The larval nervous system of the solitary tunicate Ciona is a simple model for the study of chordate neurodevelopment. The development and connectivity of the Ciona motor ganglion have been studied in fine detail, but how this important structure develops in other tunicates is not well known.
By comparing gene expression patterns in the developing MG of the distantly related tunicate Molgula occidentalis, we found that its patterning is highly conserved compared to the Ciona MG. MG neuronal subtypes in Molgula were specified in the exact same positions as in Ciona, though the timing of subtype-specific gene expression onset was slightly shifted to begin earlier, relative to mitotic exit and differentiation. In transgenic Molgula embryos electroporated with Dmbx reporter plasmids, we were also able to characterize the morphology of the lone pair of descending decussating neurons (ddNs) in Molgula, revealing the same unique contralateral projection seen in Ciona ddNs and their putative vertebrate homologs the Mauthner cells. Although Dmbx expression labels the ddNs in both species, cross-species transgenic assays revealed significant changes to the regulatory logic underlying Dmbx transcription. We found that Dmbx cis-regulatory DNAs from Ciona can drive highly specific reporter gene expression in Molgula ddNs, but Molgula sequences are not active in Ciona ddNs.
This acute divergence in the molecular mechanisms that underlie otherwise functionally conserved cis-regulatory DNAs supports the recently proposed idea that the extreme genetic plasticity observed in tunicates may be attributed to the extreme rigidity of the spatial organization of their embryonic cell lineages.
Tunicates have a long history as tractable laboratory organisms for the study of embryonic development . Most tunicate larvae develop rapidly but invariantly, according to highly stereotyped cell lineages. Furthermore, many also possess highly compact genomes and are quite amenable to a wide variety of molecular assays and perturbations. More recently, tunicates have also begun emerging as model organisms for developmental neurobiology . The complete connectome of the larva of the tunicate Ciona intestinalis, the second connectome ever mapped after that of the nematode C. elegans , revealed the synaptic connections of all 177 neurons of the central nervous system (CNS) and all 54 neurons of the peripheral nervous system (PNS) [4, 5]. With 231 total neurons (CNS and PNS combined), the Ciona larval nervous system is one of the smallest ever described, smaller than even the nervous system of the C. elegans hermaphrodite (302 neurons).
The Ciona connectome further revealed specific neural circuits that are conserved between tunicates and their sister group, the vertebrates, including a putative homolog of the Mauthner cell/C-start escape response circuit of fish . In Ciona, the putative Mauthner cell (M-cell) homologs are a single pair of descending decussating neurons (ddNs), which correspond to the A12.239 pair of cells of the motor ganglion (MG), a cluster of 30 neurons that comprise a central pattern generator for the swimming behavior of the larva , and proposed to be homologous to a combination of the vertebrate hind brain and spinal cord . The development of the ddNs and the rest of the MG have been studied in some detail, revealing gene expression patterns and transcriptional regulatory networks that are shared with hindbrain and spinal cord development in vertebrates [9–11]. These close parallels are especially striking considering the obvious, drastic reduction in size and complexity of the Ciona MG relative to the corresponding regions in vertebrates. However, it was found that cell fate specification and transcriptional patterning in the Ciona MG depends largely on cell–cell contact-dependent signaling within the neural tube by the Delta/Notch and Ephrin/Eph pathways, not on gradients of secreted, long-range morphogens as in the vertebrate spinal cord [11, 12].
Tunicates have also garnered recent attention for the fact that their extremely reduced, stereotyped cell lineages are highly conserved even between distantly related species. In spite of highly elevated mutation rates genome sequence divergence, and deep evolutionary timescales [13–16], the embryos of distantly related solitary tunicates like Ciona and Molgula (estimated divergence 390 million years apart)  are nearly indistinguishable . We previously compared the development of the cardiopharyngeal mesoderm between Ciona robusta and Molgula occidentalis and found that there were virtually no differences in cell lineage and gene expression. However, we did find that underneath this seemingly conserved developmental program, there were considerable cryptic functional differences, resulting in cis-regulatory "unintelligibility" between Ciona and Molgula. In other words, homologous cis-regulatory elements driving identical gene expression patterns were partially or completely non-functional in cross-species transgenic assays. This phenomenon was ascribed to a particularly acute form of developmental system drift (DSD) . It was recently proposed that the acute DSD observed in solitary tunicate evolution may be directly related to their mode of development, which depends primarily on invariant, contact-dependent intercellular signaling for cell fate induction. This geometric constraint would have relaxed constraints on genome evolution imposed by the relatively intricate and immutable transcriptional networks and cis-regulatory logics required for inductive events in species with larger, more variable embryos (i.e., the "Geometry vs. Genes" paradigm) .
Here we report that the phenomenon of acute DSD also extends to neurodevelopment in relatively late phases of tunicate embryogenesis. By surveying the development of the M. occidentalis MG, we show that transcription factor expression patterns, or "codes," in the developing MG are identical between Molgula and Ciona, indicating near-perfect conservation of arrangement of neuronal subtypes. However, cross-species reporter assays revealed acute DSD in the regulation of Dmbx transcription in homologous ddN precursor cells. Our results support a "Geometry vs. Genes" model to explain the emergence of DSD in the tunicate MG, given its patterning by invariant cell–cell contacts and the parsimony of its final configuration as inferred by the Ciona connectome.
Libraries prepared for the version of the Molgula occidentalis genome (ELv1.2) currently available on ANISEED (https://www.aniseed.cnrs.fr/) [17, 20, 21] were reused to reassemble the genome. The original 3 DNA libraries of paired-end reads had insert sizes of 300–400, 650–750, and 875–975 base pairs (NCBI SRA ID# PRJNA253689) . Reads were first filtered to a kmer coverage of 100X using the Khmer suite . Assembly was done using Oases (v 0.2.08)  at various word lengths (k), with k = 67 being selected as the assembly to continue the downstream analyses. After reassembly (version "k67"), additional scaffolding was done using Redundans  producing the version "k67_R" (available at https://osf.io/3crup/). The ANISEED genome (ELv1.2) was scaffolded with Redundans as well, for comparison (version "ANISEED_R"). First redundant contigs were detected and selectively removed, and next genome fragments were joined using the previously mentioned libraries from the initial assembly. Finally, gapped regions of the scaffold were filled using these 3 paired-end libraries. After reassembly and additional scaffolding, the scaffolding quality was examined using REAPR (v1.0.17)  mapping all three libraries to back to the assembly.
Gravid Molgula occidentalis (Traustedt, 1883) adults were collected and shipped by Gulf Specimen Marine Lab (Panacea, FL). Eggs were fertilized as previously described . M. occidentalis embryos and larvae were grown at ~ 24 °C and collected into three separate stage pools: 0.33 hours post-fertilization (hpf), 7, and 16 hpf. Total RNA was extracted from each pooled sample using RNAqueous Total RNA Isolation kit (ThermoFisher). PolyA+ mRNAs were selected using oligo d(T)25 magnetic beads (New England Biolabs) following two rounds of the manufacturer's protocol. Directional RNAseq libraries were prepared according to a modified version of the protocol contained at: https://wikis.nyu.edu/pages/viewpage.action?pageId=24445095 . First-strand synthesis was performed using Super Script III (ThermoFisher), primed with oligo d(T) and random hexamer primers. Second-strand synthesis was performed using dUTP instead of dTTP for directional (strand-specific) sequencing . Samples were processed and ligated to NETFLEX DNA barcode adapters (BioO Scientific) for multiplex sequencing. Adapter dimers were removed using AMPure beads (Agencourt). Samples were then treated with uracil-DNA glycosylase, amplified using 12 cycles of PCR, and purified once with a minElute kit (Qiagen) and once with AMPure beads. Samples were sequenced by Illumina 2000 2 × 50 bp runs on two lanes (all three samples were multiplex-sequenced in each lane). Resulting sequences will be deposited in the NCBI SRA database (accession number pending).
Paired-end sequences generated above were used to generate gene model. Reads were quality filtered/trimmed using trimmomatic (v0.33)  and the following parameters "MINLEN:25, and sliding window of 4 with a minimum score of 5 (ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 MINLEN:25)." Each of three embryonic stage libraries (0.33, 7, and 16 hpf) were individually mapped to the various genome versions using hisat2 (v 2.1.0) , with the default parameters. The sam files generated from hisat2 were then sorted and converted to bam using samtools (v 1.5)  and merged using PicardTools with java version 1.8 (http://broadinstitute.github.io/picard/). The merged bam file was then processed using StringTie (v 1.3.3b)  to create gene models. Gene models were then extracted using the gffread utility from cufflinks  and evaluated using BUSCO for completeness . Transcripts/gene models have been deposited at OSF (https://osf.io/3crup/).
M. occidentalis eggs were fertilized, dechorionated, and/or electroporated as previously described . Ciona robusta (intestinalis type A) adults were collected and shipped by M-REP (San Diego, CA) and eggs dechorionated, fertilized, and electroporated as previously described . Dmbx cis-regulatory DNAs from two Molgula species (M. occidentalis and M. oculata) were cloned intro reporter plasmids as illustrated in supplemental sequences files (Additional file 1). Cirobu.Dmbx>Unc-76::Venus and Cirobu.Fgf8/17/18>H2B::mCherry reporter plasmids were published previously [10, 11].
Templates for antisense riboprobes for in situ hybridization were amplified by PCR or SMARTer 3'/5'-RACE (Clontech) from cDNA libraries or from genomic DNA (see Additional file 1 for details on each sequence). Template sequences were cloned either using TOPO-TA cloning (ThermoFisher) into pCRII dual promoter vector, or using restriction enzyme cloning into pCiProbe (see Additional file 1) linearized NotI-EcoRI. In vitro transcription of labeled riboprobes and two-color fluorescent in situ hybridization were performed as previously described .
We recently sequenced the genomes of 3 species in the genus Molgula: M. occidentalis, M. oculata, and M. occulta , which can be browsed freely on the Tunicate molecular biology database ANISEED (https://www.aniseed.cnrs.fr/) . Of these, M. occidentalis emerged as a valuable species for comparative studies of tunicate development, mainly because their zygotes can be transfected with plasmid DNAs via electroporation, much like the major tunicate laboratory model species in the Ciona genus (C. intestinalis, C. robusta, and C. savignyi). To help establish additional molecular tools for developmental studies in M. occidentalis, we assembled a transcriptome based on RNAseq of 3 stages of embryonic development. These were then used to predict a new set of gene models found in the M. occidentalis genome sequence.
To do this, we also reassessed our previous M. occidentalis genome assemblies. In previous assemblies, some of the low-coverage regions were removed using khmer to reduce assembly fragmentation . To preserve the information contained in these low-coverage regions, we re-assembled the genome from the original raw sequencing reads (see "Methods" section for details). While this new assembly initially had an N50 of only 519 bp and over 680,000 scaffolds, additional scaffolding decreased the number of scaffolds to 30,188, with 20,616 of those (68%) being over 1 kb in length (Table 1). As a result of this better scaffolding, the N50 also increased from 519 to 18,312. The gap filling process decreased the number of missing bases (Ns) from 5,510,404 to 189,152. The end result is the "k67_R" genome assembly version, which we have deposited at OSF (https://osf.io/3crup/). While these procedures improved the genome assembly statistics, we wanted to ensure that the additional gene content was still preserved. To check this, we mapped mRNA reads and produced gene models to be tested using BUSCO . This allowed us to compare our new gene models against a defined collection of highly conserved sequences expected to be present in all metazoans, as a measure of assembly completeness. Our BUSCO results (Fig. 1) indicated that fewer reference genes were missing (73 missing out of 843 total) in the new "k67_R" assembly than in the ANISEED ELv1.2 version (141/843 missing) or even a version of the ANSEED assembly with additional scaffolding performed ("ANISEED_R," 79/843 missing). This indicates that these new versions of the genome assembly and their corresponding gene models should enhance the identification and cloning of sequences to use as molecular tools for M. occidentalis.
The Ciona MG is comprised of 22 neurons and includes a "core" MG of 5 morphologically and molecularly distinct left/right pairs of neurons . In the remainder of this study, we will only refer to the neurons on one side, for simplicity. The core MG is derived from the A9.30 and A9.32 blastomeres of the neural plate (Fig. 2a) that will give rise to cells along the lateral rows of the neural tube after neurulation and neural tube closure [36–39]. At the level of the MG, the neural tube of the Ciona embryo is formed by only four single-file rows of cells oriented along the anterior–posterior (A–P) axis: a dorsal row (roof plate), a ventral row (floor plate), and two neurogenic lateral rows from which most of the core MG neurons are specified (Fig. 2b). Ascending contralateral inhibitory neurons (ACINs) derived from the A9.29 blastomeres  have been traditionally excluded from the MG based on their more posterior location at the base of the tail. However, these are likely indispensable cogs in the MG central pattern generator, driving left/right alternation of tail contractions during swimming by glycinergic neurotransmission . Remaining MG neurons are poorly studied, and their development is largely unknown. In this study, we focused on the "core" MG neurons, those derived from the A9.30 and A9.32 lineages, because these lineages have been the most thoroughly studied MG lineages [9, 10, 39]. Expression patterns of developmental regulators and provisional gene regulatory networks have been documented in these lineages , and the induction events responsible for specifying the 5 distinct types of neurons in this core MG have also been elucidated [10, 11]. Thus, the MG is a perfect starting point for a comparative study on the evolution of neurodevelopment in tunicates.
We previously showed that the overall development of the M. occidentalis embryo is very similar to the development of the Ciona embryo . An in-depth study of the cardiopharyngeal mesoderm (B7.5 cell lineage) revealed perfect conservation of precise cell divisions and fate specification events, with only minimal differences in morphogenesis and timing of gene expression. However, other tissues beyond the cardiopharyngeal mesoderm were not surveyed. In this study, we sought to extend our understanding of tunicate evolution by comparing the developing nervous systems of M. occidentalis and C. robusta (intestinalis type A). In situ mRNA hybridization (ISH) for neural marker Celf3/4/5 (formerly Etr-1) in M. occidentalis neurula embryos revealed the CNS developing from the lateral rows of the dorsal hollow neural tube, as in Ciona  (Fig. 3a). Later, at tailbud stages, ISH for the neuronal transcription factors Neurogenin, Onecut, and Ebf revealed ongoing neuronal specification in the brain, MG, and bipolar tail neurons of the tail (Fig. 3b–d), also identical to the expression of these regulators in Ciona embryos [41, 42]. Two-color ISH of Ebf and the cholinergic marker Slc18a3 (also known as vesicular acetylcholine transporter, or VAChT)  revealed the earliest differentiating neurons of the larval CNS, the motor neurons of the MG (Fig. 3d). Given that no significant differences were revealed between Molgula and Ciona using these broad markers of neural fate, we focused our attention specifically to the developing MG, where we would be able to analyze more subtype-specific gene expression and cell fates.
In C. robusta, the expression patterns of mRNAs encoding transcription factors are dynamically regulated in the developing MG [9, 44]. Some of these patterns comprise a conserved "code" of homeodomain-containing genes that are differentially expressed along the dorsal–ventral (D-V) developing spinal cord of vertebrate embryos . However, in the developing Ciona MG, the neurogenic domain of neural tube is restricted to the single-file lateral rows of cells. Therefore, the D-V code of the vertebrate spinal cord is transposed along the A-P axis in Ciona. From these neural progenitors expressing different combinations, or codes, of these homeodomain proteins, the 5 distinct neuronal subtypes of the Ciona MG are born. We sought to characterize the expression patterns of homologous genes in the developing MG of M. occidentalis. We were able to identify these readily by performing BLAST against our transcriptome assembly, which revealed clear orthologs for the following genes: Mnx, Vsx, Islet, Nk6, Pax3/7, and Dmbx. An additional gene Lhx3/4.a had already been identified and characterized in our previous study .
