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Tribute to Sienna Miller: Wander Darkly
Onstage Conversation & Screening
Wednesday, April 15, 7:30 pm · Victoria Theatre
Join us for a very special onstage tribute and conversation with actor Sienna Miller, in which she will go in-depth on her work and career.
The conversation will be followed by a screening of Miller's upcoming film Wander Darkly co-starring Diego Luna and directed by Tara Miele.
With her first two starring film roles, Sienna Miller established her range. In the 18th-century-set Casanova (2005), she proved adept at period roles and established her talent for comedy. Factory Girl (2006) was a different animal altogether, a dark drama and one in which she wholly inhabited Andy Warhol's tragic and thoroughly modern muse, New York "It" girl Edie Sedgwick. Miller got her start as a model before making her screen debut at 19 opposite Rupert Everett in South Kensington (2001) and upping her profile with supporting parts in the gangster thriller Layer Cake (Festival 2005) and the dramedy remake Alfie (2004). Among other roles, she's played a soap opera actress going head to head with a contemptuous reporter in Interview (2007), for which Miller received a Film Independent Spirit Award nomination; Caitlin Thomas, Dylan Thomas's wife in The Edge of Love (2008), for which the British Independent Film Awards nominated her for Best Supporting Actress; actress Tippi Hedren in The Girl (2012), which garnered her a Golden Globe nomination; and a single mom (and grandmother!) in American Woman (2018). Join us for a conversation with Sienna Miller, followed by a screening of her latest film, Wander Darkly.
Read more about Sienna Miller in Randy Myers' essay Sienna Miller: Here to Sear Your Brain.
Wander Darkly
Tara Miele, USA, 2020, 97 min
When love goes wrong, it's often difficult to pinpoint how or where, but new parents Adrienne and Matteo have the chance to do just that after a life-changing event. What actually happens to the couple is left a mystery until the very end, and Tara Miele's shape-shifting drama depicts an otherworldly melding of past and present as the couple reevaluates their relationship through their shared, and sometimes contradictory, memories. Sienna Miller is radiant and tough as reluctant mom Adrienne while Diego Luna brings a tender bemusement to the role of Matteo.
"[Sienna] Miller is phenomenal, playing multiple emotional zones from the early days of a relationship to the fear of a horror movie heroine worried she's stuck in purgatory and the Grim Reaper is coming for her. She's legitimately always good, giving her all to everything she does, and this is one of her best performances." –Brian Tallerico, RogerEbert.com
Director Tara Miele
Tara Miele's previous features include The Lake Effect (2010), Gone Missing (2013), and Starving in Suburbia (2014). In 2016, her short film Meet a Muslim went viral, garnering over 40 million online views to date and leading to a script deal with Warner Bros.
"I really wanted to do something [with Wander Darkly] that structurally reflected the way that my brain works, which is very rarely in the present, but often in the past, or projecting into the future. The other thing was finding a way to reflect on a relationship — the best and the worst moments of it — and to try to take stock of, what is it worth, at the end of the day?" –Tara Miele, Deadline
Runtime 97
Producer Lynette Howell Taylor, Samantha Housman, Shivani Rawat, Monica Levinson
Writer Tara Miele
Editor Tamara Meem, Alex O'Flinn
Cinematographer Carolina Costa
Music Alex Weston
Cast Sienna Miller, Diego Luna, Beth Grant
Print Source 51 Entertainment
matt@51entertainment.com
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April 9, 2021 @ 12:01 am - April 18, 2021 @ 11:59 pm
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Comparative sequence and structure analysis of eIF1A and eIF1AD
Jielin Yu1 &
Assen Marintchev ORCID: orcid.org/0000-0002-9901-35001
BMC Structural Biology volume 18, Article number: 11 (2018) Cite this article
Eukaryotic translation initiation factor 1A (eIF1A) is universally conserved in all organisms. It has multiple functions in translation initiation, including assembly of the ribosomal pre-initiation complexes, mRNA binding, scanning, and ribosomal subunit joining. eIF1A binds directly to the small ribosomal subunit, as well as to several other translation initiation factors. The structure of an eIF1A homolog, the eIF1A domain-containing protein (eIF1AD) was recently determined but its biological functions are unknown. Since eIF1AD has a known structure, as well as a homolog, whose structure and functions have been extensively studied, it is a very attractive target for sequence and structure analysis.
Structure/sequence analysis of eIF1AD found significant conservation in the surfaces corresponding to the ribosome-binding surfaces of its paralog eIF1A, including a nearly invariant surface-exposed tryptophan residue, which plays an important role in the interaction of eIF1A with the ribosome. These results indicate that eIF1AD may bind to the ribosome, similar to its paralog eIF1A, and could have roles in ribosome biogenenesis or regulation of translation. We identified conserved surfaces and sequence motifs in the folded domain as well as the C-terminal tail of eIF1AD, which are likely protein-protein interaction sites. The roles of these regions for eIF1AD function remain to be determined. We have also identified a set of trypanosomatid-specific surface determinants in eIF1A that could be a promising target for development of treatments against these parasites.
The results described here identify regions in eIF1A and eIF1AD that are likely to play major functional roles and are promising therapeutic targets. Our findings and hypotheses will promote new research and help elucidate the functions of eIF1AD.
Translation initiation in eukaryotes is a multistep process involving over ten eukaryotic translation initiation factors (eIFs) (reviewed in [1,2,3,4]).
Several eIFs and the initiator Metionyl-tRNA (Met-tRNAi) bind to the small ribosomal subunit, forming the pre-initiation complex (PIC). Met-tRNAi is recruited to the ribosome in complex with the GTPase eIF2. eIF2 is an αβγ heterotrimer. eIF2γ is the actual GTPase, responsible for the bulk of the interaction with Met-tRNAi, while eIF2α and β play accessory and regulatory roles. The N-terminal tail of eIF2β (eIF2β-NTT) contains three conserved poly-lysine stretches (K-boxes) that mediate binding to eIF5, which is the GTPase-activating protein (GAP) of eIF2.
The PIC is recruited to the 5′-end of the mRNA by the Cap-binding complex, composed of eIF4E, 4G, and 4A.
The PIC then scans the mRNA until it reaches a start codon in a proper nucleotide context.
Start codon recognition (basepairing of the Met-tRNAi anticodon with the start codon) triggers major conformational rearrangements in the PIC, leading to the release of most eIFs and preparing the PIC for ribosomal subunit joining.
The last step in translation initiation is ribosomal subunit joining (binding of the large ribosomal subunit to the PIC), promoted by the GTPase eIF5B and eIF1A. eIF5B then hydrolyzes GTP and is released together with eIF1A, leaving behind a ribosome ready to translate the mRNA.
Translation initiation in bacteria is less complex, involving only three translation initiation factors (IFs), two of which, IF1 and IF2, are homologs of eIF1A and eIF5B, respectively. There is no scanning; instead, the small ribosomal subunit binds directly at the translation start site (reviewed in [3]). eIF1A is universally conserved in all Kingdoms of life. It shares with its bacterial homolog, IF1 the same binding site on the ribosome [5,6,7,8] and common functions. They both: (i) bind in the Aminoacyl-tRNA binding site (A-site) of the small ribosomal subunit and induce conformational changes in the ribosome, mimicking those caused by the binding of an aminoacyl-tRNA in the A-site [5, 7]; (ii) promote the assembly of the PIC at the start codon; and (iii) play a role in ribosomal subunit joining. Both IF1 and eIF1A have an oligonucleotide/oligosaccharide binding fold (OB) domain. eIF1A also has a helical subdomain, as well as N- and C-terminal tails (NTT and CTT, respectively) which are intrinsically disordered [9, 10] (Fig. 1a). eIF1A has acquired a number of eukaryote-specific functions and plays a role in virtually every step of the process of translation initiation. Together with other eIFs, eIF1A promotes PIC formation, mRNA binding, scanning, start codon selection, and ribosomal subunit joining. eIF1A has been reported to bind to several other eIFs: eIF2, eIF3, eIF5, and eIF5B; however, only the interaction interfaces with eIF5B have been mapped (reviewed in [1,2,3,4]). eIF1A and eIF1 were found to bind to the ribosome immediately adjacent to each other, although no productive interactions between the two proteins were observed [6, 7].
eIF1A structure and position on the ribosome. a eIF1A structure shown in ribbon. The OB-fold domain and the N- and C-termini are labeled. b Position of eIF1A, in surface representation, on the ribosome [36]. The 18S rRNA is shown in beige; eIF1 (magenta) and small ribosomal subunit protein 23 (rpS23, red) are shown and labeled in the zoomed-in box on the bottom-left. The rest of the small ribosomal subunit proteins are not shown. The Tetrahymena thermophila 40S ribosomal subunit and eIF1 are from 4bts.pdb; human eIF1A is from 1d7q.pdb
The first evidence for a second eIF1A homolog in eukaryotes, the eIF1A domain containing protein (eIF1AD), came from genome sequencing projects (see e.g. Human, Schizosaccharomyces pombe (S. pombe), Caenorhabditis elegans (C. elegans)). The protein has also been called Haponin in human [11, 12] and Obelix in chicken [13]. eIF1AD is typically annotated in databases as an RNA-binding protein and a translation initiation factor, owing to its homology to eIF1A; however, there is no available supporting experimental evidence for either. High-throughput expression, interaction and phenotype studies have provided limited information about the function of eIF1AD. Deletion of the gene in S. pombe caused abnormal cell shape, but showed that eIF1AD is not essential [14]. A number of alleles are reported in C. elegans, including an embryonic lethal (www.wormbase.org, gene ID ZK856.11), indicating that eIF1AD is essential in this organism. The protein was found to be preferentially localized to the nucleus in human [12], chicken [13], as well as S. pombe [15], which makes a role at least in canonical translation initiation unlikely. Yeast two-hybrid (Y2H) studies indicate that human eIF1AD interacts with the signal transducer and activator of transcription 1 (STAT1) transcription factor [16] and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [11]. The C. elegans eIF1AD homolog was reported to interact with ferritin heavy chains 1 and 2 in Y2H screens, while the Drosophila melanogaster (D. melanogaster) protein was found to bind to the transcription factor Extradenticle (Exd) [17, 18]. eIF1AD was found to be highly expressed in testes and ovaries in C. elegans and Drosophila (www.wormbase.org, gene ID ZK856.11; flybase.org, gene ID FBgn0051957) and upon neural induction in chicken [13]. Its overexpression in mammalian cells was reported to increase sensitivity to oxidative stress [12]. Thus, the available data fail to offer insights into the functions of eIF1AD. Recently, the Nuclear Magnetic Resonance (NMR) solution structure of human eIF1AD was solved by the Yokoyama group as part of the RIKEN Structural Genomics/Proteomics Initiative (2dgy.pdb). The structure shows significant similarity to the structure of its paralog eIF1A [9], as expected from the sequence homology between the two proteins. Like eIF1A, eIF1AD consists of a folded domain composed of an OB-fold and helical subdomains, flanked by intrinsically disordered N- and C-terminal tails.
eIF1AD is interesting in that its cellular function is unknown while at the same time its structure has been solved and it has a paralog (eIF1A) with extensively characterized functions and interactions. Based on the sequence and structure homology between eIF1A and eIF1AD, we reasoned that there may be conservation of the interaction surfaces of these two proteins. For example, eIF1A and eIF1AD could use the same surfaces for interactions with their respective ligands, or could even have a common interacting partner. If this is indeed the case, one can expect the corresponding surfaces to be conserved between the two proteins. Therefore, eIF1AD is a very promising candidate for applying bioinformatics sequence and structure analysis to generate hypotheses about its functions and interactions. In this work, we report remarkable conservation between the ribosome-binding surfaces of eIF1A and the corresponding regions in eIF1AD. These results indicate that eIF1AD may bind to the ribosome, similar to its paralog eIF1A, and could have roles in ribosome biogenesis or regulation of translation. We also identified potential protein-protein interaction motifs in eIF1AD. Our analysis of eIF1A identified a set of trypanosomatid-specific surface determinants that could be a promising target for development of treatments against these parasites.
