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Introduction: the body plan and behaviors of Hydra
Ultrastructure of the Hydra nervous system
Neurotransmitters in Hydra – small molecules and peptides
Ion channel receptors for small molecule transmitters in Hydra and their role in neuromuscular transmission
Cloning of a peptide-gated ion channel from Hydra magnipapillata
Possible function of ionotropic RFamide receptors in Hydra
Relatives of ionotropic RFamide receptors in Bilateria
Peptides and relatives of ionotropic RFamide receptors in other animal taxa
Peptide-gated ion channels and the simple nervous system of Hydra
Stefan Gründer,
Stefan Gründer *
Institute of Physiology, RWTH Aachen University
*Author for correspondence (sgruender@ukaachen.de)
Marc Assmann
S.G. drafted the manuscript and M.A. did the evolutionary analysis and helped draft the manuscript. Both authors gave final approval for publication.
The authors declare that they have no competing or financial interests
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Stefan Gründer, Marc Assmann, Peter A. V. Anderson; Peptide-gated ion channels and the simple nervous system of Hydra. J Exp Biol 15 February 2015; 218 (4): 551–561. doi: https://doi.org/10.1242/jeb.111666
Neurons either use electrical or chemical synapses to communicate with each other. Transmitters at chemical synapses are either small molecules or neuropeptides. After binding to their receptors, transmitters elicit postsynaptic potentials, which can either be fast and transient or slow and longer lasting, depending on the type of receptor. Fast transient potentials are mediated by ionotropic receptors and slow long-lasting potentials by metabotropic receptors. Transmitters and receptors are well studied for animals with a complex nervous system such as vertebrates and insects, but much less is known for animals with a simple nervous system like Cnidaria. As cnidarians arose early in animal evolution, nervous systems might have first evolved within this group and the study of neurotransmission in cnidarians might reveal an ancient mechanism of neuronal communication. The simple nervous system of the cnidarian Hydra extensively uses neuropeptides and, recently, we cloned and functionally characterized an ion channel that is directly activated by neuropeptides of the Hydra nervous system. These results demonstrate the existence of peptide-gated ion channels in Hydra, suggesting they mediate fast transmission in its nervous system. As related channels are also present in the genomes of the cnidarian Nematostella, of placozoans and of ctenophores, it should be considered that the early nervous systems of cnidarians and ctenophores have co-opted neuropeptides for fast transmission at chemical synapses.
ASIC, Degenerin, ENaC, Evolution, Ligand-gated ion channel, Nervous system, Neuropeptide
The Hydra polyp lives in fresh water and has a hollow tube-like shape with a three-layered body wall that surrounds the central gastrovascular cavity (Fig. 1). The gastrovascular cavity has a single opening at the apical end of the animal, serving as mouth and anus. The body column attaches to the substratum via the basal disk, and at its apical perioral region it extends its tentacles that harbor nematocytes for capturing prey. The body wall consists of two epithelial sheets, an ectodermal and an endodermal layer, that enclose the mesoglea, which is an acellular gel-like substance (Fig. 1B). Although Hydra does not have proper tissues, epithelial cells differentiate into specialized cell types. The most common epithelial cell type is the epitheliomuscular cell that has flat basal extensions that contain myofibrils and are contractile. Myofibrils of the ecto- (or epi-) dermal layer are longitudinally organized, whereas those of the endo- (or gastro-) dermal layer are circularly organized (Haynes et al., 1968).
Interstitial cells (I-cells) at the base of ecto- and endodermal layers give rise to nerve cells (David and Gierer, 1974) that remain at the base of the epitheliomuscular cells (Burnett and Diehl, 1964; Davis et al., 1968). Nerve cells are concentrated at the bases of the tentacle, the hypostome, the basal disk and the peduncle (the region just above the basal disk), whereas they are sparse in the body column (Burnett and Diehl, 1964; Davis et al., 1968; Westfall, 1973). Nerve cells form at least two nerve nets, an epidermal and a gastrodermal net (Davis, 1972), that might communicate with each other. The epidermal net contains more nerve cells than the gastrodermal net (Davis, 1972; Westfall, 1973). Some Hydra species have a nerve ring at the base of the tentacles (Grimmelikhuijzen, 1985; Koizumi et al., 1992). Nerve cells make synaptic contacts to each other, to nematocytes and to epitheliomuscular cells in the epi- and gastrodermis (Westfall et al., 1971; Westfall, 1973), suggesting that they coordinate movements.
Hydra has several characteristic movements. It can elongate or contract the body column to change shape or to mix fluid in the gastrovascular cavity. Contraction of the body column when the mouth is open will expel fluid from the cavity. Moreover, Hydra has several characteristic methods of locomotion (Burnett and Diehl, 1964). For example, it can bend over, transiently attach the tentacles to the substratum, detach its base and pull it forward by a contraction of its longitudinal muscles to a new contact site. Finally, Hydra has a complex feeding reaction (Burnett and Diehl, 1964). Upon contact with prey, the polyps bend their tentacles into a loop and move the prey towards the mouth opening for ingestion. The feeding reaction can be initiated simply by adding reduced glutathione (GSH) to Hydra polyps (Loomis, 1955). When conditions are favorable, Hydra reproduces asexually by budding.
A detailed electron microscopic study of Hydra littoralis revealed the presence of only two types of neurons: a peripheral sensory cell in the epithelial layer and a single type of central neuron that has features of sensory, motor and interneuronal cells of higher animals (Westfall, 1973). This sensory-motor interneuron (SMI) is probably multifunctional and may represent a primitive nerve cell from which specialized neurons in more complex nervous systems have been derived (Westfall, 1973). It localizes to the base of epitheliomuscular cells, has two or more neurites, and makes synapses at various sites en passant along a neurite (Westfall, 1973). Occasionally, neurites traverse the mesoglea (Westfall, 1973), suggesting a connection between epidermal and gastrodermal nerve cells. Interneuronal synapses are either symmetrical or asymmetrical; in symmetrical synapses, one side of the junction has clear vesicles and the other side has dense-cored vesicles (Westfall, 1973). Asymmetrical interneural, as well as neuromuscular, junctions generally have dense-cored vesicles and only occasionally have clear vesicles (Fig. 2) (Westfall et al., 1971; Westfall, 1973). These clear vesicles might represent dense-cored vesicles that have lost their cores during neurotransmission (Westfall, 1996). A neuron may synapse more than once with the same epitheliomuscular cell or with two different
acetylcholine esterase
acid-sensing ion channel
bile-acid-sensitive ion channel
BLINaC
brain liver intestine Na+ channel
CaCC
Ca2+-activated Cl− channel
degenerin
epithelial Na+ channel
FaNaC
FMRFamide-gated Na+ channel
FMRFamide-like peptide
HyNaC
Hydra Na+ channel
iRFa-R
ionotropic RFamide receptor
nicotinic acetylcholine receptor
proneuropeptide
epitheliomuscular cells (Westfall, 1973). In contrast to neuromuscular junctions, synapses with nematocytes have clear vesicles (Westfall et al., 1971; Westfall, 1973).
Several subtle differences between the synapses of Hydra and those of vertebrates have been noted (Westfall et al., 1971): (1) the presence of only a few vesicles at synaptic contacts; (2) the large size of the vesicles (120–200 nm in diameter) (Fig. 2); and (3) the occurrence of en passant synapses along neurites. Moreover, synaptic vesicles of Hydra usually have dense cores (Fig. 2) (Westfall et al., 1971) and the dense-cored vesicles appear to arise from the Golgi apparatus (Westfall, 1973), suggesting they contain neuropeptides like those in higher animals. In addition, the neuromuscular junction of Hydra lacks postsynaptic folds that are characteristic of the neuromuscular junction in higher animals (Fig. 2) (Westfall, 1996).
In summary, ultrastructural evidence suggests that neurons of Hydra stimulate muscle contraction via chemical synapses using large dense-cored vesicles (Fig. 2) (Westfall et al., 1971; Westfall, 1973), presumably containing neuropeptides. The contraction of entire muscle sheets may be coordinated by electrical synapses between epitheliomuscular cells (Westfall, 1973). Gap junctions at the bases of epitheliomuscular cells have indeed been detected by electron microscopy (Hand and Gobel, 1972; Westfall, 1973) and expression of innexin 1 at the basal membrane of epithelial cells been described (Alexopoulos et al., 2004). Innexin 2, by contrast, electrically couples nerve cells in the lower body column with each other (Takaku et al., 2014).