In Ciona, 4 of the 5 core MG neuron subtypes are marked by either Mnx or Vsx [9, 10]. These are orthologs of conserved transcription factors that specify motor neurons (HB9, MNR2, etc.) or interneurons (Chox10, CHX10, Ceh-10, etc.), respectively. Two-color ISH revealed a neatly alternating Vsx-Mnx-Vsx-Mnx pattern on one side of the developing MG of M. occidentalis, around 8.5 hours post-fertilization (hpf) (Fig. 4a). This alternating pattern closely mirrors the alternation of motor neuron (MN) and interneuron (IN) subtypes in the Ciona MG: IN1-MN1-IN2-MN2. In the Ciona MG, the latter three are the first MG neurons to differentiate, corresponding to cells A11.118 (MN1), A11.117 (IN2), and A10.64 (MN2), which are consecutively arrayed with no intervening cells between them. This appears to be the case in M. occidentalis also. However, in Ciona there are additional non-neuronal cells intercalated between IN1 and MN1, as a result of continued proliferation in the anterior MG that ultimately gives rise to IN1 (see Fig. 2b). In Ciona, Vsx expression is restricted to post-mitotic interneurons and is not observed in A11.119, the grandmother cell of IN1 . In M. occidentalis, the lack of any discernible gap between the anterior-most Vsx+ and Mnx+ cells at first suggests that Vsx transcription starts in the A11.119 progenitor cell itself, a clear example of transcriptional "priming" of cell fate . Indeed, we observed Vsx expression in two anterior cells when observed at slightly later stages, suggesting that A11.119 divided after the onset of Vsx activation (Fig. 4b). Together with evidence for earlier Dmbx expression in the A11.120 progenitor cell (see below), these data suggest that the expression of certain MG neuron subtype-specific transcription factors is already primed in M. occidentalis MG progenitors. This heterochronic shift may be related to the ~ 10% faster development of Molgula relative to Ciona .
From the Mnx ISH, MN2 appeared to be the posterior-most cell of the core MG. The identity of this cell was confirmed by two-color ISH with Islet, a marker of MN2 fate in Ciona (Fig. 4c, d). In Ciona, MN2 is the only core MG neuron that is not derived from the A9.30 lineage. Long identified as the A10.57 cell derived from the A9.29 lineage, MN2 was recently revealed in fact to be the A10.64 cell of the A9.32 lineage instead and ultimately derived from the A8.16 neuromesodermal lineage that also gives rise to the secondary tail muscles of the larva . Despite its origin from further posterior in the embryo, MN2 becomes associated with the MG thanks to a dramatic, anterior migration along the outside of the neural tube, leapfrogging over the entire A9.29 lineage . The migrating MN2 is elongated along the A-P axis due to the extension of a leading edge that ultimately contacts the A9.30 lineage. Upon contacting the A9.30 lineage, MN2 extends its axon posteriorly, retaining an elongated cell body and forming en passant synapses with the dorsal band of muscle cells down the length of the tail . In M. occidentalis, the elongated morphology of MN2 was readily apparent by ISH, in which fluorescent signal fills most of the cell bodies to reveal their shapes. Therefore, we conclude that, in M. occidentalis, the specification and morphogenesis of MN2 are highly conserved.
While MN2 controls tail contractions in a graded manner , the other major MN in Ciona is MN1, the A11.118 cell. This neuron was shown to form large, leaf-like ("frondose") motor endplates at the base of the tail . While MN2 s are proposed to exert graded motor control during maneuvering, the all-or-none flexions of the tail that drive swimming are thought to be triggered instead by MN1 s. This cell is characterized by Mnx expression without co-expression of Islet. In Ciona, MN1 is also marked by late, sustained expression of Nk6 close to hatching, even though this gene is expressed broadly throughout the posterior A9.30 lineage earlier in development . In M. occidentalis, we found this also to be true. Nk6 expression was seen in A11.119, A11.118 (MN1), and A11.117 (IN2) at the mid-tailbud stage (Fig. 4d), but later was seen only in a single pair of cells in the embryo, presumably MN1s (Fig. 4e). This further confirms the highly conserved nature of the posterior MG and the specification of MN1 by sustained Nk6 expression.
In Ciona and the stolidobranch tunicate Halocynthia roretzi, a single Lhx3/4 is transcribed as two alternative isoforms, transcript variants 1 and 2, originating from alternate promoters (Fig. 5a) [48, 49]. In both Ciona and Halocynthia, transcript variant 1 is expressed in MG precursors, while transcript variant 2 is expressed in early vegetal pole cells and is required for endoderm and cardiopharyngeal mesoderm specification [49, 50]. We previously found that in all three Molgula genomes sequenced to date, the ancestral tunicate Lhx3/4 gene was duplicated, resulting in two paralogs, termed Lhx3/4.a and Lhx3/4.b (Fig. 5a) . In M. occidentalis, their expression patterns and putative functions appear to mirror those of the two different transcript variants identified in Ciona/Halocynthia: Lhx3/4.a is expressed in the MG, while Lhx3/4.b is only expressed in the early vegetal pole cells . Lhx3/4.a is the more conservative paralog, retaining an ancestral C-terminal peptide motif, which has been lost in Lhx3/4.b . Given that Halocynthia is more closely related to Molgula than to Ciona , this suggests a Molgula-specific duplication of Lhx3/4, followed by subfunctionalization through partitioning of the expression domains of the ancestral transcript variants in addition to protein-coding changes.
Here we looked more closely at the expression of Lhx3/4.a in the developing MG of M. occidentalis. In Ciona, Lhx3/4 is initially expressed in the posterior MG (MN1, IN2, and more weakly in MN2) and later in IN1 [9, 44]. In Halocynthia, Lhx3/4 was also detected earlier in development, in the A9.30 blastomere . In M. occidentalis, we found Lhx3/4.a expressed throughout the entire MG at the mid-tailbud stage, though more strongly in MN1 and IN2 (Fig. 5b, c). Higher expression in these cells is shared with Ciona and Halocynthia, and the weaker expression in the anterior MG may reflect fading expression from earlier stages as in Halocynthia. Based on these results, we propose that Lhx3/4.a has, for the most part, conserved the MG-specific expression pattern and putative function of the ancestral Lhx3/4 gene, more specifically those of its transcript variant 1.
The anterior-most neuron in the core MG of Ciona is the descending decussating neuron (ddN), corresponding to the A12.239 cell. The recently mapped Ciona intestinalis connectome revealed that these neurons are likely homologous to giant reticulospinal Mauthner cells (M-cells) of vertebrates and situated in a neural circuit that is topologically very similar to M-cell escape circuits . In Ciona, the ddN is specified by a gene regulatory network involving the transcription factors Pax3/7 and Dmbx . To summarize, Pax3/7 is sufficient and necessary for the specification of the A11.120 progenitor, the mother cell of the ddN, and directly activates Dmbx expression in the posterior daughter cell of A11.120, which becomes the ddN (A12.239). Dmbx in turn encodes a repressor that mediates an FGF-regulated switch for differentiation . Upon differentiating, ddNs project their nascent axons across the midline, perpendicular to the A-P axis. Thus, as their name implies, they are the only core MG neurons that decussate.
We therefore sought to characterize the expression patterns of Pax3/7 and Dmbx in M. occidentalis. ISH revealed Pax3/7 and Dmbx expression in a single cell in the MG, just anterior to the Vsx-expressing cell identified as A11.119 (Fig. 6a, b). These data indicate that Pax3/7 and Dmbx are co-expressed in A11.120, the mother cell of the ddN. While Pax3/7 expression in A11.120 is shared between Molgula and Ciona, Dmbx expression in M. occidentalis appears to be shifted earlier relative to mitotic exit of the neuron. In Ciona, transcription of Dmbx is not detected in the A11.120 cell but is activated later, specifically in the ddN, by combinatorial action of Pax3/7 (inherited from the mother cell), and Neurogenin .
To confirm that Dmbx is expressed in the A11.120 cells of M. occidentalis, we sought to determine its expression pattern at cellular resolution. We turned to electroporation to transfect embryos with a reporter construct that could selectively label the A9.30 lineage and allow us to combine ISH with immunofluorescence. To this end, we used the Fgf8/17/18 promoter from C. robusta  to drive expression of histone 2B::mCherry (Cirobu.Fgf8/17/18>H2B::mCherry) in the A9.30 lineage of electroporated M. occidentalis embryos. Activity of this reporter plasmid was relatively weak and infrequent in M. occidentalis embryos, but managed to label the 4 cells of the developing MG of M. occidentalis, equivalent to the cells A11.120, A11.119, A11.118, and A11.117 (from anterior to posterior) (Fig. 6c), and ISH + immunofluorescence revealed Dmbx expression in A11.120 at this stage (Fig. 6d). Taken together, these data indicate a M. occidentalis-specific priming of Dmbx transcription in the progenitor of the ddN, similar to the priming of Vsx in the progenitor of IN1 in this species as well (see above).
Given the unique morphology and axon trajectory of the ddNs in Ciona and their likely homology to M-cells in a conserved, pan-chordate escape network , we asked whether their morphogenesis is conserved in M. occidentalis. To label differentiated ddNs, we electroporated M. occidentalis embryos with a Dmbx reporter plasmid containing ~ 1.4 Kb of genomic DNA sequence 5' to the ATG corresponding to the predicted start codon of the Dmbx mRNA from the related species M. oculata (Additional file 1), fused to the Unc-76::eGFP fluorescent reporter gene to label axons uniformly . This construct was sufficient to label the ddNs of M. occidentalis (Fig. 7a, b). Unc-76::eGFP expression in the ddNs revealed a polarization along the medial–lateral axis, perpendicular to the A-P, resulting in their unique axon trajectory initially straight across the midline, then abruptly turning 90° to descend along the outside of the neural tube toward the tail. This closely mirrors the processes of axonogenesis and axon guidance that result in the conserved axon trajectory of the ddNs in Ciona . This suggests that ddN morphogenesis is an ancient and highly conserved process in tunicates, which also shares features with M-cell morphogenesis in vertebrates .
In our comparison of cardiopharyngeal development between Ciona and Molgula, we discovered pervasive, acute developmental system drift (DSD)  that has resulted in cross-species incompatibility of orthologous cis-regulatory DNAs that regulate identical gene expression patterns in the tunicate cardiopharyngeal mesoderm . Here we tested whether DSD might also underlie the highly conserved gene expression patterns seen in the developing MG. We found that the previously identified C. robusta Dmbx driver  was sufficient to activate reporter gene expression in M. occidentalis ddNs (Fig. 7c). However, the M. oculata Dmbx reporter plasmid, used to label M. occidentalis ddNs, was non-functional in Ciona embryos (Fig. 7d). Thus, there is an asymmetry in the intelligibility of Dmbx cis-regulatory sequences: the Ciona cis-regulatory sequence works in Molgula, but not vice versa. This incompatibility is probably not due to basal promoter incompatibility since widespread non-specific M. oculata Dmbx>GFP expression was clearly visible in a variety of cell types in Ciona (Fig. 7d). Furthermore, the incompatibility is also not likely due to differences in trans-splicing (resulting in a failure to translate the fluorescent reporter protein), since we found that Dmbx is also trans-spliced in M. occidentalis (Additional file 1: Fig. S1).
One possible explanation for this asymmetry is that, in Molgula, Dmbx is initially activated in the A11.120 cell, the mother cell of the ddN (see Fig. 6d). In Ciona, Dmbx is only detected later, in the post-mitotic ddN , and is activated by a combination of Neurogenin and Pax3/7 and depends critically on the presence of a Pax3/7 binding site located in a cis-regulatory element that controls Dmbx transcription in the ddN . Therefore, it is possible that an alternate trans-regulatory logic drives the earlier activation of Dmbx that we observe in Molgula, considering that Pax3/7 and Dmbx are transcribed simultaneously in M. occidentalis MG, not sequentially as in Ciona. The required factors for this earlier Pax3/7-independent activation of Dmbx in M. occidentalis might therefore be absent from the Ciona MG, resulting in lack of activation of the Molgula reporters in Ciona. In contrast, we show that both Pax3/7 (see Fig. 6a) and Neurogenin (see Fig. 3b) expression patterns are conserved in M. occidentalis, which would allow for proper activation of the Ciona Dmbx reporter plasmid in this species.
Alternatively, the incompatibility we observed may not be due to divergent trans-regulatory logic. In fact, we previously showed that the trans-regulatory logic of cross-species incompatibility of Mesp reporter plasmids between Ciona and Molgula is in part due to coevolution of the Tbx6-related transcription factor and its target Mesp promoter . Thus, it is possible that activation of Dmbx by Pax3/7 and Neurogenin is conserved in Molgula and that the cross-species incompatibility of our Dmbx reporter plasmids is instead due to significant cis/trans coevolution between these trans-activating factors and the Dmbx cis-regulatory sequence. Regardless of the exact underlying molecular mechanism, the asymmetric intelligibility of Dmbx cis-regulatory sequences we have uncovered is yet another example of acute DSD between Ciona and Molgula, which stands in stark contrast to the overall conservation of MG development that we have documented in this study.
Here we have documented the deep conservation of regulatory gene expression patterns that establish the precise configuration of neuronal subtypes in the MG of the tunicate larva (summarized in Fig. 8). Our data suggest that the circuitry of the MG, as revealed by the recently completed C. intestinalis connectome , may be as rigidly conserved as the embryonic development of the larva itself. We did not observe any indication that certain MG neuron subtypes identified in Ciona are missing in M. occidentalis, or that additional subtypes (not observed in Ciona) are specified in M. occidentalis. Not only does there appear to be a 1-to-1 correspondence of core MG neurons between Ciona and M. occidentalis, their position in the MG is not altered. The only differences observed are precocious transcription of certain markers in M. occidentalis (e.g., Vsx, Dmbx), which may be related to the faster developmental rate of this species relative to Ciona spp. These observations suggest that the MG of the solitary tunicate larva has changed very little in nearly 400 million years, the estimated time of divergence between Ciona and Molgula . As such, the tunicate larval MG likely represents a minimal but ancient and exquisitely adapted central pattern generator for swimming behavior.
Our finding of acute, asymmetric DSD of Dmbx regulation hints at a possible cause for divergence in the regulation of seemingly identical gene expression patterns. In this case, both C. robusta and M. occidentalis specify a single pair of Dmbx+ ddNs. However, precocious transcriptional priming of Dmbx in the ddN progenitor (A11.120) in M. occidentalis may be the result of an alternate regulatory mechanism that does not operate in homologous cells of Ciona. Thus, while the spatial pattern of ddN specification is highly conserved, likely constrained by the invariance of the MG and its developmental lineages, there have been changes to the temporal dynamics of this process. Although the developmental timing of the ddN is identical between Molgula and Ciona in terms of number of mitotic generations, Molgula development is accelerated on an absolute timescale (e.g., hours post-fertilization). This accelerated developmental rate on an absolute timescale may have required very different regulatory strategies simply to maintain the same output (i.e., specification of the A12.239 pair of cells as the ddNs). Although this hypothesis remains untested, it will be interesting to investigate in the future whether the root of the many cis/trans incompatibilities observed between these geometrically identical embryos lies mostly in their different absolute developmental rates. This would be an extension of the models for how DSD arises, posited by True and Haag  and later expanded upon [55–57]. Stabilizing selection would have maintained the same exact MG neuron arrangement, while compensatory changes would be needed to account for the different absolute developmental rates. Unfortunately, the extreme divergence of tunicate noncoding sequences (Additional file 1: Fig. S2) means phylogenetic footprinting is often uninformative when searching for functional transcription factor binding sites in orthologous cis-regulatory elements. This makes comparative cis-regulatory functional studies very difficult to pursue in distantly related tunicates, such as Ciona and Molgula.
The remarkable conservation of embryonic cell lineages and geometries between distantly related tunicates has been recently proposed to also underlie the extreme divergence of tunicate genomes . Because gene expression and cell fates are induced by very precise and invariant cell–cell contact events in tunicate embryos, the gene regulatory networks involved in their regulation have been allowed to drift, freed from the constraints imposed by the mechanisms required by larger, more complex and variable embryos. In other words, precision and robustness of gene expression in tunicates arise from the precision and robustness of the embryo itself and not its gene networks. In larger animals (e.g., vertebrates), this precision and robustness can only come from more precise and robust gene regulatory networks instead. Additionally, while the invariant geometry of the tunicate embryo may have served to release the genome from these constraints, it may have imposed a different set of evolutionary constraints. Because of their invariance, tunicate embryos are unable to compensate for errors in cell fate specification and likely experienced strong selective pressure to maintain precise embryo geometries even as some evolved to develop more rapidly on an absolute timescale, like M. occidentalis. It is perhaps due to this evolutionary ratchet that Oikopleura dioica, the tunicate with the fastest rate of development and most geometrically constrained embryo, also has the most fragmented, highly derived genome as well [58–63]. Future comparative studies among the tunicates promise to shed further light on the fascinating interplay between embryonic form and genetic architecture in evolution.
EKL and AS planned the study, did the experiments, interpreted the results, and wrote the paper. The work was supported by funds awarded to AS. Both authors read and approved the final manuscript.
We thank Rodoniki Athanasiadou and David Gresham for helping us with the RNAseq. We are also grateful for the constant support from the MoEvoDevo collaborative network: Billie Swalla, C. Titus Brown, Lionel Christiaen, Filomena Ristoratore, and Claudia Racioppi. Work in the A.S. laboratory is funded by NIH award R00 HD084814. The authors have no competing interests to declare.
The datasets supporting the conclusions of this article are available in the Open Science Framework (OSF) repository, https://osf.io/3crup/, and the Sequence Read Archive (SRA), accession number pending.
Work in the A.S. laboratory is funded by NIH award R00 HD084814.
13227_2018_107_MOESM1_ESM.docx Additional file 1. Supplemental sequences, information, and figures.