The main goals of this work were to:
Identify regions on the eIF1AD surface with high degree of sequence conservation, since these are likely to be functionally important ligand-binding sites.
Compare surfaces conserved in eIF1AD with the corresponding regions in eIF1A. A surface conserved between the two proteins may serve the same function/bind to the same ligand.
Analyze the sequence conservation of regions in eIF1A and eIF1AD in individual branches of the eukaryotic domain. The goal was to obtain insights into whether any functions/interactions mapped to the respective region are conserved among all eukaryotes or are restricted to certain groups of organisms. This analysis allows determining when it is appropriate to extrapolate results obtained from one species to others. Conversely, it can also point out important functional differences between model organisms.
Sequence homology searches and sequence alignments
We used a non-redundant protein PSI-BLAST [19] tool from NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with maximum target sequences set to 20,000. The results were then curated manually based on E-value and protein length, to eliminate incomplete sequences. We extracted representative sets of sequences from the alignments, based of pairwise sequence identity and minimum coverage using HHfilter [20, 21] from the Max-Planck Institute for Developmental Biology Bioinformatics Toolkit (http://toolkit.tuebingen.mpg.de). The sequences were aligned using ClustalW [22, 23] through the Max-Planck Institute for Developmental Biology Bioinformatics Toolkit (http://toolkit.tuebingen.mpg.de/). The alignment results were checked manually. ClustalW multiple sequence alignments and protein structures were used as input for ESPript [24] (http://espript.ibcp.fr) to produce sequence alignments color-coded for sequence conservation, also showing secondary structure elements and solvent accessibility.
We used Molmol [25] for structure analysis and visualization. For homology modeling of protein structures we used Swiss Model [26], in alignment mode. The sequence alignments were obtained using ClustalW [22, 23]. Sequence conservation was mapped onto the protein structures in Molmol, using Protskin [27] (http://www.mcgnmr.mcgill.ca/ProtSkin/). The consensus sequence as well as the conservation scores were both recorded. A threshold similarity score for conservation was selected based on the distribution of scores for the particular set of sequences as well as the percent identity.
As the first step in this work, we analyzed the sequence conservation of functionally important regions in eIF1A. The goals were twofold. The first one was to provide a reference point required for the subsequent analysis of eIF1AD. The second goal was to find out whether individual eIF1A interactions are conserved among all eukaryotes or only within certain branches of the eukaryotic domain, since this type of analysis has not been previously performed on eIF1A.
Sequence conservation of surfaces in eIF1A
eIF1A is involved in a number of protein/protein and protein/RNA interactions [6,7,8, 28, 29]. Therefore, we set out to analyze whether each of the respective interaction surfaces in the protein is conserved. As described above, eIF1A is composed of a folded domain surrounded by an N-terminal and a C-terminal tails, which are intrinsically disordered (Fig. 1a). The folded domain itself consists of an OB-fold subdomain and a helical subdomain [9] (Fig. 1a). Extensive surfaces in the folded domain, as well as the NTT bind to the small 40S ribosomal subunit [6,7,8] (Fig. 1b, colored blue in Fig. 2a). On the 40S subunit, eIF1A also comes in close proximity to eIF1 (Fig. 1b, the corresponding eIF1A surface is colored cyan in Fig. 2a); however there are no obvious productive interactions [6, 7]. The extreme C-terminus of eIF1A binds to the C-terminal domain of eIF5B (eIF5B-CTD) [28, 29] (red in Fig. 2a). Another segment of eIF1A-CTT, closer to the folded domain (orange in Fig. 2a), plays a role in maintaining the stringency of start codon recognition [30], but it is not clear whether this is mediated by protein/protein interactions. The folded domain of eIF1A also contacts eIF5B (gold in Fig. 2a) [6,7,8, 31, 32]. The remaining surfaces of eIF1A (grey in Fig. 2a) were considered a separate group in the analysis.
eIF1A interactions and surface conservation. a Functionally important surfaces of eIF1A. The human eIF1A structure in the same orientation as in Fig. 1 (left), and rotated 180 degrees (right). Interaction surfaces are color-coded and labeled. b Human eIF1A structure colored by sequence conservation. The eIF1A structure in the same orientation as in A, colored by sequence conservation. The universally conserved W69 is labeled. c Human eIF1A structure colored by charge/hydrophobicity
The eIF1A sequence conservation is very high: nearly identical among all mammals; > 80% identity among vertebrates, with the zebra fish (Danio rerio) sequence, for instance, being 99% identical to that of human eIF1A. Even the sequence and length of the intrinsically disordered tails are conserved. Comparisons among the different eukaryotic kingdoms show that the NTT and the folded domain of eIF1A remain well conserved, whereas the sequence and length of the CTT are less conserved (Fig. 3, Fig. 2b). For example, human and Saccharomyces cerevisiae (S. cerevisiae) eIF1A sequences have 62% identity overall and 69% identity with no gaps over the NTT and the folded domain (excluding the CTT).
Multiple sequence alignment of eIF1A sequences. Residue numbers and secondary structure elements for human eIF1A are shown above the alignment. Solvent accessibility is shown below the alignment. Residues in the sequences from Trypanosomatida species (Phylum Euglenozoa) showing divergence from the rest of the eIF1A sequences are boxed. Abbreviations: Met, Metazoa; O, others; Ve, Vertebrates; Ar, Arthropoda; Ne, Nematoda; Mo, Mollusca; An, Annelida; Tr, Trematoda; Cn, Cnidaria; Po, Porifera; Alv, Alveolata; Str, Stramenopiles; Cho, Choanoflagellida; Amo, Amoebozoa; Rho, Rhodophyta; Eug, Euglenozoa; Cry, Cryptophyta
As expected, the ribosome-binding surfaces are most conserved, with clear conservation of positively charged residues (compare Fig. 2a, b, and c). By far the most conserved surface-exposed residue is the invariant W69 (Fig. 3, labeled in Fig. 2b, c), at the ribosome binding surface. The eIF1A surface facing eIF1 is least conserved (compare Fig. 2a, b, and c).
Overall, we did not observe differential conservation in individual branches of the eukaryotic lineage (Fig. 3), with the two notable exceptions discussed below.
In roundworms (Phylum Nematoda) and flatworms (Phylum Platyhelminthes), the eIF1A C-terminus has no discernible eIF5B-CTD binding motif and carries a positive charge. Nematodes and Platyhelminthes belong to different clades: Ecdysozoans and Lophotrochozoans, respectively. eIF1A sequences from species belonging to other phyla from both of these clades, e.g. Arthropoda (Ecdysozoans) or Annelida and Mollusca (Lophotrochozoans) have a conserved eIF5B-CTD binding site and the entire eIF1A-CTT is negatively charged (Fig. 3). Therefore, the loss of the eIF5B-CTD binding site and the added positive charges must have occurred twice in evolution.
In trypanosomatids, eIF1A shows markedly different sequence conservation pattern, compared to any other group of organisms (Fig. 3, Fig. 4a). There is a trypanosomatid-specific area with substantial hydrophobicity located on the ribosome-binding surface (compare the circled region on the Trypanosoma vivax (T. vivax) eIF1A structure, Fig. 4a, left, with the corresponding region in human eIF1A, Fig. 4a, right, and Fig. 4b, left). The eIF1A C-terminus also has a segment with high hydrophobicity unique to trypanosomatids (Fig. 3, Fig. 4a, left).
Unique features of trypanosoma eIF1A. a Homology model of Trypanosoma vivax eIF1A (left) and human eIF1A structure (right) colored by charge/hydrophobicity. The structures are in the same orientation as in Fig. 1. Trypanosoma-specific hydrophobic residues are shown in green; a unique hydrophobic surface is circled; and a hydrophobic segment in the C-terminus of T. vivax eIF1A is shown in ribbon as an inset in the left panel. b Functionally important surfaces of eIF1A are shown as a reference. Intermolecular interaction surfaces (left) are color-coded and labeled as in Fig. 2a. The intermolecular contact surface for eIF1A-CTT is shown on the right panel. The area corresponding to the Trypanosoma-specific hydrophobic surface is circled as in panel a
Homology between eIF1A and eIF1AD
eIF1AD is present only in eukaryotes. It must have been present in the last common ancestor of all eukaryotes, because some eukaryotes that have branched out early, e.g. Giardia theta, have an eIF1AD gene. At the same time, eIF1AD has been lost in a number of eukaryotes, including S. cerevisiae.
Like eIF1A, eIF1AD has an OB domain surrounded by two intrinsically disordered tails (Fig. 5a). The sequence homology between the two proteins is highest in the folded domain with 23% identity and 34% homology (Fig. 5b). While the NTT sequences are not well conserved, they both have an overall positive charge. No similarity exists between the CTT sequences of eIF1A and eIF1AD (compare Figs. 3 and 6).
Comparison between eIF1A and eIF1AD. a Center, structure alignment between the folded domains of human eIF1A (1d7q.pdb, blue) and eIF1AD (2dgy.pdb, gold). The eIF1A and eIF1AD structures are shown on the left and right, respectively. b Sequence alignment of the folded domains of eIF1A (top) and eIF1AD (bottom). Identical residues are red; similar residues are orange; and conserved hydrophobic residues are gold. Residue numbers and secondary structure elements for human eIF1A are shown above the alignment. Residue numbers and secondary structure elements for human eIF1AD are shown below the alignment
Multiple sequence alignment of eIF1AD sequences. Residue numbers and secondary structure elements for human eIF1AD are shown above the alignment. Solvent accessibility is shown below the alignment. Abbreviations: Met, Metazoa; O, others; Ve, Vertebrates; Tu, Tunicata; Ar, Arthropoda; Cn, Cnidaria; Po, Porifera; Pl, Platyhelminthes; Ne, Nematoda; Mo, Mollusca; An, Annelida; Tr, Trematoda; Str, Stramenopiles; Cho, Choanoflagellida; Amo, Amoebozoa; Cry, Cryptophyta; Rho, Rhodophyta; Hap, Haptophyta
The eIF1AD sequence conservation (Fig. 6) is not as high as that of eIF1A (see Fig. 2): > 80% identity among mammals; > 50% identity among vertebrates. As with eIF1A, the NTT and the folded domain of eIF1AD are well conserved, whereas the sequence and length of the CTT are not (Fig. 6). Analysis of surface-exposed residues in eIF1AD shows that the surfaces corresponding to ribosome-binding surfaces in eIF1A are among the best-conserved, with a substantial number of positively charged residues (Fig. 7a, b, compare with Fig. 2).
Surface conservation in eIF1AD and comparison with eIF1A. a Human eIF1AD structure colored by sequence conservation. The universally conserved W62 is labeled. b Human eIF1AD structure colored by charge/hydrophobicity. Functionally important surfaces of eIF1A. c Residues conserved between human eIF1A and eIF1AD (see Fig. 5b) mapped on the eIF1A structure in the same orientations as in panel A. Known interaction surfaces in eIF1A (see Fig. 2a) are outlined and labeled
The folded domain of eIF1AD has a conserved surface with significant hydrophobicity (circled in Fig. 7b, right), which corresponds to part of the ribosome-binding surface in eIF1A, but is less conserved. This surface is a likely site of protein-protein interactions, since solvent-exposed hydrophobic residues are both energetically unfavorable and can contribute to stability and specificity of interactions. If eIF1AD does indeed bind to the ribosome in the same way as eIF1A, this surface would become buried at the interface.