Small molecule transmitters, including catecholamines, serotonin, acetylcholine, glutamate and γ-aminobutyric acid, are common in the nervous system of higher animals. Similarly, in Hydra, there are also ample immunohistochemical, biochemical and functional data to indicate their presence (Kass-Simon and Pierobon, 2007; Pierobon, 2012) and two putative metabotropic acetylcholine receptors (mAchRs) have been identified (Collin et al., 2013). Ionotropic receptors for small-molecule transmitters have, however, not been studied at the molecular level in Hydra (see below); we will therefore concentrate our discussion of neurotransmitters on neuropeptides.
Evidence for the presence of neuropeptides in Hydra emerged more than 30 years ago. Antibodies raised against cholecystokinin, substance P, neurotensin and bombesin labeled different populations of nerve cells in Hydra (Grimmelikhuijzen et al., 1980; Grimmelikhuijzen et al., 1981c; Grimmelikhuijzen et al., 1981b; Grimmelikhuijzen et al., 1981a; Grimmelikhuijzen et al., 1996), suggesting that Hydra neurons contain related peptides. Similarly, an antibody raised against the neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) labeled nerve cells in the ectoderm of the hypostome, of the lower peduncle and of the tentacles (Grimmelikhuijzen et al., 1982). FMRFamide was first isolated from clam ganglia and has excitatory actions on heart of mollusks (Price and Greenberg, 1977). FMRFamide-like peptides (FLPs) are conserved from invertebrates to vertebrates (Jékely, 2013). Radioimmunoassays revealed that the FLPs of Hydra have roughly similar molecular weights to, but are not identical to, FMRFamide (Grimmelikhuijzen et al., 1982). Because antisera against different neuropeptides labeled different populations of neurons (Grimmelikhuijzen et al., 1980; Grimmelikhuijzen et al., 1981c; Grimmelikhuijzen et al., 1981b; Grimmelikhuijzen et al., 1981a; Grimmelikhuijzen et al., 1982; Grimmelikhuijzen, 1983), they identify specific subsets of neurons that differ from each other with respect to neuropeptide content. Antisera to RFamides label a large proportion of the Hydra nervous system and confirm neuronal centralizations in the ectoderm of the hypostome and of the lower peduncle (Grimmelikhuijzen, 1985). Finally, immunogold electron microscopy revealed that the FLPs localize to large dense-cored vesicles in neurons of the peduncle. Aggregation of labeled dense-cored vesicles at the neuromuscular junction (Fig. 3) led to the conclusion that FLPs may have a role in neuromuscular transmission (Koizumi et al., 1989).
The body plan of Hydra. (A) The Hydra polyp has a hollow tube-shaped form. The hypostome region is surrounded by tentacles and the polyp is attached to the substratum via the basal disk. The peduncle is the region just above the basal disk. (B) The body wall of Hydra polyps consists of two epithelial layers that are connected by the acellular mesogloea. Epithelial cells are contractile by virtue of myofibrils at their base, which have a longitudinal orientation in the ectoderm and a circular orientation in the endoderm. Nerve cells or ganglion neurons lie at the base of the epithelial layers. The other neuron type is a sensory neuron. From Technau and Steele (Technau and Steele, 2011), with permission.
Synapses between neurons and epitheliomuscular cells have large dense-cored vesicles. Electron micrograph of a synapse between a nerve cell (nv) and an epitheliomuscular cell (emc). Three large dense-cored vesicles are located in the nerve cell at the site of contact with the epitheliomuscular cell. Scale bar: 200 nm. From Chapman et al. (Chapman et al., 2010), with permission.
FLPs of Hydra were first isolated by a radioimmunoassay in Hydra magnipapillata, revealing four novel neuropeptides with the C-terminal sequence Arg-Phe-NH2: the Hydra-RFamides I–IV (Moosler et al., 1996) (Fig. 4A). They are proteolytically released from three different proneuropeptides (pNPs), preprohormone A, preprohormone B and preprohormone C (Fig. 4B). Preprohormone A contains one copy of all four Hydra-RFamides I–IV; preprohormone B contains one copy of Hydra-RFamide I and II, and three additional putative neuropeptide sequences; and preprohormone C contains one copy of Hydra-RFamide I and seven additional putative neuropeptide sequences (Fig. 4B) (Darmer et al., 1998). Whole-mount in situ hybridization revealed that preprohormone A is expressed in neurons of the upper gastric region, of the hypostome, of the basal region of the tentacles and of the peduncle (Fig. 5A). Preprohormone B is also expressed in neurons of the hypostome and upper gastric region, but is absent from tentacles and the foot (Fig. 5B,D). Finally, preprohormone C is exclusively expressed in neurons of the tentacles (Fig. 5C) (Darmer et al, 1998; Hansen et al., 2000). Thus, the distribution of the mRNA for preprohormones A, B and C correlates well with the distribution of neurons stained using antisera against RFamides (Grimmelikhuijzen, 1985) and reveals that Hydra-RFamides are neuropeptides. The related RFamide Antho-RFamide (pQGRFamide; Fig. 4A) from the sea anemone Calliactis parasitica (class Anthozoa) induces muscle contractions, probably by direct excitation of muscles (McFarlane et al., 1987), further suggesting that RFamides have a role in neuromuscular transmission in Cnidaria.
Immunogold labeling reveals expression of RFamides in large dense-cored vesicles close to an epitheliomuscular cell of Hydra. Gold particles preferentially label the granular core of dense-cored vesicles (arrowheads and inset). EC marks an epitheliomuscular cell surrounding the neuron. N, nucleus; C, cilium; S, stereocilia. From Koizumi et al. (Koizumi et al., 1989), with permission.
Sequence of Hydra-RFamides and structure of their preprohormones. (A) The sequence of Hydra-RFamides I–IV is shown in the one-letter code; pQ represents pyro-Glu. Conserved sequences are boxed in different colors. Note that Hydra-RFamide IV can be generated from Hydra-RFamide III by alternative processing. Antho-RFamide from Calliactis parasitica (class Anthozoa) is shown for comparison. (B) Structure of preprohormones A-C. Hydra-RFamides are in blue (Hydra-RFamides I and II) or brown (Hydra-RFamide III/IV). The black boxes indicate a signal sequence. Adapted from Hansen et al. (Hansen et al., 2000).
The Hydra peptide project revealed that Hydra contains ~500 peptide signaling molecules, ~50% of which are neuropeptides and the rest epitheliopeptides that are produced by epithelial cells (Takahashi et al., 1997; Fujisawa, 2008; Takahashi, 2013). Apart from the RFamides, the neuropeptides can be classified according to their C-terminal sequence into the Hydra-LWamides, the Hydra-KVamides (or Hym-176 family), the Hydra-RGamides (or Hym-355 family) and the Hydra-FRamides (Hayakawa et al., 2007; Fujisawa, 2008; Takahashi, 2013). Hydra-KVamide (Hym-176) is expressed by neurons in the peduncle region of Hydra and induces ectodermal muscle contractions (Yum et al., 1998), suggesting a role in neuromuscular transmission. Double-labeling in situ hybridization revealed that some of the pNPs are co-expressed with each other whereas others are not (Hansen et al., 2000; Hansen et al., 2002), confirming that Hydra has neurochemically distinct populations of neurons. In particular, it was found that preprohormone A, containing Hydra-RFamides I–IV (Fig. 4B), is co-expressed with the pNP containing Hydra-KVamide (Hym-176) in a population of neurons in the peduncle region of Hydra (Hansen et al., 2000). This co-expression might indicate that Hydra-RFamides and Hydra-KVamide cooperate in neuromuscular transmission in the peduncle region.
Whole-mount in situ hybridization of Hydra with probes coding for the three Hydra-RFamide preprohormones. (A) Preprohormone A is expressed in neurons of the tentacles, hypostome, upper gastric region and peduncle. (B) Preprohormone B is confined to neurons from the hypostome and upper gastric region. (C) Preprohormone C is expressed only in the tentacles. (D) Hybridization with a probe for preprohormone B, but with a different color from that in B. From Hansen et al. (Hansen et al., 2000), with permission.
