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History lost: Fire destroys one of Epsom's oldest homes
An official at the Epsom Fire Department confirmed that the residents, Eric Reeves, his wife Kathryn, and their pet dog all made it from the home safely.
Investigator Bill Clark from the New Hampshire Fire Marshal's office said no specific cause for the fire could be identified.
"There was too much damage to be conclusive," he said, but there are "plenty of accidental fire causes."
Although this fire has been categorized as "undetermined," Clark said the Fire Marshal's office didn't have any suspicion of criminal intent and no evidence of it being intentionally set.
The home itself dates back centuries, according to T.J. Rand of the Epsom Historical Society, although the date is not certain. He points to the discrepancy with the tax map that dates the house back to 1794, yet records indicate that the land was bought by John Yeaton in 1807. He explained that the farm originally belonged to Samuel T. Yeaton, who bought the property from William Yeaton, probably his father, with buildings in 1821. Rand said he believes that son James C. Yeaton actually built the house that burned.
Mrs. Glenna Nutter of Epsom, whose father was Pete Yeaton, said the farm had been in his family for many years. Nutter lived across the road from what was then a two-family house, she said, where her grandmother and great aunt used to live. Nutter recalls her two brothers working on the farm while she did vegetable gardening with her mother.
"It's a house that was always in the Yeaton family," she said. "There were several Yeaton families who built six or seven capes on Black Hall Road, and we lived in one of them," she said. The house she lived in was built around 1780 or 1790, she said. Back then, "the homes were all connected – houses, sheds, barns," she said, "so the people didn't have to go out at night to do the chores in the barn in the bad weather."
She said she recently drove by the farm on Saturday, Nov. 3, "and it's been completely cleaned up. It's all graded. Only the barn and silo remain."
The home is now owned by Eric Reeves, whom Nutter said is the grandson of her father's second wife.
"My father grew up there and his father, as well, and I think his grandfather and possibly his great-grandfather," she said.
Of growing up in Epsom, Nutter says, "life was much simpler then, … with no TV or that sort of thing."
She still has memories of her grandmother "baking cookies and custards and making ginger tea if you had a tummy ache."
She also recalled that they had a lot of chickens.
"Lots of people in Epsom back then had hens and chickens," she said. "We used to go out and collect the eggs at the hen house as a child."
Her grandmother also had "a very nice piano" and had "lots of music that she played at times," although she didn't like "the modern music of the '40s."
Nutter remembers the Sweet William flowers in her grandmother's garden. "I still treasure them and grow them myself," she said
Perhaps, if Reeves decides to rebuild, some of those memories may be restored. For now, however, they live in Glenna Nutter's memories, along with the fragrance of her grandmother's Sweet William flowers, tending the chickens, and ginger tea for her tummy.
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Copyright © 2012 The Authors All rights reserved.
Cell Reports, Volume 2, Issue 6, 1505-1512, 06 December 2012
Structural Basis of DNA Ligase IV-Artemis Interaction in Nonhomologous End-Joining
Pablo De Ioannes1, Shruti Malu2, 3, Patricia Cortes2, , and Aneel K. Aggarwal1, ,
1 Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA
2 Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA
Corresponding author
3 Present address: Department of Melanoma Medical Oncology, University of Texas, MD Anderson Cancer Center, Houston, TX 77054, USA
Graphical Abstract
► Crystal structure of the Ligase-IV-Artemis complex ► Ligase IV DNA binding domain contains a unique hydrophobic cavity ► The structure shows why only Ligase IV can recruit Artemis ► Insights into how activities of Ligase IV and Artemis may be coordinated in NHEJ
- DNA ligase IV (LigIV) and Artemis are central components of the nonhomologous end-joining (NHEJ) machinery that is required for V(D)J recombination and the maintenance of genomic integrity in mammalian cells. We report here crystal structures of the LigIV DNA binding domain (DBD) in both its apo form and in complex with a peptide derived from the Artemis C-terminal region. We show that LigIV interacts with Artemis through an extended hydrophobic surface. In particular, we find that the helix α2 in LigIV-DBD is longer than in other mammalian ligases and presents residues that specifically interact with the Artemis peptide, which adopts a partially helical conformation on binding. Mutations of key residues on the LigIV-DBD hydrophobic surface abolish the interaction. Together, our results provide structural insights into the specificity of the LigIV-Artemis interaction and how the enzymatic activities of the two proteins may be coordinated during NHEJ.
The adaptive immune system gains its ability to recognize a plethora of antigens by the generation of an extensive repertoire of antigen receptors on B and T lymphocytes. These numerous receptors are created by V(D)J recombination, a process during which DNA double-strand breaks (DSBs) are repaired by the nonhomologous end-joining pathway (NHEJ) (Lieber, 2010). NHEJ is one of the major pathways for the repair of DSBs in mammalian cells, playing not only a central role in V(D)J recombination and class switch recombination (CSR), but also in the repair of pathological DSBs generated by ionizing radiation and other chemical and enzymatic damage. This pathway works throughout the cell cycle and does not require the presence of a homologous sister chromatid (Lieber, 2010).
DNA ligase IV (LigIV) is an essential factor for NHEJ (Grawunder et al., 1998). Mutations in LigIV lead to a rare disease called the Lig4 syndrome, which is characterized by microcephaly, radiosensitivity, growth and developmental delay, and immunodeficiency (Chistiakov, 2010). Moreover, it has been shown that in mice the absence of LigIV increases tumor generation in nonlymphoid tissues (Sharpless et al., 2001).
LigIV is unique among other mammalian ligases in its ability to perform nuclear DNA DSB repair (Simsek et al., 2011). It exhibits a high degree of similarity with LigI and LigIII, particularly at the level of domain organization of the catalytic core (Ellenberger and Tomkinson, 2008). Thus, its unique ability to repair DSB might be partly delineated through its interacting partners. LigIV directly interacts with XRCC4 (Critchlow et al., 1997; Grawunder et al., 1997) and Ku (Chen et al., 2000; Nick McElhinny et al., 2000), and indirectly with DNA-PKcs (Drouet et al., 2005), Cernunnos-XLF (Ahnesorg et al., 2006), and the MRN complex (Chen et al., 2001), all which are necessary for NHEJ in mammalian cells. Recently, we identified Artemis as a new interaction partner of LigIV (Malu et al., 2012). Artemis, in complex with DNA-PKcs, has a diverse array of nuclease activities and is a necessary factor for the processing of DSBs during NHEJ (Ma et al., 2002, Ma et al., 2005b). Association-binding experiments indicate that the C-terminal region (C-ter) of Artemis directly interacts with the DNA binding domain (DBD) of LigIV (LigIV-DBD). Moreover, deletion analysis of Artemis C-ter identified an 11 amino acid peptide (485-DVPQWEVFFKR-495) that contains the minimal interacting region. Aromatic residues in this region are crucial for the interaction and are important for efficient V(D)J recombination (Malu et al., 2012). However, the corresponding interacting surface on LigIV-DBD remains uncharacterized due to the limited structural information for LigIV. As such, it is unclear how the interaction between Artemis C-ter and LigIV is mediated and how it facilitates NHEJ in mammalian cells.