While the eIF1AD C-terminal tail as a whole is not conserved, it contains highly conserved sequence motifs, which are likely protein-protein interactions sites (Fig. 6). One of the conserved motifs is found in most eukaryotes, but absent in Fungi and one branch of Metazoans: members of the phylum Platyhelminthes. We designate this motif here as "NTNR" (Asn-Thr-Asn-Arg), after the most conserved core of residues. The second motif is found in Fungi and the phyla Platyhelminthes, Mollusca, Anelida, and Nematoda. We designate this motif here as "LPPS" (Leu-Pro-Pro-Ser), after the most conserved core of residues. eIF1AD from Mollusca, Anelida, and Nematoda has both motifs (Fig. 6), which indicates that these are two independent sequence motifs, with independent functions. In plants, the eIF1AD-CTT contains an extended NTNR sequence motif, where the first portion of the sequence resembles the LPPS motif. It is thus possible that this motif has resulted from merging the NTNR and LPPS motifs in tandem in the plant eIF1AD sequence. Thus, the C-terminal tail of eIF1AD contains a set of sequence motifs that vary in consensus, and likely also function, among different branches of eukaryotes.
eIF1A is one of only two universally conserved translation initiation factors, found in every organism, from bacteria to human. There is also very high degree of conservation in eIF1A sequences among eukaryotes (Fig. 3). The invariant W69, at the ribosome binding surface (Fig. 3, labeled in Fig. 2b, c), is also conserved in the archaeal eIF1A homolog aIF1A, whereas the bacterial homolog IF1 has an arginine at this position (not shown). Remarkably, W69 was recently found to form a stacking interaction with a functionally important base A1709 in the Tetrahymena 18S small ribosomal RNA (rRNA), stabilizing it in a flipped-out conformation [7]. A1709 in helix 44 of the Tetrahymena 18S rRNA (A1819 in rabbit) corresponds to A1493 in Escherichia coli (E. coli), which in its flipped-out form "inspects" the proper codon-anticodon basepairing in the Aminoacyl-tRNA site (A-site) of the ribosome [33]. During translation initiation in bacteria, A1493 is also flipped out by the bacterial eIF1A homolog IF1, thus inducing conformational changes in the ribosome that can mimic the presence of a tRNA in the A-site [5]. A W69A mutant was found to cause a defect in start codon recognition (48S initiation complex formation) in vitro and appearance of aberrant 48S complexes with mRNA not positioned correctly on the ribosome [9]. The defect in 48S complex formation was not as drastic as could have been expected from the exceptionally high degree of conservation of W69 and its observed interaction with A1709, and the mutation had little effect on the assembly of the pre-initiation complex off mRNA (43S complex formation) [9]. Therefore, it appears that the main role of W69 is to induce conformational changes in the 40S ribosomal subunit, rather than in eIF1A binding to the ribosome per se.
The eIF1A surface facing eIF1 is least conserved (compare Fig. 2a, b, and c), consistent with the observation that there are no productive contacts between the two proteins in the 40S/eIF1A/eIF1 crystal structure [6, 7]. This indicates that the observed ~ 10-fold cooperativity of eIF1A and eIF1 binding to the 40S ribosomal subunit [34] is likely mediated by both proteins promoting similar conformational changes in the ribosome. It is also possible that direct contacts between eIF1 and eIF1A do contribute to the cooperativity, since binding to the ribosome places eIF1A and eIF1 in such close proximity that even weak interactions between the two proteins could have a stabilizing effect.
The eIF5B-CTD binding motif at the eIF1A C-terminus is conserved in almost all eukaryotic species, except roundworms (Phylum Nematoda) and flatworms (Phylum Platyhelminthes), where the eIF1A-CTT carries a positive charge, instead (Fig. 3). The interaction between eIF1A-CTT and eIF5B-CTD was found to be important for ribosomal subunit joining in S. cerevisiae [35]. It is thus interesting to know whether eIF1A-CTT still plays the same role in these worms. There are no compensatory changes in the respective surface on eIF5B-CTD (not shown); therefore, it is highly unlikely that the eIF1A-CTT can still bind there. Alternatively, since eIF5B-CTD interacts with the large ribosomal subunit, the positive charge of eIF1A in these species could allow it to bind to the rRNA in the vicinity of eIF5B-CTD.
As described above (Fig. 3), trypanosomatid eIF1A sequences appear to have diverged from the consensus in the rest of eukaryotes. Regions with increased hydrophobicity, conserved among trypanosomatids, but not other species, are observed in the NTT, CTT, and certain surfaces of the folded domain (Fig. 3, Fig. 4a). These regions may be sites of novel trypanosomatid-specific interactions. While trypanosomatid translation initiation is still not fully understood, there are a number of known differences from other eukaryotes. We reported recently that the eIF1A C-terminus dynamically contacts the ribosome-binding surface of eIF1A, an interaction that is disrupted when eIF1A binds to the ribosome ([36], Fig. 4b, right). Therefore, the two trypanosomatid-specific hydrophobic segments likely contact each other when eIF1A is not ribosome-bound. However, once eIF1A is bound to the ribosome, both its NTT and CTT are free to interact with other proteins [8, 30]. eIF1A interacts with eIF5B via regions adjacent to the trypanosomatid-specific hydrophobic surfaces [28, 29, 36] (see also Fig. 2a). eIF1A is known to interact with eIF2, eIF3 and the C-terminal domain of eIF5 (eIF5-CTD) [29, 37, 38], and on the ribosome, both eIF1A-NTT and -CTT are in proximity to eIF2, eIF3c, and eIF5, as well as to eIF1 [8, 30, 38,39,40]. We did not observe any trypanosomatid-specific hydrophobic surfaces in eIF1 or eIF5B (data not shown). Therefore, it is unlikely that the trypanosomatid-specific hydrophobic surfaces in eIF1A affect the interactions with eIF1 or eIF5B. The degree of sequence conservation of eIF3c and eIF5-CTD is too low for meaningful analysis and we cannot make any predictions about their interactions with eIF1A. Two of the three eIF2 subunits, α and β, have trypanosomatid-specific characteristics: eIF2α has a small N-terminal domain not found in most other eukaryotes, while eIF2β-NTT lacks the three conserved K-boxes that in other eukaryotes bind eIF5-CTD (see above). Thus, while the functional significance of these differences is not known, both eIF2α and -β could interact with the unique hydrophobic surfaces in trypanosomatid eIF1A. It is of course possible that the trypanosomatid-specific surfaces in eIF1A form novel interactions unique to trypanosomatids. For instance, trypanosomatid mRNAs are first transcribed as large polycistronic mRNAs and their maturation involves trans-splicing, adding a ~ 40 nt capped leader sequence to every mRNA. Trypanosomatids contain multiple eIF4E and eIF4G isoforms [41,42,43,44,45,46]. It was recently reported that in mammals, the PIC inspects the mRNA from the very 5′-end, placing the cap-binding complex in the vicinity of the ribosomal A- and P-sites at the beginning of scanning [47]. If this is also the case in trypanosomatids, then eIF1A could also be involved in trypanosomatid-specific interactions with eIF4A, 4E, and/or 4G. Since a number of trypanosomatids are parasites, a unique hydrophobic region on the ribosome-binding surface of an essential protein like eIF1A is a promising therapeutic target.
While eIF1AD is present in most eukaryotes, its sequence conservation s not as high as that of eIF1A (Fig. 6). Nevertheless, there is significant conservation not only among eIF1AD sequences, but also between eIF1A and eIF1AD (Fig. 5b). The majority of surface-exposed residues conserved between eIF1A and eIF1AD map to the ribosome-binding surface of eIF1A (Fig. 7c). The most conserved surface-exposed residue in eIF1AD, the almost invariant W62 (labeled in Fig. 7) also maps to the ribosome-binding surface. Remarkably, eIF1AD W62 corresponds to the most conserved residue in eIF1A, W69 (Fig. 2, Fig. 3b). As discussed above, W69 is involved in promoting conformational changes in the ribosome upon eIF1A binding [7]. The significant sequence conservation between the ribosome-binding surfaces of eIF1A and the corresponding regions of eIF1AD indicates that eIF1AD is also likely to bind to the ribosome, or rRNA. This hypothesis is further strengthened by the observation that the same tryptophan residue known to be important for eIF1A function is the most conserved surface-exposed residue in both proteins. Furthermore, the high degree of sequence conservation in the positively charged eIF1AD-NTT indicates that it may also be involved in ribosome binding, similar to eIF1A-NTT. If this is indeed the case, the function of such eIF1AD interaction remains to be determined. The eIF5B-binding regions of eIF1A are not conserved with eIF1AD, indicating that eIF1AD does not interact with eIF5B. Therefore, eIF1AD either does not act as an alternative translation initiation factor or it functions in a unique pathway that does not involve eIF5B. A number of homologs of canonical translation initiation factors have been described. In most, if not all, of the cases where the functions of these homologs are known, they are in some way or other related to translation. For example, Pdcd4, PAIP1, 5MP1, and 5MP2, which are homologs of eIF4G and eIF5, are translation regulators [48,49,50,51]. The nuclear Cap-Binding Complex, composed of CBP80 (an eIF4G homolog) and CBP20 (homologous to eIF4B and 4H) [52] plays multiple roles in transcription, mRNA maturation and export, as well as the pioneer round of translation and Nonsense-Mediated Decay (NMD) [53, 54]. eIF4A3 (a homolog of eIF4A) is part of the Exon Junction Complex and is involved in mRNA export and NMD [54,55,56], as well as in rRNA biogenesis, together with another eIF4G homolog, NOM1 [57]. Therefore, eIF1AD is likely involved in one or more of these processes. Since eIF1AD has been found to be localized predominantly in the nucleus [12, 13, 15], direct role in translation regulation is somewhat less likely. Instead, it could play roles in regulation of ribosome biogenesis or mRNA maturation.
In summary, our structure/sequence analysis of eIF1AD found significant conservation in the surfaces corresponding to the ribosome-binding surfaces of its paralog eIF1A. Remarkably, both protein families share a nearly invariant surface-exposed tryptophan residue, which plays an important role in the interaction of eIF1A with the ribosome. These results indicate that eIF1AD may bind to the ribosome, similar to its paralog eIF1A, and could have roles in ribosome biogenenesis or regulation of translation. We also identified conserved surfaces and sequence motifs in the folded domain as well as the CTT of eIF1AD, which are likely protein-protein interaction sites. The roles of these regions for eIF1AD function remain to be determined. Furthermore, our analysis of eIF1A identified a set of trypanosomatid-specific surface determinants that could be a promising target for development of treatments against these parasites. We expect that the results and hypotheses described here will promote new research and help elucidate the functions of eIF1AD.
A-site:
Aminoacyl-tRNA site
C. elegans :
CTD:
C-terminal domain
CTT:
C-terminal tail
D. melanogaster :
E. coli :
eIF1A:
Eukaryotic translation initiation factor 1A
eIF1AD:
eIF1A domain-containing protein
Exd :
Extradenticle
GAPDH:
Glyceraldehyde 3-phosphate dehydrogenase
IF1:
Initiation factor 1
NMD:
Nonsense-Mediated Decay
NMR:
NTT:
N-terminal tail
OB:
Oligonucleotide/oligosaccharide binding fold
rpS:
Small ribosomal subunit protein
rRNA:
ribosomal RNA
S. cerevisiae :
S. pombe :
Schizosaccharomyces pombe
STAT1:
Signal transducer and activator of transcription 1
T. vivax :
Trypanosoma vivax
Y2H:
Yeast two-hybrid
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The authors thank the team of scientists developing and maintaining the Bioinformatics Toolkit at the Max-Planck Institute, which was an extremely valuable resource.
This work was supported by National Institutes of Health Grant GM095720 to A.M.
The raw datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA, USA
Jielin Yu
& Assen Marintchev
Search for Jielin Yu in:
Search for Assen Marintchev in:
JY helped design the study, performed sequence and structure analysis, and helped draft the manuscript. AM conceived and designed the study, carried out homology modeling, and drafted the manuscript. Both authors contributed to analysis and interpretation of data and finalized and approved the manuscript.
Correspondence to Assen Marintchev.
Yu, J., Marintchev, A. Comparative sequence and structure analysis of eIF1A and eIF1AD. BMC Struct Biol 18, 11 (2018). https://doi.org/10.1186/s12900-018-0091-6
eIF1A
eIF1AD
Translation initiation
Protein-protein interactions
Computational analysis


