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abstract: 'The relative thermodynamic stability of surface reconstructions including vacancies, adatoms and additional layers on both CaO and MnO$_{2}$ terminations is calculated to predict the surface phase diagram of ($\sqrt{2}\times\sqrt{2}$)*R*45$^{\circ}$ CaMnO$_{3}$ (001) using *ab initio* thermodynamics. Stoichiometric and nonstoichiometric reconstructions are considered. A set of boundary conditions driven by the binary and ternary sub-phases from CaMnO$_{3}$ defines its bulk stability region, enclosing the stable surface reconstructions that can be in equilibrium with the bulk. Most of the surfaces take the magnetic ordering of the bulk ground state, G-type antiferromagnetic (AFM); however, some of the MnO$_{2}$-terminated surfaces are more stable when the surface layer spins flip. At 573 K, the stoichiometric CaO- and MnO$_{2}$-terminated surfaces are predicted to be thermodynamically stable, as well as a CaO-terminated surface reconstruction where half the Ca are replaced by Mn. The MnO$_{2}$-terminated surface reconstructions dominate the phase diagrams at high temperatures, including phases with MnO and MnO$_{2}$ adatoms per surface unit cell.'
author:
- 'Diomedes Saldana-Greco, Chan-Woo Lee, Doyle Yuan, and Andrew M. Rappe'
bibliography:
- 'rappecites.bib'
title: |
**[Structural and Magnetic Reconstruction of Thermodynamically Stable CaMnO$_{3}$ (001) Surfaces ]{}\
**
---
\[sec:level1\] I. Introduction {#seclevel1-i.-introduction .unnumbered}
==============================
Perovskite CaMnO$_{3}$ has recently been the subject of increasing interest due to its strain-assisted multiferroicity [@Bhattacharjee09p117602; @Wang12p17005], thermoelectric efficiency [@Urata07p535; @Koumoto10p363], colossal magnetoresistance [@Zeng99p8784], and catalytic properties [@Kawashima08p3439]. Its high catalytic activity for oxidation reactions and structural similarity to important biological catalysts has motivated studies on CaMnO$_{3}$ as a potential catalyst for water oxidation [@Najafpour12p1]. While atomic insight into the CaMnO$_{3}$ surface is imperative if its potential application as a catalyst is to be realized, very little has been investigated in this direction. Most of the previous studies have focused on magnetic properties [@Filippetti99p4184; @Filippetti00p11571; @Nguyen11p3613] of the stoichiometric cubic CaMnO$_{3}$ surface. In this study, we determine the atomic and magnetic low-energy surface structures of orthorhombic *Pnma* CaMnO$_{3}$ (001) as functions of environmental variables, developing surface phase diagrams by combining density functional theory (DFT) and thermodynamics [@Reuter01p035406]. Various surface reconstructions for both CaO- and MnO$_{2}$-terminated surfaces are explored within a ($\sqrt{2}\times\sqrt{2}$)*R*45$^{\circ}$ surface periodicity. For structures with Mn atoms at the surface, we considered multiple magnetic spin configurations to ensure that the surface stability accounts for the potential magnetic reconstruction at the surface. The surface free energies are computed to generate the surface phase diagram within the bulk stability range. To illustrate the surface stability at a range of temperatures, the vibrational free energy is included in the calculation of the bulk stability region. Through our surface examination, we predict thermodynamically stable surface reconstructions at a range of conditions.
\[sec:level1\] II. Methodology {#seclevel1-ii.-methodology .unnumbered}
==============================
\[sec:level2\] A. Computational Methods {#seclevel2-a.-computational-methods .unnumbered}
---------------------------------------
DFT calculations were performed using the PBEsol [@Perdew08p136406] generalized gradient approximation (GGA), as implemented in the [Quantum Espresso]{} [@Giannozzi09p395502] computer code. Perdew and collaborators have shown that the PBEsol functional describes solids and surfaces accurately [@Perdew08p136406]. PBEsol+*U* was also used to test electronic properties, with *U*$_{\textrm{eff}}$ = 7.23 eV, in line with previous work [@Luo07p036402] and determined with the linear response scheme [@Cococcioni05p035105]. However, adding strong correlations to CaMnO$_{3}$ provides unsatisfactory description of the magnetic properties, as has been established by Luo *et al.* [@Luo07p036402]. Spin-polarized electronic densities were used in all calculations, treating the magnetic moments as collinear in all cases, with additional non-collinear (NC) calculations for some Mn-terminated surfaces and for the ternary sub-phases. All atoms are represented by norm-conserving, optimized [@Rappe90p1227], designed nonlocal [@Ramer99p12471] pseudopotentials with spin-orbit interaction (for NC magnetism). The pseudopotentials are generated with the [opium]{} package [@Opium], treating the 3*s*, 3*p*, 3*d*, 4*s*, and 4*p* of Ca, the 3*s*, 3*p*, 3*d*, 4*s*, and 4*p* of Mn, and the 2*s* and 2*p* of O as valence states. An accurate description of the magnetic properties of all the systems studied is achieved when nonlinear core-valence interaction is included in the Mn pseudopotential by the partial core correction scheme [@Fuchs99p67; @Porezag99p14132]. The CaMnO$_{3}$ magnetic ground state is collinear G-type antiferromagnetic (AFM) with an observed Néel temperature of about 130 K [@Poeppelmeier82p71], estimating the magnetic interaction energy to be around 6.6 meV [@Bhattacharjee08p255229]. Therefore, all calculations are run with a 70 Ry plane-wave energy cutoff to ensure accuracy for the small relative energies among the different magnetic configurations. The Brillouin zone is sampled using a $4\times4\times1$ Monkhorst-Pack [@Monkhorst76p5188] $k$-point mesh for surface structures based on energy convergence for bulk CaMnO$_{3}$. For binary and ternary sub-phases, a dense enough set of $k$-points was used so that the total energy is converged. The ionic relaxation parameters are chosen so that the forces in the surface and bulk structures are lower than 2 meV/[Å]{}. We use density functional perturbation theory [@Baroni01p515; @Gonze95p1096] to calculate phonon frequencies and vibrational displacement vectors at the $\Gamma$ point for all binary and ternary sub-phases in order to include the vibrational free energy contribution into the definition of the free energy. Translational symmetry is ensured by enforcing the three translational acoustic sum rules requiring that the acoustic modes have zero frequency. This is accomplished through small corrections to the diagonal elements of the dynamical matrix.
\[sec:level2\] B. CaMnO$_{3}$ (001) Surface Structures {#seclevel2-b.-camno_3-001-surface-structures .unnumbered}
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The (001) surface of CaMnO$_{3}$ is the lowest energy, since it consists of alternating CaO and MnO$_{2}$ planes, each of which is neutral in an ionic picture [@Tasker79p4977]. We construct surface structures with different compositions and reconstructions for both CaO and MnO$_{2}$ terminations, with symmetrical slabs of 7-9 layers and a vacuum gap between slabs of $\approx$15 [Å]{}. All atoms are fully relaxed. Over 80 surface terminations are made by varying the stoichiometry of Ca, Mn and O including additional Ca$_{x}$O$_{y}$ and Mn$_{x}$O$_{y}$ layers in the ($\sqrt{2}\times\sqrt{2}$)*R*45$^{\circ}$ surface symmetry. The positions of adatoms and additional layers are strategically selected; Ca and Mn atoms are placed on top of O atoms, and O species on top of Ca and Mn atoms. Each surface termination is identified based on its stoichiometric termination (either CaO or MnO$_{2}$) and whether its composition involves vacancies (-) or adatoms (+). As an illustration, CaO+2.0O refers to a CaO-terminated surface with two oxygen adatoms per primitive unit cell of CaO. The number of adatoms and/or vacancies is tabulated per formula unit of either CaO or MnO$_{2}$, even though there are two formula units in the surface super cell. Similarly, $\mbox{MnO$_{2}$-0.5Mn-0.5O}$ refers to a MnO$_{2}$-terminated surface with one Mn vacancy and one oxygen vacancy per two MnO$_{2}$ units. Illustrations of the thermodynamically stable surface terminations after relaxation are shown in Fig. \[Fig.1\]. These surface phases include (a,b) the stoichiometric surfaces, (c,d) their full surface coverage with adsorbed oxygen molecules, (e) the MnO$_{2}$-terminated surface with one Mn vacancy and one oxygen vacancy, as well as (f) a CaO-terminated surface where a Mn adatom occupies a Ca vacancy. The last two surface phases (g,h) are stable at high temperatures but represent very exotic surface structures, as the $^{a}$Mn adatoms and $^{a}$O adatoms have different coordination than the stoichiometric atoms, potentially indicating active sites.
FM A-AFM C-AFM G-AFM
---------------------------------- ------ ------- ------- ------- --
Energy (meV/formula unit) 38 12 4 0
Magnetic moment ($\mu$$_{B}$/Mn) 2.75 2.68 2.64 2.61
: Energies per formula unit and magnetic moments on Mn atoms for bulk CaMnO$_{3}$ in different magnetic phases.
The ferromagnetic (FM) and three AFM phases of bulk CaMnO$_{3}$ were analyzed. The A-AFM state has FM planes of alternating direction along the *z*-axis. The C-AFM arrangement consists of a Mn with nearest neighbors of opposite spin in the plane, and parallel spins between one plane and the next, while G-type AFM arranges the spins so that all nearest neighbors have opposite spin orientation. The magnetic ground state of bulk CaMnO$_{3}$ is found to be G-AFM *Pnma* insulator, as reported in previous theoretical and experimental studies [@Pickett96p1146; @Bhattacharjee08p255229; @Poeppelmeier82p71]. Our computed electronic band gap is 0.65 eV, significantly underestimating the experimental value of 3.1 eV [@Jung97p15489] but in close agreement with previous computational work [@Tsukahara10p214108; @Pickett96p1146] The atom-projected magnetic moments of Mn atoms in each magnetic phase are shown in Table 2 as well as the relative energies with respect to the stable G-AFM phase, which are in very good agreement with those of a recent projector augmented wave calculation using the PBE functional [@Tsukahara10p214108]. The surface calculations are performed with G-AFM ordering, magnetic spin flip at the surface and other possible magnetic spin configurations for surfaces with complex Mn$_{x}$O$_{y}$ reconstructions (See Supplementary Material). The magnetic spin flip configuration is studied for all MnO$_{2}$-based surfaces since stoichiometric MnO$_{2}$ is found to be more energetically favorable when all the surface spins flip (making the top and second layers FM aligned) [@Filippetti99p4184].
\[sec:level2\] C. Thermodynamic Stability {#seclevel2-c.-thermodynamic-stability .unnumbered}
-----------------------------------------
The free energies of CaMnO$_{3}$ (001) surfaces are calculated with the thermodynamic approach described in this section. In this model, the CaMnO$_{3}$ bulk is considered to be in contact with an atmosphere composed of all its components in equilibrium. This implies that the system acts as a reservoir where removal and/or addition of any of its components at the surface leads to a range of thermodynamically stable reconstructions and compositions at different conditions. The oxygen chemical potential, $\mu$$_{\textrm{O}}$, is used to correlate the surface stability with environmental conditions such as temperature, *T*, and oxygen partial pressure, *p*(O$_{2}$). The surface free energy, $\Omega^i$, of an individual surface slab, *i*, is defined as the excess amount of free energy needed to create the surface from its bulk form [@Reuter01p035406], $$\begin{aligned}
\Omega^i &=&\frac{1}{2A}\left[G^i_{\textrm{slab}}-\sum_{j}N^i_{j}\mu_{j}\right] \nonumber \\
&=&\frac{1}{2A}\left[G^i_{\textrm{slab}}-N^i_{\textrm{Ca}}\mu^i_{\textrm{Ca}}
-N^i_{\textrm{Mn}}\mu^i_{\textrm{Mn}}-N^i_{\textrm{O}}\mu^i_{\textrm{O}}\right].\end{aligned}$$ The Gibbs free energy of the slab is denoted by G$^i_{\textrm{slab}}$. The term $\displaystyle\sum\limits_{j}N^{i}_j\mu_{j}$ represents the free energy of the material in slab *i* as the sum of the chemical potentials from the bulk of each component, $\mu_{j}$, multiplied by the number of its atoms in each slab, *N*$^{i}_{j}$ [@Cahn73p3]. The factor of $\frac{1}{2}$ is included to account for top and bottom slabs. The surface free energy is reported as energy per unit area. The $\Gamma^i_{m,n}$ term shown in Eq. (2) accounts for the off-stoichiometric atoms of component *n* with respect to component *m* in any surface slab *i* by relating the number of atoms of components *N*$_{m}$ and *N*$_{n}$ in the slab to their ratio in the bulk, $$\begin{aligned}
\Gamma^i_{m,n}=\frac{1}{2A}\left(N^i_{n}-N^i_{m}\frac{N^{\textrm{bulk}}_n}{N^{\textrm{bulk}}_m}\right).\end{aligned}$$ In our case, the non-stoichiometric surface components were defined with respect to Mn atoms. Defining $\Gamma^i_{m,n}$ with respect to Ca atoms does not change the results. Next, Eqs. (1) and (2) are merged to express the Gibbs surface free energy as $$\begin{aligned}
\Omega^i &=&\frac{1}{2A}\left[G^i_{\textrm{slab}}-N^i_{\textrm{Mn}}(\mu_{\textrm{Ca}}+\mu_{\textrm{Mn}}+3\mu_{\textrm{O}})\right] \nonumber \\
&& -\Gamma^i_{\textrm{Mn,Ca}}\mu_{\textrm{Ca}}-\Gamma^i_{\textrm{Mn,O}}\mu_{\textrm{O}}.\end{aligned}$$
Since the system acts as a thermodynamic reservoir, the chemical potentials of CaMnO$_{3}$ components are not independent; they are related to the chemical potential of CaMnO$_{3}$ crystal, $\mu$$_{\textrm{CaMnO$_{3}$}}$. The Gibbs free energy of bulk CaMnO$_{3}$ per formula unit, *g*$^{\textrm{bulk}}_{\textrm{CaMnO$_{3}$}}$, is then equal to $\mu_{\textrm{CaMnO}_{3}}$ because the slabs must be in equilibrium with the bulk as shown below: $$\begin{aligned}
\mu_{\textrm{Ca}}+\mu_{\textrm{Mn}}+3\mu_{\textrm{O}}=\mu_{\textrm{CaMnO$_{3}$}}=g^{\textrm{bulk}}_{\textrm{CaMnO$_{3}$}}.\end{aligned}$$ Inserting the relationship above into Eq. (3) simplifies the surface free energy to $$\begin{aligned}
\Omega^i &=&\frac{1}{2A}\left[G^i_{\textrm{slab}}-N^i_{\textrm{Mn}}g^{\textrm{bulk}}_{\textrm{CaMnO$_{3}$}}\right] \nonumber \\
&& -\Gamma^i_{\textrm{Mn,Ca}}\mu_{\textrm{Ca}}-\Gamma^i_{\textrm{Mn,O}}\mu_{\textrm{O}}.\end{aligned}$$
The bulk stability region is defined by a set of boundary conditions [@Lee11p115418; @Heifets11p491; @Kolpak08p036102; @Levchenko08p256101]. First, Ca and Mn are not allowed to form metallic precipitates by satisfying the following conditions: $$\begin{aligned}
\mu_{\textrm{Ca}} &\le& g^{\textrm{bulk}}_{\textrm{Ca}}, \\
\mu_{\textrm{Mn}} &\le& g^{\textrm{bulk}}_{\textrm{Mn}}. \end{aligned}$$ Second, the bulk is stable, while binary metal oxides derived from its components do not precipitate: $$\begin{aligned}
\mu_{\textrm{Ca}}+\mu_{\textrm{O}} &\le& g^{\textrm{bulk}}_{\textrm{CaO}}, \\
\mu_{\textrm{Mn}}+\mu_{\textrm{O}} &\le& g^{\textrm{bulk}}_{\textrm{MnO}}, \\
3\mu_{\textrm{Mn}}+4\mu_{\textrm{O}} &\le& g^{\textrm{bulk}}_{\textrm{Mn$_{3}$O$_{4}$}}, \\
2\mu_{\textrm{Mn}}+3\mu_{\textrm{O}} &\le& g^{\textrm{bulk}}_{\textrm{Mn$_{2}$O$_{3}$}}, \\
\mu_{\textrm{Mn}}+2\mu_{\textrm{O}} &\le& g^{\textrm{bulk}}_{\textrm{MnO$_{2}$}}. \end{aligned}$$ Third, there should be no precipitation of other ternary sub-phases (Ca$_{x}$Mn$_{y}$O$_{z}$) from the bulk CaMnO$_{3}$. The experimental phase diagram of bulk CaMnO$_{3}$ illustrates the importance of considering these ternary sub-phases, as they can co-exist with CaMnO$_{3}$ [@Horowitz78p1359; @Balakirev06pS49]. Consequently, the following conditions must be met: $$\begin{aligned}
\mu_{\textrm{Ca}}+2\mu_{\textrm{Mn}}+4\mu_{\textrm{O}} &\le& g^{\textrm{bulk}}_{\textrm{CaMn$_{2}$O$_{4}$}}, \\
\mu_{\textrm{Ca}}+7\mu_{\textrm{Mn}}+12\mu_{\textrm{O}} &\le& g^{\textrm{bulk}}_{\textrm{CaMn$_{7}$O$_{12}$}}, \\
2\mu_{\textrm{Ca}}+\mu_{\textrm{Mn}}+4\mu_{\textrm{O}} &\le& g^{\text{bulk}}_{\textrm{Ca$_{2}$MnO$_{4}$}}, \\
3\mu_{\textrm{Ca}}+2\mu_{\textrm{Mn}}+7\mu_{\textrm{O}} &\le& g^{\textrm{bulk}}_{\textrm{Ca$_{3}$Mn$_{2}$O$_{7}$}}, \\
4\mu_{\textrm{Ca}}+3\mu_{\textrm{Mn}}+10\mu_{\textrm{O}} &\le& g^{\textrm{bulk}}_{\textrm{Ca$_{4}$Mn$_{3}$O$_{10}$}}.\end{aligned}$$ The DFT energy is taken as the Helmholtz free energy at 0 K with zero-point energy, so the Gibbs free energy and the DFT energy are related as follows [@Reuter01p035406], $$\begin{aligned}
G=E^{\textrm{total}}+F^{\textrm{vib}}-TS^{\textrm{conf}}+pV\approx E+F^{\textrm{vib}}.\end{aligned}$$ The energetic contributions provided by the *pV* and the configurational entropy *TS*$^{\textrm{conf}}$ terms to the thermodynamic stability are considered to be negligible, as has been proven in previous studies [@Lee11p115418; @Heifets11p491; @Kolpak08p036102; @Levchenko08p256101]. The term *F*$^{\textrm{vib}}$, which is equal to *E*$^{\textrm{vib}}$-*TS*$^{\textrm{vib}}$, is calculated from the vibrational modes of the system. The phonon density of states, $\sigma$($\omega$), is used to express *F*$^{\textrm{vib}}$ as an integral over frequency, $\omega$: $$\begin{aligned}
F^{\textrm{vib.}}=\int d\omega F^{\textrm{vib.}}(T,\omega)\sigma(\omega).\end{aligned}$$ The analytical expression of the harmonic quantum vibrational free energy per mode as a function of temperature and frequency, *F*$^{\textrm{vib}}$(*T*,$\omega$), can be written as $$\begin{aligned}
F^{\textrm{vib.}}(T,\omega)=\frac{1}{2}\hbar\omega-k_{B}T\ln\left(1-e^{-\frac{\hbar\omega}{k_{B}T}}\right).\end{aligned}$$ Once the Gibbs free energy of each compound is calculated, the stability region of bulk CaMnO$_{3}$ as a function of *T* can be evaluated [@Lee11p115418]. The vibrational free energy is included for all bulk phases while it is neglected for all slab structures [@Heifets11p491; @Kolpak08p036102; @Levchenko08p256101]. The surface vibrational behavior is complex, and the vibrational modes could be affected by the electronic redistribution at the surface region; however, it has been shown for other systems that the phonon frequencies do not vary the order of stable surface phases [@Lee11p115418]. The chemical potential for each component of CaMnO$_{3}$ is defined as its difference from the total energy of its reference state: $$\begin{aligned}
\Delta\mu_{\textrm{Ca}}=\mu_{\textrm{Ca}}-\left(E^{\textrm{bulk}}_{\textrm{Ca}}+F^{\textrm{vib,bulk}}_{\textrm{Ca}}\right), \\
\Delta\mu_{\textrm{Mn}}=\mu_{\textrm{Mn}}-\left(E^{\textrm{bulk}}_{\textrm{Mn}}+F^{\textrm{vib,bulk}}_{\textrm{Mn}}\right), \\
\Delta\mu_{\textrm{O}}=\mu_{\textrm{O}}-\frac{1}{2}\left(E^{\textrm{gas}}_{\textrm{O$_{2}$}}+F^{\textrm{vib,gas}}_{\textrm{O$_{2}$}}\right).\end{aligned}$$ For the metals Ca and Mn, the reference energy is the DFT total energy and vibrational free energy of the elemental bulk crystal. For oxygen, the reference is the energy of an O atom in an isolated O$_{2}$ molecule. The *E*$^{\textrm{gas}}_{\textrm{O$_{2}$}}$ is corrected so that *E*$^{\textrm{gas}}_{\textrm{O$_{2}$}}$ = 2*E*$_{\textrm{O}}$+ *E*$^{\textrm{expt}}_{\textrm{binding}}$ [@Kolpak08p036102] since a known shortcoming of DFT is the overestimation of binding energies [@Furche01p195120], particularly for double bonds [@Walter99p11; @Grinberg02p2264].
Experimental Theoretical
---------------------------------------- -------------- ------------- --
CaO -6.59 -6.71
MnO (AFM-II$^{\emph{a}}$) -3.99 -4.06
Mn$_{3}$O$_{4}$ (FiM-III$^{\emph{b}}$) -14.38 -15.46
Mn$_{2}$O$_{3}$ (FM) -9.93 -11.36
MnO$_{2}$ (AFM) -5.39 -6.60
CaMn$_{2}$O$_{4}$ (AFM) — -18.23
CaMn$_{7}$O$_{12}$ (NC-AFM) — -47.01
Ca$_{2}$MnO$_{4}$ (G-AFM) — -20.56
Ca$_{3}$Mn$_{2}$O$_{7}$ (G-AFM) — -34.34
Ca$_{4}$Mn$_{3}$O$_{10}$ (G-AFM) — -48.12
CaMnO$_{3}$ (G-AFM) -12.83 -13.79
: Experimental and computed formation energies (eV/formula unit) for binary and ternary sub-phases defining the bulk stability region of CaMnO$_{3}$. The calculated formation energies for all Mn-based phases were performed with their corresponding magnetic ground-state structure, noted next to the compound. The experimental values are obtained from Ref. [@Fritsch96p1761].
FM layers in the \[111\] plane and successive antiparallel layers.
Ferrimagnetic with Mn atoms ($\uparrow$$\uparrow$$\downarrow$$\downarrow$$\uparrow$$\uparrow$) in the corresponding order as Ref. [@Fritsch96p1761].
Combining the chemical potential expressions, Eq. (21-23), and the approximation to the Gibbs free energy for the slabs and CaMnO$_{3}$ bulk leads to the following expression for the Gibbs surface free energy, $$\begin{aligned}
\Omega^i &=& \frac{1}{2A}\left[E^i_{\textrm{slab}}-N^i_{\textrm{Mn}}\left(E^{\textrm{bulk}}_{\textrm{CaMnO$_{3}$}}+F^{\textrm{vib,bulk}}_{\textrm{CaMnO$_{3}$}}\right)\right] \nonumber \\
&& -\left[\Gamma^i_{\textrm{Mn,Ca}}\left(\Delta\mu_{\textrm{Ca}}+E^{\textrm{bulk}}_{\textrm{Ca}}+F^{\textrm{vib,bulk}}_{\textrm{Ca}}\right)+\Gamma^i_{\textrm{Mn,O}}\left(\Delta\mu_{\textrm{O}}+\frac{1}{2}\left(E^{\textrm{gas}}_{\textrm{O$_{2}$}}+F^{\textrm{vib,gas}}_{\textrm{O$_{2}$}}\right)\right)\right].\end{aligned}$$ The same approach including the vibrational free energy is applied to Eq. (4), whose expression is rewritten introducing the formation energy, $\Delta$$E$$^{\textrm{bulk}}_{\textrm{f}}$, as follows, $$\begin{aligned}
\Delta\mu_{\textrm{Ca}}+\Delta\mu_{\textrm{Mn}}+3\Delta\mu_{\textrm{O}}&=&\Delta{E}^{\textrm{bulk}}_{\textrm{f,CaMnO$_{3}$}}+F^{\textrm{vib,bulk}}_{\textrm{CaMnO$_{3}$}}.\end{aligned}$$ This equation is then used to rearrange all the bulk stability boundary inequalities, Eq. (6-17), so that only $\mu_{\textrm{Ca}}$ and $\mu_{\textrm{O}}$ are kept as variables. For instance, Eq. (15) becomes $$\begin{aligned}
\Delta\mu_{\textrm{Ca}}+\Delta\mu_{\textrm{O}}&\le&\Delta{E}^{\textrm{bulk}}_{\textrm{f,Ca$_{2}$MnO$_{4}$}}
-\Delta{E}^{\textrm{bulk}}_{\textrm{f,CaMnO$_{3}$}} \nonumber \\
&& +F^{\textrm{vib,bulk}}_{\textrm{Ca$_{2}$MnO$_{4}$}}-
F^{\textrm{vib,bulk}}_{\textrm{CaMnO$_{3}$}}.\end{aligned}$$ The results from the DFT formation energies, $\Delta$$E$$^{\textrm{bulk}}_{\textrm{f}}$, for all the binary and ternary sub-phases are compared to experimental quantities in Table 2. The formation energies are slightly overestimated, which is a well-known shortcoming of DFT [@Kotomin08p4644]. The surface phase diagram dependence on *T* and *p*(O$_{2}$) provides insightful physical interpretations of the experimental range of conditions for the surface stability. The $\Delta\mu_{\textrm{O}}$ can be directly related to *T* and *p*(O$_{2}$) by the ideal gas approximation, since at equilibrium the $\mu_{\textrm{O}}$ in CaMnO$_{3}$ bulk is equal to the chemical potential of oxygen gas in the environment, $\mu_{\textrm{O}}$=$\frac{1}{2}$$\mu^{\textrm{gas}}_{\textrm{O$_{2}$}}$(*T*,*p*). This leads to $$\begin{aligned}
\Delta\mu_{\textrm{O}}(T,p) = \Delta\mu_{\textrm{O}}\left(T,p^{\textrm{0}}\right)+\frac{1}{2}kT\ln\left(\frac{p}{p^{\textrm{0}}}\right)\end{aligned}$$ where $\Delta\mu$$_{\textrm{O}}$(*T*,*p*$^0$)=$\frac{1}{2}$$\Delta$*G*$^{\textrm{gas}}_{\textrm{O$_{2}$}}$(*T*,*p*$^0$). Thermodynamic data from the NIST-JANAF thermochemical tables [@Chase98p1] are used to determine the values of $\Delta\mu$$_{\textrm{O}}$(*T*,*p*$^0$) by selecting the reference state for $\frac{1}{2}$$\Delta$*G*$^{\textrm{gas}}_{\textrm{O$_{2}$}}$(*T*,*p*$^0$) extrapolated to *T*=0 K and *p*(O$_{2}$)=1 atm. Then, the values obtained are plotted for a temperature range of 03000 K at different *p*(O$_{2}$) to correlate the stable surface phases with controlled environmental conditions.
\[sec:level1\] III. Results and Discussion {#seclevel1-iii.-results-and-discussion .unnumbered}
==========================================
\[sec:level2\] A. Magnetic Surface Reconstruction {#seclevel2-a.-magnetic-surface-reconstruction .unnumbered}
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Depending on their surface composition, some of the MnO$_{2}$-based slabs undergo a magnetic spin flip reconstruction at the surface [@Filippetti99p4184] that is energetically favorable, leading to lower total energy and greater surface stability. This magnetic spin flip reconstruction refers to a surface with G-AFM bulk order, but where the Mn spins in the surface layer align with the spins of the Mn in the second layer, generating a bilayer with C-type AFM alignment, as shown in Fig. \[Fig.2\](a,c). This occurs when the surface breaks the degeneracy of *e$_{g}$* and *t$_{2g}$* states, as the Mn cage is changed from octahedral to square pyramidal. This leads to the partial occupation of the *e$_{g}$* *d$_{z^{2}}$* bands at the surface, which favors the FM alignment of spins between the surface and subsurface MnO$_{2}$ layers. In bulk CaMnO$_{3}$, the superexchange magnetic interactions between the filled *t$_{2g}$* shells and the formally empty *e$_{g}$* shells determine the G-AFM ordering. However, this G-AFM ordering does not extend to the (001) MnO$_{2}$-terminated surfaces, since double-exchange governs the magnetic interaction between Mn subsurface and surface. The Mn 3*d* orbital-projected density of states (DOS) of both G-AFM and spin flip surface and subsurface layers of the MnO$_{2}$-terminated stoichiometric surface are shown in Fig. \[Fig.2\](b,d). The surface states of both G-AFM and spin-flip show the broken degeneracy of *e$_{g}$* $\rightarrow$ *d$_{x^{2}-y^{2}}$*, *d$_{z^{2}}$* and *t$_{2g}$* $\rightarrow$ *d$_{xy}$*, (*d$_{xz}$*,*d$_{yz}$*), as expected for square pyramidal. The G-AFM subsurface states have degenerate *e$_{g}$* and *t$_{2g}$* shells; however, the spin-flip subsurface *e$_{g}$* degeneracy is broken, as the *d$_{z^{2}}$* is favorably aligned with the *d$_{z^{2}}$* from its neighboring surface state. The alignment of the Mn spins on surface and subsurface layers is induced by a double-exchange process, which is enabled by the occupation of the *d$_{z^{2}}$* orbital, lowering the energy of MnO$_{2}$-terminated surfaces with the spin flip arrangement. Our DFT computed stoichiometric MnO$_{2}$-surface yields a spin flip structure as its magnetic ground state, with an energy lowering relative to G-AFM of 15 meV per primitive surface cell.
![Spin-flip magnetic reconstruction at the stoichiometric MnO$_{2}$-terminated surface. The atomic, electronic and magnetic structure of the stoichiometric MnO$_{2}$-terminated surface adopting (a,b) the bulk magnetic configuration, G-AFM, and (c,d) an energetically more favorable magnetic reconstruction where the spins of the surface Mn atoms are flipped. The magnetic spins are represented by the yellow arrows. The orbital-projected densities of states (DOS) for surface and subsurface Mn *d*-orbitals of the (b) G-AFM, and (d) spin-flip magnetic structures show different energetic alignment of the 3*d$_{z^{2}}$* orbital between surface and subsurface states.[]{data-label="Fig.2"}](R095_Fig2_new.eps){width="70.00000%"}
Based on the prevalence of the energetically favorable magnetic spin flip reconstruction on the stoichiometric MnO$_{2}$-terminated surface, it is important to study the effect of magnetic reconstruction on surface stability. All the MnO$_{2}$-based surface reconstructions were relaxed with both G-AFM and spin flip magnetic ordering. Different magnetic configurations were explored for CaO-terminated surfaces with Mn adatoms as well as MnO$_{2}$ terminated surfaces with Mn$_{x}$O$_{y}$ adatom reconstructions (See Supplementary Material). Half of the thermodynamically stable MnO$_{2}$-based surfaces are magnetic spin flip configurations, as shown in Fig. \[Fig.3\] (surface phases with red grid lines). For surface reconstructions with only one extra Mn adatom, the spin arrangement that makes a local FM alignment between the surface Mn adatom and the nearest Mn atom is more favorable. For example, the local FM alignment between the Mn surface adatom and the Mn on the subsurface layer in CaO-0.5Ca+0.5Mn, which is within the bulk stability region, is 12 meV per primitive surface cell more stable than a local AFM alignment (See Supplementary Material).
\[sec:level2\] B. Surface Phase Diagram {#seclevel2-b.-surface-phase-diagram .unnumbered}
---------------------------------------
The computed surface phase diagram of CaMnO$_{3}$, generated after calculating the surface free energy of the atomic and magnetic lowest energy configuration of each surface phase, is shown in Fig. \[Fig.3\]. This diagram predicts a total of fourteen thermodynamically stable surface phases in the given range of $\Delta\mu$$_{\textrm{Ca}}$ and $\Delta\mu$$_{\textrm{O}}$. The phase diagram can be divided into four different quadrants based on conditions of $\Delta\mu_{\textrm{O}}$, $\Delta\mu_{\textrm{Ca}}$ and $\Delta\mu_{\textrm{Mn}}$. Even though $\Delta\mu_{\textrm{Mn}}$ is not directly plotted, its value can always be deduced from Eq. (25). The upper left quadrant (I) shows oxygen rich and cation poor conditions; therefore, the favored phases are terminations with excess oxygen adatoms, denoted as MnO$_{2}$+2.0O, CaO+2.0O and CaO+1.0O. This quadrant also shows stability of the stoichiometric CaO- and MnO$_{2}$-terminated surfaces as well as MnO$_{2}$-based reconstructions, MnO$_{2}$-0.5Mn-0.5O and MnO$_{2}$+0.5Mn+1.0O. In quadrant (II), both O and Ca are plentiful, leading to the absence of MnO$_{2}$-terminated stable surfaces and the presence of Ca adatoms in stable CaO-based surfaces, CaO+1.0Ca+2.0O and CaO+1.0Ca+1.0O. The bottom right quadrant (III) involves oxygen-poor but relatively Ca-rich conditions. This region is characterized by stable surfaces with MnO$_{2}$ termination, denoting that even though Ca is at high concentrations, MnO$_{2}$-terminated surfaces are more favorable at oxygen poor conditions, clearly indicated by a surface phase where a Ca vacancy is filled by a Mn adatom, CaO-0.5Ca+0.5Mn. In the left bottom quadrant (IV), Mn is at its richest condition since both O and Ca are at poor levels. This promotes the large stable region for both MnO$_{2}$+1.0Mn+1.0O and MnO$_{2}$+2.0Mn+2.0O. A boundary line from the top left to mid-bottom right corners of the diagram delimits the regions of CaO- (upper) and MnO$_{2}$- (lower) terminated surfaces. Six different CaO-terminated surfaces are found, many of them covering a significant area of chemical potential space. On the other hand, MnO$_{2}$-type phases generally show smaller stable regions with a higher variety of phases, which can be explained by the multivalency of Mn. For instance, the predicted path from MnO$_{2}$+2.0Mn+2.0O to MnO$_{2}$+2.0O consists of a complex series of transitions. These transitions occur by increasing the $\Delta\mu_{\textrm{O}}$ from -3.0 eV to -0.5 eV while keeping constant the $\Delta\mu_{\textrm{Ca}}$ at $\approx$-7.0 eV. The changes in $\Delta\mu_{\textrm{O}}$ can be controlled experimentally by varying *T* or *p*(O$_{2}$). For example, these surface phase transitions are predicted to occur at a constant *p*(O$_{2}$) of 10$^{-10}$ atm, as the temperature is decreased from $\approx$1400 K to $\approx$400 K. Starting with the surface initially at $\approx$1400 K in the MnO$_{2}$+2.0Mn+2.0O phase, as the temperature is reduced to $\approx$750 K, the desorption of oxygen atoms is thermodynamically favorable, leading to the surface reconstruction MnO$_{2}$+1.0Mn+1.0O. Reducing the temperature further to $\approx$600 K should cause the surface to reconstruct into MnO$_{2}$+0.5Mn+1.0O and even co-exist with stoichiometric MnO$_{2}$-terminated surface. Once the temperature reaches $\approx$400 K, the surface phase transition to MnO$_{2}$+2.0O is thermodynamically favored. The slopes of the boundaries between phases directly relate to the changes in numbers of surface atoms of each species. The CaO+2.0O surface phase changes to CaO+1.0Ca+2.0O at a vertical boundary of $\Delta\mu_{\textrm{Ca}}$ $\approx$-5.75 eV via adsorption of Ca cations. Similarly, horizontal lines at $\Delta\mu_{\textrm{O}}$ $\approx$-0.8 eV and $\approx$-0.9 eV denotes its transition to CaO+1.0O and stoichiometric CaO, respectively, by the removal of oxygen adatoms. Between these extremes, the slope of the CaO to CaO+1.0Ca+2.0O transition, as indicated in Fig. \[Fig.3\], is -$\frac{1}{2}$, suggesting the addition of 2 Ca cations and 4 oxygen atoms per cell since the transition line will be crossed by moving toward Ca- and O-rich conditions. Taking into account the relationship shown in Eq. (25), the slope of the boundaries between surface phases can then be defined as $\frac{(\emph{a}-\emph{b})}{(3\emph{b}-\emph{c})}$, where *a*, *b*, and *c* represent the net change in number of Ca, Mn, and O atoms, respectively, between phases. For example, the removal of 2 Mn atoms and the adsorption of 2 oxygen atoms per cell results in the transition of surface phase MnO$_{2}$+2.0Mn+2.0O to MnO$_{2}$+1.0Mn+1.0O. This is in agreement with the boundary for this phase transition having slope of -$\frac{1}{4}$.
{width="35.00000%"}
### \[sec:level3\] Stability Region {#seclevel3-stability-region .unnumbered}
The surface phase diagram shows a series of stable surfaces, but only a few of them are within the bulk stability region. The ternary sub-phases illustrated in Eqs. 13-17 are included in the definition of the stability region since an extensive experimental examination of the phase relations of Ca-Mn-O bulk systems [@Horowitz78p1359; @Balakirev06pS49] found the co-existence lines for the CaMnO$_{3}$ phase with these ternary sub-phases, and correspondingly narrower stability boundaries for CaMnO$_{3}$. The boundary of bulk phase stability is defined by Ca$_{2}$MnO$_{4}$, Ca$_{3}$Mn$_{2}$O$_{7}$, and Ca$_{4}$Mn$_{3}$O$_{10}$ on the top and Mn$_{2}$O$_{3}$, MnO$_{2}$, CaMn$_{2}$O$_{4}$, and CaMn$_{7}$O$_{12}$ on the bottom (lines 9-11 and 5-8, respectively, shown in Fig. \[Fig.3\]). A confirmation of the importance of including the ternary sub-phases is the ability of this model to describe the decomposition of bulk CaMnO$_{3}$. Experimentally, decomposition of CaMnO$_{3}$ occurs at $\approx$1700 K and 0.21 atm O$_{2}$ [@Horowitz78p1359]. Using the DFT-generated stability region computed at 0 K, this decomposition temperature is predicted to be $\approx$2100 K at 0.21 atm by considering the decomposition conditions at the low $\Delta\mu_{\textrm{O}}$ end of the bulk stability region. However, if boundaries 7-11 are neglected, then the stability region is limited by MnO$_{2}$, Mn$_{2}$O$_{3}$ and Mn$_{3}$O$_{4}$ on the bottom and bordered by CaO on the top (lines 6, 5, 4, and 2, respectively, in Fig. \[Fig.2\]), artificially increasing the computed decomposition temperature to over 2500 K at 0.21 atm. These ternary sub-phases include the Ruddlesden-Popper phases CaO(CaMnO$_{3}$)$_{n}$, which are perovskite structures with double CaO layer (001) planes with *n* layers of CaMnO$_{3}$ in between [@Fawcett98p3643]. Naturally, the surface reconstruction with CaO double layer, CaO+1.0Ca+1.0O, is found on the side where the Ruddlesden-Popper phases CaO(CaMnO$_{3}$)$_{n}$ limit the stability of the CaMnO$_{3}$ bulk. The CaMn$_{2}$O$_{4}$ phase is a marokite structure [@Ling01p167], where the MnO$_{6}$ octahedra share edges and corners. This network of octahedra leads to channels along the *a*-axis which provide Ca atoms with 8-coordinated sites. It has been found that this marokite-type phase shows potential photocatalytic activity [@Wu11p107101]. This bulk phase borders the surface phase with a MnO$_{2}$-terminated layer that has an additional MnO layer, MnO$_{2}$+1.0Mn+1.0O. CaMn$_{7}$O$_{12}$ is a complex distorted double perovskite, whose chemical formula is formally written as (CaMn$^{+3}_{3}$)(Mn$^{+3}_{3}$Mn$^{+4}$)O$_{12}$. This perovskite-derived phase consists of $\frac{1}{4}$ of the *A*-sites occupied by Ca and the rest by Mn$^{+3}$, while the *B*-site charges are arranged via charge ordering below 440 K [@Bochu80p133]. All these ternary sub-phases are stable for some range of chemical potentials [@Horowitz78p1359; @Balakirev06pS49], defining realistic stability boundaries for the surface of CaMnO$_{3}$.
### \[sec:level3\] Temperature Dependence {#seclevel3-temperature-dependence .unnumbered}
The bulk stability region described above is based on DFT calculations at 0 K enclosing the CaO+2.0O, CaO+1.0O, CaO, CaO-0.5Ca+0.5Mn, MnO$_{2}$+2.0O, and MnO$_{2}$-0.5Mn-0.5O phases. A more accurate description of the stability region requires the inclusion of the vibrational free energy. This is significant, since it determines how the stability region changes as a function of temperature. The bulk stability region is recalculated incorporating the bulk vibrational free energy at 573 K, 873 K and 1173 K, shown in Fig. \[Fig.4\]. These temperatures are selected since they are within the range of experimental temperature conditions used in the synthesis and pulsed laser deposition of the CaMnO$_{3}$ crystal [@Melo01p915] and film, as well as in the annealing process for surface characterization techniques. These temperatures shift the stability region such that only MnO$_{2}$-based surfaces are favored. As temperature increases, the stability region shifts towards Ca-poor and O-poor conditions, in agreement with previous studies on other surfaces [@Lee11p115418; @Kolpak08p036102]. As the stability region moves, it encloses other phases that are predicted to be stable at those temperatures. At 573 K, the surface phases within the bulk stability region are the same as in Fig. \[Fig.3\], with the addition of the stoichiometric MnO$_{2}$-terminated surface. Increasing the temperature by 300 K leads to the stability of MnO$_{2}$+1.0Mn+1.0O surface phase. At 1173 K, the phases within the bulk stability region include the MnO$_{2}$+0.5Mn+1.0O phase. The clear trend is that the surface structures with MnO$_{2}$ terminations are more favorable at higher temperatures. Changes in the shape of the stability region reflect how binary and ternary sub-phase boundaries shift as the vibrational free energy at various temperatures is included. Overall, the bulk stability region tends to shrink as the higher temperature contributions are included. This is in good agreement with previous reports, since it is experimentally observed that CaMnO$_{3}$ crystal decomposes at temperatures higher than $\approx$1700 K [@Horowitz78p1359] and our theoretical bulk stability region vanishes around the same temperature.
\[sec:level1\] IV. Summary and Conclusions {#seclevel1-iv.-summary-and-conclusions .unnumbered}
==========================================
The energies of series of CaO- and MnO$_{2}$-terminated CaMnO$_{3}$ (001) surfaces with different combinations of vacancies, adatoms and additional layers are reported using *ab initio* thermodynamics to theoretically predict the surface phase diagram. The predicted surface phase diagram has surface structures for both terminations, with stability ranges specified by $\Delta\mu_{\textrm{O}}$ and $\Delta\mu_{\textrm{Ca}}$. The $\Delta\mu_{\textrm{O}}$ is related to *T* and *p*(O$_{2}$), linking surface stability with experimental conditions. The chemical potential region for which CaMnO$_{3}$ is the only bulk phase is bounded by coexistence lines with the Ruddlesden-Popper phases CaO(CaMnO$_{3}$)$_{\emph{n}}$ (where *n*=1, 2, and 3), as well as Mn$_{2}$O$_{3}$, MnO$_{2}$, CaMn$_{2}$O$_{4}$, and CaMn$_{7}$O$_{12}$. This shows the relevance of incorporating the ternary subphases from the experimental Ca-Mn-O compositional phase diagram [@Horowitz78p1359; @Balakirev06pS49]. The surface phases that are favored within this bulk stability region change once the vibrational free energy at different temperatures is included. The stability region shifts to lower $\Delta\mu_{\textrm{Ca}}$ for higher *T*, leading to the predominance of complex MnO$_{2}$-based surfaces at *T* above 800 K. Thermodynamically stable phases include potentially reactive surfaces, MnO$_{2}$+1.0Mn+1.0O, since it shows two unique Mn sites based on coordination. The MnO$_{2}$-based surfaces show intriguing atomic and magnetic structures which are comprehensively explored by computing the magnetic ground state and analyzing its thermodynamic stability. Some of the MnO$_{2}$-terminated surfaces are energetically more favorable when their surface and subsurface spins are aligned. This occurs when the filling of the *d$_{z^{2}}$* orbital causes a more favorable double-exchange interaction between the subsurface and surface Mn atoms. The atomic composition of the surface affects this magnetic reconstruction; for some Mn$_{x}$O$_{y}$ reconstructions, the surface reconstructs, rearranging the magnetic interactions in the system. However, this magnetic spin flip reconstruction prevails for key stable surfaces such as stoichiometric MnO$_{2}$-terminated and MnO$_{2}$+2.0O. This study addresses structures with ($\sqrt{2}\times\sqrt{2}$)*R*45$^{\circ}$ surface symmetry, leading to ordered surfaces due to periodic boundary conditions. Admittedly, real surfaces are more complex and disordered; however, by taking into account the chemical and thermal contributions to the surface stability and exploring a wide range of possible surface structures, the predicted phase diagram provides an acceptable foundation for the analysis of experimental data on this surface. Consequently, this predicted surface phase diagram could lead to experimental studies performing low-energy electron diffraction and scanning tunneling microscopy to validate the stable surface reconstructions and further explore the surface evolution.
\[sec:level1\] Acknowledgments {#seclevel1-acknowledgments .unnumbered}
==============================
D.S.-G. was supported by the Office of Naval Research under Grant No. N00014-11-1-0664. C.-W.L. was supported by the US Department of Energy Office of Basic Energy Sciences under Grant No. DE-FG02-07ER15920. D.Y. was supported by the National Science Foundation under Grant No. DMR-1124696. A.M.R. was supported by the National Science Foundation under Grant No. CMMI-1334241. Computational support was provided by the High-Performance Computing Modernization Office of the US Department of Defense, and the National Energy Research Scientific Computing Center of the US Department of Energy.
|
Q:
Unable to get values of input boxes
I have a timesheet form that auto populates the hours worked based on the times input into the start and stop input boxes. I need to take all the values for the week and total them up and populate the weekly total box with that value. I need it to dynamically populate as the values populate in the daily totals box.
I'm able to get the weeklyTotal box to populate if I manually enter the values in the dailyTotal boxes but not if they are auto populated from selecting the start and stop times.
I've tried changing change to blur, keyup, keydown, etc with no luck. What am I doing wrong?
$(function() {
function calculate() {
var start = $("#start1").val().split(':');
stop = $("#stop1").val().split(':');
var hours1 = parseInt(start[0], 10) || 0;
hours2 = parseInt(stop[0], 10) || 0;
mins1 = parseInt(start[1], 10) || 0;
mins2 = parseInt(stop[1], 10) || 0;
var hours = hours2 - hours1,
mins = 0;
if (hours < 0)
hours = 24 + hours;
if (mins2 >= mins1) {
mins = mins2 - mins1;
} else {
mins = (mins2 + 60) - mins1;
hours--;
}
mins = mins / 60; // take percentage in 60
hours += mins;
hours = hours.toFixed(2);
$(".Hours1").val(hours);
$("#dailyTotal1").val(hours);
}
$("#start1,#stop1").change(calculate);
});
$(function() {
function calculate() {
var start = $(".start2").val().split(':');
stop = $(".stop2").val().split(':');
var hours1 = parseInt(start[0], 10) || 0;
hours2 = parseInt(stop[0], 10) || 0;
mins1 = parseInt(start[1], 10) || 0;
mins2 = parseInt(stop[1], 10) || 0;
var hours = hours2 - hours1,
mins = 0;
if (hours < 0) hours = 24 + hours;
if (mins2 >= mins1) {
mins = mins2 - mins1;
} else {
mins = (mins2 + 60) - mins1;
hours--;
}
mins = mins / 60; // take percentage in 60
hours += mins;
hours = hours.toFixed(2);
$(".Hours2").val(hours);
$("#dailyTotal2").val(hours);
}
$(".start2,.stop2").change(calculate);
});
$(function() {
function calculate() {
var start = $(".start3").val().split(':');
stop = $(".stop3").val().split(':');
var hours1 = parseInt(start[0], 10) || 0;
hours2 = parseInt(stop[0], 10) || 0;
mins1 = parseInt(start[1], 10) || 0;
mins2 = parseInt(stop[1], 10) || 0;
var hours = hours2 - hours1,
mins = 0;
if (hours < 0) hours = 24 + hours;
if (mins2 >= mins1) {
mins = mins2 - mins1;
} else {
mins = (mins2 + 60) - mins1;
hours--;
}
mins = mins / 60; // take percentage in 60
hours += mins;
hours = hours.toFixed(2);
$(".Hours3").val(hours);
$("#dailyTotal3").val(hours);
}
$(".start3,.stop3").change(calculate);
});
$(function() {
function calculate() {
var start = $(".start4").val().split(':');
stop = $(".stop4").val().split(':');
var hours1 = parseInt(start[0], 10) || 0;
hours2 = parseInt(stop[0], 10) || 0;
mins1 = parseInt(start[1], 10) || 0;
mins2 = parseInt(stop[1], 10) || 0;
var hours = hours2 - hours1,
mins = 0;
if (hours < 0) hours = 24 + hours;
if (mins2 >= mins1) {
mins = mins2 - mins1;
} else {
mins = (mins2 + 60) - mins1;
hours--;
}
mins = mins / 60; // take percentage in 60
hours += mins;
hours = hours.toFixed(2);
$(".Hours4").val(hours);
$("#dailyTotal4").val(hours);
}
$(".start4,.stop4").change(calculate);
});
$(document).on("change", ".txt", function() {
var sum = 0;
$(".txt").each(function() {
sum += +$(this).val();
});
$(".total").val(sum);
});
.auto-style8 {
border-style: solid;
border-width: 1px;
background-color: #A5C4F1;
}
th {
padding: 0;
margin: 0;
border-collapse: collapse;
border-spacing: 0;
/* cellspacing */
}
td {
padding: 0;
margin: 0;
border-collapse: collapse;
border-spacing: 0;
/* cellspacing */
}
input {
text-align: center;
background-color: #CCFF99
}
<script src="https://ajax.googleapis.com/ajax/libs/jquery/2.1.1/jquery.min.js"></script>
<table>
<tr>
<th class="auto-style8" style="width: 57px; height: 22px;">START</th>
<th class="auto-style8" style="width: 52px; height: 22px;">STOP</th>
<th class="auto-style8" style="width: 87px; height: 22px;">Line Total</th>
<th class="auto-style8" style="width: 99px; height: 22px;">Daily Total</th>
</tr>
<tr>
<td><input class="txt" id="start1" name="start1" style="width: 69px" type="time" /></td>
<td><input class="txt" id="stop1" name="stop1" style="width: 66px" type="time" /></td>
<td><input class="Hours1" id="" name="lineTotal1" readonly="readonly" style="width: 89px" type="text" /></td>
<td><input class="txt" id="dailyTotal1" name="dailyTotal1" style="width: 96px" type="text" /></td>
</tr>
<tr>
<td><input class="start2" id="" name="start2" style="width: 69px" type="time" /></td>
<td><input class="stop2" id="" name="stop2" style="width: 66px" type="time" /></td>
<td><input class="Hours2" id="" name="lineTotal2" style="width: 89px" type="text" /></td>
<td><input class="txt" id="dailyTotal2" name="dailyTotal2" style="width: 96px" type="text" /></td>
</tr>
<tr>
<td><input class="start3" id="" name="start3" style="width: 69px" type="time" /></td>
<td><input class="stop3" id="" name="stop3" style="width: 66px" type="time" /></td>
<td><input class="Hours3" id="" name="lineTotal3" style="width: 89px" type="text" /></td>
<td><input class="txt" id="dailyTotal3" name="dailyTotal3" style="width: 96px" type="text" /></td>
</tr>
<tr>
<td><input class="start4" id="" name="start4" style="width: 69px" type="time" /></td>
<td><input class="stop4" id="" name="stop4" style="width: 66px" type="time" /></td>
<td><input class="Hours4" id="" name="lineTotal4" style="width: 89px" type="text" /></td>
<td><input class="txt" id="dailyTotal4" name="dailyTotal4" style="width: 96px" type="text" /></td>
</tr>
<tr>
<td></td>
<td></td>
<td>Weekly total:</td>
<td><input class="total" id="total" name="weeklyTotal" readonly="readonly" style="width: 96px" type="text" /></td>
</tr>
</table>
A:
Check this part:
$(document).on("change", ".txt", function() {
var sum = 0;
$(".dailyTotalGeneric").each(function(i,e) {
if(!isNaN($(e).val()) && $(e).val()){
console.log($(e).val());
sum += parseFloat($(e).val());
}
});
$(".total").val(sum);
});
I made some changes, like add a class to all the inputs that sum weeklyTotal => dailyTotalGeneric class.
And you have to check if the number you are trying to sum isNan or not exist to sum on variable.
$(function() {
function calculate() {
var start = $("#start1").val().split(':');
stop = $("#stop1").val().split(':');
var hours1 = parseInt(start[0], 10) || 0;
hours2 = parseInt(stop[0], 10) || 0;
mins1 = parseInt(start[1], 10) || 0;
mins2 = parseInt(stop[1], 10) || 0;
var hours = hours2 - hours1,
mins = 0;
if (hours < 0)
hours = 24 + hours;
if (mins2 >= mins1) {
mins = mins2 - mins1;
} else {
mins = (mins2 + 60) - mins1;
hours--;
}
mins = mins / 60; // take percentage in 60
hours += mins;
hours = hours.toFixed(2);
$(".Hours1").val(hours);
$("#dailyTotal1").val(hours);
}
$("#start1,#stop1").change(calculate);
});
$(function() {
function calculate() {
var start = $(".start2").val().split(':');
stop = $(".stop2").val().split(':');
var hours1 = parseInt(start[0], 10) || 0;
hours2 = parseInt(stop[0], 10) || 0;
mins1 = parseInt(start[1], 10) || 0;
mins2 = parseInt(stop[1], 10) || 0;
var hours = hours2 - hours1,
mins = 0;
if (hours < 0) hours = 24 + hours;
if (mins2 >= mins1) {
mins = mins2 - mins1;
} else {
mins = (mins2 + 60) - mins1;
hours--;
}
mins = mins / 60; // take percentage in 60
hours += mins;
hours = hours.toFixed(2);
$(".Hours2").val(hours);
$("#dailyTotal2").val(hours);
}
$(".start2,.stop2").change(calculate);
});
$(function() {
function calculate() {
var start = $(".start3").val().split(':');
stop = $(".stop3").val().split(':');
var hours1 = parseInt(start[0], 10) || 0;
hours2 = parseInt(stop[0], 10) || 0;
mins1 = parseInt(start[1], 10) || 0;
mins2 = parseInt(stop[1], 10) || 0;
var hours = hours2 - hours1,
mins = 0;
if (hours < 0) hours = 24 + hours;
if (mins2 >= mins1) {
mins = mins2 - mins1;
} else {
mins = (mins2 + 60) - mins1;
hours--;
}
mins = mins / 60; // take percentage in 60
hours += mins;
hours = hours.toFixed(2);
$(".Hours3").val(hours);
$("#dailyTotal3").val(hours);
}
$(".start3,.stop3").change(calculate);
});
$(function() {
function calculate() {
var start = $(".start4").val().split(':');
stop = $(".stop4").val().split(':');
var hours1 = parseInt(start[0], 10) || 0;
hours2 = parseInt(stop[0], 10) || 0;
mins1 = parseInt(start[1], 10) || 0;
mins2 = parseInt(stop[1], 10) || 0;
var hours = hours2 - hours1,
mins = 0;
if (hours < 0) hours = 24 + hours;
if (mins2 >= mins1) {
mins = mins2 - mins1;
} else {
mins = (mins2 + 60) - mins1;
hours--;
}
mins = mins / 60; // take percentage in 60
hours += mins;
hours = hours.toFixed(2);
$(".Hours4").val(hours);
$("#dailyTotal4").val(hours);
}
$(".start4,.stop4").change(calculate);
});
$(document).on("change", ".txt", function() {
var sum = 0;
$(".dailyTotalGeneric").each(function(i,e) {
if(!isNaN($(e).val()) && $(e).val()){
//console.log($(e).val());
sum += parseFloat($(e).val());
}
});
$(".total").val(sum);
});
.auto-style8 {
border-style: solid;
border-width: 1px;
background-color: #A5C4F1;
}
th {
padding: 0;
margin: 0;
border-collapse: collapse;
border-spacing: 0;
/* cellspacing */
}
td {
padding: 0;
margin: 0;
border-collapse: collapse;
border-spacing: 0;
/* cellspacing */
}
input {
text-align: center;
background-color: #CCFF99
}
<script src="https://ajax.googleapis.com/ajax/libs/jquery/2.1.1/jquery.min.js"></script>
<script type="text/javascript"></script>
<table>
<tr>
<th class="auto-style8" style="width: 57px; height: 22px;">START</th>
<th class="auto-style8" style="width: 52px; height: 22px;">STOP</th>
<th class="auto-style8" style="width: 87px; height: 22px;">Line Total</th>
<th class="auto-style8" style="width: 99px; height: 22px;">Daily Total</th>
</tr>
<tr>
<td><input class="txt" id="start1" name="start1" style="width: 69px" type="time" /></td>
<td><input class="txt" id="stop1" name="stop1" style="width: 66px" type="time" /></td>
<td><input class="Hours1" id="" name="lineTotal1" readonly="readonly" style="width: 89px" type="text" /></td>
<td><input class="txt dailyTotalGeneric" id="dailyTotal1" name="dailyTotal1" style="width: 96px" type="text" /></td>
</tr>
<tr>
<td><input class="txt start2" id="" name="start2" style="width: 69px" type="time" /></td>
<td><input class="txt stop2" id="" name="stop2" style="width: 66px" type="time" /></td>
<td><input class="Hours2" id="" name="lineTotal2" style="width: 89px" type="text" /></td>
<td><input class="txt dailyTotalGeneric" id="dailyTotal2" name="dailyTotal2" style="width: 96px" type="text" /></td>
</tr>
<tr>
<td><input class="txt start3" id="" name="start3" style="width: 69px" type="time" /></td>
<td><input class="txt stop3" id="" name="stop3" style="width: 66px" type="time" /></td>
<td><input class="Hours3" id="" name="lineTotal3" style="width: 89px" type="text" /></td>
<td><input class="txt dailyTotalGeneric" id="dailyTotal3" name="dailyTotal3" style="width: 96px" type="text" /></td>
</tr>
<tr>
<td><input class="txt start4" id="" name="start4" style="width: 69px" type="time" /></td>
<td><input class="txt stop4" id="" name="stop4" style="width: 66px" type="time" /></td>
<td><input class="Hours4" id="" name="lineTotal4" style="width: 89px" type="text" /></td>
<td><input class="txt dailyTotalGeneric" id="dailyTotal4" name="dailyTotal4" style="width: 96px" type="text" /></td>
</tr>
<tr>
<td></td>
<td></td>
<td>Weekly total:</td>
<td><input class="total" id="total" name="weeklyTotal" style="width: 96px" type="text" /></td>
</tr>
</table>
|
The currently emerging tuberculosis epidemic, when viewed in the context of parallel problems such as emerging drug resistance, and the high susceptibility of HIV-positive individuals, is a great cause for concern. Moreover, at the basic scientific level much is still unknown about the host-parasite relationship, including what specific antigens are recognized to initially trigger host immunity, and the mechanisms underlying the clearly complex T cell-mediated cellular response to Mycobacterium tuberculosis infection. Accordingly, in this competing continuation application, we propose to continue to perform basic research in the mouse infection model in these areas; specifically, the spectrum of antigen recognition by defined T cell subsets, including how this changes during the course of the infection, and continued work into precisely defining the role of such T cell subsets, including the kinetics of their emergence and loss, and their relative contributions to the expression of specific resistance. For the first, we will use methods standardized over the past several years within the Mycobacteria Research Laboratories, CSU, to produce highly purified subcellular fractions of native mycobacterial protein antigens, and then use these fractions in in vitro assays to measure the cytokine response of purified T cell subsets harvested from mice infected with virulent M.tuberculosis. For the second, we will continue previous work under this Program aimed at defining the precise nature of the T cell response, including the expression of protective immunity, DTH, and memory immunity, and the relative contributions of CD4, CD8, and gamma delta T cell subsets to these parameters. In addition, we will investigate the roles of chemokines and cytokines in the regulation of immunity to the infection, using modern RT-PCR technology and the realistic low dose aerosol lung infection model. Finally, the roles of specific cell subsets and cytokines will also be approached using a variety of gene disrupted mouse models, already established as breeding colonies in our laboratory. We anticipate these proposed experiments will provide useful new information regarding the relative roles of T cell subsets in acquired immunity to the infection, the spectrum of T cell subset cytokine production, the antigenic targets of this production, and how these parameters change as the course of the infection progresses from a state of active to quiescent disease. As previously in this Program, the proposed work will draw upon the broad expertise of various members of the Mycobacteria Research Laboratories, CSU, as well as a number of highly qualified consultants/collaborators. |
/**
* Represents a collection of a set of key and value pairs. Each key in the HashMap
* must be unique, the same key cannot exist twice. Access to items is provided via
* the key only. Sample usage:
*
* var map = new Ext.util.HashMap();
* map.add('key1', 1);
* map.add('key2', 2);
* map.add('key3', 3);
*
* map.each(function(key, value, length){
* console.log(key, value, length);
* });
*
* The HashMap is an unordered class,
* there is no guarantee when iterating over the items that they will be in any particular
* order. If this is required, then use a {@link Ext.util.MixedCollection}.
*/
Ext.define('Ext.util.HashMap', {
mixins: [
'Ext.mixin.Observable'
],
/**
* @private Mutation counter which is incremented upon add and remove.
*/
generation: 0,
config: {
/**
* @cfg {Function} keyFn A function that is used to retrieve a default key for a passed object.
* A default is provided that returns the `id` property on the object. This function is only used
* if the `add` method is called with a single argument.
*/
keyFn: null
},
/**
* @event add
* Fires when a new item is added to the hash.
* @param {Ext.util.HashMap} this
* @param {String} key The key of the added item.
* @param {Object} value The value of the added item.
*/
/**
* @event clear
* Fires when the hash is cleared.
* @param {Ext.util.HashMap} this
*/
/**
* @event remove
* Fires when an item is removed from the hash.
* @param {Ext.util.HashMap} this
* @param {String} key The key of the removed item.
* @param {Object} value The value of the removed item.
*/
/**
* @event replace
* Fires when an item is replaced in the hash.
* @param {Ext.util.HashMap} this
* @param {String} key The key of the replaced item.
* @param {Object} value The new value for the item.
* @param {Object} old The old value for the item.
*/
/**
* Creates new HashMap.
* @param {Object} config (optional) Config object.
*/
constructor: function(config) {
var me = this,
fn;
// Will call initConfig
me.mixins.observable.constructor.call(me, config);
me.clear(true);
fn = me.getKeyFn();
if (fn) {
me.getKey = fn;
}
},
/**
* Gets the number of items in the hash.
* @return {Number} The number of items in the hash.
*/
getCount: function() {
return this.length;
},
/**
* Implementation for being able to extract the key from an object if only
* a single argument is passed.
* @private
* @param {String} key The key
* @param {Object} value The value
* @return {Array} [key, value]
*/
getData: function(key, value) {
// if we have no value, it means we need to get the key from the object
if (value === undefined) {
value = key;
key = this.getKey(value);
}
return [key, value];
},
/**
* Extracts the key from an object. This is a default implementation, it may be overridden
* @param {Object} o The object to get the key from
* @return {String} The key to use.
*/
getKey: function(o) {
return o.id;
},
/**
* Adds an item to the collection. Fires the {@link #event-add} event when complete.
*
* @param {String/Object} key The key to associate with the item, or the new item.
*
* If a {@link #getKey} implementation was specified for this HashMap,
* or if the key of the stored items is in a property called `id`,
* the HashMap will be able to *derive* the key for the new item.
* In this case just pass the new item in this parameter.
*
* @param {Object} [o] The item to add.
*
* @return {Object} The item added.
*/
add: function(key, value) {
var me = this;
// Need to check arguments length here, since we could have called:
// map.add('foo', undefined);
if (arguments.length === 1) {
value = key;
key = me.getKey(value);
}
if (me.containsKey(key)) {
return me.replace(key, value);
}
me.map[key] = value;
++me.length;
me.generation++;
if (me.hasListeners.add) {
me.fireEvent('add', me, key, value);
}
return value;
},
/**
* Replaces an item in the hash. If the key doesn't exist, the
* {@link #method-add} method will be used.
* @param {String} key The key of the item.
* @param {Object} value The new value for the item.
* @return {Object} The new value of the item.
*/
replace: function(key, value) {
var me = this,
map = me.map,
old;
// Need to check arguments length here, since we could have called:
// map.replace('foo', undefined);
if (arguments.length === 1) {
value = key;
key = me.getKey(value);
}
if (!me.containsKey(key)) {
me.add(key, value);
}
old = map[key];
map[key] = value;
me.generation++;
if (me.hasListeners.replace) {
me.fireEvent('replace', me, key, value, old);
}
return value;
},
/**
* Remove an item from the hash.
* @param {Object} o The value of the item to remove.
* @return {Boolean} True if the item was successfully removed.
*/
remove: function(o) {
var key = this.findKey(o);
if (key !== undefined) {
return this.removeAtKey(key);
}
return false;
},
/**
* Remove an item from the hash.
* @param {String} key The key to remove.
* @return {Boolean} True if the item was successfully removed.
*/
removeAtKey: function(key) {
var me = this,
value;
if (me.containsKey(key)) {
value = me.map[key];
delete me.map[key];
--me.length;
me.generation++;
if (me.hasListeners.remove) {
me.fireEvent('remove', me, key, value);
}
return true;
}
return false;
},
/**
* Retrieves an item with a particular key.
* @param {String} key The key to lookup.
* @return {Object} The value at that key. If it doesn't exist, `undefined` is returned.
*/
get: function(key) {
var map = this.map;
return map.hasOwnProperty(key) ? map[key] : undefined;
},
/**
* @method clear
* Removes all items from the hash.
* @return {Ext.util.HashMap} this
*/
// We use this syntax because we don't want the initial param to be part of the public API
/** @ignore **/
clear: function(/* private */ initial) {
var me = this;
// Only clear if it has ever had any content
if (initial || me.generation) {
me.map = {};
me.length = 0;
me.generation = initial ? 0 : me.generation + 1;
}
if (initial !== true && me.hasListeners.clear) {
me.fireEvent('clear', me);
}
return me;
},
/**
* Checks whether a key exists in the hash.
* @param {String} key The key to check for.
* @return {Boolean} True if they key exists in the hash.
*/
containsKey: function(key) {
var map = this.map;
return map.hasOwnProperty(key) && map[key] !== undefined;
},
/**
* Checks whether a value exists in the hash.
* @param {Object} value The value to check for.
* @return {Boolean} True if the value exists in the dictionary.
*/
contains: function(value) {
return this.containsKey(this.findKey(value));
},
/**
* Return all of the keys in the hash.
* @return {Array} An array of keys.
*/
getKeys: function() {
return this.getArray(true);
},
/**
* Return all of the values in the hash.
* @return {Array} An array of values.
*/
getValues: function() {
return this.getArray(false);
},
/**
* Gets either the keys/values in an array from the hash.
* @private
* @param {Boolean} isKey True to extract the keys, otherwise, the value
* @return {Array} An array of either keys/values from the hash.
*/
getArray: function(isKey) {
var arr = [],
key,
map = this.map;
for (key in map) {
if (map.hasOwnProperty(key)) {
arr.push(isKey ? key: map[key]);
}
}
return arr;
},
/**
* Executes the specified function once for each item in the hash.
* Returning false from the function will cease iteration.
*
* @param {Function} fn The function to execute.
* @param {String} fn.key The key of the item.
* @param {Number} fn.value The value of the item.
* @param {Number} fn.length The total number of items in the hash.
* @param {Object} [scope] The scope to execute in. Defaults to <tt>this</tt>.
* @return {Ext.util.HashMap} this
*/
each: function(fn, scope) {
// copy items so they may be removed during iteration.
var items = Ext.apply({}, this.map),
key,
length = this.length;
scope = scope || this;
for (key in items) {
if (items.hasOwnProperty(key)) {
if (fn.call(scope, key, items[key], length) === false) {
break;
}
}
}
return this;
},
/**
* Performs a shallow copy on this hash.
* @return {Ext.util.HashMap} The new hash object.
*/
clone: function() {
var hash = new this.self(this.initialConfig),
map = this.map,
key;
hash.suspendEvents();
for (key in map) {
if (map.hasOwnProperty(key)) {
hash.add(key, map[key]);
}
}
hash.resumeEvents();
return hash;
},
/**
* @private
* Find the key for a value.
* @param {Object} value The value to find.
* @return {Object} The value of the item. Returns <tt>undefined</tt> if not found.
*/
findKey: function(value) {
var key,
map = this.map;
for (key in map) {
if (map.hasOwnProperty(key) && map[key] === value) {
return key;
}
}
return undefined;
}
});
|
Q:
Extract innerHTML from span[class]
so my code is something along the lines of:
<table><tr><td class="tdclass1">
$('#nameoftable tr').each(function(){
tdclass1 = $(this).find(".classname").html();
if (tdclass1:contains('classname'))
//do something here
});
What I want to do is simply extract the inner HTML of this specific span.
Any help is appreciated :)
A:
$('#nameoftable .tdclass1 span.classname').each (function (idx, elem) {
console.log ($(elem).html ());
});
A:
This should do it:
$("#nameoftable .tdclass1 span.classname").each(function () {
var html = $(this).html();
});
|
Sexual behavior of castrated boars treated with prostaglandin F(2alpha).