We report here crystal structures of the LigIV-DBD in both its apo form and in complex with a peptide derived from the Artemis C-ter. We show that LigIV-DBD interacts with Artemis through a hydrophobic surface that is much more extended than in other mammalian DNA ligases, such as LigI and LigIII. Based on the structure, the interaction surface was further evaluated by isothermal titration calorimetry (ITC) as well as by mutational analysis of critical residues. Together, these results offer structural insights into the specificity of interaction between LigIV and Artemis, important for development of the immune system and genomic stability.
Structure of apo LigIV-DBD
The crystal structure of LigIV-DBD, determined at 2.8 Å resolution (Figure S1), is composed of two helical subdomains, related by an ∼2-fold axis (Figure 1A). The first subdomain is composed of helices α1, α2, α3, α10, and α11 whereas the second subdomain consists of helices α4, α5, α6, α7, α8, and α9, and the two are connected by long loops, L1 (71–86) and L2 (191–201). This type of pseudo-symmetry is also observed in the DBDs of other mammalian DNA ligases (Ellenberger and Tomkinson, 2008), as well as in the C-terminal helix-hairpin-helix (HhH) domain of Escherichia coli LigA (Lee et al., 2000), which is functionally similar to the DBD of human DNA LigIV. As with LigI and LigIII, the helical subdomains in LigIV-DBD lend to both polar and nonpolar surfaces (Figure S2). A superimposition of LigIV-DBD onto LigI/DNA and LigIII/DNA complex structures is consistent with DNA binding to the LigIV-DBD on its positively charged surface (Figure S2) (Cotner-Gohara et al., 2010; Pascal et al., 2004). In all, even though the LigIV-DBD shares low sequence similarity with the LigI (13% sequence identity) and LigIII (15% sequence identity) DBDs (Figure S3), the structure is broadly similar, superimposing with root mean-square deviations (rmsd) of 4.1 Å (101 Cαs) and 2.5 Å (157 Cαs), respectively (Figure 1B).
The main difference between the LigIV-DBD and that of LigI and LigIII is a substantially longer helix α2 (Figure 1B). The helix is longer by approximately two turns compared to LigI and by three full-turns when compared to LigIII (Figure 1B). This extended portion of helix α2 supports an extended hydrophobic surface characterized by partially solvent exposed residues on both helices α2 (F42, W46, F49, and L53) and α1 (V10 and V14) (Figure 1C). The presence of this hydrophobic surface and the extension of helix α2 suggested that they could play a role in the interaction with Artemis.
Structure of LigIV-DBD/cArt-Pep Complex
To understand the basis of specificity between LigIV and Artemis, we determined the crystal structure of LigIV-DBD in complex with a peptide derived from the C-ter of Artemis (cArt-Pep) (Figures 2A and 2B). The structure, determined at 2.25 Å resolution, contains two complexes (A and B) in the asymmetric unit, which superimpose with an rmsd of 0.54 Å (for 218 Cαs). The cArt-Pep (485-DVPQWEVFFKR-495) is well defined in complex A, with 10 of the 11 residues built into the electron density map (Figure 2B). The cArt-Pep is less defined in complex B, with three residues traced in the electron density map. We describe below the structure of complex A.
The C-terminal portion of cArt-Pep adopts a helical conformation, supported by two intrachain hydrogen bonds (N488(O)-V491(N)) and W489(O)-F493(N)). This helical portion of the peptide fits into a shallow hydrophobic pocket on the surface of LigIV-DBD, bordered by helices α2 and α1 (Figures 2C and 2D). The N-terminal region of the peptide (residues 485–488) adopts an extended β-chain conformation that tracks the lengthened portion of helix α2.
The hydrophobic pocket on LigIV-DBD is created by an outward movement of helix α2, including a bending of the helical axis at L43 by ∼20° (Figure 2B). This results in an outward displacement of the helix α2 C terminus by ∼9 Å, when compared to the apo structure, and creates the space necessary to bind cArt-Pep (Figure 2E). In addition to this gross movement of helix α2, the side chain of F49 moves and reorients by several Angstroms to create the cavity to accommodate W489 of cArt-Pep (Figure 2E). Indeed, W489 is the most deeply buried residue of cArt-Pep and it occupies roughly the position that F49 occupied in the DBD apoenzyme. In addition to interactions with F49, the indole ring of W489 is involved in extensive hydrophobic interactions with V10, V14, F42, and W46 (Figures 2D and 2F). The F42, W46, and F49 aromatic rings pack in a herring-bone pattern against W489. V10 and V14 stem from helix α1 and they complete the hydrophobic cage around W489 of cArt-Pep. A hydrogen bond (2.74 Å) is also observed between the nitrogen of W489 indole ring and the carbonyl group of D18 of LigIV-DBD (Figure 2F). F492 and F493 are on the same side of the helical portion of cArt-Pep as W489, and they too interact with the hydrophobic pocket on the surface of LigIV-DBD; in particular, with residues F42 and F49 on helix α2. In addition, the main chain amide of F492 makes a direct hydrogen bond with the side chain of S45. Supplementing these interactions between W489, F492, and F493 on the helical portion of cArt-Pep and the LigIV-DBD are van der Waals contacts between P487 on the N-terminal extension of cArt-Pep and L53 from the extended portion of helix α2 of LigIV-DBD (Figure 2F). Taken together, LigIV and Artemis interact via an induced-fit mechanism, wherein helix α2 in LigIV-DBD rotates outward to create a hydrophobic pocket for binding a partially helical peptide from the Artemis C-ter. The mainly hydrophobic nature of the interaction is consistent with the stability of the complex at high salt concentrations, up to 500 mM NaCl (data not shown).
Structural analysis of LigIV-DBD/cArt-Pep complex shows that ∼400 Å2 of the LigIV-DBD surface area is buried at the interface. To test the interface in solution, we used ITC. Consistent with the crystal structure, wild-type LigIV-DBD binds the cArt-Pep with a Kd of 4.8 μM and a binding stoichiometry of 1:1 (n = 1.0 ± 0.1) (Figure 3A). Mutations of F49 and F42 to alanine result in a complete loss of peptide binding (Figures 3B and 3C), consistent with the central role of these phenylalanines on helix α2 in binding to the aromatic residues on cArt-Pep. By contrast, a modest effect on binding is incurred when D18 and V14 on helix α1 are mutated. D18 makes a hydrogen bond with the indole nitrogen of W489 and a D18H mutation increases the Kd to 9.1 μM (Figure 3E), whereas a V14A mutation increases the Kd to 13.64 μM (Figure 3F). Taken together, the ITC measurements are consistent with the crystal structure in showing the dominant role of F49 and F42 in binding the cArt-Pep.
To examine the role of F42 and F49 amino acids in context of full-length proteins in vivo, we tested the ability of F42A and F49A mutants to interact with Artemis by coimmunoprecipitation. Consistent with the crystal structure and ITC measurements, only wild-type LigIV interacted with Artemis. However, both the wild-type and mutant LigIV interacted equally well with XRCC4 (Figure 3H). Together, these data provide further evidence that LigIV interacts with Artemis and XRCC4 via mutually exclusive surfaces: the DBD at the N terminus interacting with Artemis, and the BRCT repeats at the C terminus engaging XRCC4 (Wu et al., 2009).