In summary, there is robust evidence that neurons of Hydra extensively use neuropeptides. Evidence for a role as a neurotransmitter is particularly strong for Hydra-RFamides, especially in neuromuscular transmission.
In vertebrates and arthropods, neuromuscular transmission uses the small molecule transmitters acetylcholine and glutamate, respectively. The genome of H. magnipapillata contains two mAchRs (Collin et al., 2013) and 17 genes coding for nicotinic acetylcholine receptor (nAchR) subunits (Chapman et al., 2010), strongly suggesting that H. magnipapillata uses acetylcholine for fast transmission. Expression of five of the nAchRs could be detected by RT-PCR, revealing a predominant expression in the head region. Expression of only one subunit could be detected by in situ hybridization, revealing expression in the ectoderm of the tentacles (Chapman et al., 2010). There is also a choline transporter for re-uptake of choline but a vesicular acetylcholine transporter is lacking (Chapman et al., 2010), suggesting that acetylcholine is not contained in, and hence not released by, the vesicles. Similarly, it appears that Hydra lacks a gene encoding a typical animal acetylcholinesterase (AchE) (Chapman et al., 2010). The authors of this study concluded that a canonical neuromuscular junction was probably not present in the last common ancestor of cnidarians and bilateria (Chapman et al., 2010). Another study identified a functional AchE in Hydra, which is predominantly expressed in the endodermal epithelium of the body column but not the tentacles or the basal disk where nerve cells are concentrated (Takahashi and Hamaue, 2010). Therefore, the authors concluded that the cholinergic system in Hydra might be non-neuronal (Takahashi and Hamaue, 2010). The presence in the Hydra genome of ionotropic receptors for other small-molecule neurotransmitters has not been reported.
Nematostella vectensis is another Cnidaria for which the whole-genome sequence is available (Putnam et al., 2007). Analysis of the Nematostella genome identified 12 genes with homology to nicotinic AchRs, a large number with homology to ionotropic glutamate receptors (six AMPA, one kainate and four NMDA), 11 with homology to GABAA receptors and one with homology to glycine receptors (Anctil, 2009). Although Nematostella belongs to anthozoans, a different class of Cnidaria than hydrozoans [to which Hydra belongs (Technau and Steele, 2011)], the presence of these ionotropic receptors in Nematostella might indicate that Hydra also uses glutamate, GABA and glycine for fast transmission. As for the nAchR from Hydra, none of the Nematostella ion channel receptors has been functionally expressed and in the absence of functional data, the ligand specificity and ion selectivity of these receptors remains uncertain, particularly as several of these genes are only distantly related to their vertebrate orthologs (Anctil, 2009). In summary, molecular evidence for a role for acetylcholine and glutamate in neuromuscular transmission in Hydra is at present incomplete and ambiguous.
In 2007, four new ion channel subunits from Hydra were cloned that belong to the degenerin/epithelial Na+ channel (DEG/ENaC) gene family (Golubovic et al., 2007). As DEG/ENaCs usually are selective Na+ channels, the novel subunits were named Hydra Na+ channels: HyNaC1–HyNaC4. HyNaC1 lacks an initiator methionine and a gating motif that is completely conserved in DEG/ENaCs (Gründer et al., 1997; Gründer et al., 1999) and is therefore most likely a nonfunctional pseudogene (Golubovic et al., 2007). Three years later, cloning of the closely related HyNaC5 was reported (Dürrnagel et al., 2010). Expression of the cDNAs coding for HyNaC2–HyNaC5 was localized by whole-mount in situ hybridization to the base of the tentacles, most likely in epitheliomuscular cells (Golubovic et al., 2007; Dürrnagel et al., 2010) (Fig. 6). Whereas HyNaC2 and HyNaC3 are uniformly expressed at the tentacle base, expression of HyNaC4 is restricted to the aboral side and expression of HyNaC5 to the oral side of the tentacle base (Fig. 6D,E). This expression pattern suggests that HyNaC2–HyNaC4 are co-expressed in epitheliomuscular cells at the aboral side whereas HyNaC2, HyNaC3 and HyNaC5 are co-expressed in epitheliomuscular cells at the oral side of the tentacle base.
Functional properties of HyNaCs were assessed by expression in a heterologous cell system: Xenopus oocytes (Golubovic et al., 2007; Dürrnagel et al., 2010). cRNAs coding for HyNaCs were injected in oocytes either alone or in combination. Expression of HyNaCs did not increase leak currents, showing they are not constitutively open channels. They were also not activated by H+, unlike related acid-sensing ion channels (ASICs; see below). Raising the intracellular concentration of Ca2+ or cAMP did also not activate HyNaCs (Golubovic et al., 2007). Hydra-RFamides I and II, however, elicited robust currents (1–10 μA) when applied to oocytes expressing HyNaC2 and HyNaC3; half-maximal activation was achieved by ~30 μmol l−1 of either peptide (Fig. 7) (Golubovic et al., 2007). Several controls were carried out to ensure that Hydra-RFamides directly activated HyNaCs rather than G-protein-coupled receptors and second messenger cascades. Co-expression of HyNaC5 with HyNaC2 and HyNaC3 strongly increased current amplitude (Fig. 7C) and apparent peptide affinity of the channel (EC50: ~5 μmol l−1 for Hydra-RFamide I and 0.3 μmol l−1 for Hydra-RFamide II) (Fig. 7D) (Dürrnagel et al., 2010). As DEG/ENaCs assemble into homo- or heterotrimers (Jasti et al., 2007; Bartoi et al., 2014), these results strongly suggest that HyNaC2/3/5 forms a peptide-gated ion channel in epitheliomuscular cells at the oral side of the tentacle base. In contrast to HyNaC5, HyNaC4 did not increase current amplitude or apparent peptide affinity of channels formed by HyNaC2 and HyNaC3 (Golubovic et al., 2007), suggesting that HyNaC4 co-assembles with other, not yet described HyNaCs. Similarly, it is possible that HyNaC2 and HyNaC3 co-assemble with another partner at the aboral side of the tentacle base, which replaces HyNaC5 there. Thus, peptide-gated channels in Hydra might be quite heterogenous. In contrast to Hydra-RFamides I and II, Hydra-RFamides III and IV did not activate HyNaCs (Fig. 7A) (Golubovic et al., 2007; Dürrnagel et al., 2010), although they differ only in their N-terminal sequence (Fig. 4A). Another unresolved issue therefore is whether Hydra-RFamides III and IV activate related channels. A complete survey of HyNaCs is necessary to address this (see below).
HyNaCs are expressed at the base of the tentacles. (A–C) Whole-mount in situ hybridization reveals strong expression of hynac2, hynac3 and hynac4 transcripts at the tentacle base in adult polyps. (D,E) hynac5 and hynac4 show complementary expression patterns. hynac5 is expressed at the oral side of the tentacle base (arrow), whereas hynac4 is expressed at the aboral side (arrow). Panels A–C reprinted from Golubovic et al. (Golubovic et al., 2007); panels D and E from Dürrnagel et al. (Dürrnagel et al., 2010). © The American Society for Biochemistry and Molecular Biology.
HyNaCs are directly activated by Hydra-RFamides I and II. (A) Hydra-RFamides I and II, but not III and IV elicit inward currents in Xenopus oocytes expressing HyNaC2/3. (B) Increasing concentrations of Hydra-RFamide I elicit currents of increasing amplitudes. (C) Co-expression of HyNaC5 with HyNaC2 and HyNaC3 potently increases current amplitudes. (D) Concentration response curves for HyNaC2/3 (open symbols) and HyNaC2/3/5 (filled symbols), and Hydra-RFamides I and II. HyNaC2/3/5 has 10 to 100-fold higher apparent affinity. Panels A and B reprinted from Golubovic et al. (Golubovic et al., 2007); panels C and D from Dürrnagel et al. (Dürrnagel et al., 2010). © The American Society for Biochemistry and Molecular Biology.
