localized in the dorso-lateral basal and olfactory lobes, the two neuropeptidergic centers controlling the activity of the gonadotropic optic gland. Immunoreactive nerve endings are also present on the glandular cells of the optic glands. These results confirm the fact that, regardless of the evolutionary distances among animal phyla, GnRH is an ancient peptide present also in invertebrates, and also reinforce the notion that, despite the name "gonadotropin releasing-hormone" was attributed according to its role in vertebrates, probably this family of peptides always had a role in the broad context of animal reproduction. The divergence and spread of several different isoforms of this peptide among animals seem to be balanced, in both invertebrates and vertebrates, by the class-specificity of the GnRH isoform involved in reproductive processes.

Pre- and postsynaptic excitation and inhibition at octopus optic lobe photoreceptor terminals; implications for the function of the 'presynaptic bags'.

Synaptic transmission was examined in the plexiform zone of Octopus vulgaris optic lobes using field-potential recording from optic lobe slices. Stimulation of the optic nerve produced pre- and postsynaptic field potentials. Transmission was abolished in calcium-free seawater, L- glutamate or the AMPA/Kainate receptor blocker CNQX (EC(50), 40 microm), leaving an intact presynaptic field potential. ACh markedly reduced or blocked and d-tubocurarine augmented both pre- and postsynaptic field potentials, while alpha-bungarotoxin and atropine were without effect. Paired-pulse stimulation showed short-term depression of pre- and postsynaptic components with a half-time of recovery of approximately 500 ms. The depression was partially relieved in the presence of d-tubocurarine (half-time of recovery, 350 ms). No long-term changes in synaptic strength were induced by repetitive stimulation. A polyclonal antibody raised against a squid glutamate receptor produced positive staining in the third radial layer of the plexiform zone. No positive staining was observed in the other layers. Taking into account previous morphological data and our results, we propose that the excitatory terminations of the photoreceptors are in the innermost layer of the plexiform zone where the transmitter is likely to be glutamate and postsynaptic receptors are AMPA/kainate-like. Thus, the function of the terminal bags is to provide a location for a presynaptic cholinergic inhibitory shunt. The results imply that this arrangement provides a temporal filter for visual processing and enhances the perception of moving vs. stationary objects.

Nitric oxide synthase expression in the central nervous system of Sepia officinalis: an in situ hybridization study.

We recently reported the molecular cloning of nitric oxide synthase (NOS) mRNA from Sepia officinalis (SoNOS) using a strategy that involves hybridization of degenerate PCR primers to highly conserved NOS regions, combined with a RACE procedure. Here, in situ hybridization study has been performed on serial sections of the cuttlefish central nervous system to reveal localized specific staining of cell bodies in several lobes of the brain. Staining was found in many lower motor centres, including cells of the inferior and superior buccal lobes (feeding centres); in some higher motor centres (anterior basal and peduncle lobes); in learning centres (vertical, subvertical and superior frontal lobes); and in the visual system [medulla and deep retina (optic lobe)]. Positive staining was also found in the olfactory lobe. NOS-expressing cells have been detected also in the interbasal lobe. Double labelling experiments, performed on consecutive sections, showed that neurons containing NOS immunoreactivity were also positive in in situ hybridization staining. All these data support the presence of NOS in several systems in the cuttlefish brain.

Neuropeptidergic control of Octopus oviducal gland.

The oviducal gland of the female of Octopus vulgaris lies about halfway along the oviduct. Progesterone and 17beta-estradiol receptors have been immunolocalized in the nuclei of the cells of the glandular compartment of previtellogenic glands. We also have evidence of FMRFamide-like and cGnRH-I-like immunoreactivity in the nerve endings that reach the oviducal gland. Moreover, we have recently shown APGWamide immunoreactivity in the glandular cells of the inner part of the oviducal gland. Here we report a review on these findings as well as our latest studies on the effect that neuropeptides may exert on the secretory activity of the oviducal gland. cAMP seems to be a possible second messenger involved in such a process. We discuss the findings of a neuropeptidergic action on the glandular cells of oviducal gland in a more complex frame of molecules, such as steroids, biogenic amines and neuromodulators, controlling the activity of the gland.