Comparison to LigI and LigIII
Figure 1B shows the structure of LigIV-DBD superimposed on the structures of LigI-DBD and LigIII-DBD. To examine whether the LigI and LigIII DBDs can also bind cArt-Pep, we performed florescence anisotropy (FA) peptide-binding experiments with purified DBDs. As shown in Figure 4A, LigI-DBD and LigIII-DBD bind cArt-Pep with substantially lower affinity than LigIV. Together, these in vitro results are in agreement with the coimmunoprecipitation experiments (Malu et al., 2012), wherein only LigIV-DBD was found to interact with Artemis. Figure 4B shows the cArt-Pep modeled on the LigI-DBD and the LigIII-DBD. In contrast to the LigIV-DBD, there is substantial steric overlap between cArt-Pep and the LigI-DBD and LigIII-DBD. Strikingly, the putative binding site in LigI-DBD is blocked by its unique N-terminal "extension," which coils around the potential binding site (Pascal et al., 2004). Also, as noted above, helix α2 is shorter by approximately two turns in LigI-DBD, which decreases the potential binding surface for cArt-Pep by ∼60 Å2. Finally, residues F49 and F42 that are essential for LigIV-DBD-cArt-Pep interaction are substituted by smaller L315 and L322 in LigI DBD (Figure S3), which likely diminishes the van der Waals interactions with W489 of cArt-Pep. In LigIII, because helix α2 is shorter by three full-turns compared to LigIV there is no residue equivalent to F49. Indeed, this region of LigIII is disordered in the crystal structure (Figure 4B) (Cotner-Gohara et al., 2010). Taken together, the LigIV-DBD differs from the LigI and LigIII DBDs in presenting a hydrophobic surface that is adapted to bind the C-terminal region of Artemis. Both the length of helix α2 and the identity of specific residues on helix α2 contribute to the specificity of LigIV-DBD-cArt-Pep interaction.
We present here molecular details of how LigIV and Artemis interact. We show that LigIV recruits Artemis via a hydrophobic surface that is more extended than in other mammalian DNA ligases. Specifically, a helix (α2) in LigIV DBD is longer than in other mammalian ligases and presents a set of hydrophobic residues that interact selectively with the C-terminal region of Artemis.
Various models for NHEJ have been proposed in recent years, from the sequential to the simultaneous recruitment of factors to the DSB (Dobbs et al., 2010; Ma et al., 2005a; Yano et al., 2009). The structure we present is consistent with the idea that, in presence of Artemis, NHEJ factors are recruited simultaneously to a DSB, leading to the formation of a large complex. Both Artemis and LigIV contain multiple domains and bind other NHEJ factors. For example, in addition of LigIV, the C-terminal region of Artemis also interacts with the kinase DNA-PKcs, leading to both phosphorylation of Artemis and autophosphorylation of the kinase itself (Goodarzi et al., 2006), which promotes DNA-PKcs release from the DNA (Hammel et al., 2010a). The N-terminal region of Artemis is composed of a metallo-β-lactamase fold and forms the catalytic core for its nuclease activity (Callebaut et al., 2002). In LigIV, the DBD at the N terminus is followed by the catalytic adenylation domain and an oligo-binding (OB) domain, whereas the C terminus contains two BRCT repeats. The LigIV's BRCT repeats are required for interaction with XRCC4 (Sibanda et al., 2001; Wu et al., 2009), which in turn recruits XLF (Cernunnos); a complex with a stoichiometry of one BRCTs, two XRCC4, and two XLF can be isolated by gel filtration (Hammel et al., 2010b). Intriguingly, from our structure, the Artemis C-terminal region (or cArt-Pep) binds to LigIV-DBD on the opposite face as the DNA, leaving the N-terminal nuclease domain of Artemis free to engage the DNA substrate in conjunction with the catalytic domain of LigIV (Figure 4C). Such coordination of the nuclease and ligation activities may help to increase the efficiency of the NHEJ reaction, though this remains to be fully tested. XRCC4 and XLF lack enzymatic activity but increase the efficiency of the ligation reaction (Grawunder et al., 1997; Gu et al., 2007). From recent small-angle X-ray scattering (SAXS) and crystallographic studies, XRCC4 and XLF can form long filaments and may serve as "holders" of the DNA for the ligation reaction (Hammel et al., 2010b, Hammel et al., 2011; Ropars et al., 2011). Although, a clearer picture of how the Artemis and LigIV enzymatic activities are coordinated will emerge from a structure of the complex with intact proteins, based on the current structure one can begin to construct an initial model of how Artemis and LigIV are recruited together to a DSB (Figure S4).
Along with other damage tolerance pathways, the NHEJ machinery is potentially an important therapeutic target for sensitizing tumor cells to chemotherapy and radiation treatment (Lord and Ashworth, 2012). The LigIV-Artemis structure will aid in the design of small molecule and peptidomimetic inhibitors for increasing the vulnerability of tumor cells to chemotherapy and radiation efficacy. The limited size of the LigIV-Artemis interface and the fact that it is largely a solvent exposed hydrophobic surface, in principle, should facilitate the design of chemical inhibitors for the LigIV-Artemis interaction, analogous to PARP inhibitors for the treatment of certain cancers (Hakimelahi et al., 2001; Lord and Ashworth, 2012).
In conclusion, despite the central role of LigIV in V(D)J recombination and DSB repair, there is limited structural information on this key enzyme. The only atomic structures currently available for LigIV are for BRCTs domains and a subdomain of the nucleotidyltransferase domain (Wu et al., 2009; Ochi et al., 2012), with low-resolution electron microscopy (EM) and SAXS studies also suggesting a degree of heterogeneity and/or flexibility when intact LigIV interacts with XRCC4 (Recuero-Checa et al., 2009; Hammel et al., 2010b). The atomic structures for LigIV-DBD we present here reveal a unique DBD that has evolved to bind both DNA and Artemis in a mutually exclusive manner.
Experimental Procedures
Protein and Peptide Production
The native and Selenomethionine (SeMet) DNA binding domain of LigIV (LigIV-DBD; residues 1–240) were expressed in E. coli. The proteins were purified by nickel affinity chromatography, cation exchange chromatography, and size exclusion chromatography. The DBDs of the human Ligase I (LigI, residues 260–535) and the human Ligase III (LigIII, residues 1–388) were expressed and purified in the same manner as LigIV-DBD. cArt-Pep encompassing the LigIV interaction motif (DVPQWEVFFKR) was synthesized by Bio-Synthesis (Lewisville, TX).
Crystallization and Data Collection
LigIV-DBD was crystallized from solutions containing 16% PEG 3350 and 200 mM (NH4)3PO4 at 20°C. SeMet LigIV-DBD crystals were obtained under similar conditions as the native protein except that (NH4)2SO4 replaced (NH4)3PO4 in the crystallization mix. The LigIV-DBD/cArt-Pep cocrystals were obtained from solutions containing 18% PEG 1000 and 200 mM Tris-HCl pH 8.0. All crystals were cryo-protected in paratone-N oil (Hampton Research), and flash cooled to 90°K. All data sets were measured at the Brookhaven National Laboratory (BNL; beamline X6A) and the Advanced Photon Source (APS; beamline 24-ID). Due to significant anisotropy in LigIV-DBD/cArt-Pep diffraction, the data were truncated and anisotropically scaled by the Diffraction Anisotropy Server (Strong et al., 2006).