HyNaCs are nonselective cation channels with a high Ca2+ permeability. (A) Replacing extracellular Na+ with Li+ or K+ does not significantly change the current amplitude of HyNaC2/3. (B) Currents elicited by Hydra-RFamide I in oocytes expressing HyNaC2/3 reverse at ~0 mV. (C) In Xenopus oocytes expressing HyNaC2/3/5, Hydra-RFamides elicit biphasic currents. A transient peak current is followed by a sustained current. (D) After intracellular injection of Ca2+ chelators such as EGTA or BAPTA, Hydra-RFamides elicit currents with a simple on-off kinetics, showing that the biphasic currents are due to secondary activation of endogenous CaCCs. (E) Activation of CaCCs by Ca2+ flux through HyNaC. Panels A and B reprinted from Golubovic et al. (Golubovic et al., 2007); panel C from Dürrnagel et al. (Dürrnagel et al., 2010). © The American Society for Biochemistry and Molecular Biology; panel D reprinted from Dürrnagel et al. (Dürrnagel et al., 2012).
Replacing extracellular Na+ by K+ does not strongly reduce the current amplitude of HyNaCs (Golubovic et al., 2007; Dürrnagel et al., 2010) and currents activated by Hydra-RFamides reverse their direction at a membrane potential close to 0 mV (Fig. 8A,B), suggesting HyNaCs are non-selective cation channels. In a more thorough analysis of ion selectivity, it was shown that HyNaC2/3/5 is indeed an unselective cation channel with a high Ca2+ permeability (relative Ca2+ permeability PCa/PNa ~4) (Dürrnagel et al., 2012). Thus, HyNaCs are clearly not selective Na+ channels, unlike all other DEG/ENaCs, and therefore the name Hydra Na+ channel is somewhat misleading; ionotropic RFamide receptor (iRFa-R) might be a more appropriate name (see also below). In Xenopus oocytes, Ca2+ influx via HyNaC activates endogenous Ca2+-activated Cl− channels (CaCCs) (Dürrnagel et al., 2012). Consequently, in oocytes expressing HyNaCs, Hydra-RFamides activate two channels: HyNaCs (directly) and CaCCs (indirectly) (Fig. 8C–E). CaCCs carry a transient Cl− current that diminishes with repetitive activation (Schroeder et al., 2008). Hydra-RFamides indeed elicit a biphasic current in oocytes: a transient peak current is followed by a sustained current (Fig. 8C). The ratio of transient and sustained current is variable and in particular the peak current amplitude diminishes with repeated application (Golubovic et al., 2007; Dürrnagel et al., 2010). Injection of oocytes with a Ca2+ chelator such as EGTA prevents activation of CaCCs and currents are then exclusively carried by HyNaCs. Under this condition, HyNaCs have a simple kinetics: they are rapidly activated by Hydra-RFamides I and II, do not desensitize and inactivate only when the peptide is washed away (Fig. 8D) (Dürrnagel et al., 2012).
Another hallmark of DEG/ENaCs is their block by the diuretic amiloride (Kellenberger and Schild, 2002). HyNaCs are also blocked by amiloride but apparent affinity is comparatively low: ~500 μmol l−1 for HyNaC2/3 (Golubovic et al., 2007) and ~100 μmol l−1 for HyNaC2/3/5 (Dürrnagel et al., 2010). Benzamil and phenamil, two amiloride analogs, however, block HyNaC2/3/5 with higher apparent affinities (Dürrnagel et al., 2010).
Expression of HyNaCs in epitheliomuscular cells in the vicinity of neurons containing Hydra-RFamides I and II strongly suggests that HyNaCs are involved in neuromuscular transmission. RFamides are contained within large dense-cored vesicles at the junction with epitheliomuscular cells (Fig. 3) and are therefore likely to be released into the synaptic cleft that separates nerve and epitheliomuscular cells. To show definitely expression of HyNaCs at the postsynaptic membrane of the neuromuscular junction, immunostaining of HyNaCs will be necessary, ideally immunogold staining combined with electron microscopy.
Activation of HyNaCs in epitheliomuscular cells at the oral base of the tentacles could induce tentacle curling and might, therefore, be involved in the feeding reaction of Hydra (Golubovic et al., 2007; Dürrnagel et al., 2010). A putative channel at the aboral base of the tentacles could induce a downward movement of the tentacles. To find indications for an involvement of HyNaCs in the feeding reaction, GSH was used to induce the reaction in the absence and presence of 100 μmol l−1 amiloride, a blocker of HyNaCs. Amiloride indeed significantly delayed the feeding reaction (Fig. 9) (Dürrnagel et al., 2010), in agreement with an involvement of HyNaCs. Amiloride is a rather unspecific blocker, however, and it will therefore be necessary to identify more potent blockers of HyNaCs and repeat these experiments. Moreover, it will be necessary to show that Hydra-RFamides can indeed induce movements of the tentacles.
Amiloride delays the feeding reaction of Hydra. Hydra magnipapillata polyps were relaxed in plain medium or medium containing 100 μmol l−1 amiloride. At time 0, the feeding reaction was induced by GSH. Amiloride significantly delayed the response to GSH. Reproduced from Dürrnagel et al. (Dürrnagel et al., 2010). © The American Society for Biochemistry and Molecular Biology.
Recently, it was suggested that striated muscles evolved independently in cnidarians and bilaterians (Steinmetz et al., 2012). Assuming that the neuromuscular junction evolved after the evolution of striated muscles, it is conceivable that the transmitter at this junction also evolved independently and that cnidarians co-opted neuropeptides instead of small molecules for fast transmission. Transmission with acetylcholine, as in vertebrates, has the advantage that the transmitter can be hydrolyzed by the AchE, quickly ending postsynaptic currents. Similarly, transmission with glutamate, as in arthropods, can be ended by fast re-uptake mechanisms. Thus, it seems that fast transmission with neuropeptides has strong disadvantages compared with small molecule transmitters. But maybe fast transmission with neuropeptides is ideally suited for the cnidarian muscle cells. Electrophysiological analysis suggests that HyNaCs do not desensitize and carry a sustained current in the presence of RFamides (Dürrnagel et al., 2012). As RFamides will probably neither be quickly hydrolyzed nor efficiently be taken up, they will activate HyNaCs for a relatively long period of time. The uniquely high Ca2+ permeability of HyNaCs could allow sufficient Ca2+ flux to induce muscle contractions directly and independently of release from intracellular stores. At the same time, the extracellular Na+ concentration in the body wall of the freshwater polyp Hydra has been estimated to be only 17 mmol kg−1 (Benos and Prusch, 1972) and inward currents will therefore be comparatively small. Thus, RFamides might induce small but sustained depolarizations of the postsynaptic membrane. In vertebrate muscle cells, sustained depolarizations of the postsynaptic membrane will inactivate voltage-gated Na+ channels, effectively paralyzing the muscle. This suggests that epitheliomuscular cells of Hydra use different voltage-gated channels than vertebrates, perhaps Ca2+ channels. All these are unresolved issues that need to be addressed by a detailed electrophysiological analysis of epitheliomuscular cells of Hydra.
The closest relatives of HyNaCs within the DEG/ENaC gene family are ASICs and the bile-acid-sensitive ion channel (BASIC; also named BLINaC or ASIC5) (Golubovic et al., 2007) (Fig. 10; see also below). ASICs are ionotropic receptors for H+ (Waldmann et al., 1997), the simplest ligand possible. At present, it seems that ASICs appeared relatively late in evolution, as they are restricted to chordates (Gründer and Chen, 2010). As mentioned before, HyNaCs are indeed not activated by H+. However, ASICs are not activated by neuropeptides, but RFamide neuropeptides, including FMRFamide, can modulate the ASIC current (Askwith et al., 2000; Catarsi et al., 2001; Deval et al., 2003; Xie et al., 2003; Ostrovskaya et al., 2004; Chen et al., 2006; Sherwood and Askwith, 2008; Sherwood and Askwith, 2009). It is believed that neuropeptides directly bind to ASICs to modulate the H+-activated current. Even Hydra-RFamides, which are not present in chordates, can modulate the ASIC current (Golubovic et al., 2007). Conservation of an RFamide binding site in ASICs and the close relationship between ASICs and ionotropic RFamide receptors (HyNaCs) suggest that ASICs evolved from an RFamide-gated channel.