It's very easy to improve your credit score. You probably would have to think in such way which is: consistency x time = a red-hot credit score! We have done some research and have finally put down the list of important factors that will gladly help all of you folks to improve the credit score over the period of time actually.
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Groups affiliated with Knitted Knockers Support Foundation, aka knittedknockers.org, have registered with KKSF and are using their patterns, approved yarns and methods of distribution. Other Countries or groups may use different patterns, registration for knitters and crocheters, yarns, and/or methods of distribution, also providing a wonderful service to their communities.
Knitted Knockers provides free knitted breast prostheses to ladies who have undergone mastectomy or lumpectomy surgery. We ship across Canada.
Our volunteer organisation "BreiBoezem" (Dutch for Knitted Knockers) supplies knitters with the material to knit and supply women who have had a mastectomy with free Knitted Knockers. Donations are very welcome. Please contact us and we will do whatever we can to help you. Onze stichting BreiBoezem verstrekt alle materiaal aan breiers om BreiBoezems te maken, en verstrekt gratis BreiBoezems aan vrouwen die een borstamputatie hebben ondergaan. Donaties zijn zeer welkom. Neem contact met ons op en we zullen doen wat we kunnen om je te helpen.
Knitted breast prostheses are provided free of charge and can be requested via our website www.mc4bc.com. We meet in the community hub of John Lewis, Birmingham, West Midlands once a month. (Above New Street station, Grand Central) Dates and times can be found on the website.
Our volunteers also craft and gift drain bags, heart shaped support cushions, 100% cotton chemo hats and 100% cotton wash cloths. These are distributed by support groups and centres directly to ladies and gents going through treatments for breast cancer. For further information email midscrafters4breastcancer@virginmedia.com or write to Joanna Dervisoglu, Midlands Crafters for Breast Cancer, 493 Tessall Lane, Birmingham, B31 5HA.
We are a Registered Charity in the UK, based on a Facebook group of pages. Everyone involved is a volunteer, and we make knockers in 100% cotton for day wear, and evening wear. We also make Aqua knockers in soft acrylic yarn for swimming, gym activities and sunbathing. Our knockers are available in a comprehensive range of 15 sizes as standard.