The objectives were to test the hypothesis that exogenous prostaglandin F(2alpha) (PGF(2alpha)) temporarily restores sexual behavior of castrated boars, and to evaluate effects of PGF(2alpha) on serum hormone concentrations. At 35 d after castration, nine lean-type adult boars were randomly assigned to three treatments in a 3x3 latin square (with three replicates). Treatments were three doses of PGF(2alpha) doses (0, 10, and 20mg) and three periods of treatment, with 5 d between each period. Serum testosterone (T) concentrations were non-detectable at the start of the experiment. Serum concentrations of estradiol (E2), LH, prolactin (PRL), and cortisol were unaffected (P>0.05) by PGF(2alpha) treatment. The interval from treatment to ejaculation in boars treated with 10mg (758s) or 20mg (660s) PGF(2alpha) did not differ, but were different (P < 0.05) from control boars (>1 800s). Ejaculation duration and false mounts differed (P < 0.05) between control boars and boars treated with 10 or 20mg PGF(2alpha). In conclusion, PGF(2alpha) treatment did not change serum concentrations of T, E2, LH, PRL, or cortisol, but restored sexual behavior. This restoration may have been due to an effect of PGF(2alpha) directly in specific areas of the brain, or indirectly via release of other hormones that stimulated areas in the brain that affected sexual behavior. |
Last week, the Dfinity Foundation distributed $35 million CHF to tens of thousands of their community members. The event was one of the largest airdrops to date. The company’s goal was to give free tokens away to raise awareness and reward the project’s members.
Tobias Batton, CEO of Signal Zero, strongly disagrees with the tactic and believes the company just gave away millions of dollars in tokens and free stock. However, Batton doesn’t completely disagree with rewarding users through airdrops.
Why is Signal Zero giving away Crypto Tokens?
Signal Zero is a leading rewards platform specifically built for games and eSports. The company announced that it has reinvented airdrops, but Batton’s goal isn’t just to raise awareness and reward the platform’s members.
Instead, he’s urging blockchain entrepreneurs to follow their lead and launch a “Proof of Concept” to establish and build an engaged community of active users, who will provide feedback, user research, and carry out beta testing. The company released a press release via Cryptoland PRand Batton had the following to say about the recent airdrop:
“I hope our initiative will spark a sea change in the way crypto-projects distribute coins to their community of supporters – completely upending the notion that individuals can receive coins for doing simple tasks like joining a Telegram group.”
What Makes Signal Zero's Upcoming Airdrop Different?
This will be the world’s first “Proof-of-Skill” blockchain platform that will reward players based on their skill level. The company has integrated with TapRivalsto give the gamers the ability to earn points, build a reputation, and show off their skills.
With the blockchain algorithm, higher ranking players will have even more access and receive greater rewards provided by TapRivals. Tobias Batton also said following about rewarding Signal Zero’s active community with the upcoming airdrop:
“Since we launched in 2014, Singal Zero has grown to a community of over 10 million global players and we have rewarded our members with more than $7 million in cash and prizes. We are excited to continue to grow the community and share even more rewards with members, no matter where they play.”
RadarZero is your daily resource for cutting-edge technologies, including blockchain & cryptocurrencies, artificial intelligence, virtual & augmented reality, autonomous vehicles and other emerging tech that is rapidly changing society and industries around the globe. Our in-depth coverage uncovers details for the experts but is also easy to understand for the layman. |
Related Links
PHILADELPHIA - The Penn men's lacrosse team still has plenty of young players in major roles this year, so a first half like they put together on Wednesday night could be expected.
Facing a Saint Joseph's team still looking for its first win this season, the Quakers looked like they might blow the Hawks out early but then allowed their hosts to draw within 6-4 by the halftime break.
Head Coach Mike Murphy must have gotten the players' attention during the break. Six goals were scored in the third quarter, and all of them came out of Quaker sticks. The fourth quarter was a chance for the bench to be cleared as the Red and Blue cruised to a 13-5 victory at SJU's Finnesey Field.
Penn is now 7-4 on the season, thanks to its second-straight road victory. Saint Joseph's is now 0-11.
Dan Savage led Penn's offense with three goals, while Al Kohart had two. Overall, the Quakers had a season-high 10 different players score. Corey Winkoff did not find the net, but dished off three assists. Penn outshot the Hawks, 40-19.
Savage got the scoring started on Wednesday night, with an unassisted effort just 1:52 into the game. Saint Joseph's answered just under six minutes later, off an unassisted goal by Ian Correia, but four minutes later long-stick defender Maxx Meyer gave Penn a lead it would not relinquish as he drained an unassisted goal.
Just 32 seconds later, Winkoff found John Conneely for a goal, and then with 1:53 left in the quarter Winkoff found Kohart for his first tally of the night. That put the Quakers up, 4-1, at the end of one. Early in the second, Will Amling made it 5-1 when he finished a Conneely feed.
The Hawks worked their way back. Kyle Mahoney had an unassisted goal with 10:59 left in the half, and then after Savage scored to make it 6-2 Saint Joseph's came right back as Bobby Hurley scored less than a minute later. A big goal came with just nine seconds left, when Kyle Williams netted an unassisted effort that drew the Hawks within two.
It did not take long for Penn to establish its dominance in the third. Zack Losco scored an unassisted goal just 2:49 into the quarter, and then 1:08 after that Greg Ives rifled a shot home off a Winkoff feed. Fifty-five seconds after that, Kohart netted an unassisted goal from distance, and then just 10 seconds later Rob Fitzpatrick scored an unassisted goal after Penn won the faceoff. Ryan Parietti added his only goal of the night, another unassisted goal, and the Quakers were up 11-4. The five-goal run covered 3:53.
Savage capped his hat trick with 2:21 left in the third, unassisted. In the fourth quarter, Correia scored with just 2:06 to play, and then Penn closed out the scoring as Losco found Kyle Mulligan with 28 seconds left.
Brian Feeney played the first three quarters in goal, making two saves. In the fourth quarter, Brendan Engelke started and made two stops without allowing a goal in 8:33 of action before Ted Rawlings closed it out and made two saves of his own while allowing one goal.
Danny Feeney had a solid game on the face-off X, winning 11 of 18 decisions and grabbing nine ground balls.
Penn returns to action on Saturday, closing out the Ivy League portion of its 2011 schedule with the final home game of the regular season. The Quakers host Dartmouth at 1 p.m. |
WASHINGTON, DC – Attorney General William Barr held a press conference on Monday to discuss the findings of an investigation into the terror attack on a Florida Naval base in December and other topics, at one point defending the Trump administration’s elimination of Iranian Major General Qasem Soleimani.
A reporter asked Barr if the Trump administration gave any “special consideration” to Soleimani’s position in the Iranian government – the head of the Islamic Revolutionary Guard Corps’ (IRGC) Quds Force, an arm dedicated to external acts of terrorism.
“Did you give any special consideration of the fact that he was a senior official of another government,” the reporter asked, “that he was a general of Iran?”
“He was the head of a terrorist organization,” Barr said, adding that that made him a legitimate military target.
A reporter with CNN asked Barr if the drone strike violated the Constitution.
“Can you explain to people in Congress, including conservatives who have questions about whether or not this was a constitutional strike given the fact that the Constitution gives Congress the right to declare war — and in this case obviously this was an act of war — and they were not consulted?” the reporter asked.
“I believe the president clearly had the authority to act as he did,” Barr said, adding that Iran instigated hostilities and the U.S. had the right to respond.
“We had a situation where the Iranians had already embarked on a series of escalating violent action taken against our allies, taken against the American people, our troops; with the avowed purpose of driving us out of the Middle East,” Barr said.
“They had attacked shipping the Strait of Hormuz; they had attacked oil fields in Saudi Arabia; they had attacked U.S. bases; they had attacked our embassy; they had killed Americans,” Barr said.
“These ongoing attacks were being planned and orchestrated by Soleimani,” Barr said. “Our ability to deter attacks had obviously broken down. The Iranians had been given a number of red lines and were crossing those lines.”
Barr said the Iranians seemed to think they could continue their aggressions “with impunity.”
“The general in charge of these operations, Soleimani, was clearly a legitimate military target,” Barr said. “We have a very brief window of time to carry out the attack when he arrived in Baghdad to plan and orchestrate follow up attacks on American interests and American people [and] personnel.”
“So this was a legitimate act of self-defense because it disrupted ongoing attacks that were being conducted,” Barr said, adding that it also acts as a deterrent to future attacks rather than starting a broader conflict.
Barr also commented on the debate on whether there was an “imminent” threat from Iran ahead of the drone attack.
“The concept of imminent attack is something of a red herring,” Barr said, given that attacks were already underway.
“I don’t think there’s a requirement, frankly, for knowing the exact time and place of the next attack,” Barr said. “And that certainly was the position of the Obama administration when it droned leaders of terrorist organizations.”
On the Pensacola shooting, Barr definitively branded it a terrorist act.
“This was an act of terrorism,” Barr said. “The evidence shows that the shooter was motivated by Jihadist ideology.”
Barr praised his agency and the FBI for its “rapid and excellent work.”
Follow Penny Starr on Twitter |
On November 6, early Uber investors, founders, and employees will be able to cash out of the ride-hailing company for the first time since it went public in May. It could be a bloodbath, or a big payday for those with a large stake in the company.
Investors aren’t the only ones anticipating the occasion, however. Uber drivers—building on momentum from recent organizing victories in California—will use the opportunity to literally get in the faces of the company’s top investors. Rideshare drivers plan to demonstrate outside at least three investors’ homes and offices, including Uber venture capitalist Bill Gurley in Silicon Valley and Uber co-founder Garrett Camp in Los Angeles.
“These guys are making billions of dollars while they’re claiming they can’t afford to pay us a fair wage,” said Jeff Perry, 39, an Uber driver from Sacramento, and one of the protest’s lead organizers. “This is a great opportunity to highlight the differences between the two classes within Uber.”
Two groups that represent rideshare drivers in California, Gig Workers Rising in Silicon Valley and Mobile Workers Alliance in Los Angeles, will lead the protests. Drivers plan to demand a pathway to unionization—and hope to highlight the differences between Uber’s billionaire stakeholders and its labor force, which isn’t guaranteed a minimum wage.
Uber has rapidly expanded into 700 cities across the world in large part by relying on a business model that preys on drivers, who do not receive basic labor protections and benefits like overtime pay, worker’s compensation, and health insurance. According to a 2018 study by the Economic Policy Institute, Uber drivers earn $9.21 an hour after accounting for the costs of gas, car maintenance and other expenses. That’s below the minimum wage in 13 of its 20 biggest urban markets in the United States.
“Our overall idea for the protest comes from the sense that this group of people and financial enterprises are getting extremely rich regardless of the way Uber treats its drivers,” said Edan Alva, 49, an Uber driver from the East Bay, also involved in organizing the November 6 protest.
Alva told Motherboard that he has had to sign up for government assistance to support himself and his son since he lost his job as a security management contractor for PayPal last year. “Uber and Lyft have cut their prices significantly. It’s literally impossible for me to exist,” he said. Other drivers he knows have been forced to live out of their cars. “We will probably show up in our cars, and it will be a ‘this is your house. This is my house’ situation,” Alva said of the protests.
Uber co-founder Garrett Camp’s $71 million Beverly Hills mansion was an obvious choice for a protest site. Camp, who has been described as the visionary behind Uber’s business model, had around $3.2 billion in the company’s stock at the time of its public offering in May.
Bay Area organizers say they selected Bill Gurley’s home in Atherton, California as another protest site because of the massive profits he has reaped off his stake in the company as a partner at Benchmark, one of Uber’s top venture capital investors when it was a startup. When the rideshare giant went public in May, Gurley received one of the fattest payouts in venture capital history, estimated at over $600 million. In a 2018 blog post called “The Thing I love Most About Uber,” Gurley wrote that “driving with Uber reverses the way we have been trained to think about labor...Instead of making labor conform to management’s notion of a ‘job,’ Uber hands control to the workers.”
But contrary to Gurley’s blog post, many drivers say that the company tightened its grasp over its workers, in particular by slashing wages and determining the share of earnings on a ride that go into the pockets of the company.
A third action will take place at Google Community Space, a free event and coworking space for nonprofits in downtown San Francisco. That action will target Google Ventures, an investment branch of Google’s parent company that had an estimated $5.2 billion in shares in Uber around the time of its IPO in May. In 2013, Google Ventures made its largest ever investment, putting $258 million into Uber, which then grew twenty-fold in value as the company expanded. (Uber’s shares have plummeted from $45 to $32 per share since May.)
Perry, the Uber driver and organizer, says he hopes that targeting specific investors will force them to grapple with how their decisions have impacted real people. “Look, I’m not a blip on a map or a little emoji driving around on a digital screen. I’m a human being, I’m a person and [Uber’s] decisions are affecting my ability to provide for my family,” he said. He says he’s seen his earnings cut in half since he began driving for Uber and Lyft four years ago.
This year, drivers in California have transformed the state into a hotbed for rideshare driver organizing. In March, Los Angeles drivers organized the first major rideshare strike in the United States, over pay cuts. In September, drivers across the state fought and helped win the passage of a historic law that will make gig workers in California the first in the country to qualify for basic labor protections and rights, including a minimum wage, overtime pay, and compensation for work-related injuries.
The organizers of the November 6 protest told Motherboard that they hope rideshare drivers in other cities and countries learn about their action and plan their own actions for that day.
“We want to send a message and say ‘hey look, there has to be some sort of moral responsibility for your investment,” said Perry. “‘You’re not going to sail off into the sunset when this is the way you profited, not without having to look me in the eyes and say it.’” |
Q:
Are Git's pack files deltas rather than snapshots?
One of the key differences between Git and most other version control systems is that the others tend to store commits as a series of deltas - changesets between one commit and the next. This seems logical, since it's the smallest possible amount of information to store about a commit. But the longer the commit history gets, the more calculation it takes to compare ranges of revisions.
By contrast, Git stores a complete snapshot of the whole project in each revision. The reason this doesn't make the repo size grow dramatically with each commit is each file in the project is stored as a file in the Git subdirectory, named for the hash of its contents. So if the contents haven't changed, the hash hasn't changed, and the commit just points to the same file. And there are other optimizations as well.
All this made sense to me until I stumbled on this information about pack files, into which Git puts data periodically to save space:
In order to save that space, Git
utilizes the packfile. This is a
format where Git will only save the
part that has changed in the second
file, with a pointer to the file it is
similar to.
Isn't this basically going back to storing deltas? If not, how is it different? How does this avoid subjecting Git to the same problems other version controls systems have?
For example, Subversion uses deltas, and rolling back 50 versions means undoing 50 diffs, whereas with Git you can just grab the appropriate snapshot. Unless git also stores 50 diffs in the packfiles... is there some mechanism that says "after some small number of deltas, we'll store a whole new snapshot" so that we don't pile up too large a changeset? How else might Git avoid the disadvantages of deltas?
A:
Summary:
Git’s pack files are carefully constructed to effectively use disk caches and
provide “nice” access patterns for common commands and for reading recently referenced
objects.
Git’s pack file
format is quite flexible (see Documentation/technical/pack-format.txt,
or The Packfile in The Git Community Book).
The pack files store objects in two main
ways: “undeltified” (take the raw object data and deflate-compress
it), or “deltified” (form a delta against some other object then
deflate-compress the resulting delta data). The objects stored in
a pack can be in any order (they do not (necessarily) have to be
sorted by object type, object name, or any other attribute) and
deltified objects can be made against any other suitable object of the same type.
Git’s pack-objects command uses several heuristics to
provide excellent locality of reference for common
commands. These heuristics control both the selection of base
objects for deltified objects and the order of the objects. Each
mechanism is mostly independent, but they share some goals.
Git does form long chains of delta compressed objects, but the
heuristics try to make sure that only “old” objects are at the ends of
the long chains. The delta base cache (whose size is controlled by the
core.deltaBaseCacheLimit configuration variable) is automatically
used and can greatly reduce the number of “rebuilds” required for
commands that need to read a large number of objects (e.g. git log
-p).
Delta Compression Heuristic
A typical Git repository stores a very large number of objects, so
it can not reasonably compare them all to find the pairs (and
chains) that will yield the smallest delta representations.
The delta base selection heuristic is based on the idea that the
good delta bases will be found among objects with similar filenames
and sizes. Each type of object is processed separately (i.e. an
object of one type will never be used as the delta base for an
object of another type).
For the purposes of delta base selection, the objects are sorted (primarily) by
filename and then size. A window into this sorted list is used to limit
the number of objects that are considered as potential delta bases.
If a “good enough”1 delta representation is not found for an object
among the objects in its window, then the object will not be delta
compressed.
The size of the window is controlled by the --window= option of
git pack-objects, or the pack.window configuration variable. The
maximum depth of a delta chain is controlled by the --depth=
option of git pack-objects, or the pack.depth configuration
variable. The --aggressive option of git gc greatly enlarges
both the window size and the maximum depth to attempt to create
a smaller pack file.
The filename sort clumps together the objects for entries with with
identical names (or at least similar endings (e.g. .c)). The size
sort is from largest to smallest so that deltas that remove data are
preferred to deltas that add data (since removal deltas have shorter
representations) and so that the earlier, larger objects (usually
newer) tend to be represented with plain compression.
1
What qualifies as “good enough” depends on the size of the object in question and its potential delta base as well as how deep its resulting delta chain would be.
Object Ordering Heuristic
Objects are stored in the pack files in a “most recently referenced”
order. The objects needed to reconstruct the most recent history are
placed earlier in the pack and they will be close together. This
usually works well for OS disk caches.
All the commit objects are sorted by commit date (most recent first)
and stored together. This placement and ordering optimizes the disk
accesses needed to walk the history graph and extract basic commit
information (e.g. git log).
The tree and blob objects are stored starting with the tree from the
first stored (most recent) commit. Each tree is processed in a depth
first fashion, storing any objects that have not already been
stored. This puts all the trees and blobs required to reconstruct
the most recent commit together in one place. Any trees and blobs that
have not yet been saved but that are required for later commits are
stored next, in the sorted commit order.
The final object ordering is slightly affected by the delta base selection
in that if an object is selected for delta representation and its base object
has not been stored yet, then its base object is stored immediately before the
deltified object itself. This prevents likely disk cache misses due to the
non-linear access required to read a base object that would have “naturally” been
stored later in the pack file.
A:
The use of delta storage in the pack file is just an implementation detail. At that level, Git doesn't know why or how something changed from one revision to the next, rather it just knows that blob B is pretty similar to blob A except for these changes C. So it will only store blob A and changes C (if it chooses to do so - it could also choose to store blob A and blob B).
When retrieving objects from the pack file, the delta storage is not exposed to the caller. The caller still sees complete blobs. So, Git works the same way it always has without the delta storage optimisation.
A:
As I mentioned in "What are git's thin packs?"
Git does deltification only in packfiles
I detailed the delta encoding used for pack files in "Is the git binary diff algorithm (delta storage) standardized?".
See also "When and how does git use deltas for storage?".
Note that the core.deltaBaseCacheLimit config which controls the default size for the pack file will soon be bumped from 16MB to 96MB, for Git 2.0.x/2.1 (Q3 2014).
See commit 4874f54 by David Kastrup (May 2014):
Bump core.deltaBaseCacheLimit to 96m
The default of 16m causes serious thrashing for large delta chains combined with large files.
Here are some benchmarks (pu variant of git blame):
time git blame -C src/xdisp.c >/dev/null
for a repository of Emacs repacked with git gc --aggressive (v1.9, resulting in a window size of 250) located on an SSD drive.
The file in question has about 30000 lines, 1Mb of size, and a history with about
2500 commits.
16m (previous default):
real 3m33.936s
user 2m15.396s
sys 1m17.352s
96m:
real 2m5.668s
user 1m50.784s
sys 0m14.288s
|
Police officers in several Canadian cities raided illegal marijuana dispensaries linked to activists Marc and Jodie Emery on Thursday, charging them and several others with drug offences as part of an investigation led by Toronto police.
The raids were the latest attempt by local police forces to shut down pot shops that have been opening in cities across the country, even as the federal government prepares to fully legalize the drug with legislation this spring. It was also notable for the involvement of Vancouver's police force, which has largely left dispensaries in the city alone, including those run by the Emerys.
The two were arrested at Toronto Pearson International Airport on Wednesday as part of a Toronto police operation called Project Gator.
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Investigation: What's in your weed?
Mr. Emery was charged with 15 counts, including trafficking, possession and possession of the proceeds of crime, while his wife was charged with five similar counts.
Chris and Erin Goodwin, of Toronto, and Britney Guerra, of Stoney Creek, Ont., also face drug-related charges.
The five are scheduled to appear in court on Friday.
On Thursday, police in Toronto, Hamilton and Vancouver searched a total of seven Cannabis Culture locations linked to the Emerys, acting on warrants from the Toronto Police Service. Search warrants were also executed at private residences in Toronto, Stoney Creek and Vancouver, for a total of 11 warrants.
Ms. Emery has told The Globe and Mail in the past that she and her husband do not own the Cannabis Culture franchises, but rather license out the brand for a marginal percentage of sales. They do not supply each location with product, she added.
The Emerys' flagship location in Vancouver adjoins a vapour lounge and office, where Pot TV and the Cannabis Culture magazine are produced. On Thursday morning, the front windows were covered with brown paper as some police officers stood guard and others removed evidence through a back entrance.
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Magazine editor Danny Kresnyak said officers seized cellphones, computers and cash, that art had been removed from the walls and that a safe had been pried open. He was among a few dozen employees and supporters who stood outside the location protesting the raid, criticizing the use of police resources.
"We're supposed to be seeing [marijuana] legalization this year," Mr. Kresnyak said. "[Justin] Trudeau ran on the idea that by the spring of 2017 [it would be legal], yet they're still raiding a shop that sells mostly glass and T-shirts that have funny slogans on them."
Kevin Bruneau, an acquaintance of the Emerys and a long-time regular at the vapour lounge, called the raid "ridiculous" and said he would rather see resources focused on the overdose epidemic. There were 22 suspected overdose deaths across Vancouver in the last two weeks, according to police.
"It's a complete waste of money," Mr. Bruneau said. "What harm does this do? This hotel right over here averages three ambulances a day. Maybe address that problem."
At an initial court appearance on Thursday, Mr. Emery strolled into the prisoner's box, hands in his jean pockets, wearing a grey sweater, and took a seat. His wife remained standing, speaking with her lawyer and, at one point, flashing a peace sign to the back of the courtroom before being scolded by Justice of the Peace Marilyn Churley.
Earlier, Chris and Erin Goodwin, the owners of one of the Emery-branded marijuana shop locations in Toronto, were arrested in the hallway at the courthouse, where supporters of the Emerys had gathered. The Goodwins were also charged with a number of drug-related offences in January, 2016, in their roles as part owners of GoodWeeds, a franchise of the Vancouver-based Weeds chain.
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Toronto Police have taken an aggressive approach to the city's growing number of marijuana dispensaries, while Vancouver police have largely turned a blind eye so long as dispensaries aren't tied to gangsters, or selling to minors. Toronto's first wave of busts came last May, after Mayor John Tory expressed concerns about the rapid proliferation of marijuana shops in the city.
Thursday's events mirror those of July, 2005, when Mr. Emery was arrested at a rally in Halifax while Vancouver police, acting on American charges, raided the Vancouver storefront. Mr. Emery ultimately served five years in U.S. federal prison for selling marijuana seeds over the Internet and was transferred back to Canada in 2014.
City of Toronto officials said that they also attended Thursday's raids in the city, laying zoning and licensing bylaw charges as they have against scores of other dispensaries over the past year.
In a separate case, city officials revealed that they are seeking a court injunction that would shut down a separate chain of seven marijuana dispensaries known as Canna Clinic – which has previously faced raids and charges.
"The operation of retail stores selling marijuana is illegal under federal law and is a contravention of the City's zoning bylaws," said Tracey Cook, head of the city's licensing and standards department, in an e-mailed statement. "The City of Toronto, in partnership with the Toronto Police Service has consistently pursued enforcement actions against these illegal marijuana stores in the city."
The federal Liberal government, which has pledged to introduce legislation to legalize marijuana this spring, has taken criticism for not decriminalizing petty cannabis-related offences in the meantime.
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"Until we have brought in legislation," Prime Minister Trudeau said earlier this month, "the current law remains the law."
Federal Health Minister Jane Philpott said Thursday the legislation will still have to go through the parliamentary process and then there are regulatory processes that would likely take place.
"People need to recognize that things take time," she said. "This is something that has to be done correctly. It's something that we feel very strongly about as a government. It's a firm commitment of our government to legalize access to cannabis, to regulate that access and to restrict it appropriately."
With a report from Gloria Galloway |
DC Update: Animal Transports to National Aquarium, Baltimore
Published October 07, 2013
The most common question we get about the closure of the National Aquarium, Washington, D.C. is “Where are all of the animals going?”.
Of the 2,500 animals that currently call our DC facility home, 1,700 will be transported to our Baltimore facility. The rest will be transported to other accredited aquariums and zoos.
The key to any successful animal move is exceptional planning and great communication between all team members. In fact, we started planning these moves as soon as the closure was announced. Everyone has a role in a big move like this, from husbandry staff to veterinarians.
Today marked the first of our transport trips, which included the move of 38 animals including a giant Pacific octopus, seven plumose anemones, a peacock wolf eel, rockfish and much more! However, on any given day in the next two months, we may be transporting 20 to 400 animals.
[gallery type="rectangular" ids="10233,10235,10237"]
Every animal that moves out of the D.C. facility will receive a veterinary exam to confirm it is healthy enough for transport. In some cases, this might be a visual examination (looking at the animal in its habitat). Most fish and invertebrates get visual exams. In other cases, such as for sharks or reptiles, we may do a complete “hands-on” physical examination including evaluating radiographs (x-rays) and blood tests.
But how do you actually move fish? First, the keepers slowly coax the animals into transport nets and then quickly move them into their transport carriers. Fish can be moved in large plastic containers or placed into individual bags, depending on their size and the number of individual fish moving that day. Water from their exhibits is used to fill their transport carriers. During transport, staff monitors temperature and dissolved oxygen levels to ensure the parameters stay where we want them.
Animals coming to Baltimore will make a stop at our Animal Care Center (ACC) before being placed on exhibit. Here they will go through at least a two week observational period to ensure they remain healthy and are eating well. If we have health concerns about an animal post-move, it’s very easy to provide medical care at the ACC. Because the transport is so short and the animals are already acclimated to human care, we expect them to do well at the Animal Care Center and quickly move into our main facility!
Stay tuned for more updates as we continue to transition our DC facility!
Featured Stories
For the first time in its history, National Aquarium Animal Rescue simultaneously released two rehabilitated seals. The two male greys, nicknamed Edwin Hubble and George Washington Carver, were released in Ocean City, Maryland, on May 23. |
Writer Thinks “War On Terror” Is American Scam
[The Brit Independent ran a translated version of part of this Egyptian Opinion piece, but I think that Goog Trans. gets the writer’s thoughts through sufficiently. Most of the article is given below, all can be accessed through the title link.]
Is it possible to believe the official version for the US government the events of September 11, 2001 ?! Is it a coincidence that the leaders of September atheist attack people received lessons in the American aeronautics institutes?! According the American government to the official version, the first attack was at about 8:46 pm New York time and targeted the north tower of the World Trade Center and after quarter of an hour at approximately 9:03, collided with another aircraft building south tower of the World Trade Center after more than half the time, hit a third plane into the Pentagon, while the fourth plane misfires and crashed … Does it make sense to move the four hijacked planes, with all this freedom and penetrate American airspace and hit the US World trade Center and the Pentagon towers one by one with a time lag first arrived more than 15 minutes the second time and the difference It has reached more than half an hour … and that is all of the above without targeting aircraft and eliminated despite intelligence, satellites and radars America … or that it was premeditated to justify the war on terror that began the series of episodes in Iraq?!
Is it possible that most of those who belong to Daesh foreign Or that Daesh are other novel and the story orchestrated to justify what is happening and what will be implemented in the Middle East and the destruction of the division and occupation?! Do you remember me from the shapes and sizes were told that they were Daesh who has broadcast video cameraman them they are being slaughtered because they were told a group of Egyptian Christians in Libya? It was it not strange that all members of the group Aldaashah featuring the same height and the same color and the whites are all dressed in the same type of foreign finest hours?!
Are they being killed, and accused of terrorist attacks in Western countries; representing the defendants leave or are there points of intelligence Western behind what is happening from the attacks and bombings are arrested Muslim citizens and liquidated later charged with simply implementing the incident to justify what is happening as the war on terrorism in the Arab states and justify as well as the persecution and expulsion of Muslims from America and Europe scheme?! … Is it already is to trick us to this point?! |
Effects of refeeding on adipocyte metabolism in the rat.
Glucose metabolism in fat cells from fasted-refed rats and ad-libitum-fed controls were studied in the postabsorptive state. Two types of experiments were performed. First, each rat donated one epididymal fat pad and the metabolic results were expressed in relation to the number and size of fat cells of these tissues. Second, the other epididymal fat pad in the rats in each experimental group was pooled for experiments in duplicate where liberated fat cells were separated in fat-cell size classes, which enabled comparisons of metabolic activities at the same fat-cell size. Glucose incorporation into carbon dioxide and triglyceride glycerol and fatty acids were about equally elevated in absolute terms in the refed rats. In relative terms the increase was much more pronounced in carbon dioxide and fatty acids because these activities were very low in control rats. These results confirm previous results showing elevated metabolism after fasting-refeeding, and also demonstrate that this is an adaptation of fat-cell metabolism rather than a consequence of a higher cellular density of adipose tissue after fasting-refeeding with smaller fat cells. Adaptations of metabolism after fasting-refeeding might be of potential importance for elucidation of the cause of the rapid relapse after weight decrease of obese subjects with diminished fat-cell size. |
Q:
Output only csv lines if column increment is 2
I have a csv file that I would like to filter. The output I need would be only to output the lines if the increment is not equal to 2. In the csv file below, I would like to compare the first line with the second line, if the increment is 2, check line 3 vs line 2, and so on. If the increment is not equal to 2, output the line. I'm looking at the 3rd cloumn values
L1,is,2.0,mins,LATE,for,Arrive,at,shop,18:07:46
L1,is,4.0,mins,LATE,for,Arrive,at,shop,18:09:46
L1,is,6.0,mins,LATE,for,Arrive,at,shop,18:11:46
L1,is,8.0,mins,LATE,for,Arrive,at,shop,18:13:46
L1,is,10.0,mins,LATE,for,Arrive,at,shop,18:15:46
L1,is,2.0,mins,LATE,for,Arrive,at,shop,18:19:49
L1,is,4.0,mins,LATE,for,Arrive,at,shop,18:21:49
L1,is,6.0,mins,LATE,for,Arrive,at,shop,18:23:49
L1,is,8.0,mins,LATE,for,Arrive,at,shop,18:25:49
L1,is,10.0,mins,LATE,for,Arrive,at,shop,18:27:49
L1,is,16.2,mins,LATE,for,Arrive,at,shop,18:34:02
L1,is,18.2,mins,LATE,for,Arrive,at,shop,18:36:02
L1,is,20.2,mins,LATE,for,Arrive,at,shop,18:38:02
L1,is,2.0,mins,LATE,for,Arrive,at,bridge,21:45:26
L1,is,4.0,mins,LATE,for,Arrive,at,bridge,21:47:26
L1,is,6.0,mins,LATE,for,Arrive,at,bridge,21:49:26
So only lines 5,10,13 and 16 would output to page.
I'm stuck on this and would appreciate any help or direction on where to look.
Thanks
A:
If your file is not too big, you can load it into memory directly, like this:
$data = array_map(function($row)
{
return explode(',', $row);
}, file('/path/to/file.csv', FILE_IGNORE_NEW_LINES));
$result = [];
$increment = 2;
$delta = 1E-13;
for($i=1; $i<count($data); $i++)
{
if(abs($data[$i][2]-$data[$i-1][2]-$increment)>$delta)
{
$result[$i] = $data[$i];
}
}
-since your column holds floats, safe comparison on equality will be using precision delta.
Your data will be gathered in $result array, so you can output it like
foreach($result as $row)
{
echo(join(',', $row).PHP_EOL);
}
-or, else, do not store rows inside $result array (if you will need them no longer) and use first cycle to output your rows.
Edit:
Sample above will work in PHP>=5.4 For PHP 5.3 you should replace array definition to
$result = array();
and if you have even older PHP version, like 5.2, then callback inside array_map() should be rewritten with using create_function()
|
Q:
Read and print from a text file N lines at a time using a generator only
Python 3.6.0
textfile = "f:\\mark\\python\\Alice_in_Wonderland.txt"
N = 60
def read_in_lines(file, n):
with open(file) as fh:
for i in range(n):
nlines = fh.readline()
if nlines:
yield nlines
else:
break
for lines in read_in_lines(textfile, x):
print(lines)
File is here: https://www.gutenberg.org/files/11/11.txt
My goal is to read in this file N lines at a time, then print the lines,
then read in the next N lines, print, repeat...
If N = 3, output should look like:
line1
line2
line3
line4
line5
line6
line7
line8
line9
line10 <-- assumes this is the last line in the file
The above print pattern should hold for any value of 'N'.
If 'N' = 4:
line1
line2
line3
line4
line5
line6
line7
line8
etc. You get the idea.
NO lists. No built in functions (islice, etc.).
I need to use a generator only.
Each iteration must contain a string containing up to the
number of lines specified by 'N'.
Two issues:
1) The above code returns 'N' lines, then stops. I assume I need to put the whole
thing in a loop, but I am unsure of how to proceed. (Newbie...)
2) The file contains A LOT of blank lines. Every single time I try to use strip()
or any of it's variants, regardless of how big I make 'N' it only ever prints one line.
nlines = fh.readline().strip <-- adding in .strip()
With N = 6000 I get:
Project Gutenberg's Alice's Adventures in Wonderland, by Lewis Carroll
Process finished with exit code 0
If I get rid of .strip() I get all the lines but not in the format I want.
I am on a Win 10 machine. In Notepad++ all of the end of file symbols are CRLF.
A:
Solved:
textfile = "f:\\mark\\python\\test.txt"
def read_n(file, x):
with open(file, mode='r') as fh:
while True:
data = ''.join(fh.readline() for _ in range(x))
if not data:
break
yield data
print()
for nlines in read_n(textfile, 5):
print(nlines.rstrip())
Output:
abc
123
def
456
ghi
789
jkl
abc
123
def
456
ghi
789
jkl
abc
123
def
456
ghi
789
jkl
abc
123
def
456
ghi
789
jkl
|
# Copyright 2012 Managed I.T.
#
# Author: Kiall Mac Innes <kiall@managedit.ie>
#
# Licensed under the Apache License, Version 2.0 (the "License"); you may
# not use this file except in compliance with the License. You may obtain
# a copy of the License at
#
# http://www.apache.org/licenses/LICENSE-2.0
#
# Unless required by applicable law or agreed to in writing, software
# distributed under the License is distributed on an "AS IS" BASIS, WITHOUT
# WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied. See the
# License for the specific language governing permissions and limitations
# under the License.
from designate.tests import TestCase
class ApiTestCase(TestCase):
pass
|
The 4 Most Stressful Work Situations for Introverts, Illustrated
Between open floor plans, team lunches, and mandatory meetings, most workplaces are set up for our more extroverted colleagues.
So how do we introverts navigate the external pressures (and stimuli!) of the workplace? And how do we create space for the alone time that makes us our most productive, fulfilled selves? Below, we’ll walk through four of the most stressful work situations for introverts — and how to stay sane while navigating them.
The 4 Most Stressful Work Situations for Introverts
1. Networking events and work parties
We can all relate to Seinfeld’s Elaine when she tells Jerry, “I’ll go if I don’t have to talk.” If you start feeling talked-out around hour two of your office holiday party or happy hour, you’re not alone.
How can you feel more at ease? Try arriving with a planned exit strategy. When you feel less like you have to stay, you might find that you’re more likely to want to stay.
“Before I’d quietly slip away whenever I felt the first tingles of ‘uh I don’t want to be here anymore.’ Now I recognize that impulse, honor it, exhale and see if I’m cool staying another 30 minutes. Once I do this check-in, I’m totally ok bouncing after 30 if that’s still the way I’m feeling, but often I’ll end up hanging out much longer without even knowing it.”
Former Yahoo CEO Marissa Meyer has a similar strategy. In an interview with Vogue magazine, she admits that she always has an urge to hide at parties. So before a party starts, she promises herself that she can leave at a certain preset time. “I will literally look at my watch and say, ‘You can’t leave until time x,’ ” she says. “ ‘And if you’re still having a terrible time at time x, you can leave.’”
2. Working with extroverted colleagues and bosses
If your boss is an extrovert, you might be familiar with MBWA, or “Management by Wandering Around.”
Next time your boss ambles by with an unprompted question (we’re guessing you won’t have to wait long), try saying, “I’m happy to give you an immediate response, but I’ll be able to give you a better response if I can take some time to think about it and then get back to you.”
Here are a few additional strategies you can suggest to your boss or team to ensure you (and your fellow introverts) have the time and space to do good work:
Heads down time: Try splitting the day between collaborative and heads down time. During heads down time, each team member has explicit permission to find a quiet place in the office and work by themselves.
Walking meetings: As Dr. Jennifer Kahnweiler explains in her book The Genius of Opposites, a walk makes it easier for introverts to talk out their ideas, since they don’t have to make constant eye contact. When they are talking or searching for words, introverts tend to look away from others to reduce visual stimuli so their brains are not as flooded with input.
Meeting rituals: Since most meetings are inherently set up for extroverted communication, introverts need to find rituals that make them feel comfortable joining in. According to author Brad Stone, before every meeting at Amazon, Jeff Bezos requires employees to write a six-page narrative detailing their points. The meeting begins in silence as everyone reads the document. Bezos does this because he believes it both encourages critical thinking and gives introverts time for reflection before the discussion begins.
3. Work travel or retreats
When Mollie is traveling for work and spends 24/7 with colleagues or clients, she often gets an introvert hangover. Shawna Courter, who coined the phrase on Introvert, Dear, describes an introvert hangover as:
“A pretty terrible thing to experience. It starts with an actual physical reaction to overstimulation. Your ears might ring, your eyes start to blur, and you feel like you’re going to hyperventilate. Maybe your palms sweat. And then your mind feels like it kind of shuts down, building barriers around itself as if you had been driving on a wide open road, and now you’re suddenly driving in a narrow tunnel. All you want is to be at home, alone, where it’s quiet.”
Introverts have a more limited source of social energy than extroverts. Once introverts run out of those reserves, the only thing that helps is retreating to be alone. What extroverts don’t always understand is that this is an actual physical sensation — not just a slight preference.
Mollie’s hangover cure is either to go for a long walk alone or lie in the fetal position on her couch and watch mindless TV, preferably a British show. Liz recovers by retreating into her minimalist, silent, white-walled apartment that offers almost no sensory stimulation.
But the best offense is a good defense. To prevent introvert hangovers, Mollie will often tell her teammates that she needs to schedule time at the end of travel days for decompression. If you know you’ll be with colleagues all day (e.g. at a conference or retreat), try to block off a few hours at the end of each day to step away from the group.
You can also figure out which parts of the day you need to attend and which are not mandatory, or talk to your manager about scheduling breaks (or if you’re a manager, schedule those breaks!).
The first company-wide retreat Genius, a music media company, hosted was a four-day, nonstop work-and-be-together offsite that left Liz exhausted. She talked to the company’s leaders, who made sure to give employees enough downtime to recharge on the second retreat.
4. Phone calls
You knew it was coming: the dreaded phone call. For introverts, phone calls mean awkward pauses, anxiously trying not to speak at the same time as the other person, and lots and lots of small talk. Receiving an unexpected phone call can also jolt us out of a period of productive creativity. Even a short conversation can derail our entire thought process.
The best way to prevent phone fatigue is to pre-arrange calls. This ensures you’ll have plenty of uninterrupted time to concentrate.
Copyright 2019 Introvert, Dear | Privacy Policy and Affiliate Disclosures All rights reserved. No part of this site may be reproduced in whole or in part in any manner without the permission of the copyright owner. |
SC Gov. Nikki Haley calls Obama a Coward
Haley Calls Obama Coward During Conference
South Carolina Gov. Nikki Haley blasted President Obama and the National Labor Relations Board today. She called on President Obama to address the NLRB’s unprecedented lawsuit against Boeing during his jobs speech on Sept. 8. The South Carolina governor made the comments during a conference call with bloggers and journalists this morning.
It’s the truth. The president won’t stand up to his gravy train. The Democratic Party gets millions of dollars every year from the AFL-CIO and other unions around the country and Obama doesn’t have the guts to do what is right, stop this ludicrous lawsuit, and piss off the unions in the process. Where are all the South Carolina Democrats at on this issue? We’re talking about a bogus lawsuit that is putting over a thousand new jobs in our state at risk and they’ve been completely mum about it. It’s for the same reason. It’s all about the Benjamins. Money speaks louder than you do. |
Q:
Android: How to make selected spinner item to TextView?
I am trying to get the selected item from the spinner to be displayed DIRECTLY after it is selected. Imagine if this is like a quiz game. If you select one of the wrong ones, a TOAST will appear(which I cannot get working). If I select the right one, a different TOAST will appear and I will link it to a new activity. How can I get the onselect to output or display its selection to the textview and how can I implement an if statement for the right/wrong answers?
package com.example.test;
import android.os.Bundle;
import android.app.Activity;
import android.view.View;
import android.widget.AdapterView;
import android.widget.AdapterView.OnItemSelectedListener;
import android.widget.ArrayAdapter;
import android.widget.Spinner;
import android.widget.TextView;
public class MainActivity extends Activity implements OnItemSelectedListener{
String[] test_array;
TextView display, selected;
String result;
Spinner spinner;
@Override
protected void onCreate(Bundle savedInstanceState) {
super.onCreate(savedInstanceState);
setContentView(R.layout.activity_main);
Spinner spinner = (Spinner) findViewById(R.id.spinner);
ArrayAdapter<CharSequence> adapter = ArrayAdapter.createFromResource(this, R.array.test_array, android.R.layout.simple_spinner_item);
adapter.setDropDownViewResource(android.R.layout.simple_spinner_dropdown_item);
spinner.setAdapter(adapter);
}
@Override
public void onItemSelected(final AdapterView<?> parent, View view, final int pos,
long id) {
// TODO Auto-generated method stub
result = (String) spinner.getSelectedItem();
display = (TextView) findViewById (R.id.display);
display.setText(result);
}
@Override
public void onNothingSelected(AdapterView<?> arg0) {
// TODO Auto-generated method stub
}
}
I REVISED ACCORDING TO GOOGLE'S SAMPLES WITHIN THE SDK. HERE IS REVISED CODE.
package com.example.test;
import android.os.Bundle;
import android.app.Activity;
import android.view.View;
import android.widget.AdapterView;
import android.widget.AdapterView.OnItemSelectedListener;
import android.widget.ArrayAdapter;
import android.widget.Spinner;
import android.widget.TextView;
public class MainActivity extends Activity{
String[] test_array;
TextView display, selected;
String result, sSelect;
Spinner spinner;
int mPos;
String mSelection;
ArrayAdapter<CharSequence> mAdapter;
@Override
protected void onCreate(Bundle savedInstanceState) {
super.onCreate(savedInstanceState);
setContentView(R.layout.activity_main);
Spinner spinner = (Spinner) findViewById(R.id.spinner);
this.mAdapter = ArrayAdapter.createFromResource(this, R.array.test_array, android.R.layout.simple_spinner_dropdown_item);
spinner.setAdapter(this.mAdapter);
OnItemSelectedListener listener = new myOnItemSelectedListener();
spinner.setOnItemSelectedListener(listener);
}
public class myOnItemSelectedListener implements OnItemSelectedListener {
@Override
public void onItemSelected(AdapterView<?> parent, View v, int pos,
long id) {
// TODO Auto-generated method stub
MainActivity.this.mPos = pos;
MainActivity.this.mSelection = parent.getItemAtPosition(pos).toString();
TextView resultText = (TextView) findViewById(R.id.display);
resultText.setText(MainActivity.this.mSelection);
}
@Override
public void onNothingSelected(AdapterView<?> parent) {
// TODO Auto-generated method stub
}
}
}
The textview now changes to the selected item, now I will attempt the if/else.
The if/else statement causes crash.
04-03 01:38:17.648: E/AndroidRuntime(817): FATAL EXCEPTION: main
04-03 01:38:17.648: E/AndroidRuntime(817): java.lang.NullPointerException
04-03 01:38:17.648: E/AndroidRuntime(817): at com.example.test.MainActivity$myOnItemSelectedListener.onItemSelected(MainActivity.java:43)
04-03 01:38:17.648: E/AndroidRuntime(817): at android.widget.AdapterView.fireOnSelected(AdapterView.java:892)
04-03 01:38:17.648: E/AndroidRuntime(817): at android.widget.AdapterView.access$200(AdapterView.java:49)
04-03 01:38:17.648: E/AndroidRuntime(817): at android.widget.AdapterView$SelectionNotifier.run(AdapterView.java:860)
04-03 01:38:17.648: E/AndroidRuntime(817): at android.os.Handler.handleCallback(Handler.java:615)
04-03 01:38:17.648: E/AndroidRuntime(817): at android.os.Handler.dispatchMessage(Handler.java:92)
04-03 01:38:17.648: E/AndroidRuntime(817): at android.os.Looper.loop(Looper.java:137)
04-03 01:38:17.648: E/AndroidRuntime(817): at android.app.ActivityThread.main(ActivityThread.java:4745)
04-03 01:38:17.648: E/AndroidRuntime(817): at java.lang.reflect.Method.invokeNative(Native Method)
04-03 01:38:17.648: E/AndroidRuntime(817): at java.lang.reflect.Method.invoke(Method.java:511)
04-03 01:38:17.648: E/AndroidRuntime(817): at com.android.internal.os.ZygoteInit$MethodAndArgsCaller.run(ZygoteInit.java:786)
04-03 01:38:17.648: E/AndroidRuntime(817): at com.android.internal.os.ZygoteInit.main(ZygoteInit.java:553)
04-03 01:38:17.648: E/AndroidRuntime(817): at dalvik.system.NativeStart.main(Native Method)
04-03 01:39:04.718: E/Trace(865): error opening trace file: No such file or directory (2)
04-03 01:39:06.028: D/gralloc_goldfish(865): Emulator without GPU emulation detected.
Implemented code
public class myOnItemSelectedListener implements OnItemSelectedListener {
@Override
public void onItemSelected(AdapterView<?> parent, View v, int pos,
long id) {
// TODO Auto-generated method stub
MainActivity.this.mPos = pos;
MainActivity.this.mSelection = parent.getItemAtPosition(pos).toString();
TextView resultText = (TextView) findViewById(R.id.display);
resultText.setText(MainActivity.this.mSelection);
if(result.equalsIgnoreCase("abc")){
// Toast in here
}else{
}
}
A:
try this
@Override
public void onItemSelected(AdapterView<?>; parent, View view, int position, long id) {
// On selecting a spinner item
String item = parent.getItemAtPosition(position).toString();
// Showing selected spinner item
Toast.makeText(parent.getContext(), "Selected: " + item, Toast.LENGTH_LONG).show();
}
try this for if-else statement
spinner = (Spinner) findViewById(R.id.test);
spinner.setOnItemSelectedListener(new OnItemSelectedListener() {
@Override
public void onItemSelected(AdapterView<?> parent, View view,
int position, long id) {
String item = "nothing";
// On selecting a spinner item
if(parent.getItemAtPosition(position).toString().equalsIgnoreCase("novels"))
item = parent.getItemAtPosition(position).toString();
else{
//do other things
}
// showing a toast on selecting an item
Toast.makeText(parent.getContext(), item, Toast.LENGTH_LONG).show();
}
@Override
public void onNothingSelected(AdapterView<?> arg0) {
// TODO Auto-generated method stub
}
});
|
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658 F.3d 299 (2011)
UNITED STATES of America,
v.
Thomas S. PENDLETON, Appellant.
No. 10-1818.
United States Court of Appeals, Third Circuit.
Argued February 16, 2011.
September 7, 2011.
*301 llana H. Eisenstein, [Argued], Office of United States Attorney, Wilmington, DE, Jennifer Leonardo, United States Department of Justice, Criminal Division, Public Integrity Section, Washington, DC, for Plaintiff-Appellee.
Eleni Kousoulis, Daniel I. Siegel, [Argued], Office of Federal Public Defender, Wilmington, DE, for Defendant-Appellant.
Before: SLOVITER and HARDIMAN, Circuit Judges and JONES, II,[*] District Judge.
OPINION OF THE COURT
HARDIMAN, Circuit Judge.
This appeal presents two questions of first impression. First, we consider whether the general criminal venue provision, 18 U.S.C. § 3238, applies when a defendant commits part of his offense inside the United States. Second, we determine whether 18 U.S.C. § 2423(c) and (f)(1), which together criminalize noncommercial illicit sexual conduct outside the United States, is a valid exercise of Congress's power under Article I, Section 8, Clause 3 of the United States Constitution (the Foreign Commerce Clause).
I
On November 25, 2005, Thomas Pendleton boarded a plane in New York City and flew to Hamburg, Germany. Six months after his arrival in Germany, Pendleton sexually molested a fifteen-year-old boy. German authorities arrested Pendleton, and a jury in Hamburg found him guilty of "engaging in sexual acts with a person incapable of resistance." After serving nineteen months in a German prison, Pendleton returned to the United States, where he was arrested and indicted by a federal grand jury in the District of Delaware on one count of engaging in noncommercial illicit sexual conduct in a foreign place, in violation of 18 U.S.C. § 2423(c) and (f)(1).
Adopted in 2003 as part of the Prosecutorial Remedies and Other Tools to End the Exploitation of Children Today Act (the PROTECT Act), § 2423(c) provides: "Any United States citizen or alien admitted for permanent residence who travels in foreign commerce, and engages in any illicit sexual conduct with another person shall be fined under this title or imprisoned not more than 30 years, or both." The statute defines "illicit sexual conduct" in two ways: (1) "a sexual act (as defined in section 2246) with a person under 18 years of age that would be in violation of chapter 109A if the sexual act occurred in the special maritime and territorial jurisdiction of the United States;" or (2) "any commercial sex act (as defined in section 1591) with a person under 18 years of age." 18 U.S.C. *302 § 2423(f). Pendleton was indicted under the first subpart of § 2423(f), which criminalizes noncommercial sex with a minor.
Pendleton moved to dismiss the indictment, challenging Congress's authority to regulate noncommercial activity outside the United States under the Foreign Commerce Clause and the Due Process Clause of the Fifth Amendment. The District Court denied Pendleton's motion, holding that 18 U.S.C. § 2423(c) was a valid exercise of Congress's power to regulate the "channels" of foreign commerce.[1]See United States v. Clark, 435 F.3d 1100, 1114 (9th Cir.2006) ("[T]he phrase `travels in foreign commerce' unequivocally establishes that Congress specifically invoked the Foreign Commerce Clause."). The District Court also held that Pendleton's due process claim was foreclosed by our decision in United States v. Martinez-Hidalgo, 993 F.2d 1052, 1056 (3d Cir.1993).[2]
Following a two-day jury trial, Pendleton was convicted of engaging in illicit sexual conduct in Germany in violation of 18 U.S.C. § 2423(c), and he was sentenced to thirty years in prison.[3] At the close of the Government's case, Pendleton moved for judgment of acquittal under Federal Rule of Criminal Procedure 29(a), claiming that he should have been tried in the Eastern District of New York. The District Court denied the motion, holding that venue was proper in the District of Delaware because Pendleton was arrested there following his return to the United States. United States v. Pendleton, 2010 WL 427230, at *6 (D.Del. Feb. 2, 2010).
Pendleton timely appealed the District Court's judgment of sentence and seeks reversal for two reasons: (1) venue was improper in the District of Delaware; and (2) the "noncommercial" prong of 18 U.S.C. § 2423(c) is facially unconstitutional. We consider each argument in turn.
II
Jurisdiction lies over Pendleton's appeal under 28 U.S.C. § 1291 and 18 U.S.C. § 3582, and we exercise plenary review over the District Court's venue determination. United States v. Perez, 280 F.3d 318, 328-30 (3d Cir.2002).
As a defendant in a criminal trial, Pendleton has a constitutional right to *303 be tried in the district where his crime was committed. Id. at 329 (citing U.S. CONST. amend. VI and U.S. Const, art. III, § 2, cl. 3). Congress may fix jurisdiction in any district where a "crucial element" of the crime is performed. Id. When Congress has "not indicate[d] where it consider[s] the place of committing the crime to be," we determine jurisdiction "from the nature of the crime alleged and the location of the act or acts constituting it." United States v. Rodriguez-Moreno, 526 U.S. 275, 279 n. 1, 119 S.Ct. 1239, 143 L.Ed.2d 388 (1999) (citations and internal quotation marks omitted). When the crime consists of distinct acts occurring in different places, venue is proper where any part of the crime occurs. Id. (citing United States v. Lombardo, 241 U.S. 73, 77, 36 S.Ct. 508, 60 L.Ed. 897 (1916)).
Although the PROTECT Act contains no express venue provision, Pendleton argues that Congress fixed venue for all crimes involving "transportation in foreign commerce" only in those districts where foreign travel commenced. For this proposition, Pendleton cites 18 U.S.C. § 3237(a), which reads in relevant part:
Any offense involving the use of the mails, transportation in interstate or foreign commerce, or the importation of an object or person into the United States is a continuing offense and, except as otherwise expressly provided by enactment of Congress, may be inquired of and prosecuted in any district from, through, or into which such commerce, mail matter, or imported object or person moves.
Because he boarded the plane to Germany in the Eastern District of New York, Pendleton claimed jurisdiction would have been proper only in that district. The District Court disagreed, writing that "the PROTECT Act contains no directive as to the appropriate venue for the prosecution of those charged under its provisions." Consequently, the Court relied on Rodriguez-Moreno's, two-pronged approach to determine venue in this case. Pendleton, 2010 WL 427230, at *6. This was not error.
Contrary to Pendleton's argument, § 3237(a) does not include a mandatory venue provision. Rather, the statute instructs that offenses involving interstate or foreign transportation "may be inquired of and prosecuted ... in the district from ... which such commerce ... moves." Id. (emphasis added). Accordingly, the Government is not statutorily barred from prosecuting Pendleton in another district if it can show that a portion of his offense was committed there. Moreover, the Constitution does not " `command a single exclusive venue.'" United States v. Goldberg, 830 F.2d 459, 466 (3d Cir.1987) (quoting United States v. Reed, 773 F.2d 477, 480 (2d Cir.1985)). "`The [c]onstitution requires only that the venue chosen be determined from the nature of the crime charged as well as from the location of the act or acts constituting it, and that it not be contrary to an explicit policy underlying venue law.'" Id. (quoting Reed, 773 F.2d at 480).
Where, as here, Congress has not designated the venue in the relevant criminal statute, we employ the two-pronged approach set forth in Rodriguez-Moreno. See 526 U.S. at 279, 119 S.Ct. 1239. "A court must initially identify the conduct constituting the offense (the nature of the crime) and then discern the location of the commission of the criminal acts." Id. To identify which conduct "constitutes the offense," we look to Pendleton's crime of conviction, which provides:
Any United States citizen or alien admitted for permanent residence who travels in foreign commerce, and engages in any illicit sexual conduct with another person *304 shall be fined under this title or imprisoned not more than 30 years, or both.
18 U.S.C. § 2423(c). The crime of conviction thus comprises three elements: (1) being a United States citizen or permanent resident; (2) traveling in foreign commerce; and (3) engaging in illicit sexual conduct. See Clark, 435 F.3d at 1105 (finding that an American citizen who traveled in foreign commerce to Cambodia and engaged in commercial sex acts with underage boys could be prosecuted under § 2423(c)).
Of these three elements, we agree with the District Court that "engaging in illicit sexual conduct" is the most critical to § 2423(c). Indeed, the title of the offense "Engaging in Illicit Sexual Conduct in Foreign Places" describes only this conduct. Moreover, while travel in foreign commerce is an element of § 2423(c), the crime itself is not complete until a person engages in illicit sex. In this regard, § 2423(c) is unlike the crime of "[t]ravel with intent to engage in illicit sexual conduct," defined in § 2423(b), which is complete as soon as one begins to travel with the intent to engage in a sex act with a minor. See United States v. Bredimus, 352 F.3d 200, 208, 210 (5th Cir.2003) ("We find ... that the criminal act under § 2423(b) is foreign travel with criminal intent; and thus, the offense is complete even if the illicit intent is never realized."). Although § 2423(c) targets the same individuals as does § 2423(b)namely, persons traveling in commerce for the purpose of engaging in illicit sexit does so by focusing the court's attention on the defendant's actual conduct in the foreign nation. See H.R.Rep. No. 108-66, at 51 (explaining that Congress enacted § 2423(c) so "the government would only have to prove that the defendant engaged in illicit sexual conduct with a minor while in a foreign country."). Thus, the locus delicti of § 2423(c) is the place where the illicit sex occurs, and notas is the case with § 2423(b)where the intent to engage in the illicit conduct is formed.
Because the crux of Pendleton's offense was "committed ... out of the jurisdiction of any ... district," the District Court held that 18 U.S.C. § 3238 was the controlling venue provision. Section 3238 provides that an offense "begun or committed" outside the United States "shall be [prosecuted] in the district in which the offender ... is arrested." Pendleton argues that § 3238 does not apply to him because part of his offense occurred in the Eastern District of New York and the title of § 3238 describes only those "offenses not committed in any district." Id. (emphasis added). This argument has some persuasive force, as two of our sister courts of appeals have held that "[s]ection 3238 does not apply unless the offense was committed entirely on the high seas or outside the United States." United States v. Pace, 314 F.3d 344, 351 (9th Cir.2002); United States v. Gilboe, 684 F.2d 235 (2d Cir.1982); see also United States v. Perlitz, 728 F.Supp.2d 46 (D.Conn.2010) (stating in dicta that § 3238 cannot apply, "by its terms," to a § 2423(c) offense because "an essential conduct element" of the offense, i.e., foreign travel, occurs within a district of the United States).
On the other hand, the Courts of Appeals for the Fourth and Fifth Circuits have held that § 3238 applies even when some of a defendant's offense conduct takes place in the United States. See, e.g., United States v. Levy Auto Parts, 787 F.2d 946, 950-952 (4th Cir.), cert. denied, 479 U.S. 828, 107 S.Ct. 108, 93 L.Ed.2d 56 (1986) (finding venue proper under § 3238 when conspiracy was "essentially foreign," even when some overt acts occurred inside the United States); United States v. Erwin, 602 F.2d 1183, 1185 (5th Cir.1979), *305 cert. denied, 444 U.S. 1071, 100 S.Ct. 1014, 62 L.Ed.2d 752 (1980) ("That venue may also be appropriate in another district will not divest venue properly established under § 3238."); see also United States v. Bin Laden, 146 F.Supp.2d 373, 381 n. 17 (S.D.N.Y.2001) (criticizing the Second Circuit's narrow reading of § 3238 in Gilboe as "myopic" and "directly in conflict with the clear language of the statute," and noting that the decision "has never been favorably cited or relied upon" by district courts in the Second Circuit).
Although the title of § 3238 includes only "offenses not committed in any district," it is a "well-settled rule of statutory interpretation that titles and section headings cannot limit the plain meaning of statutory text where that text is clear." M.A. ex rel. E.S. v. State-Operated Sch. Dist, 344 F.3d 335, 348 (3d Cir.2003). Here, the plain language of § 3238 supports the Fourth and Fifth Circuits' interpretation of the statute. Section 3238 applies, by its terms, to any offense "begun or committed" outside the United States. Pendleton would have us read the term "committed" to mean "wholly committed." But this cannot be correct, because crimes that are "wholly committed" outside the United States are, by definition, "begun" abroad. For the term "committed" to have independent meaning, it must refer to crimes that begin inside the United States but that are in their essence committed abroad. See Rosenberg v. XM Ventures, 274 F.3d 137, 141 (3d Cir.2001) ("[W]hen interpreting a statute, courts should endeavor to give meaning to every word which Congress used and therefore should avoid an interpretation which renders an element of the language superfluous.").
Here, although Pendleton's offense began when he initiated foreign travel by boarding a plane bound for Germany in the Eastern District of New York, he "committed" the offense when he engaged in an illicit sex act in Germany. Because Pendleton's criminal conduct was "essentially foreign," Levy Auto Parts, 787 F.2d at 950, the District Court did not err in applying § 3238 to hold that venue was proper in the district of arrest.
III
Having found that venue was proper in Delaware, we turn to Pendleton's substantive claim, namely, his assertion that the "noncommercial prong" of 18 U.S.C. § 2423(c) is facially unconstitutional. Pendleton's constitutional claim is subject to plenary review. United States v. Singletary, 268 F.3d 196,199 (3d Cir.2001). Because Pendleton raises a facial challenge, we will invalidate the statute only if we find "that no set of circumstances exists under which the Act would be valid, i.e., that the law is unconstitutional in all of its applications." Wash. State Grange v. Wash. State Republican Party, 552 U.S. 442, 449, 128 S.Ct. 1184, 170 L.Ed.2d 151 (2008) (citations and internal quotation marks omitted). The Supreme Court has noted that a facial challenge is the "most difficult challenge to mount successfully." United States v. Salerno, 481 U.S. 739, 745, 107 S.Ct. 2095, 95 L.Ed.2d 697 (1987).
A
The Constitution authorizes Congress "to regulate Commerce with foreign Nations, and among the several States, and with the Indian Tribes." U.S. CONST. art. I, § 8, cl. 3. In the early days of the Republic, the Supreme Court defined "commerce" broadly to include "every species of commercial intercourse" between two parties. Gibbons v. Ogden, 22 U.S. 1, 193-94, 9 Wheat. 1, 6 L.Ed. 23 (1824). More recently, the Supreme Court has recognized "three general categories of *306 regulation in which Congress is authorized to engage under its commerce power." Gonzales v. Raich, 545 U.S. 1, 5, 125 S.Ct. 2195, 162 L.Ed.2d 1 (2005). These include: (1) the use of the channels of interstate commerce; (2) the instrumentalities of interstate commerce, or persons or things in interstate commerce; and (3) activities that substantially affect interstate commerce. Lopez, 514 U.S. at 558-59, 115 S.Ct. 1624. In its pathmarking decision in Lopez, the Supreme Court held unconstitutional a statute criminalizing the possession of a firearm in a school zone because it did not fall within one of the three aforementioned categories. Five years later, in United States v. Morrison, the Court struck down portions of the Violence Against Women Act on similar grounds. 529 U.S. 598, 617, 120 S.Ct. 1740, 146 L.Ed.2d 658 (2000) ("The concern ... that Congress might use the Commerce Clause to completely obliterate the Constitution's distinction between national and local authority seems well founded.").
The three-category framework outlined in Lopez and Morrison applies, on its face, to statutes enacted pursuant to the Interstate Commerce Clause. The Supreme Court has yet to determine whether this framework applies to cases involving Congress's power to regulate pursuant to the Foreign Commerce Clause. Early opinions of the Court suggest that the three subparts of the Commerce Clause should be interpreted similarly. Notably, in Gibbons v. Ogden, Chief Justice Marshall suggested that "commerce, as the word is used in the constitution, is a unit ... [and] it must carry the same meaning throughout the sentence, and remain a unit, unless there be some plain intelligible cause which alters it." 22 U.S. at 194; see also Reno v. Bossier Parish Sch. Bd., 528 U.S. 320, 329, 120 S.Ct. 866, 145 L.Ed.2d 845 (2000) ("refus[ing] to adopt a construction that would attribute different meanings to the same phrase in the same sentence, depending upon which object it is modifying"); Saikrishna Prakash, Our Three Commerce Clauses and the Presumption of Intrasentence Uniformity, 55 ARK. L.REV. 1149, 1173 (2003) ("In practice, we have three different Commerce Clauses when text and history indicate that we ought to have but one.").
Notwithstanding Chief Justice Marshall's statement in Gibbons, the three subclauses of Article 1, § 8, cl. 3 have acquired markedly different meanings over time. Whereas the Interstate Commerce Clause has been constrained by state sovereignty concerns, see, e.g., Morrison, 529 U.S. at 615, 120 S.Ct. 1740, the Indian Commerce Clause has been interpreted so broadly as to grant Congress "plenary and exclusive" authority to regulate nearly every aspect of Indian life. United States v. Lara, 541 U.S. 193, 200, 124 S.Ct. 1628, 158 L.Ed.2d 420 (2004) (citing United States v. Wheeler, 435 U.S. 313, 319, 98 S.Ct. 1079, 55 L.Ed.2d 303 (1978)); see also Cotton Petroleum Corp. v. New Mexico, 490 U.S. 163, 109 S.Ct. 1698, 104 L.Ed.2d 209 (1989) ("While the Interstate Commerce Clause is concerned with maintaining free trade among the States even in the absence of implementing federal legislation, the central function of the Indian Commerce Clause is to provide Congress with plenary power to legislate in the field of Indian affairs.").
The Foreign Commerce Clause likewise has "followed its own distinct evolutionary path," Clark, 435 F.3d at 1113, having been used primarily as a tool to limit the ability of the several states to intervene in matters affecting international trade. See, e.g., Barclays Bank PLC v. Franchise Tax Bd., 512 U.S. 298, 114 S.Ct. 2268, 129 L.Ed.2d 244 (1994); Japan Line, Ltd. v. County of Los Angeles, 441 U.S. 434, 99 S.Ct. 1813, 60 L.Ed.2d 336 (1979); Antilles *307 Cement Corp. v. Acevedo Vila, 408 F.3d 41 (1st Cir.2005). For example, in Japan Line, the Supreme Court held that California could not impose an ad valorem tax on Japanese shipping containers that were stored temporarily in the state because the scheme could restrict the federal government's ability to "speak with one voice" in foreign affairs. 441 U.S. at 448, 99 S.Ct. 1813. Recognizing that the purpose of the Foreign Commerce Clause was to establish national uniformity over commerce with foreign nations,[4] the Court held that, "[a]lthough the Constitution, Art. I § 8, cl. 3, grants Congress power to regulate commerce `with foreign Nations' and `among the several States' in parallel phrases, there is evidence that the Founders intended the scope of the foreign commerce power to be the greater." Id.
Although jurisprudence on the so-called "dormant" Foreign Commerce Clause is well-developed, "[c]ases involving the reach of ... congressional authority to regulate our citizens' conduct abroad are few and far between." Clark, 435 F.3d at 1102. Courts have consistently held that the Foreign Commerce Clause requires a jurisdictional nexus "with" the United States, see, e.g., U.S. v. Weingarten, 632 F.3d 60 (2d Cir.2011) (stating that a person who travels from one foreign nation to another to commit an illicit sex act may not be punished pursuant to Congress's foreign commerce power); Cheng v. Boeing Co., 708 F.2d 1406, 1412 (9th Cir.1983) ("The Federal Aviation Act does not apply to the activities of a foreign carrier operating between two foreign points without contact in the United States."), but there is precious little case law on how to establish the requisite link to commercial interests in the United States. In the absence of Supreme Court precedent on the issue, the Court of Appeals for the Ninth Circuit determined that the Lopez framework which developed to "reconcile[] ... the conflicting claims of state and national power"has little analytical value in the Foreign Commerce Clause context. Clark, 435 F.3d at 1118. Rather than applying Lopez's three-part framework to determine whether a statute has a "constitutionally tenable nexus with foreign commerce," the Ninth Circuit proposed a "global, commonsense approach," which considers "whether the statute bears a rational relationship to Congress's authority under the Foreign Commerce Clause."[5]Id.
The Government urges us to adopt the Ninth Circuit's approach to the Foreign *308 Commerce Clause. Although we agree with Clark that the Interstate Commerce Clause developed to address "unique federalism concerns" that are absent in the foreign commerce context, we are hesitant to dispose of Lopez's "time-tested" framework without further guidance from the Supreme Court. See id. at 1119 (Ferguson, J., dissenting). The Supreme Court has not yet held that Congress has greater authority to regulate activity outside the United States than it does within its borders; in fact, the language used to describe its extraterritorial jurisdiction is quite similar to that used in Lopez. See, e.g., Hartford Fire Ins. Co. v. California, 509 U.S. 764, 795, 113 S.Ct. 2891, 125 L.Ed.2d 612 (1993) (recognizing that the Sherman Antitrust Act applies extraterritorially, and stating that a jurisdictional nexus exists when "foreign conduct was meant to produce and did in fact produce some substantial effect in the United States"). In any case, we need not reach the fundamental question of whether the Supreme Court will adopt the Ninth Circuit's broad articulation of the Foreign Commerce Clause because, as we shall explain, § 2423(c) is a valid congressional enactment under the narrower standard articulated in Lopez.
B
"[T]he authority of Congress to keep the channels of interstate commerce free from immoral and injurious uses has been frequently sustained, and is no longer open to question." Heart of Atlanta Motel, Inc. v. United States, 379 U.S. 241, 256, 85 S.Ct. 348, 13 L.Ed.2d 258 (1964) (quoting Caminetti v. United States, 242 U.S. 470, 491, 37 S.Ct. 192, 61 L.Ed. 442 (1917)); see also Morrison, 529 U.S. at 612, 120 S.Ct. 1740 (describing the Court's holding in Lopez, and noting that although 18 U.S.C. § 922(g) contains "no express jurisdictional element which might limit its reach to a discrete set of firearm possessions that additionally have an explicit connection with or effect on interstate commerce,... [s]uch a jurisdictional element [would have] establish[ed] that the enactment is in pursuance of Congress's regulation of interstate commerce") (citations and internal quotation marks omitted). Unlike Congressional authority to regulate activities affecting interstate commerce under the third category in Lopez, Congress's authority to regulate the channels of commerce is not confined to regulations with an economic purpose or impact. See, e.g., Caminetti, 242 U.S. at 491, 37 S.Ct. 192 (criminalizing the interstate transportation of a woman or girl for prostitution); Perez v. United States, 402 U.S. 146, 150, 91 S.Ct. 1357, 28 L.Ed.2d 686 (1971) (banning the interstate shipment of kidnapped persons); United States v. Cummings, 281 F.3d 1046, 1049-51 (9th Cir.2002) (holding *309 that the International Parental Kidnapping Crime Act regulates the channels of foreign commerce by prohibiting the removal or retention of a child outside the United States "with intent to obstruct the lawful exercise of parental rights").
In United States v. Tykarsky, we held that 18 U.S.C. § 2423(b), which criminalizes interstate travel with intent to engage in illicit sexual conduct with a minor, is a valid exercise of Congress's power to regulate the channels of commerce. 446 F.3d 458, 470 (3d Cir.2006); accord United States v. Hawkins, 513 F.3d 59, 61 (2d Cir.2008) (per curiam); United States v. Buttrick, 432 F.3d 373, 374 (1st Cir.2005); Bredimus, 352 F.3d at 205-207. Pendleton attempts to distinguish Tykarsky by noting that unlike § 2423(b), § 2423(c) includes no intent requirement. Citing United States v. Rodia for the proposition that "[t]he mere presence of a jurisdictional element ... does not in and of itself insulate a statute from judicial scrutiny under the Commerce Clause," Pendleton claims the District Court should have inquired whether "the jurisdictional component in this case limits the statute to items that have an explicit connection with, or effect upon, [foreign] commerce." 194 F.3d 465, 472 (3d Cir.1999) (finding that 18 U.S.C. § 2252(a)(4)(B)'s jurisdictional requirement that materials like film and cameras move in interstate commerce "is only tenuously related to the ultimate activity regulated: intrastate possession of child pornography"). No such connection exists here, Pendleton argues, because his conviction under § 2423(c) would stand even if he traveled through the channels of commerce for an entirely lawful purpose and only later formed the intent to engage in illicit sex with a minor. Contrary to Pendleton's assertions, however, a statute need not include an element of mens rea to trigger the first prong of Lopez.