Tubulin nitration in human gliomas.

Immunohistochemical and biochemical investigations showed that significant protein nitration occurs in human gliomas, especially in grade IV glioblastomas at the level of astrocytes and oligodendrocytes and neurones. Enhanced alpha-tubulin immunoreactivity was co-present in the same elements in the glioblastomas. Proteomic methodologies were employed to identify a nitrated protein band at 55 kDa as alpha-tubulin. Peptide mass fingerprinting procedures demonstrated that tubulin is nitrated at Tyr224 in grade IV tumour samples but is unmodified in grade I samples and in non-cancerous brain tissue. These results provide the first characterisation of endogenously nitrated tubulin from human tumour samples.

Nitric oxide synthase in the nervous system and ink gland of the cuttlefish Sepia officinalis: molecular cloning and expression.

Nitric oxide (NO) signaling is involved in numerous physiological processes in mollusks, e.g., learning and memory, feeding behavior, neural development, and defence response. We report the first molecular cloning of NOS mRNA from a cephalopod, the cuttlefish Sepia officinalis (SoNOS). SoNOS was cloned using a strategy that involves hybridization of degenerate PCR primers to highly conserved NOS regions, combined with RACE procedure. Two splicing variants of SoNOS, differing by 18 nucleotides, were found in the nervous system and the ink gland of Sepia. In situ hybridization shows that SoNOS is expressed in the immature and mature cells of the ink gland and in the regions of the nervous system that are related to the ink defence system.

Calcium currents correlate with oocyte maturation during the reproductive cycle in Octopus vulgaris.

Using the whole-cell voltage clamp technique, we have studied the Ca2+ currents and the steady-state conductance during different oocyte growth stages and during the reproductive cycle of the female of Octopus vulgaris. Evidence is presented that L-type Ca2+ currents are high in small pre-vitellogenic oocytes (80-150 microm diameter) and significantly lower in early vitellogenic oocytes (180-300 microm diameter). Similarly, a significant decrease of the steady-state conductance occurred from the pre to early- vitellogenic oocytes. Octopus oocytes showed larger Ca2+ currents in the reproductive rather than non-reproductive periods. These data indicates that ion and L-type Ca2+ currents play a role in oocyte growth and cytoplasmic maturation, and possibly in preparing the plasma membrane to the interaction with the spermatozoon. By using fluorescent microscopy, we show that oocytes from 80 to 400 microm diameter have the large germinal vesicle characteristic of the immature oocytes. In subsequent stages of growth (up to 1000 microm diameter) the nucleus is no more visible and the metaphase spindle appears. These data demonstrate that Octopus vulgaris oocytes are arrested in the first meiotic prophase up to the early-vitellogenic stage and resume meiosis at this stage up to a second block presumably in metaphase I. We discuss a possible role for progesterone as the hormonal stimulus for the first prophase-metaphase meiotic transition.

The presence of APGWamide in Octopus vulgaris: a possible role in the reproductive behavior.

The concerted action of many neuropeptides has been implicated in the nervous control of specific behaviors in many molluscs. In the present study, the presence of amidated tetrapeptide Ala-Pro-Gly-Trp-NH2 (APGWamide) in those lobes that are involved in the control of reproductive behavior in Octopus vulgaris has been investigated. APGWamide immunoreactivity was mainly confined to the posterior olfactory lobule and in the inferior frontal system. These areas are involved in Octopus in the processing of either chemotactile sense or olfaction. From these lobes, immunoreactive fibers reached other lobes of the central nervous system (CNS) which could be indirectly involved in the reproductive behavior. APGWamide immunoreactivity was also present in the glandular cells of the oviducal gland in the female reproductive system. These results constitute the first detailed immunolocalization of APGWamide in cephalopods and open a new insight into the possible effects that both distant and close chemical stimuli can exert on neuropeptidergic circuitries, which may affect the reproductive behavior of cephalopods.