Mar 2002 [act] 89108 USAF 4 FS 'HL'	 F-16C Block 40G "Lets Roll"
I Was Assigned To The 421 FS 388 EMS Working Phase When We Got The Word That This Jet Went Down And The Unfortunate News That Pilot Cpt.Hougton Did Not Make It... I Worked On This Jet And We Finished The Phase On It The Week Prior.
This was the 308 FS Commanders Aircraft when I had it.


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Science Says: Are poinsettias poisonous? Some…
Science Says: Are poinsettias poisonous? Some holiday truths
By Lindsey Tanner and The Associated Press |
PUBLISHED: December 26, 2017 at 12:00 a.m. | UPDATED: September 6, 2018 at 12:00 a.m.
CHICAGO (AP) — Are poinsettias really poisonous? Are snowflakes really pure as the driven snow? Does feasting really put on the pounds? Sure as sugarplums, myths and misconceptions pop up every holiday season. Here"s what science says about some of them:
Poinsettias, those showy holiday plants with red and green foliage, are not nearly as harmful as a persistent myth says. Mild rashes from touching the plants or nausea from chewing or eating the leaves may occur but they aren"t deadly, for humans or their pets. Poinsettias belong to the same botanical family as rubber plants that produce latex, so some skin rashes occur in people allergic to latex. According to a Western Journal of Emergency Medicine research review, the plants" toxic reputation "stems from a single unconfirmed death of a 2-year-old in Hawaii in 1919."
Dr. Rachel Vreeman, an Indiana University pediatrician who has researched holiday myths, cited a study on more than 20,000 poison control center reports involving contact with poinsettias.
"In none of those cases were there deaths or serious injury. In fact, more than 95 percent of them required zero medical care," she said.
The anglicized name comes from Joel Poinsett, a 19th century U.S. diplomat who brought the plant back from Mexico.
THE WHITE STUFF
To form snowflakes, moisture high in the atmosphere is frozen by clinging to particles that may include dust specks or soot. Add germs to that list. University of Florida microbiologist Brent Christner has found that bacteria commonly found on plants are surprisingly abundant ice "nucleators" present in snow from populated areas, barren mountain peaks and even Antarctica.
So is catching snowflakes on your tongue a bad idea?
"There"s a yuck factor," Christner said. "It"s better than yellow snow."
He said the number of bacteria in snow would probably be about 100-fold less than in the same amount of bottled water.
"There are a lot more things to be worried about in making you sick than ingesting snowflakes," he said.
The same things that can make holidays merry — great expectations and family time — can also be stressful. Holiday blues are a real thing for many people grieving loss or absence of a loved one, and wintertime can trigger true but transient depression in some people, a condition sometimes called seasonal affective disorder. It"s linked with lack of sunlight in winter and some scientists think affected people overproduce the sleep-regulating hormone melatonin. Research suggests it affects about 6 percent of the U.S. population and rates are higher in Scandinavia. But contrary to popular belief, suicides peak in springtime, not winter. No one has figured out why.
HAIR OF THE DOG
Forget that bloody mary. If extra shots of bourbon in your eggnog have you feeling lousy the next day, drinking more alcohol — hair of the dog — won"t cure you.
Here"s what George Koob, director of the National Institute on Alcohol Abuse and Alcoholism, has to say about that:
"You are in a sense self-medicating a mild withdrawal syndrome by drinking more. The problem is that"s going to wear off and you"re going to have an even worse hangover."
Alcohol is dehydrating so replenishing with lots of water or other non-alcoholic drinks can help relieve the symptoms. But experts emphasize that prevention is the healthiest cure.
Says Koob: "It all boils down to, don"t drink too much."
So what about that saying, "hair of the dog?" According to an old folk remedy, a dog bite could be cured by putting the animal"s hair in the wound.
DOUGHN"T EAT IT
Bakers beware: sampling holiday cookie dough, or any raw dough, can make you sick. And recent research says it"s not just because dough often contains raw eggs, which may harbor salmonella bacteria. Flour is another culprit. A study published last month in the New England Journal of Medicine details a 2016 E. coli outbreak that hit dozens of people in 24 states that was linked with flour. Some patients had eaten or handled raw dough made with flour contaminated with that bacteria. Authorities recalled 10 million pounds of flour, some of which had been sold to restaurants that allow children to play with raw dough while waiting for their meals. Baking generally kills any bacteria.
A headline on a Food and Drug Administration consumer update sums up the agency"s advice: "Raw dough"s a raw deal."
The truth about holiday weight gain depends on whether your Champagne glass is half empty or half full. One often-cited study says it"s commonly assumed that the average American gains 5 pounds between Thanksgiving and New Year"s Day. But the study authors found the average was a little less than 1 pound. Other studies have found it"s closer to 2 pounds, still barely enough to make your pants feel tight. An extra piece of pie or one gigantic holiday feast won"t doom you, says Indiana University"s Vreeman. The problem, she says, is that the extra pound or two at holiday time becomes a pattern year after year and adds up.
Follow AP Medical Writer Lindsey Tanner on Twitter at @LindseyTanner. Her work can be found here.
This Associated Press series was produced in partnership with the Howard Hughes Medical Institute"s Department of Science Education. The AP is solely responsible for all content.
Lindsey Tanner