Structure Determination and Refinement
LigIV-DBD apo structure was solved by multiwavelength anomalous diffraction (MAD) phasing, followed by density modification. The initial model was refined in program Phenix with strict geometric and B-factor restraints (Adams et al., 2010), followed by several rounds of refinement that included simulated annealing, energy minimization, geometry optimization, TLS refinement, and manual rebuilding as needed. The final apo LigIV-DBD model contains 212 amino acids and 2 phosphate ions with an Rfactor of 21.7% and Rfree of 25.6% at 2.8 Å resolution (Table S1. Data Collection, Phasing, and Refinement Statistics, Related to Figures 1 and 2).
The structure of LigIV-DBD/cArtPep complex was solved by molecular replacement by the program PHASER (McCoy et al., 2007), using the apo LigIV-DBD as a search model. The structure was refined in the same manner as the apo LigIV-DBD. The final model contains two LigIV-DBD/cArt-Pep complexes in the asymmetric unit, complex A (aa 7–112 and 122–237 of LigIV-DBD and aa 485–494 of Artemis) and complex B (aa 8–113 and 123–237 of LigIV-DBD and 487–489 of Artemis).
Interestingly, because of crystal contacts, helices α10 and α11 at the C terminus take different paths in the apo LigIV-DBD and the LigIV-DBD/cArt-Pep structures. The topology α10 and α11 in apo LigIV-DBD differs from that observed in the LigI/DNA, LigIII/DNA, and LigIV/cArt-Pep crystal structures due to the crystal contacts these helices in apo LigIV-DBD structure make with symmetry-related molecules.
Peptide Binding Experiments
Peptide binding experiments were performed by mixing 6-FAM (6-caboxyfluorescein)-labeled cArt-Pep (5 nM final concentration) with increasing concentrations of Lig-DBDs (0.1 nM–300 μM) in a peptide dilution buffer.
ITC Measurements
Calorimetric measurements were performed with the MicroCal ITC200 instrument (GE Healthcare) in the temperature range of 5°C–25°C.
Transfections and Flag Immunoprecipitation from 293T Cells
HEK293T cells were transfected using the calcium phosphate method. Cellular extracts were incubated with 200 μg/ml ethidium bromide at 4°C. The cell lysate (supernatant) obtained after centrifugation was used for Flag IP using anti Flag M2-agarose beads (Sigma-Aldrich) and proteins were eluted with 0.2 μg/ml Flag peptide (Sigma-Aldrich) in Buffer C (150 mM KCl) (Ma et al., 2002). Western blot analysis was performed at least two times from independent transfections and representative results are shown.
Protein and Peptide Production
The DNA binding domain of LigIV (LigIV-DBD; residues 1-240) was cloned in a pET-15b vector (Novagen) and expressed in E. coli BL21(DE3) CodonPlus RPL cell strain (Stratagene) (Malu et al., 2012). Selenomethionine (SeMet) LigIV-DBD was expressed by the metabolic repression expression protocol (Van Duyne et al., 1993). The domain was purified by the following steps: nickel affinity chromatography (QIAGEN), cation exchange chromatography by SP-Sepharose (GE healthcare) and size exclusion chromatography using Superdex 75 10/300 (GE healthcare). The protein was stored in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT and concentrated to 52 mg/ml with a 10 kDa BIOMAX centrifugal device (Millipore). The DBDs of the human Ligase I (LigI, residues 260 - 535) and the human Ligase III (LigIII, residues 1 - 388) were expressed and purified in the same manner as LigIV-DBD. Point mutations were introduced by the QuickChange Site-Directed Mutagenesis Kit (Stratagene). cArt-Pep encompassing the LigIV interaction motif (DVPQWEVFFKR) was synthesized by Bio-Synthesis Inc (Lewisville, Texas).
Crystallization and Data Collection
LigIV-DBD was crystallized from solutions containing 16% PEG 3350 and 200 mM (NH4)3PO4, at 20°C. SeMet LigIV-DBD crystals were obtained under similar conditions as the native protein except that (NH4)2SO4 replaced (NH4)3PO4 in the crystallization mix. The LigIV-DBD/cArt-Pep co-crystals were obtained from solutions containing 18% PEG 1000 and 200 mM Tris-HCl pH 8.0. All crystals were cryo-protected in paratone-N oil (Hampton research), and flash cooled to 90° K. All data sets were measured from single frozen crystals at the Brookhaven National Laboratory (BNL; beamline X6A) and the Advanced Photon Source (APS; beamline 24-ID). The data were processed by the HKL2000 software (Otwinowski and Minor, 1997). Due to significant anisotropy in the LigIV-DBD/cArt-Pep diffraction, the data were truncated and anisotropically scaled by the Diffraction Anisotropy Server (Strong et al., 2006).
Structure Determination and Refinement
LigIV-DBD apo structure was solved by MAD phasing, where the initial experimental map was calculated by Phenix (Adams et al., 2010), followed by density modification. An initial poly-alanine model was constructed by building alpha helices in the electron density, using Coot (Emsley and Cowtan, 2004). SeMet sites and bulky amino-acids side chains served as guides in fitting the amino acid sequence. The initial model was refined in Phenix with strict geometric and B-factor restraints (Adams et al., 2010), followed by several rounds of refinement that included simulated annealing, energy minimization, geometry optimization, TLS refinement, and manual rebuilding as needed. The final apo LigIV-DBD model contains 212 amino acids and 2 phosphate ions with an Rfactor of 21.7% and Rfree of 25.6% at 2.8 Å resolution (Table S1. Data Collection, Phasing, and Refinement Statistics, Related to Figures 1 and 2).
The structure of LigIV-DBD/cArtPep complex was solved by molecular replacement (MR) by the program PHASER using the apo LigIV-DBD as a search model (RF Z-score = 9.5, TF Z-score = 38.1 and LLG = 1112) (McCoy et al., 2007). The structure was refined in the same manner as the apo LigIV-DBD. The final model contains 2 LigIV-DBD/cArt-Pep complexes in the asymmetric unit, complex A (aa 7-112 and 122-237 of LigIV-DBD and aa 485-494 of Artemis) and complex B (aa 8-113 and 123-237 of LigIV-DBD and 487-489 of Artemis). The refined model has excellent geometry (Table S1. Data Collection, Phasing, and Refinement Statistics, Related to Figures 1 and 2), as calculated by the Molprobity server (Chen et al., 2010).
Interestingly, because of crystal contacts, helices α10 and α11 at the C terminus take different paths in the apo LigIV-DBD and the LigIV-DBD/cArt-Pep structures. The topology α10 and α11 in apo LigIV-DBD differs from that observed in the LigI/DNA, LigIII/DNA, and LigIV/cArt-Pep crystal structures due to the crystal contacts these helices in apo LigIV-DBD structure make with symmetry-related molecules.