In United States v. Shenandoah, we upheld portions of the Sex Offender Registration and Notification Act (SORNA), 18 U.S.C. § 2250(a)(1) and (2) and 42 U.S.C. § 14072(i)(1), making it illegal for a sex offender to fail to properly register after traveling in interstate commerce. 595 F.3d 151, 161 (3d Cir.2010); accord United States v. Ambert, 561 F.3d 1202,1211 (11th Cir.2009); United States v. May, 535 F.3d 912, 921 (8th Cir.2008). Like the provision at issue here (§ 2423(c)), SORNA does not require that a sex offender intend, at the time of travel, to later violate federal registration requirements. Nor does SORNA require the Government to demonstrate a temporal connection between the time of travel and a sex offender's failure to register. United States v. Husted, 2007 U.S. Dist. LEXIS 56662, at *9 (W.D. Okla. June 29, 2007) (citing H.R.Rep. No. 109-218 (Sept. 7, 2005)) ("[T]he legislative history of the statute shows Congress chose not to incorporate a temporal requirement but, instead, intended to encompass all sex offenders."). For instance, a "tier I sex offender" who moves from one state to another and, years later, violates SORNA's provisions by failing to update his information on an annual basis can be convicted under the statute. 18 U.S.C. § 2250(a)(1); see Carr v. United States, ___ U.S. ___, ___, 130 S.Ct. 2229, 2235, 176 L.Ed.2d 1152 (2010) (observing in dicta that "[a] sequential reading [of the statute] ... helps to assure a nexus between a defendant's interstate travel and his failure to register as a sex offender").[6]
*310 Nevertheless, SORNA was specifically enacted to address "one of the biggest problems in our current sex offender registry," 152 CONG. REC. S8012-14 (daily ed. July 20, 2006), 2005 WL 2034118, namely, sex offenders who go "missing" from the national registry by moving from one state to another, H.R. REP. No. 109-218(1) (2005), 2005 WL 2210642. Finding that "over 10,000 sex offenders, or nearly one-fifth in the Nation ... are `missing,'" id., Congress chose to regulate the behavior of all sex offenders who cross state lines. Because Congress invoked its authority to regulate "the use of interstate commerce to facilitate forms of immorality," Shenandoah, 595 F.3d at 161 (citing Brooks v. United States, 267 U.S. 432, 436, 45 S.Ct. 345, 69 L.Ed. 699 (1925)), it was not obliged to include an express intent or temporal element in its definition of the offense. Accord United States v. Dixon, 551 F.3d 578 (7th Cir.2008) (summarily rejecting defendant's Commerce Clause argument, noting that the defendant "must in the heat of argument have forgotten the Mann Act"); United States v. Hann, 574 F.Supp.2d 827, 833 (M.D.Tenn.2008) ("[Lopez] encompasses § 2250(a) because the statute regulates sex offenders who travel in interstate commerce even though the threat Congress was attempting to addressfailure to register as a sex offenderis an intrastate activity.") (citations omitted).
The same rationale applies to Pendleton's case. Just as SORNA's "failure to report" provision was intended to prevent convicted sex offenders from "us[ing] the channels of interstate commerce in evading a State's reach," Carr, 130 S.Ct. at 2238, Congress enacted § 2423(c) to close "significant loopholes in the law that persons who travel to foreign countries seeking sex with children are currently using to their advantage in order to avoid prosecution," H.R. REP. NO. 107-525, at 3 (summarizing the purpose of adopting language similar to § 2423(c) in the Sex Tourism Prohibition Improvement Act). Specifically, Congress found that American citizens were using the channels of foreign commerce to travel to countries where "dire poverty and ... lax enforcement" would allow them to "escape prosecution" for their crimes of child sexual abuse. 148 CONG. REC. 3884; id. at 3885 ("Sadly, we know that many Americans go abroad to prey on young girls in other countries because laws protecting women are very weak, non-existent, or not enforced."); H.R. REP. NO. 107-525, at 4 ("According to the National Center for Missing and Exploited Children, child-sex tourism is a major component of the worldwide sexual exploitation of children and is increasing. There are more than 100 web sites devoted to promoting teenage commercial sex in Asia alone."); see also 109 H.R. 2012, 109th Cong. § 2 (2005) ("The United Nations estimates that sex trafficking, including sex tourism, generates approximately $5,000,000,000 a year in revenues. There are a number of United States-based companies that overtly and explicitly facilitate sex tours, often involving the sexual exploitation of children. According to some estimates, up to 1/4 of international sex tourists are American.").
*311 Members of Congress also expressed concern that § 2423(b) would not adequately deter child-sex tourists because prosecutors were having an "extremely difficult" time "proving intent in such cases." 148 CONG. REC. 3884 (stating that intent is particularly "difficult to prove without direct arrangement booked through obvious child sex-tour networks."). This, in turn, "creat[ed] a loophole in the law for men who go abroad to have sex with minors, which in the United States is considered statutory rape." Id. Section 2423(c) was enacted to close the enforcement gap and to "send a message to those who go to foreign countries to exploit children that no one can abuse a child with impunity." Id. Thus, as it did with SORNA, Congress enacted § 2423(c) to regulate persons who use the channels of commerce to circumvent local laws that criminalize child abuse and molestation. And just as Congress may cast a wide net to stop sex offenders from traveling in interstate commerce to evade state registration requirements, so too may it attempt to prevent sex tourists from using the channels of foreign commerce to abuse children. Id.; Clark, 435 F.3d at 1116 ("Congress legitimately exercises its authority to regulate the channels of commerce where a crime committed on foreign soil is necessarily tied to travel in foreign commerce, even where the actual use of the channels has ceased."); N. Am. Co. v. SEC, 327 U.S. 686, 705, 66 S.Ct. 785, 90 L.Ed. 945 (1946) ("Congress may impose relevant conditions and requirements on those who use the channels of interstate commerce in order that those channels will not become the means of promoting or spreading evil, whether of a physical, moral or economic nature.").
In sum, because the jurisdictional element in § 2423(c) has an "express connection" to the channels of foreign commerce, Morrison, 529 U.S. at 612, 120 S.Ct. 1740, we hold that it is a valid exercise of Congress's power under the Foreign Commerce Clause.[7]
IV
For the foregoing reasons, we will affirm the District Court's judgment of conviction and sentence.
NOTES
[*] The Honorable C. Darnell Jones, II, District Judge for the United States District Court for the Eastern District of Pennsylvania, sitting by designation.
[1] The legislative history of the PROTECT Act does not include a statement regarding the source of Congress's authority to enact § 2423(c). See generally H.R. REP. NO. 108-66, at 51, reprinted in 2003 U.S.C.C.A.N. 683, 686 (Apr. 9, 2003). However, the language of § 2423(c) was adopted verbatim from an earlier billthe Sex Tourism Prohibition Improvement Act of 2002which relied on the Foreign Commerce Clause as the basis for its constitutional authority. See H.R. REP. No. 525, at 5, 2002 WL 1376220, at *5 (June 24, 2002).
[2] Pendleton asks us to reexamine Martinez-Hidalgo's holding that no due process violation occurs when Congress criminalizes conduct abroad that is "condemned universally by law-abiding nations." Id. at 1056. We will not do so because a panel of this Court has no authority to overrule a precedential opinion of the Court. See Mariana v. Fisher, 338 F.3d 189, 201 (3d Cir.2003).
[3] Pendleton also was sentenced to a concurrent term of ten years in prison for failing to register as a sex offender, in violation of the Adam Walsh Child Protection and Safety Act of 2006. See United States v. Pendleton, 636 F.3d 78 (3d Cir.2011). Pendleton was first convicted of sexually abusing children in Michigan in 1981 and was sentenced to 24 months probation. In 1993, a New Jersey jury found Pendleton guilty of various sex crimes against a 12-year-old boy and he was sentenced to seven years in prison. About three years after his release from prison, Pendleton traveled to Latvia and was convicted there for sex crimes against two children, ages 9 and 13. A little over a year after Pendleton was released from a Latvian prison he committed the offense at issue in this case.
[4] Indeed, this was a principal reason for assembling the Constitutional Convention of 1787. Gibbons, 22 U.S. at 225 (Johnson, J., concurring) (quoting the preamble of James Madison's draft resolution at the Virginia Ratifying Convention, stating that "the relative situation of the United States has been found, on trial, to require uniformity in their commercial regulations, as the only effectual policy for obtaining, in the ports of foreign nations, a stipulation of privileges reciprocal to those enjoyed by the subjects of such nations in the ports of the United States"); see also Michelin Tire Corp. v. Wages, 423 U.S. 276, 283, 96 S.Ct. 535, 46 L.Ed.2d 495 (1976) ("[A] compelling reason for the calling of the Constitutional Convention of 1787 ... was the fact that the Articles [of Confederation] essentially left the individual States free to burden commerce both among themselves and with foreign countries very much as they pleased."); United States v. The William, 28 F.Cas. 614, 620 (D.Mass.1808) ("It is well understood, that the depressed state of American commerce, and complete experience of the inefficiency of state regulations, to apply a remedy, were among the great, procuring causes of the federal constitution.").
[5] The Ninth Circuit in Clark claims to borrow this "rational basis" test from the Supreme Court's holding in Gonzales v. Raich. See 545 U.S. at 5, 125 S.Ct. 2195 (holding that Congress had a "rational basis" for believing that intrastate possession and manufacture of marijuana had a substantial effect on interstate commerce). As the dissent in Clark rightly notes, however, the "rational basis" analysis in Raich went to Congress's "substantial effects" determination. The Supreme Court has articulated several factors to be weighed in determining whether an activity "substantially affects" interstate commerce: (1) whether the regulated activity is economic in nature; (2) whether the statute contains an "express jurisdictional element" linking its scope in some way to interstate commerce; (3) whether Congress made express findings regarding the effects of the regulated activity on interstate commerce; and (4) attenuation of the link between the regulated activity and interstate commerce. See Morrison, 529 U.S. at 611-12, 120 S.Ct. 1740.
The "rational basis" standard articulated by the Ninth Circuit in Clark does not consider any of these factors. Rather, its open-ended inquiry seems to borrow more heavily from the Supreme Court's pre-Lopez jurisprudence, which held that a court's "investigation . . . end[s]" once it determines that "legislators. . . have a rational basis for finding a chosen regulatory scheme necessary to the protection of commerce." Katzenbach v. McClung, 379 U.S. 294, 303-04, 85 S.Ct. 377, 13 L.Ed.2d 290 (1964).
[6] In this respect, SORNA's "failure to register" provision is similar to the federal felon-in-possession law, 18 U.S.C. § 922(g), enacted pursuant to Congress's authority under the Commerce Clause. Section 922(g) makes it unlawful for a felon to "possess in or affecting commerce, any firearm or ammunition . . . which has been shipped or transported in interstate or foreign commerce." In United States v. Singletary, we held that the transport of a weapon through the channels of interstate commercehowever remote in the distant pastprovides a sufficient jurisdictional nexus to satisfy Lopez's first prong. 268 F.3d 196, 200 (3d Cir.2001) (citing Scarborough v. United States, 431 U.S. 563, 564, 97 S.Ct. 1963, 52 L.Ed.2d 582 (1977)). Similarly, under § 2423(c), a person's travel through foreign commerce continues to provide a link to his illicit sexual conduct long after his travel is complete.
[7] Having found that the statute is constitutional under the first prong of Lopez, we need not address Pendleton's contention that § 2423(f)(1) does not survive Morrison's stringent "substantial effects" test. See United States v. MacEwan, 445 F.3d 237, 245 (3d Cir.2006) ("[W]e need not proceed to an analysis of Lopez's third category when Congress clearly has the power to regulate such an activity under the first two."); United States v. May, 535 F.3d 912, 922 (8th Cir.2008) (upholding SORNA under the first and second prongs of Lopez, and thus finding that it "need not address [the defendant's] contention SORNA was not accompanied by findings that the activity in question exerted a `substantial influence on interstate commerce' similar to those in support of the Controlled Substances Act regulation considered and upheld in Gonzales v. Raich, 545 U.S. 1, 125 S.Ct. 2195, 162 L.Ed.2d 1 (2005)").
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Great service, food was okay
Average Rating
60
We had great service, very personable and attentive. The pancake was delicious but the eggs and sausage lacked any real taste. The sausage tasted more like a small hamburger patty. Disappointed but willing to try it again.
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The store is actually "Another Broken Egg Cafe". I got the smoked salmon benedict, which was wonderful. Five stars. The service gets maybe three stars. I was in a little after seven am on a weekday, it was not busy yet it took twenty minutes for my food to come out. The server never checked on me again until it...
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Another Broken Egg makes another eggs-travagant splash in Mountain Brook. Ron E. Green shed his nine-to-five corporate skin to manifest his longstanding dream of opening a restaurant. Disappointed with the lack of creativity in brunch menus, he opened his flagship cafe in Louisiana that guarantees a delicious and healthy start to the day. The quirky name was born during a conversation with a... |
sp {arithmetic*propose*finish-problem
(state <s> ^name arithmetic
^count <count>
^arithmetic-problem.current-column <c>)
(<c> ^result
^next-column nil)
-->
(<s> ^operator <op> + =)
(<op> ^name finish-problem
^count <count>)
}
sp {arithmetic*apply*finish-problem*decrement-count
(state <s> ^name arithmetic
^operator <o>
^arithmetic-problem <ap>
^count <c>)
(<o> ^name finish-problem
^count <c>)
-->
(<s> ^arithmetic-problem <ap> -
^count <c> -
(- <c> 1))
}
########## Print out problem and answer #############
#sp {arithmetic*apply*finish-problem*1column*result
# (state <s> ^name arithmetic
# ^output yes
# ^operator <o>
# ^arithmetic-problem <ap>)
# (<o> ^name finish-problem)
# (<ap> ^one-column <oc>
# ^operation-symbol <as>)
# (<oc> ^digit1 <n1>
# ^digit2 <n2>
# ^result <n3>
# ^next-column nil)
#-->
# (write (crlf) (crlf) | | <n1> )
# (write (crlf) <as> <n2> )
# (write (crlf) |--|)
# (write (crlf) | | <n3> )
#}
#sp {arithmetic*apply*finish-problem*2column*result
# (state <s> ^name arithmetic
# ^output yes
# ^operator <o>
# ^arithmetic-problem <ap>)
# (<o> ^name finish-problem)
# (<ap> ^one-column <oc>
# ^operation-symbol <as>)
# (<oc> ^digit1 <n1>
# ^digit2 <n2>
# ^result <a1>
# ^next-column <tc>)
# (<tc> ^digit1 <n3>
# ^digit2 <n4>
# ^result <a2>
# ^next-column nil)
#-->
# (write (crlf) (crlf) | | <n3> <n1> )
# (write (crlf) <as> <n4> <n2> )
# (write (crlf) |---|)
# (write (crlf) | | <a2> <a1>)
#}
#
#sp {arithmetic*apply*finish-problem*3column*result
# (state <s> ^name arithmetic
# ^output yes
# ^operator <o>
# ^arithmetic-problem <ap>)
# (<o> ^name finish-problem)
# (<ap> ^one-column <oc>
# ^operation-symbol <as>)
# (<oc> ^digit1 <n1>
# ^digit2 <n2>
# ^result <a1>
# ^next-column <tc>)
# (<tc> ^digit1 <n3>
# ^digit2 <n4>
# ^result <a2>
# ^next-column <vc>)
# (<vc> ^digit1 <n5>
# ^digit2 <n6>
# ^result <a3>
# ^next-column nil)
#-->
# (write (crlf) | |)
# (write (crlf) | | <n5> <n3> <n1> )
# (write (crlf) <as> <n6> <n4> <n2> )
# (write (crlf) |----|)
# (write (crlf) | | <a3> <a2> <a1>)
#}
#
#sp {arithmetic*apply*finish-problem*4column*result
# (state <s> ^name arithmetic
# ^output yes
# ^operator <o>
# ^arithmetic-problem <ap>)
# (<o> ^name finish-problem)
# (<ap> ^one-column <oc>
# ^operation-symbol <as>)
# (<oc> ^digit1 <n1>
# ^digit2 <n2>
# ^result <a1>
# ^next-column <tc>)
# (<tc> ^digit1 <n3>
# ^digit2 <n4>
# ^result <a2>
# ^next-column <vc>)
# (<vc> ^digit1 <n5>
# ^digit2 <n6>
# ^result <a3>
# ^next-column <uc>)
# (<uc> ^digit1 <n7>
# ^digit2 <n8>
# ^result <a4>
# ^next-column nil)
#-->
# (write (crlf) | |)
# (write (crlf) | | <n7> <n5> <n3> <n1> )
# (write (crlf) <as> <n8> <n6> <n4> <n2> )
# (write (crlf) |----|)
# (write (crlf) | | <a4> <a3> <a2> <a1>)
#}
|
---
abstract: 'In a recent paper (M. Barash, A. Okhotin, “Defining contexts in context-free grammars”, LATA 2012), the authors introduced an extension of the context-free grammars equipped with an operator for referring to the left context of the substring being defined. This paper proposes a more general model, in which context specifications may be two-sided, that is, both the left and the right contexts can be specified by the corresponding operators. The paper gives the definitions and establishes the basic theory of such grammars, leading to a normal form and a parsing algorithm working in time $\mathcal{O}(n^4)$, where $n$ is the length of the input string.'
author:
- Mikhail Barash
- Alexander Okhotin
title: 'Grammars with two-sided contexts[^1]'
---
Introduction
============
The context-free grammars are a logic for representing the syntax of languages, in which the properties of longer strings are defined by concatenating shorter strings with known properties. Disjunction of syntactic conditions is represented in this logic as multiple alternative rules defining a single symbol. One can further augment this logic with conjunction and negation operations, leading to *conjunctive grammars* [@Conjunctive] and *Boolean grammars* [@BooleanGrammars]. These grammars are context-free in the general sense of the word, as they define the properties of each substring independently of the context, in which it occurs. Furthermore, most of the practically important features of ordinary context-free grammars, such as efficient parsing algorithms, are preserved in their conjunctive and Boolean variants [@BooleanGrammars; @BooleanMatrix]. These grammar models have been a subject of recent theoretical studies [@AizikowitzKaminski_LR0; @EsikKuich; @Jez; @Kountouriotis_et_al; @Ziervogel]. Not long ago, the authors [@grammars_with_contexts_lata; @grammars_with_contexts] proposed an extension of the context-free grammars with special operators for expressing the form of the *left context*, in which the substring occurs. For example, a rule $A \to BC {\mathop{\&}}{{\lhd} D}$ asserts that every string representable as $BC$ in a left context of the form described by $D$ therefore has the property $A$. These grammars were motivated by Chomsky’s [@Chomsky p. 142] well-known idea of a phrase-structure rule applicable only in some particular contexts. Chomsky’s own attempt to implement this idea by string rewriting resulted in a model equivalent to linear-space Turing machines, in which the “nonterminal symbols”, meant to represent syntactic categories, could be freely manipulated as tape symbols. In spite of the name “context-sensitive grammars”, the resulting model was unsuitable for describing the syntax of languages, and thus failed to represent the idea of a rule applicable in a context.
Taking a new start with this idea, the authors [@grammars_with_contexts] defined *grammars with one-sided contexts*, following the logical outlook on grammars, featured in the work of Kowalski [@Kowalski Ch. 3] and of Pereira and Warren [@PereiraWarren], and later systematically developed by Rounds [@Rounds]. A grammar defines the truth value of statements of the form “a certain string has a certain property”, and these statements are deduced from each other according to the rules of the grammar. The resulting definition maintains the underlying logic of the context-free grammars, and many crucial properties of grammars are preserved: grammars with one-sided contexts have parse trees, can be transformed to a normal form and have a cubic-time parsing algorithm [@grammars_with_contexts]. However, the model allowed specifying contexts only on one side, and thus it implemented, so to say, only one half of Chomsky’s idea.
This paper continues the development of formal grammars with context specifications by allowing contexts in both directions. The proposed *grammars with two-sided contexts* may contain such rules as $A \to BC {\mathop{\&}}{{\lhd} D} {\mathop{\&}}{{\rhd} E}$, which define any substring of the form $BC$ preceded by a substring of the form $D$ and followed by a substring of the form $E$. If the grammar contains additional rules $B \to b$, $C \to c$, $D \to d$ and $E \to e$, then the above rule for $A$ asserts that a substring $bc$ of a string $w=dbce$ has the property $A$. However, this rule will not produce the same substring $bc$ occurring in another string $w'=dbcd$, because its right context does not satisfy the conjunct ${{\rhd} E}$. Furthermore, the grammars allow expressing the so-called *extended right context* (${{\trianglerighteqslant} \alpha}$), which defines the form of the current substring concatenated with its right context, as well as the symmetrically defined *extended left context* (${{\trianglelefteqslant} \alpha}$).
In Section \[section\_deduction\], this intuitive definition is formalized by deduction of propositions of the form ${A\big({u \langle w \rangle v}\big)}$, which states that the substring $w$ occurring in the context between $u$ and $v$ has the property $A$, where $A$ is a syntactic category defined by the grammar (“nonterminal symbol” in Chomsky’s terminology). Then, each rule of the grammar becomes a schema for deduction rules, and a string $w$ is generated by the grammar, if there is a proof of the proposition ${S\big({{\varepsilon}\langle w \rangle {\varepsilon}}\big)}$. A standard proof tree of such a deduction constitutes a parse tree of the string $w$.
The next Section \[section\_examples\] presents basic examples of grammars with two-sided contexts. These examples model several types of cross-references, such as declaration of identifiers before or after their use.
The paper then proceeds with developing a normal form for these grammars, which generalizes the Chomsky normal form for ordinary context-free grammars. In the normal form, every rule is a conjunction of one or more *base conjuncts* describing the form of the current substring (either as a concatenation of the form $BC$ or as a single symbol $a$), with any context specifications (${{\lhd} D}$, ${{\trianglelefteqslant} E}$, ${{\trianglerighteqslant} F}$, ${{\rhd} H}$). The transformation to the normal form, presented in Section \[section\_normal\_form\], proceeds in three steps. First, all rules generating the empty string in any contexts are eliminated. Second, all rules with an explicit empty context specification (${{\lhd} {\varepsilon}}$, ${{\rhd} {\varepsilon}}$) are also eliminated. The final step is elimination of any rules of the form $A \to B {\mathop{\&}}\ldots$, where the dependency of $A$ on $B$ potentially causes cycles in the definition.
Once the normal form is established, a simple parsing algorithm for grammars with two-sided contexts with the running time $\mathcal{O}(n^4)$ is presented in Section \[section\_parsing\_algorithm\]. While this paper has been under preparation, Rabkin [@Rabkin] has developed a more efficient and more sophisticated parsing algorithm for grammars with two-sided contexts, with the running time $\mathcal{O}(n^3)$.
Definition {#section_deduction}
==========
Ordinary context-free grammars allow using the concatenation operation to express the form of a string, and disjunction to define alternative forms. In conjunctive grammars, the conjunction operation may be used to assert that a substring being defined must conform to several conditions at the same time. The grammars studied in this paper further allow operators for expressing the form of the left context (${{\lhd} }$, ${{\trianglelefteqslant} }$) and the right context (${{\rhd} }$, ${{\trianglerighteqslant} }$) of a substring being defined.
A grammar with two-sided contexts is a quadruple $G=(\Sigma, N, R, S)$, where
- $\Sigma$ is the alphabet of the language being defined;
- $N$ is a finite set of auxiliary symbols (“nonterminal symbols” in Chomsky’s terminology), which denote the properties of strings defined in the grammar;
- $R$ is a finite set of grammar rules, each of the form $$\label{eq:rule}
\begin{split}
A \to \alpha_1 {\mathop{\&}}\ldots {\mathop{\&}}\alpha_k {\mathop{\&}}&{{\lhd} \beta_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\lhd} \beta_m} {\mathop{\&}}{{\trianglelefteqslant} \gamma_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglelefteqslant} \gamma_n} {\mathop{\&}}\\ {\mathop{\&}}&{{\trianglerighteqslant} \kappa_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglerighteqslant} \kappa_{m'}} {\mathop{\&}}{{\rhd} \delta_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\rhd} \delta_{n'}},
\end{split}$$ with $A \in N$, $k \geqslant 1$, $m,n,m',n' \geqslant 0$ and $\alpha_i, \beta_i, \gamma_i, \kappa_i, \delta_i \in (\Sigma \cup N)^*$;
- $S \in N$ is a symbol representing well-formed sentences of the language.
If all rules in a grammar have only left contexts (that is, if $m'=n'=0$), then this is a grammar with one-sided contexts [@grammars_with_contexts]. If no context operators are ever used ($m=n=m'=n'=0$), this is a conjunctive grammar, and if the conjunction is also never used ($k=1$), this is an ordinary context-free grammar.
For each rule (\[eq:rule\]), each term $\alpha_i$, ${{\lhd} \beta_i}$, ${{\trianglelefteqslant} \gamma_i}$, ${{\trianglerighteqslant} \kappa_i}$ and ${{\rhd} \delta_i}$ is called a *conjunct*. Denote by ${u \langle w \rangle v}$ a substring $w \in \Sigma^*$, which is preceded by $u \in \Sigma^*$ and followed by $v \in \Sigma^*$, as illustrated in Figure \[f:substring\_with\_contexts\]. Intuitively, such a substring is generated by a rule (\[eq:rule\]), if
- each *base conjunct* $\alpha_i=X_1 \ldots X_\ell$ gives a representation of $w$ as a concatenation of shorter substrings described by $X_1, \ldots, X_\ell$, as in context-free grammars;
- each conjunct ${{\lhd} \beta_i}$ similarly describes the form of the *left context* $u$;
- each conjunct ${{\trianglelefteqslant} \gamma_i}$ describes the form of the *extended left context* $uw$;
- each conjunct ${{\trianglerighteqslant} \kappa_i}$ describes the *extended right context* $wv$;
- each conjunct ${{\rhd} \delta_i}$ describes the *right context* $v$.
![A substring $w$ of a string $uwv$: four types of contexts.[]{data-label="f:substring_with_contexts"}](substring_with_contexts)
The semantics of grammars with two-sided contexts are defined by a deduction system of elementary propositions (items) of the form “a string $w \in \Sigma^*$ written in a left context $u \in \Sigma^*$ and in a right context $v \in \Sigma^*$ has the property $X \in \Sigma \cup N$”, denoted by ${X\big({u \langle w \rangle v}\big)}$. The deduction begins with axioms: any symbol $a \in \Sigma$ written in any context has the property $a$, denoted by ${a\big({u \langle a \rangle v}\big)}$ for all $u,v \in \Sigma^*$. Each rule in $R$ is then regarded as a schema for deduction rules. For example, a rule $A \to BC$ allows making deductions of the form $$\begin{aligned}
{B\big({u \langle w \rangle w'v}\big)},
{C\big({uw \langle w' \rangle v}\big)}
\vdash_G
{A\big({u \langle ww' \rangle v}\big)}
&& (\text{for all $u,w,w',v \in \Sigma^*$}),\end{aligned}$$ which is essentially a concatenation of $w$ and $w'$ that respects the contexts. If the rule is of the form $A \to BC {\mathop{\&}}{{\lhd} D}$, this deduction requires an extra premise: $$\begin{aligned}
{B\big({u \langle w \rangle w'v}\big)},
{C\big({uw \langle w' \rangle v}\big)},
{D\big({{\varepsilon}\langle u \rangle ww'v}\big)}
&\vdash_G
{A\big({u \langle ww' \rangle v}\big)}.
\intertext{And if the rule is $A \to BC {\mathop{\&}}{{\trianglerighteqslant} F}$,
the deduction proceeds as follows:}
{B\big({u \langle w \rangle w'v}\big)},
{C\big({uw \langle w' \rangle v}\big)},
{F\big({u \langle ww'v \rangle {\varepsilon}}\big)}
&\vdash_G
{A\big({u \langle ww' \rangle v}\big)}.
$$ The general form of deduction schemata induced by a rule in $R$ is defined below.
\[def:deduction\_system\] Let $G=(\Sigma, N, R, S)$ be a grammar with two-sided contexts. Define the following deduction system of items of the form ${X\big({u \langle w \rangle v}\big)}$, with $X \in \Sigma \cup N$ and $u,w,v \in \Sigma^*$. There is a single axiom scheme $\vdash_G {a\big({u \langle a \rangle v}\big)}$, for all $a \in \Sigma$ and $u,v \in \Sigma^*$. Each rule (\[eq:rule\]) in $R$ defines the following scheme for deduction rules: $$I \vdash_G {A\big({u \langle w \rangle v}\big)},$$ for all $u, w, v \in \Sigma^*$ and for every set of items $I$ satisfying the below properties:
- For every base conjunct $\alpha_i=X_1 \ldots X_\ell$, with $\ell \geqslant 0$ and $X_j \in \Sigma \cup N$, there should exist a partition $w=w_1 \ldots w_\ell$ with ${X_j\big({u w_1 \ldots w_{j-1} \langle w_j \rangle w_{j+1} \ldots w_\ell v}\big)} \in I$ for all $j \in \{1, \ldots, \ell\}$.
- For every conjunct ${{\lhd} \beta_i}={{\lhd} X_1 \ldots X_\ell}$ there should be such a partition $u=u_1 \ldots u_\ell$, that ${X_j\big({u_1 \ldots u_{j-1} \langle u_j \rangle u_{j+1} \ldots u_\ell w v}\big)} \in I$ for all $j \in \{1, \ldots, \ell\}$.
- Every conjunct ${{\trianglelefteqslant} \gamma_i}={{\trianglelefteqslant} X_1 \ldots X_\ell}$ should have a corresponding partition $uw=x_1 \ldots x_\ell$ with ${X_j\big({x_1 \ldots x_{j-1} \langle x_j \rangle x_{j+1} \ldots x_\ell v}\big)} \in I$ for all $j \in \{1, \ldots, \ell\}$.
- For every conjunct ${{\rhd} \delta_i}$ and ${{\trianglerighteqslant} \kappa_i}$, the condition is defined symmetrically.
Then the language generated by a symbol $A \in N$ is defined as $$L_G(A) = {\{ \, {u \langle w \rangle v} \mid u,w,v \in \Sigma^*, \: \vdash_G {A\big({u \langle w \rangle v}\big)} \, \}}.$$ The language generated by the grammar $G$ is the set of all strings with empty left and right contexts generated by $S$: $L(G)={\{ \, w \mid w \in \Sigma^{*}, \: \vdash_G {S\big({{\varepsilon}\langle w \rangle {\varepsilon}}\big)} \, \}}$.
The following trivial example of a grammar is given to illustrate the definitions.
\[example:abca\_grammar\] Consider the grammar with two-sided contexts that defines the singleton language $\{abca\}$: $$\begin{aligned}
S &\to& a S \ | \ S a \ | \ B C \\
A &\to& a \\
B &\to& b {\mathop{\&}}{{\lhd} A} \\
C &\to& c {\mathop{\&}}{{\rhd} A}\end{aligned}$$
The deduction given below proves that the string $abca$ has the property $S$. $$\begin{aligned}
& \vdash {a\big({{\varepsilon}\langle a \rangle bca}\big)}
&& (axiom) \\
& \vdash {b\big({a \langle b \rangle ca}\big)}
&& (axiom) \\
& \vdash {c\big({ab \langle c \rangle a}\big)}
&& (axiom) \\
& \vdash {a\big({abc \langle a \rangle {\varepsilon}}\big)}
&& (axiom) \\
{a\big({{\varepsilon}\langle a \rangle bca}\big)}
& \vdash {A\big({{\varepsilon}\langle a \rangle bca}\big)}
&& (A \to a) \\
{b\big({a \langle b \rangle ca}\big)}, {A\big({{\varepsilon}\langle a \rangle bca}\big)}
& \vdash {B\big({a \langle b \rangle ca}\big)}
&& (B \to b {\mathop{\&}}{{\lhd} A}) \\
{a\big({abc \langle a \rangle {\varepsilon}}\big)}
& \vdash {A\big({abc \langle a \rangle {\varepsilon}}\big)}
&& (A \to a) \\
{c\big({ab \langle c \rangle a}\big)}, {A\big({abc \langle a \rangle {\varepsilon}}\big)}
& \vdash {C\big({ab \langle c \rangle a}\big)}
&& (C \to c {\mathop{\&}}{{\rhd} A}) \\
{B\big({a \langle b \rangle ca}\big)}, {C\big({ab \langle c \rangle a}\big)}
& \vdash {S\big({a \langle bc \rangle a}\big)}
&& (S \to BC) \\
{a\big({{\varepsilon}\langle a \rangle bca}\big)}, {S\big({a \langle bc \rangle a}\big)}
& \vdash {S\big({{\varepsilon}\langle abc \rangle a}\big)}
&& (S \to a S) \\
{S\big({{\varepsilon}\langle abc \rangle a}\big)}, {a\big({abc \langle a \rangle {\varepsilon}}\big)}
& \vdash {S\big({{\varepsilon}\langle abca \rangle {\varepsilon}}\big)}
&& (S \to S a)\end{aligned}$$
![A parse tree of the string $abca$ according to the grammar in Example \[example:abca\_grammar\].[]{data-label="f:abca_tree"}](simple_parse_tree)
Another possible definition of grammars with contexts is by directly expressing them in first-order logic over positions in a string [@Rounds]. Nonterminal symbols become *binary predicates*, with the arguments referring to positions in the string. Each predicate $A(x, y)$ is defined by a formula $\varphi_A(x, y)$ that states the condition of a substring delimilited by positions $x$ and $y$ having the property $A$. There are built-in unary predicates $a(x)$, for each $a \in \Sigma$, which assert that the symbol in position $x$ in the string is $a$, and binary predicates $x<y$ and $x=y$ for comparing positions. Arguments to predicates are given as *terms*, which are either variables ($t=x$) or constants referring to the first and the last positions ($t={\ensuremath{ \underline{\mathrm{begin}} }}$, $t={\ensuremath{ \underline{\mathrm{end}} }}$), and which may be incremented ($t+1$) or decremented ($t-1$). Each formula is constructed from predicates using conjunction, disjunction and first-order existential quantification.
The grammar from Example \[example:abca\_grammar\] is expressed by the following formulae defining predicates $S(x,y)$, $B(x,y)$, $A(x,y)$ and $C(x,y)$. $$\begin{aligned}
S(x,y) &=&
\left( a(x) \land S(x+1,y) \right)
\,\lor\,
\left( S(x,y-1) \land a(y) \right)
\,\lor\,
\left( \exists z \left( x < z < y \land B(x,z) \land C(z,y) \right) \right) \\
A(x,y) &=&
a(x) \land x+1 = y \\
B(x,y) &=&
b(x) \land x+1 = y \land A({\ensuremath{ \underline{\mathrm{begin}} }}, x) \\
C(x,y) &=&
c(x) \land x+1 = y \land A(y, {\ensuremath{ \underline{\mathrm{end}} }})\end{aligned}$$ The membership of a string $w$ is expressed by the statement $S({\ensuremath{ \underline{\mathrm{begin}} }}, {\ensuremath{ \underline{\mathrm{end}} }})$, which may be true of false.
Examples {#section_examples}
========
This section presents several examples of grammars with two-sided contexts generating important syntactic constructs. All examples use ordinary context-free elements, such as a grammar for ${\{ \, a^nb^n \mid n \geqslant 0 \, \}}$, and combine these elements using the new context operators. This leads to natural specifications of languages in the style of classical formal grammars.
Consider the problem of checking declaration of identifiers before their use: this construct can be found in all kinds of languages, and it can be expressed by a conjunctive grammar [@BooleanSurvey Ex. 3]. The variant of this problem, in which the identifiers may be declared *before or after* their use, is also fairly common: consider, for instance, the declaration of classes in C++, where an earlier defined method can refer to a class member defined later. However, no conjunctive grammar expressing this construct is known.
A grammar with one-sided contexts for declarations before or after use has recently been constructed by the authors [@grammars_with_contexts]. That grammar used context specifications, along with iterated conjunction, to express what would be more naturally expressed in terms of two-sided contexts. In the model proposed in this paper, the same language can be defined in a much more natural way.
\[declarations\_before\_or\_after\_use\_example\] Consider the language $$\label{declarations_before_or_after_use_language}
{\{ \, u_1 \ldots u_n \mid \text{for every $u_i$,
\textbf{either}
$u_i \in a^* c$,
\textbf{or}
$u_i = b^k c$ and
there exists $j \in \{1, \ldots, n\}$
with $u_j=a^k c$} \, \}}.$$ Substrings of the form $a^k c$ represent declarations, while every substring of the form $b^k c$ is a reference to a declaration of the form $a^k c$.
This language is generated by the following grammar. $$\begin{array}{rcl@{\quad \qquad}rcl}
S &\to& AS \ | \ CS \ | \ DS \ | \ {\varepsilon}& C &\to& B {\mathop{\&}}{{\trianglelefteqslant} EFc} \\
A &\to& a A \ | \ c
& D &\to& B {\mathop{\&}}{{\trianglerighteqslant} HcE} \\
B &\to& b B \ | \ c
& F &\to& a F b \ | \ c E \\
E &\to& AE \ | \ BE \ | \ {\varepsilon}& H &\to& b H a \ | \ c E
\end{array}$$
The idea of the grammar is that $S$ should generate a substring ${u_1 \ldots u_\ell \langle u_{\ell+1} \ldots u_n \rangle {\varepsilon}}$, with $0 \leqslant \ell \leqslant n$ and $u_i \in a^* c \cup b^* c$, if and only if every reference in $u_{\ell+1} \ldots u_n$ has a corresponding declaration somewhere in the whole string $u_1 \ldots u_n$. The rules for $S$ define all substrings satisfying this condition inductively on their length, until the entire string ${{\varepsilon}\langle u_1 \ldots u_n \rangle {\varepsilon}}$ is defined. The rule $S \to {\varepsilon}$ defines the base case: the string ${u_1 \ldots u_n \langle {\varepsilon}\rangle {\varepsilon}}$ has the desired property. The rule $S \to CS$ appends a reference of the form $b^*c$, restricted by an extended left context ${{\trianglelefteqslant} EFc}$, which ensures that this reference has a matching *earlier* declaration; here $E$ represents the prefix of the string up to that earlier declaration, while $F$ matches the symbols $a$ in the declaration to the symbols $b$ in the reference. The possibility of a *later* declaration is checked by another rule $S \to DS$, which adds a reference of the form $b^*c$ with an extended right context ${{\trianglerighteqslant} HcE}$, where $H$ is used to match the $b$s forming this reference to the $a$s in the later declaration.
The next example abstracts another syntactic mechanism—*function prototypes*—found in the C programming language and, under the name of *forward declarations*, in the programming language Pascal.
\[example:prototypes\] Consider the language
$$\begin{aligned}
\label{eq:prototype_declarations__prototype}
\big\{
u_1 \ldots u_n
\: \big| \:
\text{for every $u_i$}, \;
& \text{\textbf{either} $u_i = a^k c$ and there exists $j > i$,
such that $u_j = d^k c$,} \\
\label{eq:prototype_declarations__reference}
& \text{\textbf{or} $u_i = b^k c$ and there exists $j < i$,
for which $u_j = a^k c$}\big\}.
$$
A substring of the form $a^k c$ represents a function prototype and a substring $d^k c$ represents its body. Calls to functions are expressed as substrings $b^k c$. Condition (\[eq:prototype\_declarations\_\_prototype\]) means that every prototype must be followed by its body, and restriction (\[eq:prototype\_declarations\_\_reference\]) requires that references are only allowed to declared prototypes. This language can be generated by the following grammar with two-sided contexts. $$\begin{array}{rcl@{\quad\qquad}rcl@{\quad\qquad}rcl}
S &\to& U S \ | \ V S \ | \ D S \ | \ {\varepsilon}& D &\to& d D \ | \ c
& E &\to& A E \ | \ B E \ | \ D E \ | \ {\varepsilon}\\
A &\to& a A \ | \ c
& U &\to& A {\mathop{\&}}{{\trianglerighteqslant} H c E}
& H &\to& a H d \ | \ c E\\
B &\to& b B \ | \ c
& V &\to& B {\mathop{\&}}{{\trianglelefteqslant} E F c}
& F &\to& a F b \ | \ c E\\
\end{array}$$
The rules $S \to US$ and $U \to A {\mathop{\&}}{{\trianglerighteqslant} HcE}$ append a prototype $a^k c$ and the extended right context of the form $a^k c \ldots d^k c \ldots$ ensures that this prototype has a matching body somewhere *later* within the string. The rules $S \to VS$ and $V \to B {\mathop{\&}}{{\trianglelefteqslant} EFc}$ append a reference $b^k c$, and the context specification $\ldots a^k c \ldots b^k c$ checks that it has a matching prototype *ealier* in the string. Function bodies $d^k c$ are added by the rule $S \to DS$. Using these rules, $S$ generates substrings of the form ${u_1 \ldots u_\ell \langle u_{\ell+1} \ldots u_n \rangle {\varepsilon}}$, with $0 \leqslant \ell \leqslant n$ and $u_i \in a^*c \cup b^*c \cup d^*c$, such that every prototype $u_i = a^k c$ in $u_{\ell+1} \ldots u_n$ has a corresponding body $d^k c$ in $u_{i+1} \ldots u_n$ and every reference $u_i = b^k c$ in $u_{\ell+1} \ldots u_n$ has a corresponding prototype $a^k c$ in $u_1 \ldots u_{i-1}$.
The next example gives a grammar with contexts that defines reachability on graphs. Sudborough [@Sudborough] defined a linear context-free grammar for a special encoding of the graph reachability problem on acyclic graphs, in which every arc goes from a lower-numbered vertex to a higher-numbered vertex. The grammar presented below allows any graphs and uses a direct encoding. This example illustrates the ability of grammars with contexts to define various kinds of cross-references.
\[graph\_example\] Consider encodings of directed graphs as strings of the form $b^s \:
a^{i_1} b^{j_1} \: a^{i_2} b^{j_2} \: \ldots \: a^{i_n} b^{j_n} \:
a^t$, with $s, t \geqslant 1, \: n \geqslant 0, \: i_k,j_k \geqslant 1$, where each block $a^i b^j$ denotes an arc from vertex number $i$ to vertex number $j$, while the prefix $b^s$ and the suffix $a^t$ mark $s$ as the source vertex and $t$ as the target. Then the following grammar defines all graphs with a path from $s$ to $t$. $$\begin{array}{rcl@{\quad \qquad}rcl}
S &\to& F D C A \ | \ F
& &&\\
A &\to& a A \ | \ c
& D &\to& B {\mathop{\&}}{{\trianglelefteqslant} B C E} \ | \ B {\mathop{\&}}{{\trianglerighteqslant} FDCA} \ | \ B {\mathop{\&}}{{\trianglerighteqslant} F} \\
B &\to& b B \ | \ c
& E &\to& a E b \ | \ D C A \\
C &\to& A B C \ | \ {\varepsilon}& F &\to& b F a \ | \ b C a
\end{array}$$
The grammar is centered around the nonterminal $D$, which generates all such substrings ${b^s a^{i_1} b^{j_1} \ldots a^{i_k} \langle b^{j_k} \rangle a^{i_{k+1}}b^{j_{k+1}} \ldots a^{i_n} b^{j_n} a^t}$ that there is a path from $j_k$ to $t$ in the graph. If this path is empty, then $j_k = t$. Otherwise, the first arc in the path can be listed either to the left or to the right of $b^k$. These three cases are handled by the three rules for $D$. Each of these rules generates $b^{j_k}$ by the base conjunct $B$, and then uses an extended left or right context operator to match $b^{j_k}$ to the tail of the next arc or to $a^t$.
The rule $D \to B {\mathop{\&}}{{\trianglelefteqslant} B C E}$ considers the case when the next arc in the path is located to the left of $b^{j_k}$. Let this arc be $a^{i_\ell}b^{j_\ell}$, for some $\ell<k$. Then the extended left context $BCE$ covers the substring $b^s a^{i_1}b^{j_1} \ldots a^{i_\ell}b^{j_\ell} \ldots a^{i_k}b^{j_k}$. The concatenation $BC$ skips the prefix $b^s a^{i_1}b^{j_1} \ldots a^{i_{\ell-1}}b^{j_{\ell-1}}$, and then the nonterminal $E$ matches $a^{i_\ell}$ to $b^{j_k}$, verifying that $i_\ell=j_k$. After this, the rule $E \to DCA$ ensures that the substring $b^{j_\ell}$ is generated by $D$, that is, that there is a path from $j_\ell$ to $t$. The concatenation $CA$ skips the inner substring $a^{i_{\ell+1}}b^{j_{\ell+1}} \ldots a^{i_k}$.
The second rule $D \to B {\mathop{\&}}{{\trianglerighteqslant} FDCA}$ searches for the next arc to the right of $b^{j_k}$. Let this be an $\ell$-th arc in the list, with $\ell>k$. The extended right context $FDCA$ should generate the suffix $b^{j_k} \ldots a^{i_\ell}b^{j_\ell} \ldots a^{i_n}b^{j_n} a^t$. The symbol $F$ covers the substring $b^{j_k} \ldots a^{i_\ell}$, matching $b^{j_k}$ to $a^{i_\ell}$. Then, $D$ generates the substring $b^{j_\ell}$, checking that there is a path from $j_\ell$ to $t$. The concatenation $CA$ skips the rest of the suffix.
Finally, if the path is of length zero, that is, $j_k=t$, then the rule $D \to B {\mathop{\&}}{{\trianglerighteqslant} F}$ uses $F$ to match $b^{j_k}$ to the suffix $a^t$ in the end of the string.
Once the symbol $D$ checks the path from any vertex to the vertex $t$, for the initial symbol $S$, it is sufficient to match $b^s$ in the beginning of the string to any arc $a^{j_k} b^{j_k}$, with $j_k=s$. This is done by the rule $S \to FDCA$, which operates in the same way as the second rule for $D$. The case of $s$ and $t$ being the same node is handled by the rule $S \to F$.
All the above examples use identifiers given in unary, which are matched by rules of the same kind as the rules defining the language ${\{ \, a^n b^n \mid n \geqslant 0 \, \}}$. These examples can be extended to use identifiers over an arbitrary alphabet $\Sigma$, owing to the fact that there is a conjunctive grammar generating the language ${\{ \, w\#w \mid w \in \Sigma^* \, \}}$, for some separator $\# \notin \Sigma$ [@Conjunctive; @BooleanSurvey].
Normal form {#section_normal_form}
===========
An ordinary context-free grammar can be transformed to the Chomsky normal form, with the rules restricted to $A \to BC$ and $A \to a$, with $B,C \in N$ and $a \in \Sigma$. This form has the following generalization to grammars with contexts.
A grammar with two-sided contexts $G=(\Sigma,N,R,S)$ is said to be in the binary normal form, if each rule in $R$ is of one of the forms $$\begin{aligned}
A &\to B_1 C_1 {\mathop{\&}}\ldots {\mathop{\&}}B_k C_k {\mathop{\&}}{{\lhd} D_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\lhd} D_m} {\mathop{\&}}{{\trianglelefteqslant} E_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglelefteqslant} E_n} {\mathop{\&}}{{\trianglerighteqslant} F_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglerighteqslant} F_{n'}} {\mathop{\&}}{{\rhd} H_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\rhd} H_{m'}}, \\
A &\to a {\mathop{\&}}{{\lhd} D_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\lhd} D_m} {\mathop{\&}}{{\trianglelefteqslant} E_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglelefteqslant} E_n} {\mathop{\&}}{{\trianglerighteqslant} F_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglerighteqslant} F_{n'}} {\mathop{\&}}{{\rhd} H_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\rhd} H_{m'}},\end{aligned}$$ where $k \geqslant 1$, $m,n,n',m' \geqslant 0$, $B_i, C_i, D_i, E_i, F_i, H_i \in N$, $a \in \Sigma$.
The transformation to the normal form consists of three stages: first, removing all *empty conjuncts* ${\varepsilon}$; secondly, eliminating *empty contexts* (${{\lhd} {\varepsilon}}$, ${{\rhd} {\varepsilon}}$); finally, getting rid of *unit conjuncts* of the form $B$, with $B \in N$.
The first step is the removal of all rules of the form $A \to {\varepsilon}{\mathop{\&}}\ldots$, so that no symbols generate ${\varepsilon}$, while all non-empty strings are generated as before. As generation of longer strings may depend on the generation of ${\varepsilon}$, already for ordinary context-free grammars, such a transformation requires adding extra rules that simulate the same dependence without actually generating any empty strings.
\[example:nullable\_context\_free\] Consider the following context-free grammar, which defines the language $\{abc, ab, ac, a, bcd, bd, cd, d\}$. $$\begin{aligned}
S &\to& a A \ | \ A d \\
A &\to& BC\\
B &\to& {\varepsilon}\ | \ b \\
C &\to& {\varepsilon}\ | \ c\end{aligned}$$ Since $B$ generates the empty string, the rule $A \to BC$ can be used to generate just $C$; therefore, once the rule $B \to {\varepsilon}$ is removed, one should add a new rule $A \to C$, in which $B$ is omitted. Similarly one can remove the rule $C \to {\varepsilon}$ and add a “compensatory” rule $A \to B$. Since both $B$ and $C$ generate ${\varepsilon}$, so does $A$ by the rule $A \to BC$. Hence, extra rules $S \to a$ and $S \to d$, where $A$ is omitted, have to be added.
An algorithm for carrying out such a transformation first calculates the set of nonterminals that generate the empty string, known as $\textsc{Nullable}(G) \subseteq N$, and then uses it to reconstruct the rules of the grammar. This set is calculated as a least upper bound of an ascending sequence of sets $\textsc{Nullable}_i(G)$. The set $\textsc{Nullable}_1(G) = {\{ \, A \in N \mid A \to {\varepsilon}\in R \, \}}$ contains all nonterminals which directly define the empty string. Every next set $\textsc{Nullable}_{i+1}(G) = {\{ \, A \in N \mid A \to \alpha \in R, \: \alpha \in \textsc{Nullable}_i^*(G) \, \}}$ contains nonterminals that generate ${\varepsilon}$ by the rules referring to other nullable nonterminals. This knowledge is given by the Kleene star of $\textsc{Nullable}_i(G)$.
For the grammar in Example \[example:nullable\_context\_free\], the calculation of the set $\textsc{Nullable}(G)$ proceeds as follows: $$\begin{aligned}
\textsc{Nullable}_0(G) &=& {\varnothing},\\
\textsc{Nullable}_1(G) &=& \big\{ B, C \big\},\\
\textsc{Nullable}_2(G) &=& \big\{ B, C, A \big\},\end{aligned}$$ and $\textsc{Nullable}(G) = \textsc{Nullable}_2(G)$.
The same idea works for conjunctive grammars as well [@Conjunctive]. For grammars with contexts [@grammars_with_contexts], the generation of the empty string additionally depends on the left contexts, in which the string occurs. This requires an elaborated version of the set $\textsc{Nullable}(G)$, formed of nonterminals along with the information about the left contexts in which they may define ${\varepsilon}$.
In order to eliminate null conjuncts in case of grammars with two-sided contexts, one has to consider yet another variant of the set $\textsc{Nullable}(G)$, which respects both left and right contexts.
\[example:nullable\_grammar\_with\_two\_sided\_contexts\] Consider the following grammar with two-sided contexts, obtained by adding context restrictions to the grammar in Example \[example:nullable\_context\_free\]; this grammar defines the language $L = \{abc, ac, bcd, bd\}$. $$\begin{aligned}
S &\to& a A \ | \ A d\\
A &\to& B C\\
B &\to& {\varepsilon}{\mathop{\&}}{{\lhd} D} \ | \ b\\
C &\to& {\varepsilon}{\mathop{\&}}{{\rhd} E} \ | \ c\\
D &\to& a\\
E &\to& d\end{aligned}$$ In this grammar, the nonterminal $B$ generates the empty string only in a left context of the form defined by $D$, while $C$ defines the empty string only in a right context of the form $E$. In those contexts where *both* $B$ and $C$ generate ${\varepsilon}$, so can $A$, by the rule $A \to BC$.
The information about the left and right contexts, in which a nonterminal generates the empty string, is to be stored in the set $\textsc{Nullable}(G)$, which is defined as a subset of $2^N \times N \times 2^N$. An element $(U, A, V)$ of this set represents an intuitive idea that $A$ defines ${\varepsilon}$ in a left context of the form described by each nonterminal in $U$, and in a right context of the form given by nonterminals in $V$.
For the grammar in Example \[example:nullable\_grammar\_with\_two\_sided\_contexts\], such a set $\textsc{Nullable}(G)$ is constructed as follows. $$\begin{aligned}
\textsc{Nullable}_0(G) &=& {\varnothing}\\
\textsc{Nullable}_1(G) &=& \big\{ (\{D\}, B, {\varnothing}), ({\varnothing}, C, \{E\}) \big\} \\
\textsc{Nullable}_2(G) &=& \big\{ (\{D\}, B, {\varnothing}), ({\varnothing}, C, \{E\}), (\{D\}, A, \{E\}) \big\}\end{aligned}$$ Then $\textsc{Nullable}(G) = \textsc{Nullable}_2(G)$. The elements $(\{D\}, B, {\varnothing})$ and $({\varnothing}, C, \{E\})$ are obtained directly from the rules of the grammar, and the element $(\{D\}, A, \{E\})$ represents the “concatenation” $BC$ in the rule for $A$. Note the similarity of this construction to the one for the ordinary grammar in Example \[example:nullable\_context\_free\]: the construction given here is different only in recording information about the contexts.
The above “concatenation” of triples $(\{D\}, B, {\varnothing})$ and $({\varnothing}, C, \{E\})$ should be defined to accumulate both left and right contexts. This can be regarded as a generalization of the Kleene star to sets of triples, denoted by $\textsc{Nullable}^\star(G)$. Formally, $\textsc{Nullable}^\star(G)$ is the set of all triples $(U_1 \cup \ldots \cup U_\ell, \: A_1 \ldots A_\ell, \: V_1 \cup \ldots \cup V_\ell)$ with $\ell \geqslant 0$ and $(U_i, A_i, V_i) \in \textsc{Nullable}(G)$. The symbols $A_i$ are concatenated, while their left and right contexts are accumulated. In the special case when $\ell = 0$, the concatenation of zero symbols is the empty string, and thus ${\varnothing}^\star = \big\{ ({\varnothing}, {\varepsilon}, {\varnothing}) \big\}$.
Before giving a formal definition of the set $\textsc{Nullable}(G)$, assume, for the sake of simplicity, that context operators are only applied to single nonterminal symbols, that is, every rule is of the form $$\label{eq:rule_alpha_D_E_F_H}
\begin{split}
A \to \alpha_1 {\mathop{\&}}\ldots {\mathop{\&}}\alpha_k {\mathop{\&}}{{\lhd} D_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\lhd} D_m} {\mathop{\&}}{{\trianglelefteqslant} E_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglelefteqslant} E_n} {\mathop{\&}}{{\trianglerighteqslant} F_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglerighteqslant} F_{m'}} {\mathop{\&}}{{\rhd} H_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\rhd} H_{n'}},
\end{split}$$ with $A \in N$, $k \geqslant 1$, $m,n,m',n' \geqslant 0$, $\alpha_i \in (\Sigma \cup N)^*$ and $D_i, E_i, F_i, H_i \in N$. As will be shown in Lemma \[lemma:almost\_normal\_form\], there is no loss of generality in this assumption.
\[def:Nullable\] Let $G = (\Sigma, N, R, S)$ be a grammar with two-sided contexts with all rules of the form (\[eq:rule\_alpha\_D\_E\_F\_H\]).
Construct the sequence of sets $\textsc{Nullable}_i(G) \subseteq 2^N \times N \times 2^N$, for $i \geqslant 0$, as follows.
Let $\textsc{Nullable}_0(G) = {\varnothing}$. Every next set $\textsc{Nullable}_{i+1}(G)$ contains the following triples: for every rule (\[eq:rule\_alpha\_D\_E\_F\_H\]) and for every $k$ triples $(U_1, \alpha_1, V_1)$, …, $(U_k, \alpha_k, V_k)$ in $\textsc{Nullable}_i^\star(G)$, the triple $\big(
\{D_1,\ldots,D_m, E_1, \ldots, E_n\}
\cup \{U_1, \ldots, U_k\},
\; A, \;
\{F_1,\ldots,F_{m'}, H_1, \ldots, H_{n'}\}
\cup \{V_1, \ldots, V_k\}
\big)$ is in $\textsc{Nullable}_{i+1}(G)$. Finally, let $\textsc{Nullable}(G) = \bigcup_{i \geqslant 0} \textsc{Nullable}_{i}(G)$.
The next lemma explains how exactly the set $\textsc{Nullable}(G)$ represents the generation of the empty string by different nonterminals in different contexts.
\[lemma:nullable\] Let $G = (\Sigma,N,R,S)$ be a grammar with contexts, let $A \in N$ and $u, v \in \Sigma^*$. Then, ${u \langle {\varepsilon}\rangle v} \in L_G(A)$ if and only if there is a triple $(\{J_1, \ldots, J_s\}, A, \{K_1, \ldots, K_t\})$ in $\textsc{Nullable}(G)$, such that ${{\varepsilon}\langle u \rangle v} \in L_G(J_i)$ for all $i$ and ${u \langle v \rangle {\varepsilon}} \in L_G(K_j)$ for all $j$.
The plan is to reconstruct the grammar, so that for every triple $(\{J_1, \ldots, J_s\}, A, \{K_1, \ldots, K_t\})$ in $\textsc{Nullable}(G)$, and for every occurrence of $A$ in the right-hand side of any rule, the new grammar contains a companion rule, in which $A$ is omitted and context operators for $J_i$ and $K_i$ are introduced.
The following case requires special handling in the new grammar. Assume that $A$ generates ${\varepsilon}$ in the empty left context (that is, $u={\varepsilon}$ in Lemma \[lemma:nullable\]). This is reflected by a triple $(\{J_1, \ldots, J_s\}, A, \{K_1, \ldots, K_t\})$ in $\textsc{Nullable}(G)$, in which all symbols $J_i$ also generate ${\varepsilon}$ in the left context ${\varepsilon}$. The latter generation may in turn involve some further right context operators. In the new grammar, the left context will be explicitly set to be empty (${{\lhd} {\varepsilon}}$), whereas all those right contexts should be assembled together with the set $\{K_1, \ldots, K_t\}$, and used in the new rules, where $A$ is omitted. This calculation of right contexts is done in the following special variant of the set $\textsc{Nullable}$.
\[def:left\_Nullable\] Let $G = (\Sigma, N, R, S)$ be a grammar. Define sets ${\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}_i(G) \subseteq N \times 2^N$, with $i \geqslant 0$: $$\begin{aligned}
{\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}_0(G) &= {\{ \, (A, V) \mid ({\varnothing}, A, V) \in \textsc{Nullable}(G) \, \}},
\\
{\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}_{i+1}(G) &=
\big\{
(A, V \cup V_1 \cup \ldots \cup V_s)
\: \big| \:
(\{J_1, \ldots, J_s\}, A, V) \in \textsc{Nullable}(G), \\
&\hspace*{4cm}
\exists \: V_1, \ldots, V_s \subseteq N:
(J_i, V_i) \in {\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}_i(G) \big\}.\end{aligned}$$ Let ${\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G) = \bigcup_{i \geqslant 0} {\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}_i(G)$.
\[lemma:left\_nullable\] Let $G = (\Sigma, N, R, S)$ be a grammar, let $A \in N$ and $v \in \Sigma^*$. Then ${{\varepsilon}\langle {\varepsilon}\rangle v} \in L_G(A)$ if and only if there is a pair $(A, \{K_1, \ldots, K_t\})$ in ${\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$, such that ${{\varepsilon}\langle v \rangle {\varepsilon}} \in L_G(K_i)$ for all $i$.
There is a symmetrically defined set ${\ensuremath{ {{\rhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G) \subseteq 2^N \times N$, which characterizes the generation of ${\varepsilon}$ in an empty right context. With the generation of the empty string represented in these three sets, a grammar with two-sided contexts is transformed to the normal form as follows. First, it is convenient to simplify the rules of the grammar, so that every concatenation is of the form $BC$, with $B, C \in N$, and the context operators are only applied to individual nonterminals. For this, base conjuncts $\alpha$ with ${\ensuremath{ |\alpha| }} > 2$ and context operators ${{\lhd} \alpha}$, ${{\trianglelefteqslant} \alpha}$, ${{\trianglerighteqslant} \alpha}$ and ${{\rhd} \alpha}$ with ${\ensuremath{ |\alpha| }} > 1$ are shortened by introducing new nonterminals.
\[lemma:almost\_normal\_form\] For every grammar $G_0 = (\Sigma, N_0, R_0, S_0)$, there exists and can be effectively constructed another grammar $G = (\Sigma, N, R, S)$ generating the same language, with all rules of the form:
\[eq:rules\_almost\_normal\_form\] $$\begin{aligned}
\label{eq:almost_a} A &\to a\\
\label{eq:almost_BC} A &\to BC\\
\label{eq:almost_B_D_E_F_H}
A &\to B_1 {\mathop{\&}}\ldots {\mathop{\&}}B_k {\mathop{\&}}{{\lhd} D_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\lhd} D_m} {\mathop{\&}}{{\trianglelefteqslant} E_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglelefteqslant} E_n} {\mathop{\&}}{{\trianglerighteqslant} F_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglerighteqslant} F_{m'}} {\mathop{\&}}{{\rhd} H_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\rhd} H_{n'}}\\
\label{eq:almost_e} A &\to {\varepsilon},\end{aligned}$$
with $a \in \Sigma$ and $A, B, C, D_i, E_i, F_i, H_i \in N$.
\[Construction:epsilon\_elimination\] Let $G = (\Sigma, N, R, S)$ be a grammar with two-sided contexts, with all rules of the form (\[eq:rules\_almost\_normal\_form\]). Consider the sets $\textsc{Nullable}(G)$, ${\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$ and ${\ensuremath{ {{\rhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$, and construct another grammar with two-sided contexts $G' = (\Sigma, N, R', S)$, with the following rules.
1. All rules of the form (\[eq:almost\_a\]) in $R$ are added to $R'$.
2. Every rule of the form (\[eq:almost\_BC\]) in $R$ is added to $R'$, along with the following extra rules, where a nullable nonterminal is omitted and the fact that it generates ${\varepsilon}$ is expressed by context operators. $$\begin{aligned}
A &\to B {\mathop{\&}}{{\trianglelefteqslant} J_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglelefteqslant} J_s} {\mathop{\&}}{{\rhd} K_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\rhd} K_t},
\quad \text{for $(\{J_1, \ldots, J_s\}, C, \{K_1, \ldots, K_t\}) \in \textsc{Nullable}(G)$} \\
A &\to B {\mathop{\&}}{{\trianglelefteqslant} J_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglelefteqslant} J_s} {\mathop{\&}}{{\rhd} {\varepsilon}},
\quad \text{for $(\{J_1, \ldots, J_s\}, C) \in {\ensuremath{ {{\rhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$ with $s \geqslant 1$} \\
A &\to C {\mathop{\&}}{{\lhd} J_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\lhd} J_s} {\mathop{\&}}{{\trianglerighteqslant} K_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglerighteqslant} K_t},
\quad \text{for $(\{J_1, \ldots, J_s\}, B, \{K_1, \ldots, K_t\}) \in \textsc{Nullable}(G)$} \\
A &\to C {\mathop{\&}}{{\trianglerighteqslant} K_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglerighteqslant} K_t} {\mathop{\&}}{{\lhd} {\varepsilon}},
\quad \text{for $(B, \{K_1, \ldots, K_t\}) \in {\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$
with $t \geqslant 1$}\end{aligned}$$ In the first case, $C$ defines ${\varepsilon}$ in left contexts $J_i$ and right contexts $K_i$, and this restriction is implemented by context operators in the new rule. Since the left context of $C$ includes $B$, extended context operators (${{\trianglelefteqslant} J_i}$) are used on the left, whereas the right context operators are proper (${{\rhd} K_i}$).
The second case considers the possibility of a nullable nonterminal $C$, which defines ${\varepsilon}$ in an empty right context. This condition is simulated by the conjunct ${{\rhd} {\varepsilon}}$ and extended left contexts ${{\trianglelefteqslant} J_i}$.
The two last rules handle symmetrical cases, when the nonterminal $B$ defines the empty string.
3. Every rule of the form (\[eq:almost\_B\_D\_E\_F\_H\]) is preserved in $R'$. In the original grammar, this rule (\[eq:almost\_B\_D\_E\_F\_H\]) may generate strings in empty contexts, as long as symbols in the context operators (${{\lhd} D_i}$, ${{\rhd} H_i}$) are nullable. For any collection of pairs $(D_1, V_1)$, …, $(D_m, V_m) \in {\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$, with $m \geqslant 1$, add the rule $$A \to
B_1 {\mathop{\&}}\ldots {\mathop{\&}}B_k {\mathop{\&}}E_1 {\mathop{\&}}\ldots {\mathop{\&}}E_n {\mathop{\&}}{{\trianglerighteqslant} K_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglerighteqslant} K_t} {\mathop{\&}}{{\trianglerighteqslant} F_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglerighteqslant} F_{m'}} {\mathop{\&}}{{\rhd} H_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\rhd} H_{n'}}
{\mathop{\&}}{{\lhd} {\varepsilon}},$$ where $\{K_1, \ldots, K_t\} = \bigcup_{i=1}^{m} V_i$. Nonterminals $D_1$, …, $D_m$ define ${\varepsilon}$ in the right contexts given in the set ${\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$. This is represented by conjuncts ${{\lhd} {\varepsilon}}$ and ${{\trianglerighteqslant} K_i}$. Extended left contexts ${{\trianglelefteqslant} E_i}$ are replaced with base conjuncts $E_i$, because in the empty left context they have the same effect.
Symmetrically, if $(U_1, H_1)$, …, $(U_{n'}, H_{n'}) \in {\ensuremath{ {{\rhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$, with $n' \geqslant 1$, then there is a rule $$A \to
B_1 {\mathop{\&}}\ldots {\mathop{\&}}B_k {\mathop{\&}}F_1 {\mathop{\&}}\ldots {\mathop{\&}}F_{m'} {\mathop{\&}}{{\lhd} D_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\lhd} D_m} {\mathop{\&}}{{\trianglelefteqslant} E_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglelefteqslant} E_n} {\mathop{\&}}{{\trianglelefteqslant} K_1} {\mathop{\&}}\ldots {\mathop{\&}}{{\trianglelefteqslant} K_t} {\mathop{\&}}{{\rhd} {\varepsilon}},$$ where $\{K_1, \ldots, K_t\} = \bigcup_{i=1}^{n'} U_i$.
Finally, if with $m$, $n' \geqslant 1$ and $(D_1, V_1)$, …, $(D_m, V_m) \in {\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$, $(U_1, H_1)$, …, $(U_{n'}, H_{n'}) \in {\ensuremath{ {{\rhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$, then the set $R'$ contains a rule $$A \to
B_1 {\mathop{\&}}\ldots {\mathop{\&}}B_k {\mathop{\&}}E_1 {\mathop{\&}}\ldots {\mathop{\&}}E_n {\mathop{\&}}F_1 {\mathop{\&}}\ldots {\mathop{\&}}F_{m'} {\mathop{\&}}K_1 {\mathop{\&}}\ldots {\mathop{\&}}K_t {\mathop{\&}}{{\lhd} {\varepsilon}} {\mathop{\&}}{{\rhd} {\varepsilon}},$$ where $\{K_1, \ldots, K_t\} = \bigcup_{i=1}^{m} V_i \cup \bigcup_{j=1}^{n'} U_j$. In this case, both left and right contexts of a string are empty. All the symbols $D_i$ and $H_i$ define ${\varepsilon}$ in the contexts specified in ${\ensuremath{ {{\lhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$ and ${\ensuremath{ {{\rhd} {\varepsilon}}\text{-}\textsc{Nullable} }}(G)$. These contexts apply to the entire string and are explicitly stated as $K_1 {\mathop{\&}}\ldots {\mathop{\&}}K_t$ in the new rule. The null contexts ${{\lhd} {\varepsilon}}$, ${{\rhd} {\varepsilon}}$ limit the applicability of this rule to the whole string. Again, as in the two previous cases, the base conjuncts are used instead of extended context operators.
\[epsilon\_conjunct\_elimination\_lemma\] Let $G = (\Sigma, N, R, S)$ be a grammar with two-sided contexts. Then the grammar $G' = (\Sigma, N', R', S)$ obtained by Construction \[Construction:epsilon\_elimination\] generates the language $L(G') = L(G) \setminus \{{\varepsilon}\}$.
The above construction eliminates the empty string in all base conjuncts, but the resulting grammar may still contain null context specifications (${{\lhd} {\varepsilon}}$ and ${{\rhd} {\varepsilon}}$), which state that the current substring is a prefix or a suffix of the whole string. These operators are eliminated by the following simple transformation. First, define a new nonterminal symbol $U$ that generates all non-empty strings in the empty left context. This is done by the following three rules: $$\begin{aligned}
U &\to U a
&& \text{(for all $a \in \Sigma$)} \\
U &\to a {\mathop{\&}}{{\trianglelefteqslant} X}
&& \text{(for all $a \in \Sigma$)} \\
X &\to a
&& \text{(for all $a \in \Sigma$)}\end{aligned}$$ Another symbol $V$ generates all non-empty strings in the empty right context; it is defined by symmetric rules. Then it remains to replace left and right null context operators (${{\lhd} {\varepsilon}}$, ${{\rhd} {\varepsilon}}$) with $U$ and $V$, respectively.
The third stage of the transformation to the normal form is removing the *unit conjuncts* in rules of the form $A \to B {\mathop{\&}}\ldots$ Already for conjunctive grammars [@Conjunctive], the only known transformation involves substituting all rules for $B$ into all rules for $A$; in the worst case, this results in an exponential blowup. The same construction applies verbatim to grammars with contexts.