N-methyl-D-aspartate receptor-like immunoreactivity in the brain of Sepia and Octopus.

Ionotropic glutamate receptors have been subdivided into N-methyl-D-aspartate (NMDA) and AMPA/kainate classes. NMDA receptor subunit 2A and 2B immunoreactivity is shown to be present in specific regions of the central nervous system (CNS) of the cephalopod molluscs Sepia officinalis and Octopus vulgaris. An antibody that recognizes both mammalian NMDAR2A and NMDAR2B subunits equally was used. SDS-PAGE/Western blot analysis performed on membrane proteins revealed an immunoreactive band at 170 kDa for both species. Immunoreactive bands from both Octopus and Sepia brains disappeared when the antibody was preabsorbed with membrane proteins from rat hippocampus or from their own brains. The same antibody was then used for immunohistochemical staining of serial sections of the CNS to reveal localized specific staining of cell bodies and fibers in several lobes of the brain. Staining was found in lower motor centers, in some higher motor centers, in learning centers, and in the optic lobes. Immunopositivity was also found in the areas of brain that control the activity of the optic gland, a gonadotropic endocrine gland. These findings suggest that glutamate, via NMDA receptors, may be involved as a signaling molecule in motor, learning, visual, and olfactory systems in the cephalopod brain.

Role of FMRFamide in the reproduction of Octopus vulgaris: molecular analysis and effect on visual input.

As a part of continuous research on the neurobiology of the cephalopods in general, and the neuroendocrine control of reproduction in Octopus vulgaris in particular, the presence, the molecular analysis and the effect of FMRFamide on the screening-pigment migration in the visual system have been analysed. FMRFamide immunoreactive fibres are present in the outer plexiform layer of the retina as well as in the plexiform zone of the deep retina. These fibres presumably come from optic and olfactory lobes. We isolated an incomplete Octopus FMRFamide cDNA which encodes an amino terminal truncated precursor containing several FMRFamide-related peptides (FaRPs) showing a high degree of identity with the FaRPs encoded in the precursor of Sepia officinalis, except for the presence of an Rpamide related peptide, present only in cnidarians. Finally, stimulation of isolated retina demonstrated that the effect of this tetrapeptide, coupled with dopamine, is the induction of an extreme adaptation of the retina to the light condition. This situation de facto inhibits sexual maturation. Our results on the effect of FMRFamide on the retina confirm the suggested hypothesis that this peptide plays an inhibitory role on the activity of optic gland.

Histone H1-like protein and a testis-specific variant in the reproductive tracts of Octopus vulgaris.

In this study, we have identified a 28-kDa protein resembling the linker H1 in the testis and prostate of the reproductive system of Octopus vulgaris. This protein, OvH1, was partially purified by reverse phase high-pressure liquid chromatography (HPLC) of the perchloric acid extract from testis nuclei. It showed electrophoretic mobility, CD spectrum and amino acid composition highly comparable with those of the mammalian histone. Moreover, it was microheterogeneous, as resulted from prostate and testis HPLC and mass spectrometry analyses. Such analysis showed that in testis there are two H1 subfractions, which do not appear in the prostate. Amino acid composition of the major testis specific variant (OvH1t) showed high similarity with rat testis specific H1t. The histone-like nature of OvH1 was confirmed by its ability to bind DNA as tested both by circular dichroism and protection of the nucleic acid toward deoxyribonuclease I activity. The circular dichroism spectra of Octopus DNA in the absence and presence of increasing amounts of the protein showed a dose-dependent effect, leading to a progressive compactness of the polynucleotide. OvH1/DNA complexes were also resistant to nuclease digestion. The presence of H1 in the testis and prostate of the reproductive system of Octopus is discussed in light of the fact that there is a similarity between its behavior and that of vertebrates.