Virginia Woolf, Alice Walker and Chimamanda Ngozi Adichie were among the iconic writers read and discussed by 20 high school students at Newcomb College Institute's (NCI) first pre-college program titled, Newcomb Summer Session: Women's Literature.
This intensive program, directed by Molly Pulda, administrative assistant professor of women's literature, brought in students from as far as New Jersey, Kentucky and Texas, as well as the greater New Orleans area.
The five-day session was packed with programming and projects that fostered critical thinking and promoted leadership. The group enjoyed daily "Leadership Lunch" lectures by visiting writers and scholars, including NCI's director Sally J. Kenney, as well as field trips to the Amistad Research Center and Newcomb Art Museum. Pulda shared strategies for writing the college application essay, using Woolf's essay A Room of One's Own as a model, and staff-led interactive workshops that ranged from an introduction to key leadership skills to a guided writing session.
The students also wrote, designed, and produced Ours, an original zine or feminist magazine, to commemorate the inspiring week. They viewed examples of feminist zines from the Newcomb Archives and learned how to create their own content.
The goal for the Newcomb Summer Session is to become a residential program that offers a full college campus experience. As the program grows it will stay true to its original goal—to form a strong community through the exploration of gender in literature.
Registration for next summer will open in spring 2018.


text:
Michigan International Speedway Race Report 8.12.18
Almirola Finishes Seventh at Michigan
Smithfield Ford Driver Earns His First Top-10 at Brooklyn Track
Date: Aug. 12, 2018
Event: Consumers Energy 400 (Round 23 of 36)
Series: Monster Energy NASCAR Cup Series
Location: Michigan International Speedway in Brooklyn (2.0-mile oval)
Format: 200 laps, broken into three stages (60 laps/60 laps/80 laps)
Start/Finish: 11th/7th (Running, completed 200 of 200 laps)
Point Standing: 12th (639 points, 347 out of first)
Race Winner: Kevin Harvick of Stewart-Haas Racing (Ford)
Stage 1 Winner: Kevin Harvick of Stewart-Haas Racing (Ford)
Stage 1 Recap (Laps 1-60):
● Aric Almirola started 11th and finished Stage 1 in the seventh spot to earn four stage points.
● Through the first two green-flag runs, Almirola reported he was "getting hurt" on the restarts because of starting on the inside lane and competitors against the door of the No. 10 machine.
● On lap 27 Almirola pitted during the competition caution for fuel and two right-side tires. He restarted 11th and worked his way up to the seventh spot on lap 53.
● The Tampa native pitted at the conclusion of the stage for four tires and fuel. Almirola came down pit road in seventh and came out 12th.
Stage 2 Recap (Laps 61-120):
● Almirola started Stage 2 in 12th and completed it eighth to earn three bonus points.
● During the fifth caution on lap 75, Almirola pitted from ninth for fuel, two right-side tires and adjustments. He restarted 16th after different pit strategies played out.
● The caution was displayed on lap 85; Almirola stayed out on the track and restarted 11th on lap 87.
● The balance of the stage was under the green flag, and Almirola ultimately completed it in eighth.
● Before the start of the final stage, Almirola visited pit road for fuel, four tires and adjustments to his Smithfield Ford.
Final Stage Recap (Laps 121-200):
● Almirola started the final stage in the seventh position and ended it 25th.
● The eighth caution was on lap 134. Almirola stayed out and restarted seventh on lap 143.
● The 34-year-old made a scheduled green-flag pit stop on lap 172 for fuel and four tires.
● The remainder of the event ran under green, and Almirola finished seventh.
● This was Almirola's 10th top-10 of 2018.
● Almirola earned his first top-10 in 13 career Monster Energy NASCAR Cup Series starts at Michigan.
● Almirola finished seventh in Stage 1 to earn four bonus points and eighth in Stage 2 to earn an additional three bonus points.
● Harvick's victory in the Consumers Energy 400 marked the 53rd overall win for Stewart-Haas Racing (SHR). It was the organization's 48th points-paying Monster Energy NASCAR Cup Series win and its third at Michigan International Speedway in Brooklyn.
● SHR swept the Monster Energy NASCAR Cup Series races at Michigan in 2018. Clint Bowyer won in the series' June visit to the track.
● Harvick's win marked SHR's series-leading ninth points-paying victory 23 races into the 2018 season. SHR also won the 2018 Monster Energy NASCAR All-Star Race via Harvick.
● This was SHR's 12th Monster Energy NASCAR Cup Series victory with Ford. The team won its first race with Ford when Kurt Busch captured the 2017 Daytona 500.
Aric Almirola, driver of the No. 10 Smithfield Ford Fusion for Stewart-Haas Racing:
"It was a solid day. We ran top-10 in both stages and finished top-10. It was finally a normal weekend for us where we had good speed and executed all day. We had some bad restarts and had a couple stops on pit road that we struggled on, but overall it was a decent day and a good way to build our momentum back as we head into the playoff stretch."
The next event on the Monster Energy NASCAR Cup Series schedule is the Bass Pro Shops NRA Night Race on Saturday, Aug. 18 at Bristol (Tenn.) Motor Speedway. The race starts at 7:30 p.m. EDT with live coverage provided by NBCSN beginning at 6 p.m.