This three-stage transformation proves the following theorem.
\[thm\_normal\_form\] For each grammar with two-sided contexts $G=(\Sigma,N,R,S)$ there exists and can be effectively constructed a grammar with two-sided contexts $G'=(\Sigma,N',R',S)$ in the binary normal form, such that $L(G)=L(G') \setminus \{{\varepsilon}\}$.
Parsing algorithm {#section_parsing_algorithm}
=================
Let $G=(\Sigma,N,R,S)$ be a grammar with two-sided contexts in the binary normal form, and let $w=a_1 \ldots a_n \in \Sigma^+$, with $n \geqslant 1$ and $a_i \in \Sigma$, be an input string to be parsed. For every substring of $w$ delimited by two positions $i,j$, with $0 \leqslant i < j \leqslant n$, consider the set of nonterminal symbols generating this substring. $$T_{i,j} = {\big\{ \: A \; \big| \; A \in N, \;
{a_1 \ldots a_i \langle a_{i+1} \ldots a_j \rangle a_{j+1} \ldots a_n} \in L_G(A) \: \big\}}$$ In particular, the whole string $w$ is in $L(G)$ if and only if $S \in T_{0,n}$.
In ordinary context-free grammars, a substring $a_{i+1} \ldots a_j$ is generated by $A$ if there is a rule $A \to BC$ and a partition of the substring into $a_{i+1} \ldots a_k$ generated by $B$ and $a_{k+1} \ldots a_j$ generated by $C$, as illustrated in Figure \[f:dependencies\_with\_contexts\](left). Accordingly, each set $T_{i,j}$ depends only on the sets $T_{i', j'}$ with $j'-i'<j-i$, and hence all these sets may be constructed inductively, beginning with shorter substrings and eventually reaching the set $T_{0,n}$: this is the Cocke–Kasami–Younger parsing algorithm. For conjunctive grammars, all dependencies are the same, and generally the same parsing algorithm applies [@Conjunctive]. In grammars with only left contexts, each set $T_{i,j}$ additionally depends on the sets $T_{0,i}$ and $T_{0,j}$ via the conjuncts of the form ${{\lhd} D}$ and ${{\trianglelefteqslant} E}$, respectively, which still allows constructing these sets progressively for $j=1, \ldots, n$ [@grammars_with_contexts].
The more complicated structure of logical dependencies in grammars with two-sided contexts is shown in Figure \[f:dependencies\_with\_contexts\](right). The following example demonstrates how these dependencies may form circles.
![How the membership of $A$ in $T_{i,j}$ depends on other data, for rules (a) $A \to BC$ and (b) $A \to BC {\mathop{\&}}{{\lhd} D} {\mathop{\&}}{{\trianglelefteqslant} E} {\mathop{\&}}{{\trianglerighteqslant} F} {\mathop{\&}}{{\rhd} H}$.[]{data-label="f:dependencies_with_contexts"}](dependencies_with_contexts_two_sided)
Consider the grammar with the rules $$\begin{aligned}
S &\to AB\\ A &\to a {\mathop{\&}}{{\rhd} B}\\ B &\to b {\mathop{\&}}{{\lhd} C}\\
C &\to a $$ and the input string $w=ab$. It is immediately seen that $C \in T_{0,1}$. From this, one can infer that $B \in T_{1,2}$, and that knowledge can in turn be used to show that $A \in T_{0,1}$. These data imply that $S \in T_{0,2}$. Thus, none of the sets $T_{0,1}$ and $T_{1,2}$ can be fully constructed before approaching the other.
The proposed algorithm for constructing the sets $T_{i,j}$ works as follows. At the first pass, it makes all deductions $\vdash_{G} {A\big({a_1 \ldots a_i \langle a_{i+1} \ldots a_j \rangle a_{j+1} \ldots a_n}\big)}$ that do not involve any contexts, and accordingly puts $A$ to the corresponding $T_{i,j}$. This pass progressively considers longer and longer substrings, as done by the Cocke–Kasami–Younger algorithm for ordinary grammars. During this first pass, some symbols may be added to any sets $T_{0,j}$ and $T_{i,n}$, and thus it becomes known that some contexts are true. This triggers another pass over all entries $T_{i,j}$, from shorter substrings to longer ones, this time using the known true contexts in the deductions. This pass may result in adding more elements to $T_{0,j}$ and $T_{i,n}$, which will require yet another pass, and so on. Since a new pass is needed only if a new element is added to any of $2n-1$ subsets of $N$, the total number of passes is at most $(2n-1) \cdot |N| + 1$.
These calculations are implemented in Algorithm \[alg:parsing\_cky\], which basically deduces all true statements about all substrings of the input string. For succinctness, the algorithm uses the following notation for multiple context operators. For a set $\mathcal{X} = \{X_1, \ldots, X_\ell\}$, with $X_i \in N$, and for an operator $Q \in \{{{\lhd} }, {{\trianglelefteqslant} }, {{\trianglerighteqslant} }, {{\rhd} }\}$, denote $Q \mathcal{X} := Q X_1 {\mathop{\&}}\ldots {\mathop{\&}}Q X_\ell$.
\[alg:parsing\_cky\] Let $G=(\Sigma,N,R,S)$ be a grammar with contexts in the binary normal form. Let $w=a_1 \ldots a_n \in \Sigma^+$ (with $n \geqslant 1$ and $a_i \in \Sigma$) be the input string. Let $T_{i,j}$ with $0 \leqslant i < j \leqslant n$ be variables, each representing a subset of $N$, and let $T_{i,j}={\varnothing}$ be their initial values.
\[cubic\_time\_parsing\_\_loop\_while\_changes\] \[cubic\_time\_parsing\_\_loop\_j\] \[cubic\_time\_parsing\_\_a\_j\] $T_{j-1,j} = T_{j-1,j} \cup \{A\}$ \[cubic\_time\_parsing\_\_T\_jminus1\_j\] \[cubic\_time\_parsing\_\_loop\_i\] let $P = {\varnothing}$ ($P \subseteq N \times N$) \[cubic\_time\_parsing\_\_loop\_k\] $P = P \cup (T_{i,k} \times T_{k,j})$ \[cubic\_time\_parsing\_\_cartesian\_product\] \[cubic\_time\_parsing\_\_loop\_T\_i\_j\] $T_{i,j} = T_{i,j} \cup \{A\}$ \[cubic\_time\_parsing\_\_T\_i\_j\]
\[cubic\_time\_parsing\_theorem\] For every grammar with two-sided contexts $G$ in the binary normal form, Algorithm \[alg:parsing\_cky\], given an input string $w=a_1 \ldots a_n$, constructs the sets $T_{i,j}$ and determines the membership of $w$ in $L(G)$, and does so in time $\mathcal{O}(|G|^2 \cdot n^4)$, using space $\mathcal{O}(|G| \cdot n^2)$.
While this paper was under preparation, Rabkin [@Rabkin] developed a more efficient and more sophisticated parsing algorithm for grammars with two-sided contexts, with the running time $\mathcal{O}(|G| \cdot n^3)$, using space $\mathcal{O}(|G| \cdot n^2)$. Like Algorithm \[alg:parsing\_cky\], Rabkin’s algorithm works by proving all true statements about the substrings of the given string, but does so using the superior method of Dowling and Gallier [@DowlingGallier]. Nevertheless, Algorithm \[alg:parsing\_cky\] retains some value as the elementary parsing method for grammars with two-sided contexts—just like the Cocke–Kasami–Younger algorithm for ordinary grammars remains useful, in spite of the asymptotically superior Valiant’s algorithm [@Valiant].
Conclusion
==========
This paper has developed a formal representation for the idea of phrase-structure rules applicable in a context, featured in the early work of Chomsky [@Chomsky]. This idea did not receive adequate treatment at the time, due to the unsuitable string-rewriting approach. The logical approach, adapted from Rounds [@Rounds] and his predecessors, brings it to life.
There are many theoretical questions to research about the new model: for instance, one can study the limitations of their expressive power, their closure properties, efficient parsing algorithms and subfamilies that admit more efficient parsing. Another possibility for further studies is investigating Boolean and stochastic variants of grammars with contexts, following the recent related work [@EsikKuich; @Kountouriotis_et_al; @Ziervogel].
On a broader scope, there must have been other good ideas in the theory of formal grammars that were inadequately formalized before. They may be worth being re-investigated using the logical approach.
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[^1]: Supported by the Academy of Finland under grant 257857.
|
Okada heads to court to regain Wynn shares for board fight
LOS ANGELES (Reuters) - Lawyers for dissident Wynn Resorts (WYNN.O) shareholder Kazuo Okada’s will urge a Nevada court on Tuesday to overturn the casino company’s forced redemption of the Japanese billionaire’s $2.7 billion stake, a ruling which would allow him to vote at its November 2 shareholder meeting to unseat two board members.
Okada said on September 17 that he would nominate Yale law professor Jonathan Macey and former CBS Corp (CBS.N) Chief Financial Officer Fredric Reynolds for the company’s 12-person board. Okada, who remains a board member, held a 20 stake in Wynn when the board voted in February to rescind the shares.
Wynn has rejected the nominations as invalid, calling it an attempt to divert attention from the issues facing Okada and his holding company, Aruze USA Inc.
The high-stakes legal battle pits Okada, formerly Wynn’s largest shareholder, against Wynn CEO Steve Wynn in a nearly year-long struggle. Each billionaire claims the other made improper payments to win favor in their respective Asian markets.
Wynn forcibly bought back Okada’s stake, valued at $2.7 billion, at a 30 percent discount after an internal probe by former FBI director Louis Freeh revealed that Okada had allegedly violated U.S. anti-corruption laws.
“Given the ticking clock aspect, I expect this litigation will be a full-time job up until November 2 and that any ruling by the judge is highly likely to be appealed,” said Jacob Frenkel, a partner with Shulman Rogers in Maryland.
Okada’s lawyers say the contract under which Okada first bought his stake in Wynn precluded a forced redemption. They also say Wynn’s rationale, based on concerns the company’s standing with gaming regulators is threatened due to Okada’s alleged conduct, was without merit since no charges were proven.
Wynn argued its actions were justified because Okada’s alleged payoffs to regulators at the Philippines Amusement and Gaming Corp (PAGCOR) were potential violations of the Foreign Corrupt Practices Act (FCPA), therefore threatening Wynn’s standing with gaming regulators in Nevada and Macau.
In his lawsuit, Okada “disputes that any redemption has occurred” and alleges that Wynn “undertook a secret investigation” to force him off the board and “committed a series of predicate acts of racketeering, which include fraud.”
Okada’s countersuit alleges that Steve Wynn “has run Wynn Resorts as a personal fiefdom,” and that his shares were worth $2.7 billion, well above the $1.9 billion Wynn paid him in a 10-year note that was a 30 percent discount to the market.
In court filings, Okada said shareholders have lost confidence in Wynn’s management and board, citing the stock’s decline of 30 percent this year. He has claimed that only seven of the 12 board members are independent, using Nasdaq listing standards.
In August, Okada filed a defamation lawsuit in Japan against the casino company related to the forced redemption of his shares in February.
Okada claimed $140 million (11.2 billion yen) in damages, alleging Wynn’s actions led to a fall in Universal’s stock price and new business opportunities, and damaged his reputation. |
TimescaleDB is the first open-source time-series database that natively supports full SQL and is deployed in production across industries all around the world for powering applications in DevOps, IoT, SaaS, and Machine Learning. It is packaged as a Postgres extension. As the Hasura GraphQL engine gives you GraphQL APIs instantly on any Postgres database, it also works with TimescaleDB.
For example, you can use create powerful views using TimescaleDB functions and instantly build real-time dashboards using GraphQL live-queries that Hasura will automatically provide. You can leverage TimescaleDB’s fast ingestion and use GraphQL mutations from your app to insert data quickly and easily.
For this post, we are going to set this up on a Digital Ocean VM.
TL;DR
These are the topics we’ll cover in this guide:
Create a Digital Ocean droplet and install docker and docker-compose
Run Hasura with TimescaleDB using docker-compose
Learn what hypertables are
Run GraphQL queries on hypertables
Run GraphQL subscriptions and get real-time data
Create a Digital Ocean droplet
Open Digital Ocean console, and click on the “Create” button, and select “Droplet” from the menu:
In the next screen, choose the image to be “Ubuntu 16.04”:
Then choose a machine size and choose a region of your preference.
Next, make sure to add a SSH key (you can add your existing SSH key on your computer), so that we can login to our server. Finally, click on the “Create” button.
Installing Docker
Get the public IP of your droplet, and then SSH to the server:
Once we are logged in, install Docker and Docker compose:
$ apt-get update $ apt-get install docker.io docker-compose # verify docker is working $ docker info
Run TimescaleDB with Hasura
To run TimescaleDB stand-alone, we can just run their docker image:
$ docker run -d -p 5432:5432 timescale/timescaledb:latest-pg10
This will pull the timescale/timescaledb docker image and run it, binding the host’s port 5432 to the container’s port 5432 .
But instead of this let’s use docker compose to run TimescaleDB and Hasura GraphQL together.
Copy and paste the following in a file, name it docker-compose.yaml :
Change the HASURA_GRAPHQL_ACCESS_KEY and POSTGRES_PASSWORD to your preference.
Then run:
$ docker-compose up -d
Check if everything is running, by:
$ docker ps CONTAINER ID IMAGE ... CREATED STATUS PORTS ... 097f58433a2b hasura/graphql-engine ... 1m ago Up 1m 8080->8080/tcp ... bccb3af615fd timescale/timescaledb ... 1m ago Up 1m 5432/tcp ...
This starts both TimescaleDB and Hasura.
Now in your browser open: http://your-droplet-public-ip:8080 , and you should see the Hasura console. It will prompt for the access key you have entered before.
Setup the TimescaleDB extension
We have to setup the timescaledb extension on our database:
We need to use psql to create the extension. To do that, we need to exec into out container.
Find out the container id from:
$ docker ps
Then exec into the container with psql , and create the extension:
# exec into the container, with psql # once we are connected to the db, we get a psql prompt # create the timescale extension $ docker exec -it <container-id> psql -U postgrespostgres-# postgres-# CREATE EXTENSION IF NOT EXISTS timescaledb CASCADE;
Now, we are all set to use TimescaleDB.
First steps with TimescaleDB
SQL tables in Timescale are called hypertables. They are an abstraction over standard SQL tables for efficient managing and querying of timescale data. For the end user, they can use hypertables as normally they would with standard SQL statements.
Note: hypertables are created by first creating a normal SQL table, then converting it into a hypertable via the function create_hypertable
Let’s create our first hypertable via the Hasura console:
-- We start by creating a regular SQL table CREATE TABLE conditions ( time TIMESTAMPTZ NOT NULL, location TEXT NOT NULL, temperature DOUBLE PRECISION NULL, -- in celsius humidity DOUBLE PRECISION NULL -- in percentage );
Create a table in the SQL tab
We are using raw SQL to create the table because we have data types that are not available in the console directly. We will also track the table, so that we can perform GraphQL queries on it.
Next, convert it into a hypertable:
-- This creates a hypertable that is partitioned by time -- using the values in the `time` column. SELECT create_hypertable('conditions', 'time');
Convert the table into a hypertable
-- This creates a hypertable that is partitioned by time -- using the values in the `time` column.
Let’s insert some sample data:
INSERT INTO conditions(time, location, temperature, humidity) VALUES (NOW(), 'office', 27.24, 48.15); INSERT INTO conditions(time, location, temperature, humidity) VALUES (NOW(), 'home', 32.21, 77.23);
We can use standard SQL to query our tables. TimescaleDB also offers specialized functions to query time-series data.
The following query will fetch last three hours data, grouped in 15 minute windows with their max temperature and humidity, and grouped by each location:
SELECT time_bucket('15 minutes', time) AS fifteen_min, location, COUNT(*), MAX(temperature) AS max_temp, MAX(humidity) AS max_hum FROM conditions WHERE time > NOW() - interval '3 hours' GROUP BY fifteen_min, location ORDER BY fifteen_min DESC, max_temp DESC;
Hasura GraphQL with TimescaleDB
To query our hypertables with TimescaleDB-specific functions like time_bucket , we can just create a view, track the view and make GraphQL queries on the view.
We can create this view:
CREATE VIEW last_fifteen_mins_stats AS ( SELECT time_bucket('15 minutes', time) AS fifteen_min, location, COUNT(*), MAX(temperature) AS max_temp, MAX(humidity) AS max_hum FROM conditions WHERE time > NOW() - interval '3 hours' GROUP BY fifteen_min, location ORDER BY fifteen_min DESC, max_temp DESC );
Now we can make GraphQL queries on our view:
query { last_fifteen_mins_stats { location, fifteen_min, max_temp, max_hum } }
Which will result in:
{ "data": { "last_fifteen_mins_stats": [ {"max_temp": 32.21, "location": "home", "max_hum": 77.23, "fifteen_min": "2018-08-22T11:15:00+00:00"}, {"max_temp": 27.24, "location": "office", "max_hum": 77.23, "fifteen_min": "2018-08-22T11:15:00+00:00"} ] } }
Real-time data with TimescaleDB & Hasura GraphQL
We can make a simple Python script which ingests data into TimescaleDB. We can run a subscription query to fetch our data in real-time.
As our conditions table is also tracked in Hasura GraphQL, we can make insert mutations !
Our script looks like this:
If we save this script in a file timescale_ingest.py , we can run this script by:
$ python3 timescale_ingest.py
This will run in an infinite loop and ingest random data into our TimescaleDB instance via Hasura GraphQL mutations.
We can run subscribe to this query by using Hasura GraphQL subscription:
subscription { last_fifteen_mins_stats { location, fifteen_min, max_temp, max_hum } }
Hasura subscriptions with TimescaleDB ingestion in action
Summary
Using TimescaleDB and Hasura GraphQL engine, we could query our Postgres database for real-time data using GraphQL APIs in under 10 minutes. This makes building powerful, real-time dashboards extremely simple.
To get started with TimescaleDB, check out their GitHub and feel free to join their Slack community.
You can get started with Hasura here. We’re very active on our community chat on Discord. You can also DM me on Discord @rayanon if you are trying this out and have any questions! |
Full List of 2012 Oscar Presenters Includes Christian Bale, Emma Stone, Zach Galifianakis and the Cast of BRIDESMAIDS
We’re only a few short days away from the 84th Academy Awards, which means all that prognosticating and “fake controversy” business will finally come to an end on Monday morning. Until next year, that is. Nevertheless, a number of presenters have been announced over the past few weeks and we figured we’d bring you a full list of all the pretty celebrities scheduled to appear during Sunday night’s telecast. Zach Galifianakis, Will Ferrell, Tina Fey, Emma Stone, The Muppets and the leading ladies from Bridesmaids will (hopefully) be bringing the funny, and today the Academy announced that last year’s winners in the acting categories will return to present. I really, really liked the way the awards were given out a few years back with past winners speaking directly to each of the nominees, so I’m hoping Christian Bale, Melissa Leo, Natalie Portman, and Colin Firth will be doing something similar.
Hit the jump to read the full list of presenters and performers, and be sure to check back this weekend to see how yours truly and Matt Goldberg think the awards will stack up in our predictions feature. Billy Crystal hosts the 84th Academy Awards on Sunday, February 26th.
Here’s the full list of presenters as of February 23rd:
Christian Bale
Bale received an Oscar® for his supporting role in “The Fighter.”
Halle Berry
Berry won an Oscar® in 2001 for her lead performance in “Monster’s Ball.”
The “Bridesmaids” Cast
Six actresses – Rose Byrne, Ellie Kemper, Melissa McCarthy, Wendi McLendon-Covey, Maya Rudolph and Kristen Wiig – from the hit comedy “Bridesmaids” will be presenters at the 84th Academy Awards. McCarthy received her first Oscar® nomination this year for her supporting role in the film, and Wiig also became a first-time nominee for the film’s original screenplay.
Cirque du Soleil®
The troupe Will Pay Tribute to Hollywood’s Biggest Night with Its Largest Ensemble Cast Ever Assembled in One Act
Bradley Cooper
Cooper will be seen next in “The Words” and, later this year, in “The Place Beyond the Pines” and “The Silver Linings Playbook.”
Tom Cruise
Cruise was nominated for his lead performances in “Born on the Fourth of July” and “Jerry Maguire.” He also was nominated for his supporting role in “Magnolia.”
Penélope Cruz
Cruz won an Oscar in 2008 for her supporting role in “Vicky Cristina Barcelona.” She was also nominated for her lead performance in “Volver” (2006) and for her supporting role in “Nine” (2009).
Cameron Diaz
Diaz will be seen next in “What to Expect When You’re Expecting” and “Gambit.”
Michael Douglas
Douglas won an Oscar for his lead performance in “Wall Street” (1987) and a Best Picture award as a producer of “One Flew over the Cuckoo’s Nest” (1975).
Will Ferrell
Ferrell’s film credits include “Everything Must Go,” “Blades of Glory,” “Stranger Than Fiction,” “Talladega Nights: The Ballad of Ricky Bobby,” “The Producers,” “Bewitched,” “Anchorman: The Legend of Ron Burgundy,” “Elf” and “Old School.”
Tina Fey
Fey came to prominence as a regular on “Saturday Night Live” and currently stars in, produces and writes for the Emmy®-winning comedy series “30 Rock.”
Colin Firth
Two-time nominee Firth took home an Academy Award for his lead performance in “The King’s Speech.”
Zach Galifianakis
Galifianakis, who was recently seen in “The Muppets,” will be making his first Oscar show appearance. His other film credits include “Puss in Boots,” “Due Date,” “Dinner for Schmucks,” “Up in the Air,” “What Happens in Vegas,” “Into the Wild” and the two “Hangover” films.
Tom Hanks
Hanks, who is a governor of the Academy, won consecutive Oscars® in 1993 and 1994 for his lead performances in “Philadelphia” and “Forrest Gump.”
Angelina Jolie
In 1999 Jolie took home the Oscar for Best Supporting Actress for her performance in “Girl, Interrupted,” and she received a nomination in 2008 for her lead performance in “Changeling.”
Milla Jovovich
Jovovich hosted the Academy’s Scientific and Technical Awards on Saturday, February 11
Melissa Leo
Leo received an Oscar® for her supporting role in “The Fighter.”
Jennifer Lopez
Lopez, who serves as a judge on “American Idol,” will be seen next in the feature “What to Expect When You’re Expecting” and recently completed work on “Parker” and “Ice Age: Continental Drift.”
Natalie Portman
Two-time nominee Portman took home an Academy Award for her lead performance in “Black Swan.”
Chris Rock
In 2005 Rock served as host of the 77th Academy Awards. He will be seen next in “What to Expect When You’re Expecting” and “2 Days in New York.”
Ben Stiller
Stiller will be playing the title character in “The Secret Life of Walter Mitty,” which he is also directing, and will be seen next in “Neighborhood Watch.”
Emma Stone
Stone appears in the Best Picture nominee “The Help.” Her other film credits include “Crazy, Stupid, Love.,” “Friends with Benefits,” “Easy A” and “Superbad.”
Meryl Streep
Streep, who is nominated for her lead performance in “The Iron Lady,” has been nominated a total of 17 times – the most for any performer in Oscar history.
COLLIDER participates in various affiliate marketing programs, which means COLLIDER gets paid commissions on purchases made through our links to retailer sites. Our editorial content is not influenced by any commissions we receive. |
Lately I have been spotting more and more cargo bikes and trikes "in the wild" in the Boston Metro area. Bakfiets, Christiania, Nihola, Xtracycle, Yuba Mundo, Gazelle, all sorts of neat models. Interestingly, almost none of them could have been purchased locally, because no local bike shops carry them. This seems to be based on the bizarre notion held by local bike stores and importers/distributors, that Boston would not make a good market for cargo bikes. I have personally heard this rhetoric many times from various members of the bicycle industry: Cargo bikes in Boston? Oh no, there is no market. Terrible place for cycling. Awful drivers, dense car traffic, narrow streets with no room for bike lanes, rude people. It's a good place for fixies and such, but cargo bikes? No way.
No market for them, eh? I must have seen half a dozen over the past week alone. And since local shops won't sell them, the cargo-bike-starved population of Boston is forced to travel to Portland ME or NYC to shop for them, or else order online, or else attempt to get them direct from the distributor with lots of behind the scenes begging. Odd really.
Larry vs Harry Bullitt, At this point you might be wondering why I care. In fact, why do I have reviews of cargo bikes here at all (see my test ride reports of the Bakfiets Christiania and the Maderna Cycle Truck )? I don't have children and I don't really have enough stuff to transport on a regular basis to need a dedicated cargo bike. So why the interest?
I think cargo bikes are important in that they indicate how far a city has come in embracing and normalising transportational cycling. They equate cycling with safety and comfort, as opposed to danger and athletic skill. The mere sight of cargo bikes suggests:
"Look, people here must feel comfortable carting around their children by bike!"
"Look, there are people here who even replace their trucks with bikes!"
"Look, it must be okay here for bicycles to take up lots of room on the road!"
And of course when people see signs that something is okay to do because it looks like others do it, they are more likely to consider doing it themselves. So even if they do not need or want a cargo bike per se, they may be more likely to look into cycling with their children, cycling with baggage, cycling on the road, and other aspects of transportational cycling because these ideas are suggested by the mere existence of cargo bikes.
I suppose a simpler way of saying what I am trying to say might be that cargo bikes are symbolic of a strong, healthy "bike culture." Going with this premise, I am pleased that more of them are popping up in Boston and wish the industry would take note. What about your city?
But clearly there is a lag between how Boston is perceived by the bicycle industry and what is actually happening here - especially in the lively suburbs (more like boroughs) of Cambridge and Somerville. Beacon Street - a main road that acts as a border between the two - features parade-like processions of cyclists during morning and evening rush hour of almost Copenhagenesque proportions. Women in skirts, men in smart blazers, child seats strapped to rear racks, baskets on the handlebars, enormous panniers, and even - that's right - cargo bikes. |
The National Network of Libraries of Medicine program is intended to provide health science practitioners, investigators, educators, and administrators with timely, convenient access to information. The program is coordinated by the National Library of Medicine and carried out through a nation-wide network of more than 3,300 health science libraries and information centers. It incorporates the individual health practitioner within the institutional network by involving all of the libraries in the Network in establishing direct contact with health professionals. The Network also includes eight Regional Medical Libraries, or RMLs. These are major institutions designated by NLM to administer and provide backup services in each of eight geographical regions. Objectives for the RMLs are: to promote awareness of and access to biomedical information resources for health professionals through expanded and targeted outreach programs, to develop and improve the biomedical information resources in the regions, and to support the sharing of these resources within the regions and throughout the nation. Region 7 provides service to the states of Arizona, California, Hawaii, and Nevada, and U.S. Territories in the Pacific Basin. |
About The Author Alex Holt is a professional interactive designer and web developer who has worked successfully for a variety of clients in Australia, UK, USA and most recently … More about Alex Holt …
jQuery and JavaScript Coding: Examples and Best Practices
Smashing Newsletter Every week, we send out useful front-end & UX techniques. Subscribe and get the Smart Interface Design Checklists PDF delivered to your inbox. Your (smashing) email Subscribe →
When used correctly,jQuery can help you make your website more interactive, interesting and exciting. This article will share some best practices and examples for using the popular Javascript framework to create unobtrusive, accessible DOM scripting effects.
When used correctly, jQuery can help you make your website more interactive, interesting and exciting. This article will share some best practices and examples for using the popular Javascript framework to create unobtrusive, accessible DOM scripting effects. The article will explore what constitutes best practices with regard to Javascript and, furthermore, why jQuery is a good choice of a framework to implement best practices.
You may want to take a look at the following newer posts:
1. Why jQuery?
jQuery is ideal because it can create impressive animations and interactions. jQuery is simple to understand and easy to use, which means the learning curve is small, while the possibilities are (almost) infinite.
Javascript and Best Practices
Javascript has long been the subject of many heated debates about whether it is possible to use it while still adhering to best practices regarding accessibility and standards compliance.
The answer to this question is still unresolved, however, the emergence of Javascript frameworks like jQuery has provided the necessary tools to create beautiful websites without having to worry (as much) about accessibility issues.
Obviously there are cases where a Javascript solution is not the best option. The rule of thumb here is: use DOM scripting to enhance functionality, not create it.
Unobtrusive DOM Scripting
While the term “DOM scripting” really just refers to the use of scripts (in this case, Javascripts) to access the Document Object Model, it has widely become accepted as a way of describing what should really be called “unobtrusive DOM scripting”—basically, the art of adding Javascript to your page in such a way that if there were NO Javascript, the page would still work (or at least degrade gracefully). In the website world, our DOM scripting is done using Javascript.
The Bottom Line: Accessible, Degradable Content
The aim of any web producer, designer or developer is to create content that is accessible to the widest range of audience. However, this has to be carefully balanced with design, interactivity and beauty. Using the theories set out in this article, designers, developers and web producers will have the knowledge and understanding to use jQuery for DOM scripting in an accessible and degradable way; maintaining content that is beautiful, functional AND accessible.
2. Unobtrusive DOM Scripting?
In an ideal world, websites would have dynamic functionality AND effects that degrade well. What does this mean? It would mean finding a way to include, say, a snazzy Javascript Web 2.0 animated sliding news ticker widget in a web page, while still ensuring that it fails gracefully if a visitor’s browser can’t (or won’t) run Javascripts.
The theory behind this technique is quite simple: the ultimate aim is to use Javascript for non-invasive, “behavioural” elements of the page. Javascript is used to add or enhance interactivity and effects. The primary rules for DOM scripting follow.
Rule #1: Separate Javascript Functionality
Separate Javascript functionality into a “behavioural layer,” so that it is separate from and independent of (X)HTML and CSS. (X)HTML is the markup, CSS the presentation and Javascript the behavioural layer. This means storing ALL Javascript code in external script files and building pages that do not rely on Javascript to be usable.
For a demonstration, check out the following code snippets:
Bad markup:
Never include Javascript events as inline attributes. This practice should be completely wiped from your mind.
<a onclick="doSomething()" href="#">Click!</a>
Good markup:
All Javascript behaviours should be included in external script files and linked to the document with a <script> tag in the head of the page. So, the anchor tag would appear like this:
<a href="backuplink.html" class="doSomething">Click!</a>
And the Javascript inside the myscript.js file would contain something like this:
... $('a.doSomething').click(function(){ // Do something here! alert('You did something, woo hoo!'); }); ...
The .click() method in jQuery allows us to easily attach a click event to the result(s) of our selector. So the code will select all of the <a> tags of class “doSomething” and attach a click event that will call the function. In practice, this
In Rule #2 there is a further demonstration of how a similar end can be achieved without inline Javascript code.
Rule #2: NEVER Depend on Javascript
To be truly unobtrusive, a developer should never rely on Javascript support to deliver content or information. It’s fine to use Javascript to enhance the information, make it prettier, or more interactive—but never assume the user’s browser will have Javascript enabled. This rule of thumb can in fact be applied to any third-party technology, such as Flash or Java. If it’s not built into every web browser (and always enabled), then be sure that the page is still completely accessible and usable without it.
Bad markup:
The following snippet shows Javascript that might be used to display a “Good morning” (or “afternoon”) message on a site, depending on the time of day. (Obviously this is a rudimentary example and would in fact probably be achieved in some server-side scripting language).
<script language="javascript"> var now = new Date(); if(now.getHours() < 12) document.write('Good Morning!'); else document.write('Good Afternoon!'); </script>
This inline script is bad because if the target browser has Javascript disabled, NOTHING will be rendered, leaving a gap in the page. This is NOT graceful degradation. The non-Javascript user is missing out on our welcome message.
Good markup:
A semantically correct and accessible way to implement this would require much simpler and more readable (X)HTML, like:
<p title="Good Day Message">Good Morning!</p>
By including the “title” attribute, this paragraph can be selected in jQuery using a selector (selectors are explained later in this article) like the one in the following Javascript snippet:
var now = new Date(); if(now.getHours() >= 12) { var goodDay = $('p[title="Good Day Message"]'); goodDay.text('Good Afternoon!'); }
The beauty here is that all the Javascript lives in an external script file and the page is rendered as standard (X)HTML, which means that if the Javascript isn’t run, the page is still 100% semantically pure (X)HTML—no Javascript cruft. The only problem would be that in the afternoon, the page would still say “Good morning.” However, this can be seen as an acceptable degradation.
Rule #3: Semantic and Accessible Markup Comes First
It is very important that the (X)HTML markup is semantically structured. (While it is outside the scope of this article to explain why, see the links below for further reading on semantic markup.) The general rule here is that if the page’s markup is semantically structured, it should follow that it is also accessible to a wide range of devices. This is not always true, though, but it is a good rule of thumb to get one started.
Semantic markup is important to unobtrusive DOM scripting because it shapes the path the developer will take to create the DOM scripted effect. The FIRST step in building any jQuery-enhanced widget into a page is to write the markup and make sure that the markup is semantic. Once this is achieved, the developer can then use jQuery to interact with the semantically correct markup (leaving an (X)HTML document that is clean and readable, and separating the behavioural layer).
Terrible markup:
The following snippet shows a typical list of items and descriptions in a typical (and terribly UNsemantic) way.
<table> <tr> <td onclick="doSomething();">First Option</td> <td>First option description</td> </tr> <tr> <td onclick="doSomething();">Second Option</td> <td>Second option description</td> </tr> </table>
Bad markup:
The following snippet shows a typical list of items and descriptions in a more semantic way. However, the inline Javascript is far from perfect.
<dl> <dt onclick="doSomething();">First Option</dt> <dd>First option description</dd> <dt onclick="doSomething();">Second Option</dt> <dd>Second option description</dd> </dl>
Good markup:
This snippet shows how the above list should be marked up. Any interaction with Javascript would be attached at DOM load using jQuery, effectively removing all behavioural markup from the rendered (X)HTML.
<dl id="OptionList"> <dt>First Option</dt> <dd>First option description</dd> <dt>Second Option</dt> <dd>Second option description</dd> </dl>
The <id> of “OptionList” will enable us to target this particular definition list in jQuery using a selector—more on this later.
3. Understanding jQuery for Unobtrusive DOM Scripting
This section will explore three priceless tips and tricks for using jQuery to implement best practices and accessible effects.
Understanding Selectors: the Backbone of jQuery
The first step to unobtrusive DOM scripting (at least in jQuery and Prototype) is using selectors. Selectors can (amazingly) select an element out of the DOM tree so that it can be manipulated in some way.
If you’re familiar with CSS then you’ll understand selectors in jQuery; they’re almost the same thing and use almost the same syntax. jQuery provides a special utility function to select elements. It is called $.
A set of very simple examples of jQuery selectors:
$(document); // Activate jQuery for object $('#mydiv') // Element with ID "mydiv" $('p.first') // P tags with class first. $('p[title="Hello"]') // P tags with title "Hello" $('p[title^="H"]') // P tags title starting with H |
Carnage in stock markets as I write — and all of the headlines I see attribute it to S&P’s downgrade.
They really are trying to make my head explode, aren’t they?
Once again: S&P declared that US debt is no longer a safe investment; yet investors are piling into US debt, not out of it, driving the 10-year interest rate below 2.4%. This amounts to a massive market rejection of S&P’s concerns.
The “signature” of debt concerns should be stock and bond prices both falling; what we actually see is those prices moving in opposite directions. And that’s normally the signature of concerns about a weak economy and deflation risk (see Japan, decline of).
What triggered economy fears? To some extent I think this is a Wile E. Coyote moment, with investors suddenly noticing just how weak the fundamentals are. Also, the mess in Europe.
And maybe, maybe there is an S&P story — but not the one you think. Arguably, that downgrade will bully policy makers into even more deflationary, contractionary policies than they would have undertaken otherwise, which has the perverse effect of making US debt more attractive, since the alternatives are worse.
But all the Very Serious People, having totally misdiagnosed our problems so far, will probably double down on that wrong diagnosis as markets fall. |
Since the first report of Zwilling and coworkers[@b1], vertically aligned titanium dioxide (TiO~2~) nanotube (NT) arrays by anodic oxidation have been largely investigated as active element for dye-sensitized solar cells[@b2][@b3][@b4], Li-ions batteries[@b5][@b6], water photoelectrolysis[@b7], artificial photosynthesis[@b8], biomedical devices[@b9][@b10], molecular sensors[@b11][@b12][@b13], gas sensors[@b14], and photocatalytic degradation of pollutants[@b15]. Thanks to their quasi one-dimensional arrangement, TiO~2~ NTs are able to provide high surface area and superior electron transport properties resulting in a performance enhancement in the different fields of application. The electrochemical oxidation of Ti foil in a fluorine-based electrolyte (see [Figure 1a](#f1){ref-type="fig"}) is a simple, cheap and green process, easy to be scaled up towards massive production and with a low energy payback time if used for energy harvesting applications. However, the as-grown NTs are fully amorphous, while crystalline TiO~2~ is highly desired for most applications[@b16]. Thus, the nanotube synthesis is usually followed by a thermal annealing process (at around 450°C)[@b17] or by a hydrothermal treatment (at about 200°C)[@b18] to obtain a nanocrystalline material in the anatase phase. These additional treatments drastically affect the energetic balance of the overall process and dictate some constrains for the applicability of this material. In particular, the involved temperatures hinder the integration of ordered NT carpets on the most common polymeric substrates, which is a crucial step for the fabrication of flexible and lightweight devices.
For these reasons low-temperature water-assisted treatment to crystallize amorphous TiO~2~ nanotubes have attracted enormous interest in the scientific community in the last years. All the proposed approaches are based on the interaction of the amorphous TiO~2~ NT with water molecules and, depending on the interface that is formed, they can be divided in two main groups: solid-liquid and solid-gas interface reactions[@b19]. The former have been deeply investigated[@b18][@b20][@b21][@b22][@b23], revealing a crystallization rate and morphology modification which depend on the water temperature and incubation time. On the contrary, the latter approach is up to now less explored, even if the recent report of Liu and co-workers show that outstanding material properties can be obtained following this direction[@b19]. However, all the above mentioned methods required hydrothermal reaction conditions (high pressure and temperatures greater than 100°C)[@b18][@b20][@b21][@b22] or time-consuming incubation at room temperature (more than 72 hours in ambient atmosphere)[@b23] to obtain the desired anatase phase.
Herein we demonstrate for the first time to the best of our knowledge the near-room temperature (50°C) crystallization process of anodic NTs from amorphous TiO~2~ to anatase phase after only 30 minutes of sample exposure to water vapor in ambient condition. The water vapor treated samples were deeply characterized in order to gain a whole understanding of their structural, physical and chemical properties, while their functional properties were evaluated in terms of their photocatalytic response during dye degradation experiments. The photocatalytic activity is a crucial characteristic, since it is the basis of a huge number of applications including for instance water splitting for the production of molecular hydrogen, photoreduction of CO~2~, self-cleaning coatings, degradation or oxidization of organic contaminants, elimination of heavy metal ions, electrochromic devices, sensors, antibacterial surfaces and electrochemical solar cells. The obtained results confirm the effectiveness of the proposed ultrafast near-room temperature crystallization method.
Results and Discussion
======================
The interaction of a solid surface exposed to water vapor is a highly complex interfacial system, since three phases (gas, liquid, and solid) and their interfaces (solid/liquid, liquid/gas and solid/gas) are involved. The low temperature crystallization approach proposed up to now can be described taking into account only the solid/liquid or solid/gas interfaces, and neglecting the presence of gas and liquid phases at the same time. Recently, Wang and coworkers showed that the conversion of amorphous nanotube array into polycrystalline TiO~2~ is possible at room temperature exploiting spontaneous water-assisted reaction, based on sample incubation in DI-water for more than 72 hours[@b23]. This crystallization mechanism involves the dissolution and precipitation processes in which a spatial atomistic order is induced by water molecules. Nevertheless very long incubation time is required to obtain the full crystallization of the material. Other research groups investigated the effect of the hydrothermal treatment showing that faster conversion can be obtained increasing the reaction temperature under pressure[@b18][@b20][@b21][@b22]. On the contrary, our approach is based on the simple exposure in ambient atmosphere of as-grown amorphous TiO~2~ NTs to the steam evolving by hot water (see [Figure 1b](#f1){ref-type="fig"}). In general, the exposure of the metal oxide surface to water vapor leads to the formation of a thin water adlayer with a thickness on the order of nanometers that corresponds to the reversible adsorption and desorption of water from the surface. This aqueous film is the dynamic medium in which the crystallization process occurs.
It is necessary to specify that the as-grown TiO~2~ NTs are composed by two layers (see [Figure 1c](#f1){ref-type="fig"} step I): an outer part (shell) of pure and dense TiO~2~ and an inner porous part (core) that contains incorporated electrolyte components[@b24]. In [Figure 2](#f2){ref-type="fig"} and [Figure 3](#f3){ref-type="fig"} we show the Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM) images respectively, taken at different times of steam exposure. By increasing the exposure time, the NTs morphology evolves from an amorphous tubular shape up to a polycrystalline rod-like form. The possible conversion mechanism has been previously described by Wang et al.[@b23], but in our case it is accelerated by water vapor adsorption/condensation kinetics thanks to the formation of a solid/liquid/gas dynamic equilibrium. Two parallel events occur during the crystallization process. The first takes place at the inner tube walls, where the water vapor can condensate as hot water, acting as catalyzer in the crystallization process. Water molecules act as catalyzers favoring the rearrangement of TiO~6~^2−^ octahedra in the amorphous TiO~2~ nanotubes through hydration/dehydration reactions, as previously reported by Yu and coworkers[@b18]. The hot water cools down because of the lower temperature (equal to 50°C, accurately measured by Pt100 temperature detector) of the sample and it is substituted by new condensated molecules. The second process is the crystallization at the outer wall, where small crystals form at the NT surface at the very beginning.
In [Figure 2](#f2){ref-type="fig"}, the image at 0 minutes shows the morphology of the as-grown NTs (corresponding to step I in [Figure 1c](#f1){ref-type="fig"}). The amorphous nature of this material is demonstrated by the TEM characterization, image at 0 minutes in [Figure 3](#f3){ref-type="fig"}. After 5 minutes of exposure, some crystallites appear at the outer surface of the tubes that increase after 10 minutes, when the formation of crystals in the inner shell of the tube becomes also evident.
After 20 minutes, the formation of a core-shell structure is the ultimate step (step II in [Figure 1c](#f1){ref-type="fig"}) before the dissolution of the shell by the thickness reduction of the pristine amorphous wall in favor of the consolidation of the inner rod-like structure. This consolidation process is well visible starting from 30 minutes of exposure time, where the conversion process into the porous rod-like structure is almost completed, even if a residual thin wall remains (step III in [Figure 1c](#f1){ref-type="fig"}). This residual wall decreases in thickness as the exposure time increases, until its complete dissolution (step IV in [Figure 1c](#f1){ref-type="fig"}). The grain size, evaluated from FESEM characterization, seems to be unvaried starting from the sample exposed for 30 minutes, up to the one exposed for 4 hours. A more precise estimation of the grain size is given by the High Resolution TEM (HRTEM) image reported in [Figure 3](#f3){ref-type="fig"}, and it is in the range of 5--10 nm. The Fast Fourier Transform (FFT) in the inset of the same image demonstrates the polycrystalline-randomly oriented nature of the anatase rod-like structure obtained after 2 hours of exposure time, in agreement with the X-Ray Diffraction (XRD) results reported below and in the [Supporting Information](#s1){ref-type="supplementary-material"} ([Figure S3](#s1){ref-type="supplementary-material"}).
The porous nature of the converted TiO~2~ is also confirmed by BET (Brunauer--Emmett--Teller) measurements. In fact the calculated specific area, evaluated by nitrogen sorption isotherm, increases with the water vapor treatment according to the FESEM observation. The amorphous nanotube array exhibits about 38 m^2^/g surface area, while after 2 hours of steam exposure its value is increased up to 106 m^2^/g, with a reduction of the pore size from about 12 nm down to about 4 nm (see [Figure S4](#s1){ref-type="supplementary-material"}).
Crystallographic data confirm that just after anodization the TiO~2~ nanotubes are amorphous, but a rapid structural evolution emerges during the solid/liquid/vapor interaction. As evidenced in the XRD spectra reported in [Figure 4a](#f4){ref-type="fig"}, the crystallization of the material in the anatase form starts rapidly, and after a 10 min treatment the main diffraction peak at 25.3 deg (2 theta), related to the (101) crystal face, begins to be visible. For longer vapor exposure, an ultrafast crystallization kinetic is evidenced, and the crystallization process saturates after 30 min of treatment. This aspect is well evidenced by the evolution of the average crystallite size, estimated through the Scherrer formula, as a function of the vapor exposure time, reported in [Figure 4b](#f4){ref-type="fig"}. The average crystallite dimension is approximately equal to 7 nm after 30 min treatment and does not evolve significantly for longer water vapor exposure, in line with the HRTEM analysis.
It is important to underline that no intentional heating was provided to the substrates but, due to the interaction with the water vapor, a slight increase of the temperature of the substrate was evidenced, reaching the maximum value of 50°C. In order to exclude this mild heating of the substrate as potential responsible of the fast crystallization process, other as-grown TiO~2~ NT sample were prepared on purpose and incubated into hot water at 50°C for different times up to 4 hours (see [Supporting Information](#s1){ref-type="supplementary-material"}). Neither the FESEM characterization ([Figure S1](#s1){ref-type="supplementary-material"}) nor the XRD analysis ([Figure S2](#s1){ref-type="supplementary-material"}) evidenced any morphological or crystallographic evolution during this treatment. These results are in line with the observation reported by Krengvirat and co-workers that demonstrated a minimum temperature of crystallization equal to 70°C with 3 hours of treatment[@b22]. Our empirical evidence supports the hypothesis that the rapid formation of anatase phase can be only due to the interaction of water vapor molecules with the amorphous TiO~2~ nanotube walls. Unlike what happens in the liquid water, the molecules in the gas are not in strong interaction with each other. This allows greater freedom to the water molecules to fill better and faster a nanoporous structure as the TiO~2~ NTs. Consequently they can increase the adsorption rate onto the amorphous titania surface, dramatically increasing the rate of crystallization.
Contact angle (CA) measurements were performed to study the evolution of TiO~2~ nanotubes wettability depending on the water vapor exposure time (see [Figure 4c](#f4){ref-type="fig"}). The mean value of the water CA exponentially decreases from slightly hydrophobic for the amorphous nanotubes to super hydrophilic in the water-crystallized material. This trend follows and confirms the increase of the surface roughness revealed by FESEM and BET investigations. The enhanced wettability can be ascribed to the water vapor interaction, which is responsible for the introduction of a high number of surface defects, acting as adsorption sites for --OH groups. Furthermore, this increased wetting behaviour can be considered as a co-responsible for the rapid crystallization of the material during the interaction with the water vapor. In fact, the steam exposure significantly increases the wettability and consequently the interaction between water (catalyzing agent of crystallization) and the surface of TiO~2~ (gradually more and more crystalline) gets stronger. As counterproof also the contact angle of the nanotube sample incubated in water at 50°C was measured and the results show that no considerable variations of wettability can be observed.
The influence of vapor treatment on the chemical structure of TiO~2~ NT arrays was investigated by Fourier Transform-InfraRed (FT-IR) analysis, and the related spectra are shown in [Figure 5a](#f5){ref-type="fig"}. The main results are well in line with those shown by Krengvirat and coworkers[@b22]. A broad band at about 3400 cm^−1^ and a weak band at 1626 cm^−1^ are present at different extent in all the samples, and they can be ascribed to chemisorbed H~2~O. In the as-grown material several peaks are present, that can be related to the chemicals used during the anodization step. A weak band associated to --OH groups is visible at 1433 cm^−1^. Stretching vibrations consistent with amino-acids (--NH~3~^+^) were detected at 1072 cm^−1^, and N--H deformation vibration is well visible at 1558 cm^−1^. The weak bands at 854 and 610 cm^−1^ are attributed to the stretching vibration of Ti--O in the TiO~6~ octahedron. After water vapor treatment, a broad band appears in all the samples at about 2900 cm^−1^, and it is consistent with the degradation of organic compounds associated to the dissolution of residual ethylene glycol. The disappearance of the bands located at 1558 and 1072 cm^−1^ confirmed the removal of adsorbed ammonium. In the treated samples the water and --OH related bands, at 1626 cm^−1^ and 1433 cm^−1^ respectively, become stronger with increasing treatment time, which is in line with the progressively increased wettability of the material.
Moreover, to further analyze the crystallization process, Raman spectroscopy was employed. [Figure 5b](#f5){ref-type="fig"} displays Raman spectra of the TiO~2~ nanotubes in the as-grown condition and after the water vapor treatment for different exposure times. The as-grown sample exhibits three very broad bands confirming the presence of amorphous structure of TiO~2~. Such broad features are assigned to the Ti--O bending (190 cm^−1^, 450 cm^−1^) and Ti--O stretching (610 cm^−1^) vibrations. An additional broad shoulder appears at 890 cm^−1^, followed by a series of vibrational bands of variable intensity at 1075, 1136, 1205, 1283, 1370, 1456 and 1595 cm^−1^. These bands existing in the range 1000--1700 cm^−1^ as well as the broad shoulder at 895 cm^−1^ are closely related to the Raman modes of ethylene glycol molecules as observed by Likodimos et al.[@b25] and after 30 minutes of water vapor exposure are found disappeared. The obtained Raman active modes at \~150 cm^−1^, 200 cm^−1^, 399 cm^−1^, 513 cm^−1^ and 633 cm^−1^ are related to the tetragonal structure of anatase TiO~2~ with D4h space group[@b26].
The influence of water vapor treatment on the optical properties of TiO~2~ NTs was investigated and the results are reported in [Figure 5c](#f5){ref-type="fig"}. Absorbance of as-grown nanotubes increases in visible region from higher wavelengths to lower ones and the absorption edge is not well-defined and less sharp, a characteristic behavior of amorphous oxides[@b27]. Nevertheless after 30 minutes of water vapor treatment, nanotube arrays showed comparatively flat curve in visible region and a sharp absorption edge indicating the improved crystallinity of the material.
The indirect energy band gap (E~g~) for the water-vapor crystallized TiO~2~ nanotubes, has been determined by extrapolation of the plot of *(F(R)\*hυ)^1/2^* against the photon energy, *hυ*, according to the following equation: *F(R)\*hυ = const (hυ- E~g~)^2^*. The obtained value of the semiconductor band gap, i.e. 3.27 ± 0.01 eV, is very close to the values (3.26--3.30 eV) reported by other researchers for anatase phase[@b28][@b29][@b30], which is considered the best phase of titania for photocatalytic applications[@b31]. This result shows that the optical properties analyzed by UV-Vis spectroscopy are clearly related to the crystalline phase obtained from Raman and XRD measurements.
Finally we evaluated the decomposition of methylene blue (MB) dye under UV light as test to assess the photocatalytic activity of the prepared samples. The photocatalytic degradation and mineralization of the dye in aqueous solution is a well-known process[@b32][@b33][@b34], thus it could be considered a valuable protocol to test the properties of the synthesized material.
In [Figure 6a](#f6){ref-type="fig"} the trend of the ratio C/C~0~ versus time is reported, where C~0~ is the concentration of starting MB solution in water and C is the one after the illumination. In general, the degradation of dyes depend on several parameters such as solution pH, catalyst concentration, substrate morphology, etc. In our case, we compared the decomposition kinetics under the same experimental conditions, revealing a first-order Langmuir--Hinshelwood process, as witnessed by the linear plot reported in the [Supporting material](#s1){ref-type="supplementary-material"} ([Fig. S5](#s1){ref-type="supplementary-material"}). Since the decomposition rate is proportional to the surface coverage, we argued that the improved photocatalytic properties of the water vapor- treated TiO~2~ with respect to the other nanocrystalline titania-based materials are related with the higher exposed surface area (as measured by BET). Given that for an effective applicability a photocatalyst should be stable under repeated uses, the photocatalytic stability of water vapor crystallized TiO~2~ NTs was verified testing the samples repeatedly five times. As shown in [Figure 6b](#f6){ref-type="fig"}, after being cleaned, dried, and then recycled, the photocatalysts exhibit almost the same degradation of MB dye under the selected reaction conditions, with only a 5% reduction after 5 consecutive cycles.
Conclusions
===========
In summary, we demonstrated for the first time that amorphous TiO~2~ NTs can be crystallized into anatase phase after exposure to water vapor in ambient condition. This facile approach provides a cost effective strategy to allow the integration of ordered NT carpets on temperature-sensitive substrates, as crucial step for the fabrication of flexible and lightweight devices. We found that the crystallographic conversion is complete after only 30 minutes, with physical and chemical properties of the NTs that remains unvaried for prolonged treatment. Finally the photocatalytic activity of the converted material was tested by dye degradation experiments, revealing an improvement with respect to thermally treated samples, and demonstrating the effectiveness of the here proposed crystallization method.
Experimental
============
Materials and methods
---------------------
TiO~2~ nanotube arrays were grown by anodic oxidation of Ti foil (250 μm thick, 99.6% purity, Goodfellow) in an electrolytic solution consisting of 0.5 wt% NH~4~F (98%, Sigma Aldrich) and 2.5 vol% deionized water in ethylene glycol (98%, Sigma Aldrich). Samples were cleaned by ultrasonication in acetone, rinsed in ethanol and pretreated into a 1wt% HF aqueous solution to remove the native oxide layer obtaining a fresh metal surface for the NTs growth. A Pt sheet (250 μm thick, 99.99% purity, Goodfellow) was employed as a cathode in a two-electrode configuration Teflon cell. The electrochemical process was conducted under continuous stirring at ambient temperature applying an anodization potential of 60 V using a DC power supply (GW Instek SPD-3606). After the anodization, the oxidized Ti foils were abundantly rinsed in DI-water to remove the impurities eventually adsorbed from the electrolytic solution during the growth.
The NT arrays were subsequently fixed into a clamping system and exposed to the water vapor evolving by DI-water (18.2 MΩ·cm^−1^ @ 25°C) previously heated at 90°C by an hot plate for different times (0--240 min). The temperature of the substrate was measured by a Pt100 temperature detector connected to a dedicated controller.
Characterization
----------------
The evolution of the morphology on the different samples was characterized by a ZEISS Auriga FESEM. The TEM was performed with a FEI Tecnai F20ST operating at 200 kV, to evaluate the quality of the crystalline TiO~2~ in Bright Field and High Resolution modes. For the TEM characterization, the sample was scratched from the substrate and dispersed in HPLC ultrapure ethanol for 2 minutes by sonication. Then, a drop of dispersion was put on a lacey carbon copper grid and let dry in ambient atmosphere.
X-ray diffraction patterns were acquired with a PANalytical X\'Pert Pro equipment in Bragg-Brentano configuration (Cu Kα X-ray source). The average anatase crystallite size was estimated through the Scherrer equation using the main anatase peak, related with the (101) crystal planes. Its full width at half maximum was estimated with a pseudo-Voigt fitting and corrected taking into account the instrumental broadening.
Fourier transformed infrared spectra were recorded using a Nicolet 5700 FTIR Spectrometer in attenuated total reflectance (ATR) mode with 4 cm^−1^ resolution and an average of 64 scans.
The room temperature Raman spectra of NT arrays were collected using Renishaw Invia spectrometer with the 514 nm Ar-Kr excitation laser beam. Optical absorbance of the water vapor treated TiO~2~ NT arrays was measured by a Varian Cary 5000 spectrophotometer employing integrating sphere.
Brunauer--Emmett--Teller specific surface area was measured from N~2~ sorption isotherms (Quantachrome Autosorb1) by multipoint method within the relative pressure range of 0.1--0.3 P/P~0~.
Contact angle measurements were performed using a OCA H200 Dataphysics equipment in ambient conditions. The sessile drop method was implemented employing DI-water droplets with 1.5 μl volume.
The photocatalytic activity of converted TiO~2~ NT array was investigated by the incubation of a sample (with 1 cm^2^ active area and thickness equal to 6 μm) into 20 mL methylene blue water solution (2 × 10^−5^ M) under UV illumination (30 mW/cm^2^, LC8 Lightningcure, Hamamatsu) and measuring the absorbance spectra each 15 min up to 1 hour. Heat treated (450°C for 1 hour) TiO~2~ NT arrays with the same area and thickness were used as comparison. The residual concentration of MB was evaluated by its optical absorbance peak intensity at 663 nm with a Varian Cary 5000 spectrophotometer.
Author Contributions
====================
A.L. conceived the idea, performed the growth and the crystallization of NT arrays, performed the contact angle characterizations and the photochatalitic degradation experiments, and wrote the paper. A.C. performed FESEM and TEM characterization. N.S. performed Raman and Optical measurements. S.B. performed XRD measurements and the Sherrer analysis. M.Q. performed FTIR measurements. C.F.P. supervised the activity. All authors contributed to the scientific discussion and revision of the article, giving approval to the final version of the manuscript.
Supplementary Material {#s1}
======================
###### Supplementary Information
Supporting Information
Authors would like to thank Miss C. Ottone for the aid in BET measurements and Dr. A. Sacco and Dr. G. Cicero for the fruitful discussions.
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A comparison of the palmaroproximal-palmarodistal view of the isolated navicular bone to other views.
A collection of 69 isolated navicular bones, normal or affected with navicular disease, was examined radiographically using dorsopalmar, lateromedial and palmaroproximal-palmarodistal views. Radiographic findings on each view were recorded. A radiographic diagnosis of normality or navicular disease was made, first on each view separately and afterwards based on the combination of the 3 views. Forty-four navicular bones were considered radiographically normal and 25 navicular bones had distinct signs of radiographic navicular disease. The dorsopalmar and the lateromedial views of each navicular bone were assessed together and compared with the palmaroproximal-palmarodistal view. In not a single bone was the palmaroproximal-palmarodistal view considered indispensable to make the final diagnosis. |
Introduction
============
Aromatic compounds are included among the most widespread organic compounds in nature, and some of them are man-made environmental pollutants ([@B1][@B2][@B4]). Microorganisms play a fundamental role in the degradation of these aromatic compounds in diverse ecological niches ([@B3], [@B5][@B6][@B8]). Many habitats containing large amounts of aromatic compounds are often anoxic. In the last decades, biochemical studies concerning the anaerobic degradation of aromatic compounds have been steadily accumulating, with benzoyl-CoA representing the intermediate to which most monocyclic aromatic compounds are converted ([@B3][@B4][@B5], [@B9][@B10][@B12]). On the contrary, the study on the specific regulatory systems controlling the expression of the gene clusters involved in the anaerobic degradation of aromatic compounds has been mainly restricted to the characterization of a few transcriptional regulators.
Anaerobic benzoate degradation via benzoyl-CoA was shown to be controlled by the two-component BamVW regulatory system ([@B13]) or the BgeR regulator ([@B14]) in the obligate anaerobes *Geobacter* strains, and by the BadR/BadM ([@B15], [@B16]) and BzdR/BoxR ([@B17][@B18][@B20]) regulators in the facultative anaerobes *Rhodopseudomonas palustris* and *Azoarcus* strains, respectively. Moreover, a few global regulators, *e.g.* AadR, AcpR, and AccR, that influence the anaerobic expression of the benzoyl-CoA central pathway have been reported ([@B15], [@B21], [@B22]). A TdiSR (TutC1B1) two-component regulatory system was described for the regulation of the *bss/bbs* genes encoding the peripheral pathway that converts toluene into benzoyl-CoA in denitrifying bacteria ([@B4], [@B23][@B24][@B25]). It was also reported that the regulation of the peripheral routes that funnel 4-hydroxybenzoate and *p*-coumarate into the benzoyl-CoA central pathway in the phototrophic *R. palustris* strain is accomplished by the HbaR and CouR proteins, respectively ([@B26], [@B27]). However, no specific-transcriptional regulators that control anaerobic degradation pathways, other than that of benzoyl-CoA and some peripheral routes that converge to the latter, have been described so far.
*Azoarcus* sp. CIB is a denitrifying β-proteobacterium able to anaerobically degrade different aromatic compounds, including some hydrocarbons such as toluene, via benzoyl-CoA, and *m*-xylene, via 3-methylbenzoyl-CoA ([@B28]). The *Azoarcu*s sp. CIB *bzd* genes responsible for the anaerobic degradation of benzoate are clustered and consist of the *P~N~* promoter-driven *bzdNOPQMSTUVWXYZA* catabolic operon and the *bzdR* regulatory gene ([@B29]). BzdR-mediated repression of *P~N~* is alleviated by the inducer molecule benzoyl-CoA, the first intermediate of the catabolic pathway ([@B17], [@B18]). In addition, the *P~N~* promoter is also subject to control by the benzoyl-CoA-dependent BoxR repressor, a BzdR paralog that regulates the expression of the *box* genes responsible for the aerobic degradation of benzoate in *Azoarcus* sp. CIB ([@B20]). The *mbd* cluster of *Azoarcus* sp. CIB encodes the central pathway responsible for the degradation of the 3-methylbenzoyl-CoA formed during the anaerobic degradation of *m*-xylene and 3-methylbenzoate ([Fig. 1](#F1){ref-type="fig"}) ([@B28]). The *mbd* cluster is organized in at least three operons, *i.e.* the *mbdO-orf9*, *mbdB1-mbdA,* and *mbdR* operons ([Fig. 1](#F1){ref-type="fig"}*A*). The *mbdB1-mbdA* operon is driven by the *P~B~*~1~ promoter and encodes a putative 3-methylbenzoate ABC transporter (MbdB1B2B3B4B5) and the 3-methylbenzoate-CoA ligase (MbdA) that activates 3-methylbenzoate to 3-methylbenzoyl-CoA (peripheral pathway) ([Fig. 1](#F1){ref-type="fig"}*B*). The *mbdO-orf9* operon is regulated by the *P~O~* promoter and encodes the enzymes for the anaerobic conversion of 3-methylbenzoyl-CoA to a hydroxymethylpimelyl-CoA (MbdMNOPQWXYZ) (upper central pathway) and the further degradation of the latter to the central metabolism (Orf1--9) (lower central pathway) ([Fig. 1](#F1){ref-type="fig"}) ([@B28]). The *mbdR* gene was proposed to encode a transcriptional regulator of the TetR family that might regulate the inducible expression of the catabolic *mbd* genes ([@B28]). The efficient expression of the *bzd* and *mbd* genes required the oxygen-dependent AcpR activator, and it was under the control of AccR-mediated carbon catabolite repression by some organic acids and amino acids ([@B22], [@B28]).
In this work we have characterized the promoters of the *mbd* cluster and demonstrated the 3-methylbenzoyl-CoA/MbdR-dependent transcriptional control of the *mbd* genes in *Azoarcus* sp. CIB. The studies on the structural-functional relationships of the MbdR protein expand our current view on the transcriptional regulation of anaerobic pathways, and highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.
EXPERIMENTAL PROCEDURES
=======================
###
#### Bacterial Strains, Plasmids, and Growth Conditions
Bacterial strains and plasmids used are listed in [Table 1](#T1){ref-type="table"}. *Escherichia coli* strains were grown in lysogeny broth (LB) medium ([@B31]) at 37 °C. When required, *E. coli* cells were grown anaerobically in M63 minimal medium ([@B40]) at 30 °C using the corresponding necessary nutritional supplements, 20 m[m]{.smallcaps} glycerol, as carbon source, and 10 m[m]{.smallcaps} nitrate, as terminal electron acceptor. *Azoarcus* sp. CIB strains were grown anaerobically in MC medium at 30 °C, using the indicated carbon source(s) and 10 m[m]{.smallcaps} nitrate as the terminal electron acceptor, as described previously ([@B29]). For aerobic cultivation of *Azoarcus* strains, the same MC medium was used but without nitrate. When appropriate, antibiotics were added at the following concentrations: ampicillin (100 μg ml^−1^), gentamicin (7.5 μg ml^−1^), and kanamycin (50 μg ml^−1^).
######
**Bacterial strains and plasmids used in this study**
Strain or plasmid Description[*^a^*](#TF1-1){ref-type="table-fn"} Ref. or source
------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------- ----------------
***E. coli* strains**
B834 (DE3) F^−^, *ompT*, *hsdS~B~*(*r~B~^−^m~B~^−^*), *gal*, *dcm*, *met*, λDE3 [@B30]
BL21 (DE3) F^−^, *ompT*, *hsdS~B~*(*r~B~^−^m~B~^−^*), *gal*, *dcm*, λDE3 [@B31]
S17-1λpir Tp^r^, Sm^r^, *recA*, *thi*, *hsdRM*+, RP4::2-Tc::Mu::Km, Tn*7*, λ*pir* phage lysogen [@B32]
MC4100 *araD139* Δ(*argF-lac*)U169 *rpsL*150 (Sm^r^) *relA1 flbB*5301 *deo*C1 *ptsF25 rbsR* [@B33]
***Azoarcus* sp. strains**
CIB Wild-type strain [@B29]
CIBd*mbdR* Km^r^, CIB mutant strain with a disruption of the *mbdR* gene This work
CIBd*mbdB1* Km^r^, CIB mutant strain with a disruption of the *mbdB1* gene This work
CIBΔ*P~A~* CIB mutant strain with a deletion of the *P~A~* promoter This work
**Plasmids**
pK18*mob* Km^r^, *ori*ColE1, Mob^+^, *lac*Zα, used for directed insertional disruption [@B34]
pK18mbdRnew Km^r^, pK18*mob* containing a 524-bp HindIII/EcoRI *mbdR* internal fragment This work
pK18mbdB1 Km^r^, pK18*mob* containing a 728-bp EcoRI/XbaI *mbdB1* internal fragment This work
pK18*mobsacB* Km^r^, *ori*ColE1, Mob^+^, *lac*Zα. Vector with a *sacB* selection marker for gene replacement by double site homologous recombination [@B34]
pK18*mobsacB*Δ*P~A~* Km^r^, pK18*mobsacB* containing a chimeric 2.6-kb XbaI/HindIII fragment carrying the Δ*P~A~* This work
pUC19 Ap^R^, *oriColE*1, *lacZ*α [@B31]
pUCmbdA Ap^R^, pUC19 derivative expressing *mbdA* gene under *Plac* control [@B28]
pIZ1016 Gm^r^, *ori*pBBR1, Mob^+^, *lacZ*α, *Ptac*/*lacI^q^*, broad host range cloning and expression vector [@B35]
pIZP~B1~ Gm^r^, pIZ1016 derivative expressing the *P~B~*~1~::*lacZ* fusion [@B28]
pIZP~A~ Gm^r^, pIZ1016 derivative expressing the *P~A~*::*lacZ* fusion This work
pIZP~3R~ Gm^r^, pIZ1016 derivative expressing the *P*~3~*~R~*::*lacZ* fusion This work
pIZmbdA Gm^r^, pIZ1016 derivative expressing the *mbdA* gene under control of *Ptac* This work
pCK01 Cm^r^, *ori*pSC101, low copy number cloning vector [@B36]
pCKmbdR Cm^r^, pCK01 derivative expressing *mbdR* gene under the control of *Plac* promoter This work
pET-29a(+) Km^r^, *ori*ColE1, *P~T~*~7~, cloning and overexpression vector Novagen
pETmbdR Km^r^, pET-29a (+) expressing *mbdR*-His~6~ under *P~T~*~7~ This work
pEHISTEV Km^r^, *ori*ColE1, *P~T~*~7~, coding 6His, TEVpro, cloning, and overexpression vector [@B37]
pEHISTEVMbdR Km^r^, pHISTEV derivative expressing TEV protease-cleavable His~6~ *mbdR* under *P~T~*~7~ This work
pSJ3 Ap^r^, *ori*ColE1, *′lacZ* promoter probe vector [@B38]
pSJ3P~A~ Ap^r^, pSJ3 derivative carrying the *P~A~*::*lacZ* fusion This work
pSJ3P~3R~ Ap^r^, pSJ3 derivative carrying the *P*~3~*~R~*::*lacZ* fusion This work
pSJ3P~B1~ Ap^r^, pSJ3 derivative carrying the *P~B~*~1~::*lacZ* fusion [@B28]
pSJ3P~O~ Ap^r^, pSJ3 derivative carrying the *P~O~*::*lacZ* fusion This work
pJCD01 Ap^r^, *ori*ColE1, polylinker of pUC19 flanked by *rpoC* and *rrnBT*~1~*T2* terminators [@B39]
pJCDP~O~ Ap^r^, pJCD01 derivative harboring a 271-bp ScaI/EcoRI fragment that includes the *P~O~* promoter This work
pJCDP~B1~ Ap^r^, pJCD01 derivative harboring a 251-bp ScaI/EcoRI fragment that includes the *P~B~*~1~ promoter This work
*^a^* The abbreviations used are as follows: Ap^r^, ampicillin-resistant; Cm^r^, chloramphenicol-resistant; Gm^r^, gentamicin-resistant; Km^r^, kanamycin-resistant; Sm^r^, streptomycin- resistant; TEV, tobacco etch virus.
#### Molecular Biology Techniques
Standard molecular biology techniques were performed as described previously ([@B31]). Plasmid DNA was prepared with a High Pure plasmid isolation kit (Roche Applied Science). DNA fragments were purified with Gene-Clean Turbo (Q-biogene). Oligonucleotides were supplied by Sigma. The oligonucleotides employed for PCR amplification of the cloned fragments and other molecular biology techniques are summarized in [Table 2](#T2){ref-type="table"}. All cloned inserts and DNA fragments were confirmed by DNA sequencing with fluorescently labeled dideoxynucleotide terminators ([@B41]) and AmpliTaq FS DNA polymerase (Applied Biosystems) in an ABI Prism 377 automated DNA sequencer (Applied Biosystems). Transformation of *E. coli* cells was carried out by using the RbCl method or by electroporation (Gene Pulser; Bio-Rad) ([@B31]). The proteins were analyzed by SDS-PAGE and Coomassie-stained as described previously ([@B31]). The protein concentration was determined by the method of Bradford ([@B42]) using bovine serum albumin as the standard. Nucleotide sequence analyses were done at the National Center for Biotechnology Information (NCBI) server ([www.ncbi.nlm.nih.gov](http://www.ncbi.nlm.nih.gov)). Pairwise and multiple protein sequence alignments were made with the ClustalW program ([@B43]) at the EMBL-EBI server.