redpajama_set_name: RedPajamaCommonCrawl


a. An administrative officer in charge of a college, faculty, or division in a university.
b. An officer of a college or high school who counsels students and supervises the enforcement of rules.
2. Ecclesiastical The head of the chapter of canons governing a cathedral or collegiate church.
3. Roman Catholic Church A priest appointed to oversee a group of parishes within a diocese.
4. The senior member of a body or group: the dean of the Washington diplomatic corps.
Dean, Jay Hanna Known as "Dizzy." 1911?-1974.
American baseball player. A pitcher for the St. Louis Cardinals (1930-1937), he is best remembered for his 1934 season, in which he won 30 regular-season games, led the league in strikeouts, and won two World Series games.
Dean (dēn), James Byron 1931-1955.
American actor remembered for his moody, rebellious persona in films such as East of Eden and Rebel Without a Cause (both 1955).


The idea that it's not where you start but where you finish makes for a nice soundbite — or lead graph in a baseball preview story — but it won't fly on the Flats. Not anymore.
In 2012, it would have fit after injuries decimated the pitching staff, and actually became something of a moral for the season when the Yellow Jackets qualified for the ACC Tournament on the final day of the season, tore through the ACC Tournament, qualified for the NCAA Tournament and advanced to the Gainesville Regional title game, where they fell to top-seeded Florida.
But 2013 is not 2012 and the Yellow Jackets have no plan to wait as long as they did to earn a spot in the ACCs, or stop playing as early as they did in the NCAAs.
This year's squad is determined to make a statement right off the bat — starting Friday afternoon at 4:00 p.m., when they host the Akron Zips.
Junior Daniel Palka will play a big part in supplying the exclamation point on the opening-weekend statement and pretty much any statements made this year. He can't wait to get started.
Tech baseball head coach Danny Hall is putting a lot on the shoulders of the 6-4, 234-pound Palka, a Second Team All-ACC performer last year, one of four All-ACC honorees from last year, three of whom are returning (pitcher Buck Farmer and outfielder Brandon Thomas were First-Teamers, and are returning, while Jake Davies also was Second Team All-ACC). Hall feels that the slugger is ready to carry the burden of being a team leader as well as being the big bat in the heart of the order.
Nobody on the current roster has turned pitches around as often as Palka, whose 24 career home runs more than double the nearest returnee — shortstop Mott Hyde has 11 — and whose 97 RBIs and 37 doubles also are team-highs. Palka also is one of four players with a career .300 batting average (.302, third on the team), he's third in career triples (five), and fourth in hits (141), and runs (85).
The Lyman, S.C., native likes the chances of those numbers going up as the Yellow Jackets' offense is loaded with speed in front of him in center fielder Kyle Wren, left fielder Brandon Thomas, and jack-of-all-trades Sam Dove, and pop behind him in catcher Zane Evans. Palka and Evans may switch places in the order, but regardless of whether it's Palka-Evans or Evans-Palka, there are going to be good pitches and RBI opportunities aplenty.
Palka's proved in his first two years that there's no park that can hold him and very little pitching that can stop him. He reinforced his image as a national slugger over the summer, when he competed in the TD Ameritrade College Home Run Derby, was named MVP of the Cape Cod League All-Star Game, then helped lead the Wareham Gatemen to the Cape Cod League championship (he teamed with Hyde).
But Palka is not satisfied with being one of the ACC's premiere power hitter. He's out to prove that he's one of its premiere hitters, period. He cut his strikeouts from 71 as a freshman to 50 as a sophomore and this season plans to be even more consistent about putting the ball in play when he's not hitting it out of the park.
The biggest change facing Palka this season probably will be in his position defensively. He'll see time at first base but also in the outfield.
He'll even pitch if needed, but he feels that's one area the team is set.


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text:
First New York FCU Makes History with First In-School Branch
SCHENECTADY, N.Y. - First New York Federal Credit Union and Schenectady High School have broken new ground here by opening the Capital District's first in-school branch office. The "Patriot Branch" is housed in Schenectady High School and will be run by student volunteers drawn from the Global Commerce House classes...
By Staff Writer | April 05, 2005 at 08:00 PM | The original version of this story was published on Credit Union Times Magazine
SCHENECTADY, N.Y. – First New York Federal Credit Union and Schenectady High School have broken new ground here by opening the Capital District's first in-school branch office. The "Patriot Branch" is housed in Schenectady High School and will be run by student volunteers drawn from the Global Commerce House classes to serve as an introduction to the business world while giving students hands on experience in marketing and the credit union environment. The experience is part of the curriculum, with students being graded for their participation. The branch will open one day a week on Fridays from 10:45 a.m. to 1:15 p.m., and will be available for use by students and staff members to open new accounts and conduct financial transactions. To spread the word students recently conducted a "March Madness Membership Drive" promotion.
The District CU Names Steve Bouras as New CEO
Michael Ogden | January 13, 2022
Bouras takes over the new position from retiring CEO Brad Johnson.


Studies in Time Division by Larry Teal. Encore Publications, 1955, SS, 24 pages. Subtitled "A Practical Approach to Accurate Rhythm Perception Concepts." These studies are aimed at developing an accurate rhythmic sense built up from the sixteenth-note time value. They can be used for both private and classroom study and for any instrument (the examples are in treble clef). These exercises start with basic rhythms and conclude with those that might be found in upper intermediate level music.


text:
Microsoft Adds Advert Game To Xbox Live Arcade
Two games will hit the Arcade this week, Microsoft announced. The RPG puzzler Puzzle Quest: Challenge of the Warlords will be the spearhead, followed closely by the advert game Yaris.
Puzzle Quest: Challenge of the Warlords will become available tomorrow and will cost 1200 Microsoft Points. The game will have the players fight against the evil side once again. Among its top feature we can mention support for widescreen, HD display and LIVE features such as camera support, Achievements, leaderboards and Gamerscore.
In the mean time, Yaris will come free of charge, but only if you're living is the US or Canada. Otherwise, it won't come at all. According to Microsoft's John Porcaro, the Yaris game is a North America-only offer because:
"the game's portrayal and model options of the Yaris aren't available in all global markets, and the specific brand positioning of the Yaris is unique to the U.S. and Canada."
The player will take the wheel of a customizable Toyota Yaris and will get busy destroying enemies and avoiding obstacles, hoping to get to finish the mad race, be in single player mode or in multiplayer over Xbox Live.
Yaris will be available on Xbox Live Arcade for one year.
Categories: Games Tags: adds, advert, arcade, game, live, microsoft, xbox
40GB PlayStation 3 Model Takes The Plane To The US. Spiderman Follows
Fable II Hits The Stores This October
Burnout Paradise Ready To Race On PC
BioShock Not Shocking Enough For Gears OF War
PSP Goes Red In Europe And Japan
Ikaruga Arrives On Xbox Live Arcade
Disney Launches Online Nintendo DS Service: DGamer
EA Pulls Over The Need for Speed Series?
Microsoft Trims Prices On Three Xbox Live Arcade Titles
Sony Prepares New Battery For PlayStation Portable
Fallout 3 Arrives On Steam
PC Sales Charts: A Tale Of Two Sims