Phylogenetic relationship between DEG/ENaCs from different animal taxa. (A) Circular cladogram of a Bayesian analysis of 115 known and predicted members of the DEG/ENaC gene family. Sequences were collected from a BLAST search for DEG/ENaC protein sequences in distinct animal genomic databases. The focus was on invertebrate organisms. During our Bayesian analysis, we ran 10 million generations with one chain without Metropolis coupling. After a burn-in of 10,000 generations, every 100th generation was sampled. The estimated average s.d. was below 0.0001%. Placozoa are represented by a single species (yellow, T. adhaerens), Ctenophora by two species (orange, M. leidyi and P. bachei) and Cnidaria also by two species: one hydrozoan (dark blue, H. magnipapillata) and one anthozoan (light blue, N. vectensis). Bilateria are represented by species from four phyla: Mollusca (pink, FaNaC from four species, Aplysia kurodai, Helisoma trivolvis, Helix aspersa and Lymnea stagnalis), Arthropoda (green, D. melanogaster), Nematoda (brown, C. elegans) and Chordata (red, ASICs from R. norvegicus). The position of HyNaC2–HyNaC5 is indicated by dark-blue dots. The four FaNaCs (pink) are from four different molluscan species. From chordates, only ASICs but not BASIC and ENaC were included. (B) Phylogenetic relation of the different taxa included in our analysis is shown on the left. The relation of porifera, placozoa and ctenophores to the other animals is controversial. Cnidaria are a sister group to Bilateria. The number of known or predicted DEG/ENaC genes from different species included in our analysis is indicated on the right.
BASIC is closely related to ASICs and seems to be restricted to mammals. It is activated by millimolar concentrations of bile acids (Wiemuth et al., 2012; Wiemuth et al., 2013a; Wiemuth et al., 2013b), but at present it is not clear whether bile acids are the natural stimulus to open the channel. In rodents, BASIC is also expressed in the brain (Sakai et al., 1999; Boiko et al., 2014) where concentrations of bile acids are not high enough to activate the channel. It is therefore possible that BASIC is activated by an unknown neuropeptide.
The DEG/ENaC gene family also contains the only other known peptide-gated ion channel, the FMRFamide-activated Na+ channel, FaNaC, from mollusks (Lingueglia et al., 1995). A FMRFamide-activated channel was first described by Cottrell (Cottrell et al., 1990) and was the first peptide-gated ion channel that was cloned (Lingueglia et al., 1995). Interestingly, FaNaC is a more distant relative of HyNaCs than ASICs or BASIC (Golubovic et al., 2007) (Fig. 10). The existence of a bilaterian peptide-gated channel and a related cnidarian channel suggests that the common ancestor of bilaterians and cnidarians already contained an RFamide-gated channel. Perhaps the whole DEG/ENaC gene family evolved from RFamide-gated channels, as is likely for ASICs (see above). Irrespective of the exact relationship between different DEG/ENaCs, the common ancestor of DEG/ENaCs gave rise to two types of ionotropic RFamide-receptors, one of the HyNaC type and one of the FaNaC type that evolved independently. Moreover, the presence of iRFa-Rs in bilaterians and cnidarians suggest that FLPs co-evolved with DEG/ENaCs for several hundred million years.
Other DEG/ENaC channels in bilaterians are the degenerins, mechanosensitive ion channels from C. elegans (O'Hagan et al., 2005; Árnadóttir and Chalfie, 2010), the FLR-like channels from C. elegans (Take-uchi et al., 1998), and ripped pocket (RPK) and pickpockets (PPKs) from Drosophila (Zelle et al., 2013) (Fig. 10). The function of many of these channels is unknown, but peptide-mediated activation has not been shown for any of them so far.
The relationship between animal taxa is still controversial. One view suggests that the three taxa with nervous system and muscle cells (Cnidaria, Ctenophora and Bilateria) form a eumetazoan clade, whereas Porifera and Placozoa have a more basal position (Philippe et al., 2009). Others suggest that ctenophores are the most basal animal lineage and that a nervous system evolved independently in this taxon (Moroz et al., 2014). But there seems to be consensus that Cnidaria and Bilateria are sister groups to one another. A global view of proneuropeptides (pNPs) revealed that cnidarian RFamides are orthologs of bilaterian R[FY]amides and that, therefore, FMRFamide-like peptides (FLPs) are evolutionarily old and were already present in the last common ancestor of Cnidaria and Bilateria (Jékely, 2013). In the ctenophore Pleurobrachia bachei, 72 putative prohormones have been predicted (Moroz et al., 2014). Interestingly, several pNPs are already present in the neuronless placozoan Trichoplax adhaerens, which probably give rise to several amidated peptides, including Famides (Jékely, 2013; Nikitin, 2014). The presence of pNPs in Trichoplax indicates that neuropeptide signaling may predate the origin of nervous systems (Jékely, 2013; Nikitin, 2014). In fact, very recently it has been shown that gland cells of Trichoplax are secretory cells and that a subset of them is stained by an antibody against FMRFamide (Smith et al., 2014), suggesting that they secrete FLPs. Although gland cells do not make synapses, it has been suggested that they control the behavior of surrounding cells in a paracrine fashion (Smith et al., 2014). In contrast to animals from other taxa, pNPs have so far not been found in the sponge Amphimedon queenslandica (Jékely, 2013), indicating that Porifera do not use peptides for paracrine communication. An amidating enzyme involved in pNP maturation, however, has been predicted in A. queenslandica (Attenborough et al., 2012), hinting at the possible presence of pNPs in sponges.
To analyze the presence of DEG/ENaCs in different animal taxa, we carried out a BLAST search for DEG/ENaC protein sequences in the genomic databases of animals at the base of the animal kingdom. Placozoans were represented by T. adhaerens (Srivastava et al., 2008), porifera by A. queenslandica (Srivastava et al., 2010), ctenophores by P. bachei (Moroz et al., 2014) and Mnemiopsis leidyi (Ryan et al., 2013), and cnidarians by the hydrozoan H. magnipapillata (Chapman et al., 2010) and the anthozoan N. vectensis (Putnam et al., 2007). The genome of the choanoflagellate Monosiga brevicollis (King et al., 2008) was also analyzed as choanoflagellates are the closest known relatives of metazoans. We could not find any DEG/ENaC related proteins in choanoflagellates or sponges, suggesting that DEG/ENaCs evolved in metazoans after the split of sponges; interestingly, sponges also do not seem to have pNPs (Jékely, 2013). By contrast, we found a large number of predicted DEG/ENaCs in the genomes of all other animals. In the genome of T. adhaerens (Placozoa), we found eight putative DEG/ENaCs. Furthermore, we found 20 putative DEG/ENaCs in M. leidyi and six in P. bachei (Ctenophora), 11 in H. magnipapillata (Hydrozoa) and eight in N. vectensis (Anthozoa). Thus, we identified seven additional DEG/ENaCs in Hydra.
We made a sequence alignment of the DEG/ENaCs we identified in placozoans, ctenophores and cnidarians with DEG/ENaCs of bilaterians, represented by arthropods (RPK and PPKs from D. melanogaster), mollusks (FaNaC from four snail species: Aplysia kurodai, Helisoma trivolvis, Helix aspersa and Lymnea stagnalis), nematodes (degenerins and FLR-like proteins from C. elegans) and chordates (ASICs from R. norvegicus), and analyzed their phylogenetic relation by Bayesian analysis (Fig. 10) and maximum likelihood analysis (supplementary material Fig. S1). With a few exceptions mentioned below, both types of analysis gave very similar results.
With one exception, the seven additional DEG/ENaCs from Hydra are all closely related to HyNaC2–HyNaC5 (Fig. 10; supplementary material Fig. S1), strongly suggesting that they might also contribute to the formation of ionotropic RFamide-receptors of the HyNaC type. Strikingly, the majority of DEG/ENaCs from Trichoplax and most DEG/ENaCs from Nematostella are also closely related to HyNaCs and ASICs (Fig. 10; supplementary material Fig. S1), forming a monophyletic group within the DEG/ENaC gene family. Assuming that ASICs evolved from a peptide-gated channel, this branch of the DEG/ENaC gene family likely arose from an iRFa-receptor of the HyNaC type and, therefore, all channels of this branch are candidates for iRFa-Rs of the HyNaC type.