For structural analysis, all structure superpositions were performed by the SSM software in the CCP4 suit (Winn et al., 2011). The electrostatic surface potentials were calculated by the Adaptive Poisson-Boltzman Solver Software (APBS) (Baker et al., 2001), implemented as a plug-in in Pymol. All structural figures were prepared using the Pymol software (DeLano, 2008).
Peptide Binding Experiments
Peptide binding experiments were performed by mixing 6-FAM (6-Caboxyfluorescein) labeled cArt-Pep (5 nM final concentration) with increasing concentrations of Lig-DBDs (0.1 nM – 300 μM) in a peptide dilution buffer. The binding reactions were incubated for 15 min (at 20°C) prior to measurement. Fluorescence anisotropy data collection and processing were performed as described in Malu et al., 2012.
Isothermal Titration Calorimetry (ITC) Measurements
Calorimetric measurements were performed with the MicroCal ITC200 instrument (GE healthcare) in the temperature range of 5 - 25°C. The LigIV-DBD samples (WT and mutants domains) were prepared in buffer 25 mM Tris-HCl pH 7.5, 150 mM NaCl. All titrations were performed with a fixed concentration of LigIV-DBD (200 μM in 200 μl) into which concentrated peptide samples (∼1.5 mM × 40 μl) were injected in fifteen successive injections. Acquired calorimetric titration data were analyzed using software Origin 7.0 (Microcal).
Transfections and Flag Immunoprecipitation from 293T Cells
HEK293T cells were transfected using the Calcium Phosphate method. 48 hr post transfection; the cells were harvested and extracted in Buffer A (150 mM KCl) with protease inhibitors as described (Ma et al., 2002). Cellular extracts were incubated with 200 μg/ml EtBr at 4°C. The cell lysate (supernatant) obtained after centrifugation was used for Flag IP using anti Flag M2-agarose beads (Sigma) and proteins were eluted with 0.2 μg/ml Flag peptide (Sigma) in Buffer C (150 mM KCl) (Ma et al., 2002). The IP proteins were separated on 4%–20% SDS PAGE followed by Western blotting. Western blot analysis was performed at least two times from independent transfections and representative results are shown. Antibodies used for this experiment were as described in Malu et al., 2012.
We thank the staff at BNL (beamline X6A) and APS (beamline 24-ID) for facilitating X-ray data collection. We thank W. Hendrickson and F. Ferrage for discussions and C. Escalante for help with the ITC experiments. Work in P.C.'s laboratory is supported in part by grants R01 AI080755 and R01 AI070532.
Accession Numbers
The Protein Data Bank (PDB) accession numbers for the atomic coordinates and structure factors reported in this paper are 4HTO (LigIV-DBD) and 4HTP (LigIV-DBD/Artemis).
Supplemental Information
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Publication Information
Received: September 25, 2012
Revised: November 5, 2012
Accepted: November 9, 2012
Published: December 6, 2012


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Parables of a Violent World
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Expelled from Eden: A William T. Vollmann Reader
Vollmann, born in 1959, has published eight novels (four of which are part of his Seven Dreams series, a historicofictional account of the settlement of North America), three collections of stories (including The Atlas), a memoir about his experiences in Afghanistan, and an extraordinary seven-volume, two-thousand-plus-page meditation on violence called Rising Up and Rising Down (2003). In 2004, he produced an abridgment of this last work (just 726 pages, not including the acknowledgments) and the tone of his preface makes clear why it is so easy to like him and also why you might not want his company all the time. "The longer version of Rising Up and Rising Down," he says, "took me twenty-three years, counting editorial errands. The abridgment took me half an hour." And:
The single justification which I can offer [for the length of the original version] is that I believe it needed to be that long. This abridgment likewise has only one justification: I did it for the money. In other words, I can't pretend (although you may disagree) that a one-volume reduction is any improvement upon the full version. All the same, it's not necessarily worse. For one thing, the possibility now exists that someone might read it.
Vollmann is a writer who is both stylish and garrulous, a combination I thought impossible until I started to read him. He is also both tough and sentimental, but this is a more familiar mix: he's seen it all but he still hasn't lost his innocence. Here he is—as the narrator of a story in The Atlas—left holding the jacket of a prostitute who has gone out onto the streets of San Francisco to look for crack. He riffles
through the lining, into which all kinds of objects have fallen: lighters, Vaseline, tissues, a hamburger wrapping, a broken cigarette, some matches, "and finally, like some sweet secret, a little Tootsie Roll." He is touched by the Tootsie Roll, and says he doesn't know why. But in the next sentence he knows why. "It was like her, the dearness of her hidden inside all the greed and the lies, the goodness of her that the badness drew on and exhibited and used for its own selfish work."
And here he is—he or another avatar in another story—reading a letter from a Japanese girl. He has written a passionate missive to her, and she says in reply, among more mundane things, "Please come back alive from Burma. Because I love you, too." He treasures the letter and its reciprocation of passion, reads it again and again, but gradually its potency fades. "One night the letter was used up. Instead of tacit it seemed lukewarm." When he looks at the letter again a week later it is "not lukewarm but sisterly, loving, enthusiastic, not at all erotic."
He said to himself: How can the meaning of these words squirm and wriggle so much on my mind's hook?
But then …


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Google spends record $5 million on lobbying
Google continues to up the ante on the money it spends each quarter on Washington lobbying.
The search giant spent a record $5.03 million (PDF) last quarter, according to the company's lobbying report. That figure compares with $3.76 million spent in the fourth quarter and just $1.48 million in 2011's first quarter.
Among the lobbying issues grabbing Google's attention were the regulation of online advertising, privacy and competition issues in online advertising, openness and competition in online services, and International tax reform.
The search giant devoted lobby dollars to HR 1389 - Global Online Freedom Act of 2011, … Read more


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MCW07 Simple Models for Molecular Transport Junctions
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VIDEO: A world with heterophobia
What if the tables were turned and people were heterophobic instead of homophobic?
"Love Is All You Need?" is a short movie that turns the tables, showing what it would be like if men and women being together was taboo through a young girl's eyes.
While it may seem dramatic to some, similar scenarios happen to LGBT youth around the world.


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If you've ever been required to take a radiation safety course, you may be familiar with the outdated teaching video that begins with the soft "click, click, click" of a Geiger counter, illustrating the presence of ambient radiation to which we are constantly exposed. Indeed, we are exposed to low doses of radiation on a daily basis, whether it is naturally occurring, as a result of occupation, or due to medical diagnostic tests. The Low Dose Radiation Research Program through the U.S. Department of Energy funds research projects that look at the biological response to low levels of radiation exposure (as you might have gathered by the title of the program). The website not only details a number of the research projects, but it also provides a wealth of information about radiation and its biological effects in general. The "Slide Shows" tab is especially educational, offering PowerPoint presentations that cover topics such as background radiation, genomic instability, and radiation dose.
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Tasty Tuesday is Tasty Thursday this week but we have the goods - ready to eat! Tasty Tuesday is vegetarian this week! We have a great choice of fun and delicious meals that will make your family beg for more veggies and sneaking Super Foods into the meals is always a win-win for body and health.... More »
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Quinoa, Fennel & Pomegranate Salad
From the Hungry Goddess via Chef Ottolenghi - a vegetarian salad that has layers of flavor in its delivery! You will be addicted!