WHEN preparing these verses for the press, the first question which met me was this: Should Boake be treated from a literary standpoint or from a personal standpoint—as poet or as man and poet? I chose the personal standpoint. To consider Boake as a literary figure, with an eye chiefly to his literary reputation, meant omission of whatever herein is crude or weak, in order that readers should judge him only by his best. Such a course implied loss and discontent to many persons who care little for the niceties of style, and find in vigorous picturing and natural emotion ample amends for bad rhymes and false accents And, what is more important, such a course would have given a wrong impression of Boake. Some of his most valuable work lies in fragments of poems which as wholes seem comparatively ineffective. These fragments could not be divorced from the context, yet were not lightly to be discarded. Further, Boake's least remarkable compositions, with two or three exceptions, are as characteristic of Australia and of himself as are the most remarkable. So, instead of trying to exalt the Poet by his work, I have tried rather to show the Man in his poetry.
This decision brought an easy answer to the second question: How far was it justifiable to prune or polish Boake's verses: how far was it desirable? Plainly, the less trimming the better; since his errors are a part of Boake, and every one removed helps to misrepresent him. Nor was it certain that a foreign hand would not mar as much as it mended. Sometimes Boake has gained variety and force at the expense of metre and rhyme; and to alter is to plunge deep in the old controversy whether gain of mechanical correctness balances loss of picturesque vigour. So I have not attempted to patch Boake's garment with alien cloth. I have carefully compared the printed poems with available originals, correcting errors of the press and adopting a few alternative readings sanctioned by MS.; and occasionally I have strengthened a line or changed a word where the advantage seemed obvious or the necessity great.
The order of the verses is that of convenience. There is in many cases no clue to the precise dates of composition, which did not coincide with the dates of publication: hence exact chronological order is unattainable. And Boake's poetic harvest was reaped in so brief a time—some eighteen months—that there is little material difference of merit between his earlier and his later verses.
Of the thirty-one indexed poems which follow, twenty-five were printed in The Bulletin, and two in The Sydney Mail: four now appear for the first time. The Bulletin Newspaper Company, Limited, is acting in the matter of this publication as trustee for Boake's near relatives, who share with the publishers. Messrs. Angus and Robertson, any profits accruing after expenses have been paid.
I have to tender warm thanks to Messrs. F. P. Mahony, A. J. Fischer, and G. W. Lambert, the artists who have illustrated the volume, and to whose talent and pains is due much of any interest it may possess. I gratefully acknowledge, also, the care and skill of the engravers, Messrs. Benton and Bacon, Mr. Irwin (of The Daily Telegraph), and Mr. Hamilton (of The Bulletin); and especially of the printers, Messrs. Websdale, Shoosmith, and Co.
And, identifying myself as nearly as may be with Barcroft Boake, to the memory of his mother and to Australia I dedicate this book.
The Land of Dumb Despair.
That meets the March of Hell.
The Land of Dumb Despair!
And fling the goblets down.
The grief of all the world.
Which draught such thirst would slake.
So many ways to Death!
Would faint in such a pall.
Each leaves beside the board.


University of Arkansas, has received a $650,000 award from the National Science Foundation.
Colin Heyes, an assistant professor in the department of chemistry and biochemistry in the institute was instrumental in receiving the award.
The Faculty Early Career Development Program award was given to further his investigation of the interfaces between the core and shell of colloidal quantum dots.
Colloidal quantum dots are microscopic semiconductor crystals that are grown in solution.
the structures of the core-shell or shell-ligand interfaces and the trap states will allow us to more precisely control these excitons that underlie the optical and electrical properties," Heyes says.
The grant will support Heyes' research in this area for the next five years and will encourage and promote the participation of graduate, undergraduate and minority students. As part of the grant, a two-week, hands-on workshop will be held each summer on the U of A campus.


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List of courses offered in the University of Ghana 2020/2021
List of courses offered in the University of Ghana 2020/2021 University of Ghana (UG Legon) Admission 2020/2021 Applications are invited from qualified applicants for admission into regular, sandwich and distance programmes at University of Ghana, Legon for the 2020/2021 academic year. Don't undergo the strain of going back and forth browsing other websites which can end you nothing. Trainingforms.com is favorite students' portal with the aim of providing only unique updates. [expand title = "COLLEGE OF HEALTH SCIENCES"] School of Medicine and Dentistry
School of Biomedical and Allied Health Sciences
Bachelor of Science in Dietetics
Bachelor of Science in Medical Laboratory
Bachelor of Science in Occupational Therapy
Bachelor of Science in Physiotherapy
Bachelor of Science in Radiography
School of Nursing & Midwifery
Bachelor of Science in Midwifery [/expand]
[expand title = "COLLEGE OF BASIC AND APPLIED SCIENCES"] Groupings Group 1: Physical Sciences Physics, Chemistry, Geophysics Group 2: Mathematical Sciences Mathematics, Statistics, Actuarial Science, Computer Science, Biomathematics, Physics Group 3: Earth Sciences Geology, Applied Geology, Applied Geophysics Group 4: Information Technology Group 5: Biological Sciences Animal Biology and Conservation Science, Biochemistry Cell and Molecular Biology, Nutrition, Food Science, Plant and Environmental Biology, Marine Science, Fisheries Science, Psychology, Microbiology. Group 6: Agriculture Animal Science, Crop Science, Soil Science, Agriculture Economics, Agribusiness, Agriculture Extension, Post-Harvest Technology. Group 7: Family and Consumer Science (Food and Clothing Option) Group 8: Family and Consumer Science (Family and Child Studies Option) Group 9: Engineering Sciences Material Science and Engineering, Computer Engineering, Biomedical Engineering, Food Process Engineering, Agriculture Engineering Group 10: Doctor of Veterinary Medicine [/expand] [expand title = "COLLEGE OF HUMANITIES"] University of Ghana Business School
University of Ghana School of Law
Bachelor of Laws (LLB)
School of Arts – Bachelor of Arts –Available in the following Departments:
Department of Religions
Department of Philosophy and Classics
Department of Archaeology and Heritage Studies
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Department of French
Department of Modern Languages
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Department of Geography and Resource Development
Department of Social Work
School of Performing Arts – Bachelor of Fine Arts/Bachelor of Arts –Available in the following Departments:
Department of Dance Studies
Department of Theatre Arts
Department of Music [/expand]
[expand title = "COLLEGE OF EDUCATION"] School of Information and Communication Studies
Bachelor of Arts in Information Studies
School of Education and Leadership
Bachelor of Arts in Education
Bachelor of Science in Education
Bachelor of Arts in Sports and Physical Culture Studies
School of Continuing and Distance Education
Bachelor of Arts – Available in the following Departments:
Department of Information Studies
Department of Adult Education and Human Resource Studies
Bachelor of Science in Information Technology
Bachelor of Science in Nursing (For professional nurses who already have a Diploma in Nursing from a recognized Nursing Training College)
Diploma Programmes The following diploma programmes are now run at the Accra, Kumasi and Tamale Learning Centres. Applicants should note that the all distance education programmes are non-residential.
Diploma in Public Administration
Diploma in Statistics
Diploma in Librarianship
Diploma in Archives Administration
Diploma in Adult Education
Diploma in Youth Development Work (CYP) [/expand]
University of Ghana College of Education Entry Requirements
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API Marine Participation in SMM 2016 – The Best Ever!
From 6th to 9th of September 2016 API Marine participated in the outstanding Shipbuilding, Machinery and Marine technology exhibition (SMM). We had the pleasure of being visited by many of our existing customers and partners, as well as new and potential clients and partners.
The exhibition is held in the maritime heart of Europe, Hamburg, and it draws visitors and exhibitors from all around the globe. This makes it an excellent place to attract the attention of large number potential local and international companies.
API Marine presented its range of high quality sensors and solutions. In addition, several new products and solutions were launched – expanding and strengthening solutions being offered to our clients.
Particularly the new API Marine Tank Level Radar received a high level of interest from shipyards and ship owners – the API Marine Tank Level Radar is a natural addition to the high quality sensors being offered by API Marine, and will greatly enhance our ability to offer attractive solutions to our customers.
Furthermore, API Marine launched at SMM new Valve Remote Control System (VRC) – a complete and flexible solution for various types of vessels – The first orders for VRC combined with Cargo monitoring and control system have already been booked.
With over 50,000 trade visitors, 2,100 exhibitors from 67 countries SMM is one of the most important events in the maritime industry calendar – this was a truly successful event for our company!


Panels come individual and we supply a white surround to hold panels together. The panels are available in a range of colours on the face side including anthracite and white on the back. Rollers, tracks and automation available but not included in this listing. We can cut panels to any size you require. Want something different please ask.
The item "Modern Metal Drive Gates Sliding panels Wooden Gates Sheds garden Entrance Thick" is in sale since Wednesday, April 25, 2018. This item is in the category "Garden & Patio\Garden Fencing\Garden Gates".
The seller is "tradegaragedoorsuk" and is located in Cannock. This item can be shipped to United Kingdom.