Working in the Tourism Industry Part A and Part B provides an overview of the tourism Industry highlighting all the exciting areas students may wish to work in. This subject includes industry structures, the interrelationships of the different sectors as well as the technology, laws and ethical issues specifically relevant to a career in travel and tourism. Students will integrate this knowledge to work effectively in the industry.
Students will also conduct research in order to develop a general destination knowledge database on local Australian or International tourist locations for use in the selling process. Students will then develop the skills and knowledge to provide customer information and advice on tourist attractions and products available in their customers chosen destination. The subject will also cover the health and safety requirements of overseas travel.
Travel Agency Sales I, II and III provide the skills and knowledge required to sell travel and tourism products to customers in a travel agency. It requires the ability to identify specific customer needs, suggest a range of products to meet those needs, provide current and accurate product information, provide a quotation and close the sale.
Travel Agency Sales II and III follows on from Travel Agency Sales I, where students are required to make and administer customer bookings for products and services with suppliers. It requires the ability to identify customer booking requirements from previous quotations or offers issued and then reconfirm them with suppliers. The sale must be recorded by raising invoices and receipts and then issuing documents to customers such as airline tickets or accommodation vouchers. Travel Sales III also covers dealing with diversity issues that may arise when dealing with a broad array of customers and their special needs or characteristics.
In this module students will learn what is required to create a customer's flight itinerary when calculating and constructing normal or special promotion airfares that may be available for international or domestic airfares. This module requires the ability to interpret flight information and conditions that apply to specific fares and to construct these airfares according to International Air Transport Association (IATA) regulations.
Students will learn to use 'Galileo', a computerised reservations system used to create, administer and maintain airline bookings. In addition to airline reservations, the Galileo CRS is also used to book train travel, cruises, car rental, and hotel rooms.
This module describes the performance outcomes, skills and knowledge required to work as a Tour Guide. Tourism has been identified as a growth area for employment and opportunities over the 2015-2018 period. Tour Guiding offers a wonderful small business opportunity for foreign students to develop markets amongst their own country people in Australia, or to guide foreigners in their home countries, either way there are great opportunities by learning this subject. This subject also covers key legal, ethical, safety, environmental and professional development issues that Guides must consider in their day‑to‑day work…..enjoy!
In this module student will develop the skills and knowledge required to correctly create and use spreadsheets and charts using spreadsheet software. The unit will include formatting a spreadsheet, develop and test formula, create chart and text, save and send.
This module covers the skills and knowledge required to incorporate safe work practices into your own workplace activities. It requires the ability to follow health, safety and security procedures and to participate in organisational work health and safety (WHS) management practices. Students will learn how to recognize hazards and potential emergency situations and then report WHS issues as they arise .