Degenerins and FaNaC are on a separate but neighboring branch than ASICs/HyNaCs (Fig. 10; supplementary material Fig. S1). Assuming that HyNaCs and FaNaC evolved from the same peptide-gated ion channel, the degenerin/FaNaC branch likely arose from an iRFa-receptor of the FaNaC type and therefore, all channels of this branch are candidates for iRFa-Rs of the FaNaC type. The only DEG/ENaC from Trichoplax that does not cluster with HyNaCs/ASICs is closely related to FaNaCs (Fig. 10; supplementary material Fig. S1), making it an especially good candidate for an iRFa-R of the FaNaC type. Thus, Trichoplax might have ionotropic RFamide-receptors of the HyNaC and the FaNaC type.
In Bayesian analysis, all DEG/ENaCs from ctenophores form an independent large branch (Fig. 10), indicating a long independent evolution. If this branch had split off after the first RFamide-gated DEG/ENaC appeared, then it could potentially also contain RFamide receptors. In this case they would be of a third type. However, in maximum likelihood analysis, three DEG/ENaCs from ctenophores belong to the degenerins/FaNaC branch (supplementary material Fig. S1) and are, therefore, candidates for iRFa-Rs of the FaNaC type. Thus, ctenophores might also have two types of ionotropic RFamide-receptors. It should be emphasized that these conclusions are based on the assumption that HyNaCs and FaNaC evolved from the same peptide-gated ion channel. Moreover, ASICs and degenerins are not gated by peptides, showing that not all DEG/ENaCs on the HyNaC/ASIC and the degenerin/FaNaC branch are indeed peptide gated. Peptide gating of a channel always needs experimental validation and cannot be predicted solely by phylogenetic analysis of protein sequences. Nevertheless, as the appearance of pNPs in placozoans and ctenophores coincides with the appearance of DEG/ENaCs, DEG/ENaCs from these species should be evaluated for activation by peptides, in particular RFamides. A role for neuropeptides as a fast transmitter is also suggested by the fact that placozoans and ctenophores apparently only have ionotropic glutamate receptors, DEG/ENaCs and, in the case of Trichoplax, P2X receptors, but no pentameric ligand-gated ion channels (Jorgensen, 2014; Moroz et al., 2014).
FLR-like proteins from C. elegans and all DEG/ENaCs from Drosophila are isolated from other DEG/ENaCs (Fig. 10; supplementary material Fig. S1), suggesting they adopted new functions and activation mechanisms. It is known that C. elegans and Drosophila have high rates of molecular evolution and that they diverged more from the common bilaterian ancestor than did chordates (Raible and Arendt, 2004). The one DEG/ENaC from Hydra that is not closely related to HyNaCs has an uncertain relation (clustering on the degenerin/FaNaC branch in Bayesian analysis and on the FLR-like branch in maximum likelihood analysis; Fig. 10; supplementary material Fig. S1) and is unlikely to be a peptide-gated channel.
Interestingly, the organization of DEG/ENaCs in the anthozoan Nematostella is different to that in Hydra (Fig. 10; supplementary material Fig. S1). There is only one DEG/ENaC that seems to be a direct ortholog of HyNaCs; four or five others (depending on the algorithm used for phylogenetic analysis; Fig. 10; supplementary material Fig. S1) belong to the HyNaC/ASIC branch but are more distantly related to HyNaCs than to ASICs. The remaining two or three belong to the degenerins/FaNaC branch. We provisionally conclude that DEG/ENaCs in Nematostella serve more diverse functions than in Hydra. As the degenerin/FaNaC branch also contains DEG/ENaCs from placozoa and ctenophores (Fig. 10), this branch must have an evolutionarily old origin and consequently representatives from this branch have been lost in Hydra. Hydra diverged from anthozoans more than 540 million years ago and the amino acid substitution rate in the Hydra lineage is enhanced relative to the Nematostella lineage (Chapman et al., 2010), suggesting that its genome is more derived than the genome of Nematostella. Thus, loss of the degenerin/FaNaC branch and the expansion of the HyNaC branch seem to be derived features of hydrozoans.
In summary, phylogenetic analysis reveals a large variety of HyNaCs, likely contributing to iRFa-Rs in Hydra, and related channels in Nematostella. In addition, it uncovers a large variety of DEG/ENaCs in cnidarians, placozoans and ctenophores. Trichoplax is likely to have one iRFa-R of the FaNaC type and has a variety of DEG/ENaCs with a relatively close relationship to the iRFa-Rs of the HyNaC type. Ctenophores have no DEG/ENaCs that are closely related to iRFa-Rs of the HyNaC type, but like placozoans and cnidarians they have DEG/ENaCs that are candidates for peptide-gated ion channels. A large variety of iRFa-Rs of the HyNaC type, however, seem to be a derived feature of hydrozoans.
We thank Y. Moran (Hebrew University, Jerusalem) for advice on phylogenetic analysis. This work was presented at the 'Evolution of the First Nervous Systems II' meeting, which was supported by the National Science Foundation (NSF).
Note added in proof
In a recent report (Assmann et al., 2014), the seven additional DEG/ENaC subunits from Hydra magnipapillata shown in Fig. 10 have been functionally characterized and the results support their role in neuromuscular transmission.
Work in the lab of S.G. was supported by the Deutsche Forschungsgemeinschaft [GR1771/7-1].
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Supplementary Material- pdf file


text:
Posted inNational News, News
Odisha: Good Samaritan rescues 6-day-old abandoned baby
The 42-year-old Dalit woman took the baby home, tended him and later handed him over to Childline officials
by Press Trust of India February 2, 2021 Updated February 23, 2021
She found the infant, who was crying and shivering in the cold though wrapped in a piece of cloth, on the roadside Credit: Representational image
Phulbani: A woman rescued a six-day-old boy abandoned on a road in a cold winter night in Odisha's Kandhamal district, an official said on Monday.
She found the infant, who was crying and shivering in cold though wrapped in a piece of cloth, on the roadside near a school in Phulbani town on late Sunday night.
The 42-year-old Dalit woman took the baby home, tended him and later handed him over to Childline officials.
The boy, who was apparently six days old, was hospitalised, said Suprava Nayak, a counsellor of Childline.
The woman and her daughter were returning home from a relative's place at around 11.30 pm when they found the child.
After taking him home, they kept him warm by lighting a fire and called up the Childline number 1098 to report the matter, Nayak said.
A Childline team reached her home at around 2.30 am, took the baby from her and admitted him to a government hospital.
The baby has been kept in a ventilator and his condition is critical, attending doctors said.
Following a complaint lodged by Childline, the police are investigating the case.
It is suspected that after the birth of the child, his mother kept him with her for five days, before abandoning him.
Arunachal: Three-day-old baby, sold by parents for Rs 50,000, rescued
Assam: Nine minors, one infant rescued by NF Railways
NF Railway rescues 3 minors, 9 destitute women from stations
What to do if your kid is biting, hitting and kicking? January 31, 2023
Assam: GP Singh takes charge as DGP, pledges to make force citizen-centric January 31, 2023
Nagaland 2023: Two legislators resign from state Assembly session January 31, 2023

redpajama_set_name: RedPajamaCommonCrawl


text:
The Story of Cirrus Flux
Matthew Skelton
When Cirrus Flux was just a baby he was brought to the Foundling Hospital and left there. Cirrus does not know what it means to have parents or a family, and he fully expects that he, like all the other orphans at the hospital, will eventually find a place in the world when some tradesman takes him on as an apprentice. Surely there is no reason to think that his future will be different. But it is.
Instead Cirrus soon finds himself running through the streets of London, trying to get away from two terrifying villains. One is a beautiful woman, Madame Orrery, who also happens to be a skilled mesmerist. The other is a man called Mr. Sidereal who has built a system that allows him to spy on the lives of just about everyone in the great city of London. Both these people believe that Cirrus has a small metal pendent in his possession, something that Cirrus' father, James Flux, left with his baby son when he left him at the Foundling Hospital. The pendent contains a scrap of something called the Breath of God, a powerful force that James Flux collected at the ends of the earth. Both Madame Orrery and Mr. Sidereal want to possess the Breath of God, and it is up to Cirrus and his new found friends to make sure that they fail in their attempts.