Roasted Cauliflower Salad
Guilty Kitchen completely removes the guilt but delivers on taste with the roasted cauliflower salad - I may never eat cauliflower any other way now!
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Per Foodies Unite - If this soup can tempt a 15 year old to make it a midnight snack - it can tempt anyone! Great flavors and easy to make!
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Gluten Free Girl always delivers yummy and gluten free recipes that are sure to delight! This recipe is a combo of some of our go-to "greens" - broccoli, brussel sprouts and celery with a light dressing.
Baked French Fries with Cumin and Coriander
This recipe from Cookin' Canuck spices up french fries which are always a family fave! Dip them in a yogurt-based dressing for added fun!
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Carrot Soup with Miso and Sesame
Smitten Kitchen offers aromatic and flavorful combos to get carrots in front of the family - can you say umami?
Pesto and Cream Cheese Stuffed Cherry Tomatoes
Mother Thyme has fabulous recipes for changing veggies up - this dish would be great as an appetizer or paired with a salad or soup. Bet the kids would love to help stuff the tomatoes too!
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Flower Gift (Ability)
From Bulbapedia, the community-driven Pokémon encyclopedia.
Flower Gift | フラワーギフト | ||||||||||||||||||
Flower Gift | |||||||||||||||||||
Flavor text
Flower Gift (Japanese: フラワーギフト Flower Gift) is an Ability introduced in Generation IV. It is the signature Ability of Cherrim.
In battle
Flower Gift raises all party Pokémon's Attack and Special Defense stats by 50% in sunshine. Cherrim also changes forms in sunlight, from a closed flower (Overcast Form) to one with spread petals (Sunshine Form); however, this is not caused by Flower Gift in Generation IV.
Outside of battle
Flower Gift has no effect outside of battle.
Pokémon with Flower Gift
# | Pokémon | Types | First Ability | Second Ability | Hidden Ability | |
Cherrim | Grass | Flower Gift | None | None | ||
Please note that this is only 100% accurate to Generation VI games.
In other languages
This article is part of Project Moves and Abilities, a Bulbapedia project that aims to write comprehensive articles on two related aspects of the Pokémon games. |


text:
Availability: Pullman Paris La Défense
Please enter your travel dates.
Classic Double Room
- FREE Breakfast
Breakfast is included in your stay.
- FREE Wifi
Wifi is included in your stay.
Classic Twin Room, 2 Single Beds
- FREE Breakfast
Breakfast is included in your stay.
- FREE Wifi
Wifi is included in your stay.
Superior Twin Room, 2 Single Beds
- FREE Breakfast
Breakfast is included in your stay.
- FREE Wifi
Wifi is included in your stay.
Superior Double Room
- FREE Breakfast
Breakfast is included in your stay.
- FREE Wifi
Wifi is included in your stay.
Fully renovated 5-star hotel 15 minutes from Paris city center
By conference center
Fully renovated 5-star glass hotel set in the Paris business district of La Défense, facing the CNIT conference center and less than 10 minutes walk from Les Quatre Temps shopping center and cinema.
Hotel restaurant
Guests at the Pullman Paris La Défense can unwind in the Turkish bath, and enjoy the 5 vibrant, fresh, delicate and simply inspired cuisines served the modern restaurant.
Satellite television
Renovated rooms with Pullman bed, free wireless Internet throughout the hotel, laptop safe, TV with international channels, minibar, 2 free bottles of water, tea and coffee, 24-hour room service, and laundry service.
Information missing or incorrect? Tell us!Pullman Paris La Défense Amenities
- General
After a day's business, sightseeing, or attending a game at the sports stadium, guests at Pullman Paris La Défense can tone up on cardio-vascular and weights machines in the fitness facility or take a stroll in green neighborhood parks.
The hotel houses 10 meeting rooms with complimentary wireless Internet access, computer station, color printer, headphones, technology helpdesk and large integrated cameras. The largest can accommodate conferences and seminars of up to 90 people. The concierge can advise on local sights and attractions, and other amenities include currency exchange and front-desk safe. Motorists will find secure parking and valet parking (surcharges may apply). Smoking is permitted in designated areas only.
- Translation services
- Number of buildings/towers - 1
- Babysitting or childcare (surcharge)
- Business center
- Fitness facilities
- Concierge services
- Currency exchange
- Steam room
- Air-conditioned public areas
- Bar/lounge
- Restaurant
- Total number of rooms - 382
- 24-hour front desk
- Porter/bellhop
- Express check-out
- Multilingual staff
- Audiovisual equipment
- Ballroom(s)
- Banquet facilities
- Number of floors - 19
- Breakfast available (surcharge)
- Business services
- Coffee shop or café
- Complimentary newspapers in lobby
- Doorman/doorwoman
- Dry cleaning/laundry service
- Elevator/lift
- Event catering
- Exhibit space
- Express check-in
- Free Wi-Fi
- Wired (high-speed) Internet access - surcharge
- Designated smoking areas
- Internet point
- Computer station
- Luggage storage
- One meeting/conference room
- Valet parking (surcharge)
- Parking nearby (surcharge)
- Parking nearby
- Room service (24 hours)
- Safe-deposit box at front desk
- Year Built 2000
- Meeting facilities size (feet) - 8073
- Secure covered parking (surcharge)
- Street parking (metered)
- Conference center
- Snack bar/deli
- Secretarial services
- Security guard
- Technology helpdesk
- Technology support staff
- Tours/ticket assistance
- Meeting facilities size (meters) - 750
- Smoke-free property
- Internet
Available in all rooms: Free Wi-Fi, Wired high-speed Internet access (surcharge)
Available in some public areas: Free Wi-Fi, Wired (high-speed) Internet access - surcharge
- Parking
Secure covered parking (surcharge), Street parking (metered), Valet parking (surcharge), Parking nearby, Parking nearby (surcharge)
- Room Amenities
- Connecting / adjoining rooms available
- Free Wi-Fi
- Air conditioning
- Private bathroom
- Pay movies
- Hypo-allergenic bedding available
- Premium bedding
- Blackout drapes/curtains
- Climate control
- Complimentary newspaper
- Voice mail
- Direct-dial phone
- Complimentary bottled water
- Minibar
- Coffee/tea maker
- Wake-up calls
- Daily housekeeping
- Extra towels/bedding
- Turndown service (on request)
- Designer toiletries
- Hair dryer
- Bathrobes
- Bathroom phone
- Handheld showerhead
- Makeup/shaving mirror
- Bathtub or shower
- Satellite TV service
- Electronic/magnetic keys
- Iron/ironing board
- In-room safe
- Desk
- Cribs/infant beds available
- Wired high-speed Internet access (surcharge)
- Soundproofed rooms
- Complimentary toiletries
- Slippers
- City view
- Dining
Quinte & Sens - Inspired cuisine in the heart of la Défense designed to awaken the senses. Serving fresh, vibrant yet simple dishes. Open daily 6:30-1 AM.
- Accessibility
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
- Accessibility equipment for the deaf
- Accessible bathroom
- Accessible path of travel
- Accessible parking
- In-room accessibility
- Roll-in shower
Pullman Paris La Défense Policies & Fees
- Check-in
Check-in time starts at 2 PM
- Check-out
Check-out time is noon
- Payment types
Accepted at this hotel: American Express, Diners Club, MasterCard, Visa
- Pets
Pets allowed
- General
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
- Fees
- The following fees and deposits are charged by the property at time of service, check-in, or check-out.