Everybody is very very friendly, management are very open, salary is great and the stocked kitchens are a big plus.
Management are being quite indecisive at the moment.
"Great environment, bad salary, bad location"
"Great people, culture and values."
- Guidewire offices are clean, have a casual atmosphere, and employees are provided with free snacks and drinks.
- Management encourages and facilitates employees to progress their professional and leadership skills with clear career paths outlined.
- Employees have control over their own career path with a lot of internal opportunities available.
- Guidewire is solving important and complex problems with their products and services.
- There are office based positions or 80% travel based positions.
- Methodologies are sometimes outdated and it can be difficult to change the internal status quo.
Review project methodologies and processes more frequently and give the development teams on the ground level more of a voice in this area.
It's a good Company and good culture. Very friendly people around and work environment is really good.
Less pay. Very less chance to learn new technologies and very less development work involved in day-to-day work.
"I've lost 2 years of my life there"
Proprietary technology - so it can be really hard to find a job if you spend too much time at Guidewire, especially if it's your first experience.
"Wear what you want". Loads of activities. snacks, soda, fruit, showers, bike shed, parking, shuttle from the Rotunda.
Most people are friendly here.
As many monitors as you want.
Training is long, rigid and very hard.
Toilets can be filthy at times. * Cleaners are excellent, it's just that some workers aren't hygienic.
Ryu seems to care about people. Managers are also supportive.
Glassdoor has 698 Guidewire reviews submitted anonymously by Guidewire employees. Read employee reviews and ratings on Glassdoor to decide if Guidewire is right for you.