######
**Oligonucleotides used in this study**
Primers Sequence (5′ → 3′)[*^a^*](#TF2-1){ref-type="table-fn"} Use
------------------------ ----------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------
CIB+1P~mbdO~3′ `CATTTGACGTTCTCCTCCTCACTTG` Primer extension *P~O~* promoter
CIB+1P~mbdB1~3′ `CATCTCTCCCTCCTGGACGATGAAG` Primer extension *P~B~*~1~ promoter
PmbdOF1 `GCTGGTATGTTGTGCGGAGTGG` Amplification of 203-bp *P~O~* fragment for RT-PCR assays
bcrBR2 `TGCGCCATCGTACACTCCTCG`
PmbdB1F1 `CGCCGTTTTCCGCAATGACTG` Amplification of 278-bp *P~B~*~1~ fragment for RT-PCR assays
mbdB1R1 `GGCAAAGTGGGCGGGCAGC`
PmbdREcoRI 3′ `CGGAATTCGTTCCAATGGATTTGCCTCTCGG (EcoRI)` Primer extension *P*~3~*~R~* promoter
PmbdAEcoRI 3′ `CGGAATTCCCTCAATGCGCATCAACATAGTG (EcoRI)` Primer extension *P~A~* promoter
5′ mbdRmut2 `GCGAAGCTTACCGTGCGACAACGAT (HindIII)` 524-bp *mbdR* internal fragment cloned into double-digested pK18*mob* to generate pK18mbdRnew
3′ mbdRmut2 `CGGAATTCGCCATTGAGAAGTACCG (EcoRI)`
mbdB1mutEcoRI 5′ `GGAATTCCGGCCGCGAGGTTGAGTACG (EcoRI)` 728-bp *mbdB1* internal fragment cloned into double-digested pK18*mob* to generate pK18mbdB1
mbdB1mutXbaI 3′ `GCTCTAGACCTGCACCGCGTACACGTCG (XbaI)`
P~A~ del. Z1 mbdB4 5′ `GCTCTAGACATTTACGGTATTCGAGAACGCG (XbaI)` 1191-bp *P mbd~B~* flanking region (Z1) cloned together with Z2 into double-digested pK18*mobsacB* to generate pK18*mobsacB*Δ*P~A~*
P~A~ del. Z1 mbdB5 3′ `GGGGTACCTCAAACGCCGAGAAAATTTTTCAAC (KpnI)`
P~A~ del. Z2 Inter. 5′ `GGGGTACCGTCACTATGTTGATGCGCATTGAG (KpnI)` 1451-bp *P~A~* flanking region (Z2) cloned together with Z1 into double-digested pK18*mobsacB* to generate pK18*mobsacB* Δ*P~A~*
P~A~ del. Z2 mbdA 3′ `CCCAAGCTTCAATCTTGAGTACGATCCATGCCTC (HindIII)`
Inter. mbdB5-A 5′ `GGGGTACCAAGTTTTCATTATCTCTAGTACCGG (KpnI)` 238-bp *mbdB5-mbdA* intergenic fragment including *P~A~* promoter cloned into double-digested pSJ3 to generate pSJ3P~A~
Inter. mbdB5-A 3′.2 `GCTCTAGACCCATGGTCGGTTTCCTCAATGCGC (XbaI)`
PmbdRKpnI5′ `GGGGTACCATGCTCGAAGTCAGGTATCCATC (KpnI)` 451-bp *tdiR-mbdR* intergenic fragment including *P*~3~*~R~* promoter cloned into double-digested pSJ3 to generate pSJ3P~3~*~R~*
PmbdRXbaI3′ `GCTCTAGAGGCATGATGTCTGGAGATGTTCC (XbaI)`
PmbdOKpnI5′ `GGGGTACCCATCTCTCCCTCCTGGACGATGAAG (KpnI)` 563-bp *mbdO-mbdB1* intergenic fragment including *P~O~* promoter cloned into double-digested pSJ3 to generate pSJ3P~O~
PmbdOXbaI3′ `GCTCTAGAGGCATTTGACGTTCTCCTCCTCACTTG (XbaI)`
mbdRSalI 5′ `ACGCGTCGACTGACCTAAGGAGGTAAATAATGAGAAAGCTGAACAAGAAGGAAG (SalI)` 676-bp fragment including *mbdR* gene plus a consensus RBS sequence (double underline) for its cloning into double-digested pCK01 to generate pCKmbdR
mbdRPstI 3′ `AACTGCAGTCAGAATGTCGGATTTTTGCAGG (PstI)`
mbdRNdeI 5′ `GGAATTCCATATGAGAAAGCTGAACAAGAAGGAAGAGCAGAG (NdeI)` 651-bp *mbdR* fragment for its cloning into double-digested pET-29 to generate pETmbdR
mbdRXhoI 3′ `CCGCTCGAGGAATGTCGGATTTTTGCAGGAGCC (XhoI)`
mbdRBspHI 5′ `GGCGTCATGAGAAAGCTGAACAAGAAG (BspHI)` 659-bp *mbdR* fragment for its cloning into double-digested pHISTEV to generate pHISTEVMbdR
mbdRBamHI 3′ `ATTCGGATCCTCAGAATGTCGGATTTTTG (BamHI)`
mbdAQ-RT-PCRF3 `CCTTAACACCATGCTGACATCG` 167-bp *mbdA* fragment amplified in real-time RT-PCR
mbdAQ-RT-PCRR5 `CCAGACTTCCGGCAACGTG`
Pdiv\>OScaI 5′.2 `AAAAGTACTGGTATTACGGTAAGTGCTCCACG (ScaI)` 271-bp *mbdO-mbdB1* intergenic fragment including *P~O~* promoter. *P~O~* probe for *in vitro* assays
Pdiv\>OEcoRI 3′ `CCGGAATTCGCTCCCGCGGCTCTTCCAC (EcoRI)`
Pdiv\>B1ScaI 5′.2 `AAAAGTACTCGTGGAGCACTTACCGTAATACC (ScaI)` 251-bp *mbdO-mbdB1* intergenic fragment including *P~B~*~1~ promoter. *P~B~*~1~ probe for *in vitro* assays
Pdiv\>B1EcoRI 3′ `CCGGAATTCCCTGCGCGCGGCACTATG (EcoRI)`
PmbdAScaI 5′ `AAAAGTACTGAGGCCCCGCCCAAGTTTTC (ScaI)` 225-bp *mbdB5-mbdA* intergenic fragment including *P~A~* promoter. *P~A~* probe for *in vitro* assays
PmbdAEcoRI 3′ `CGGAATTCCCTCAATGCGCATCAACATAGTG (EcoRI)`
PmbdRScaI 5′ `AAAAGTACTCACAACTCTTCACCACCAACGCG (ScaI)` 352-bp *tdiR-mbdR* intergenic fragment including *P*~3~*~R~* promoter. *P*~3~*~R~* probe for *in vitro* assays
PmbdREcoRI 3′ `CGGAATTCGTTCCAATGGATTTGCCTCTCGG (EcoRI)`
5′POLIIIHK `GGACGCAGTCTTTTGCGTGGTAAC` 220-bp internal fragment of housekeeping gene *dnaE* (DNApol III α subunit)
3′POLIIIHK `GTGCGTCAAAGTCGCTGCTGTCG`
*^a^* Engineered restriction sites are underlined, and the corresponding restriction enzyme is shown in parentheses.
#### Synthesis and Purification of 3-Methylbenzoyl-CoA
The 3-methylbenzoyl-CoA was synthesized from the corresponding carboxylic acid via its succinimide ester as described ([@B44]). The CoA ester formed was purified by preparative reversed phase HPLC on a 1525 Binary HPLC Pump system (Waters) equipped with a NUCLEOSIL®100--7 C~18~ column (Macherey-Nagel, 50 ml total volume) using acetonitrile in 50 m[m]{.smallcaps} potassium phosphate buffer, pH 6.8, at a flow rate of 8 ml min^−1^. The column was equilibrated with 5% acetonitrile; elution was at 25% acetonitrile in buffer. For removal of phosphate, the freeze-dried CoA ester was suspended in 2% aqueous acetonitrile; elution was with 25% aqueous acetonitrile. The purity was checked by reversed phase HPLC as described above and by the UV-visible spectrum. 3-Methylbenzoyl-CoA was stored at −20 °C as freeze-dried powder.
#### Construction of Azoarcus sp. CIBdmbdR and Azoarcus sp. CIBdmbdB1 Mutant Strains
For insertional disruption of *mbdR* and *mbdB1* through single homologous recombination, an internal region of each gene was PCR-amplified with the primer pairs 5′mbdRmut2/3′mbdRmut2 and mbdB1mutEcoRI5′/mbdB1mutXbaI3′ ([Table 2](#T2){ref-type="table"}). The obtained fragments were double-digested with the appropriate restriction enzymes and cloned into double-digested pK18*mob* vector, generating the pK18mbdRnew and pK18mbdB1 recombinant plasmids ([Table 1](#T1){ref-type="table"}). These plasmids were transferred from *E. coli* S17-1λ*pir* (donor strain) to *Azoarcus* sp. CIB (recipient strain) by biparental filter mating ([@B32]), and exconjugant strains *Azoarcus* sp. CIBd*mbdR* and *Azoarcus* sp. CIBd*mbdB1* were isolated aerobically on kanamycin-containing MC medium harboring 10 m[m]{.smallcaps} glutarate as the sole carbon source for counterselection of donor cells. The mutant strains were analyzed by PCR to confirm the disruption of the target genes.
#### Construction of Azoarcus sp. CIBΔP~A~ Mutant Strain
The *P~A~* promoter was deleted by allelic exchange through homologous recombination using the mobilizable plasmid pK18*mobsacB,* which allows positive selections of double-site recombinants using the *sacB* gene of *Bacillus subtilis* ([@B34]). In summary, two primer pairs ([Table 2](#T2){ref-type="table"}) were used to PCR-amplify the 1191-bp (Z1 fragment) and 1451-bp (Z2 fragment) flanking regions of the *P~A~* promoter. Both fragments were digested with restriction endonuclease KpnI and ligated, and the chimeric DNA harboring a deleted *P~A~* promoter was PCR-amplified, double-digested, and cloned into the pK18*mobsacB* plasmid. The resulting pK18*mobsacB*Δ*P~A~* plasmid was transformed into the *E. coli* S17-1λ*pir* strain (donor strain) and then transferred to *Azoarcus* sp. CIB (recipient strain) by biparental filter mating ([@B32]). Exconjugants containing first site recombination were selected on kanamycin-containing MC medium harboring 10 m[m]{.smallcaps} glutarate as the sole carbon source for counterselection of donor cells. Second site recombination was selected by growth on the same medium supplemented with 5% sucrose and by plating on glutarate-containing MC plates supplemented with 5% sucrose. Correct allelic exchange in sucrose-resistant and kanamycin-sensitive *Azoarcus* sp. CIBΔ*P~A~* was verified by PCR with the appropriate primers ([Table 2](#T2){ref-type="table"}).
#### Construction of a P~A~::lacZ Translational Fusion
The intergenic region between *mbdB5* and *mbdA* genes that includes the *P~A~* promoter was PCR-amplified using the primers Inter.mbdB5-A5′ and Inter.mbdB5-A3′.2 ([Table 2](#T2){ref-type="table"}). The resulting 238-bp fragment was KpnI/XbaI double-digested and cloned upstream of the *lacZ* gene into the double-digested pSJ3 promoter probe vector, generating plasmid pSJ3*P~A~* ([Table 1](#T1){ref-type="table"}). The recombinant pSJ3*P~A~* plasmid was KpnI/HindIII double-digested, and the 3.3-kb fragment containing the *P~A~*::*lacZ* translational fusion was then cloned into the broad host-range pIZ1016 cloning vector ([Table 1](#T1){ref-type="table"}). To this end, pIZ1016 was KpnI/HindIII double-digested and its *Ptac* promoter and polylinker region were replaced by the *P~A~*::*lacZ* translational fusion, generating plasmid pIZP*~A~* ([Table 1](#T1){ref-type="table"}).
#### Construction of a P~3R~::lacZ Translational Fusion
The intergenic region between *tdiR* and *mbdR* genes that includes the *P*~3~*~R~* promoter was PCR-amplified using the primers PmbdRKpnI5′ and PmbdRXbaI3′ ([Table 2](#T2){ref-type="table"}). The resulting 451-bp fragment was KpnI/XbaI double-digested and cloned upstream of a *lacZ* gene into the double-digested pSJ3 promoter probe vector, generating plasmid pSJ3P~3R~ ([Table 1](#T1){ref-type="table"}). The recombinant pSJ3P~3R~ plasmid was KpnI/HindIII double-digested, and the 3.5-kb fragment containing the *P*~3~*~R~*::*lacZ* translational fusion was then cloned into the broad host range pIZ1016 cloning vector ([Table 1](#T1){ref-type="table"}). To this end, pIZ1016 was KpnI/HindIII double-digested and its *Ptac* promoter and polylinker region were replaced by the *P*~3~*~R~*::*lacZ* translational fusion, generating plasmid pIZP~3R~ ([Table 1](#T1){ref-type="table"}).
#### Construction of the pIZmbdA and pCKmbdR Plasmids
The pIZmbdA plasmid is a broad host range plasmid that expresses the *mbdA* gene under the control of the *P~tac~* promoter ([Table 1](#T1){ref-type="table"}). For the construction of pIZmbdA, the 1.7-kb HindIII/XbaI fragment containing the *mbdA* gene from pUCmbdA ([@B28]) was cloned into HindIII/XbaI double-digested pIZ1016 plasmid. The pCKmbdR plasmid ([Table 1](#T1){ref-type="table"}) expresses the *mbdR* gene under control of the *Plac* promoter in the pCK01 cloning vector. To this end, the *mbdR* gene was PCR-amplified as a 676-bp fragment using mbdRSalI5′ and mbdRPstI3′ oligonucleotides ([Table 2](#T2){ref-type="table"}). The SalI/PstI double-digested PCR fragment was then cloned into double-digested pCK01 plasmid to generate pCKmbdR.
#### Overproduction and Purification of MbdR
The recombinant pETmbdR plasmid ([Table 1](#T1){ref-type="table"}) carries the *mbdR* gene, which was PCR-amplified (651-bp) with primers mbdRNdeI5′ and mbdRXhoI3′ ([Table 2](#T2){ref-type="table"}), with a His~6~ tag coding sequence at its 3′-end, under control of the *P~T~*~7~ promoter that is recognized by the T7 phage RNA polymerase. The gene encoding T7 phage RNA polymerase is present in monocopy in *E. coli* BL21(DE3), and its transcription is controlled by the *Plac* promoter and the LacI repressor, making the system inducible by the addition of isopropyl 1-thio-β-[d]{.smallcaps}-galactopyranoside (IPTG).[^5^](#FN5){ref-type="fn"} *E. coli* BL21 (DE3) (pETmbdR) cells were grown at 37 °C in 100 ml of kanamycin-containing LB medium until the culture reached an *A*~600~ of 0.5. Overexpression of the His-tagged protein was then induced during 5 h by the addition of 0.5 m[m]{.smallcaps} IPTG. Cells were harvested at 4 °C, resuspended in 10 ml of 20 m[m]{.smallcaps} imidazole-containing working buffer (50 m[m]{.smallcaps} NaH~2~PO~4~, pH 8, 300 m[m]{.smallcaps} KCl), and disrupted by passage through a French press operated at a pressure of 20,000 p.s.i. Cell debris was removed by centrifugation at 16,000 × *g* for 20 min at 4 °C, and the resulting supernatant was used as crude cell extract. The MbdR-His~6~ protein was purified from the crude cell extract by a single-step nickel-chelating chromatography (nickel-nitrilotriacetic acid spin columns, Qiagen). The column was equilibrated with resuspension buffer, loaded with the crude extract, and washed four times with working buffer plus increasing concentrations of imidazole (20, 75, and 100 m[m]{.smallcaps}). The MbdR-His~6~ protein was eluted in three steps adding to the column working buffer plus increasing concentrations of imidazole (250 and 500 m[m]{.smallcaps} and 1 [m]{.smallcaps}). The purity of MbdR-His~6~ protein was analyzed by SDS-12.5% PAGE. When necessary, the protein solutions were dialyzed against working buffer plus 20 m[m]{.smallcaps} imidazole, concentrated using Vivaspin 500 columns (Sartorius, 10,000 molecular weight cutoff membrane), and stored at 4 °C where they maintained their activity for at least 6 months.
#### Analytical Ultracentrifugation Methods
Sedimentation velocity and equilibrium were performed to determine the state of association of MbdR-His~6~. The analytical ultracentrifugation analysis was performed using several protein concentrations (from 11 to 46 μ[m]{.smallcaps}). All samples were equilibrated in buffer containing 50 m[m]{.smallcaps} NaH~2~PO~4~, 300 m[m]{.smallcaps} KCl, 20 m[m]{.smallcaps} imidazole, pH 8. The sedimentation velocity experiments were carried out at 48,000 rpm and 20 °C in an Optima XL-A analytical ultracentrifuge (Beckman-Coulter Inc.) equipped with UV-visible optic detection system, using an An50Ti rotor and 12-mm double sector centerpieces. Sedimentation profiles were registered every 1--5 min at 260 and 275 nm. The sedimentation coefficient distributions were calculated by least squares boundary modeling of sedimentation velocity data using the *c*(*s*) method ([@B45]), as implemented in the SEDFIT program. These *s* values were corrected to standard conditions (water at 20 °C and infinite dilution) using the SEDNTERP program ([@B46]) to get the corresponding standard *s* values (*s*~20~,*~w~*). Sedimentation equilibrium assays were carried out at speeds ranging from 5000 to 15,000 rpm (depending upon the samples analyzed) and at several wavelengths (260, 280, and 290 nm) with short columns (85--95 μl), using the same experimental conditions and instrument as in the sedimentation velocity experiments. After the equilibrium scans, a high speed centrifugation run (40,000 rpm) was done to estimate the corresponding baseline offsets. The measured low speed equilibrium concentration (signal) gradients of MbdR-His~6~ were fitted using an equation that characterizes the equilibrium gradient of an ideally sedimenting solute (using a MATLAB program, kindly provided by Dr. Allen Minton, National Institutes of Health) to obtain the corresponding buoyant signal average molecular weight.
#### Crystallization and X-ray Crystal Structure Determination of MbdR
To determine the three-dimensional structure of MbdR, the *mbdR* gene from *Azoarcus* sp. CIB was cloned into pEHISTEV vector ([@B37]). To this end, the *mbdR* gene was PCR-amplified with primers mbdRBspHI5′ and mbdRBamHI3′ ([Table 2](#T2){ref-type="table"}) by using genomic DNA of *Azoarcus* sp. CIB as template, digested with BspHI and BamHI, and then ligated into the NcoI/BamHI double-digested pEHISTEV vector, giving rise to plasmid pEHISTEVMbdR. Protein expression of the selenomethionine (SeMet)-substituted recombinant MbdR protein was carried out in *E. coli* B834(DE3) strain ([Table 1](#T1){ref-type="table"}) transformed with pEHISTEVMbdR, and purification was carried out essentially as described previously ([@B47]). The purified SeMet MbdR protein has an extra glycine and alanine at the N terminus resulting from cleavage of the engineered hexa-histidine tag. Crystallization of SeMet MbdR was carried out as described previously ([@B47]), and the MbdR crystals were finally grown in the optimized condition of 0.1 [m]{.smallcaps} MOPS, pH 7.0, 28% PEG3550, and 0.08% (NH~4~)~2~PO~4~. Structure was determined using SeMet MAD data and refined using CCP4 package ([@B48]). The atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession number [4uds](4uds). Crystallization of MbdR·inducer complex was tried out using the purified MbdR protein with 3-methylbenzoyl-CoA either by co-crystallization or crystal socking, but in both cases the production of crystals failed.
#### RNA Extraction and RT-PCR Assays
*Azoarcus* cells grown in MC medium harboring the appropriate carbon source were harvested at the mid-exponential phase of growth and stored at −80 °C. Pellets were thawed, and cells were lysed in TE buffer (10 Tris-HCl, pH 7.5, 1 m[m]{.smallcaps} EDTA) containing 50 mg ml^−1^ lysozyme. Total RNA was extracted using the RNeasy mini kit (Qiagen), including a DNase treatment according to the manufacturer\'s instructions (Ambion), precipitated with ethanol, washed, and resuspended in RNase-free water. The concentration and purity of the RNA samples were measured by using a, ND1000 spectrophotometer (Nanodrop Technologies) according to the manufacturer\'s protocols. Synthesis of total cDNA was carried out with 20 μl of reverse transcription reactions containing 400 ng of RNA, 0.5 m[m]{.smallcaps} concentrations of each dNTP, 200 units of SuperScript II reverse transcriptase (Invitrogen), and 5 μ[m]{.smallcaps} concentrations of random hexamers as primers in the buffer recommended by the manufacturer. Samples were initially heated at 65 °C for 5 min then incubated at 42 °C for 2 h, and the reactions were terminated by incubation at 70 °C for 15 min. In standard RT-PCRs, the cDNA was amplified with 1 unit of AmpliTaq DNA polymerase (Biotools) and 0.5 μ[m]{.smallcaps} concentrations of the corresponding primer pairs ([Table 2](#T2){ref-type="table"}). Control reactions in which reverse transcriptase was omitted from the reaction mixture ensured that DNA products resulted from the amplification of cDNA rather than from DNA contamination. The *dnaE* gene encoding the α-subunit of DNA polymerase III was used to provide an internal control cDNA that was amplified with oligonucleotides 5′POLIIIHK/3′POLIIIHK ([Table 2](#T2){ref-type="table"}). The expression of the internal control was shown to be constant across all samples analyzed. For real time RT-PCR assays, the cDNA was purified using the GENECLEAN® Turbo kit (MP Biomedicals), and the concentration was measured using an ND1000 spectrophotometer (Nanodrop Technologies). The IQ5 Multicolor Real Time PCR Detection System (Bio-Rad) was used for real time PCR in a 25-μl reaction containing 10 μl of diluted cDNA (5 ng in each reaction), 0.2 μ[m]{.smallcaps} primer 5′, 0.2 μ[m]{.smallcaps} primer 3′, and 12.5 μl of SYBR Green Mix (Applied Biosystems). The oligonucleotides used to amplify a fragment of *mbdA* were mbdAQ-RT-PCRF3 and mbdAQ-RT-PCRR5 ([Table 2](#T2){ref-type="table"}). PCR amplifications were carried out as follows: 1 initial cycle of denaturation (95 °C for 4 min) followed by 30 cycles of amplification (95 °C, 1 min; test annealing temperature, 60 °C, 1 min; elongation and signal acquisition, 72 °C, 30 s). Each reaction was performed in triplicate. After the PCR, a melting curve was generated to confirm the amplification of a single product. For relative quantification of the fluorescence values, a calibration curve was constructed by 5-fold serial dilutions of an *Azoarcus* sp. CIB genomic DNA sample ranging from 0.5 to 0.5 × 10^−4^ ng. This curve was then used as a reference standard for extrapolating the relative abundance of the cDNA target within the linear range of the curve. Results were normalized relative to those obtained for the *dnaE* internal control.
#### Gel Retardation Assays
DNA probes containing *P~O~*, *P~B~*~1~, *P~A~*, and *P*~3~*~R~* promoters were PCR-amplified with the corresponding primers indicated in [Table 2](#T2){ref-type="table"}. The amplified DNA was then digested with ScaI and EcoRI restriction enzymes and single end-labeled by filling in the overhanging EcoRI-digested end with \[α-^32^\]dATP (6000 Ci/mmol; PerkinElmer Life Sciences) and the Klenow fragment of *E. coli* DNA polymerase I as described previously ([@B31]). The labeled fragments (*P~O~*, *P~B~*~1~, *P~A~*, and *P*~3~*~R~* probes) were purified using GENECLEAN Turbo (Qbiogen). The retardation reaction mixtures contained 20 m[m]{.smallcaps} Tris-HCl, pH 7.5, 10% glycerol, 50 m[m]{.smallcaps} KCl, 0.05 n[m]{.smallcaps} DNA probe, 250 μg/ml bovine serum albumin, 50 μg/ml unspecific salmon sperm DNA, and purified MbdR-His~6~ protein in a 9-μl final volume. After incubation of the retardation mixtures for 20 min at 30 °C, mixtures were fractionated by electrophoresis in 5% polyacrylamide gels buffered with 0.5× TBE (45 m[m]{.smallcaps} Tris borate, 1 m[m]{.smallcaps} EDTA). The gels were dried onto Whatman 3MM paper and exposed to Hyperfilm MP (Amersham Biosciences) accompanied by amplifier screens (Cronex Lightning Plus, DuPont). The radioactivity present in the retardation complexes and free probes was quantified by using a densitometer with the Quantity One software (Bio-Rad).
#### DNase I Footprinting Assays
The DNA ^32^P-probes used for these experiments were labeled as indicated for the gel retardation assays. The reaction mixture contained 2 n[m]{.smallcaps} DNA probe (*P~O~*, *P~B~*~1~, or *P~A~*), 500 μg/ml bovine serum albumin, and purified MbdR-His~6~ protein in 15 μl of buffer (20 m[m]{.smallcaps} Tris-HCl, pH 7.5, 10% glycerol, 50 m[m]{.smallcaps} KCl). This mixture was incubated for 20 min at 30 °C, after which 3 μl (0.05 units) of DNase I (Roche Applied Science) (prepared in 10 m[m]{.smallcaps} CaCl~2~, 10 m[m]{.smallcaps} MgCl~2~, 125 m[m]{.smallcaps} KCl, and 10 m[m]{.smallcaps} Tris-HCl, pH 7.5) was added, and the incubation was continued at 37 °C for 20 s. The reaction was stopped by the addition of 180 μl of a solution containing 0.4 [m]{.smallcaps} sodium acetate, 2.5 m[m]{.smallcaps} EDTA, 50 μg/ml salmon sperm DNA, and 0.3 μl/ml glycogen. After phenol extraction, DNA fragments were precipitated with absolute ethanol, washed with 70% ethanol, dried, and directly resuspended in 90% (v/v) formamide-loading gel buffer (10 m[m]{.smallcaps} Tris-HCl, pH 8, 20 m[m]{.smallcaps} EDTA, pH 8, 0.05% w/v bromphenol blue, 0.05% w/v xylene cyanol). Samples were then denatured at 95 °C for 3 min and fractionated in a 6% polyacrylamide-urea gel. A+G Maxam and Gilbert reactions ([@B49]) were carried out with the same fragments and loaded in the gels along with the footprinting samples. The gels were dried onto Whatman 3MM paper and visualized by autoradiography as described previously.
#### Primer Extension Analyses
*Azoarcus* sp. CIB cells were grown anaerobically on MC medium plus 3-methylbenzoate (inducing conditions) or benzoate (control condition) until mid-exponential phase. For the primer extension analysis of *P~O~* and *P~B~*~1~ promoters, total RNA was isolated by using RNeasy mini kit (Qiagen) according to the instructions of the supplier. In the case of *P~A~* and *P*~3~*~R~* promoters, the procedure was the same but *Azoarcus* sp. CIB strains harboring pIZP~A~ or pIZP~3R~ plasmids were used instead of the parental strain due to the weaker nature of these promoters. Primer extension reactions were carried out with the avian myeloblastosis virus reverse transcriptase (Promega) and 15 μg of total RNA as described previously ([@B17]), using oligonucleotides CIB+1P~mbdO~3′, CIB+1P~mbdB1~3′, PmbdREcoRI3′, and PmbdAEcoRI3′ ([Table 2](#T2){ref-type="table"}), which hybridize with the coding strand of the *mbdO*, *mbdB1*, *mbdR,* and *mbdA* genes, respectively. These oligonucleotides were labeled at their 5′-end with phage T4 polynucleotide kinase and \[γ-^32^P\]ATP (3000 Ci/mmol; PerkinElmer Life Sciences). To determine the length of the primer extension products, sequencing reactions of plasmids pSJ3P~O~, pSJ3P~B1~, pIZP~A~, and pIZP~3R~ ([Table 1](#T1){ref-type="table"}) were carried out with oligonucleotides CIB+1P~mbdO~3′, CIB+1P~mbdB1~3′, PmbdAEcoRI3′, and PmbdREcoRI3′, respectively, using the T7 sequencing kit and \[α^32^P\]dATP (PerkinElmer Life Sciences) as indicated by the supplier. Products were analyzed on 6% polyacrylamide-urea gels. The gels were dried on Whatman 3MM paper and exposed to Hyperfilm MP (Amersham Biosciences).
#### In Vitro Transcription Experiments
Multiple-round *in vitro* transcription assays were performed as published previously ([@B50]). Plasmids pJCDP~O~ and pJCDP~B1~ ([Table 1](#T1){ref-type="table"}) were used as supercoiled *P~O~* and *P~B~*~1~ templates. Reactions (50-μl mixtures) were performed in a buffer consisting of 50 m[m]{.smallcaps} Tris-HCl, pH 7.5, 50 m[m]{.smallcaps} KCl, 10 m[m]{.smallcaps} MgCl~2~, 0.1 m[m]{.smallcaps} bovine serum albumin, 10 m[m]{.smallcaps} dithiothreitol (DTT), and 1 m[m]{.smallcaps} EDTA. Each DNA template (0.25 n[m]{.smallcaps}) of supercoiled plasmids pJCDP~O~ or pJCDP~B1~ was premixed with 30 n[m]{.smallcaps} σ^70^-containing *E. coli* RNA polymerase (1 unit/μl; United States Biochemical Corp.), different amounts of purified MbdR-His~6~ protein, and different concentrations of the 3-methylbenzoyl-CoA inducer. For multiple-round assays, transcription was then initiated by adding a mixture of 500 μ[m]{.smallcaps} (each) ATP, CTP, and GTP, 50 μ[m]{.smallcaps} UTP, and 2.5 μCi of \[α-^32^P\]UTP (3000 Ci/mmol; PerkinElmer Life Sciences). After incubation for 15 min at 37 °C, the reactions were stopped with an equal volume of a solution containing 50 m[m]{.smallcaps} EDTA, 350 m[m]{.smallcaps} NaCl, and 0.5 mg/ml carrier tRNA. The mRNA produced was then precipitated with ethanol, dissolved in loading buffer (7 [m]{.smallcaps} urea, 1 m[m]{.smallcaps} EDTA, 0.6 [m]{.smallcaps} glycerol, 0.9 m[m]{.smallcaps} bromphenol blue, and 1.1 m[m]{.smallcaps} xylene cyanol), electrophoresed on a denaturing 7 [m]{.smallcaps} urea, 4% polyacrylamide gel, and visualized by autoradiography.
#### β-Galactosidase Assays
The β-galactosidase activities from promoter-*lacZ* reporter fusions were measured with permeabilized cells when cultures reached mid-exponential phase, as described by Miller ([@B40]).
RESULTS
=======
###
#### mbdR Gene Encodes a Specific Repressor of the P~O~ and P~B1~ Promoters in Azoarcus sp. CIB
*In silico* analysis at the 3′-end of the *mbd* cluster revealed a gene, *mbdR*, that encodes a putative specific transcriptional regulator ([Fig. 1](#F1){ref-type="fig"}) ([@B28]). To analyze the role of the *mbdR* gene in the expression of the catabolic and transport *mbd* genes, an *mbdR* disruptional insertion mutant (*Azoarcus* sp. CIBd*mbdR* strain; [Table 1](#T1){ref-type="table"}) was constructed. Because *Azoarcus* sp. CIBd*mbdR* mutant strain grew normally on minimal medium containing 3-methylbenzoate as the only carbon source, the *mbdR* gene does not seem to function as a transcriptional activator of the *mbd* genes. Wild-type *Azoarcus* sp. CIB strain and *Azoarcus* sp. CIBd*mbdR* mutant strain were grown anaerobically on minimal medium containing benzoate (control condition) or 3-methylbenzoate (inducing condition) as the only carbon sources, and the expression from *P~O~* and *P~B~*~1~ promoters was analyzed by RT-PCR experiments. Whereas the wild-type strain showed a clear induction of the *P~O~* and *P~B~*~1~ promoters when grown in 3-methylbenzoate, the MbdR mutant exhibited expression from the *P~O~* and *P~B~*~1~ promoters when growing both in benzoate or 3-methylbenzoate ([Fig. 2](#F2){ref-type="fig"}, *A* and *B*). Hence, these results support the idea that MbdR acts as a specific transcriptional repressor of the *P~O~* and *P~B~*~1~ promoters.
{#F1}
![**MbdR protein controls the *P~O~* and *P~B~*~1~ promoters.** *A* and *B*, activity of the *P~O~* and *P~B~*~1~ promoters in wild-type *Azoarcus* sp. CIB and the *Azoarcus* sp. CIBd*mbdR* mutant strain. Agarose gel electrophoresis of RT-PCR products obtained from the divergent promoters *P~O~* (*A*) and *P~B~*~1~ (*B*). Total RNA was extracted from *Azoarcus* sp. CIB (*wt*) and *Azoarcus* sp. CIBd*mbd*R (d*mbd*R) cells grown under denitrifying conditions using 3 m[m]{.smallcaps} benzoate (*lane B*) or 3 m[m]{.smallcaps} 3-methylbenzoate (*lane 3M*) as sole carbon sources. The primer pairs used to amplify the *mbdO* (*P~O~*) and *mbdB1* (*P~B~*~1~) gene fragments as described under "Experimental Procedures" are detailed in [Table 2](#T2){ref-type="table"}. *Lane M*, molecular size markers (HaeIII-digested ΦX174 DNA). *Numbers* on the *left* represent the sizes of the markers (in base pairs). *C* and *D*, the MbdR protein binds to the *P~O~* and *P~B~*~1~ promoters. Gel retardation assays were performed as indicated under "Experimental Procedures." *C* shows the interaction between increasing concentrations of purified MbdR-His~6~ protein and a DNA probe (271-bp) containing the *P~O~* promoter. *D* shows the interaction between increasing concentrations of purified MbdR-His~6~ protein and a DNA probe (251-bp) containing the *P~B~*~1~ promoter. *Lane numbers* refer to the MbdR-His~6~ protein concentration (nanomolar) used for each reaction. *P~O~* and *P~B~*~1~ probes as well as the major *P~O~*·MbdR and *P~B~*~1~·MbdR complexes are marked with *arrows*.](zbc0231515780002){#F2}
#### MbdR Is a New Member of the TetR Family of Transcriptional Regulators
Analysis of the primary structure of MbdR shows an overall low amino acid sequence similarity to members of the TetR family of transcriptional regulators ([Fig. 3](#F3){ref-type="fig"}) ([@B51], [@B52]). To determine the structure of the MbdR repressor, we cloned and expressed in the pETmbdR plasmid ([Table 1](#T1){ref-type="table"}) a C-terminally His-tagged version of the MbdR protein. The MbdR protein (24.9 kDa) was overproduced in *E. coli* BL21 (DE3) cells harboring plasmid pETmbdR and purified from the soluble protein fraction by a single-step affinity chromatography (data not shown). The oligomeric state of MbdR protein in solution was determined by analytical ultracentrifugation experiments carried out at different concentrations (11--46 μ[m]{.smallcaps}) of MbdR. Sedimentation velocity analysis of 11 μ[m]{.smallcaps} MbdR revealed a single species with a sedimentation (*s*) value of 2.9 ± 0.1 (data not shown). The molecular mass of the 2.9 S species, as measured by sedimentation equilibrium, is compatible with the mass of the MbdR dimer (data not shown). Because the frictional ratio *f/f*~0~ was 1.46, the shape of the MbdR dimer deviates from that expected for a globular protein and suggests a slightly elongated dimer.
) are drawn as *bars* at the *top* of the alignment. The N-terminal α1--α3 helices that constitute the DNA binding domain are shown as *filled bars*, with the helix-turn-helix motif indicated in *bold* and *italics*. The C-terminal α4--α9 helices that constitute the dimerization and ligand binding domain are shown as *open bars*.](zbc0231515780003){#F3}
The crystal structure of MbdR was determined using multiple wavelength anomalous diffraction data, and it was refined to 1.76 Å resolution. A summary of the crystallographic statistics is shown in [Table 3](#T3){ref-type="table"}. The crystal structure reveals that the crystallographic asymmetric unit contains a monomer of the protein ([Fig. 4](#F4){ref-type="fig"}*A*). The N-terminal 14 amino acids, residues Thr-46 and Lys-47, and the C-terminal 10 residues in the structure are disordered. Helices α1 to α3 (Ala-13 to Phe-54) make up the N-terminal DNA binding domain and contain the helix-turn-helix motif ([Fig. 3](#F3){ref-type="fig"}). The larger C-terminal ligand binding domain of MbdR ([Fig. 3](#F3){ref-type="fig"}) consists of helices α4 to α9 (Lys-57 to Val-204) ([Fig. 4](#F4){ref-type="fig"}*A*). The long axis of helices α4, α5, α7, α8, and α9 are approximately parallel and at right angles to α1. The short helix α6 lies approximately parallel to α1 and bisects the C-terminal domain with α4 and α7 on the one side and α5, α8, and α9 on the other side ([Fig. 4](#F4){ref-type="fig"}*A*). A 2-fold crystallographic symmetry operator (arises in space group I222) sits parallel to α4 and generates a dimeric arrangement. The dimer interface is formed mainly by helices α8 and α9 with small contributions from helices α6 and α7. In total, the dimer buries 1759 Å^2^/monomer of surface area with mostly hydrophobic residues ([Fig. 4](#F4){ref-type="fig"}*B*).
######
**X-ray crystallographic phasing and refinement statistics**
Values in parentheses relate to the highest resolution shell.
X-ray source Diamond Io3
--------------------------------------------------------- -----------------------------------------------------
Wavelength (Å) 0.9792
Resolution (Å) 52.35-1.76 (1.8-1.76)
Space group I222
Unit cell (Å) *a* = 47.0, *b* = 56.2, *c* = 143.8; α = β = γ = 90
Unique reflections 17,349
Completeness (%) 99.3 (76.7)
Redundancy 7 (6.3)
*R*~merge~ (%) 10.8 (83.5)
*I/*σ (*I*) 17.1 (4.1)
*V~m~* (Å/Da) 1.98 (1mol)
Solvent (%) 37.9
**Refinement**
*R*~work~/*R*~free~ 17.61/22.61 (18.53/24.17)
Figure of merit[*^a^*](#TF3-1){ref-type="table-fn"} 0.8
**Root mean square deviation**
Bonds (Å)/angle (°) 0.022/0.74
Average *B*-factor
All atoms (1677, A^2^) 24.0
**Ramachandran**
Preferred regions (%) 98.92
Allowed regions (%) 1.08
Outlier (%) 0
PDB code [4uds](4uds)
*^a^* The figure of merit is calculated after density modification.
![**Three-dimensional structure of MbdR.** *A*, *ribbon* diagram of the three-dimensional structure of the MbdR monomer, which belongs to space group I222. The refined structure has *R*~work~ of 0.185 and *R*~free~ of 0.242 with % completeness of 99.3. *B*, *ribbon* diagram of the MbdR dimer generated using two neighboring monomers, showing the interface and the buried residues. *C*, molecular surface representation of the MbdR dimer with rotation of 90° backward to show the MbdR-DNA interaction surface. *Red* and *blue* surfaces represent negative and positive electrostatic potentials. *D*, similarity (superposition) of MbdR (*red*) to the structures of other TetR-like regulators such as AcrR (3LHQ, *gold*), EthR (3G1O, *tan*), HapR (2PBX, *yellow*), IcaR (2ZCN, *green*), QacR (3BTL, *blue*), and TetR (3LWJ, *cyan*). *E*, superimposition of the MbdR apo-structure (*gold*) and the QacR·4,4′\[1,6-hexanediylbis(oxy)\]bisbenzenecarboximidamide (*red*) complex structure (*blue*) (3BTJ) to show the proposed internal cavity of MbdR induced by 3-methylbenzoyl-CoA binding. *F,* putative key residues comprising the ligand-binding pocket of MbdR are shown as *sticks.* Figures were drawn using the PyMOL program.](zbc0231515780004){#F4}
Taken together, all these results indicate that the MbdR homodimer shows the characteristic structure of the TetR family regulators. The members of the TetR family are mostly repressors ([@B51], [@B52]), and MbdR behaves also as a transcriptional repressor of the *mbd* genes responsible for the anaerobic catabolism of 3-methylbenzoate.
#### MbdR Binds to Palindrome Operator Sites within P~O~ and P~B1~ Promoters
To confirm *in vitro* that the MbdR regulator directly interacts with the *P~O~* and *P~B~*~1~ promoters, gel retardation experiments were carried out with purified MbdR and a 271-bp DNA harboring *P~O~* or a 251-bp DNA containing *P~B~*~1~ as probes. The MbdR protein was able to retard the migration of both DNA probes in a protein concentration-dependent manner ([Fig. 2](#F2){ref-type="fig"}, *C* and *D*). The affinity of MbdR for both *P~O~* and *P~B~*~1~ probes was very similar, showing a relative *K~d~* of 1.71 ± 0.18 and 3.72 ± 0.03 n[m]{.smallcaps}, respectively. To further study the interaction of the MbdR protein with the *P~O~* and *P~B~*~1~ promoters, we mapped the transcription start sites of both promoters. Primer extension analyses were performed with total RNA isolated from *Azoarcus* sp. CIB cells grown exponentially in benzoate (control condition) or 3-methylbenzoate (inducing condition). Whereas no transcript was observed from cells growing in benzoate, a transcript band was visible from cells growing in 3-methylbenzoate ([Fig. 5](#F5){ref-type="fig"}, *A* and *B*), confirming a 3-methylbenzoate-dependent activation of the *P~O~* and *P~B~*~1~ promoters. The transcription start site at the *P~O~* and *P~B~*~1~ promoters was mapped at a guanine located 137 and 138 bp upstream of the ATG translation initiation codon of the *mbdO* and *mbdB1* genes, respectively.
![**MbdR protein interacts with the *P~O~* and *P~B~*~1~ promoter regions.** *A* and *B*, determination of the transcription start site in the *P~O~* and *P~B~*~1~ promoters. Total RNA was isolated from *Azoarcus* sp. CIB cells growing on 3-methylbenzoate (inducing condition) or benzoate (control condition) as sole carbon sources as described under "Experimental Procedures." The size of the extended products under inducing conditions (*lane 3M*) or noninducing conditions (*lane B*) was determined by comparison with the DNA sequencing ladder (*lanes A, T, C,* and *G*) of the *P~O~* (*A*) and *P~B~*~1~ (*B*) promoter regions. Primer extension and sequencing reactions of the *P~O~* and *P~B~*~1~ promoters were performed with primers CIB+1P~mbdO~3′ and CIB+1P~mbdB1~3′, respectively, as described under "Experimental Procedures." An expanded view of the nucleotides surrounding the transcription initiation site (*circled*) in the noncoding strand is shown. The longest extension product is shown by an *arrow. C* and *D*, DNase I footprinting analyses of the interaction of MbdR with the *P~O~* and *P~B~*~1~ promoter regions. The DNase I footprinting experiments were carried out using the *P~B~*~1~ (*C*) and *P~O~* (*D*) probes labeled as indicated under "Experimental Procedures." *Lanes AG*, show the A+G Maxam and Gilbert sequencing reaction. *Lanes A--G* show footprinting assays containing increasing concentrations of MbdR-His~6~. *Lanes F* (*C*) and *G* (*D*) show footprinting assays containing MbdR-His~6~ plus 250 μ[m]{.smallcaps} 3-methylbenzoyl-CoA (*3MCoA*). Phosphodiester bonds hypersensitive to DNase I cleavage are indicated by *asterisks*. On the *left side* of each panel, an expanded view of the promoter region is shown. Protected regions are *shaded* in *gray* over the promoter sequences. The −10/−35 regions are *boxed,* and the transcription initiation sites (+1) are *underlined*. The predicted MbdR operators are flanked by palindrome sequences indicated by convergent *dotted arrows*.](zbc0231515780005){#F5}
To characterize the DNA-binding sites of MbdR within the *P~O~* and *P~B~*~1~ promoters, we performed DNase I footprinting assays. As shown in [Fig. 5](#F5){ref-type="fig"}, *C* and *D*, MbdR protected DNA regions spanning from positions +18 to −16 and from −4 to −34 with respect to the transcription start sites of the *P~O~* and *P~B~*~1~ promoters, respectively. The protected regions contained a conserved palindromic sequence (ATAC*N*~10~GTAT) that is suggested to be the operator sequence recognized by MbdR. The MbdR operator in *P~O~* and *P~B~*~1~ promoters spans the transcription initiation sites as well as the −10 and −35 (only in *P~B~*~1~) sequences for recognition of the σ^70^-dependent RNA polymerase ([Fig. 5](#F5){ref-type="fig"}, *C* and *D*). Therefore, the characterization of the MbdR operator supports the observed repressor role of MbdR at the *P~O~* and *P~B~*~1~ promoters ([Fig. 2](#F2){ref-type="fig"}, *A* and *B*).
#### 3-Methylbenzoyl-CoA Is the Inducer That Alleviates the MbdR-dependent Repression of the mbd Genes
To identify the inducer molecule that alleviates the specific repression exerted by MbdR on the expression of the *mbd* genes, we first accomplished an *in vivo* approach. Thus, the activity of a *P~B~*~1~::*lacZ* translational fusion in plasmid pIZP~B1~ ([Table 1](#T1){ref-type="table"}) was measured in *E. coli* cells harboring also the pCKmbdR plasmid that expresses the *mbdR* gene under the IPTG-controlled *Plac* promoter ([Table 1](#T1){ref-type="table"}). As shown in [Fig. 6](#F6){ref-type="fig"}*A*, the β-galactosidase activity levels of recombinant *E. coli* cells expressing the *mbdR* gene and grown anaerobically in minimal medium with glycerol as sole carbon source were significantly lower than those obtained in *E. coli* control cells lacking the *mbdR* gene. This result confirms in a heterologous host the role of MbdR as a transcriptional repressor of the *mbd* genes. Interestingly, the addition of 3-methylbenzoate to the culture medium of recombinant *E. coli* cells unable to metabolize this aromatic acid did not alleviate the repression exerted by MbdR ([Fig. 6](#F6){ref-type="fig"}*A*), suggesting that 3-methylbenzoate, the substrate of the mbd pathway, is not the specific inducer of the *P~B~*~1~ promoter. It has been described previously that the transcriptional activation of benzoate degradation operons in *Azoarcus* sp. CIB requires benzoyl-CoA, the first intermediate of the anaerobic/aerobic degradation pathways, as inducer molecule ([@B17], [@B20]). Thus, we checked whether 3-methylbenzoyl-CoA, the first CoA-derived intermediate of the mbd pathway, could be the specific inducer molecule of the *mbd* genes. To this end, we expressed the *mbdA* gene encoding the 3-methylbenzoate-CoA ligase (MbdA) that catalyzes the transformation of 3-methylbenzoate to 3-methylbenzoyl-CoA ([@B28]), in the reporter *E. coli* strain containing plasmids pIZP~B1~ and pCKmbdR. As shown in [Fig. 6](#F6){ref-type="fig"}*A*, the activity of the *P~B~*~1~ promoter increased after the addition of 3-methylbenzoate to the culture medium, suggesting that 3-methylbenzoyl-CoA is the specific inducer of the MbdR repressor.
{ref-type="table"}) were grown anaerobically in glycerol-containing minimal medium, supplemented with 0.5 m[m]{.smallcaps} IPTG to allow expression of the *mbdR* and *mbdA* genes, in the absence (−3MBz) or presence (+3MBz) of 3 m[m]{.smallcaps} 3-methylbenzoate until they reached mid-exponential phase. Values for β-galactosidase activity were determined as indicated under "Experimental Procedures," and they are represented as a percentage of the activity from *E. coli* MC4100 (pIZP~B1~) cells (4000 Miller units). Each value is the average from three separate experiments (*error bars* indicate S.D.). *B*, interaction of MbdR with the *P~O~* and *P~B~*~1~ promoters in the presence of 3-methylbenzoyl-CoA. Gel retardation assays were performed as indicated under "Experimental Procedures," and they show the interaction between MbdR-His~6~ protein (30 n[m]{.smallcaps}), the *P~O~* (271-bp) or *P~B~*~1~ (251-bp) DNA probes, and increasing concentrations of 3-methylbenzoyl-CoA (*3MBzCoA*). *Lane* −, free *P~O~* and *P~B~*~1~ probes. *Lane numbers* refer to the 3-methylbenzoyl-CoA concentration (μ[m]{.smallcaps}) used for each assay. *P~O~* and *P~B~*~1~ probes, as well as the *P~O~*·MbdR and *P~B~*~1~·MbdR complexes are marked with *arrows. C,* interaction of MbdR with the *P~O~* promoter in the presence of different aromatic compounds. Gel retardation assays were performed as indicated under "Experimental Procedures," and they show the interaction between purified MbdR-His~6~ protein (30 n[m]{.smallcaps}) and the *P~O~* probe in the absence (*lane A*) or presence (*lanes B--F*) of different aromatic compounds: *lane B*, 250 μ[m]{.smallcaps} 3-methylbenzoyl-CoA; *lane C*, 2 m[m]{.smallcaps} benzoyl-CoA; *lane D*, 2 m[m]{.smallcaps} phenylacetyl-CoA; *lane E*, 2 m[m]{.smallcaps} 3-methylbenzoate; *lane F*, 2 m[m]{.smallcaps} 3-methylbenzoate + 2 m[m]{.smallcaps} CoA. *Lane* −, free *P~O~* probe. The *P~O~* probe and the *P~O~*·MbdR complex are marked with *arrows. D*, effect of MbdR and 3-methylbenzoyl-CoA on *in vitro* transcription from *P~O~* and *P~B~*~1~. Multiple-round *in vitro* transcription reactions were performed as indicated under "Experimental Procedures" by using pJCDP~O~ and pJCDP~B1~ plasmid templates ([Table 1](#T1){ref-type="table"}) that produce 227- and 224-nucleotide mRNAs (*arrows*) from *P~O~* and *P~B~*~1~ promoters, respectively, and 30 n[m]{.smallcaps} *E. coli* RNA polymerase. The transcription reactions were carried in the absence of repressor (*lanes* −) or in the presence of 100 n[m]{.smallcaps} MbdR-His~6~ with increasing concentrations of 3-methylbenzoyl-CoA (*3MBzCoA*). *Lane numbers* refer to the 3-methylbenzoyl-CoA concentration (μ[m]{.smallcaps}) used for each assay.](zbc0231515780006){#F6}
*In vitro* experiments were then performed to confirm the direct role of 3-methylbenzoyl-CoA as the inducer molecule of the *mbd* cluster. First, gel retardation experiments showed that the presence of 3-methylbenzoyl-CoA inhibited the interaction of MbdR with the *P~O~* and *P~B~*~1~ probes ([Fig. 6](#F6){ref-type="fig"}*B*). On the contrary, 3-methylbenzoate or some 3-methylbenzoyl-CoA analogs, such as benzoyl-CoA or phenylacetyl-CoA, did not avoid the interaction of MbdR with its target promoters ([Fig. 6](#F6){ref-type="fig"}*C*), suggesting that MbdR recognizes 3-methylbenzoyl-CoA specifically. The inducing effect of 3-methylbenzoyl-CoA was also observed in footprinting assays where the addition of 3-methylbenzoyl-CoA reverted the protection of MbdR against the DNase I digestion on the *P~O~* and *P~B~*~1~ promoters ([Fig. 5](#F5){ref-type="fig"}, *C* and *D*).
The role of MbdR as a specific transcriptional repressor of the *P~O~* and *P~B~*~1~ promoters and 3-methylbenzoyl-CoA as the cognate inducer was also demonstrated by *in vitro* transcription assays using supercoiled DNA templates bearing each of the two promoters. Thus, [Fig. 6](#F6){ref-type="fig"}*D* shows the MbdR-dependent repression of the *P~O~* and *P~B~*~1~ promoters, and it also reveals how the addition of increasing amounts of 3-methylbenzoyl-CoA leads to formation of the expected transcripts from both promoters.
#### Identification of Additional MbdR-dependent Promoters in the mbd Cluster, the P~3R~ and P~A~ Promoters
Nucleotide sequence analysis of the intergenic regions of the *mbd* cluster revealed putative MbdR binding regions that contain the conserved (ATAC*N*~10~GTAT) palindromic sequence in the *P*~3~*~R~* promoter that drives the expression of *mbdR* ([Fig. 1](#F1){ref-type="fig"}) ([@B28]) and upstream of the *mbdA* gene encoding the 3-methylbenzoate-CoA ligase (putative *P~A~* promoter). To experimentally validate that *P*~3~*~R~* and *P~A~* are functional promoters of the *mbd* cluster, the upstream region of *mbdR* and the *mbdB5-mbdA* intergenic region were cloned into the promoter probe vector pSJ3, rendering plasmids pSJ3P~3R~ and pSJ3P~A~ that contain the *P*~3~*~R~*::*lacZ* and *P~A~*::*lacZ* translational fusions, respectively ([Table 1](#T1){ref-type="table"}). Both translational fusions were then subcloned into the broad host range vector pIZ1016 giving rise to plasmids pIZP~3R~ (*P*~3~*~R~*::*lacZ*) and pIZP~A~ (*P~A~*::*lacZ*) ([Table 1](#T1){ref-type="table"}). *E. coli* cells containing plasmids pIZP~3R~ or pIZP~A~ were grown in M63 minimal medium, and they showed 75 and 50 Miller units of β-galactosidase activity, respectively, suggesting that *P*~3~*~R~* and *P~A~* are functional but weak promoters. Primer extension experiments revealed that the transcription initiation sites (+1) of *P*~3~*~R~* and *P~A~* promoters are located 120 bp (data not shown) and 117 bp ([Fig. 7](#F7){ref-type="fig"}*A*) upstream of the *mbdR* and *mbdA* start codons, respectively.
{ref-type="table"}), as described under "Experimental Procedures." An expanded view of the nucleotides surrounding the transcription initiation site (*circled*) in the noncoding strand is shown. The longest extension product is pointed by an *arrow. B*, DNase I footprinting analyses of the interaction of purified MbdR protein and the *P~A~* promoter region. The DNase I footprinting experiments were carried out using the *P~A~* probe labeled as indicated under "Experimental Procedures." *Lane A*+*G* shows the A+G Maxam and Gilbert sequencing reaction. *Lanes A--D* show footprinting assays containing increasing concentrations of MbdR-His~6~. *Lane E* shows a footprinting assay containing MbdR-His~6~ (25 n[m]{.smallcaps}) in the presence of 250 μ[m]{.smallcaps} 3-methylbenzoyl-CoA. *Left side*, an expanded view of the *P~A~* promoter region is shown. The protected region is *shaded in gray* over the promoter sequence. The −10/−35 regions are *boxed,* and the transcription initiation site (+1) is *underlined*. The predicted MbdR operator is flanked by palindrome sequences indicated by *convergent arrows*.](zbc0231515780007){#F7}
To demonstrate the direct interaction of MbdR with the *P*~3~*~R~* and *P~A~* promoters, gel retardation assays were performed. To this end, purified MbdR was incubated either with a 352-bp DNA probe carrying the *P*~3~*~R~* promoter or with a 225-bp DNA fragment containing the *P~A~* promoter. [Fig. 8](#F8){ref-type="fig"}, *A* and *C*, shows that MbdR was able to retard the migration of both DNA probes in a protein concentration-dependent manner. The binding was specific, because the addition of unlabeled heterologous DNA did not affect the protein-DNA binding, but the addition of unlabeled specific DNA inhibited the retardation of the probes (data not shown). Several *P*~3~*~R~*-MbdR retardation bands were observed ([Fig. 8](#F8){ref-type="fig"}*C*), which agrees with the fact that several MbdR operator regions were suggested in *P*~3~*~R~* ([Fig. 8](#F8){ref-type="fig"}*E*). As observed previously with the *P~O~* and *P~B~*~1~ promoters, 3-methylbenzoyl-CoA behaved as the inducer of MbdR because binding of this protein to the *P~A~* and *P*~3~*~R~* promoters was significantly diminished in the presence of this aromatic CoA ester ([Fig. 8](#F8){ref-type="fig"}, *B* and *D*).
![**MbdR protein binds to the *P~A~* and *P*~3~*~R~* promoters and 3-methylbenzoyl-CoA acts as inducer.** Gel retardation assays were performed as indicated under "Experimental Procedures." *A* shows the interaction between increasing concentrations of purified MbdR-His~6~ protein and a DNA probe (225-bp) containing the *P~A~* promoter. *B* shows the interaction between MbdR-His~6~ protein (30 n[m]{.smallcaps}), the *P~A~* DNA probe, and increasing concentrations of 3-methylbenzoyl-CoA (*3MBzCoA*). *Lane* −, free *P~A~* probe. *Lanes 0* to *100* (*A*) and *0* to *50* (*B*) refer to the MbdR-His~6~ protein concentration (n[m]{.smallcaps}) and the 3-methylbenzoyl-CoA concentration (μ[m]{.smallcaps}) used for each assay, respectively. *P~A~* probe as well as the major *P~A~*·MbdR complex are marked with *arrows. C* shows the interaction between increasing concentrations of purified MbdR-His~6~ protein and a DNA probe (352-bp) containing the *P*~3~*~R~* promoter. *Lanes 0* to *1* refer to the MbdR-His~6~ protein concentration (μ[m]{.smallcaps}) used for each reaction. *P*~3~*~R~* probe as well as the *P*~3~*~R~*·MbdR complexes are marked with an *arrow* and a *bracket*, respectively. *D* shows the interaction between MbdR-His~6~ protein (0.5 μ[m]{.smallcaps}), the *P*~3~*~R~* DNA probe, and 0 or 250 μ[m]{.smallcaps} of 3-methylbenzoyl-CoA (*3MBzCoA*). *Lane* −, free *P*~3~*~R~* DNA probe. *E*, nucleotide sequence of the predicted MbdR operator regions in promoters *P~A~* (*O~A~*) and *P*~3~*~R~* (*O1*~3~*~R~*, *O2*~3~*~R~*, and *O3*~3~*~R~*). The flanking ATAC and GTAT palindrome regions are indicated by *convergent arrows*, and the nonconserved nucleotides are *boxed*. Nucleotides that extend the palindromic regions are indicated by *triangles*.](zbc0231515780008){#F8}
Although the role of *P*~3~*~R~* driving the expression of the *mbdR* regulatory gene is obvious, the role of the *P~A~* promoter located within the *P~B~*~1~-driven operon ([Fig. 1](#F1){ref-type="fig"}) is puzzling, and therefore, it was further investigated.
#### P~A~ and P~B1~ Promoters Are Essential for Growth of Azoarcus sp. CIB on 3-Methylbenzoate
As described previously, the *P~B~*~1~ promoter drives the expression of the *mbdB1B2B3B4B5mbdA* operon ([Fig. 1](#F1){ref-type="fig"}) ([@B28]). We have shown above ([Fig. 8](#F8){ref-type="fig"}*A*) that a new MbdR-dependent promoter, the *P~A~* promoter, is located upstream of *mbdA* within the *P~B~*~1~-driven operon ([Fig. 1](#F1){ref-type="fig"}). To explore whether both promoters share a similar MbdR-dependent regulation, the sequence of the *P~A~* promoter recognized by MbdR was experimentally determined by DNase I footprinting assays. [Fig. 7](#F7){ref-type="fig"}*B* shows that the region of *P~A~* protected by MbdR against the DNase I digestion includes the predicted (ATAC*N*~10~GTAC) operator region ([Fig. 8](#F8){ref-type="fig"}*E*), and it spans the −35 sequence for recognition of the σ^70^-dependent RNA polymerase. Moreover, the addition of 3-methylbenzoyl-CoA released the MbdR-dependent protection ([Fig. 7](#F7){ref-type="fig"}*B*), confirming the role of this molecule as inducer. All these data support the hypothesis that MbdR behaves also as a transcriptional repressor for the *P~A~* promoter. To confirm *in vivo* the repressor role of MbdR on the *P~A~* promoter, the activity of a *P~A~*::*lacZ* translational fusion in plasmid pIZP~A~ ([Table 1](#T1){ref-type="table"}) was measured in *E. coli* MC4100 cells harboring also the pCKmbdR and pUCmbdA plasmids that express the *mbdR* and *mbdA* genes under the IPTG-controlled *Plac* promoter, respectively ([Table 1](#T1){ref-type="table"}). The β-galactosidase activity levels (5 Miller units) of recombinant *E. coli* cells expressing the *mbdR/mbdA* genes and grown anaerobically were significantly lower than those obtained in *E. coli* control cells expressing the *P~A~*::*lacZ* translational fusion but lacking the *mbdR/mbdA* genes (50 Miller units). However, the addition of 3-methylbenzoate to the culture medium, which is transformed to 3-methylbenzoyl-CoA by the MbdA activity, alleviated the repression exerted by MbdR, and values of β-galactosidase activity of about 40 Miller units were obtained. Therefore, these results show that MbdR behaves as a functional repressor of the *P~A~* promoter, and 3-methylbenzoyl-CoA acts as the inducer molecule.
As suggested above by comparing the β-galactosidase values in *E. coli* cells expressing *P~A~*::*lacZ* (50 Miller units) and *P~B~*~1~::*lacZ* (4000 Miller units) fusions, the *P~A~* promoter appears to be significantly weaker than *P~B~*~1~. To confirm the major role of *P~B~*~1~ in the expression of the *mbdA* gene in the homologous system, we checked by real time RT-PCR the expression of *mbdA* in the wild-type *Azoarcus* sp. CIB strain and in *Azoarcus* sp. CIBd*mbdB1*, a mutant strain that contains an insertion within the *mbdB1* gene that should block transcription from the *P~B~*~1~ promoter but maintains a functional *P~A~* promoter ([Table 1](#T1){ref-type="table"}). The expression levels of the *mbdA* gene in *Azoarcus* sp. CIBd*mbdB1* grown in the presence of 3-methylbenzoate were similar to the basal levels observed with the wild-type CIB strain grown in the absence of 3-methylbenzoate, and they were more than 47 times lower than those observed in the wild-type CIB strain grown in 3-methylbenzoate (data not shown). These data suggested that *P~B~*~1~, but not *P~A~*, has indeed a major contribution to the *mbdA* expression in *Azoarcus* sp. CIB. In agreement with this observation, the *Azoarcus* sp. CIBd*mbdB1* mutant strain was unable to use 3-methylbenzoate as sole carbon source ([Fig. 9](#F9){ref-type="fig"}*A*), and growth was restored when the *mbdA* gene was provided in *trans* in plasmid pIZmbdA ([Fig. 9](#F9){ref-type="fig"}*A*). In contrast, *Azoarcus* sp. CIBd*mbdB1* mutant strain was still able to use *m*-xylene as a sole carbon source (data not shown), which is in agreement with the fact that the Bss-Bbs peripheral pathway for the anaerobic degradation of *m*-xylene generates 3-methylbenzoyl-CoA without the need of a specific CoA ligase activity ([Fig. 1](#F1){ref-type="fig"}) ([@B53][@B54][@B56]). Taken together, all of these results indicated that *P~B~*~1~ is essential for growth of *Azoarcus* sp. CIB in 3-methylbenzoate by providing an efficient expression of the *mbdA* gene rather than by transcribing the *mbdB1-B5* genes encoding a putative 3-methylbenzoate ABC transporter.
{ref-type="table"}) growing anaerobically in MC medium containing 3 m[m]{.smallcaps} 3-methylbenzoate as detailed under "Experimental Procedures." *B*, β-galactosidase activity of *Azoarcus* sp. CIB cells harboring plasmids pIZP~B1~ (*P~B~*~1~::*lacZ*) (*closed bars*) or pIZP~A~(*P~A~*::*lacZ*) (*open bars*). Cells were grown anaerobically in MC minimal medium with 7 m[m]{.smallcaps} glutarate until the cultures reached an *A*~600~ ∼0.6, and then diluted to *A*~600~ ∼0.17 and induced with 3 m[m]{.smallcaps} 3-methylbenzoate. β-Galactosidase activity (in Miller units) was measured at different time points (h) after induction as described under "Experimental Procedures." *Error bars* represent standard deviation of at least three independent experiments.](zbc0231515780009){#F9}
Nevertheless, the presence of the *P~A~* promoter within the *P~B~*~1~-driven operon raised a question about the role of this weak promoter in 3-methylbenzoate degradation. To confirm whether *P~A~* is essential for the anaerobic degradation of 3-methylbenzoate, an *Azoarcus* sp. CIBΔ*P~A~* mutant strain harboring a deletion of the *P~A~* promoter but maintaining a complete *mbdA* gene and the native *P~B~*~1~ promoter was constructed ([Table 1](#T1){ref-type="table"}). Interestingly, *Azoarcus* sp. CIBΔ*P~A~* was not able to grow anaerobically in 3-methylbenzoate ([Fig. 9](#F9){ref-type="fig"}*A*), suggesting that *P~A~* is also necessary for an efficient expression of the *mbdA* gene, which in turn supports the presence of *P~A~* within the *P~B~*~1~-driven operon.
Because *P~B~*~1~ accounts for most of the *mbdA* expression, the role of the weak *P~A~* promoter might be related to the initial induction of the *mbdA* expression when the cells start to grow in 3-methylbenzoate. To check this hypothesis, the activity of the *P~B~*~1~ and *P~A~* promoters was analyzed by β-galactosidase assays along the growth curve of *Azoarcus* sp. CIB harboring pIZ*P~B~*~1~ (*P~B~*~1~::*lacZ*) and *Azoarcus* sp. CIB harboring pIZP~A~ (*P~A~*::*lacZ*) grown in the presence of 3-methylbenzoate. The activity of the weak *P~A~* promoter was always higher than that of *P~B~*~1~ up to 6 h after the addition of 3-methylbenzoate, and then *P~B~*~1~ showed a significant induction and reached values about 20-fold higher than those of *P~A~* ([Fig. 9](#F9){ref-type="fig"}*B*). Therefore, these results suggest that the fast and modest induction of the *P~A~* promoter will be critical to provide the required amount of the inducer molecule 3-methylbenzoyl-CoA for triggering the induction of the *P~B~*~1~ promoter and to allow growth on 3-methylbenzoate.
DISCUSSION
==========
Bacterial metabolism of some compounds that usually are nonpreferred carbon sources, *e.g.* aromatic compounds, is generally strictly regulated at the transcriptional level ([@B8]). In this work, we have characterized the specific regulation of the *mbd* central cluster, which is responsible for anaerobic 3-methylbenzoate degradation in *Azoarcus* sp. CIB, by the MbdR transcriptional repressor. MbdR is an efficient repressor of the *mbd* genes whose expression can only be switched on when the *Azoarcus* sp. CIB cells grow anaerobically on 3-methylbenzoate ([@B28]) but not on benzoate ([Fig. 2](#F2){ref-type="fig"}, *A* and *B*). This finding provides an explanation to the fact that *Azoarcus* sp. CIBd*bzdN*, a strain lacking a functional benzoate degradation (*bzd*) pathway, cannot use benzoate anaerobically despite the Mbd enzymes that can activate benzoate to benzoyl-CoA and further metabolize this CoA-derived compound ([@B28]). On the other hand, it is worth noting that the *bzd* genes are not induced when *Azoarcus* sp. CIB grows anaerobically in 3-methylbenzoate (data not shown). Therefore, these results reveal that there is no cross-induction between the bzd and mbd pathways, supporting the existence of devoted BzdR- and MbdR-dependent regulatory systems that control, respectively, each of these two central catabolic pathways in *Azoarcus* sp. CIB.
Analytical ultracentrifugation and crystallographic data indicate that MbdR is a homodimer in solution, a common feature shared by most TetR-like regulators ([Fig. 4](#F4){ref-type="fig"}*D*) ([@B51], [@B52]). Like other members of the TetR family, *e.g.* TetR ([@B57]), QacR ([@B58]), ActR ([@B59]), FadR ([@B60], [@B61]), PfmR ([@B62]), and the MbdR monomeric structure includes two domains with nine helices (α1 to α9) linked by loops ([Fig. 4](#F4){ref-type="fig"}*A*). The N-terminal DNA binding domain (helices α1 to α3) contains the helix-turn-helix motif whose amino acid sequence is rather conserved in other TetR-like transcriptional regulators ([Fig. 3](#F3){ref-type="fig"}). An electrostatic surface representation of the MbdR dimer structure shows a positively charged patch at the N-terminal domain of both monomers ([Fig. 4](#F4){ref-type="fig"}*C*), which might contact the phosphate backbone of the target operator region as in the cases of other TetR family proteins ([@B52]). An 18-bp conserved palindromic sequence (ATAC*N*~10~GTAT) was suggested to be the operator region recognized by MbdR in *P~O~* and *P~B~*~1~ promoters ([Fig. 5](#F5){ref-type="fig"}). The MbdR binding regions in *P~O~* and *P~B~*~1~ promoters span the transcription initiation sites as well as the −10 and −35 (only in *P~B~*~1~) sequences for recognition of the σ^70^-dependent RNA polymerase ([Fig. 5](#F5){ref-type="fig"}, *C* and *D*), which is in agreement with the observed repressor role of MbdR at both promoters ([Fig. 2](#F2){ref-type="fig"}, *A* and *B*), and it supports MbdR as a transcriptional repressor of the *mbd* cluster. Although the length of the MbdR operator region is similar to that of other TetR operators, their different consensus sequences agree with the fact that the DNA-binding mechanisms differ among the TetR family proteins ([@B52]).
*In vivo* ([Fig. 6](#F6){ref-type="fig"}*A*) and *in vitro* ([Fig. 6](#F6){ref-type="fig"}, *B* and *D*) experiments revealed that 3-methylbenzoyl-CoA, the first intermediate of the mbd catabolic pathway, is the cognate inducer molecule that interacts with the MbdR repressor allowing transcription from the *P~O~* and *P~B~*~1~ promoters. There is an increasing number of regulators, *i.e.* PaaR ([@B63]) (TetR family), CouR, FerC, HcaR, FerR, and GenR (MarR family) ([@B27], [@B64][@B65][@B67]), PaaX (GntR family) ([@B38]), and BzdR and BoxR(XRE family) ([@B17], [@B20]), that control aromatic degradation pathways and recognize aromatic CoA thioesters as inducers. Thus, FerR/FerC recognize feruloyl-CoA; CouR/HcaR recognize *p*-coumaroyl-CoA; BzdR/BoxR/GenR recognize benzoyl-CoA; and PaaX/PaaR recognize phenylacetyl-CoA. In this work, we show that MbdR constitutes the first member of this group of regulators that belongs to the TetR family and controls the anaerobic catabolism of aromatic compounds.