With a story that shifts between the past and the present, this exciting and intriguing novel will delight readers who enjoy stories that are full of adventure, colorful villains, and fantastical occurrences. Throughout the book author gives his readers a fascinating and extremely vivid picture of what life was like in England in the 1700's.


Automatic air vent valve with removable cover for inspection. Body and cover in brass CW617N. Corrosion-resistant polyethylene float. Nominal pressure: 10 bar.
INT/R: Automatic air vent valve like INT but complete with automatic shut-off valve RIA.


The NatTable 1.3.0 release is intended to be a bugfix release. It contains mainly bugfixes and just small enhancements requested by the community in order to make the NatTable API more extensible.
Almost every change in code is tracked via ticket in Bugzilla, so if you are curious about the details and all the bugs that are fixed and enhancements that were added with the 1.3.0 release, have a look here.
Introduced the ICalculatedValueCache interface and added corresponding getter and setter on SummaryRowLayer and GroupByDataLayer in order to be able to exchange the implementation for caching calculated values.
Added AbstractTextPainter#setTrimText(boolean) to be able to configure if the text that is rendered by an AbstractTextPainter is trimmed or not.
Reworked column reordering in combination with column grouping. This caused the introduction of several new command handler and the modification of several existing command handler related to column reordering. The update of column groups on column reordering is now performed in ColumnGroupReorderLayer#updateColumnGroupModel().
Changed visibility of ColumnResizeDragMode#getColumnWidthMinimum() to protected in order to support specifying a custom min width for columns on drag resizing.
Replaced every occurence of SWT.CTRL in the NatTable default bindings with SWT.MOD1 to make the bindings work correctly on Mac OS.
Introduced the usage of an API Baseline and using the API tooling in order to ensure to have correct versions regarding the OSGi semantic versioning.


Contrary to general impression it is not so difficult to explain the emergence and the function of god in human history. We can easily disregard god as a kind of pre-scientific concept, but we cannot but to accept the role that god features in almost any society. Even if you are ateist you relate yourself to something that is supposed to be a god.
But there is even more! I will prove that there is less need for a god in pre-scientific societies that in a modern, formal democracy based cultures. We could name this paradox as "Dawkins paradox": with his vigorous attacks on any instance of god and with his plea for atheistic society, Dawkins in fact positioned religious realm more precisely than almost any other religious discourse. Why it is that atheism can only be positioned as antithesis to theism while theism can survive regardless any opposition?
Although human brains are complex entities, their emergent property, consciousness, is fairly simple and linear. As such, simple and linear, this consciousness could not and cannot directly correspond to complex, non-linear processes that brains are structured upon. Brains – consciousness relation is only one of many that underpin unique human identity. In fact, each and every simple human (emergent) property depends on (comes out of) complex structure.
One example can be a legislation in relation to life. We do intuitively know, that cause and effect in life is not linear. We thus "know" that life is much too complex to be reflected through legislation that tries to reposition life in linear law structures and linear legislative processes. In ancient, non-democratic days, it was a word of tribe leader, that was himself a part of a complex structure, that ruled over the community. There was an oral taboo system that represented a social cohesion system, but the tribe leader did not refer to taboo system in a way that contemporary judge is referring to law in a contemporary society. In those days, the truth was equal to formal ruling while today we say that the realm of truth has nothing to do with rulings within legal system. What we want to say with this is that truth is a part of a complex system that our legal apparatus (emergent property of human society) is not capable to be in touch with. What is ruled as against the law can be at the same time true or not true, and vice versa. Legal act as a part of linear, simple legal structure does not want to govern in the domain of life, complexity, while older, oral, tribal societies still perceived tribal leader ruling as not only formally correct but also true.
The surprising paradox lies in the fact, that more than human civilization developed emergent properties like law, written books, procedures… more the gap between complex nature and linear properties are seen and felt. More than we rely on consciousness, more than we know about it, more than evolution developed it, more we feel disconnected from the complex realm that this consciousness is based upon. More than we develop (formalize) legal system, more we feel that it is detached from the questions of truth. More than we understand the nature of complex systems, so beautifully explained by Peer Bak sand pile and avalanches explanation, more we see that trying to find one sand that could be blamed for an avalanche (law, linear mind, … is pushing us to find one and only culprit) is not only against human nature, but obviously also against physical nature.
And since simple, linear properties like consciousness, law… cannot develop complex solutions (common belief), another simple property, which aim was to fill the gap between two realms, emerged: god. God is simple emergent property of human civilization that aim was to link unsatisfying solutions of linear world with what humans felt but could not understand as complexities of life. Consequently, the need for a god expands with the development of properties like consciousness and law! The highest paradox lies in a fact that more than one another emergent property is developed, science, more humans need a god to connect them (their consciousness, their science…) with complexity of life. That is why Higgs particle is in fact "god particle", but not because it would unlock doors towards complexity, but because it will double the need for a god as an intermediator between Keep It Stupid Simple (KISS) realm of human mind (legislation and even physics) and life. That is why more than humans formalize democracy, more we need a god to saturate it.
There is of course another answer to this separation that proved to be wider and wider with each KISS step of human society. God as himself a part of KISS strategy is apparently false (but explicable) answer. The alternative answer is found on many parts of this blog. As I am more and more convinced that the solution lies in de-formalization, in disrupting KISS strategy, in personal experience of complexity and personal allowance that complexity enters into everyday practice, I should not make it simple and repeat here, what was already written somewhere else, only for the reason of accepting your commodity, my dear reader. But perhaps a hint only: individual as a cornerstone of wisdom, power and beauty. Openess to quantum like entanglement. From a point of social, political and philosophical sciences, this is called: Homonism.
When we say that a formal judicial ruling can declare truth and lie at the same time, we in fact follow one of core quantum laws: uncertainty on one side and entanglement on the other side. In analogue, Einstein world, the locality principle rules. Two bodies cannot be on one place in the same time. One judicial ruling is local, linear; it should be such because there is not a person that would in our eyes represent both formal (linear) and life (complex) world, but the law, that is objective (repeatable). So, we have a formal ruling coming from analogue world on one side and quantum like position of truth, that can never be localized in relation to analogue formal ruling, on another.
If we apply these finding to public procurement system, we see clearly the gloomy future of the system if it is going to develop towards higher formalization. More than you develop rules to rule out the god's presence in procurement system, more you call for its intervention in the end.


A waterproof surface or structure is a treatment which prevents water from crossing under water pressure. Waterproofing Bayer system can be placed on a positive or negative side. In the absence of hydrostatic pressure, it is advisable to treat a surface or structure to pass water. A moist proofing Bayer system is used to perform similar functions of waterproofing system, but it can not be used to protect against water pressure. Water can be forced through building members by hydrostatic pressure, water vapor shield, capillary action, wind-rain, or any of these combinations. This movement is growing with porous concrete, cracks or structural defects or joints which are improperly designed or installed. Leakage of water in the structure can lead to structural damage, and can always damage the material of the structure.
The new roofs RB or RCC slab specified by the designer should be constructed. Roof waterproofing is a wide-ranging topic. During the construction of RB or RCC roof slab, often leads to inadequate attention, waterproof and wrong products used in inadequate treatment, leakage. Due to structural deflection, settlement etc., to detect movement and vertical temperature variation, develop cracks in roof slab and water is starting to start with these cracks.
While producing RCC roof slab, it should be kept in mind that the practice of using concrete is not indisputable and is not desirable to have more reliance on water resistant measures. Concrete should be made indisputably in its own right and the waterproof method should be seen as additional protection devices.
Grade of roof slab concrete will be strictly specified by the designer. The solid materials must be properly proportionately to maintain the maximum water, cement ratio, minimum cement material and essential functionality. Concrete should be mixed with a superplastic engine.