Today I want to share a quick and easy treat!
I love the way these look laying about on the table.
and you gotta love the ears and tail!
In a microwave melt chocolate bark. I used white and dark chocolate. Dip cherry in melted chocolate and set on wax paper. Lean a chocolate kiss on the cherry and place two almonds in between to make the ears and let cool. That's it! Make as many as you want.
Scatter then around the table at your party so they can be picked up and eaten by the tail of course!
Halloween is fast approaching and they are calling for rain!
Poor little trick or treaters!
I was hoping to greet them with this sweet arrangement.
One thing I love about this time of year are all the pretty fall colors. Whether it is Halloween or not, pumpkins can be used through Thanksgiving!
That is why I decided to make the Owl Succulent Pumpkin.
I wanted to use my Lady Baltimore Luggage!
What a beautiful weekend to be at the fair!
This was the first time I had been to the Country Living Fair. I happen to win a ticket from Country Living so it made going that much easier.
This was a Hometalk meetup too!
Linda from It All Started with Paint is here helping me out today!
If you haven't noticed I have a thing for hardware!
Decorative knobs on furniture is the icing on the cake for me.
It is what sets off a great piece.
Hometalk asked me to curate a board of some great knob posts to get your creative ideas flowing!
I love all that are out there and now you can pin them and have them all in one place!
I would love for you to go check them out!
Leave me a comment and let me know which one is your favorite!
Also congratulations to Kelli Fox for winning the Country Living Fair 3-day passes!!
I have been to several barn sales and fairs but I have never been to the Country Living Fair!
But I am going NOW!
I will be traveling to Atlanta next weekend to shop and enjoy the beauty of Stone Mountain!
One lucky winner will be chosen randomly to win 2 3-day passes for the Fair!
Read bottom to see how to enter!
I want to meet you so please enter!
NEW YORK (August 20, 2013) – Country Living magazine announces the 2013 Country Living Fair in Atlanta, taking placeOct. 25-27 at Stone Mountain Park. The Fair is expected to draw more than 20,000 visitors from across the country.
The Country Living Fair in Atlanta will give attendees the chance to enjoy the Country Living lifestyle in a fun, festival atmosphere with cooking, crafting, and other DIY demonstrations, as well as fantastic local and artisanal food. In addition, guests will have access to a unique shopping experience featuring more than 200 vendors offering antiques, gifts, home décor, jewelry and more.
Attendees will be able to engage with Country Living through several social media initiatives at the Country Living Fair, including Facebook, Twitter, Instagram, Google+ and Pinterest. The Country Living Fair hashtag for all platforms is #clfair.
Go RVing and Mahatma Rice are sponsors of this year's Country Living Fair in Atlanta. The Fair is produced by Stella Show Management Company. For additional details visit www.countryliving.com/fair; to buy tickets visitwww.stellashows.com or call (866) 500-FAIR.
· Children ages 16 and under are free.
Held rain or shine outdoors under tents, the Fair takes place on the grounds of the Stone Mountain Park, a scenic recreation area 16 miles east of downtown Atlanta [Exit 8 off of U.S. Highway 78]. Regular parking fees apply daily.
Country Living (www.countryliving.com) is a shelter-lifestyle magazine focusing on a variety of topics including decorating, antiques, cooking, travel, remodeling and gardening. In addition to its U.S. flagship, Country Living publishes a United Kingdom edition. Country Living is published by Hearst Magazines, a unit of Hearst Corporation (www.hearst.com), one of the nation's largest diversified media and information companies with interests in magazines, newspapers, digital media, business media and television. Hearst Magazines is a unit of Hearst Corporation, one of the nation's largest diversified media and information companies. With 20 titles in the U.S., Hearst is the leading publisher of monthly magazines in terms of total paid circulation (ABC 2012) and reaches 82 million adults (Spring 2013 MRI gfk). Follow Country Living on Twitter, Facebook, Pinterest and Instagram.
Stone Mountain Park is home to Georgia's largest campground with over 400 sites for everything from full size RVs to tents. Stone Mountain Park is a green oasis where families and friends can enjoy the outdoors together. Listed in Frommer's 500 Places to Visit with Your Kids Before They Grow Up and cited by the Atlanta Journal-Constitution as one of "35 Natural Wonders in Georgia You Must See Before You Die," Stone Mountain Park is located just 16 miles east of downtown Atlanta. The five square mile park is Georgia's most visited attraction. Home to the world's largest piece of exposed granite, Stone Mountain Park offers a variety of attractions, entertainment and recreation.
This is what you have to do!
Good Luck and please share on your social media pages!!
Congratulations Kelli Fox! Please email me for details. If you are unable to attend I will pick another winner. Thanks!!
Sharing my dream trip with you has been so helpful in keeping my memories vivid. I will always be able to look back here and relive this!
You see, being an artist I was fascinted by great pieces of art and sculpture.
Do you still love succulents?
How about a Succulent Owl Pumpkin?
I am over at Inspiration Cafe at Silo Hill Farm. Come by and check it out!
I hope you have enjoyed the few things that I shared. Please stop and look around at some more. You can search on the side bar.
Thanks again Danni! I love that our blogs have brought us together and started a friendship!


There is an air of excitement among the hackerspaces of Europe, because this month is hacker camp season. In Denmark they have Bornhack beginning on Thursday, in Italy IHC was held earlier in the month, while here in the UK we are looking forward to Electromagnetic Field. We're excited be at Eastnor Castle for Electromagnetic Field at the cusp of August and September for several days under canvas surrounded by our community's best and brightest work. We'll even have a Hackaday Readers' Village this year!
If you've never been to a hacker camp before, this is one that's not to be missed. Technically this is camping, but where every structure from the smallest tent upwards has mains power and gigabit Ethernet. It's the equivalent of a music festival if you replace the music with technology and other cool stuff from our world. There are talks on a huge variety of fascinating subjects, the chance to see up close some of the things you'll have read about here on Hackaday, and best of all, a significant proportion of Europe's hackerspace communities all together in one place. They are a uniquely stimulating and exciting environment.


1 checked in bag 5 carry on cases 6 backpacks 2 buggies 1 baby carrier 4 children 2 very tired parents.
Foremost of all, I felt this holiday was one that precisely was not meant to go on. With several people close to the family getting seriously ill in hospital and we had no passports for the children, even though, we applied for them a month before we were due to depart.
I've just made out a few tips to help you learn from my mistakes about going on a plane with kids.
Do not book the earliest flight… It may be cheaper, but your sanity isn't worth it. We were flying out at 7.40 am, so that meant we had to be up at 4.15 to go to the airport, we did pack the car the night before, best tip ever as it causes less stress. So, we got to Quick park in Dublin airport at 5 am to then park the car, unload the car. (Remember 1 checked in bag 5 carry on cases 6 backpacks 2 buggies 1 baby carrier 4 children, 2 very tired parents) get the stuff to the bus stop while a bus is waiting for us. Kids crying because its cold and they are tired. If I had booked a later flight we could have gotten to airport earlier, hubby could of dropped me and the kids (or half of them) to the airport with the bags, drove back to the car park and park it while we waited for him, by the time we are on the flight the kids were wired and over tired it's a 4 hour flight to Fuerteventura and they were like a pack of wired cats that got high on skittles. Then they were too excited to be staying in a new place and to see their grandparents and brother.
One big thing is if you are flying with Ryanair don't try to save the money by not booking in luggage. Its €70 return for a bag, but it's well worth it because as we thought we'd save a bit of money and only book in a 15-kilo bag for the 6 of us and carry on the 5 carry-ones that we are allowed to. But completely forgot about the fact that the two-year-old and the five-year-old cannot manage the cases and so Mammy and Daddy needed to try to wheel all these as well as try to mind the three younger kids.
If you are bringing a child under 4 I'd highly recommend bringing a buggy for each child. We brought two as even though our two-year-old won't sit in a buggy at home, she really needed it when we were away it's a lot of walking and also late nights. We used one for the baby (5 months) also and I brought my baby carrier so I could strap him on. This came in handy as when the five-year-old got tired we put her in the baby buggy and I carried him. Less moaning and when Daddy was sunburned he didn't have to carry her much on his shoulders. If you are doing this and don't need it in the airport you can ask them to check it in with your luggage, you just need a sticker label of them and bring it to oversize baggage.
Security is a nerve-racking place even for people who don't suffer anxiety. I just need to see a guard and I panic that I'm breaking the law. I've watched enough of banged up a board and border control to make me think awful things will happen to me. So, make sure all liquids are in the bags that are required by the airport. But baby food, bottles of milk, baby medicine, yoghurts, etc. don't need to be under the 100 ml and you can carry as much as you like. But try to have them all handy in the changing bag so you can take them out and put them into the tray. The security needs to test these by dipping a swab into them you must agree to this. Also, make sure there is nothing in the buggy basket or in any pockets. This need to be all emptied out. And they also swab the buggy for drugs, I bought it only a day or two before and had never used it that was a major eek moment.
With 3 kids at that hour of morning say goodbye to any airport, shopping you think you might get done. Just head straight to the gate. And if you are flying with Ryanair it's a long, long walk to the gate. Probably 20 minutes for an adult with two wandering and easily distracted girls its about 40 minutes.
Make sure you have tablets charged earphones that work, different colours and books and teddies whatever your child uses to keep quiet, I had these, but none kept mine quiet, the five-year-old decided to sing the song from tangled at the top of her voice I had to keep telling her to keep quiet. Also, bring soothers bottles and hard sucky sweets for taking off and landing.
I must say Corralejo was a beautiful place and we stayed in Oasis Dunas .Click Here. It was perfect for the children, with 3 pools 4 water slides and 2 playgrounds. They also have a kid's club; however, we didn't avail of this. A pool bar that served lunch and drinks all day and a separate bar that opened in the evening a supermarket and a restaurant. The apartment was lovely there were two rooms with both having two beds, fan, wardrobe and dressing table and a real wooden cot for the baby The kitchen had the standard Cupboards, fridge, 2 rings ceramic hob, toaster, microwave, kettle and coffee maker, There were a sofa bed and a terrace with patio furniture and a sun lounger and where we where it was a real suntrap.
The town is lovely it has a lot of modern shops like Mango, Bershka and Zara as well as the usual tourist spots where you can get your rip of goods like fake MK bags and Super dry T-shirts. There are tonnes of restaurants my favourite while I was there were INFUSIONS and loved Toro Beach but couldn't enjoy it as the kids were misbehaving and Most bars accept kids but the friendly bar is Murphy's Irish Bar.
I came home for the holiday with 3 Spanish looking kids, they all have my shallow skin and are golden in colour and the baby is snow white & hubby is a lobster. The flight home was as much stress as the flight over, but with an added random drug swab for my five-year-old daughter, also she had a Build a Bear Teddy bear had wires in it and the security asked me if it was a bomb. I didn't even know it did anything, but if you press his ears they light up.
So after the Stressful week of a holiday I couldn't wait to get back into the norm of school runs, Think the next time we decide to go away as a family the kids will have to be a lot older than now. I need a holiday after it.


text:
Ке́мбриджские пра́вила — первый унифицированный свод футбольных правил.