The C-terminal domain of TetR-like regulators is highly variable, with its specific surfaces required for the dimerization of the protein and for the interaction with the inducer ([@B51], [@B52], [@B57]). Based on the previously published studies of other TetR-like regulators, ligand binding usually induces a conformational change in the protein that leads to changes in DNA recognition and interaction, causing the dissociation of the repressor from the cognate promoter ([@B52]). To date, all ligands bind in the same general location at or near the dimer interface. However, it has been shown that in some members of the TetR family, for example AcrR ([@B68]), the ligand binds in a large internal cavity in the C-terminal region, surrounded by helices α4 through α8 of each monomer. In contrast, MbdR and other members of TetR family, such as QacR ([@B58]), do not have such a cavity ([Fig. 4](#F4){ref-type="fig"}, *A* and *C*). By superimposing the apo-MbdR structure with the structure of the QacR·diamidine hexamidine complex ([@B69]), we could suggest the binding site of 3-methylbenzoyl-CoA in MbdR and a model of the MbdR-3-methylbenzoyl-CoA interaction ([Fig. 4](#F4){ref-type="fig"}*E*). Binding of 3-methylbenzoyl-CoA would require the movements of helices α5, α6, α8, and α9 in MbdR, similar to that described as the "induced fit" mechanism of QacR bound to its ligand ([@B69], [@B70]). Similar to what has been observed in the QacR·ligand complex structure, the movement of α6 after 3-methylbenzoyl-CoA binding to MbdR would induce a rotation of the helix-turn-helix domain ([Fig. 4](#F4){ref-type="fig"}*E*), and as a consequence, this DNA binding domain would lose its DNA binding ability. Sequence comparison of MbdR and PaaR ([Fig. 3](#F3){ref-type="fig"}), another member of the TetR family which uses phenylacetyl-CoA as inducer ([@B63]), shows two MbdR-specific hydrophobic clusters, Gln-107 to Gly-123 within α6 and the α6/α7 linkage loop, and Ser-165 to Ile-176 within α8. Some residues within these two clusters could be involved in discriminating between the 3-methylbenzoyl group of 3-methylbenzoyl-CoA and the phenylacetyl group of phenylacetyl-CoA ([Fig. 4](#F4){ref-type="fig"}*F*). Nevertheless, further experiments are needed to determine the structure of the MbdR·3-methylbenzoyl-CoA complex for understanding the inducer specificity determinants and the molecular mechanism of transcriptional de-repression at the target promoters.
*P~A~* and *P*~3~*~R~* are two additional promoters within the *mbd* cluster whose activity levels are lower than those of *P~O~* and *P~B~*~1~ but that share with the latter the 3-methylbenzoyl-CoA/MbdR-dependent control ([Fig. 8](#F8){ref-type="fig"}). The *P*~3~*~R~* promoter drives the expression of the regulatory *mbdR* gene ([Fig. 1](#F1){ref-type="fig"}). Interestingly, the amount of MbdR needed for the retardation of 50% of the *P*~3~*~R~* probe was at least 1 order of magnitude higher than that needed for the retardation of the *P~A~* ([Fig. 8](#F8){ref-type="fig"}*A*), *P~O~* ([Fig. 2](#F2){ref-type="fig"}*C*), and *P~B~*~1~ ([Fig. 2](#F2){ref-type="fig"}*D*) promoters. The fact that the activity from the *P*~3~*~R~* promoter is under auto-repression by MbdR at high protein concentrations underlines the importance of a negative feedback loop that would restrict the intracellular concentration of the transcriptional repressor when it reaches a given concentration. The *P~A~* promoter is located within the *P~B~*~1~-driven operon ([Fig. 1](#F1){ref-type="fig"}). The predicted MbdR operator region (ATAC*N*~10~GTAT) ([Fig. 8](#F8){ref-type="fig"}*E*) spans the −35 sequence for recognition of the σ^70^-dependent RNA polymerase in the *P~A~* promoter ([Fig. 7](#F7){ref-type="fig"}*B*), thus supporting the observed repressor role of MbdR on this promoter. Whereas the role of *P*~3~*~R~* driving the expression of the *mbdR* regulatory gene is obvious, the role of the *P~A~* promoter was puzzling, and therefore, it was further investigated.
Inactivation of either the strong (*P~B~*~1~) or the weak (*P~A~*) promoters in *Azoarcus* sp. CIBd*mbdB1* and *Azoarcus* sp. CIBΔ*P~A~* mutant strains, respectively, revealed that both promoters are essential for the anaerobic growth of strain CIB in 3-methylbenzoate ([Fig. 9](#F9){ref-type="fig"}*A*). However, whereas *P~B~*~1~ accounts for most of the *mbdA* expression when the cells are actively growing in 3-methylbenzoate, the *P~A~* promoter allows the initial induction of the *mbdA* expression when the cells start to grow in this aromatic compound ([Fig. 9](#F9){ref-type="fig"}*B*). Therefore, these results suggest that the fast and modest induction of the *P~A~* promoter in the presence of 3-methylbenzoate leads to an increase of *mbdA* expression that, in turn, would enhance the amount of the inducer molecule 3-methylbenzoyl-CoA triggering the induction of the *P~B~*~1~ promoter. The expression of the *mbdA* gene driven by the induced *P~B~*~1~ promoter will provide the required amount of MbdA for the efficient degradation of 3-methylbenzoate and thus will allow growth on this aromatic compound. In summary, these studies highlight the main role of some minor regulatory loops that control the expression of CoA ligases for triggering the efficient expression of aromatic catabolic pathways that use aryl-CoA compounds as central intermediates.
Mbd enzymes are able to activate benzoate and further convert benzoyl-CoA *in vitro* ([@B28]). We have shown here that MbdR is an efficient repressor of the *mbd* genes, and it recognizes 3-methylbenzoyl-CoA, but not benzoyl-CoA, as inducer. These results suggest that the broad substrate range *mbd* catabolic genes have recruited a regulatory system based on the MbdR regulator and its target promoters to evolve to a distinct central aromatic catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as central metabolite. Thus, the existence in *Azoarcus* sp. CIB of two different central pathways, *i.e.* the *bzd* pathway, for the anaerobic degradation of aromatic compounds that generate benzoyl-CoA as central intermediate, and the *mbd* pathway, for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as central intermediate, could be mainly driven by the high specificity of the corresponding repressors, *i.e.* BzdR and MbdR, for their cognate inducers, *i.e.* benzoyl-CoA and 3-methylbenzoyl-CoA, respectively. If correct, this highlights the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.
The studies presented in this work expand our knowledge on the specific regulation of anaerobic pathways for the catabolism of aromatic compounds ([@B4], [@B9], [@B14], [@B17], [@B20], [@B27], [@B28]). Moreover, it worth noting that 3-methylbenzoyl-CoA is an uncommon metabolite in living cells, and MbdR-responsive promoters are likely to be also very infrequent in nature. Therefore, the *P~B~*~1~ promoter, *mbdR* regulator, and *mbdA* genes become potential BioBricks for creating new conditional expression systems that respond to 3-methylbenzoate in a fashion minimally influenced by the host and that has no impact on the host physiology (biological orthogonality), two major desirable traits in current synthetic biology approaches ([@B71]).
This work was supported in part by Ministry of Economy and Competitiveness of Spain Grants BIO2009-10438, BIO2012-39501, and CSD2007-00005 and European Union FP7 Grant 311815. Crystallography was supported by a Biotechnology and Biological Sciences Research Council grant and a Wellcome Trust award.
The atomic coordinates and structure factors (code [4uds](http://www.pdb.org/pdb/explore/explore.do?structureId=4uds)) have been deposited in the Protein Data Bank ([http://wwpdb.org/](http://www.pdb.org/)).
The abbreviations used are: IPTGisopropyl 1-thio-β-[d]{.smallcaps}-galactopyranosideSeMetselenomethionine.
We thank A. Valencia for technical assistance, Secugen S.L. for DNA sequencing, and C. A. Botello for ultracentrifugation experiments.
[^1]: Supported by a predoctoral fellowship from the Comunidad Autónoma de Madrid. Present address*:* Dept. of Genetics, Harvard Medical School, 77 Louis Pasteur Ave., Boston, MA 02155.
[^2]: Present address: College of Plant Protection, Shandong Agricultural University, 61 Daizong Rd., Taian, Shandong 271018, China.
[^3]: A Royal Society Wolfson Merit Award holder.
|
The Cooling Off Rule provides that it is unfair and deceptive for sellers engaged in “door-to-door” sales valued at more than $25 to fail to provide consumers with disclosures regarding their right to cancel the sales contract within three business days of the transaction. |
Guild Wars 2 coming in June
Guild Wars 2 is set for release on June 28th -- or is it? NCSoft says that no official release date has been announced, and they are still waiting on feedback from beta testing before they announce a release date. Beyond saying that Guild Wars 2 will be released when it is ready, NCSoft isn’t saying much else.
Kotaku has, however, obtained a photo of an advertisement seen at Fry’s retail stole in Texas that says Guild Wars 2 is coming on June 28th. It could be nothing but a placeholder, but our sources say that it is more truth than fiction. (GameStop is saying that the game will be release on June 26th, if that means anything, and another independent retailer we spoke with says his distributor is telling him that the game is set for release on June 26th, as well.)
We think that the big takeaway here is that Guild Wars 2 is getting closer to release; and if you want the Collector’s Edition, you might want to get your pre-order in now. We suspect that a late June release for the game is likely, based on our discussions with a number of sources, but you just never know. We do think that we should know soon with an expected announcement at E3 by NCSoft. |
INTRODUCTION
============
Osteopetrosis was first published in 1904 by Albers-Schoenberg \[[@hnx032-B1]\] and later by Karschner \[[@hnx032-B2]\]. It is based on osteoclast dysfunction leading to failure of bone resorption and to increase of bone mass. Persistance of chondroid and primitive bone formation makes bone brittle \[[@hnx032-B3]\]. In human osteopetrosis the main variants are (i) the malignant autosomal recessive infantile form, (ii) the intermediate autosomal recessive form and (iii) the autosomal dominant form \[[@hnx032-B4], [@hnx032-B5]\]. Patients with the malignant infantile form present pathological fractures at birth or early in infancy combined with severe bone marrow dysfunction. Life expectance is severely reduced due to haemorrage and/or sepsis \[[@hnx032-B6]\]. The intermediate autosomal recessive form is rare and usually diagnosed from pathological fractures in childhood; patients survive into adulthood \[[@hnx032-B6]\]. Prevalent and of primary orthopaedic interest is the autosomal dominant osteopetrosis, which can be further subdivided into Types I and II according to the radiological sclerosis pattern of the axial skeleton; type I is supposed to be slightly more benign \[[@hnx032-B4]\]. Most manifestations of the condition are seen in the area of the hip \[[@hnx032-B5]\]. Approximately, one-third of hip prostheses implanted in osteopetrosis are for failed fracture fixation and yet information about the handling of osteopetrosis bone, or any special technical precautions required, are rare and non-specific \[[@hnx032-B7]\]. Osteotomies in the presence of osteopetrosis are generally confined to subtrochanteric valgization for non-union, or for coxa vara in children. Other osteotomies around the hip are not mentioned, nor are difficulties addressed which may appear during metal removal. In this article, we provide this information and illustrate it with four different examples.
GENERAL REMARKS
===============
The dense structure of osteopetrosis bone is associated with a low modulus of elasticity, allowing little bony deformation. This facilitates fractures when grasping instruments and implants are used. Drills and saw blades may not grasp but nevertheless fill surprisingly fast with bone dust and heat the instrument and surrounding bone. During repetitive irrigation and cleaning it is difficult to maintain instruments in the desired direction. Blunt instruments substantially increase such difficulties. Meanwhile, repeated overheating may lead to breakage while drilling. Tapping a female screw thread can lead to the instrument displacing from its desired track, as well as a tendency to jam with deeper penetration; in the femoral head this may even lead to splitting of the bone, especially when a motorized tap is used.
The high friction between implants and osteopetrosis bone may lead to jamming and destruction of hexagonal screwheads, especially for long screws or those with a small diameter. Powered screwdrivers may predispose to fracture. Implants made from titanium clearly cause higher friction.
Bone healing in osteopetrosis can take longer than normal, especially in cortical bone where osteoclasts are insufficient to create channels in the existing bone and thereby allow osteoblasts to bridge and fill any gaps with new bone.
In addition, the removal of implants can be very laborious and breakage of screw heads is common, so the indications and techniques for surgery must carefully be considered. ([Table I](#hnx032-T1){ref-type="table"}) Table I.Specific recommendationsPay attention! Bone in osteopetrosis is brittleIf possible, do not use grasping instruments and implants like blade plates or bolt plates.Always keep drills and saw blades cool and cleanDo not insert screws with powered assistanceAvoid long screws with a small diameterAvoid titanium screwsTry to avoid removing metalworkWhen removal is necessary, have special tools available
INTERTROCHANTERIC OSTEOTOMY
===========================
A search of the literature yielded three publications, each about paediatric hips. Two reported coxa vara treated by a subtrochanteric valgus osteotomy \[[@hnx032-B8], [@hnx032-B9]\]. No further details were given. The third, by Armstrong, Newfield and Gillespie \[[@hnx032-B10]\], was a survey and review paper \[[@hnx032-B8]\]: they reported that of seven fractures, the four that had undergone internal fixation had healed; the three treated non-operatively had failed to heal and developed a varus non-union. In 20 coxa vara hips, valgus osteotomy was consistently reliable, while pinning failed in two out of three cases.
[Figure 1A--F](#hnx032-F1){ref-type="fig"} demonstrates the course of an intertrochanteric osteotomy in which technical problems led to serious complications. This 15-year-old male patient acquired a right femoral neck fracture ([Fig. 1A](#hnx032-F1){ref-type="fig"}). The operating note describes a difficult insertion and orientation of K-wires for the preliminary fixation. Similar problems occurred while attempting to maintain accurate drill direction, leading to a suboptimal placement of two short-threaded 6.5 mm screws and one bolt. Tightening of the screws led to rotation of the head and redisplacement of the fracture rather than to the desired interfragmentary compession. Ten months later there was non-union, varus and retrotilting of the head as well as breakout of one screw; a revision procedure was required ([Fig. 1B](#hnx032-F1){ref-type="fig"}). In an era when screw plates for osteotomies were not yet popularized, preoperative paper planning determined that a double angle blade plate with an 80 mm blade would bridge the non-union and allow normal leg length to be regained ([Fig. 1C](#hnx032-F1){ref-type="fig"}) \[[@hnx032-B11]\]. Neutralization of any displacing effect of the seating chisel was obtained with careful pushing and pulling manoeuvres. However, as the instrument was advanced into the femoral head a sound change was followed by easier propagation. A fracture gap leading from the non-union area towards the head was then identified; closure of the gap was observed if the seating chisel was pulled back to the 70 mm mark. Consequently, a blade length 10 mm shorter than had been planned preoperatively was used. With a new saw blade, the intertrochanteric osteotomy was uneventful. The postoperative radiographs were identical with the planning and showed leg length had been restored ([Fig. 1C](#hnx032-F1){ref-type="fig"}). Postoperative CT imaging revealed a residual gap caused by a split in the femoral head but without a step ([Fig. 1D](#hnx032-F1){ref-type="fig"}). The patient's records note that a lengthy period was needed until the osteotomy line disappeared but by 2 years after surgery the radiographs showed full consolidation with a good joint space. However, small ring osteophytes of the femoral head indicated that the joint was compromised ([Fig. 1E](#hnx032-F1){ref-type="fig"}).
{#hnx032-F1}
SUBCAPITAL REALIGNEMENT IN SEVERE CHRONIC SLIPPED CAPITAL FEMORAL EPIPHYSIS
===========================================================================
Again, the literature reveals three case reports that deal with slipped capital femoral epiphysis (SCFE) in the context of osteopetrosis \[[@hnx032-B12]\]. In only one, pinning *in situ* is mentioned but without further comment \[[@hnx032-B13]\].
[Figure 2A--D](#hnx032-F2){ref-type="fig"} shows the subcapital realignment of a severe left-sided SCFE in a 12-year-old boy. As the slip was thought to be unstable ([Fig. 2A](#hnx032-F2){ref-type="fig"}), the surgical dislocation approach \[[@hnx032-B15]\] was extended and the epiphysis pinned before dislocation. Drilling of the epiphysis was followed by brisk bleeding. Inspection after full joint dislocation did not show substantial damage to the acetabular labrum and adjacent articular cartilage but did reveal rupture of the femoral neck periosteum, albeit limited to the anterior surface. Even after extension of the retinacular flaps \[[@hnx032-B16]\], the epiphysis was not detached, although separation was easy. After removal of callus formation at the proximal part of the posterior neck, and debridement of growth plate material, the epiphysis was manually reduced and fixed with a retrograde K-wire through the fovea capitis \[[@hnx032-B16]\]. Advancing the wire until it perforated at the lateral subtrochanteric cortex was only possible after laborious antegrade predrilling. Similar difficulties were experienced during the antegrade insertion of a second K-wire. For definitive fixation, antegrade insertion of predrilled, fully threaded 3 mm wires was performed. Nevertheless penetration of the wire tip into the epiphysis led to distraction and rotation of the epiphysis, even with the previous K-wires in place. Consequently, retrograde insertion helped avoid interfragmentary distraction ([Fig. 2B](#hnx032-F2){ref-type="fig"}). Healing time was longer than with normal bone ([Fig. 2C](#hnx032-F2){ref-type="fig"}). By 3 years after this procedure there were no signs of femoral head necrosis ([Fig. 2D](#hnx032-F2){ref-type="fig"}).
{#hnx032-F2}
PERIACETABULAR OSTEOTOMY
========================
It was not possible to find any literature about the use of pelvic osteotomy in osteopetrosis. However, [Figure 3A--D](#hnx032-F3){ref-type="fig"} shows a periacetabular osteotomy for acetabular dysplasia \[[@hnx032-B17]\] in a 21-year-old female, with Grade 1--2 cartilage degeneration and osteopetrosis. Execution of the surgical procedure was less laborious than expected, although the resistance of the bone was higher than normal. This may have been because the osteotomy surfaces were smaller and cortices thinner compared with the proximal femur. It proved helpful to use double cuts for the first cortex of the pubis osteotomy and for the osteotomy at the turning point of the supraacetabular to retroacetabular cut. As with the standard procedure, the supraacetabular cut was undertaken with an oscillating saw, the remainder with especially thin and sharp osteotomes. In addition, it was anticipated that osteopetrosis bone might not follow a predetermined breaking line, as seen with normal bone. Consequently, all osteotomies were completed by using hand instruments. After the procedure, although the healing time seemed somewhat prolonged, by 5 years the joint space showed only minor additional narrowing.
{#hnx032-F3}
METAL REMOVAL
=============
It was not possible to identify any literature that discussed the special difficulties which can occur during the removal of metalwork from osteopetrosis hips.
[Figure 4A and B](#hnx032-F4){ref-type="fig"} demonstrates the laborious and time-consuming efforts which may be needed, including the use of special instrumentation. This 37-year-old female patient suffered first from femoral shaft fractures which were fixed with steel plates. Shortly after consolidation she then sustained a subtrochanteric fracture, first on the right then on the left. The level of the fractures made removal of the existing plates necessary. Surgery to remove the plates was described as time consuming and complicated by breakage of some screwheads. For optimal bridging of the new fracture a special plate was chosen, allowing screw placement into the femoral neck and head while the long shaft covered and protected the screw holes of the former plate. For best positioning of the new plate, two portions of residual screw had to be overdrilled in order to be removed. While fixation and healing on the right side was uneventful, surgery on the left was complicated by a split in the femoral head during final insertion of the screw. The screw was thus replaced by a shorter one and the split femoral head fixed with two small screws. One year later the patient complained of substantial hip pain, possibly related to necrosis and collapse of the femoral head fragment although the subtrochanteric fracture had consolidated ([Fig. 4A](#hnx032-F4){ref-type="fig"}). A total hip replacement (THR) was thus recommended but to do this metal removal was necessary and special instruments were required, e.g. high speed diamond saw to cut metal and hollow drills to remove broken portions of screw were held available. Although the screws had to be removed for a distance corresponding to the length of the prosthetic stem, it was considered that if the plate and screws were left in place more distally they would provide better protection against a new fracture. Reaming of the femoral shaft to allow insertion of a prosthetic stem was anticipated to be difficult. In the event, this required laborious predrilling to allow the insertion of a small stem size ([Fig. 4B](#hnx032-F4){ref-type="fig"}).
{#hnx032-F4}
DISCUSSION
==========
The purpose of this paper is to demonstrate that even complex osteotomies to preserve the natural hip joint are feasible in osteopetrosis, when a number of technical matters are taken into consideration. Osteopetrosis bone is not very tolerant towards grasping instruments and implants. This is well demonstrated by the split femoral head in two of the cases we present here. The risks of fracture can be reduced if the surgeon prepares the area properly, for example, with a drill or saw. Techniques have also changed. For example, today, for a situation such as Case 2 (above) a screw plate implant would likely be better instead of the displacing blade plate that was used at the time. Conversely, non-displacing tools create high friction and heat, so constant irrigation is imperative as is repetitive irrigation of bone dust. High friction is also an issue during screw implantation, especially when small-diameter and/or long screws have to be inserted. It is possible that the high rate of failure after screw fixation of femoral neck fractures is in part related to the difficulty in applying interfragmentary compression with lag screws. For this same reason titanium implants should not be used in osteopetrosis bone.
One constant observation is that bone healing in osteopetrosis takes longer; therefore, limited weight-bearing should be recommended for a lengthier period than for normal bone. Experience with implant removal comes mainly from the failure of fixation of proximal femoral fractures when the hip is to be replaced by an artificial joint. Implant removal may require special instruments to overdrill any portions of broken screw. While any holes in the lateral femoral cortex may be splinted by a prosthesis stem, distal to this stem such holes may become a stress riser and can lead to a new fracture. Surgeons should remain aware of this and consider cutting the plate, removing its proximal portion, and leaving the distal part, beyond the tip of the prosthesis, still attached to the bone. Metal should not be removed routinely. If removal is unnecessary for inserting a prosthesis, metalwork can be left where it is. Implants left *in situ* in the femoral head and/or neck might also shield against femoral neck fracture, a rather frequent complication in osteopetrosis.
The authors thank Selina Nauer, research assistant for technical support during preparation of this work.
FUNDING
=======
No funding was used for the purposes of this study.
CONFLICT OF INTEREST STATEMENT
==============================
None declared.
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Daphnia revisited: local stability and bifurcation theory for physiologically structured population models explained by way of an example.
We consider the interaction between a general size-structured consumer population and an unstructured resource. We show that stability properties and bifurcation phenomena can be understood in terms of solutions of a system of two delay equations (a renewal equation for the consumer population birth rate coupled to a delay differential equation for the resource concentration). As many results for such systems are available (Diekmann et al. in SIAM J Math Anal 39:1023-1069, 2007), we can draw rigorous conclusions concerning dynamical behaviour from an analysis of a characteristic equation. We derive the characteristic equation for a fairly general class of population models, including those based on the Kooijman-Metz Daphnia model (Kooijman and Metz in Ecotox Env Saf 8:254-274, 1984; de Roos et al. in J Math Biol 28:609-643, 1990) and a model introduced by Gurney-Nisbet (Theor Popul Biol 28:150-180, 1985) and Jones et al. (J Math Anal Appl 135:354-368, 1988), and next obtain various ecological insights by analytical or numerical studies of special cases. |
1 Magellan Boulevard, London, E16
1 Bedroom property for sale
Description
Situated in Bawley Court, Royal Dockside is this recently completed one bedroom, one bathroom first floor apartment.
Located in the Royal Dockside development is this stunning apartment which spans a sizeable 544 square feet. This property will offer a spacious open plan kitchen/reception room leading on to a private balcony offering river facing views.
Transport Links include Gallions reach DLR station offering an excellent commute to Canning town station Jubilee line, Canary Wharf and Bank. |
1. A Simplified Model of Genome Replication in Higher Eukaryotes {#sec1-genes-10-00199}
================================================================
1.1. Completeness of Genome Replication Requires Small Distances between Origins and Reliable Origin Firing {#sec1dot1-genes-10-00199}
-----------------------------------------------------------------------------------------------------------
Genome replication must be complete and accurate to generate two faithful copies of the DNA in order to inherit a full set of genes to both daughter cells. Completeness of genome duplication is a particularly complex issue in eukaryotes that require multiple replication origins to duplicate their large genomes within a typical replication period that can last from several minutes in yeasts to some hours in vertebrates. With multiple origins comes the risk of gaps between two replication initiation sites that are too large to be replicated by two replication forks within the duration of an S phase. In order to keep the number of large inter-origin gaps minimal to guarantee completeness of genome duplication two requirements must be met ([Figure 1](#genes-10-00199-f001){ref-type="fig"}A): (1) origins must be placed regularly, not too far from each other in the genome and (2) origins must fire reliably as any unfired origin increases gap size.
In most eukaryotes including metazoa the large-gap problem becomes aggravated by the fact that origin positions are not strictly determined by DNA sequence. This generates an element of randomness in the location of origins and, thus, a certain size distribution of small and large inter-origin distances \[[@B1-genes-10-00199]\]. The larger the genome, the higher the number of origins must be at a given replication fork speed and mammalian genomes have several ten thousand origins. Because larger genomes have more origins the total number of very large inter-origin distances that can potentially not be completely replicated rises with genome size.
One important mechanism utilized by eukaryotes to reduce large inter-origin distances that is not discussed in detail in this article is the use of dormant origins \[[@B2-genes-10-00199]\]. Eukaryotic cells generate many more potential origins by origin licensing (discussed below) than actually fire in normal S phases \[[@B3-genes-10-00199],[@B4-genes-10-00199],[@B5-genes-10-00199],[@B6-genes-10-00199],[@B7-genes-10-00199],[@B8-genes-10-00199]\]. These are dormant origins that fire only if they have enough time, as shown in yeast and vertebrates \[[@B9-genes-10-00199],[@B10-genes-10-00199],[@B11-genes-10-00199]\]. Unfired dormant origins will normally get inactivated if their DNA is replicated by an incoming fork generated by a neighbouring origin. If this does not occur, for example when no origin in the vicinity fires or when DNA damage stalls incoming forks, dormant origin firing helps complete duplication of the DNA in their genome region.
In addition to dormant origins that help reduce size of many inter-origin gaps eukaryotes have also evolved a complex genome replication program to ensure complete genome replication, which is discussed in detail in this review.
1.2. Domain-Wise Replication May Help Complete Genome Duplication in Limited Origin Firing Conditions {#sec1dot2-genes-10-00199}
-----------------------------------------------------------------------------------------------------
Some origin firing factors are present in limiting amounts in somatic eukaryotic cells \[[@B12-genes-10-00199],[@B13-genes-10-00199],[@B14-genes-10-00199],[@B15-genes-10-00199]\]. Hence, not all origins can fire at the same time at the beginning of S phase but must fire distributed over the duration of S phase. If the relatively rare firing events were scattered throughout the genome extremely large inter-origin distances would ensue. Consequently, the likelihood of persistently stalled forks that cannot be rescued by an incoming opposing fork would be high and so would be the rate of fork breakdown and genomic re-arrangements \[[@B9-genes-10-00199],[@B10-genes-10-00199],[@B11-genes-10-00199],[@B16-genes-10-00199]\]. Presumably to avoid this, higher eukaryotic cells concentrate origin firing locally, which leads to genome replication in the form of separate genomic domains, called replication domains/factories \[[@B17-genes-10-00199]\]. This domain-wise replication may represent a way to make replication locally efficient within replication domains, despite low overall origin firing.
1.3. Timing of Domain Replication {#sec1dot3-genes-10-00199}
---------------------------------
The timing with which replication domains replicate is an important aspect of eukaryotic genome duplication. Replication timing changes with cell differentiation, as shown by genome-wide replication timing analyses and these timing changes are conserved between different human and mouse cell types, indicating that replication timing is evolutionarily important \[[@B18-genes-10-00199],[@B19-genes-10-00199]\]. Moreover, late replication is associated with elevated levels of genetic alterations \[[@B20-genes-10-00199],[@B21-genes-10-00199]\] and chromosome fragility \[[@B22-genes-10-00199]\].
Replication domains juxtaposed in the genome appear to mostly replicate successively, meaning once a domain has replicated its neighbour starts (dominos model) ([Figure 1](#genes-10-00199-f001){ref-type="fig"}B) \[[@B23-genes-10-00199],[@B24-genes-10-00199],[@B25-genes-10-00199]\]. This may minimise the frequency with which domains that replicate in isolation occur, that is domains replicating at vastly different times from their neighbours. Such isolated domains inevitably generate two forks, the two outmost forks, that will find opposing forks for termination only with a significant time delay. These forks will therefore have a high probability of permanent stalling. Exceptions to the dominos-like propagation of replication are timing transition regions (TTRs), the regions between two neighbouring domains that replicate with a significant delay. Indeed, TTRs were found to be replicated unidirectionally, consistent with an outmost fork travelling out of its domain (slightly more complex scenarios were suggested) \[[@B18-genes-10-00199],[@B26-genes-10-00199]\]. Whether TTRs pose a particular problem for accurate genome replication is a matter of debate.
From this dominos model of higher eukaryotic genome replication follows that the timing with which replication domains replicate is dictated by the replication time of the first replicating domain combined with the rate of spreading of replication through the neighbouring domains ([Figure 1](#genes-10-00199-f001){ref-type="fig"}B). At TTRs, blocks to replication spreading must exist. The replication timing of the later replicating domain at TTRs must be determined anew ([Figure 1](#genes-10-00199-f001){ref-type="fig"}C).
This model of genome replication presented is highly simplified and leaves open many questions. What determines the timing of the first domain in a row (the first domino)? How is spreading of replication achieved and how is it blocked at TTRs? Which chromatin processes and parameters influence replication timing?
Some fundamental aspects of replication timing in eukaryotes have been characterised. It has long been known that transcriptionally active open euchromatin replicates in early S phase, whereas the structurally compact heterochromatin replicates late (with some exceptions). Microscopically, early replication becomes visible in mammalian cells as hundreds of foci of active replication scattered throughout the nucleus after pulse-labelling with detectable nucleotide analogues or when expressing labelled PCNA (proliferating cell nuclear antigen) \[[@B17-genes-10-00199],[@B27-genes-10-00199],[@B28-genes-10-00199],[@B29-genes-10-00199],[@B30-genes-10-00199]\]. Mid and late S phase replication sites are localised close to heterochromatin, at the nuclear periphery and around nucleoli. Very late S phase replication is visible as few large bright foci. The microscopic replication domains were estimated to contain about 1 Mbp of DNA based on the number of foci per cell and genome size \[[@B29-genes-10-00199]\]. At least six replicons (12 forks) are required to replicate such domains, as calculated from the average replication fork speed of 1.2 kbp/min and a life time of a typical factory of about 45 min \[[@B29-genes-10-00199]\]. Genome-wide replication timing analyses confirmed similar mean replication domain sizes of 1.4--3.6 Mbp, albeit with a high variability \[[@B18-genes-10-00199]\]. Domains that changed replication timing during differentiation had a lower average size of 0.4--0.8 Mbp. Active replication and transcription occur largely separately from each other, as co-labelled active replication and transcription foci showed little overlap in S phase nuclei \[[@B31-genes-10-00199],[@B32-genes-10-00199],[@B33-genes-10-00199]\]. This separation could help minimise replication-transcription interference to avoid genome instability \[[@B34-genes-10-00199],[@B35-genes-10-00199],[@B36-genes-10-00199],[@B37-genes-10-00199]\].
Interestingly, some genomic regions are subject to specific timing regulation. The heterochromatin at pericentromeres replicates very early in budding and fission yeasts, as does the heterochromatic mating type locus in fission yeast, whereas other heterochromatic regions replicate late. There are specific molecular mechanisms to determine these early and late replication times \[[@B38-genes-10-00199],[@B39-genes-10-00199]\], providing first examples that eukaryotes use active regulation to facilitate the appropriate replication timing. We will discuss these examples in detail below.
2. Complete and Well-Timed Domain-Wise Genome Replication Requires Coherent Origin Firing within Domains and Proper Control of Origin Firing Time {#sec2-genes-10-00199}
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Two types of control of replication origin firing are necessary in order to avoid large inter-origin gaps and ensure appropriately timed replication in the described domain-wise replication mode: (1) The timing with which the origins within individual domains fire needs to be controlled and (2) once a domain is set to replicate its origins must fire coherently---efficiently and in a narrow time window---to be fully replicated in the typical 45--60 min \[[@B17-genes-10-00199],[@B30-genes-10-00199],[@B40-genes-10-00199]\] a domain takes to replicate. To illustrate that coherent firing matters: A typical 1 Mbp replication domain can be copied by about 16--22 forks from 8--11 origins at a fork speed of 1 kb/min in 45--60 min if all origins fire reliably at the same time. If 50% of the origins fired with a delay of 30 min of a 60 min domain replication period 240 kbp will be left unreplicated if not compensated by firing of at least four more origins after 30 min.
3. Molecular Mechanisms of Origin Firing {#sec3-genes-10-00199}
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All temporal and spatial regulations of replication origin firing must ultimately control the activity of essential origin firing factors. The molecular mechanisms of eukaryotic origin firing are relatively well-known in budding yeast, where genetic screening, cell biology and biochemistry identified the essential origin firing factors and characterised fundamental activities and regulations. Recent biochemical re-constitution experiments then defined the set of core firing factors sufficient for in vitro origin firing, the establishment of bi-directional DNA replication with leading and lagging strand synthesis out of pre-replicative complex (pre-RCs) \[[@B41-genes-10-00199]\]: the Mcm2-7 helicase, the cell cycle kinases CDK and DDK, Sld2, Sld3, Sld7, Dpb11, Cdc45, the GINS complex, DNA polymerases epsilon and α/primase and Mcm10.
Eukaryotic replication initiation has two steps, origin licensing and origin firing. Licensing is the loading of the replicative helicase comprising the six Mcm2-7 subunits onto origin DNA. Licensing occurs exclusively under low-CDK conditions during mitotic exit and in the G1 phase of the cell cycle \[[@B42-genes-10-00199]\]. The loaded helicase forms a head-to-head dimer of two Mcm2-7 hexamers that encircle double-stranded DNA \[[@B43-genes-10-00199],[@B44-genes-10-00199]\] ([Figure 2](#genes-10-00199-f002){ref-type="fig"}A). The loaded Mcm2-7 double hexamer is called pre-RC. The core licensing machinery sufficient for pre-RC formation on naked origin DNA in vitro constitutes ORC (origin recognition complex), Mcm2-7, Cdt1 and Cdc6 \[[@B43-genes-10-00199],[@B44-genes-10-00199]\]. Other factors influence licensing of chromatin in living cells, such as the chromatin factors Hbo1 \[[@B45-genes-10-00199],[@B46-genes-10-00199]\], SNF2 \[[@B47-genes-10-00199]\], GRWD1 \[[@B48-genes-10-00199]\] and PR-Set7 \[[@B49-genes-10-00199],[@B50-genes-10-00199],[@B51-genes-10-00199],[@B52-genes-10-00199],[@B53-genes-10-00199]\] or Mcm-BP \[[@B54-genes-10-00199],[@B55-genes-10-00199]\]. In the pre-RC the helicase is not active, as no DNA unwinding seems to occur. Origin firing activates the helicase and converts pre-RCs into two active bi-directional replication forks or replisomes \[[@B56-genes-10-00199],[@B57-genes-10-00199],[@B58-genes-10-00199],[@B59-genes-10-00199]\]. Because origin firing requires high CDK and DDK activities it can only occur from G1-S transition when these kinases become active. In vitro, the first step of origin firing is phosphorylation of pre-RCs by DDK, which leads to the binding of the Sld3-Sld7 protein complex \[[@B60-genes-10-00199],[@B61-genes-10-00199],[@B62-genes-10-00199],[@B63-genes-10-00199],[@B64-genes-10-00199]\] ([Figure 2](#genes-10-00199-f002){ref-type="fig"}B,C). In vivo, the situation might be slightly more complicated as Sld3 and Cdc45 recruitment to origins depend on each other \[[@B65-genes-10-00199],[@B66-genes-10-00199]\]. CDK phosphorylates Sld3 and Sld2 that constitute the minimal set of CDK substrates sufficient for replication \[[@B67-genes-10-00199],[@B68-genes-10-00199],[@B69-genes-10-00199],[@B70-genes-10-00199]\] ([Figure 2](#genes-10-00199-f002){ref-type="fig"}D). Phospho-Sld3-Sld7 and phospho-Sld2 bind to separate phosphorylation and sequence-specific protein binding domains in the Dpb11 protein, BRCT repeat domains ([BR]{.ul}CA1 [C]{.ul}-[t]{.ul}erminal repeats) \[[@B69-genes-10-00199],[@B70-genes-10-00199]\] ([Figure 2](#genes-10-00199-f002){ref-type="fig"}D). As a result, the so-called SDS complex (Sld3/7-Dpb11-Sld2) may form. Although phospho-Sld2 and phospho-Sld3 interactions with Dpb11 events are essential for origin firing \[[@B69-genes-10-00199],[@B70-genes-10-00199]\] it is possible that, in vivo, they do not bind simultaneously or bind transiently because a stable SDS complex in cells has not been reported. Sld2 associates with and recruits GINS and DNA polymerase epsilon to pre-ICs \[[@B71-genes-10-00199]\], whilst Sld3 recruits Cdc45 \[[@B63-genes-10-00199]\]. We here call the SDS complex, Cdc45, GINS and polymerase epsilon bound to pre-RCs the [p]{.ul}re-[i]{.ul}nitiation [c]{.ul}omplex (pre-IC) \[[@B66-genes-10-00199],[@B72-genes-10-00199]\], although the exact structure of the pre-IC remains unclear. Pre-IC can be stably assembled in vitro \[[@B41-genes-10-00199],[@B64-genes-10-00199]\] and can be detected by ChIP in vivo \[[@B66-genes-10-00199]\] ([Figure 2](#genes-10-00199-f002){ref-type="fig"}E). Cdc45 and GINS assemble stably \[[@B73-genes-10-00199]\] with the Mcm2-7 helicase to form the CMG complex (Cdc45-Mcm2-7-GINS) \[[@B56-genes-10-00199],[@B74-genes-10-00199]\], the active replicative helicase \[[@B57-genes-10-00199],[@B75-genes-10-00199]\] ([Figure 2](#genes-10-00199-f002){ref-type="fig"}F).
During CMG formation the SDS complex proteins dissociate from origins \[[@B73-genes-10-00199]\]. In contrast to the other origin firing factors the SDS proteins are thought to have no role in active replisomes but constitute a regulation platform of origin firing. Once they have mediated CDK and DDK-dependent origin firing they are not needed any more and can help fire the next origin.
After CMG formation active replisomes assemble in an Mcm10 dependent manner \[[@B41-genes-10-00199],[@B76-genes-10-00199],[@B77-genes-10-00199],[@B78-genes-10-00199]\], which involves additional replication proteins and complex re-arrangements of the CMG helicase and the origin DNA \[[@B58-genes-10-00199],[@B79-genes-10-00199]\] ([Figure 2](#genes-10-00199-f002){ref-type="fig"}G). Bi-directional DNA synthesis can commence.
Although little mechanistic detail of replication initiation in metazoa is known, many of the basic principles of origin firing appear to be conserved, including those of the SDS firing regulation platform. Like in yeast, metazoan Sld3, Treslin/TICRR^Sld3^ \[[@B80-genes-10-00199],[@B81-genes-10-00199],[@B82-genes-10-00199]\], mediates CDK-dependency of origin firing by phosphorylation-mediated interaction with the Dpb11 orthologue TopBP1 \[[@B83-genes-10-00199],[@B84-genes-10-00199]\]. Vertebrate Sld2, RecQL4, is an essential firing factor \[[@B85-genes-10-00199],[@B86-genes-10-00199]\] but does not seem to depend on CDK. At first, an orthologue of the Sld3^Treslin^ interactor Sld7 \[[@B87-genes-10-00199]\] could not be identified in metazoa. Instead, an essential origin firing factor, the MTBP protein, for which no orthologue in yeast could be found and that binds to Treslin/TICRR^Sld3^ was described \[[@B88-genes-10-00199]\]. Only later remote homology of MTBP with Sld7 in two protein regions could be detected \[[@B89-genes-10-00199]\]. MTBP^Sld7^ completed the list of core origin firing factors in higher eukaryotes that had been defined in yeast. A stable protein complex containing Treslin/TICRR^Sld3^, MTBP^Sld7^ and TopBP1^Dpb11^ could be detected in cell lysates \[[@B88-genes-10-00199]\]. We will call it TMT complex (Treslin-MTBP-TopBP1) from here.
4. Pre-IC Formation is a Main Regulation Step of Origin Firing {#sec4-genes-10-00199}
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Many cellular molecular pathways are regulated at an early step to guarantee an economical use of resources. Naturally, the regulated activity in a molecular pathway must be rate limiting. The pre-IC factors DDK, Sld2/RecQL4, Sld3/Treslin, Dpb11/TopBP1 and Cdc45, have been shown to be limiting in yeast and/or vertebrate model systems \[[@B12-genes-10-00199],[@B13-genes-10-00199],[@B14-genes-10-00199],[@B15-genes-10-00199]\]. Consistent with pre-IC formation ([Figure 2](#genes-10-00199-f002){ref-type="fig"}B--E) being a regulation step of origin firing, the described cell cycle regulations by CDK and DDK occur at the pre-IC formation stage in yeasts and metazoa \[[@B60-genes-10-00199],[@B62-genes-10-00199],[@B63-genes-10-00199],[@B64-genes-10-00199],[@B69-genes-10-00199],[@B70-genes-10-00199],[@B83-genes-10-00199],[@B84-genes-10-00199],[@B90-genes-10-00199]\]. Regulation at the level of pre-IC formation is not a cell cycle-specific feature but rather a principle of origin firing control, as exemplified by the fact that firing regulation upon DNA damage also invokes pre-IC factors. Under DNA damage conditions, origin firing becomes inhibited by the S phase checkpoint to prevent genetic alterations due to copying damaged DNA templates. In yeast, the checkpoint inhibits CDK and DDK action \[[@B91-genes-10-00199],[@B92-genes-10-00199],[@B93-genes-10-00199]\]. The Rad53 checkpoint kinase phosphorylates Sld3^Treslin^, suppressing the CDK-dependent binding of Sld3^Treslin^ to Dpb11^TopBP1^. Rad53 also phosphorylates the Dbf4 subunit of DDK, which inhibits this kinase. As discussed in detail below, the origin firing timing mechanisms uncovered to date also involve the regulation of pre-IC factors.
The metazoan orthologues of the SDS complex, TMT complex proteins Treslin/TICRR^Sld3^, MTBP^Sld7^ and TopBP1^Dpb11^, have metazoa-specific protein domains in addition to the core domains conserved with yeast \[[@B88-genes-10-00199],[@B89-genes-10-00199]\]. These metazoa-specific domains may well constitute regulatory units that mediate proper control of origin firing in the complex metazoan cells, in which appropriately controlled origin firing is probably particularly important. Consistently, the metazoa-specific C-terminus of Treslin/TICRR^Sld3^ has non-essential roles in replication \[[@B94-genes-10-00199],[@B95-genes-10-00199]\], as discussed below and the metazoa-specific MTBP^Sld7^ middle region is required for replication \[[@B89-genes-10-00199]\]. This MTBP^Sld7^ middle domain role partly depends on binding the Cdk8/19-cyclin C kinase, although the exact role of this interaction is unclear.
The SDS/TMT regulation hub alongside CDK and DDK kinases will certainly prove to have a key role in controlling both timing and coherence of origin firing to achieve complete and well-timed domain-wise genome replication.
5. Origin Firing Control to Determine Replication Timing {#sec5-genes-10-00199}
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How is the timing of origin firing in individual replication domains determined? Two principle possibilities appear plausible: (1) chromatin organisation in structural units determines if and when a domain fires its origins. For this, origin firing factors, for example TMT, CDK or DDK, need to be granted access to or allowed to be active within the confines of the domain at the right time ([Figure 3](#genes-10-00199-f003){ref-type="fig"}A). (2) Firing of clusters of origins is regulated at the origin level rather than being a consequence of a structural chromatin unit. For this, the origins in a cluster need to be regulatorily coupled. Coupling could involve that the origins in a cluster are subject to the same control pathway, be it inhibitory so that origins are kept inactive until a signal relieves the inhibition or activating so that the firing of neighbouring origins is simultaneously stimulated ([Figure 3](#genes-10-00199-f003){ref-type="fig"}B).
Intuitively, origin coupling could involve that neighbouring origins influence each other. The unfired origins in a cluster could inhibit each other until at least one origin fires, relieving the inhibitory signal. Alternatively, firing of the first origin in a cluster could create an activating signal and stimulate firing of its neighbours.
Alternatively, timing could be controlled by an intrinsic property of origins rather than by active regulation by external factors. For example, origins of early domains could respond more sensitively than late origins to limiting origin firing factors, for example due to higher accessibility or higher affinity and therefore fire earlier. It was proposed that the different firing probabilities between origin clusters resulting from such specific sensitivities to firing factors could largely explain replication timing programs like those seen in eukaryotes \[[@B96-genes-10-00199]\].
Models 1 and 2 ([Figure 3](#genes-10-00199-f003){ref-type="fig"}) are not mutually exclusive. For example, origin coupling could involve elements of chromatin structure and modification. Conversely, origins could modify their chromatin environment depending on their firing activity.
5.1. Structural Chromatin Units May Underlie Replication Timing {#sec5dot1-genes-10-00199}
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That stable chromatin units may underlie replication timing was proposed based on the finding that replication in metazoan cells occurs in form of individual physical chromatin units, today called replication domains. Early studies analysing isolated DNA fibres from mammalian cells on which replication had been labelled using radioactive nucleotides showed that origins do not fire scattered over the DNA but that juxtaposed origins often fire nearly simultaneously in clusters (reviewed in Reference \[[@B17-genes-10-00199]\]). Microscopic work mentioned above using fluorescence labelling of active replication sites established that replication occurs in discrete replication foci in mammalian nuclei. The foci represent a single or a small number of origin clusters. Replication domains were proposed to form stable physical units as labelled domains were stable over many successive cell divisions \[[@B29-genes-10-00199],[@B40-genes-10-00199],[@B97-genes-10-00199],[@B98-genes-10-00199]\].
A specific replication timing program was proposed based on the observation that the same replication foci patterns (specific for early, mid and late S phase) re-occurred in every S phase \[[@B17-genes-10-00199],[@B27-genes-10-00199],[@B28-genes-10-00199],[@B30-genes-10-00199]\]. Elegant nuclei transfer experiments then drew a link between chromatin domain formation and replication timing \[[@B28-genes-10-00199]\]. This work showed that a time point in early G1 phase exists at which replication timing is determined, the timing decision point (TDP). For these experiments, nuclei isolated from synchronised mammalian cells were replicated in *Xenopus* egg extracts. In nuclei isolated from cells in mitosis or G1 before the TDP (up to 1 h after anaphase onset), the different genome regions did not replicate in a defined order but in a random fashion typical for *Xenopus* embryonic extracts. In contrast, chromatin isolated more than 2 h after mitosis replicated in the same order as in the cells of origin. They had passed the TDP. The TDP coincided with the time of re-establishment of an interphase-like chromatin architecture out of the mitotic chromatin. The authors therefore suggested that the establishment of interphase chromatin domains in G1 may specify replication timing in the subsequent S phase. Later genome-wide proximity studies of genome regions in cells by HiC showed a correlation of genome structure with replication timing \[[@B19-genes-10-00199],[@B99-genes-10-00199]\]. It turned out that replication domains largely overlap with stable chromatin folding units, topologically associated domains (TADs) \[[@B100-genes-10-00199]\]. Re-formation of these TADs after mitosis coincided with the TDP \[[@B101-genes-10-00199]\].
However, direct poof that the structuring of chromatin into folding units underlies the determination of replication timing has not been provided. It has also not been proven that the formation of the microscopically visible replication foci that reflect structural chromatin domains is required to determine replication timing. In fact, genome structure and replication timing do not always correlate: G2 cells retain the overall TAD organisation but replication timing is random when G2 nuclei are forced to replicate either in *Xenopus* egg extracts or by inducing a second replication round in G2 cells \[[@B101-genes-10-00199],[@B102-genes-10-00199]\]. Conversely, G0 cells whose chromatin undergoes great changes in organisation retain replication timing. Taken together, it seems that even if the formation of stable chromatin folding units is required to determine replication timing it is not sufficient. One or more activities that are absent in G2 chromatin are required at the TDP for establishment of replication timing.
5.2. How Could the Folding of Chromatin into Physical Units Determine Origin Firing Time? {#sec5dot2-genes-10-00199}
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A chromatin domain could form a confined space that excludes or concentrates origin firing factors, thereby controlling firing timing. However, there is little direct evidence to verify this idea.
A well-established concept is that chromatin structure determines the accessibility of its DNA to DNA binding proteins. Controlled accessibility of DNA for firing factors within a chromatin domain could regulate firing timing. Correlations between high DNA accessibility and early replication activity have been drawn. Genome-wide HiC analysis in cultured cells revealed a good correlation between the nuclear compartment containing open, transcriptionally active chromatin and early S phase replication, whereas the compartment containing closed heterochromatin replicates late \[[@B19-genes-10-00199]\]. Moreover, opening chromatin structure by deletion of histone deacetylases from yeast cells, by recruiting acetylases to chromatin in human cells or by induction of transcription in *Drosophila* can lead to earlier origin firing \[[@B103-genes-10-00199],[@B104-genes-10-00199],[@B105-genes-10-00199],[@B106-genes-10-00199],[@B107-genes-10-00199]\]. Recently, it was suggested that more open chromatin induced by preventing methylation of lysine 4 of histone 4 in cultured mammalian cells increases origin firing \[[@B108-genes-10-00199]\]. Here, origin licensing in addition to origin firing was elevated upon induced chromatin opening, indicating that the amount of licensing could affect whether and how efficiently an origin fires. Perhaps increased pre-RC levels locally increase the concentration of firing factors.
Another model for how chromatin domain formation determines firing timing is that domains could constitute structural units to control DNA position in the nucleus. Re-positioning of domains could move DNA between nuclear regions with high or low concentrations of firing factors. It was suggested that localisation of late replicating telomeric DNA close to the nuclear periphery may withdraw it from regions with high firing factor concentrations in the nuclear interior \[[@B109-genes-10-00199]\]. However, artificial peripheral localisation is not always sufficient to mediate late replication of a genome region that is normally located in the nuclear interior \[[@B110-genes-10-00199]\]. Folding of DNA into chromatin domains could also control firing timing by bringing origins in close proximity to each other, as suggested for how forkhead transcription factors mediate early origin firing \[[@B111-genes-10-00199]\] (discussed in detail below).
5.3. In Yeast, Several Origin Firing Control Pathways in Combination with Competition between Origins for Firing Factors Determine Replication Time {#sec5dot3-genes-10-00199}
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It has emerged in recent years that yeast cells utilize distinct molecular pathways to control origin firing timing of different genome regions. There is even more than one pathway to control the firing of separate groups of early and late origins, respectively. For example, both pericentromeric heterochromatin and a large proportion of the transcribed euchromatin replicate early. Yet pericentromeric origins require the kinetochore protein Ctf19 for early firing whereas many euchromatic origins rely on forkhead transcription factors \[[@B38-genes-10-00199],[@B111-genes-10-00199]\]. Different late origins require Rif1 and/or Taz1 and/or shelterin for late firing, depending on whether they are in chromosome arms, subtelomeres or telomeres \[[@B112-genes-10-00199],[@B113-genes-10-00199],[@B114-genes-10-00199]\]. These pathways are discussed in detail below.
In addition to such active firing control pathways, competition between origins for limiting firing factors is important to determine firing time. In mutant yeast and mammalian cells, in which firing of early origins is delayed, the firing of later origins is advanced and vice versa \[[@B111-genes-10-00199],[@B115-genes-10-00199]\]. Apparently, early origin firing delays late origins in wild-type conditions. In the delay mutants, the early origins do not effectively compete with late origins for firing factors, giving late origins better access to these factors.
Together, these insights suggest that eukaryotes employ several distinct regulation pathways specific for the different genome regions to advance the firing of some origins and delay others. Combined with competition for limiting firing factors these pathways establish the replication timing program ([Figure 4](#genes-10-00199-f004){ref-type="fig"}). What necessitates several specific molecular mechanisms to determine firing time? To date, we can only speculate that the distinct chromatin structures and the different ongoing chromatin processes in the different genome regions, for example transcription activity, require distinct regulatory pathways.
5.4. Forkhead Transcription Factors Advance Origin Firing Timing in Budding Yeast {#sec5dot4-genes-10-00199}
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The forkhead transcription factors Fkh1 and Fkh2 are required in budding yeast for early replication of about 30% of origins \[[@B111-genes-10-00199]\] ([Figure 4](#genes-10-00199-f004){ref-type="fig"}). This was revealed by analysis of genome-wide replication dynamics using purified nascent DNA in yeast mutants lacking Fkh1/2. The control of replication timing by forkhead proteins appears to be independent of their classic role in transcription. To mediate early firing the binding of forkhead to DNA in the vicinity of the origin was required. This forkhead-DNA interaction may facilitate the formation of conglomerates of early firing origins, as suggested by DNA proximity analysis using the 4C technique to compare wild-type and forkhead mutant cells \[[@B111-genes-10-00199]\]. An interaction between Fkh1/2 bound to origins and the licensing factor ORC bound to neighbouring origins may bring juxtaposed origins into proximity. How origin proximity advances firing time is unknown. A conceivable model is that in conglomerates origins cooperatively concentrate limiting firing factors.
In addition to these structural effects of forkhead factors on chromatin Fkh1/2 also directly recruit DDK kinase to early origins to control their firing \[[@B116-genes-10-00199]\]. Early firing required a direct interaction between Fkh1/2 and the Dbf4 subunit of the DDK kinase. A binding-deficient Dbf4 mutant delayed origin firing. Origin localisation of DDK was the only function of the interaction domain of Dbf4 required for early replication since the firing delay of this mutant was rescued by fusing it to forkhead. Whether and how the structural and DDK recruitment roles of Fkh1/2 cooperate to mediate early firing remains to be investigated.
5.5. Early Firing of Pericentromeric Heterochromatin Origins Depends on the Recruitment of DDK by the Ctf19 Kinetochore Protein or Swi6 in Budding and Fission Yeast, Respectively {#sec5dot5-genes-10-00199}
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Despite the typically late replication of heterochromatin, the heterochromatin at pericentromeres replicates in early S phase in yeasts ([Figure 4](#genes-10-00199-f004){ref-type="fig"}). In fission yeast, pericentromeric heterochromatin as well as the heterochromatic *matK* locus but not the subtelomeric heterochromatin, show early origin firing. Pericentromeres and *matK* recruit DDK dependently on the heterochromatin protein Swi6, the fission yeast orthologue of metazoan heterochromatin protein 1 (HP1) \[[@B39-genes-10-00199]\]. DDK recruitment is mediated by an interaction between Swi6 and Dfp1, the fission yeast Dbf4 orthologue. Although subtelomeres have also Swi6 bound, their origins fire late. This late firing appears to be due to limiting activity of DDK because artificial recruitment of DDK to subtelomeres advanced replication timing. Whether low concentration of DDK at subtelomeres (despite Swi6) or high subtelomeric activity of the DDK counteracting activity of Rif1-PP1 (discussed below) are responsible for late firing of subtelomeric origins is unclear.
In budding yeast, early replication of centromeres relies on DDK recruitment to pericentromeric origins dependently on the Ctf19 kinetochore protein \[[@B38-genes-10-00199]\]. Thus, fission and budding yeasts utilise separate molecular mechanisms to fire pericentromeric origins early, both culminating in the recruitment of DDK.
Ctf19-dependent DDK recruitment to pericentromere origins occurs in telophase and G1 phase, indicating that DDK recruitment precedes the firing of the respective origins in the subsequent S phase \[[@B38-genes-10-00199]\]. This is reminiscent of the above-mentioned TDP in G1 phase described in higher eukaryotes \[[@B28-genes-10-00199]\].
The presence of DDK at origins in G1 potentially poses a problem. If the G1-recruited DDK was active, the cell would run the risk of firing these DDK-bound origins prematurely in G1, inevitably leading to re-licensing and re-firing. Eukaryotes control origin firing by DDK and CDK that both increase in activity upon S phase entry to mediate S phase specificity of firing. The requirement for two distinct S phase kinase pathways is considered a safety mechanism against premature firing and bypassing one control pathway likely compromises robustness of this regulation. This means either that origin-bound DDK needs to be kept inactive or that other firing factors need to be strictly suppressed in G1. It was suggested that a protein complex formed by Rif1 and protein phosphatase 1 (PP1) may keep DDK bound to G1 origins in check until the time is right for firing in S phase \[[@B117-genes-10-00199]\]. This was concluded from the observation that phosphorylation of the DDK substrate Mcm4 increased in G1 phase upon attenuation of PP1 action by inactivating its targeting factor Rif1.
Despite this Rif1-dependent DDK inhibition in G1, there is indication that DDK is active in G1 phase at some early firing origins in budding yeast. Thorough ChIP experiments showed that Sld3^Treslin^ is bound to early origins in G1 phase \[[@B65-genes-10-00199],[@B73-genes-10-00199]\]. This suggests that DDK is active at these origins as DDK is required for Sld3^Treslin^ interaction with pre-RCs \[[@B13-genes-10-00199],[@B63-genes-10-00199],[@B64-genes-10-00199]\]. The issue of regulating DDK activity in G1 cells and how origin firing is prevented in G1 to avoid re-replication and genome instability clearly requires further investigation.
Ctf19-mediated early origin firing may have evolved to meet a specific requirement of the pericentromeric genome region. The recruited DDK does not only allow pericentromeric origins to fire early it also recruits the Scc2-Scc4 cohesion loading complex to pericentromeres \[[@B38-genes-10-00199]\]. In the absence of DDK-dependent Scc2-Scc4 recruitment pericentromeric sister chromatid cohesion was compromised. Thus, Ctf19-mediated DDK recruitment couples early replication with cohesion establishment at centromeres. Why this coupling is necessary is less clear. It is conceivable that the limited time between chromosome duplication and segregation due to short G2 phase in budding yeast necessitates particularly effective control of cohesion establishment at sister centromeres.
5.6. Rif1 and Taz1 Facilitate Late Origin Firing in Yeasts {#sec5dot6-genes-10-00199}
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In theory, the late firing of the heterochromatic origins that are not regulated by Swi6/Ctf19 could be a passive process resting on two elements: the competition between origins for limiting firing factors and the low accessibility of heterochromatin for firing factors. These could combine into a delay of firing time. Although this passive process likely contributes to late firing, yeasts employ active regulation to mediate late firing of origins, for example in subtelomeric heterochromatin ([Figure 4](#genes-10-00199-f004){ref-type="fig"}). The late timing of these origins depends on the proteins Rif1 and Taz1, as genome-wide replication timing analyses of fission yeast mutants showed \[[@B112-genes-10-00199],[@B113-genes-10-00199]\]. Budding yeast Rif1 binds PP1 directly through a conserved interaction motif and this is thought to target PP1 to late replicating chromatin \[[@B117-genes-10-00199],[@B118-genes-10-00199],[@B119-genes-10-00199]\]. A model was proposed that binding of fission yeast Rif1-Taz1 complexes to telomeres (Taz1 is homologous to metazoan telomere repeat factors TRF1/2) creates a cloud of PP1 activity around telomeres, delaying the firing of origins in the vicinity, including those in subtelomeres, by counteracting DDK \[[@B114-genes-10-00199]\]. Supporting this model, genetic interaction studies in budding yeast showed that Rif1 counteracts DDK in delaying origin firing \[[@B113-genes-10-00199],[@B117-genes-10-00199]\].
Another class of late firing origins also depends on Taz1 \[[@B112-genes-10-00199]\]. These origins are located away from telomeres in the chromosome arms and contain telomere repeat DNA for binding Taz1 ([Figure 4](#genes-10-00199-f004){ref-type="fig"}). About 50% of late origins in chromosome arms belong to this class. Mutant yeast strains showed that the late firing of these origins, unlike subtelomeric origins, requires the shelterin telomere binding complex that interacts with Taz1 \[[@B114-genes-10-00199]\]. The model developed proposes that Taz1-shelterin interaction brings Taz1-bound chromosome arm origins in close proximity to telomeres, where their firing is delayed by the high local activity PP1 enriched there by Rif1. Consistently, fluorescently labelled Taz1-dependent arm origins are located at the nuclear periphery \[[@B114-genes-10-00199]\], where telomeres reside due to their anchoring to the inner nuclear membrane \[[@B120-genes-10-00199]\]. Peripheral localisation of origins is dispensable for late firing as shown using yeast mutants that cannot tether telomeres to the nuclear membrane \[[@B114-genes-10-00199]\]. Instead, it is probably the proximity of the origins to telomeres mediated by the Taz1-shelterin interaction that delays firing.
Yet another class of late firing origins in chromosome arms depend on Rif1 but not Taz1 \[[@B112-genes-10-00199],[@B113-genes-10-00199],[@B121-genes-10-00199]\]. Rif1 localises in the vicinity of these origins and prevents DDK action, presumably by recruiting PP1 \[[@B112-genes-10-00199],[@B113-genes-10-00199]\] ([Figure 4](#genes-10-00199-f004){ref-type="fig"}).
Together, it appears that yeast Rif1, Taz1 and PP1 constitute a set of versatile firing time regulators that integrate various signals to mediate late firing of different classes of origins.
5.7. Metazoa May Utilise Rif1-Dependent and Independent Pathways to Mediate Late Replication {#sec5dot7-genes-10-00199}
--------------------------------------------------------------------------------------------
Our picture of the molecular pathways that control origin firing timing in higher eukaryotes is far less complete than in yeasts. Rif1 could be confirmed as an important determinant of late replication. Mouse Rif1 knock-out cells and Rif1 siRNA-treated cultured human cells showed gross changes in genome-wide replication timing profiles \[[@B115-genes-10-00199],[@B122-genes-10-00199]\]. The fraction of the genome that changed replication timing was significant: it was greater than the fraction that changes timing during differentiation of embryonic stem cells into neural progenitor cells \[[@B115-genes-10-00199]\]. A subpopulation of usually late replicating domains shifted to earlier timing and some early domains replicated later. The primary effect of Rif1 inactivation is probably the relieving of firing suppression in late domains, resulting in their earlier replication, whereas the replication delay of early domains is indirect due to increased competition for limiting firing factors. Mouse Rif1 was shown to associate with late replicating domains by genome-wide ChIP and fluorescence microscopy \[[@B115-genes-10-00199]\]. In a subset of these domains, in chromocenters whose replication dynamics can be particularly well observed microscopically, it appeared that replication only started after Rif1 had left this region. This is consistent with a model where Rif1 inhibits origin firing until its timed departure allows firing.
Two molecular mechanisms have been suggested for how Rif1 mediates late firing in metazoa. (1) Rif1 may delay origin firing by targeting PP1 to late firing origins, similar to yeast. In line with this model, mouse Rif1 associates with PP1 \[[@B123-genes-10-00199]\]. Moreover, human and *Xenopus* Rif1 and PP1 cooperate in counteracting DDK phosphorylation of Mcm2-7 subunits at replication origins, decreasing their firing efficiency \[[@B124-genes-10-00199]\]. However, it has not been finally proven that metazoan Rif1 controls firing timing by targeting PP1 to origins to reverse DDK phosphorylation. (2) Rif1 may---alternatively or additionally---control firing time by affecting chromatin domain organisation. 4C-seq analysis revealed that Rif1 deletion in mouse embryonic stem cells and embryonic fibroblasts leads to robust changes in chromatin domain organisation \[[@B125-genes-10-00199]\]. After Rif1 inactivation, replication domains interacted weaker with neighbouring domains that have the same replication timing in wild-type conditions. Instead, the domains showed a higher number but weak interactions with domains of different replication timing. This change in domain organisation upon Rif1 inactivation was independent of and preceded the change in replication timing, as observed after acute inactivation of Rif1 in synchronised cells. The data was interpreted such that Rif1 is required to define and constrain chromatin domains as physical units of replication timing. In Rif1 knock-out cells these physical units become less defined and as a result the timing of replication changes. This work showed for the first time that the controls of chromatin domain organisation and replication timing are combined in the same molecule, Rif1. Together these observations are consistent with but do not prove, the model that domain structure underlies replication timing.
Also metazoa seem to have more than one molecular pathway to determine replication timing, since upon Rif1 deletion not all late replication domains advance replication time \[[@B115-genes-10-00199]\]. Two classes of late replicating genomic domains could be defined, one characterised by high levels of Rif1 and association with lamin B (Rif1/LB^+^), the other one characterised by high Rif1 levels but low lamin B (Rif1/LB^−^) \[[@B125-genes-10-00199]\]. Rif1/LB^-^ domains were the late regions that changed their replication time to early upon Rif1 inactivation, whereas Rif1/LB^+^ domains did not. This means that Rif1 is required for late replication of one class of domains, Rif1/LB^−^. In Rif1/LB^+^ domains, Rif1 is either not required to regulate timing or it acts redundantly with another timing control mechanism.
A third class of late domains may be controlled by polymerase theta \[[@B126-genes-10-00199]\]. In polymerase theta depleted cells, earlier replication of a subset of late domains was observed. This was accompanied by higher levels of Mcm2-7 helicase on chromatin, suggesting that increased licensing may underlie earlier replication. It is conceivable that excess pre-RCs advance replication timing by increasing the local concentration of firing factors or by inducing a more open chromatin structure.
In conclusion, a model is emerging that, like yeasts, higher eukaryotes utilise distinct mechanisms to control replication timing of different genome regions.
5.8. The TMT Complex May Help Control Firing Timing in Metazoa {#sec5dot8-genes-10-00199}
--------------------------------------------------------------
The TMT complex, Treslin/TICRR^Sld3^-MTBP^Sld7^-TopBP1^Dpb11^, is a regulation platform for origin firing in metazoa. Its counterpart in yeast, the SDS complex, Sld3^Treslin^-Sld7^MTBP^-Dpb11^TopBP1^ together with Sld2^RecQL4^, mediates cell cycle and DNA damage checkpoint regulations and these factors are subject to subtle control of levels and activity to ensure complete and accurate genome replication \[[@B12-genes-10-00199],[@B13-genes-10-00199],[@B127-genes-10-00199],[@B128-genes-10-00199]\]. In metazoa, TMT mediates conserved cell cycle and checkpoint regulations \[[@B83-genes-10-00199],[@B84-genes-10-00199]\] and TopBP1^Dpb11^ and Treslin/TICRR^Sld3^ have also been implicated in other regulations, for example the coupling of genome replication to sufficiently high nutrient levels \[[@B129-genes-10-00199]\]. The TMT proteins are well-equipped to integrate signals from many regulatory pathways. Apart from the conserved core domains Treslin/TICRR^Sld3^ contains metazoa-specific N- and C-terminal domains that do not perform essential core origin firing functions but are required for normal DNA replication in human cells \[[@B82-genes-10-00199],[@B94-genes-10-00199],[@B95-genes-10-00199]\] (Ferreira and Boos, unpublished). Moreover, MTBP whose yeast orthologue Sld7^MTBP^ does not seem to be a very important signal integrator has apparently evolved into an origin firing regulator in metazoa. Apart from the Sld7-homologous functions, it contains a metazoa-specific domain that is important for complete genome duplication in human cells \[[@B89-genes-10-00199]\]. The molecular functions of this domain are only partially understood.
The metazoa-specific C-terminus of Treslin/TICRR^Sld3^ has been implicated in controlling replication timing \[[@B95-genes-10-00199]\]. A short motif in the large C-terminus mediates the binding of Treslin/TICRR^Sld3^ to the BRD2 and BRD4 proteins that associate with acetylated chromatin via bromodomains. This interaction recruits Treslin/TICRR^Sld3^ to chromatin, presumably at acetylated genome regions. Treslin/TICRR^Sld3^ mutants that cannot bind to BRD2/4 replicated at relatively normal overall speed but showed aberrant execution of the replication program. EdU labelling and fluorescence microscopy of human cells revealed that early and mid S phase replication foci patterns merge in cell lines expressing BRD2/4 binding-deficient Treslin/TICRR^Sld3^. These aberrant patterns suggest changed replication timing. Genome-wide timing analysis in these mutants will be required to confirm and specify the timing changes. A feasible model of how Treslin/TICRR^Sld3^ mediates correct replication timing is that its appropriately timed acetylation-dependent recruitment to specific chromatin sites induces replication locally. How the appropriate timing of Treslin/TICRR^Sld3^ recruitment is regulated in the cell is a key question. Because acetylation is often associated with open euchromatin that replicates early Treslin/TICRR^Sld3^ recruitment could be important for early replication and, as a consequence of competition for limiting firing factors, help to delay replication of late domains, which could explain the observed merging of early and late patterns in cells lacking this regulation.
6. Firing Coherence Mechanisms {#sec6-genes-10-00199}
==============================
As already mentioned, it is important for cells to fire enough origins in a short time window once a replication domain is set to replicate. How such coherent origin firing is achieved has been little investigated. It must involve a local, switch-like transition from an origin firing-incompetent to a competent state to ensure that, before the switch is turned, not a single origin fires and, after turning, many origins fire nearly synchronously. Thus, the rate-limiting step(s) of the origin firing reaction must be switched on both sharply and locally. This switch could involve any of the molecular mechanisms used to control the localisation or activity of firing factors discussed above. Intuitively, linking the origins of a domain, for example, by physical clustering, like suggested for forkhead factors \[[@B111-genes-10-00199]\], could be the basis to create coherent firing. Such physical linkage between origins could help control the firing threshold of origins in a cluster. It was proposed that it would suffice to have a higher threshold for the firing of the very first origin of a replication domain than for the others to create nearly synchronous firing in a replication domain \[[@B130-genes-10-00199]\].
Mathematical modelling suggested that, in yeast, the multisite phosphorylation of Sld2^RecQL4^ and Sld3^Treslin^ could induce their switch-like binding to Dpb11^TopBP1^ in a manner, mediating coherent origin firing \[[@B131-genes-10-00199]\]. This is reminiscent of a famous example of biological switches, the CDK inhibitor Sic1. In response to rising CDK levels at the G1-S phase transition, six CDK phosphorylation sites of Sic1 act cooperatively to induce the switch-like degradation of Sic1 by the ubiquitin-proteasome pathway \[[@B132-genes-10-00199]\]. In analogy, the origins of a replication domain could be switched on when local CDK activity passes a threshold between an intermediate level that does not promote firing but inhibits licensing and a high level that allows firing. Although the CDK phosphorylation of only two sites in Treslin/TICRR^Sld3^ is sufficient for relatively high affinity binding to TopBP1^Dpb11^, Treslin/TICRR^Sld3^ contains dozens more CDK consensus sites that could regulate this binding event.
7. Conclusions {#sec7-genes-10-00199}
==============
Eukaryotes have evolved distinct molecular pathways to control firing timing of distinct sets of origins. This indicates that appropriate replication timing serves to adapt genome replication to the specific requirements of the different genomic regions. These active timing control pathways combine with the competition of origins for limiting firing factors to establish the temporal program of genome replication. During competition between origins the much-discussed intrinsic firing propensity of individual origins likely plays an important role in determining which origins win the competition and fire first.