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Bishop Directs Parishes To Help St. Peter's
The Catholic Church is involved in helping those in the East End whose homes have been hit by flooding. Bishop Fred Collie says among other things they have allowed St. Peter's Church to be one of the emergency focal points and he's asking other parish churches in the city to get involved by helping St. Peter's through donations or volunteering.
Labels: Photo: diocese of thunder bay
LU Students Graduate
Hundreds of Lakehead University students are starting new lives after this weekend. They are graduating in separate ceremonies at the Community Auditorium today and tomorrow.
Labels: Photo: Sheri Leviski
Health Unit Worries About Public Safety
Thunder Bay's Health Unit doesn't want homeowners staying in their flood damaged homes. Spokesperson Chris Beveridge says it concerns him because it's dangerous with risk of electrocution and mould. He adds residents also need to take precautions by wearing rubber gloves, goggles and boots during clean up.
Documentary Stars Summer Beaver Resident
A 22 year old former Summer Beaver First Nation resident is the star of a new documentary. Thunder Bay's Handicapped Action Group, Skyworks Charitable Foundation and the Provincial Government are funding the new film called "Ordinary Woman, Extraordinary Dreams". Maggie Sofea says she wants to be a role model for area youth. Crews will follow Sofea home to Summer Beaver this summer and visit the site of her diving accident. Sofea was unable to live on reserve after her accident and had to adapt to a life living in a wheelchair in an urban mostly non-native community.
Tax Dollars For Local Businesses
Two local businesses are on the receiving end of Provincial Government funds. Kakabeka Falls based business "Amanda's Green Barn" will use 100 thousand dollars to expand and include a new indoor horse riding area, new box stalls for the horses and feeding rooms. Sumac Forest Information Services will be using 11 thousand dollars to buy new equipment and software as well as hire four new employees. The money came from the Northern Ontario Heritage Fund.
Transit Plan Back Before Council
City councillors will be asked to approve Thunder Bay Transit's new Master Plan at their meeting Monday night. Manager Brad Loroff says the plan proposes changes to streamline the system including major route changes
Public Meetings For Flood Victims
If your home has been affect by this week's devastating flood you have a chance to ask questions. The city is holding two public meetings tomorrow, one at the Slovak Legion in the East End and the second at the Moose Hall on Fort William Road. The meetings take place at 2 o'clock at the Slovak and 4 o'clock at the Moose Hall.
Salvation Army Swims In Cash
The Salvation Army is swimming with cash after raising a quarter million dollars for disaster relief efforts from the community. Major Merv Halvorsen says that money will go to to help the flood victims who do not have insurance to cover their losses. The donations included 50 thousand dollars from the Fort William Rotary Club.
City Still Working On Sewage Treatment Plant
The City of Thunder Bay continues to work on alleviating the flood situation in the east end and the surrounding communities. General Manager Darryl Matson says they've been able to send crews into part of the sewage treatment plant. They have removed 2 of the 5 motors that drive the main pumps, so they can be dried. Matson adds crews are looking at the electrical side of the treatment plant and figuring out how best to power the pumps back up.
State Of Emergency News Conference June 1, 2012 10 AM
Streaming Live by Ustream
M.T.O. Update Friday
Highway 11-17 between the Thunder Bay Expressway and Highway 102 remains closed. Highway 102, however is open to east-west traffic. Rural roads in the area remain closed due to the flooding and washouts: In Ware Township, Portage Creek Road is closed at the first bridge. Silver Falls Road from Ellis Road to Shields Road is closed and Forbes River Road is currently open but motorists are asked to drive with caution. At Dawson Road Lots, Aitken Road at Sunshine Loop is open but motorist are asked to drive with caution.
Cats Earn First Win
The Thunder Bay Border Cats earned their first win of the Northwoods League season with an 8-5 win over Alexandria. The Cats are in Rochester tonight to begin a 3 game series. Their home opener is on Monday at Subway field at 7:05 p.m.
State Of Emergency News Conference June 1, 2012 10...


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NBA Preview: Washington Wizards at Los Angeles Lakers Predictions, Betting Picks
November 29, 2019 by Jared Johnson
The NBA was off on Thanksgiving Day, giving all those associated with the league a chance to spend time with loved ones. Today is Black Friday and the NBA is back with a massive slate of 13 games. The finale is a contest at Staples Center between the 6-10 Washington Wizards and 16-2 Los Angeles Lakers (10.30pm ET Friday).
Washington has been one of the NBA's best offensive teams and also one of the worst defensive teams. The Lakers are much more balanced and have been the league's most consistent team so far.
How will these teams fare against each other today? Read this preview from USAbetting before we give our Washington Wizards vs Los Angeles Lakers predictions from the pick of the betting lines from the elite U.S. bookmakers in our conclusion.
Will the Wizards' Former Lakers Bigs Play With a Vengeance?
There is no question that the Lakers have a talent advantage in this matchup. LeBron James and Anthony Davis for Los Angeles are clearly the two best players in this matchup and the Lakers also have a host of strong role players. This game is also being played on their home court.
The Wizards need a lot of things to go right if they are to win this game. One of those potential things that could go well is highly motivated performances from two former Lakers now on the Wizards: Thomas Bryant and Mo Wagner.
LA gave up on both players after one season of very little playing time, not truly giving either a chance at proving themselves. Now, both guys have blossomed into strong rotation players. Bryant is averaging close to 15 points, nine rebounds and three assists per game, and Wagner is averaging around 12 points and five rebounds per game while leading all qualified players in true-shooting percentage.
Both sweet-shooting big men need to hit their outside jumpers to pull out the Lakers' defense. They also need to use any feelings of vengeance to translate to strong rebounding and defensive play against a long, athletic Lakers frontcourt.
Will the Lakers Fall into the Trap of Frequent Anthony Davis Isolations?
As great as Anthony Davis is for the Lakers, Los Angeles is best on offense when the team isn't running everything through its superstar big man. The Lakers are 3-2 with a point differential of minus-0.2 per game when Davis takes at least 20 field-goal attempts. In games where Davis takes fewer than 20 shots, the Lakers are 13-0 with a point differential of plus-12.
Davis is certainly capable of a massive offensive game against Washington. The Wizards don't really have anyone who can consistently guard him. If Los Angeles is truly going to thrive on offense, the squad needs to prioritize a perimeter-centric ball movement offense that takes advantage of Washington's mismatches and lack of established chemistry across the lineup.
A more balanced attack would do a better job of keeping everyone on the Lakers' roster in rhythm and contributing, rather than just tossing the ball into the post for Davis to operate on his defender one-on-one.
Does Bradley Beal Have a Scoring Explosion in Him?
The Wizards are likely going to have trouble guarding the Lakers. Washington is the NBA's worst defense by points allowed per 100 possessions. The Lakers have the league's No. 7 offense that has been even better during their nine-game winning streak.
When the Lakers are defending in the half court, the Wizards may also have trouble generating offense as a team against a stiff Los Angeles defense.
If Washington is going to keep this game close or win, Beal is going to have to go off and be the top offensive player in the game and create his own shots. The 26-year-old has certainly done so plenty of times this year, putting up performances of 46, 44, 44, 35, 34, 33, 30, 30 and 30 points all in his last 12 games. An effort of 30 to 40 points will be needed from him.
Our Preview's Washington Wizards vs LA Lakers Betting Picks & Predictions Verdict
The Lakers are a much better team and will very likely win this game to extend their winning streak. However, I wouldn't be surprised to see Washington and its four former Lakers players (Mo Wagner, Thomas Bryant, Isaiah Thomas and Isaac Bonga) play more motivated basketball than its opponent in this contest.
I see this game being surprisingly competitive for much of the game due to the motivation of the Wizards' players, leading to the Wizards playing relatively solid defense. Given those comments, these Washington Wizards vs LA Lakers predictions should be no surprise:
The Lakers are favored by a large margin at 11 or 11.5 points. I think going with the Wizards +11.5pts @ -113 with BetOnline is a good play. It is +11pts @ -110 with Bovada, Bookmaker or Intertops.
In the regular season, motivation to win is almost as important as teams' strengths and weaknesses. I like Washington's chances at competing hard on defense and in the rebounding battle. Take the under 235 total points to be scored @ -105 with Bovada.
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