- Breakfast fee: EUR 26 per person (approximately)
- Self parking fee: EUR 25 per day
- Pet fee: EUR 16 per pet, per night
- Babysitting/childcare fee: EUR 15 per hour, per child
- Rollaway bed fee: EUR 55 per night
The above list may not be comprehensive. Fees and deposits may not include tax and are subject to change.
- Additional Details
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
- Details
Complimentary Accommodations/Breakfast for 1 Child
Book a stay in any room or rate category and receive the following:
- Complimentary accommodations and daily breakfast for 1 child younger than age 12 who stays in the same room as parents or guardians using existing bedding
National Rating
- Details
This hotel has received its official star rating from the French Tourism Development Agency, ATOUT France.


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Revealed: Compensation Spreads of The Am Law 200
Which firms have the widest variances in partner pay?
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It appears that the spread is increasing. In early 2011, the legal media was aghast at ratios of 12:1. At the top end, that's now doubled, and the average ratio is now not far off the headline-grabber level of two years ago.
For lawyers not at that rarified level, the message should be clear: Either develop a book of business that makes you irreplaceable, or finance the stars' wealth. Here's a link to my Feb 2011 blog post, which could be republished today, unchanged.
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Definition of palolo worm in English:
palolo worm
Syllabification: pa·lo·lo wormPronunciation: /pəˈlōlō ˌwərm
- A marine bristle worm that swarms in response to changes in light intensity, particularly that of the moon. The worm's posterior segments detach themselves and swim to the surface where the reproductive cells are released into the sea.
- Several species in Eunicidae and other families, in particular the Samoan palolo worm (Palola (or Eunice) viridis), which occurs on South Pacific reefs
More example sentences- The palolo worm makes its home, according to Anja Schulze of the Smithsonian Marine Station, in the shallow reef, where it uses its sizeable jaws to dig itself a burrow in the limestone substrate.
- All around them palolo worms are thrashing in vast numbers.
- Palolo worms from the South Pacific are relatively well studied.
late 19th century: palolo from Samoan or Tongan.
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Pronunciation: ˌɛksə(ʊ)ˈtɛrɪk
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Seminars & Webcasts
On-Demand Learning
From the program: Acquiring or Selling the Privately Held Company 2013
Released on: Jun. 21, 2013
In this perennial favorite, our experienced faculty will walk you through all of the steps associated with acquiring and selling a privately held company, whether it is a large independent corporation, a division or subsidiary of a large public company, or a smaller venture capital-backed or family-owned entrepreneurial enterprise. You will learn about the special issues that apply when a private equity firm is the buyer or owns the target company, ...
From the program: Advanced Compliance and Ethics Workshop 2013
Released on: Dec. 3, 2013
Legal drivers for corporate compliance and ethics programs continue to grow in complexity and importance. Compliance programs are now widely expected by enforcement personnel and key stakeholders, and good practices continue to evolve. In addition, the costs of non-compliance have never been greater. Enforcement risks increase as governments work together more than ever before, and harsh penalties are increasingly becoming the norm both inside and ...
From the program: Advanced Copyright Law Annual Review 2013
Released on: May. 3, 2013
Copyright law has become an increasingly complex area in the face of new technologies and new ways of doing business. To help you keep up-to-date and provide the sophisticated, pragmatic legal advice your clients expect, PLI has brought together a group of leading experts on copyright law. The speakers at this advanced seminar will address critical issues in copyright law, drawing on their extensive experience and judgment.Current issues will be examined ...
From the program: Advanced Deposition Techniques 2014
Released on: Feb. 4, 2014
Depositions provide a unique forum to put forth your best case and get a glimpse at your adversary's evidence. Whether you've done one deposition or 100, you need to be prepared to handle the special situations that arise and know how to identify and exploit the opportunities that exist. This program covers a range of topics and scenarios that can challenge even the most seasoned litigators. Our faculty of experienced litigators and deposition ...
From the program: Advanced Licensing Agreements 2013
Released on: Apr. 4, 2013
Licensing transactions are more complex than ever. The constantly evolving legal, regulatory and technical landscape drives the need to stay current in all of these key areas. Whether utilized to develop technology, expand or create market opportunities, or generate returns from existing assets, managing complex licensing transactions requires a broad and varied toolkit. Additionally, whether licensing patents, copyrights, trade secrets or trademarks, ...
From the program: Advanced Licensing Agreements 2014
Released on: Feb. 12, 2014
Licensing transactions are more complex and more important than ever. The constantly evolving legal, regulatory and technical landscape drives the need to stay current in a wide variety of key areas. Whether utilized to develop technology, expand or create market opportunities, or generate returns from existing assets, managing complex licensing transactions requires a broad and varied toolkit. Additionally, whether licensing patents, copyrights, ...
From the program: Advanced Patent Licensing 2013: How to Make Money Without Getting Sued
Released on: Nov. 8, 2013
In the current economy, the heat is on to monetize patent portfolios. Thus, understanding patent transactions has never been more important. Experts in patent licensing will tell you how to get the most money from patent licensing, how to negotiate patent license agreements, and discuss "The Good, The Bad and The Ugly" examples from actual patent license agreements. The program will emphasize practical tips for structuring, negotiating and drafting ...
From the program: Advanced Swaps & Other Derivatives 2013
Released on: Nov. 1, 2013
The Dodd-Frank Act establishes a novel, comprehensive framework for the regulation of over-the-counter (OTC) derivatives and the market participants who transact in these products. That regulatory framework is now being implemented and its effect are becoming clear.The new regulatory regime touches the activities of all participants in the swaps markets, from end users to major swap participants to swap dealers. The new regulations will ...
From the program: Advanced Trademark Law Annual Review 2013
Released on: May. 2, 2013
Up-to-date knowledge of current trends and case law involving trademarks is essential for corporate and law firm attorneys who specialize in, or often find themselves involved in, this critical area of intellectual property. The 2013 Advanced Trademark Law Annual Review brings together an outstanding faculty of practitioners and industry experts who will provide up-to-date developments in trademark law and practice. This seminar is also noted for ...
From the program: Advanced Venture Capital 2013
Released on: Dec. 17, 2013
In this program, our expert faculty of lawyers, venture capitalists and business executives will analyze the legal and business issues relevant to navigating today's evolving venture capital world. Geared toward the experienced venture capital practitioner who knows the basics of venture capital and is seeking an in-depth analysis of current market conditions and the changing legal environment, this program will also address the latest ...
From the program: Advocating for Veterans: The Basics on VA Benefits, Discharge Upgrades and Veteran Cultural Competency 2013 (Free)
Released on: Nov. 15, 2013
There are over 21 million veterans in the U.S.; many of them are not aware of the benefits to which they are entitled. Medical and financial assistance are available to men and women who have served in the military, but some who can qualify are not receiving the benefits that they need. Attorneys can assist veterans with the problems that preclude them from receiving benefits, such as: Lack of proper paper trails to "service-connect" ...