Pre-IC formation is the step at which firing timing regulation occurs. In yeasts, the control of DDK activity is a common theme among firing control pathways. In higher eukaryotes, the TMT regulation platform is a good candidate to mediate firing time control. Rif1 is conserved from yeast to man as a mediator of late firing. Much research is required, particularly in metazoa, to uncover and understand how origin firing time is determined.
Once the molecular mechanisms are known it will be possible to directly address how important replication timing control is by manipulating these pathways using genetics and chemical inhibitors. As mentioned, replication timing changes with cell differentiation and tumorigenesis and has been linked to genome instability but the nature of these links---e.g., if timing changes are cause or consequence and what the cellular mechanisms are---is mostly unknown. Thus, investigating the relevance of replication timing control is important for our understanding of how cells work and also for human health.
We would like to thank Doris Hellerschmied-Jelinek for critical comments on the manuscript and all members of the Dominik Boos and Stefan Westermann labs for scientific discussions.
Conceptualization, D.B.; writing---original draft preparation, D.B., P.F.; visualisation, P.F.; funding acquisition, D.B.
NRW Rückkehrerprogramm fellowship Ministry of Culture and Science North Rhine-Westphalia, Germany. Core funding of the University of Duisburg-Essen.
The authors declare no conflict of interest.
{#genes-10-00199-f001}
{#genes-10-00199-f002}
{#genes-10-00199-f003}
{#genes-10-00199-f004}
|
VANCOUVER, May 7, 2013 /PRNewswire/ - WesternOne Inc. (TSX: WEQ, WEQ.DB,
WEQ.DB.B, WEQ.DB.C) today announced that its modular building division
Britco has signed a memorandum of understanding with the Lax Kw'alaams
Band to establish a modular building rental business.
The focus of this innovative partnership will be to lease and sell
modular buildings within a territory that stretches from Prince Rupert
to Terrace and north to Stewart.
"Britco has been investing in people in British Columbia for over 36
years and they are very clearly focused on generating meaningful,
long‐term partnerships," stated Lax Kw'alaams Mayor, Garry Reece. There
will be an office established in Prince Rupert with a member of Lax
Kw'alaams employed by Britco to develop business opportunities in the
region. This will provide a long term career management opportunity in
sales and marketing for the Lax Kw'alaams individual who will receive
significant training and support from Britco.
"We are very excited about the potential of our partnership with Lax
Kw'alaams and we are looking forward to building opportunities with the
Lax Kw'alaams in this region," said Mike Ridley, President of Britco.
The memorandum of understanding was signed by Lax Kw'alaams Mayor Garry
Reece and Britco President Mike Ridley in Prince Rupert, B.C.
About Lax Kw'alaams Band
The Coast Tsimshian village of Lax Kw'alaams is located north of Prince
Rupert on the northwestern coast of British Columbia.
Lax Kw'alaams owns and operates a number of businesses including Lax
Kw'alaams Fishery and Coast Tsimshian Resources. Lax Kw'alaams Fishery
is the leading canned food producer in the region. The plant has
overseas customers in China and the United States of America and
products from this cannery are sold under several brands. Lax Kw'alaams
Fishery is 100% owned by the Lax Kw'alaams Band. Coast Tsimshian
Resources holds 3 forest tenures in Northwestern BC.
With over 1,000 employees in Australia, Canada, and the United States,
Britco is one of the largest modular construction companies in the
industry.
Britco provides temporary and permanent residential and commercial
modular buildings and offers, not only leading design-build
capabilities but also turn-key construction project management services
for a wide range of customers in the infrastructure, construction,
energy and resource sectors. Britco also has one of the largest lease
fleets of office complexes, construction site offices, sales centres
and storage containers in British Columbia. Britco is headquartered in
Langley, British Columbia.
WesternOne Inc. seeks to acquire and grow businesses in the construction
and infrastructure services sector in order to generate stable and
growing dividends to its shareholders and to achieve capital
appreciation. |
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Disclaimer: Information provided through RealAdventures website, newsletters, emails or other services has been provided directly by the companies and/or individuals owning or offering the products/services. RealAdventures does not guarantee or warranty the accuracy of the information contained herein. It is the sole responsibility of the user to ensure the accuracy and clarity of any posted material and to determine the suitability of any service for their particular needs or requirements. Likewise, it is the responsibility of advertisers to determine the suitability and credit worthiness of potential customers prior to any transaction. |
Expert diagnostic system for moving-coil loudspeakers using nonlinear modeling.
This work aims at the development of an expert diagnostic system for moving-coil loudspeakers. Special emphasis is placed on the defects resulting from loudspeaker nonlinearities. As a loudspeaker operates in the large signal domain, nonlinear distortions may arise and impair sound quality. Analysis of nonlinear responses can shed light on potential design faults of a loudspeaker. By exploiting this fact, this expert diagnostic system enables classification of design faults using a defect database alongside an intelligent fault inference module. Six types of defects are investigated in this paper. A large signal model based on electromechanical analogous circuits is employed for generating the defect database, through which a neural-fuzzy network is utilized for inferring the defect types. Numerical simulations and experimental investigations were undertaken for validating the loudspeaker diagnostic system. |
The use of a power takeoff device (herein referred to by its common abbreviation “PTO”) in association with truck or other vehicle (or stationary engine) transmissions is generally known. Generally speaking, such PTO's often include an input gear, an output gear and a mechanism for engaging the input gear with the output gear so as to rotate the output shaft of the PTO to power an auxiliary device to perform useful work when desired. The mechanism also provides a means for disengaging the input gear and the output gear.
In certain conventional designs, the input gear of the PTO is constantly engaged with the appropriate gear in the vehicle's transmission when installed on the vehicle and is, thus, continuously turning whenever that gear in the vehicle's transmission is turning. In order to provide for selective rotation of the output shaft of the PTO (associated with the output gear), a clutch mechanism is frequently provided between this input gear of the PTO and the output gear. When this output shaft is rotated, useful auxiliary work can be performed. For example, the output shaft may be connected to a hydraulic pump that may be used to operate auxiliary equipment, such as garbage compacting bodies, dump bed bodies (a/k/a, “dump trucks”), garbage trucks or trailers, winches, post hole diggers, and the like. Example PTOs are disclosed in U.S. Pat. No. 5,542,306, issued Aug. 6, 1996; U.S. Pat. No. 7,070,036, issued Jul. 4, 2006; and U.S. Pat. No. 7,159,701, issued Jan. 9, 2007.
One type of conventional PTO clutch is operated through an aligned multi-disc stack arrangement located between the shaft of the PTO and the gears of the PTO which engage with the gears of the truck's transmission. When an axial force is applied to the disc stack, the individual discs in the stack are forced together such that the interfacial friction between the discs cause the shaft to be rotationally joined to the rotating gears. Engagement and disengagement of the clutch is frequently effected by an electrically operated solenoid valve arrangement, the switch for which is frequently located (along with an appropriate warning light) in the cab of the truck. |
Chris and his girlfriend Rose go upstate to visit her parents for the weekend. At first, Chris reads the family's overly accommodating behavior as nervous attempts to deal with their daughter's interracial relationship, but as the weekend progresses, a series of increasingly disturbing discoveries lead him to a truth that he never could have imagined. |
NEW YORK (Reuters) - On Oct. 10 last year, eight North Korean vessels - several carrying illicit coal shipments - were anchored in Chinese waters off the port of Ningbo-Zhoushan, according to a photo in a U.N. report published online on Friday.
A photo from the annual report to the U.N. Security Council by independent experts monitoring the implementation of U.N. sanctions on North Korea shows a North Korean-flagged vessel conducting a ship-to-ship transfer of coal near the Chinese port of Lianyungang with help from a floating crane in this August 20, 2019 photo provided to the U.N. experts by an unidentified U.N. member state. United Nations/Handout via REUTERS
That appears to be a lax enforcement by China of U.N. sanctions aimed at curbing Pyongyang’s nuclear program under which countries are required to inspect cargo destined to or coming from North Korea that is within their territory or being transported on North Korean-flagged vessels.
The annual report to the U.N. Security Council by independent sanctions monitors said North Korea continued to flout council resolutions “through illicit maritime exports of commodities, notably coal and sand” in 2019, earning Pyongyang hundreds of millions of dollars.
A U.N. Security Council diplomat, speaking to Reuters on condition of anonymity, said China has the capacity to stop sanctions busting by its ally North Korea but “is simply choosing not to implement the Security Council resolutions.”
“According to a Security Council member, Chinese warships have received notification in real time on multiple occasions of vessels entering its territorial waters who have been observed to be in violation of the U.N. Security Council resolutions,” the diplomat said.
China’s policing of neighbor North Korea’s adherence to U.N. sanctions, first put in place in 2006, is considered crucial to the effectiveness of the measures, spearheaded by the United States and unanimously adopted by the Security Council.
Reuters first reported in February that the sanctions monitors report found North Korea continued to enhance its nuclear and ballistic missile programs last year.
CHINA SAYS IMPLEMENTS SANCTIONS
When asked if China had received the notifications and acted on them, the office of the spokesperson at the Chinese foreign ministry said in a statement on Thursday that China implemented U.N. sanctions.
Slideshow ( 4 images )
“China has always earnestly fulfilled its international obligations and dealt with relevant issues in accordance with the resolutions,” it said.
The office said: “The report clearly states that this is information provided by ‘a member state’ and not the opinion of the expert group.”
North Korea’s mission to the United Nations in New York did not immediately respond to a request for comment.
A second photo from the U.N. report shows ten North Korean vessels anchored in Chinese waters on Sept. 15 last year near the port of Lianyungang. The photos were provided to the U.N. sanctions monitors by an unidentified member state.
The monitors reported that North Korea conducts ship-to-ship transfers of illicit cargo such as coal, which has been banned since 2017 in a bid to cut off funding to Pyongyang.
More photos in the report show floating cranes supporting “illicit ship-to-ship coal transfers.”
Under U.N. sanctions imposed in 2017 all countries were also required to repatriate North Koreans working abroad by the end of last year to stop them earning foreign currency for leader Kim Jong Un’s government.
A senior U.S. official in January accused China of failing to send home all North Korean workers.
The United States has said it believed Pyongyang was earning some $500 million a year from nearly 100,000 workers abroad, 50,000 of whom were in China.
In a March 20 letter to the council’s North Korea sanctions committee, China - which requested that its note remain private - said it had completed the repatriation by the Dec. 22 deadline.
“China will continue to implement Security Council resolutions and fulfill its international obligations,” China’s U.N. Ambassador Zhang Jun wrote in the note, seen by Reuters. |
According to conventional practice, joints in lead anodes used in the mining industry are formed using traditional lead burning procedures (e.g., torch welding) in which lead is melted by hand to form the welds or joints. There are, however, a number of disadvantages associated with conventional lead burning procedures as will be discussed below.
Conventional lead burning procedures involve a high degree of risk to the worker. First, the heat generated during creation of the weld can result in worker injury. Second, during melting of the lead, the ambient levels of lead can rise to toxic levels. If inhaled, it could lead to lead poisoning, one of the oldest forms of occupational hazards. As a result, a variety of bodily processes are affected, as well as the deterioration of numerous organs such as the heart, bones, intestines, kidneys, and reproductive and nervous systems.
Conventional lead burning procedures often produce anodes having defective welds which have repeatedly lead to product failure. By training and using a specially skilled work force, the potential for defective welds can be reduced, but is not eliminated, as incomplete welds can occur despite use of skilled workers. In these incomplete welds, the joint visually appears sound. However, the two melt pools created to form the weld have never merged, thereby greatly reducing the material available in the anode joint for current transport and creep (i.e., deformation) resistance.
Even when an anode having a defect-free joint is created, conventional lead burning procedures create an undesirable heat affected zone in the base metal structure around the region of the anode joint. In this heat affected region, the grain structure of the materials in the anode is altered by the heat used to create the joint. The altered grain structure can be a further source of reduced corrosion resistance and decreased creep resistance.
Moreover, lead and copper (used to create anodes) do not naturally weld together. As such, various techniques, such as soldering, are utilized in conventional lead burning procedures to join the lead and copper, thereby constructing the anode. Therefore, in addition to the foregoing disadvantages, conventional anode manufacturing processes are more costly due to the increased manufacturing time and materials involved in joining the lead and copper.
Accordingly, since conventional lead burning techniques used to produce anodes are rife with problems, companies must devote costly resources to safety, worker training, quality control testing, and manufacturing. To date, conventional lead burning techniques have failed to adequately address these issues in the anode industry.
In view of the foregoing, there is a need in the art for an anode manufacturing technique which greatly reduces or eliminates the disadvantages of the prior art. |
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<DebugType>full</DebugType>
<Optimize>false</Optimize>
<OutputPath>bin\Debug\</OutputPath>
<DefineConstants>DEBUG;TRACE</DefineConstants>
<ErrorReport>prompt</ErrorReport>
<WarningLevel>4</WarningLevel>
<StartAction>Program</StartAction>
<StartProgram>C:\Program Files %28x86%29\Microsoft Visual Studio\2017\Enterprise\Common7\IDE\devenv.exe</StartProgram>
<StartArguments>/rootsuffix Exp</StartArguments>
<Prefer32Bit>false</Prefer32Bit>
</PropertyGroup>
<PropertyGroup Condition=" '$(Configuration)|$(Platform)' == 'Release|AnyCPU' ">
<DebugType>pdbonly</DebugType>
<Optimize>true</Optimize>
<OutputPath>bin\Release\</OutputPath>
<DefineConstants>TRACE</DefineConstants>
<ErrorReport>prompt</ErrorReport>
<WarningLevel>4</WarningLevel>
<StartAction>Program</StartAction>
<StartProgram>C:\Program Files %28x86%29\Microsoft Visual Studio\2017\Enterprise\Common7\IDE\devenv.exe</StartProgram>
<StartArguments>/rootsuffix Exp</StartArguments>
<Prefer32Bit>false</Prefer32Bit>
</PropertyGroup>
<ItemGroup>
<Compile Include="ImageVisualizerPackage.cs" />
<Compile Include="Properties\AssemblyInfo.cs" />
</ItemGroup>
<ItemGroup>
<None Include="Key.snk" />
<None Include="source.extension.vsixmanifest">
<SubType>Designer</SubType>
</None>
</ItemGroup>
<ItemGroup>
<Reference Include="Microsoft.CSharp" />
<Reference Include="PresentationCore" />
<Reference Include="PresentationFramework" />
<Reference Include="System" />
<Reference Include="System.Data" />
<Reference Include="System.Design" />
<Reference Include="System.Drawing" />
<Reference Include="System.Net.Http" />
<Reference Include="System.Windows.Forms" />
<Reference Include="System.Xml" />
<Reference Include="WindowsBase" />
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<ItemGroup>
<Content Include="Logo_128x.png">
<CopyToOutputDirectory>Always</CopyToOutputDirectory>
<IncludeInVSIX>true</IncludeInVSIX>
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<CopyToOutputDirectory>Always</CopyToOutputDirectory>
<IncludeInVSIX>true</IncludeInVSIX>
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<EmbeddedResource Include="VSPackage.resx">
<MergeWithCTO>true</MergeWithCTO>
<ManifestResourceName>VSPackage</ManifestResourceName>
</EmbeddedResource>
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<ItemGroup>
<PackageReference Include="Microsoft.VisualStudio.Shell.14.0">
<Version>14.3.25407</Version>
</PackageReference>
<PackageReference Include="Microsoft.VSSDK.BuildTools">
<Version>15.9.3039</Version>
<IncludeAssets>runtime; build; native; contentfiles; analyzers</IncludeAssets>
<PrivateAssets>all</PrivateAssets>
</PackageReference>
</ItemGroup>
<Import Project="$(MSBuildToolsPath)\Microsoft.CSharp.targets" />
<Import Project="$(VSToolsPath)\VSSDK\Microsoft.VsSDK.targets" Condition="'$(VSToolsPath)' != ''" />
<!--<PropertyGroup>
<PostBuildEvent>xcopy /y $(TargetPath) $(SolutionDir)aeiscontroller\bin\Release</PostBuildEvent>
</PropertyGroup>-->
<!-- To modify your build process, add your task inside one of the targets below and uncomment it.
Other similar extension points exist, see Microsoft.Common.targets.
<Target Name="BeforeBuild">
</Target>
<Target Name="AfterBuild">
</Target>
-->
</Project> |
/**
* ## Project: infra-security-groups
*
* Manage the security groups for the entire infrastructure
*/
variable "aws_environment" {
type = "string"
description = "AWS Environment"
}
# Resources
# --------------------------------------------------------------
terraform {
backend "s3" {}
required_version = "= 0.11.14"
}
provider "aws" {
region = "${var.aws_region}"
version = "2.46.0"
}
# used by the fastly ip ranges provider.
# an API key is needed but 'fake' seems to work.
provider "fastly" {
api_key = "fake"
version = "0.1.2"
}
provider "github" {}
data "fastly_ip_ranges" "fastly" {}
data "github_ip_ranges" "github" {}
data "terraform_remote_state" "infra_vpc" {
backend = "s3"
config {
bucket = "${var.remote_state_bucket}"
key = "${coalesce(var.remote_state_infra_vpc_key_stack, var.stackname)}/infra-vpc.tfstate"
region = "${var.aws_region}"
}
}
|
Systems are needed for the removal of unwanted material from wastewater and other fluids. For example, bar screens are often used to protect a wastewater plant or pumping station against the entry of large objects that are likely to cause blockage in different parts of the installation, and to separate and extract bulky matter carried in the raw influent that is likely to interfere with subsequent operation or to create complications in the treatment process.
Screening is typically performed either with manually-cleaned bar screens or (when the plant is sufficiently large) with an automatically-cleaned bar screen system called a mechanical bar screen. These bar screen systems are typically installed in a fluid channel prior to entry of the influent to the treatment system to physically remove debris from the fluid as it travels along the channel.
These systems may utilize a hoist rope, pin rack or other such system to which a rake is attached. The bar screen extends down from the rack into the fluid channel to collect the debris. An electric motor, and possibly a hydraulic fluid pump, is typically used to drive the rake, forcing it down into the fluid, where it scoops up debris, dragging it up along the bar screen and up to a discharge apron, where the debris falls into a disposal unit, such as a cart or other conveyor.
During this process, the flow of the fluid through the channel does not need to be interrupted, and continues to flow during the cleaning process. While the mechanical bar screen normally operates at predetermined speed based upon the flow rate of the influent, systems are also known that may operate at two predetermined speeds; one for the off-peak flow rate and one for the peak flow rate. The system may be switched between these two speeds by the operator.
However, such systems cannot be adaptively controlled to account for ongoing variations in the flow rate of the fluid through the channel, and therefore cannot operate at maximum efficiency when fluctuations in flow rate occur. A system is therefore needed that improves upon these other designs. |
Architecture
Anyone can buy a used shipping container for pretty cheap, but it takes a certain amount of creative vision and skill to make it into a place to call home. And yet, this 355-square-foot trio of containers built by Joseph Dupuis proves that not only can you use them to build a home, but a beautiful one at that. Joseph works at Algonquin College in Otttawa as a researcher on renewable energy and built this masterpiece as a sort of experiment. While he currently lives here, he has the home listed for sale at a remarkably low price of $58k, not including shipping.
1 of 10
The cabin is fully insulated, heated by an in-floor radiant heating system (propane fired hotwater tank for heating purposes and domestic water), and a wood stove. The cabin is designed to be dismantled, moved and erected in a new location with limited resources and time.”
photos courtesy S7vn Photography
Demolition of an old house made room for this elegant new home/studio complex in the Barton Heights neighborhood of Austin. Not a lot of room, because there was already a swimming pool on the property, as well as oak and cypress trees that owner Laurie Frick didn’t want to knock down. But designer KRBD still found a way to give Laurie 1,600 square feet of residential space in addition to the 700-square-foot art studio she asked for by building around a gallery of steel bays.
Around the home, that steel is covered with tigerwood; the studio has a stucco exterior.
Once you get inside you’re met with a long hallway displaying rugs, books, and some of Laurie’s own art pieces. Such an outstanding collection really cries out for a place to show it off, making the hallway a very nice touch for this particular owner.
After you pass through, you’re in a large, open living room/dining room/kitchen area which gives way to the pool in the back. An enclosed glass walkway connects the house to the studio, which is naturally lit by a skylight and clerestory windows (Laurie needed the wall space for her paintings, so ground-level windows were out of the question).
A clerestory window also features in the master bedroom, which is located at the other end of the entry hall. The ensuite bathroom has one too, and there’s a skylight in the hallway guest bathroom. The home office to the front of the house has a more conventional picture window which looks out on a very pleasant street view.
Accessory Dwelling Units (ADUs) are becoming more and more popular in cities that are trying to increase density in single family zones, without incentivising the demolition of historic architecture. As most of these strucutres are being build in backyards and limited to modest footprints, designers must get creative in terms of getting the most out of their small stature. This bright and spacious ADU shows just how much functionality one can get out of a small footprint, which also happened to be a conversion of an existing detached garage.
Susan Moray had large aspirations when she began her journey to create new space on her small lot in Portland, Oregon. She had 550 square feet to work with and was looking to build-out the garage into a guest house, even with potential to rent out to long-term or short term tenants.
Historic neighborhood standards kept Susan from going design-crazy on the exterior. The form of the existing garage provided a solid framework to build on, adding contemporary touches like fully glazed sliding doors, a patio, and garden features.
The kitchen occupies a nicely lit corner in the ADU, with functional open shelving and a funky orange refrigerator.
2022 24th Avenue E of Montlake in Seattle, Washington seems like any other tiny home recently inspired by the Tiny House Movement. The home is 850 square feet, barely five feet wide, is currently on the market for $519,000 – and is in the heart of local legend.
In 1925, a judge presiding over a vicious and bitter divorce ruled that the husband would keep the marital home, and his wife was awarded a sliver of land just in front of the home. To add insult to injury, the husband offered what was to her an unacceptably low offer to purchase the land. As a result, she began construction of what has now come to be known as “The Montlake Spite House” by the community. She even went as far as to paint the wall facing their formal marital home black as to further obstruct his view!
In this photo, you can see just how narrow this house really is!
A view of the living room. While it may be too small to hold a television set, it has plenty of space to seat guests comfortably!
The master bedroom. Believe it or not, this home has two bedrooms, and two full baths!
The place where you really begin to feel cramped may be the kitchen – but it’s worth it just to be able to look at your ex-now-neighbor through the kitchen window with a look of triumph!
There’s even a two-car garage!
She proved her point to her ex-husband, and since then the house has only ever steadily increased in worth: The Montlake Spite House sold in 1996 for $140,550; $235,500 in 2000; and $375,000 in 2014. How much of a factor the notoriety and legends surrounding the of home plays into that increase is up for speculation.
Welcome to the Acacia, the fifth home built by French/Canadian company Minimaliste. This home features some seriously eye-catching design elements, and borrows a lot of “big home” feel for such a small space.
There’s a 4-burner stove, a couch that converts to a bed, fold down dining/work table, and much more!
That thick green film that collects on rocks at the floor of a body of water may just be the solution to the excess CO2 in our atmosphere. In the past two decades researchers have been searching for ways to apply the positive atmospheric attributes of algae to design technology. EcoLogic Studio has done just that with the Urban Algae Canopy.
The structure is currently in the prototype stage of development and will be the first of its kind on display at the Expo Milano 2015. The canopies are completely reactive, and can produce and move energy and oxygen based on a number of inputs, including weather patterns and user movement. EcoLogic Studio claims that the canopies have the capability of producing the oxygen equivalent of four hectares of woodland area.
The ambitious project presents an innovative intersection between technology and biology, and fits in with the ever-growing movement of integrating natural processes into man-made structures.
Before Cathy Calhoun eventually became a jewelry store owner, she worked as a bank teller at The National Bank of Spring City in Spring City, PA. In 1989, the old bank went up for sale and Cathy jumped at the opportunity to own it, paying a paltry $52,000 for the vacant building. She had a vision. A vision that would take 24 years of tireless renovation to become what it is today: a Pennsylvania girl’s dream home.
Scroll down to watch a video tour of the up-cycled old building, which includes a trip through the 2-story boardroom-turned-master suite complete with seating, a fireplace, jacuzzi and a big-screen TV. What she did with both the original vault and the newer one is truly unique, but we’ll let the video do the talking for that one.
A spiral staircase adds a dramatic curve to the otherwise boxed layout of the bank.
Photo Credits: YouTube
Cathy’s modern updates are extensive and eclectic, but have managed to preserve, even celebrate the bank’s historic aesthetic. All said, it’s clear that the money she put into this bank was a very wise investment, indeed.
The American Institute of Architects has announced its 10 Best Green Buildings of 2015. The following structures make a point to champion sustainability and energy conservation without sacrificing cutting-edge design or functionality.
1. The Bullitt Center. Seattle, Washington
This office building, opened on earth day in 2013, is the largest certified Living Building in the United States. Designed by The Miller Hull Partnership, the Bullitt Center features 100% renewable energy, water, and waste-management. The entire shed roof canopy is composed of one large solar panel array.
2. The CANMET Materials Technology Laboratory. Hamilton, Ontario
This LEED Platinum certified laboratory contains 174,300 square feet of research, office and lab space focusing on innovations in material technology. Green features include sun shading on the south facade, green roofs, and renewable energy systems integrated into the building’s exterior shell.
3. Collaborative Life Sciences Building. Portland, Oregon.
ERA Architects and CO Architects collaborated on this LEED Platinum office and research building. The stark grey exterior gives the building a feeling of cleanliness and sterility, something that the integrated building systems exhibit themselves. Stormwater management, green roofs, atrium heat recovery, and low ventilation fume hoods are features that scream sustainability.
photo courtesy Jeremy Bitterman
4. E+ Townhouses. Boston, Massachusetts
The E+ Townhouses were built as prototypes for energy efficient living in affordable housing. The replicable model homes were the brain child of a collaboration between Interface Studio Architects (ISA) and Urbanica Design, and were built under Boston’s Energy Plus (E+) Green Building Program. In the prototype, versatility is shown in how the structures march down the slope of the natural terrain.
photo courtesy Sam Oberter
5. Hughes Warehouse Adaptive Reuse. San Antonio, Texas
Adaptive reuse projects have a prominent role in transitioning into the green era. Overland Partners face-lifted this early 1900’s warehouse was into contemporary studios with state of the art sustainability features. The project features flexible interior spaces and a public courtyard to promote user health and public engagement.
Photo courtesy Dror Baldinger
6. San Antonio Military Medical Center. San Antonio, Texas
“Doing it Bigger” in Texas appears to apply to sustainability as well! RTKL designed this massive complex that focuses on medical research and care for our nation’s military. A enveloping screen traces around the southern facade, protecting the interiors from heat gain while providing ample natural light.
photo Charles Davis Smith
7.New Orleans BioInnovation Center. New Orleans, Louisiana
This 65,000 square foot biotech lab achieved a LEED Gold certification, making it the first of it’s kind in New Orleans. The building features a 3,000 square foot central courtyard is prominently visible from famed Canal Street through the building’s transparent, yet UV protected facade. Designed by Eskew+Dumez+Ripple.
photo courtesy Will Crocker
8. Homes For Adults With Autism. Sonoma, California
LEDDY MAYTUM STACY Architects built four homes in a complex that champions sustainability and energy conservation. Each of the homes is fit with a powerful rooftop solar array, making good use of the persistent California sun. In addition to the homes, the complex boasts a community center, therapy pools, and even an urban garden.
photo courtesy Tim Griffith
9. Sustainable Housing Development. Oakland, California
Why build one sustainable building when you can construct an entire neighborhood? That’s exactly what David Baker Architects had in mind when they designed this complex that contains 60 affordable apartments, 77 attached townhouses and 20 additional apartments. The homes are well insulated and promote passive cooling techniques such as natural ventilation.
photo courtesy Brian Rose
10. University Center – The New School. New York, New York
Famous architecture firm Skidmore, Owings and Merrill (SOM) designed this university that was conceptualized on the platform of combined heat and power systems that were specifically designed for water management. It was enough to afford the building a LEED Gold certification. The facade seen here seamlessly shades glazed openings, providing UV protection and reducing energy consumption.
You’ve heard of modern-rustic, and now you’ll see what happens when sheer decadence collides with a rustic design. This spectacular modern home was designed by Swaback Partners and is situated on a large lot in the private community of Camp Martis, located on the shores of Lake Tahoe.
“A home for the high Sierra’s that does not fall in line with the traditional regional architecture that mostly is a dark and heavy composition. Instead, the concept was to celebrate the light and airy feeling of snow and the effects that it can bring to the interiors.”
For years we’ve heard about “man caves” and seen them in all their glory, the wooden walls adorned with pennants from their favorite sports teams and poster of pin-up girls, big screen television in one corner and a fridge stocked with Coors Light next to the leather sofa. But what about the women in their life? Surely they must have an equivalent type of space to find solace right?
They do, and we call them “she sheds”, a phenomenon which has undoubtedly existed in one form or another for centuries. Unlike their male counterparts, women tend to have a bit more decorating sense, and infuse their personal space with a unique style that’s purposeful. Today we share some of our favorite she-sheds with you, and perhaps you’ll draw inspiration from these to create your own backyard haven. |
Effect of intergranular versus intragranular cornstarch on tablet friability and in vitro dissolution.
The effect of blending dry cornstarch versus wet granulation with the drug and other excipients on friability and in vitro dissolution of a ticlopidine hydrochloride tablet formulation was studied. The friability of the tablets was reduced by wet granulating cornstarch with the drug and other excipients compared with the dry blending. The dissolution rate and the tablet-to-tablet variability was improved by incorporating cornstarch in the wet-granulation stage. The lactose placebo tablets, which were wet granulated with either a binder solution or without a binder, also showed reduced tablet friability due to the incorporation of cornstarch in the wet-granulation step. Examination of the tablet cross sections under the scanning electron microscope indicated clumping of starch grains when starch was blended in the dry form. Starch grains were well embedded in the other materials of the tablet and not readily visible when starch was wet granulated with the other excipients. This results in better bonding, fewer weak points, and better homogeneity of the starch disintegrator within the tablet, which accounts for better friability and improved dissolution. |
American democracy is under assault.
In one super-PAC alone, Karl Rove and the Enron grifter Ed Gillespie, have assembled $200 million from big polluters and Wall Street moguls to buy the 2012 election.
Two of the Koch Brothers, Charles and David, pledged $130 million to elect candidates who favor unrestrained corporate profiteering.
The senators and congressmen they fund and elect are not representing the United States -- they are representing Koch and its oil industry cronies, Big Pharma, and the Wall Street banksters currently mounting a hostile takeover of our government.
I have no problem characterizing these corporate-centric super-PACs as treasonous. We are now in a free fall toward old-fashioned oligarchy; noxious, thieving and tyrannical.
The most corporate-friendly Supreme Court since the Gilded Age had declared in its notorious Citizens United decision that corporations are people and that money is speech. Those who have the most money now have the loudest voices in our democracy while poor Americans are mute.
And the money is talking; in 97 percent of federal elections over the past two decades, the best-funded candidates were victorious.
America, the world's proud template for democracy and a robust middle class, is now listing toward oligarchy and corporate kleptocracy.
America today is looking more and more like a colonial economy, with a system increasingly tilted toward enriching the wealthy 1 percent and serving the mercantile needs of multinational corporations with little allegiance to our country.
These radical forces already dominate the national press, with Fox News and talk radio snugly in the pocket of the corporate Right.
This is the first time in American history that corporate and media interests have been so clearly and so perilously aligned.
With the media in their hands, and unlimited money, the final strategy of Rove, Koch, the Chamber of Commerce, and others of that ilk is to permanently cripple representative democracy by stopping Americans from voting.
A boatload of new Jim Crow laws target Democrats by erecting impediments that deter poor and minority communities, senior citizens, and students from exercising their franchise.
Voter suppression is a crime.
In Billionaires & Ballot Bandits, Greg Palast details each of these devious scams for disenfranchising vulnerable voters ... but also, crucially, Palast here follows the money that powers the machinery of democracy's destruction.
This is not a partisan issue. Clearly the GOP agenda is to suppress votes, as Karl Rove has repeatedly and unashamedly signaled. But Billionaires & Ballot Bandits exposes the vote-count blindness, biases, venality, and ballot gaming by Democrats as well. I don't believe there are Republican children or Democratic children.
Every American citizen ought to have the right to vote and everybody ought to have the right to clean air and clean water, to integrity and transparency in the marketplace, and to a functioning democracy. |
Learn About This Location
Founded in 1792, Raleigh is still a staid government town. The charming downtown has some neat museums and galleries, and the foodie and music scene is on the come-up. Raleigh is home to North Carolina State University and constitutes part of the Research Triangle, along with Durham and Chapel Hill. The "Triangle" originated after the creation of the Research Triangle Park in 1959, located between the three cities and their respective universities.
Raleigh is an example of an early American planned city, chosen to be the location of the state capital in 1788. Originally laid out in a grid with the State Capitol in the middle, the city was spared from any major battle in the Civil War, though it did have to endure economic hardships during Reconstruction. The twentieth century saw a large influx of people due to jobs created from the Research Triangle area, and it was among the fastest growing communities in the US by the early 2000s. |
Q:
Android Bluetooth Where can I get UUID?
I want to connect 3 devices via Bluetooth.
As for example I use BluetoothChat. So How I understand I should use different UUID for this devices. I have been trying to connect via such UUID=766c82f0-e1b4-11df-85ca-0800200c9a66, which I 've get it from Web UUID generator. But it doesn't work at all.
I have succesfully connected (to 1 device) if I used UUID=00001101-0000-1000-8000-00805F9B34FB
Where can I get UUID?
A:
if you are using linux or mac, enter "uuidgen" this command without quotes in terminal, you will get an unique UUID, use that in your android project.
|
We learned about place value. We made "No Davids" after reading the book to learn about school rules. We talked about Character Traits. We played Homeworkopoly for turning in our homework for the week. We played the place value game after learning about place value, the value of a digit, expanded notation and the word form. We are now learning how to rename a number. The first week we learned about story elements (characters, setting and plot) and the beginning, middle and end. By Ellie and Aryanna |
Learn more about how we calculate nutritional information, and always consult a registered dietician or your physician before embarking on any diet plan which relies on these numbers, and for any other questions.
8 ounces (229g)canned diced green chiles
1 lb (456g)monterey jack cheese
1 lb (454g)cheddar cheese
3/4 cup (189g)evaporated milk
4 (200g)eggs
1 tablespoon (8g)flour
1/2 teaspoon (3g)salt
1/8 teaspoon (0g)pepper
2 (246g)tomatoes
Total
1 serving
Alanine (g)
0.1
3.1
3.2
0.4
1.5
0.0
0.0
0.0
0.1
8
1
Alcohol (g)
0.0
0.0
0.0
n/a
0.0
0.0
0.0
0.0
0.0
0
0
Arginine (g)
0.1
4.2
4.3
0.5
1.6
0.0
0.0
0.0
0.1
11
1
Ash (g)
3.0
16.2
17.9
2.9
2.1
0.0
3.0
0.0
1.2
46
6
Aspartic acid (g)
0.2
7.2
7.3
1.0
2.7
0.0
0.0
0.0
0.3
19
2
Caffeine
n/a
0.0
0.0
n/a
0.0
0.0
0.0
0.0
0.0
0
0
Calcium (mg)
82.3
3,400.4
3,276.2
493.3
112.0
1.2
0.7
1.3
24.6
7,392
924
Calories
48.0
1,700.2
1,831.2
253.3
286.0
28.4
0.0
0.7
44.3
4,192
524
Calories (kj)
201.1
7,119.9
7,652.1
1,062.2
1,198.0
119.0
0.0
3.0
182.0
17,537
2,192
Carbohydrates (g)
10.5
3.1
5.8
19.0
1.4
6.0
0.0
0.2
9.6
56
7
Cholesterol (mg)
0.0
405.7
477.1
54.8
744.0
0.0
0.0
0.0
0.0
1,682
210
Copper
n/a
0.1
0.1
0.0
0.1
0.0
0.0
0.0
0.1
1
0
Cystine (g)
0.0
0.6
0.6
0.1
0.5
0.0
0.0
0.0
0.0
2
0
Dietary Fiber (g)
3.9
0.0
0.0
0.0
0.0
0.2
0.0
0.1
3.0
7
1
Folate (mcg)
123.4
82.0
81.8
15.1
94.0
14.3
0.0
0.0
36.9
448
56
Folate, DFE (mcg)
123.4
82.0
81.8
15.1
94.0
22.7
0.0
0.0
36.9
456
57
Folate, food (mcg)
123.4
82.0
81.8
15.1
94.0
2.3
0.0
0.0
36.9
436
54
Folic acid (mcg)
0.0
0.0
0.0
0.0
0.0
12.0
0.0
0.0
0.0
12
2
Fructose
n/a
n/a
n/a
n/a
0.0
n/a
n/a
0.0
3.4
3
0
Galactose
n/a
n/a
n/a
n/a
0.0
n/a
n/a
0.0
0.0
0
0
Glucose (dextrose)
n/a
n/a
n/a
n/a
0.7
n/a
n/a
0.0
3.1
4
0
Glutamic acid (g)
0.2
27.3
27.7
2.7
3.3
0.3
0.0
0.0
1.1
63
8
Glycine (g)
0.1
1.9
1.9
0.3
0.9
0.0
0.0
0.0
0.0
5
1
Histidine (g)
0.0
3.9
4.0
0.3
0.6
0.0
0.0
0.0
0.0
9
1
Hydroxyproline
n/a
n/a
n/a
n/a
n/a
n/a
n/a
0.0
n/a
0
0
Iron (mg)
3.0
3.3
3.1
0.4
3.5
0.4
0.0
0.0
0.7
14
2
Isoleucine (g)
0.1
6.9
7.0
0.8
1.3
0.0
0.0
0.0
0.0
16
2
Lactose
n/a
n/a
1.0
n/a
0.0
n/a
n/a
0.0
0.0
1
0
Leucine (g)
0.1
10.7
10.8
1.3
2.2
0.1
0.0
0.0
0.1
25
3
Lysine (g)
0.1
9.3
9.4
1.0
1.8
0.0
0.0
0.0
0.1
22
3
Magnesium (mg)
9.1
123.1
127.2
45.4
24.0
1.7
0.0
0.5
27.1
358
45
Maltose
n/a
n/a
0.7
n/a
0.0
n/a
n/a
0.0
0.0
1
0
Manganese
n/a
0.1
0.0
0.0
0.1
0.1
0.0
0.0
0.3
1
0
Methionine (g)
0.0
2.9
3.0
0.3
0.8
0.0
0.0
0.0
0.0
7
1
Monounsaturated Fat (g)
0.0
39.9
42.7
4.4
7.3
0.0
0.0
0.0
0.1
94
12
Niacin (mg)
1.4
0.4
0.4
0.4
0.2
0.5
0.0
0.0
1.5
5
1
Pantothenic acid (mg)
0.2
1.0
1.9
1.2
3.1
0.0
0.0
0.0
0.2
8
1
Phenylalanine (g)
0.1
5.9
6.0
0.6
1.4
0.0
0.0
0.0
0.1
14
2
Phosphorus (mg)
25.1
2,023.8
2,326.5
383.7
396.0
8.4
0.0
0.5
59.0
5,223
653
Phytosterols
n/a
n/a
n/a
n/a
n/a
n/a
0.0
0.3
17.2
17
2
Polyunsaturated Fat (g)
0.3
4.1
4.3
0.5
3.8
0.0
0.0
0.0
0.2
13
2
Potassium (mg)
258.3
369.2
445.3
572.7
276.0
8.4
0.2
3.8
583.0
2,517
315
Proline (g)
0.1
12.6
12.8
1.2
1.0
0.1
0.0
0.0
0.0
28
3
Protein (g)
1.6
111.6
113.1
12.9
25.1
0.8
0.0
0.0
2.2
267
33
Retinol (mcg)
0.0
875.2
1,172.4
n/a
320.0
0.0
0.0
0.0
0.0
2,368
296
Riboflavin (mg)
0.1
1.8
1.7
0.6
0.9
0.0
0.0
0.0
0.0
5
1
Saturated Fat (g)
0.1
86.9
95.8
8.7
6.3
0.0
0.0
0.0
0.1
198
25
Selenium (mcg)
0.7
66.1
63.2
4.3
61.4
2.6
0.0
0.0
0.0
198
25
Serine (g)
0.1
6.5
6.6
0.7
1.9
0.0
0.0
0.0
0.1
16
2
Sodium (mg)
907.4
2,443.2
2,821.8
200.3
284.0
0.2
1,162.7
0.1
12.3
7,832
979
Starch
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
0.0
0
0
Sucrose
n/a
n/a
1.1
n/a
0.0
n/a
n/a
0.0
0.0
1
0
Sugar
n/a
2.3
2.4
n/a
0.7
0.0
0.0
0.0
6.5
12
1
Theobromine
n/a
0.0
0.0
n/a
0.0
0.0
0.0
0.0
0.0
0
0
Thiamin (mg)
0.0
0.1
0.1
0.1
0.1
0.1
0.0
0.0
0.1
1
0
Threonine (g)
0.1
4.0
4.0
0.6
1.1
0.0
0.0
0.0
0.1
10
1
Tocopherol, alpha
n/a
1.2
1.3
n/a
2.1
0.0
0.0
0.0
1.3
6
1
Tocopherol, beta
n/a
n/a
n/a
n/a
0.0
0.0
n/a
0.0
0.0
0
0
Tocopherol, delta
n/a
n/a
0.0
n/a
0.1
0.0
n/a
0.0
0.0
0
0
Tocopherol, gamma
n/a
n/a
0.0
n/a
1.0
0.0
n/a
0.0
0.3
1
0
Total Fat (g)
0.6
138.0
150.6
14.3
19.0
0.1
0.0
0.0
0.5
323
40
Trans-monoenoic Fat
n/a
n/a
n/a
n/a
0.1
n/a
n/a
0.0
n/a
0
0
TransFatty acids
n/a
n/a
n/a
n/a
0.1
n/a
n/a
0.0
n/a
0
0
Tryptophan (g)
0.0
1.4
1.5
0.2
0.3
0.0
0.0
0.0
0.0
3
0
Tyrosine (g)
0.0
5.4
5.5
0.6
1.0
0.0
0.0
0.0
0.0
13
2
Valine (g)
0.1
7.5
7.6
0.9
1.7
0.0
0.0
0.0
0.0
18
2
Vitamin A, IU (IU)
288.0
3,505.3
4,553.1
750.3
1,080.0
0.0
0.0
1.6
2,049.2
12,227
1,528
Vitamin A, RAE (mcg)
13.7
902.5
1,204.2
n/a
320.0
0.0
0.0
0.1
103.3
2,544
318
Vitamin B-12 (mcg)
0.0
3.8
3.8
0.3
1.8
0.0
0.0
0.0
0.0
10
1
Vitamin B-6 (mg)
0.3
0.4
0.3
0.1
0.3
0.0
0.0
0.0
0.2
2
0
Vitamin C (mg)
78.2
0.0
0.0
3.6
0.0
0.0
0.0
0.0
33.7
115
14
Vitamin D (IU)
0.0
100.3
109.1
n/a
164.0
0.0
0.0
0.0
0.0
373
47
Water (g)
213.1
186.9
167.0
139.9
152.3
0.9
0.0
0.0
232.5
1,093
137
Zinc (mg)
0.2
13.7
14.1
1.5
2.6
0.1
0.0
0.0
0.4
33
4
Our nutritional database is not complete and serving size information for many recipes is unknown, which leads to incomplete nutritional data. Thus, we encourage any user to submit corrections or add information to any recipe on the site. |
asics sneakers charlottetown 2016
The asics sneakers charlottetown 2016 liberal and concise design can make you look brighter and energetic shop by - American Museum Of Beat Art .They are high quality but a very cheap price! You cannot miss the vital time and shop.
12/10/2016 01:22 am ET
Tim Wimborne / Reuters
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To help you tackle any challenges posed by mother nature we have put together a range of fact sheets that will assist you in the fruit garden with pest and disease management. ??Services & Support Log In My Account Shopping Cart Shipping & Payment Shipping & Returns Privacy Policy Sitemap Why choose us Fast Shipping 100% Guarantee Superior Quality More Information Checkout Advanced Search Nike Free Run 5. I'll be honest though, as a conference organiser it really upsets me that within half an hour of one of our events starting someone will tweet only 30% women here total sausage fest.
Choosing your profile From par to a bespoke moulding Brooks Bros can supply precisely what you want: from stock in some cases or to your precise requirement. It's a close up glimpse at those wonderful Katsuya Kamo's headpieces and then some inner circle workroom shots that shows the team working hard at precise paper cut outs. Your call back is scheduled for First Name Last Name Telephone Number Invalid Number Where can we take you. |
#region License NUnit.Specifications
/* From https://raw.githubusercontent.com/derekgreer/NUnit.Specifications/master/license.txt
Copyright(c) 2015 Derek B.Greer
Permission is hereby granted, free of charge, to any person obtaining a copy
of this software and associated documentation files (the "Software"), to deal
in the Software without restriction, including without limitation the rights
to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
copies of the Software, and to permit persons to whom the Software is
furnished to do so, subject to the following conditions:
The above copyright notice and this permission notice shall be included in
all copies or substantial portions of the Software.
THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT.IN NO EVENT SHALL THE
AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN
THE SOFTWARE.
*/
#endregion
using System;
using System.Diagnostics;
using System.Threading.Tasks;
namespace Paramore.Brighter.Core.Tests
{
[DebuggerStepThrough]
public static class Catch
{
public static Exception Exception(Action action)
{
Exception exception = null;
try
{
action();
}
catch (Exception e)
{
exception = e;
}
return exception;
}
public static async Task<Exception> ExceptionAsync(Func<Task> action)
{
Exception exception = null;
try
{
await action();
}
catch (Exception e)
{
exception = e;
}
return exception;
}
}
}
|
Leaching and efficiency of six organic zinc fertilizers applied to navy bean crop grown in a weakly acidic soil of Spain.
Zinc contamination of groundwater from fertilizers applied to pulse crops is a potential problem, but the use of different types of organic chelates can minimize the contamination potential while still adequately feeding the crops. The objective of this study was to compare the leaching, distribution in fractions and availability, and relative effectiveness of Zn from six organic Zn fertilizers (zinc-ethylenediaminetetraacetate- N-2-hydroxyethylethylenediaminetriacetate (Zn-EDTA-HEDTA), Zn-HEDTA, zinc- S, S'-ethylenediaminedisuccinate (Zn- S, S-EDDS), zinc-polyhydroxyphenylcarboxylate, Zn-EDTA, and zinc-ethylenediaminedi(2-hydroxy-5-sulfophenylacetate) (Zn-EDDHSA)) applied to a navy bean ( Phaseolus vulgaris, L.) crop cultivated by applying different Zn levels, in a weakly acidic soil under greenhouse conditions. Zinc soil behavior was evaluated by diethylenetriaminepentaacetic acid-triethanolamine (DTPA-TEA), DTPA-ammonium bicarbonate (DTPA-AB), Mehlich-3, and BaCl 2 extractions and sequential fractionation. In all the fertilizer treatments, the percentage of labile Zn that remained in the soil was high with respect to the quantity of Zn applied, with values respectively ranging from 42 to 80% for Zn-EDDHSA and Zn-EDTA sources. A positive correlation with a high level of significance existed between the micronutrient concentration in the navy bean crop (total and soluble) and labile Zn fractions, available Zn, and easily leachable Zn ( r ranged from 0.89 to 0.95, P < 0.0001). The relatively high quantity of total Zn leached by applying Zn-EDTA and Zn-S,S-EDDS sources (11.9 and 6.0%, respectively, for the rate 10 mg of Zn kg(-1) of soil) poses a potential pollution risk for neighboring waters. It would seem recommendable to apply Zn-HEDTA or Zn-EDDHSA sources, even applied at the low rate (5 mg of Zn kg(-1) of soil), because they produced available Zn concentrations in the soil that were above the critical concentration and also produced high Zn concentrations in plants (139 and 106 mg of Zn kg(-1) of dry matter, respectively). |
My Nonprofit Reviews
ScottyD1111
Volunteering with TheACY the last three years has been instrumental in helping me heal through any residual weight of guilt and grief related to an accidental death that I was directly involved with three years prior. Their recognition that everyone has a unique healing process was appreciated, and their emphasis on art therapy is effective and empowering. The organization provides valuable tools to the many people working through the death of someone close, both children and adults alike. |
Announcements
2017 SC Hurricane Preparedness Guide
The S.C. Emergency Management Division (SCEMD) has released the official 2017 South Carolina Hurricane Guide. SCEMD and partner agencies have updated the Guide for the 2017 hurricane season. It details useful information on what residents should do before, during and after the landfall of a major hurricane.
Updated sections of the Guide include new storm surge watch and warning information, tips on preparing for an evacuation, ways to stay connected during an emergency and steps to keep in mind when returning home after a major storm. The Guide also features imagery and artwork from last year’s Hurricane Matthew.
The online edition of the Hurricane Guide is available for download HERE.
SCEMD, county emergency managers and the National Weather Service urge citizens to take time now to prepare for major emergency like hurricanes by reviewing their family emergency plans, developing a disaster supplies kit and talking with family members about what could happen during a crisis. |
Compression of visual space in natural scenes and in their photographic counterparts.
Classical theories of space perception posit continuous distortions of subjective space. These stand in contrast to the quantitatively and qualitatively different distortions experienced in space that is represented pictorially. We challenge several aspects of these theories. Comparing real-world objects with depictions of the same objects, we investigated to what extent distortions are introduced by the photographic medium. Corners of irregularly shaped buildings had to be judged in terms of the vertical dihedral angles subtended by two adjacent walls. Across all conditions, a robust effect of viewing distance was found: Building corners appear to flatten out with distance. Moreover, depictions of corners produce remarkably similar results and should not receive a different theoretical treatment than do real-world scenes. The flattening of vertical angles cannot be explained by a linear distortion of the entire visual space. We suggest that, for natural scenes, compression of space is local and dependent on contextual information. |
#ubuntu-papercuts 2013-04-03
<Dedunu> Hi guys good morning
|
Q:
Parsing Multidimensional Array in PHP
Apologies if this is a duplication of another answer, I have spent a considerable amount of time looking through this and other forums without finding an explanation that makes sense to my very small brain.
I have a multi-dimensional array that I need to parse through using PHP:
Array
(
[dataSetOut] => Array
(
[diffgram] => Array
(
[anonymous] => Array
(
[maindb_productionhistory] => Array
(
[0] => Array
(
[z_internal_sequence] => 1
[z_internal_groupid] => 0
[z_internal_colorbg] => 15461355
[z_internal_colorfg] => 0
[maindb_productionhistory_operation] => 0402D04
[maindb_productionhistory_date] => 2014-02-19T00:00:00+00:00
[maindb_productionhistory_shift] => 1
[maindb_productionhistory_transcode] => 33
[maindb_productionhistory_qty] => 153
[!diffgr:id] => maindb_productionhistory1
[!msdata:rowOrder] => 0
)
[1] => Array
(
[z_internal_sequence] => 2
[z_internal_groupid] => 0
[z_internal_colorbg] => 16777215
[z_internal_colorfg] => 0
[maindb_productionhistory_operation] => 0402D04
[maindb_productionhistory_date] => 2014-02-19T00:00:00+00:00
[maindb_productionhistory_shift] => 1
[maindb_productionhistory_transcode] => 34
[maindb_productionhistory_qty] => 6
[!diffgr:id] => maindb_productionhistory2
[!msdata:rowOrder] => 1
)
)
)
)
)
[errorMessage] =>
)
I am looking to output a table like:
Operation | Date | Qty
0402D04 | 2014-02-19 | 153
0402D04 | 2014-02-19 | 6
A:
<table>
<thead>
<tr>
<th>Operation</th>
<th>Date</th>
<th>Qty</th>
</tr>
</thead>
<tbody>
<?php
foreach($array['dataSetOut']['diffgram']['anonymous']['maindb_productionhistory'] as $val){
echo "<tr>";
echo "<td>".$val['maindb_productionhistory_operation']."</td>";
echo "<td>".date('Y-m-d',strtotime($val['maindb_productionhistory_date']))."</td>";
echo "<td>".$val['maindb_productionhistory_qty']."</td>";
echo "</tr>";
}
?>
</tbody>
</table>
|
Just one Republican—Rep. Brian Fitzpatrick of Pennsylvania—joined a united House Democratic caucus on Friday to pass what rights groups hailed as "historic" legislation to restore and expand voter protections that were gutted by the Supreme Court in 2013.
Lisa Gilbert, vice president of legislative affairs for Public Citizen, said passage of the Voting Rights Advancement Act (H.R. 4) is a "critical step" in combating Republican voter suppression efforts that have proliferated in the six years since the Supreme Court's infamous decision in Shelby County v. Holder.
"Numerous state legislatures have undertaken targeted and deliberate steps to limit or impede the right to vote for communities of color, students, the elderly, and people with disabilities," said Gilbert. "Americans who are eligible to vote but are denied that right due to fabricated or illegal barriers are being deprived of the full privilege of our democracy."
BREAKING: The House just passed #HR4, one of the most important bills to emerge during this Congress. As voter suppression continues to spread across the US, this bill would breathe life back into the Voting Rights Act. A win for the people & a win for democracy! #RestoreTheVote pic.twitter.com/TR14b3ltxQ — Kristen Clarke (@KristenClarkeJD) December 6, 2019 BREAKING: @RepBrianFitz is the one and only House Republican that believes in voting rights. https://t.co/37aqHMYIMO — Public Citizen (@Public_Citizen) December 6, 2019
"If we want a true democracy, we must protect the right to vote for all," said Rep. Pramila Jayapal (D-Wash.), co-chair of the Congressional Progressive Caucus. "The Voting Rights Advancement Act is critical to getting there."
The legislation now heads to the Senate, which is controlled by Republicans. Senate Majority Leader Mitch McConnell (R-Ky.) has refused to allow a vote on Democrats' For the People Act (H.R. 1), to which the Voting Rights Advancement Act was previously attached.
"To Majority Leader McConnell, we ask: why are you afraid of all Americans having their full right to vote?" Sylvia Albert, director of voting and elections at Common Cause, said in a statement.
As Ari Berman of Mother Jones reported, H.R. 4—sponsored by Rep. Terri Sewell (D-Ala.)—would "initially cover 11 states: nine in the South, plus California and New York, which have more recently been found to discriminate against Latinos and Asian Americans."
"The bill would also require all states to get federal approval for election changes that are known to disproportionately affect voters of color, such as strict voter ID laws, tighter voter registration requirements, and polling place closures in areas with large numbers of minority voters," Berman noted.
Vanita Gupta, president and CEO of The Leadership Conference on Civil and Human Rights, applauded the House for passing H.R. 4 and demanded that McConnell bring the bill up for a vote in the Senate as soon as possible.
"The passage of H.R. 4 in the House of Representatives today brings us one step closer to restoring the Voting Rights Act and undoing the tremendous damage of Shelby," Gupta said in a statement. "The VRAA is too important—and the right to vote is too fundamental—to end up buried in the McConnell legislative graveyard." |
1998 Internazionali Femminili di Palermo – Doubles
Silvia Farina and Barbara Schett were the defending champions but only Schett competed that year with Patty Schnyder.
Schett and Schnyder lost in the final 6–4, 6–2 against Pavlina Stoyanova and Elena Wagner.
Seeds
Champion seeds are indicated in bold text while text in italics indicates the round in which those seeds were eliminated.
Barbara Schett / Patty Schnyder (Final)
Kristie Boogert / Miriam Oremans (Quarterfinals)
Radka Bobková / Caroline Schneider (First Round)
Pavlina Stoyanova / Elena Wagner (Champions)
Draw
External links
1998 Internazionali Femminili di Palermo Doubles Draw
Category:Internazionali Femminili di Palermo
Category:1998 WTA Tour |
Hundreds protested in Puerto Rico on Thursday in a demonstration reminiscent of those that ousted the island’s former governor last year, as anger grows over emergency aid that until recently sat unused in a warehouse amid ongoing earthquakes.
Demonstrators gathered under the heavy rain at the governor’s mansion as they waved flags, banged on pots, with even a guillotine carried aloft, although it appeared to be purely symbolic.
Um, the people of Puerto Rico are carrying a guillotine to the Governor’s mansion right now. pic.twitter.com/uAeH1WoLcN — Joshua Potash (@JoshuaPotash) January 23, 2020
The newest protest, organized by the Puerto Rican singer René Pérez of Calle 13 fame, has unleashed debate about whether Puerto Ricans should try to oust Governor Wanda Vázquez as the US territory struggles to recover from a recent 6.4 magnitude earthquake that killed one person and damaged hundreds of homes in the island’s southern region. Officials are still awaiting millions of dollars in federal funds for Hurricane Maria, a Category 4 storm that hit in September 2017.
“We’ve had enough,” said 82-year-old Iris Guardiola as she waved a tiny Puerto Rican flag. “The people are tired of the abuse … of the lack of humanity. I am here helping those who cannot be here.”
One former protest leader, Rey Charlie, who jumped to fame after leading hundreds of motorcycle riders in a demonstration last summer, said he will not participate in the most recent one.
“A massive demonstration like the one last year would cause great damage, damage that I think would be irreparable,” he said. “You are holding up the economy, you are holding up aid supplies to victims in the southern region, you are paralyzing the country … You have to think of the consequences.”
Charlie and other critics of the recent protests argue that the most effective strategy is to vote in the upcoming general elections in November, in which Vázquez is seeking a second term as governor. Vázquez has only briefly addressed the recent protests, warning of people who want to talk about “negative issues”.
Protestors carried a guillotine aloft as they marched in Old San Juan on 23 January. Photograph: Jose Jimenez/Getty Images
“We cannot allow groups with other interests to divert our attention,” she told reporters on Wednesday. “We’re in a moment of helping people in the south, not of creating controversy.”
Vázquez came under fire on Saturday after an online blogger posted a live feed of a warehouse in the southern coastal city of Ponce filled with water, cots, baby food and other supplies dating from Hurricane Maria. A group of people broke into the warehouse and began distributing supplies to those affected by the recent quake, which prompted Donald Trump to approve a major disaster declaration for more than a dozen municipalities in the island’s southern region. More than 4,500 people remain in shelters.
The governor fired the director of Puerto Rico’s emergency management agency shortly after the incident, as well as the secretaries of the department of housing and family, as she blamed them in part for not distributing the aid.
René Pérez said people should not wait until the general elections to express their discontent.
“We’re not going to wait until November because the politicians in this country are not going to wait until November to steal. They’re going to steal starting now,” he said.
Concerns over alleged corruption and mismanagement have delayed federal funds to Puerto Rico and led US officials to impose new restrictions as the island struggles to emerge from a 13-year recession.
Carmen Velázquez, a 71-year-old retiree, said she doubts the protests will accomplish what they did last year, “but I can’t remain sitting at home. The lies and the cheating are still going on.” |
Rumors of the death of New Orleans Saints offense are greatly exaggerated.
Just look at this graphic from Fox Sports One’s Colin Cowherd:
Misleading graphic much? To quote our friend, Mark Ingram, “Look at the details!”
For one, look at the text under “Remaining Teams.” It says, “Since Week 15 with starting QB.” Why Week 15? No reason in particular. Why “with starting QB?” Because they’re trying to be fair and exclude the game Drew Brees didn’t play in Week 17. That means for the Saints, you’re looking at a three-game sample size (Week 15, Week 16, and the divisional round). For the other three teams, it’s a four-game sample size, as each starting QB played in Week 17 for those clubs, and each has only had one playoff game thus far.
But let’s take a look at some of those details behind the numbers.
For one, in Week 15, of the four teams listed above, the Saints were the only team to actually go on and win their game. The Chiefs lost - at home - to the Los Angeles Chargers. The Rams lost - at home - to the Philadelphia Eagles. The New Orleans Saints played on the road at Carolina, and beat the Panthers. The Patriots were the only other of the four teams to play on the road, and they lost to the Pittsburgh Steelers.
In Week 16, that same Steelers team that beat the Patriots the week before, took the “L” against the Saints back home in the Dome. The Chiefs lost back-to-back games, this time to the Seattle Seahawks on the road. Sure, the Patriots and Rams were able to win their games, but it was against the mighty juggernaut Arizona Cardinals and Buffalo Bills.
Week 17 is largely irrelevant for the graphic because Drew Brees and the starting Saints offense didn’t play (outside of Michael Thomas), but this graphic includes the games from the other three teams. Again, sure, those teams won, but the Chiefs, Patriots, and Rams faced off against the Raiders, Jets, and 49ers respectively. It’s not like they were putting up big numbers against world-beaters.
Bottom line, here is what you need to know about the Saints offense:
Yes, the New Orleans Saints “only” put up 20 points of offense up against the Philadelphia Eagles in the divisional round. Understand, though, that the Rams offense (an offense the graphic is not trying to dismiss) “only” put up 23 points against that same Eagles teams, at home in Week 15, in a loss.
The Saints offense did enough to win the game last week, and they played exactly the type of game they needed to to win. After the momentum swung back to the Saints midway through the second quarter, the Saints absolutely dominated the ball. The Eagles could do nothing on offense, and the Saints offense was able to wear out the Philadelphia defense. On the back of an 18-play scoring drive to take the lead in the third quarter, the Saints offense held the ball for a whopping 37 minutes and 50 seconds. The Saints didn’t want to, or have to, score quickly in hopes of winning a shootout. They played exactly the game they wanted to play.
In the Rams’ loss to the Eagles in Week 15, the Rams - who scored 23 points - held the ball for only 28:24. That is half of an entire quarter of football less than the Saints in the divisional round. The Rams offense couldn’t put in work against the Eagles defense, couldn’t sustain drives, and couldn’t come out with the win.
I know the law of syllogism (if A > B, and B > C, then A > C) doesn’t apply in the NFL. Just because Team A beat Team B, and Team B beat Team C, doesn’t not guarantee Team A will beat Team C. But consider this:
The vaunted #Bears defense held Nick Foles and the Eagles offense to 16 points at home in the divisional round. The #Saints were even better, holding PHI to only 14 points Sunday. That same Bears D held the Rams offense to only 6 pts just over a month ago. — Christopher Dunnells (@dunnellz) January 16, 2019
The Saints defense was able to do something the Chicago Bears defense, for all their credit, couldn’t do: hold the Eagles offense to under 15 points at home. Granted, weather was a factor, but those same Bears held the Rams offense to only 6 points at home in Week 14 (yeah, Cowherd didn’t include Week 14 in his graphic). If the Saints defense can perform in the playoffs to the same caliber as the Bears, why couldn’t the Saints run a similar game plan against LA as they did against Philadelphia - dominate on defense and control time of possession?
Frankly, I’m not worried at this point about the Chiefs and Patriots. Only one of them will make the Super Bowl, and the Saints have to beat the Rams before that even matters. But as far as the Rams go, Rams fans - not Saints fans - should be the ones worried about the Saints offense. |
So April 22 was Earth Day and after doing nothing to celebrate it I decided to make a new holiday called Anti-People Day. And not because ‘we’re destroying the Earth’. We aren’t. Get over it you stupid...
Hard to believe another week has passed already. OK, so that’s a total lie. I completely expected it. But despite that, we did have a whole week of shit happen that the crack reporting staff here at the Gallery did not... |
1. Field of the Invention
The invention relates to an information carrier and processes for producing an original copy of the carrier, which comprises a recording layer deposited on a carrier material. A relief image containing the information is embossed in the recording layer.
2. Description of the Prior Art
Images with grating-like screening have been produced by the ZOD (zero order diffraction) technique described in the LASER u. Elektro-Optik Journal No. 3/1976, pages 16-17. Three nickel matrices are produced from the relief images which correspond, for example, to three primary-color grating patterns in a photoresist, and colorless thermoplastic films of, for example, polyvinyl chloride are embossed with these matrices. These films are mechanically superimposed, and, on projection with conventional projectors, colored projection images are obtained from the colorless relief images. The grating-shaped screening is effected with relief gratings of rectangular cross-section, the grating periods being approximately 1.5 .mu.m. A separate nickel matrix with different relief depths is made for each color separation (red, yellow and blue), from which separate embossed images are produced. The relief depths differ depending on the color separation. The greatest relief depths are used for the red color separation while the smallest are used for the blue color separation. The color separation images are screened. The embossed images are then superimposed to form a three-layered relief image which can be used to project colored images. The technique described yields very bright color images with high resolution. The relief images can be duplicated relatively cheaply and rapidly by an embossing process.
A disadvantage, which has hindered the introduction of this technique, is the expensive production process, incurred by performing three completely separate operations for producing the individual, embossed relief images, corresponding to the color separations. A further disadvantage ensues from the necessity of aligning the three separate relief images to form the duplicate image required for the colored projection. |
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SKT 2013 Team Jersey on Sale Now ! Text by disciple Front Side Back
Vestments of the Dynasty SK Telecom T1 need little introduction. They are the New York Yankees, the Real Madrid, the Los Angeles Lakers of esports. For over a decade, they have been the golden standard to which all other teams in professional gaming aspire to. They have won dozens of championships, been home to some of the most popular progamers in the world, and played a crucial role in making esports what it is today.
So when SKT send you an email that says 'would you be interested in partnering with us on a project?' you check your Inception token to make sure you are not dreaming. For TeamLiquid, it is a great honor and privilege to offer the T1 team jersey for the first time to fans around the world. For T1 fans like me, this is a moment we've been waiting on for years.
But even to fans you who have no particular allegiance to SK Telecom, this day marks an important milestone for all of us as a community. We're no longer a small group fanatics across the ocean, so isolated that we even called ourselves 'foreign.' By reaching out to the international scene, SK Telecom T1 has shown they recognize the importance of their fans around the world. Korean players, coaches, and industry insiders have all expressed how impressed they were with the fans at international tournaments. Now, they've made an important step toward making us a part of their future.
We only have 750 shirts and we won't be producing any more of this specific model. So the chance for you to own one of these is lower than the chance to own a Ferrari F40. I want to thank SK Telecom T1 for being so cooperative and great to work with. I hope this is the beginning of a beautiful friendship, and that we might work to bring the fans even more great products.
- disciple, TeamLiquid store manager. SK Telecom T1 need little introduction. They are the New York Yankees, the Real Madrid, the Los Angeles Lakers of esports. For over a decade, they have been the golden standard to which all other teams in professional gaming aspire to. They have won dozens of championships, been home to some of the most popular progamers in the world, and played a crucial role in making esports what it is today.So when SKT send you an email that says 'would you be interested in partnering with us on a project?' you check your Inception token to make sure you are not dreaming. For TeamLiquid, it is a great honor and privilege to offer the T1 team jersey for the first time to fans around the world. For T1 fans like me, this is a moment we've been waiting on for years.But even to fans you who have no particular allegiance to SK Telecom, this day marks an important milestone for all of us as a community. We're no longer a small group fanatics across the ocean, so isolated that we even called ourselves 'foreign.' By reaching out to the international scene, SK Telecom T1 has shown they recognize the importance of their fans around the world. Korean players, coaches, and industry insiders have all expressed how impressed they were with the fans at international tournaments. Now, they've made an important step toward making us a part of their future.We only have 750 shirts and we won't be producing any more of this specific model. So the chance for you to own one of these is lower than the chance to own a Ferrari F40. I want to thank SK Telecom T1 for being so cooperative and great to work with. I hope this is the beginning of a beautiful friendship, and that we might work to bring the fans even more great products.
SK Telecom T1 2013 Team Shirt $64.95 USD Sizing Information Buy Now
SK Telecom T1 2013 Team Shirt €59.95 EUR Sizing Information Buy Now
International Shipping has been as always a tricky situation. Because the limited stock and how valuable the shirts are we decided to go for the most reliable shipping option we have on our hands. That's why all international orders from the NA Store will be shipped with Fedex International which is incredibly fast, reliable and includes a detailed tracking. Fedex is not cheap but we absolutely cannot risk using USPS in this case and if we were to do that we were not in a position to guarantee the delivery of your order. We did the same with our hoodies and I believe this is the best way to do this.
Also, because of the limited stock we have to change our story policy for that specific product - we do not offer size exchanges ! Make sure you get the right size because we won't be able to replace your shirt should you pick a shirt that doesn't fit you. We will still accept returns as we normally do for a full refund.
Finally the maximum number of shirts per order is limited to 2 so people can't buy a bunch in order to resell for a profit and further speculate with the price.
About that Price
We understand that a lot of you aren't going to be happy with the price. The shirt is by no means cheap, and I don't intend to argue with you about its value. However, I do think we owe you an explaination for the price.
As we started hashing out the details with SK Telecom several months ago, it became apparent that the team uniform would be expensive for two major reasons.
Production costs: This shirt is manufactured by the same Nike supplier that makes the official uniforms SKT players wear in the booth. I can't reveal the exact number, but I will say that it's a high quality product that has a production cost similar to the TL hoodie (sold for around 55$ on our store). The reason why the product we are selling in the store doesn't have a Nike logo is because SKT and Nike were not able to reach a licensing agreement. Other than that, the product we are offering is the exact same quality and model as the official uniform.
This shirt is manufactured by the same Nike supplier that makes the official uniforms SKT players wear in the booth. I can't reveal the exact number, but I will say that it's a high quality product that has a production cost similar to the TL hoodie (sold for around 55$ on our store). The reason why the product we are selling in the store doesn't have a Nike logo is because SKT and Nike were not able to reach a licensing agreement. Other than that, the product we are offering is the exact same quality and model as the official uniform. Import taxes and Shipping: Unlike other TL store products which are produced by our partners in North America and Europe, the SK Telecom T1 uniforms were produced in Korea. We had to pay to ship them to our distribution facilities in NA and EU, on top of which we had to pay import taxes. Just for the 375 shirts in Europe we paid 4000 GBP in taxes and shipping, which added 11-12€ per shirt. And of course, there's the additional 20% VAT for Europe.
These shirts are not great business for us. A TeamLiquid product that had the exact same costs and conditions would have been scrapped before making it to the store. Yet, having the SKT shirts on our store and making them available to the fans around the world is a dream for us, and we just couldn't decline when SKT came knocking. Also, we think that opening our doors in this way could be an important step toward offering more merchandise from Korean teams in the future.
Okay, so I lied about not selling you on the value. Even considering the high prices, I hope that some of you might see this opportunity for what it is: a rare chance to get the uniform of one of the greatest progaming teams of all time.