How September flew! I ended my job at the hospital on September 9th, and started my job at the credit union on September 12th. So my whole routine has been turned upside down, but I think it's going to be a good thing once the dust settles. Unfortunately, all that upheaval caused a lack of reading on my part for September.
The Death of Bees by Lisa O'Donnell was unique. It had three narrators and revolves around two sisters growing up in the slums of Scotland. Their loser parents wind up dead, and the girls decide not to tell anyone because they'd rather continue fending for themselves than be put in foster care. Their elderly neighbor knows something is up, but can't quite put his finger on it. He's determined to help the girls out anyways. There's a wide assortment of wrenches in their plan, including a drug dealer their dad owed money to, an over zealous grandfather who shows up, and a truancy officer. Goodreads describe it as "coming of age" and I suppose it is. And it was good, but there was just something missing for me. I can't quite put my finger on it.
Silas Marner by George Eliot was my only other read for the month. This book took me way longer than it should have, but by the time I finished it, I wasn't really sure what all the hoopla was about. It's a classic, but it doesn't really have anything exciting, or suspenseful happen. Silas Marner goes from being a miser to a nice fatherly type figure who cares for Eppie's happiness above his own, but I felt like this could have been knocked down to a short story no problem. Blasphemous I suppose, but I think it's by far the most disappointing classic I have read to date.
and A Tale for the Time Being by Ruth Ozeki were all part of my Books n Bloggers Swap with Jenni King. You can read more about it here.
Stone Mattress by Margaret Atwood was found on a trip to Goodwill. It's a collection of her short stories, so I'm looking forward to giving it a read.
Children of God by Mary Doria Russell, which is the sequel to The Sparrow- my favorite read of 2016!
So that brings my year to date totals up to 30 books read, 46 books acquired. I'm really going to have to have a good fourth quarter to get my goal of 48 books read accomplished, but I do think it's still possible! How bout you? Are you on track with your yearly reading goals?