From the program: Antitrust Counseling & Compliance 2013
Lawyers counseling businesses must possess a solid foundation in principles of antitrust law and an understanding of the latest antitrust developments. Running afoul of antitrust rules can result in class actions and treble damage litigation as well as government enforcement and adverse publicity. Are you prepared to provide practical answers to the antitrust questions your clients will ask? You will be after this program, developed with the purpose ...
From the program: Antitrust Institute 2013: Developments & Hot Topics
Released on: May. 21, 2013
From mergers and acquisitions to enforcement priorities, resale price maintenance to international competition, you can be certain to hear about all of the developments in antitrust law at this program led by top experts in the field. Hear from leading antitrust practitioners, senior government officials, and state regulators about how to manage all of the challenges new developments might throw your way. Whether you are a seasoned antitrust ...
From the program: Appellate Advocacy 2013
Released on: Apr. 3, 2013
Appellate Advocacy will give you real-world insight into effective appellate advocacy. You will learn about all aspects of the appellate process, from things to do (and not to do) in the trial court, to strategies and techniques for persuasive brief writing and oral argument, to the rehearing and certiorari processes. You will also hear the "View from the Bench" - what judges find persuasive and effective. Lecture Topics ...
From the program: Approaching Veterans Issues: A Legal Overview Including an Update of California Penal Code Section 1170.9
Released on: Jun. 7, 2013
Certain laws and courtroom practices have the potential to impact veterans differently than the rest of the population. This three hour overview will provide useful information to attorneys, judicial officers and others who interact with veterans about the implications of certain laws and practices with regards to veterans' pasts and futures. The program will focus on Penal Code 1170.9, especially with regard to the 2013 enhancements and will address ...
From the program: Asset Based Financing Strategies 2013
Released on: Mar. 25, 2013
Asset-based financing is a key source of credit for small and medium-size companies and for some larger, publicly traded companies as well. In contrast to equity financing where ownership in the company is diluted, asset-based financing allows companies to use their own assets to generate cash flow. In structuring these loans, lenders and their counsel must consider all senior interests in the collateral, as well as the practical difficulties in ...
From the program: Audit Committees and Financial Reporting 2013: Recent Developments and Current Issues
Released on: Jul. 5, 2013
As we continue to see many new regulations from the SEC, PCAOB and financial institution regulators, we also continue to see numerous challenges emerge for the audit committee. If you are a director or member of an audit committee, or if you advise audit committees, this program will help you understand what is required of the audit committee, and those who advise them, in this rapidly changing environment. You will hear from an expert faculty of ...
From the program: Bankruptcy, Mortgages and Foreclosure: What Bankruptcy Can and Can't Do for Borrowers in Distress
Released on: Apr. 12, 2012
In this training you will get an overview of how consumer bankruptcy interacts with mortgage debt and foreclosures. During the session, practitioners working at the intersection of these two crucial areas in consumer law will provide a basic overview of consumer bankruptcy; share tools for assessing whether bankruptcy might help - or harm - a delinquent borrower; and discuss how bankruptcy can be used to address mortgage ...
From the program: Bankruptcy Basics for Low-Income Clients 2013 (Free)
Released on: Oct. 10, 2013
Although the recession is beginning to even out and foreclosures in many parts of the country are in the decline, the recession continues to affect financially marginalized people in disproportionate numbers. Many debtors are unrepresented and bankruptcy clinics are still in great demand. There is a critical need for pro bono attorneys to assist low-income clients at all levels. The practice of bankruptcy law changed dramatically with the Bankruptcy ...
From the program: Bankruptcy & Reorganizations 2013: Current Developments
Released on: Apr. 26, 2013
The credit markets continue to be volatile and in today's weak economy, staying abreast of the current developments in restructuring, bankruptcy and reorganizations is essential. This advanced conference assembles a remarkable faculty of bankruptcy judges and leading practitioners who will provide you with a thorough review and analysis of hot topics, developments, and recent decisions in diverse industries. They will cover the ramifications of the ...
From the program: Basic Immigration Law 2013
Released on: Mar. 28, 2013
This program will explain the basic concepts of U.S. immigration law and procedure, and reflect current developments and trends, including developments relating to the new prosecutorial discretion policies. Lecture Topics [Total time 06:33:57] Segments with an asterisk (*) are available only with the purchase of the entire program. Opening Remarks and Introduction* [00:00:50] Cyrus D. Mehta Overview of the U.S. Immigration ...
From the program: Basic Negotiation Skills 2014
Released on: Jan. 27, 2014
Negotiation is part of any lawyer's everyday life. You negotiate with colleagues, clients, and staff about an array of issues, ranging from simple project deadlines to complex contractual language. This program is designed to help you gain confidence in every negotiation you conduct. The panelists represent academicians, sole practitioners, and members of leading local law firms.Lecture Topics [Total time 06:10:39]Segments ...
From the program: Basics of Accounting for Lawyers 2013: What Every Practicing Lawyer Needs to Know
Released on: Aug. 2, 2013
Accounting is a language of business. Because attorneys encounter accounting information in a wide range of legal issues, it is critical for lawyers to understand financial statements and core accounting concepts. This practical course, specifically designed for the non-accountant attorney, will provide an understanding of financial statements and the key principles of accounting, using "real life" examples of accounting issues that attorneys commonly ...
From the program: Basics of International Taxation 2013 (New York)
Released on: Aug. 7, 2013
In a global business, the tax laws of the United States may impact transactions regardless of where the transactions take place. A working knowledge of the U. S. international tax rules is, therefore, of paramount importance to a wide variety of tax and corporate professionals.In this program, some of the world's leading tax experts will discuss the current rules of U.S. international taxation. Each panel will focus upon a distinct international ...
From the program: Basics of International Taxation 2013 (San Francisco)
Released on: Oct. 8, 2013
Nathan A. Schachtman ~ Nathan A. Schachtman, Esq., PC
Gary A. Adler ~ Bingham McCutchen LLP
Sabine Chalmers ~ Chief Legal & Corporate Affairs Officer, Anheuser-Busch InBev
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text: Public Information Office NORTHERN OKLAHOMA COLLEGE BOISE CITY COMPETES IN NOC QUIZ BOWL Following a disappointing 315-50 loss to Cimarron in the Northern Oklahoma College High School Academic Quiz Bowl, the Boise City Quiz Bowl team battled back in the loser's bracket, romping over Perry, 270-45; squeaking by Deer Creek-Lamont, 195-185 and trouncing Drumright, 310-135 before finally falling to Pioneer, 180-115. Boise City team members were juniors Jonathan Faulkner, Summer James and Tori Willett and sophomores Destry Elms, Denisse Hernandez and Kyle Wilson. Tammy Crabtree coached the team. Twenty-two high schools competed in the double elimination Quiz Bowl tourney on the Tonkawa campus Jan. 5. Winners were Drummond, first; Edmond Home School second; and Kremlin-Hillsdale, third. Selected as All-Stars were Sam Engleman, Beaver; Shawn Hollopeter, Copan ; Shannon Rodenberg and Tyler Hoeltzel, Drummond; Brady Juengel, Edmond Home School ; and Bobbi Lakin, Kremlin -Hillsdale. Boise City News |