История
В 1846 году Х. де Уинтон и Джон Чарльз Тринг из Кембриджского университета встретились с представителями частных школ с целью сформулировать и принять свод единых правил. Дискуссия длилась 7 часов и 55 минут, и в результате возник документ, опубликованный под названием «Кембриджские правила». Они были одобрены большинством школ и клубов, и позднее (лишь с незначительными изменениями) их приняли за основу правил Футбольной ассоциации Англии.

Правила 1862 года
Копии первоначального свода «Кембриджских правил» не сохранились. Есть правила Университетского Футбольного клуба от 1856 года. Самый ранний из существующих документов, к которым восходят современные правила Футбольной ассоциации это свод правил, опубликованный мистером Трингом в 1862 году:

 Гол считается забитым, когда мяч прошёл в створ ворот и под перекладиной, за исключением тех случаев, когда он заброшен в ворота рукой.
 Мяч разрешается останавливать руками лишь для того, чтобы установить его перед собой для удара.
 Удары должны быть направлены только в мяч.
 Запрещается бить ногами по летящему мячу.
 Запрещается ставить подножки, делать подсечки и бить противника по ногам.
 Когда мяч выбит за боковые флажки, игрок, выбивший мяч, возвращает его в игру с того же места, где мяч пересёк боковую линию, в направлении по прямой линии к середине поля.
 Когда мяч выбит за линию ворот, его возвращают в игру игрок той команды, через линию ворот которой прошёл мяч.
 Игроки команды соперников должны находиться на расстоянии не ближе шести шагов от футболиста, выбивающего мяч в игру с боковой линии или с линии ворот.
 Игрок считается находящимся в положении «вне игры», как только он оказался впереди мяча, и должен немедленно занять положение позади мяча. Если мячом владеет его команда, игрок в положении «вне игры» не имеет права трогать мяч или продвигаться вперёд до тех пор, пока кто-либо из другой команды не дотронется до мяча или кто-либо из его собственной команды не выбьет мяч на одну линию с ним или вперёд.
 В случае, если игрок находится в положении «вне игры», ему запрещается атаковать соперника. Он может начать атаку только тогда, когда выйдет из положения «вне игры».

Это были правила игры, которую сам Тринг определил как «самая простая игра». Кембриджские правила стали основой для свода правил Тринга.

Ссылки
 The Cambridge Rules

Правила футбола
1848 год в Великобритании

redpajama_set_name: RedPajamaWikipedia


text:
Sampdoria is back in Seria A!!!
On June 12, 2012 By mouseytongIn Celebrations, Italy, Misc..., Musings
If you followed my twitter or even my update on "Life in Photos", you will see that I had a great Saturday. I am not an avid football fan but in a land of football (soccer for Americans) you get swept into the celebrations. What's more if your in-laws are ALL Sampdoria fans. Except for my husband, they are all Sampdoria fans. The hardcore fans are my FIL, BIL and nephew. FIL NEVER misses a game at the stadium if it is played at home. Now, if you are a football fan, you might recognise this team. I am not a hardcore football fan so I didn't till I came here but my brother did. He follows most of the leagues especially those teams that have been in the EUFA like the other more famous teams like Juvetus, Milan AC and Inter Milan. Some cities have 2 football teams much like Manchester City and Manchester United. Truthfully, I never knew there was a Manchester City till they got into the Premier League a few year back but Man U? It would be blasphemy if I didn't! My brother is a great fan of Man U!! I had an ex who is an Arsenal fan so, Arsenal is also known to me. To really irritate him, I will cheer for Chelsea always, doing a little dance and singing "Chelsea, Chelsea!" LOL!! All in good fun! I also never miss the World Cup. I watch it whenever I can. Suffice to say, I know more about football than M and that is not a feat. M has no interest in football except to maybe watch Italy in the World Cup every 4 years.
As I was saying, Genoa like major cities here in Italy, has 2 football teams. It seems Genoa is the older and first football team, and supposedly in Italy too. I don't know how true this is. Then there is Sampdoria. Easily identifiable bu their colours (duh!) and the emblems (double duh!). I know, I know…. Anyway, Genoa has the Gryphon and its colours are dark blue and red affectionally known as "Rosso Blu" (Red Blue) whereas Sampdoria has the eagle and red, white, blue and black also known as "Blue Cerchiati" (Blue circle?). All Sampdorianis do not like gryphons and I say this because when we started the vespa restoration company and designed a logo with a gryphon on it, my FIL was visibly upset. It was funny! 😀 We incorporated a gryphon because the original Piaggio (the company that makes vespas) has a gryphon on it. We changed it to a dragon.
Why the big celebration? Well, last year, Sampdoria played really badly and got relegated to Seria B. Something that was unfathomnable to most. In the last 6 years here and maybe more (since my brother has heard of Sampdoria means they were quite a team) they have always been near the top of the table. And for them to do so badly and get relegated was devastating. The constant playful taunting from the Genoanas was heartbreaking for my FIL. 🙂 Poor fella! It was worse because just 2 years ago, Genoa was in Seria B and when they got up to Seria A they blazed their way to the top of the table while Sampdoria slipped down the rankings and eventually fell off the table.
But within 1 season, they are back!!! This was what all the celebrations was about. Last Saturday was the determining game and we made it!!!!!!! Wohoo!!! I am not a true fan but a fan none the less. I went with M to the historic centre to celebrate with the family. Paid a little homage to Sampdoria and saw the crazy crowd in action. Celebrations went on till wee hours of the morning. At 5 am, the horns were still honking on the streets. Whoever who watched the game at home all come out in full force to celebrate. Streets were packed, there was traffic especially near the expo centre at Foce where they have set up a open air cinema-like area for the fans to watch the game. To give you an idea how big that area is, they usually have the travelling circus or theme parks set up there complete with the area for all their caravans and 18 wheeler trucks. That is how big that area is. There was fireworks, drinking, singing, horns and loads of flag wavings! It was fun!! Even the children were out with their parents. From babies to toddlers. Some were even fast asleep among all the ruckus! I don't know how they could sleep through ALL that noise.
It was a fun Saturday night. A different twist to the usual Saturdays. 🙂
I will leave you with videos of that night's madness.You can even see me in one of them giving my tribute. hahahaha! 😛
Toodeloos!
footballSampdoria
Life In Photos – Day 6


Get the latest Yu-Yu Hakusho: Spirit Detective cheats, codes, unlockables, hints, Easter eggs, glitches, tips, tricks, hacks, downloads, hints, guides, FAQs, walkthroughs, and more for Game Boy Advance (GBA). CheatCodes.com has all you need to win every game you play!
Use the above links or scroll down see all to the Game Boy Advance cheats we have available for Yu-Yu Hakusho: Spirit Detective.
Both Hiei and Kurama need to get to at least lv.15 to learn there 2nd move and around 20 to get there 3rd move.
(Note: If these hints don't work, beat your enemies the way you did in story mode.) I hope this hint helps you.
Here are the directions on how to beat Suzaku: First, start hitting him until he splits into 7 guys. Use shotgun until he's 1 guy again. When you have low health and energy, go to the bottom left and right of the area. You will find power-ups. Repeat. Power-ups will regenerate every few minutes.
To beat Hiei, face the top platform and dodge his blasts. When he comes out, hit him with spirit gun. (Note: If you get hit by his blasts, you won't be able to move for a couple seconds and he will get on your platform and hit you. However, when you can move again you can hit Hiei with spirit punch.
To get the shotgun (Yusuke's best attack), you need to beat Rando. Then you will fight Gen Kai. She has all of Yusuke's moves(including the shotgun), and is slow. After that you'll get the shotgun and go off to fight the Saint Beasts.
To unlock extras mode, beat the game. To unlock minigames mode, beat tournament mode.
We have no cheats or codes for Yu-Yu Hakusho: Spirit Detective yet. If you have any unlockables please submit them.
We have no unlockables for Yu-Yu Hakusho: Spirit Detective yet. If you have any unlockables please submit them.
We have no easter eggs for Yu-Yu Hakusho: Spirit Detective yet. If you have any unlockables please submit them.
We have no glitches for Yu-Yu Hakusho: Spirit Detective yet. If you have any unlockables please submit them.
We have no guides or FAQs for Yu-Yu Hakusho: Spirit Detective yet. If you have any unlockables please submit them.


text:
Jose Mourinho Names His Favourite Team to Win The 2022 Europa League. (READ)
AS Roma football club manager, Jose Mourinho has named his favourite team to win the Europa League competition this season.
When he was asked which teams are the favourites to win the Europa League trophy after AS Roma's 1-1 draw with Real Betis on Thursday, Mourinho told reporters that he sees "Betis as candidates, but the failed sharks of the Champions League will arrive, and they will come in hard."
According to reports Mourinho is not happy with the rule that allows teams that finish third in the Champions' League group stage to drop into the Europa League knockout stage.
"They shouldn't come but it's the reality. It's going to be fun. The failures of the Champions League", the Portuguese football manager told reporters.
Previous Wizkid, Tems Bag Nominations at The American Music Awards 2022. (See Full List)
Next Blaqbonez Says He Was Unable to Achieve a Planned Scene of Davido in 'Back In Uni' Video.
Frank Lampard Sacked As Everton Manager After Defeat At West Ham.
"Don't Always Pass To Ronaldo"- Al-Nassr Coach Advises Players.
Antonio Conte Set to Leave Tottenham by the End of the Season.


K-5 Math Teaching Resources specializes in offering math professional development services focused on the development of teacher content and pedagogical knowledge. We are an approved sponsor of Continuing Teacher and Leader Education (CTLE). Click on the underlined event title for more details. If you have questions regarding any of the events below please contact us by phone or email.


Here's an animated music video that merges two eras. Visually it hearkens back to the 1930's with its style. The narrative addresses modern behavior that's emerged through the use of personal communication devices. London based Steve Cutts did a superb job blending both together to make a strong statement concerning what we're becoming socially. Music by Moby and the Void Pacific Choir.
Video of Are You Lost In The World Like Me?


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