Rone is back with his fourth album called "Mirapolis", released on the 3rd of November.
Rone is back with his fourth album called "Mirapolis", released on the 3rd of November. Twelve tracks featuring some of the best indie artists like Bryce Dessner (The National guitarist, Battles), spoken-word poet Saul Williams, Blonde Redhead vocalist Kazu Makino and London singer-songwriter Baxter Dury.
I highly recommend the Fnac Edition (french reseller) with his precious release coming with a silkscreen printing by movie director Michel Gondry for a similar price than the standard edition.
Infiné's favorite provides us a brilliant album, slightly more pop than his previous ones. Highly recommended!


Shop girls pajama sets and girls robes at Lands' End to find the girls flannel pajamas and girls pajama pants your child needs! From cute graphic cotton girls pajama sets to cozy fleece girls pajama bottoms, Lands' End has it.
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We carry girls pajama sets and girls flannel pajamas in sizes from Toddler to Big Kid, so we have something for just about everyone. If yours is the kind of family that likes to do matching pajamas for holidays or occasions, we can outfit everyone! Check this page for kids Christmas pajamas or girls pajama sets that will coordinate perfectly with holiday pajamas in other departments! Holiday jammies are a fun tradition lots of different families embrace, and we are thrilled to help you make those precious memories.
We're excited to see the new arrivals in girls pajamas! As always, you can shop with confidence at Lands' End, because all of our girls pajama sets, including girls pajama pants and girls flannel pajamas are Guaranteed. Period.®, just like everything we sell. If you and your child are not delighted with her new kids pajama pants, you can return them for a refund or exchange at any time, for any reason. Shop girls pajama sets and girls pajama bottoms today, and remember to check back for new arrivals!


We remind you that the road of the Stelvio Pass will remain closed to traffic on June 3rd from 9.00 am until 4.00 pm.
Family and friends who don't participate to the Granfondo can use the STELVIO BUS SERVICE to reach the top of the Stelvio.
BORMIO to STELVIO – 8.30 am from Perego Station. Via Milano, Bormio.
STELVIO to BORMIO – 4.00 pm from Passo dello Stelvio bus parking.
You must book your bus ticket at race pack collection. There is a limited availability (40 seats per service). Price per round trip ticket € 8,00.
The BUS service is not intended for the participants of the Granfondo Stelvio Santini who will have to ride back to Bormio.


The Capcom-Unity Blog has revealed that the previously announced PS4 and Xbox One port of Resident Evil 4 is releasing August 30.
The PS4 and Xbox One ports feature all of the game's previous bonus content, which includes Separate Ways, Assignment: Ada, and The Mercenaries.
You can look at some screenshots of the port below. It will be $19.99 when it launches.


Buskers, renovated in February 2016, is a lively contemporary bar, and a well-known landmark in the vibrant and hugely popular Temple Bar area. Located on Fleet Street, Buskers is literally a stone's throw away from O'Connell Bridge and Grafton Street. Trading since 1993, Buskers remains a favourite for locals and tourists alike and its popularity continues to grow. Buskers is one of Dublin`s best late night bars with live music 7 nights a week. There's no better place in Dublin city for live music.
Join them 7 nights a week for their free irish show. The shows take place from 7pm – 9pm during which professional irish dancers bring you a unique and intimidate performance alongside their popular live irish music two-piece band.
Buskers specialised lunch and evening menus are a fusion of modern contemporary Irish with an emphasis on light, healthy eating in a casual relaxed setting. The lunch menu is available from noon to 5pm featuring daily specials, light bites, and all the firm favourites, including their gourmet sandwiches. From 5pm – 9pm you can order from the Evening menu which includes Buskers famous Burger and Beer Battered Fresh Cod and chips; all made using fresh local ingredients. Group bookings are welcome and they endeavour to meet all dietary requirements.
Buskers has all the right ingredients offering high quality, great value food daily, fabulous cocktails and an extensive wine list. Buskers Bar offers a large sports bar, showing all major sporting events and providing live music seven nights a week. Cocktail lessons are also available in their designated cocktail bar. It is the perfect venue for a girl's night out or group celebrations. Buskers has something to offer everyone and with its smooth, sleek décor.


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text:
package de.ks.strudel.route.handler;

import de.ks.strudel.HandlerNoReturn;
import io.undertow.util.CopyOnWriteMap;

import javax.inject.Singleton;
import java.util.Map;
import java.util.function.Supplier;

/**
 * Stores handlers for exceptions.
 * Each handler is a supplier which should generate a new instance of the exception handler.
 */
@Singleton
public class ExceptionMappingRegistry {
  Map<Class<? extends Exception>, Supplier<HandlerNoReturn>> exceptionMappings = new CopyOnWriteMap<>();

  public Map<Class<? extends Exception>, Supplier<HandlerNoReturn>> getExceptionMappings() {
    return exceptionMappings;
  }
}

redpajama_set_name: RedPajamaGithub


It is no secret that our winters can be very unpredictable. Whether it is the drastic changes in temperature we experience almost daily in the winter, or the snowstorms that materialize out of nowhere, it pays to hire a snow and ice management company with experience and a proven track record.
Martins 4 Seasons has both, as well as an unrelenting dedication to providing quality snow removal and ice management services. We have a number of employees who have worked in the snow industry for decades, as well as dependable equipment that is maintained to meet or exceed industry standards.
Having snow troubles? Don't spend hours shoveling. Call Martins 4 Seasons in Milton, Ontario and let us take care of it for you!
We know that spending your time shoveling, salting, and plowing is not something that you want to do, let our professionals take care of it for you!
Commercial snow removal and de-icing services are our specialties. Our customers require their properties to be accessible and safe during inclement winter weather. Every situation is different and we use common sense and communication to make sure we cater our service to your needs.
Commercial snow removal and de-icing services are our specialties. Our customers require their properties accessible and safe during inclement winter weather. Every situation is different and we use common sense and communication to make sure we cater our service to your needs.