International Shipping has been as always a tricky situation. Because the limited stock and how valuable the shirts are we decided to go for the most reliable shipping option we have on our hands. That's why all international orders from the NA Store will be shipped with Fedex International which is incredibly fast, reliable and includes a detailed tracking. Fedex is not cheap but we absolutely cannot risk using USPS in this case and if we were to do that we were not in a position to guarantee the delivery of your order. We did the same with our hoodies and I believe this is the best way to do this.Also, because of the limited stock we have to change our story policy for that specific product - we do not offer size exchanges ! Make sure you get the right size because we won't be able to replace your shirt should you pick a shirt that doesn't fit you. We will still accept returns as we normally do for a full refund.Finally the maximum number of shirts per order is limited to 2 so people can't buy a bunch in order to resell for a profit and further speculate with the price.We understand that a lot of you aren't going to be happy with the price. The shirt is by no means cheap, and I don't intend to argue with you about its value. However, I do think we owe you an explaination for the price.As we started hashing out the details with SK Telecom several months ago, it became apparent that the team uniform would be expensive for two major reasons.These shirts are not great business for us. A TeamLiquid product that had the exact same costs and conditions would have been scrapped before making it to the store. Yet, having the SKT shirts on our store and making them available to the fans around the world is a dream for us, and we just couldn't decline when SKT came knocking. Also, we think that opening our doors in this way could be an important step toward offering more merchandise from Korean teams in the future.Okay, so I lied about not selling you on the value. Even considering the high prices, I hope that some of you might see this opportunity for what it is: a rare chance to get the uniform of one of the greatest progaming teams of all time. Administrator "I'm a big deal." - ixmike88 |
Duterte threatened by US rebuff while mass movement advances against him
February 6’s Senate hearing about the long-demanded termination of the Visiting Forces Agreement (VFA) left the Filipino people dissatisfied, if not unsurprised. Short of articulating their opposition to Rodrigo Duterte’s statement who supposedly pushes to end the VFA, the Upper Chamber’s deliberation of the issue ran along vague terms of ‘negative impact to US-Philippine ties’ but parrots that the decision rests on Duterte.
It thus becomes a waiting game. The Filipino people is anxious to see if an unjust bilateral military agreement that has undermined Philippine sovereignty and independence for 21 years will be terminated at the whim of an unhinged president and his favourite lapdog Ronald ‘Bato’ Dela Rosa. Or will Duterte use the Senate’s ambivalence as an excuse to renegade on a declared change on major foreign policy?
Wrong and Right Reasons
It started with a sad Bato whose US visa was cancelled. Duterte then threatened to terminate the VFA if the issue was not corrected within a month.
But the struggle to abrogate the VFA, along with its annexations Mutual Defense Treaty and Enhanced Defense Cooperation Agreement, actually started the moment it was passed. It is a primary indication of neocolonialism where imperialist powers do not directly inhabit a weaker nation but still subjugates it through political and economic intervention, military affairs included.
Since the VFA was passed in August 1998, various mass organizations have opposed it because it allows the US military to bring in nuclear weapons. Such results in greater risk of war involvement and far greater damage during wartime. It also exempts US forces from taxes and tariffs while clearing American personnel from criminal liability. Both Lance Corporals Daniel Smith and Joseph Pemberton of the US Marines were tried in the country for raping Filipino individuals only due to the public pressure led by anti-imperialist organizations. They were also both found guilty but avoided serving their sentences in any Philippine correctional facility because of the VFA. These are the most valid of reasons why the Filipino people oppose the VFA and campaign for its cessation.
Advancing Mass Movement
It might seem like Duterte’s most recent outburst is just another item from his long list of arbitrary orders given verbally. Yet, upon closer analysis, Duterte and his minions do need to worry. It is assumed that the US government applied the Global Magnitsky Human Rights Accountability Act against Bato. This is a US law sanctioning foreign government officials implicated in extensive violations by barring them entrance and freezing their US assets. Bato could be just the first among a string of officials, Duterte included, who would experience the omnipresent Big Brother’s display of power and control. It is no wonder that the megalomaniac Duterte is antagonized and irked.
This also corresponds to the growing infamy of Duterte and his administration. The US government is known to withdraw support from its puppets when the latter becomes irrevocably unpopular amongst the people. The organized mass movement against the present regime is steadily rising and expanding and the threat of an ouster is almost palpable.
All organizations and forces under the National Democratic Front – Ilocos must intensify the people’s struggle not just against the VFA and all unfair agreements but also against all forms of imperialism, feudalism and bureaucrat capitalism. Every allied organization in the united front is expected to contribute to the further weakening of this despotic but desperate regime. Link regional and sectoral issues to the most urgent national issues now gripping the Filipino people. Support the escalation of the armed revolution in the countryside. The sound of thousands chanting and marching in the streets must reverberate with the sound of the revolutionary army’s winning gunfire against the military troops of this isolated regime. ### |
Well, that’s exactly what has happened today
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1. Field of the Invention
The present invention relates to a dump valve apparatus for dumping water from a firetruck.
More specifically, the present invention relates to a dump valve apparatus which enables the rapid dumping of several thousand gallons of water from a firetruck at the site of a fire.
2. Information Disclosure Statement
Dump valves are installed in firetrucks in order to permit the rapid dumping of large quantities of water from the firetruck within a few second so that such water can be pumped to a holding tank for subsequent pumping to firehoses and the like for fighting a fire.
The dump valve apparatus of the present invention greatly simplifies the attachment of such dump valve apparatus to a firetruck thereby reducing the installation costs thereof.
Therefore, it is a primary objective of the present invention to provide a dump valve apparatus that overcomes the problems associated with the prior art dump valves and which makes a considerable contribution to the art of firefighting.
Other objects and advantages of the present invention will be readily apparent to those skilled in the art by a careful consideration of the detailed description contained hereinafter taken in conjunction with the annexed drawings. |
Introduction {#s1}
============
Thymineless death (TLD), the rapid loss of viability in cultures deprived of thymine, occurs in *E. coli*, yeast and human cells (reviewed [@pgen.1000865-Ahmad1]). Cancer chemotherapeutic drugs methotrexate, 5-fluorouracil (5-FU), and fluorodeoxyuridine, and the antibiotic trimethoprim, work by inducing TLD by targeting thymidylate synthase and/or interfering with *de novo* synthesis of thymidine monophosphate. Whereas 5-FU kills cells both TLD-dependently and TLD-independently (reviewed [@pgen.1000865-Longley1]), newer drugs are being developed that target thymidylate synthase specifically [@pgen.1000865-Jackman1]. Despite its relevance to problems of chemotherapy resistance, and although studied extensively, the mechanism(s) responsible for TLD remain unclear.
Work by Sat et al. suggested that TLD in *E. coli* was a form of cell suicide induced by the MazF toxin gene, an RNase that can induce cell death under various stresses coincident with destruction of mRNAs [@pgen.1000865-Sat1],[@pgen.1000865-Godoy1] by a mechanism not fully understood. Though intriguing, this is probably not the full story of TLD. Whereas inhibition of transcription by various drugs relieved TLD [@pgen.1000865-Nakayama1]--[@pgen.1000865-Morganroth1], MazF is repressed under active transcription by the presence MazE anti-toxin, and becomes available specifically when transcription is inhibited and MazE is degraded [@pgen.1000865-Aizenman1], such that inhibiting transcription would have been expected to exacerbate TLD (discussed [@pgen.1000865-Morganroth1]).
TLD also requires proteins involved in homologous recombination (HR) and repair, such as RecF and RecO which load RecA recombinase onto single-strand (ss)DNA [@pgen.1000865-Nakayama2],[@pgen.1000865-Nakayama3], RecQ DNA helicase [@pgen.1000865-Nakayama4],[@pgen.1000865-Nakayama5], and RecJ exonuclease [@pgen.1000865-Nakayama3]. TLD is exacerbated in cells lacking the UvrD helicase [@pgen.1000865-Siegal1], which acts in nucleotide excision repair (NER), and mismatch repair, and dismantles RecA filaments on single strand DNA, and so opposes HR [@pgen.1000865-Veaute1]. The UvrD anti-TLD role appears not to be *via* its role in NER, because NER-defective *uvrA* cells are not TLD hypersensitive [@pgen.1000865-Morganroth1]. TLD is also exacerbated in cells lacking RecBCD, the main double--strand exonuclease and catalyst of double-strand-break repair by homologous recombination in *E. coli* [@pgen.1000865-Nakayama2]. Chromosomal abnormalities/damage are associated with TLD in that cells undergoing TLD exhibit DNA breaks [@pgen.1000865-Freifelder1] and degradation [@pgen.1000865-Breitman1]. Further, abnormal DNA structures detected during TLD are reduced in cells lacking RecF, RecJ, RecQ or RecA [@pgen.1000865-Nakayama6]. Despite this evidence supporting a mechanism for TLD involving HR proteins, surprisingly, RecA, the central HR protein and activator of the SOS DNA-damage response, was reported not to be required for TLD [@pgen.1000865-Nakayama2],[@pgen.1000865-Anderson1]. In these studies non-null *recA* alleles were used: missense mutations *recA1*, *recA13* and *recA56* encode proteins with diminished strand exchange and SOS induction *in vitro* (measured by LexA cleavage), while retaining the ability to bind ssDNA [@pgen.1000865-Lauder1]; and *recA99* is an amber nonsense mutation that results in expression of a 7-amino-acid peptide [@pgen.1000865-Mount1],[@pgen.1000865-Dutreix1]. A single conflicting report using an undefined *recA* allele [@pgen.1000865-Inouye1] drew the opposite conclusion, that RecA was involved in TLD. Moreover, the SOS response, which is controlled by RecA, was also reported not to affect TLD [@pgen.1000865-Morganroth1]. This might have seemed to contradict a previous report that SulA, a protein made only during SOS, promoted TLD [@pgen.1000865-Huisman1]. However, that study tested *sulA* effects only in *lon* (protease-negative) cells, which have abnormally high SulA expression, leaving open the possibility that normally (in *lon^+^* cells) SulA and SOS were not involved.
The evidence that numerous HR proteins promote TLD and the conflicting *recA* literature led us to reinvestigate the roles of RecA, HR proteins, and the SOS response in TLD. We show that RecA, is required for much of TLD, and that its major role is *via* the SOS response. We find that the SOS-controlled inhibitor of cell division, SulA, accounts for most of the requirement for the SOS response in TLD, implicating irreversible checkpoint activation, causing a block to cell division, as a major contributor to TLD. We find that HR proteins previously shown to be required for TLD promote TLD by both SOS-dependent and SOS-independent pathways involving chromosome-segregation failure and apparent chromosome-region-specific DNA destruction.
Results {#s2}
=======
Roles of RecA in TLD {#s2a}
--------------------
In contrast with previous results obtained with non-null *recA* alleles [@pgen.1000865-Nakayama2],[@pgen.1000865-Anderson1], we find that cells carrying a deletion of *recA* are initially more sensitive to thymine deprivation than *rec^+^* cells ([Figure 1A](#pgen-1000865-g001){ref-type="fig"}, before 180 min.), but are ultimately more resistant to TLD ([Figure 1A](#pgen-1000865-g001){ref-type="fig"}, after 180 min.). The magnitude of the effect of the *recA* deletion is somewhat variable between experiments (e.g., [Figure 1A](#pgen-1000865-g001){ref-type="fig"} *versus* [Figure 1B](#pgen-1000865-g001){ref-type="fig"}), but we observed the same trend in a second genetic background KL742 ([Figure S1](#pgen.1000865.s001){ref-type="supplementary-material"}). Most of the work presented uses the AB2497 genetic background because, first, it has been used commonly in the *E. coli* TLD literature (e.g., [@pgen.1000865-Nakayama2],[@pgen.1000865-Nakayama5],[@pgen.1000865-Howe1]), and second, it shows greater sensitivity to thymine deprivation than KL742.
{ref-type="sec"} for statistical methods.](pgen.1000865.g001){#pgen-1000865-g001}
The shape of the Δ*recA* curve ([Figure 1A](#pgen-1000865-g001){ref-type="fig"}, ▪s) implies that early during thymine deprivation RecA protects against TLD, but at later times RecA contributes to TLD. We do not know why in some instances, Δ*recA* cultures show an increase in colony forming units (cfu) during TLD (e.g., [Figure 1C](#pgen-1000865-g001){ref-type="fig"} between 120 and 180 min.). Perhaps in the absence of RecA some cells complete an additional round of cell division because some cells lyse, releasing thymine used by the remainder.
In the following section, we show that activation of the SOS DNA-damage response is required for much of TLD. To determine whether the apparent dual roles of RecA in TLD correspond to its two known functions in HR *versus* induction of the SOS response, we examined cells carrying the *recA430* allele, which encodes a RecA protein that is competent for HR but defective for induction of the SOS response [@pgen.1000865-Ennis1]. We find that *recA430* cells display the increased TLD resistance seen with the Δ*recA* allele late in thymine starvation, but do not show the increased TLD sensitivity early in TLD seen with the Δ*recA* null allele ([Figure 1A](#pgen-1000865-g001){ref-type="fig"}). This implies that the early protective role of RecA in TLD is not via SOS-induction, and so could be via HR, whereas the later TLD-promoting role is via SOS induction (discussed below).
MazF is an RNase expressed during stress that leads to programmed cell death (reviewed [@pgen.1000865-EngelbergKulka1]) and was implicated in TLD [@pgen.1000865-Sat1],[@pgen.1000865-Godoy1]. The previously reported requirement for MazF in TLD was variable (complete [@pgen.1000865-Sat1] *versus* 4- to 5-fold [@pgen.1000865-Godoy1]) and was not tested in AB2497, the strain used for much previous work on TLD. We wished to understand whether the role of RecA might be, for example, activating expression of MazF. To determine whether the observed role for RecA ([Figure 1A](#pgen-1000865-g001){ref-type="fig"}, [Figure S1](#pgen.1000865.s001){ref-type="supplementary-material"}) is part of the same pathway as the MazF RNase in TLD, we tested the magnitude of the *mazF* effect in the AB2497 strain used here. We find that Δ*mazF* caused a slight, but insignificant, increase in TLD resistance ([Figure S2](#pgen.1000865.s002){ref-type="supplementary-material"}, see [Materials and Methods](#s4){ref-type="sec"} for statistical methods), indicating that the MazF RNase is not a major mechanism contributing to TLD in this strain. Thus, the role of RecA in promoting TLD is likely to be independent of MazF.
SOS response and SulA in TLD {#s2b}
----------------------------
RecA functions both in HR and in induction of the SOS response to DNA damage (reviewed [@pgen.1000865-Lusetti1],[@pgen.1000865-Butala1]). The SOS response is induced when single-stranded (ss)DNA, the SOS-inducing signal, accumulates at sites of DNA damage or blocked replication forks. RecA binds the ssDNA, becomes activated as a co-protease and facilitates auto-proteolytic cleavage of the LexA transcriptional repressor, thus upregulating expression of about 40 damage-inducible SOS genes.
We found that blocking the ability of cells to induce SOS with either of two special "SOS-off" mutations conferred resistance to TLD: *lexA3*(Ind^−^), which encodes an uncleavable LexA/SOS repressor; and *recA430*, the recombination-proficient, SOS-induction-deficient *recA* allele ([Figure 1A](#pgen-1000865-g001){ref-type="fig"}, orange s and grey s). We conclude that induction of the SOS response is required for TLD.
Both the *lexA3*(Ind^−^) and *recA430* results reported here contradict a previous report that *lexA3*(Ind^−^) did not affect TLD-sensitivity [@pgen.1000865-Morganroth1]. Experiments summarized in [Figure S3](#pgen.1000865.s003){ref-type="supplementary-material"} and legend indicate that the strain used previously contained the *lexA3*(Ind^−^) mutation but additionally carried another genetic element(s) that suppressed the TLD-resistance phenotype.
Addition of an operator-constitutive *recA*o allele, which constitutively produces SOS-induced levels of RecA, to the *lexA3*(Ind^−^) cells did not overcome the resistance to TLD conferred by *lexA3*(Ind^−^) ([Figure S4](#pgen.1000865.s004){ref-type="supplementary-material"}), and is significantly different from the AB2497 parental strain only after 300 minutes of thymine deprivation (p = 0.012). We conclude that SOS-induced levels of a LexA-controlled function other than, or in addition to, RecA is required for TLD.
SulA is an inhibitor of cell division that is expressed only during an SOS response [@pgen.1000865-Friedberg1]. We find that *sulA* cells are nearly as resistant to TLD as *lexA3*(Ind^−^) or *recA430* cells ([Figure 1A](#pgen-1000865-g001){ref-type="fig"}), indicating that SulA can account for most or nearly all of the role of the SOS response in TLD. These data imply that a large fraction of TLD results from an irreversible block to cell division caused by SOS induction and SulA expression.
The following data indicate that much of the role of RecA in promoting TLD results from its role in induction of SOS and SulA. First, we see that after 150 min Δ*recA* cells are only slightly more resistant to TLD than *sulA* cells ([Figure 1C](#pgen-1000865-g001){ref-type="fig"}). Because SulA induction requires RecA [@pgen.1000865-Friedberg1], this implies that most of the contribution of RecA to TLD is via the same pathway as SulA. Second, *ΔrecA sulA* cells show slightly, but statistically insignificantly greater TLD resistance than the *ΔrecA* single mutant ([Figure 1C](#pgen-1000865-g001){ref-type="fig"}) indicating, as expected, that *sulA* functions completely in the same pathway as *recA*. Third, *ΔrecA sulA* is slightly but significantly more resistant than the *sulA* single mutant after 210 minutes of thymine deprivation ([Figure 1C](#pgen-1000865-g001){ref-type="fig"}), indicating that SulA accounts for most, but not all, of the RecA role in TLD. Fourth, Δ*recA* and Δ*recA lexA3*(Ind^−^) cells are also slightly but insignificantly more TLD resistant late in TLD than *lexA3*(Ind^−^) single mutant cells ([Figure 1B](#pgen-1000865-g001){ref-type="fig"}). This indicates that most of the Δ*recA* phenotype late in TLD occurs via the same pathway (SOS induction) as that blocked in the *lexA3*(Ind^−^) "SOS-off" mutant. The slightly greater TLD resistance of Δ*recA* single mutants and of both *ΔrecA sulA* and Δ*recA lexA3*(Ind^−^) double mutants compared with *sulA* and *lexA3*(Ind^−^) single mutants suggest that there is a small SOS/SulA-independent role for RecA, however, most of the requirement for RecA in TLD appears to occur *via* the same pathway that leads to SOS/SulA induction. This could be because the important role of RecA in TLD is in inducing SOS directly or that, *e.g*., RecA-promoted HR intermediates cause SOS/SulA induction that leads to death, or both.
Holliday-junction-resolution prevents TLD {#s2c}
-----------------------------------------
As noted above, there is a small RecA-dependent but SOS/SulA-independent component of TLD (previous paragraph, and [Figure 1B and 1C](#pgen-1000865-g001){ref-type="fig"}). We hypothesized that this segment of TLD might result from "death-by-recombination" (per [@pgen.1000865-Magner1]), caused when interchromosomal HR intermediates (IRIs) accumulate and prevent chromosome segregation, thereby killing cells. Thymine deprivation could lead to ssDNA gaps in replicating DNA. Perhaps while some thymine remains, repair by HR with a sister chromosome is possible and protective, explaining the early part of the Δ*recA* curve; but later in the complete absence of thymine, the cellular capacity to resolve RecA-promoted IRIs might be inhibited and accumulated IRIs could cause chromosome-segregation failure and death (model discussed below).
The RuvABC resolvasome constitutes a major pathway of IRI resolution in *E. coli* [@pgen.1000865-Zerbib1]. The death-by-recombination hypothesis for TLD predicts that RuvABC would protect cells from TLD by reducing levels of IRIs that cause death. Indeed, we find that deletion of *ruvABC* makes cells more sensitive to TLD ([Figure 1D](#pgen-1000865-g001){ref-type="fig"}). As predicted, this sensitivity is completely dependent on RecA activity ([Figure 1D](#pgen-1000865-g001){ref-type="fig"}), implying that accumulation of unresolved RecA-promoted IRIs in cells lacking RuvABC promotes TLD. In support of the interpretation that Δ*ruvABC* exacerbated TLD because of excess unprocessed HJs/IRIs, expression of an unrelated HJ resolvase, RusA, partially compensated for the lack of RuvABC ([Figure S5A](#pgen.1000865.s005){ref-type="supplementary-material"}). RusA is encoded in a cryptic prophage and is expressed if cells carry the *rus-1* mutation, which restores partial resistance to UV light to *ruvC* strains [@pgen.1000865-Bolt1]. These experiments do not address whether in wild-type (RuvABC^+^) cells TLD *normally* results from excess IRIs. To test this one would ideally provide *more* resolution capacity than in wild-type cells, and ask whether TLD was reduced. However, because of toxicity effects upon overproduction, interpretations of results from overproduction experiments are inconclusive, and we have not presented those here.
SOS--dependent and --independent roles of RecF in TLD {#s2d}
-----------------------------------------------------
Having established a role for RecA in TLD via SulA/SOS response activation, we sought to determine whether other HR proteins previously shown to be required for TLD promote TLD by the same pathway.
RecF loads RecA onto ssDNA, a precursor to both HR and SOS induction [@pgen.1000865-Friedberg1], and is required for replication restart [@pgen.1000865-Courcelle1] apparently *via* activating SOS, in that SOS-constitutive-mutant cells no longer require RecF [@pgen.1000865-Rangarajan1]. We find that both Δ*recF lexA3*(Ind^−^) ([Figure 2A](#pgen-1000865-g002){ref-type="fig"}) and Δ*recF sulA* ([Figure 2B](#pgen-1000865-g002){ref-type="fig"}) cells show somewhat greater TLD resistance than *lexA3*(Ind^−^) and *sulA* single mutants, respectively. The difference is significant in both cases ([Figure 2 legend](#pgen-1000865-g002){ref-type="fig"}). The data imply that most of role of RecF in TLD is in the SOS/SulA-dependent pathway leading to TLD, but that RecF also promotes TLD SOS/SulA-independently either *via* HR or another route.
{#pgen-1000865-g002}
RecQ and RecJ promote TLD SOS/SulA- and RecA- independently {#s2e}
-----------------------------------------------------------
Under some conditions RecQ is required for SOS induction [@pgen.1000865-Hishida1]. To test whether the role of RecQ in TLD is *via* SOS/SulA induction, we examined Δ*recQ lexA3*(Ind^−^) ([Figure 3A](#pgen-1000865-g003){ref-type="fig"}) and Δ*recQ sulA* ([Figure 3B](#pgen-1000865-g003){ref-type="fig"}) cells. Both double mutants were significantly more resistant than their respective single-mutant controls indicating a wholly or partly additive TLD resistance when both SOS/SulA and RecQ are inactivated. We conclude that RecQ promotes TLD *via* a pathway wholly or partly independent of and additive with the SOS/SulA TLD pathway.
{#pgen-1000865-g003}
If the sole role of RecQ in TLD were to assist RecA-mediated accumulation of IRIs leading to death by recombination, then loss of RecQ would be expected to provide no further resistance to TLD above that already seen in Δ*recA* cells. However, we observed greater TLD resistance of Δ*recQ* Δ*recA* double mutants than Δ*recA* cells ([Figure 3C](#pgen-1000865-g003){ref-type="fig"}). Similarly, Δ*recQ* Δ*recF* double mutants showed greater resistance to TLD than Δ*recF* or Δ*recQ* ([Figure 3D](#pgen-1000865-g003){ref-type="fig"}). We conclude that although RecQ might catalyze death-by-recombination in TLD in a minor pathway, it must also promote TLD by a RecA- RecF-independent, and thus HR-independent mechanism.
RecJ exonuclease is thought to work closely with RecQ to unwind and degrade nascent DNA at stalled replication forks [@pgen.1000865-Hishida1],[@pgen.1000865-Courcelle2], and *recJ* and *recQ* have similar phenotypes in TLD ([Figure 4A](#pgen-1000865-g004){ref-type="fig"} and [@pgen.1000865-Nakayama3]), and also in a "death-by-recombination" pathway in which cells that accumulate unresolved interchromosomal recombination intermediates (IRIs) die from chromosome-segregation failure [@pgen.1000865-Magner1]. We find that the double *recQ recJ* mutant is as resistant to TLD as *recJ* alone ([Figure 4A](#pgen-1000865-g004){ref-type="fig"}) indicating that these two proteins promote TLD *via* the same pathway. Interestingly *recJ* has a greater resistance to TLD than *recQ* ([Figure 4A](#pgen-1000865-g004){ref-type="fig"}), possibly because RecQ helicase can create the 5′-ssDNA-end substrate degraded by RecJ exonuclease (e.g., [@pgen.1000865-Hishida1],[@pgen.1000865-Courcelle2]), but RecJ can also degrade 5′-ssDNA ends that arise via means other than RecQ. Like RecQ, RecJ promotes TLD *via* a pathway that is wholly or partly additive with, and thus wholly or partly independent of, SOS induction ([Figure 4B](#pgen-1000865-g004){ref-type="fig"}); we find that Δ*recJ lexA3*(Ind^−^) cells are significantly more TLD resistant than either Δ*recJ* or *lexA3*(Ind^−^) single mutants. These data show that at least two pathways contribute to TLD, a RecA-, RecF-, and LexA-dependent one requiring SOS/SulA induction and another involving HR proteins RecQ and RecJ without SOS induction or RecA.
{#pgen-1000865-g004}
Topoisomerase III plays no role in TLD {#s2f}
--------------------------------------
Homologues of RecQ have been shown to work with Topoisomerase III in a "dissolvasome" complex to resolve converging replication [@pgen.1000865-Suski1] or recombination intermediates [@pgen.1000865-Raynard1]. We tested the possibility that Topoisomerase III was necessary for TLD, similarly to RecQ, but did not find significant resistance to TLD in cells lacking *topB*, the gene encoding Topoisomerase III ([Figure S6](#pgen.1000865.s006){ref-type="supplementary-material"}).
Chromosome-segregation and -replication defects and DNA loss during TLD {#s2g}
-----------------------------------------------------------------------
We found that the majority of cells undergoing TLD exhibit severe chromosome-segregation defects ([Figure 5A](#pgen-1000865-g005){ref-type="fig"}). Whereas most cells grown in the presence of thymine appear small and have discreet, segregated nucleoids (bacterial chromosomes), one hour after thymine deprivation most cells appear elongated with a single, small central DNA mass which appears to contain less DNA than normal nucleoids ([Figure 5A and 5B](#pgen-1000865-g005){ref-type="fig"} 90 min). "Guillotining" of DNA during cell division (see [Figure 5A](#pgen-1000865-g005){ref-type="fig"}) occurs early during TLD, whereas anucleate cells, which may result from degradation of broken/guillotined DNA or septum formation at the ends of elongated cells, appear later ([Figure 5A](#pgen-1000865-g005){ref-type="fig"}).
{#pgen-1000865-g005}
DNA content of the cells undergoing TLD appeared diminished with respect to both normal cells ([Figure 5A](#pgen-1000865-g005){ref-type="fig"}) and cells dying the death-by-recombination observed previously [@pgen.1000865-Magner1]. We examined chromosome replication and integrity using fluorescent in-situ hybridization (FISH) with probes homologous to the chromosomal replication origin (*ori*) (green) and terminus (ter) (red, [Figure 5B](#pgen-1000865-g005){ref-type="fig"}). At time 0, cells were small with an average of 2.2±0.1:1 labeled *ori*:ter foci. Per [Figure 5C](#pgen-1000865-g005){ref-type="fig"}, 45% had 2 *ori* and 1 ter focus, expected in replicating DNA, 23% had 1 of each, and 17% and 11% had only one *ori* or ter focus, respectively. The 17% and 11% with only one *ori* or ter focus presumably reflect the imperfect efficiency of the FISH probes to reveal their targets, as reported previously [@pgen.1000865-Magner1],[@pgen.1000865-Bates1], which is a constant for each probe set against which deviations are compared and normalized ([@pgen.1000865-Magner1],[@pgen.1000865-Bates1], [Figure 5C](#pgen-1000865-g005){ref-type="fig"}).
The profile of *ori* and ter foci changed dramatically with prolonged thymine deprivation ([Figure 5C](#pgen-1000865-g005){ref-type="fig"}). At 90 min ([Figure 5C](#pgen-1000865-g005){ref-type="fig"}), only 3% had 2 *ori*:1 ter, whereas 64% had 1 of each. Although it is formally possible that many chromosomes completed replication but did not re-initiate, this is highly unlikely given the absence of thymine. A more likely explanation is that replication halted mid-chromosome. In this second (more likely) instance, the subsequent shift from the majority of cells containing 2 *ori* and 1 ter to the majority containing a single *ori* and ter over the first 90 min of thymine deprivation may indicate that *ori-*containing DNA was specifically lost or destroyed. Significantly, those with 1 *ori*:0 ter focus increased to 28%, implying loss of *ter*-containing DNA. Supporting this interpretation, the fraction with 1 ter:0 *ori* decreased to 1.6%. This pattern was more pronounced at 210 min ([Figure 5C](#pgen-1000865-g005){ref-type="fig"}), at which time 2 *ori*:1 ter cells fell further to 2.3%; 1 *ori*:1 ter cells dropped to 46%; while cells with 1*ori*:0ter increased correspondingly to 48%. During normal segregation of daughter chromosomes, first, *ori\'*s segregate to the distal cell poles away from the cell-division septum while the ter sequences localize at the septum and are replicated and segregated last [@pgen.1000865-Bates1] (illustrated [Figure 6E](#pgen-1000865-g006){ref-type="fig"}). During TLD, first, all of the foci stayed mid-cell, where the cell-division septum would form in nonarrested cells ([Figure 5B](#pgen-1000865-g005){ref-type="fig"}); second, the number of foci per cell is fewer than normal ([Figure 5B and 5C](#pgen-1000865-g005){ref-type="fig"}); and third, the accumulation of 1 *ori*:1 ter cells replacing 2 *ori*: 1 ter cells, followed by depletion of 1 *ori*:1 ter and concurrent accumulation of 1 *ori*:0 ter cells implies that chromosomes either completed replication then lost their ter sequences, or, more likely given the general DNA reduction seen ([Figure 5A and 5B](#pgen-1000865-g005){ref-type="fig"}), lost one of their two *ori*s, then subsequently lost ter-containing DNA. We failed to observe a significant fraction of cells containing a single ter and no *ori* (discussed below). The apparent degradation of DNA near ter (which is probably preceded by degradation near one of the two *ori*s) could be caused by chromosome tearing as cells try to segregate unresolved chromosomes, perhaps unsegregated because of IRIs per death-by-recombination models ([Figure 6E](#pgen-1000865-g006){ref-type="fig"}), or by RecQ/J-promoted DNA degradation, discussed below.
![Models for TLD by SOS, death-by-recombination, and RecQ/J-promoted DNA destruction.\
(A) Gaps in DNA result from insufficient thymine. (B) Gap extension by RecQ and RecJ. (C) RecF-assisted RecA loading at ssDNA gaps promotes strand exchange and IRI formation. (D) Some portion of IRIs can be resolved by RuvABC to yield segregated chromosomes. (E) "Death-by-recombination," a death caused by failed chromosome segregation, occurs when more IRIs accumulate than can be processed by Ruv and other IRI-resolution pathways [@pgen.1000865-Magner1]. (F) SOS induction and SulA production create an irreversible cell-cycle checkpoint which prevents cells from recovering upon addition of thymine. Note that all ssDNA substrates drawn, including those in *G*, *H* and *I* could lead to SOS-induction. (G) RecQ/J-mediated DNA fragmentation, a possible mechanism for the RecA-independent contribution of RecQ/J to TLD. This degradation towards the *ori* from stopped forks along the chromosome, then (not shown) RecBCD-mediated degradation of the double-stranded DNA end back, past *ori*, to the next stalled fork, would lead to specific loss of *ori*-containing DNA. (H) RecQ/RecJ-promoted DNA destruction. Nascent-strand removal from stopped forks to *ori* and past to the next stopped fork, might be used to restore arrested replication bubbles to the duplex state, but creates extensive ssDNA. (I) Breakage of an old strand in regions of extensive ssDNA, shown here to occur if a hairpin forms and is cleaved by a hairpin endonuclease (but possible with other secondary structures), opens the whole chromosome up to degradation by RecQ and RecJ. This model accounts for apparent loss of first *ori-* then ter-containing DNAs. Lines, strands of DNA except in bottommost schemes (F, and H left) in which solid and dashed circles represent whole bacterial chromosomes; arrow heads, 3′ ends; IRI, interchromosomal recombination intermediate.](pgen.1000865.g006){#pgen-1000865-g006}
Discussion {#s3}
==========
The data presented establish a prominent role for RecA, the SOS response and the SOS-controlled SulA inhibitor of cell division in TLD. They further show that at least three pathways of TLD operate concurrently with a remarkable pattern of chromosome-segregation failure and chromosome-region-specific loss of FISH-detectable foci, in which first apparent replication-origin-containing then terminus-proximal DNA disappeared.
Death by SOS {#s3a}
------------
First, a major TLD pathway, constituting ≥1 of the 2--3 logs of loss of colony-forming ability observed by 300 min of thymine starvation, is attributable to RecA- and RecF-dependent activation of the SOS DNA-damage response turning on the SulA inhibitor of cell division ([Figure 1](#pgen-1000865-g001){ref-type="fig"}, [Figure 2](#pgen-1000865-g002){ref-type="fig"}). This implies, surprisingly, that a significant fraction of TLD results from an irreversible cell-cycle checkpoint such that when returned to medium with thymine in the cfu assay, cell division does not resume. Simply removing *sulA* allowed these cells to form colonies ([Figure 1](#pgen-1000865-g001){ref-type="fig"}, [Figure 2](#pgen-1000865-g002){ref-type="fig"}, [Figure 3](#pgen-1000865-g003){ref-type="fig"}), as if many of the underlying DNA problems that caused SOS induction and SulA expression were not themselves lethal. Irreversible SOS-induction causing apparent cell stasis or senescence has been reported previously in a study from one of our laboratories of spontaneous SOS induction in growing *E. coli* populations which showed that only about 30% of spontaneously SOS-induced cells recover to a proliferating state [@pgen.1000865-Pennington1]. The discovery that at least a log of TLD results from inhibition of cell division raises the possibility that some TLD might reflect cell stasis rather than death. A model for the SOS/SulA dependent component of TLD is shown in [Figure 6F](#pgen-1000865-g006){ref-type="fig"}. SOS is activated when RecA binds ssDNA [@pgen.1000865-Friedberg1]. In [Figure 6](#pgen-1000865-g006){ref-type="fig"} we consider potential sources of ssDNA that might activate SOS during TLD, some of which would not otherwise kill cells (discussed below).
Death by recombination {#s3b}
----------------------
A minor second TLD pathway appeared to require RecA but not SulA ([Figure 1C](#pgen-1000865-g001){ref-type="fig"}) and so might reflect a lethal role of HR. In [Figure 6A--6E](#pgen-1000865-g006){ref-type="fig"} we consider a "death-by-recombination" model for this component of TLD, based on the observations of death by recombination in Holliday-junction-resolution-defective cells ([@pgen.1000865-Magner1] and references therein). In it we hypothesize that ssDNA gaps caused by inability to replicate in the absence of thymine provoke the RecQ, J, F, A-dependent initiation of HR with a sister chromosome ([Figure 6A--6E](#pgen-1000865-g006){ref-type="fig"}) creating interchromosomal recombination intermediates (IRIs, [Figure 6D and 6E](#pgen-1000865-g006){ref-type="fig"}). IRIs are normally resolved by RuvABC allowing chromosome separation ([Figure 6D](#pgen-1000865-g006){ref-type="fig"}) [@pgen.1000865-Zerbib1], but we suggest that when the number of gaps and resulting IRIs exceeds resolution capacity, their failure to be resolved will cause death by failed chromosome segregation ([Figure 6E](#pgen-1000865-g006){ref-type="fig"}). Death by failed chromosome segregation caused by excessive IRI accumulation ("death-by-recombination") was seen in cells lacking Ruv resolution and UvrD anti-RecA proteins [@pgen.1000865-Magner1] and cells lacking UvrD and RecG Holliday-junction-processing proteins [@pgen.1000865-Fonville1], and, like TLD, required RecA, RecF, RecQ and RecJ (SOS independently). As predicted by this model, TLD is associated with failed chromosome segregation ([Figure 5](#pgen-1000865-g005){ref-type="fig"}) and is exacerbated by removal of RuvABC ([Figure 1D](#pgen-1000865-g001){ref-type="fig"}), implying that a mechanism like this can occur at least in Ruv-deficient cells. A possible death-by-recombination component of TLD might underlie the minor RecA-dependent SulA-independent fraction of TLD ([Figure 1C](#pgen-1000865-g001){ref-type="fig"}).
Death by RecQ and RecJ {#s3c}
----------------------
Yet a third TLD pathway requires RecQ and RecJ but is dependent upon neither RecA nor SOS induction, and thus is also HR-independent ([Figure 3](#pgen-1000865-g003){ref-type="fig"}, [Figure 4](#pgen-1000865-g004){ref-type="fig"}).
In [Figure 6G--6I](#pgen-1000865-g006){ref-type="fig"} we suggest two HR-independent ways by which RecQ and RecJ could cause TLD and the DNA destruction suggested by our cytological and FISH results ([Figure 5](#pgen-1000865-g005){ref-type="fig"}). In [Figure 6G](#pgen-1000865-g006){ref-type="fig"}, RecQ helicase and RecJ 5′ exonuclease are shown degrading DNA at a 5′ end at a replication fork lagging strand [@pgen.1000865-Hishida1], leading to DNA fragmentation when the next fork upstream is reached. Because this mechanism degrades newly replicated DNA from a stopped fork towards the *ori*, this might cause the observed loss of *ori*-containing foci early during TLD ([Figure 5B and 5C](#pgen-1000865-g005){ref-type="fig"}, assuming degradation of the double-strand DNA end created, see [Figure 6G](#pgen-1000865-g006){ref-type="fig"}), and could explain RuvC-independent linearization of *E. coli* chromosomes during TLD reported by Guzman and colleagues [@pgen.1000865-Guarino1], but does not explain ter-specific DNA loss.
Similarly, when replication forks stop in thymine-starved cells, RecQ 5′ helicase and RecJ 5′ single-strand-dependent exonuclease might degrade DNA extensively from the forks\' 5′-ending lagging strands back towards the *ori* ([Figure 6H](#pgen-1000865-g006){ref-type="fig"}), removing both nascent strands from arrested replication bubbles so that cells unable to complete replication return their chromosomes to a simple double-stranded circular starting point allowing re-initiation of replication later, when replication precursors are available (not an apparently death-promoting activity).
Although this appears to predict only *ori*-proximal DNA loss, extensive nascent-strand degradation would expose long tracts of ssDNA, which would induce SOS and might also be susceptible to further breakage upon exposure of secondary-structure-forming sequences in the extensive ssDNA regions ([Figure 6I](#pgen-1000865-g006){ref-type="fig"}). Digestion of secondary structures would break an "old" strand in these replication bubbles which would then open up the whole chromosome to degradative activities, including the ter ([Figure 6I](#pgen-1000865-g006){ref-type="fig"}). This might underlie the initial loss of *ori*-containing FISH foci, and later loss of terminus-proximal FISH foci because after an old strand is broken, single-strand degradation can pass a stopped fork and proceed towards the terminus ([Figure 6I, right](#pgen-1000865-g006){ref-type="fig"}). Although simple removal of nascent strands ([Figure 6H](#pgen-1000865-g006){ref-type="fig"}) would be expected not to be lethal, breaking an old strand followed by chromosome degradation ([Figure 6I](#pgen-1000865-g006){ref-type="fig"}) could be lethal. Both models 6G and 6I can explain why there is first loss of only one of two *ori* foci.
Another possibility for ter-specific DNA loss is that chromosome dimers formed by HR that are not resolved will accumulate as shown in [Figure 6E](#pgen-1000865-g006){ref-type="fig"}, with ters at the cell-division septum and *ori*\'s away from it [@pgen.1000865-Bates1]. Tearing of unresolved chromosomes might be expected to occur terminus proximally and this could set off degradation specific to the ter region. Perhaps chromosome dimers formed by HR, which are usually resolved at the septum by XerCD [@pgen.1000865-Ip1], cannot be resolved when cell division is blocked by SulA, and this could result in such tearing ([Figure 6E](#pgen-1000865-g006){ref-type="fig"}).
Other TLD pathway(s) {#s3d}
--------------------
In addition to the TLD pathways listed, our data indicate that at least one more must operate because *recA recQ* cells which are defective for SOS, HR, and the SOS/HR-independent roles for RecQ in TLD, still suffer ≥1 log of TLD by 300 min ([Figure 4C](#pgen-1000865-g004){ref-type="fig"}).
Cancer chemotherapies and resistance {#s3e}
------------------------------------
Thymineless death is the mode of action of important cancer chemotherapeutic drugs methotrexate, 5-fluorouracil and 5-fluorodeoxyuridine, as well as the antibiotic trimethoprim. The results presented here catalogue a series of proteins and pathways that if disrupted could be expected to confer some level of resistance to those drugs in bacteria and humans. Humans have several RecA homologues including RAD51, whose function in double-strand-break repair by HR is disrupted in *BRCA*-defective cells including in some breast and ovarian cancers (reviewed [@pgen.1000865-Powell1]). Humans possess five RecQ homologues, defects in three of which are known to be associated with cancer-predisposition syndromes (reviewed [@pgen.1000865-Singh1]), any of which might also be defective in sporadic cancers. Cancers with the homologues and analogues of these bacterial DNA repair pathways disrupted might be resistant to TLD, and so to treatment with TLD-inducing drugs. Similarly disruption of the eukaryotic DNA-damage responses and checkpoints might also confer resistance as seen for the SOS response here. The DNA repair, replication and metabolism pathways are very well conserved from bacteria to humans (reviewed [@pgen.1000865-Friedberg1]) making application of these mechanisms to human cancer treatment plans and investigations practical and imperative.
Materials and Methods {#s4}
=====================
Strains and TLD assays {#s4a}
----------------------
Origins of strains used in this study are given in [Table S1](#pgen.1000865.s007){ref-type="supplementary-material"}. P1 transductions were as described [@pgen.1000865-Miller1]. TLD experiments were as described [@pgen.1000865-Sat1] with minor variations. Thymine auxotrophs were grown at 37°C with shaking in M9 minimal medium with 50 µg/ml thymine, 0.1% glucose and 0.5% casamino acids, and for strains containing pGB2 or pGBruvABC, 100 µg/ml spectinomycin. Saturated cultures were diluted 25-fold into the same medium and grown to early/mid-log (OD~450~ of 0.5). 1.0 ml samples were centrifuged, washed twice with M9 saline solution, and resuspended in 2.0 ml of M9 with glucose and casamino acids (no thymine), then returned to 37°C, with shaking, for up to five hours with aliquots taken at intervals for cfu assays on LBH thymine plates. Cfu were scored on a Microbiology International ProtoCOL colony counter after 24 h at 37°C. Longer incubations verified that all cfu were apparent at 24 h. Because the absolute extent of killing varied widely between experiments, whereas the relative effect of the mutations used did not, data presented show curves that are means of sets of independent experiments in which absolute extents of killing were similar.
Statistical analyses {#s4b}
--------------------
Error bars represent ± one SEM of ≥3 independent experiments. Statistical analyses were performed using SigmaStat. For TLD assays significance was determined as p\<0.05 using repeated measure ANOVA to analyze the curve data, and Tukey post-hoc analysis.
Microscopy {#s4c}
----------
Chromosome-segregation analyses were as described [@pgen.1000865-Magner1] with minor changes. Cells were fixed by adding an equal volume of PBS with 4% paraformaldehyde for 10 min at room temperature and 20 min on ice, washed three times with cold PBS and stored in an appropriate volume of PBS. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1 µg/ml), placed on slides, and photographed with an Olympus B×51 microscope equipped with an Uplan Fluorite 100× oil objective, DAPI filter (U-N31000, Olympus), and an Olympus MagnaFire CCD digital camera.
Fluorescence in situ hybridization {#s4d}
----------------------------------
FISH was as described [@pgen.1000865-Magner1]. Probes were 6 kb DNA fragments PCR amplified (Phusion DNA polymerase, New England Biolabs) from MG1655 DNA. Primers for the *ori* and ter probes were as described [@pgen.1000865-Bates1]. Probes were visualized using a Zeiss Axio Imager microscope equipped with 100× oil immersion objective, DAPI filter, Oregon Green filter, Rhodamine filter, and Hamamatsu EMCCD camera. Foci were scored on each channel prior to RGB merging of the images. Images were processed using Axiovision digital image processing software and ImageJ.
Supporting Information {#s5}
======================
######
RecA is required for TLD in the KL742 strain background. Δ*recA* cells (SMR10432, ▪) are significantly more resistant to TLD than KL742 (♦) at t≥180 min, and Δ*recQ* cells (SMR10435, ▴) are significantly more resistant at t≥90 min. The results recapitulate those shown in [Figure 1A](#pgen-1000865-g001){ref-type="fig"}, [Figure 3A](#pgen-1000865-g003){ref-type="fig"} using the AB2497 strain background. Mean ± SEM of 3 experiments.
(0.09 MB TIF)
######
Click here for additional data file.
######
MazF is not the predominant cause of TLD in the AB2497 strain background. A strain lacking the MazF toxin (SMR10685, ▪) of the MazEF toxin/antitoxin pair is slightly, but not significantly, more resistant to TLD than the parental strain (♦). Mean ± SEM of 3 experiments.
(0.09 MB TIF)
######
Click here for additional data file.
######
The *lexA3*(Ind^−^) mutation causes TLD resistance in the HL353 strain background. This was observed when the strain HL353 *lexA3*(Ind^−^) was reconstructed (SMR10675, ▴), but not with the originally published construction: HL354 (▪). SMR10675 is significantly different from HL353 (♦) at t≥180 min. Mean ± SEM of 3 experiments. To understand why Morganroth and Hanawalt saw no TLD-resistance in a *lexA3*(Ind^−^) strain relative to its *lexA^+^* parent [@pgen.1000865-Morganroth1], whereas we observed TLD resistance of both a *lexA3*(Ind^−^) strain and *recA430* strain relative to their isogenic *lexA^+^ recA^+^* parent ([Figure 1A](#pgen-1000865-g001){ref-type="fig"}), we first repeated their result with their strains HL353 (Parental) and HL354 (*lexA*Ind^−^) (this figure). Next, we reintroduced the *lexA3*(Ind^−^) allele by phage P1-mediated transduction into the HL353 genetic background used by Morganroth and Hanawalt, thus creating strain SMR10675. We observed that SMR10675, but not the originally published *lexA3*(Ind^−^) strain HL354, was TLD resistant (this figure), confirming our finding that an inducible SOS response is required for TLD. We sequenced the *lexA* gene and verified the presence of the *lexA3*(Ind^−^) mutation (G to A at position 355 \[Markham, et al\]) and the absence of any other mutation in the *lexA* gene or ≥500 bp up- or downstream of *lexA* in all three putative *lexA3*(Ind^−^) strains: ours in the AB2497 strain background (SMR10669), SMR10675 and HL354. Because the *lexA3*(Ind^−^) allele confers TLD-resistance in both genetic backgrounds, including when moved afresh into HL353, because a different SOS-off mutation, *recA430*, also confers TLD resistance, and because SulA is required for TLD ([Figure 1A and 1C](#pgen-1000865-g001){ref-type="fig"}) and is expressed only during SOS \[Courcelle, et al\], we conclude that induction of SOS is required for TLD. It seems most likely that some other, unknown mutation(s) is present in HL354 which suppresses the TLD-resistance phenotype conferred by *lexA3*(Ind^−^) in that strain. \[Markham BE, Little JW, Mount DW (1981) Nucleotide sequence of the lexA gene of *Escherichia coli* K-12. Nucleic Acids Res 9: 4149-4161.\] \[Courcelle J, Khodursky A, Peter B, Brown PO, Hanawalt PC (2001) Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient *Escherichia coli*. Genetics 158: 41-64.\]
(0.10 MB TIF)
######
Click here for additional data file.
######
SOS--induced levels of RecA do not compensate for an uninducible SOS/LexA regulon in TLD. The *recAo* strain SMR10673 (▴) was not significantly different from the isogenic parent AB2497 (♦) except for at 300 minutes of thymine deprivation (p = 0.012) and *recAo lexA3*(Ind^−^) cells (SMR10676, ) were no more TLD sensitive than *lexA3*(Ind^−^) (SMR10669, ▪) or *sulA* (SMR10674, •) cells. Mean ± SEM of 3 experiments.
(0.13 MB TIF)
######
Click here for additional data file.
######
RusA expression partially reverses the hyper-TLD-sensitivity of Δ*ruvABC* cells. (A) The RusA resolvase, expressed in *rus-1* cells, partially restores TLD resistance to Δ*ruvABC* cells. In the Δ*ruvABC* (SMR10689, ▴) background, the *rus-1* allele (SMR10690, ) increased resistance to TLD, but in the *thy* ^−^ *rus* ^+^ parental background (SMR10687, ▪) activating RusA *via rus-1* mutation (SMR10686, ♦) did not have a significant effect. We cannot rule out the possibility that the lack of effect in Ruv^+^ cells is due to an inability of RusA to function when RuvABC are present (*in vitro*, RusA was inhibited by RuvA \[McGlynn, et al\]). Also, there is no reason to believe that *rus-1* creates more resolution capacity than in wild-type cells, such that restoration to Ruv^+^ levels might be expected. (B) Possible RusA effects on TLD are not masked by SulA. Similar results to those in (A) are obtained even when RusA is activated in the absence of SulA. RusA activation partially suppressed the TLD hypersensitivity of Δ*ruvABC sulA* cells (SMR10719, •, and SMR10718, ▴, respectively), but activating RusA in the absence of SulA (SMR10717, ▪) conferred no additional TLD-resistance over that conferred by *sulA* alone (SMR10716, ♦). This rules out the possibility that SulA expression might mask increased TLD-resistance of *rus-1* cells by preventing cell division. Means ± SEM of 3 experiments (A,B). \[McGlynn P, Lloyd RG, Marians KJ (2001) Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled. Proc Natl Acad Sci U S A 98: 8235-8240.\]
(0.20 MB TIF)
######
Click here for additional data file.
######
Topoisomerase III is not required for TLD. Cells lacking *topB* (SMR10672, ▪) are not significantly more resistant to TLD than their isogenic parental strain (AB2497; ♦), indicating that Topoisomerase III is not required for the RecQ-pathway of TLD in *E. coli*. Mean ± SEM of 3 experiments.
(0.09 MB TIF)
######
Click here for additional data file.
######
*E. coli* strains and plasmids used.
(0.26 MB DOC)
######
Click here for additional data file.
We thank M. Blankschien, R. Galhardo, J. Gibson, C. Gonzales, J. Halliday, C. Herman, D. Magner, and Z. Vaksman for helpful suggestions and comments and R.G. Lloyd for the gift of a *rus-1* strain.
The authors have declared that no competing interests exist.
Supported by National Institutes of Health (<http://www.nih.gov>) grants R01-GM64022 (PJH) and R01-CA85777 (SMR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[^1]: Conceived and designed the experiments: NCF SMR. Performed the experiments: NCF. Analyzed the data: NCF DB PJH PCH SMR. Contributed reagents/materials/analysis tools: DB PCH SMR. Wrote the paper: NCF PJH PCH SMR.
|
Autologous fat transfer to the cranio-maxillofacial region: updates and controversies.
Autologous fat grafts have gained popularity among Cranio-Maxillofacial surgeons within the past years. Most publications report favourable outcomes but lack quantifiable evidence of graft survival. To assess autologous fat transfer for facial asymmetry, and review the literature focusing on current indications, techniques, complications, fat survival and patient satisfaction. Nine patients presented facial asymmetry due to onchological resection, congenital anomaly, or craniofacial traumatism. A total of 11 autologous fat transfers were performed. Four procedures followed the Coleman technique; in the other seven procedures, fat centrifugation was obviated. No significant complications derived. Progressive volumetric decrease was evidenced for up to 6 months after surgery. A second procedure was performed in two patients. No clinical differences in cosmetic outcomes or graft survival were observed between centrifuged and non-centrifuged grafts. Patients reported high satisfaction. Facial recontouring with autologous fat transfer restores volumetric defects with high patient satisfaction. The scientific literature offers inconsistent results. The authors did not find clinical differences between centrifuged and non-centrifuged grafts. Volume gain may result from induced fibrosis, inflammation and native adipocyte growth or differentiation. The heightened interest in these procedures should instigate further investigation to refine surgical procedures and improve predictability. |
August 2013
I’ve started gathering a few quotes that I like about dogs and cats and their owners. I especially like this one by Mahatma Gandhi:
The greatness of a nation and its moral progress can be judged by the way its animals are treated.
Enjoy!
Dogs are not our whole life, but they make our lives whole.
Roger Caras
I have felt cats rubbing their faces against mine and touching my cheek with claws carefully sheathed. These things, to me, are expressions of love.
James Herriot
He who is cruel to animals becomes hard also in his dealings with men. We can judge the heart of a man by his treatment of animals.
Immanuel Kant
Dogs are our link to paradise. They don’t know evil or jealousy or discontent.
Milan Kundera
Some people talk to animals. Not many listen though. That’s the problem.
A.A. Milne (Winnie-the-Pooh)
I care not much for a man’s religion whose dog and cat are not the better for it.
Abraham Lincoln
Whoever said you can’t buy happiness forgot little puppies.
Gene Hill
How many legs does a dog have if you call the tail a leg? Four. Calling a tail a leg doesn’t make it a leg.
Abraham Lincoln
All of the animals except for man know that the principle business of life is to enjoy it.
Samuel Butler
If having a soul means being able to feel love and loyalty and gratitude, then animals are better off than a lot of humans.
James Herriot
The greatest pleasure of a dog is that you may make a fool of yourself with him and not only will he not scold you, but he will make a fool of himself, too. |
RIO DE JANEIRO, September 12. /TASS/. The Chinese national team has won 23 medals in the fourth day of the 2016 Summer Paralympic Games in Rio de Janeiro, leading in the unofficial team standing.
In total, the Chinese team has already won 39 golds, 30 silvers and 23 bronze medals. The second place is taken by the UK as its para athletes won 23 golds, 14 silvers and 19 bronze medals. The Ukrainian team is at the third spot with 49 medals (18-13-18) followed by the US team with 39 medals (13-15-11).
Russian para athletes do not take part in the 2016 Paralympics. The International Paralympic Committee (IPC) disqualified the Russian Paralympic Committee (RPC) and banned all Russian para athletes from taking part in the Rio Games. Attempts to challenge this decision at the Court of Arbitration for Sport (CAS) in Lausanne proved unsuccessful. IPC also denied 175 Russian para athletes individual participation in the Games in Brazil.
The IPC made this decision following the report of the World Anti-Doping Agency’s (WADA) Independent Commission chaired by Richard McLaren. On July 22, IPC opened a case against RPC after the McLaren report mentioned 35 Russian para athletes.
The 2016 Summer Paralympic Games in Rio de Janeiro will end on September 18. |
CLEVELAND — Winning four games in the NBA Finals isn’t easy. The Golden State Warriors only made it look that way.
That’s what happens when four All-Stars — Kevin Durant, Steph Curry, Klay Thompson and Draymond Green — and a collection of talented reserves play team basketball on offense and defense.
The Warriors are champions for the third time in four seasons, joining just three other NBA franchises to win at least three titles in four year. The talk of a Warriors dynasty can begin now.
Golden State completed the sweep — the ninth sweep in Finals history — against Cleveland with a 108-85 victory in Game 4 on Friday in what was possibly LeBron James’ last game as a Cavalier. For the second consecutive year, Kevin Durant was named Finals MVP.
CLOSE
The party was on for the Warriors after winning their third NBA championship in four years.
USA TODAY Sports
James had 23 points, eight assists and seven rebounds. It wasn’t his best game of the series — James also had six turnovers and five fouls — and there wasn’t much offense that worked against these Warriors, who led for much of the game. By the middle of the third quarter, the Warriors led 77-60.
It’s difficult to come back against Golden State, and the Cavs didn’t have a comeback in them after losing the first three games of the series.
Curry scored 20 of his team-high 37 points in the first half, Durant had a triple-double: 20 points, 12 rebounds and 10 assists, and he also had three blocks and one steal.
Asked before the game if he wanted to win a title in four games in Cleveland or in five games at home, Warriors coach Steve Kerr said, “They both sound awesome to me. I'll take either one.” |
It’s been nearly a year since a two-pump Electric Vehicle (EV) charging station was installed at the Hanover Mall and not only is the station used regularly, but National Grid representatives expect the charges to continue stacking up given charted trends and the expanding electric car market.
“It is being used right on pace with what we expected,” said John Gilbrook, transportation project manager for National Grid. “The mall station is up there with some of our more active stations.”
From the station’s installation on Feb. 10 of 2012 until Jan. 1 of this year, the station saw 135 uses with 350 total kilowatt-hours of electricity dispensed. Gilbrook said the first year was marked with ups and downs, which produced an eventual upward trend.
“We saw a huge spike at first,” he said. “Late July had a big hop as well and it is common to have a drop-off in the winter.”
Although the numbers fluctuated last year, Gilbrook said this year has shown a more consistent trend towards increased use. Moreover, he expects the numbers to come in much higher this year than during the station’s first year. He said the trend is clear when looking at the station’s use over last year.
“We can see the information and trend it,” he said. “Last year, over the last six months, frequency [of use] doubled.”
Aside from the upward trend in the numbers, Gilbrook pointed to one other factor which could signify a major uptick in station use.
“Last year, [the station use was] trending up with only two potential vehicles available,” said Gilbrook, referring to the Chevrolet Volt and the Nissan Leaf. “Several new models are being released this year.”
Gilbrook said he wouldn’t be surprised to see the numbers double this year and expects a significant increase. He referred to 2012 as the “reintroduction” of electric vehicles to the general public and said the opportunity for the station’s use to rise is going to come together.
“People are getting the cars and the prices [for the cars] are starting to trend down,” he said. “People will rediscover the station.”
Ed Callahan, general manager of the Hanover Mall, said there are consistent efforts on the part of mall leadership to bring people to the station.
“We consistently have a message on the Route 3 message board and on the website,” Callahan said. “We’re doing everything we can to bring people in.”
Mall leadership agreed to not only allow National Grid to install the station a year ago but also agreed to foot the bill for the first two years. With that in mind, Callahan said promotions for the station always include this fact: energy charges at the station are free.
Gilbrook said the mall stations have been considered a huge success and, of the 33 stations National Grid currently operates, the mall station is near the top of the list in terms of activity.
He said that, although the station has been busy, National Grid hasn’t had to do very much to maintain it. Also, with the mall under contract through the year, both sides agreed the deal had been mutually beneficial.
“It’s well worth the program,” Callahan said. “Our hope is consumers come into the mall while charging the car outside.”
[…] 3 Ways to Save Money on Printer InkVapors of the bathtub refinishing – how can his own shieldUSA: Hanover Mall sees success with electric vehicle charging stations Read more: Hanover Mall sees …Top 3 Ways to Save Money on Printer InkVapors of the bathtub refinishing – how can his own […] |
Engineering Library Closing
The Engineering Library is scheduled to close permanently on Friday, December 18th, 2015. Any changes and progress will be posted here.
Since this is an ongoing project, there will be times when the Engineering Library is noisy. Alternatively, quiet study and group study spaces are available in the Main, Math, and Business Libraries.
Beginning Fall Semester 2015, Engineering Course Reserves will be housed in and checked out from the Main Library Reserves (the Circulation Desk on the 1st floor).
Many Engineering books and the current issues of the print journals have been moved to the Main Library. Low-use materials will be moved to our Remote Storage facility. Daily runs to Remote Storage allow prompt retrieval of materials. Please see the library catalog for locations and availability. |
---
title: IHostPolicyManager::OnDefaultAction (Método)
ms.date: 03/30/2017
api_name:
- IHostPolicyManager.OnDefaultAction
api_location:
- mscoree.dll
api_type:
- COM
f1_keywords:
- IHostPolicyManager::OnDefaultAction
helpviewer_keywords:
- OnDefaultAction method [.NET Framework hosting]
- IHostPolicyManager::OnDefaultAction method [.NET Framework hosting]
ms.assetid: 071e73bd-4795-470f-9373-cfaef553b7f2
topic_type:
- apiref
ms.openlocfilehash: e6aa8cb814e509d310c2f5b5524e0fd6727fc43f
ms.sourcegitcommit: d223616e7e6fe2139079052e6fcbe25413fb9900
ms.translationtype: MT
ms.contentlocale: es-ES
ms.lasthandoff: 05/22/2020
ms.locfileid: "83804282"
---
# <a name="ihostpolicymanagerondefaultaction-method"></a>IHostPolicyManager::OnDefaultAction (Método)
Notifica al host que el Common Language Runtime (CLR) está a punto de realizar la acción predeterminada establecida por una llamada al método [ICLRPolicyManager:: SetDefaultAction (](iclrpolicymanager-setdefaultaction-method.md) en respuesta a una anulación o descarga del subproceso <xref:System.AppDomain> .
## <a name="syntax"></a>Sintaxis
```cpp
HRESULT OnDefaultAction (
[in] EClrOperation operation,
[in] EPolicyAction action
);
```
## <a name="parameters"></a>Parámetros
`operation`
de Uno de los valores de [EClrOperation](eclroperation-enumeration.md) , que indica el tipo de evento al que responde el CLR.
`action`
de Uno de los valores de [EPolicyAction](epolicyaction-enumeration.md) , que indica la acción que el CLR está llevando a cabo en respuesta al evento.
## <a name="return-value"></a>Valor devuelto
|HRESULT|Descripción|
|-------------|-----------------|
|S_OK|`OnDefaultAction`se devolvió correctamente.|
|HOST_E_CLRNOTAVAILABLE|CLR no se ha cargado en un proceso o CLR está en un estado en el que no puede ejecutar código administrado ni procesar la llamada. successfully|
|HOST_E_TIMEOUT|Se agotó el tiempo de espera de la llamada.|
|HOST_E_NOT_OWNER|El autor de la llamada no posee el bloqueo.|
|HOST_E_ABANDONED|Se canceló un evento mientras un subproceso o fibra bloqueados estaba esperando en él.|
|E_FAIL|Se produjo un error grave desconocido. Cuando un método devuelve E_FAIL, CLR ya no se puede usar en el proceso. Las llamadas subsiguientes a métodos de hospedaje devuelven HOST_E_CLRNOTAVAILABLE.|
## <a name="requirements"></a>Requisitos
**Plataformas:** Vea [Requisitos de sistema](../../get-started/system-requirements.md).
**Encabezado:** MSCorEE. h
**Biblioteca:** Se incluye como recurso en MSCorEE. dll
**.NET Framework versiones:**[!INCLUDE[net_current_v20plus](../../../../includes/net-current-v20plus-md.md)]
## <a name="see-also"></a>Consulte también
- [EClrOperation (Enumeración)](eclroperation-enumeration.md)
- [EPolicyAction (Enumeración)](epolicyaction-enumeration.md)
- [ICLRPolicyManager (Interfaz)](iclrpolicymanager-interface.md)
- [IHostPolicyManager (Interfaz)](ihostpolicymanager-interface.md)
|
Q:
History implementation in a simple shell program in c
i'm new and learning c on my own. i managed to write a simple shell code in c but my problem now is to have commands entered stored so that when entering a command "history" the recently entered commands are displayed on screen. a sample code or any materials to read to help me have my shell have history will be appreciated.
A:
There are many ways to achieve this. You could use the GNU readline library, which is really nice for that sort of thing. This would provide much more than a simple history command.
But just implementing a simple history would be easier. If you have a fixed limit for commands in the history a simple array would be sufficient, maybe like this:
static const char *history[HISTORY_MAX_SIZE];
static const unsigned history_count = 0;
void add_command_to_history( const char *command )
{
if (history_count < HISTORY_MAX_SIZE) {
history[history_count++] = strdup( command );
} else {
free( history[0] );
for (unsigned index = 1; index < HISTORY_MAX_SIZE; index++) {
history[index - 1] = history[index];
}
history[HISTORY_MAX_SIZE - 1] = strdup( command );
}
}
|
'use strict';
const get = require('lodash.get');
const utils = require('../../utils');
/*!
* Gather all indexes defined in the schema, including single nested,
* document arrays, and embedded discriminators.
*/
module.exports = function getIndexes(schema) {
let indexes = [];
const schemaStack = new WeakMap();
const indexTypes = schema.constructor.indexTypes;
const collectIndexes = function(schema, prefix) {
// Ignore infinitely nested schemas, if we've already seen this schema
// along this path there must be a cycle
if (schemaStack.has(schema)) {
return;
}
schemaStack.set(schema, true);
prefix = prefix || '';
const keys = Object.keys(schema.paths);
const length = keys.length;
for (let i = 0; i < length; ++i) {
const key = keys[i];
const path = schema.paths[key];
if (path.$isMongooseDocumentArray || path.$isSingleNested) {
if (get(path, 'options.excludeIndexes') !== true &&
get(path, 'schemaOptions.excludeIndexes') !== true) {
collectIndexes(path.schema, prefix + key + '.');
}
if (path.schema.discriminators != null) {
const discriminators = path.schema.discriminators;
const discriminatorKeys = Object.keys(discriminators);
for (const discriminatorKey of discriminatorKeys) {
collectIndexes(discriminators[discriminatorKey]._originalSchema,
prefix + key + '.');
}
}
// Retained to minimize risk of backwards breaking changes due to
// gh-6113
if (path.$isMongooseDocumentArray) {
continue;
}
}
const index = path._index || (path.caster && path.caster._index);
if (index !== false && index !== null && index !== undefined) {
const field = {};
const isObject = utils.isObject(index);
const options = isObject ? index : {};
const type = typeof index === 'string' ? index :
isObject ? index.type :
false;
if (type && indexTypes.indexOf(type) !== -1) {
field[prefix + key] = type;
} else if (options.text) {
field[prefix + key] = 'text';
delete options.text;
} else {
field[prefix + key] = 1;
}
delete options.type;
if (!('background' in options)) {
options.background = true;
}
indexes.push([field, options]);
}
}
schemaStack.delete(schema);
if (prefix) {
fixSubIndexPaths(schema, prefix);
} else {
schema._indexes.forEach(function(index) {
if (!('background' in index[1])) {
index[1].background = true;
}
});
indexes = indexes.concat(schema._indexes);
}
};
collectIndexes(schema);
return indexes;
/*!
* Checks for indexes added to subdocs using Schema.index().
* These indexes need their paths prefixed properly.
*
* schema._indexes = [ [indexObj, options], [indexObj, options] ..]
*/
function fixSubIndexPaths(schema, prefix) {
const subindexes = schema._indexes;
const len = subindexes.length;
for (let i = 0; i < len; ++i) {
const indexObj = subindexes[i][0];
const keys = Object.keys(indexObj);
const klen = keys.length;
const newindex = {};
// use forward iteration, order matters
for (let j = 0; j < klen; ++j) {
const key = keys[j];
newindex[prefix + key] = indexObj[key];
}
indexes.push([newindex, subindexes[i][1]]);
}
}
};
|
Evolution from primordial oligomeric repeats to modern coding sequences.
It seems as though nature was most innovative at the very beginning of life on this Earth a few billion years ago. For example, the functional competence of most, if not all, of the sugar-metabolizing enzymes was clearly established before the division of eukaryotes from prokaryotes eons ago, each critical active-site amino acid sequence being conserved ever since by bacteria as well as by mammals. I contend that this initial innovativeness was due to the first set of coding sequences being repeats of base oligomers, thus encoding polypeptide chains of various periodicities; such periodical polypeptide chains can easily acquire alpha-helical and beta-sheet-forming segments. In fact, the entire length of sugar-metabolizing enzymes is comprised of alternating alpha-helical and beta-sheet-forming segments. In the prebiotic (therefore nonenzymatic) replication of nucleic acids, what was in short supply was long templates, for there apparently was no inherent obstacle in copying of long templates, if such existed, in the presence of Zn2+. I submit that in this prebiotic condition, only those nucleotide oligomers that were internal doubles were automatically assured of progressive elongation to become long templates. For example, a decamer that was a pentameric repeat and its complementary sequence may pair unequally to initiate the next round of replication: first unit pairing with second, and a paired segment serving as a primer. As a consequence of this unequal pairing, decameric templates managed to become pentadecameric templates only after one round of replication, and this elongation process had no inherent limit. |
#!/bin/sh
# Copyright 2017-2020 Viktor Szakats <https://vsz.me/>
# See LICENSE.md
_BRANCH="${APPVEYOR_REPO_BRANCH}${TRAVIS_BRANCH}${CI_COMMIT_REF_NAME}${GITHUB_REF}${GIT_BRANCH}"
cat /etc/*-release
export _CCSUFFIX=''
[ "${CC}" = 'mingw-clang' ] && _optpkg="clang${_CCSUFFIX}"
[ "${_BRANCH#*dev*}" != "${_BRANCH}" ] && _optpkg="${_optpkg} autoconf automake libtool"
dpkg --add-architecture i386
apt-get -qq -o=Dpkg::Use-Pty=0 update
# shellcheck disable=SC2086
apt-get -qq -o=Dpkg::Use-Pty=0 install \
curl git gpg python3-pip make cmake \
libssl-dev \
gcc-mingw-w64 g++-mingw-w64 ${_optpkg} \
zip zstd time jq dos2unix wine64 wine32
./_build.sh
|
Scaling, genetic drift, and clonal interference in the extinction pattern of asexual population.
We investigate the dynamics of loss of favorable mutations in an asexual haploid population. In the current work, we consider homogeneous as well as spatially structured population models. We focus our analysis on statistical measurements of the probability distribution of the maximum population size N(sb) achieved by those mutations that have not reached fixation. Our results show a crossover behavior which demonstrates the occurrence of two evolutionary regimes. In the first regime, which takes place for small N(sb) , the probability distribution is described by a power law with characteristic exponent theta(d) =1.8 +/- 0.01. This power law is not influenced by the rate of beneficial mutations. The second regime, which occurs for intermediate to large values of N(sb), has a characteristic exponent theta(c) which increases as the rate of beneficial mutations grows. These results establish where genetic drift and clonal interference become the main underlying mechanism in the extinction of advantageous mutations. |
Conventionally, nanometer-sized particles (nanoparticles) are used in various industrial applications including energy applications.
For example, a method has been proposed in which an electrolyte membrane is obtained by applying and drying a composition prepared by blending inorganic nanoparticles having a proton conductive group bound thereto with a polymer and a solvent (see, for example, Patent Document 1 below). |
The effects of zilpaterol hydrochloride and shade on blood metabolites of finishing beef steers.
The effects of feeding zilpaterol hydrochloride (ZH) and shade were evaluated on blood metabolites in finishing beef steers ( = 480). Cattle were fed 0 or 8.33 mg/kg of diet DM ZH for 21 d with a 3- or 4-d withdrawal before harvest and were housed in open or shaded pens. Blood samples were collected the day before ZH was fed and on the day the cattle were shipped to the commercial abattoir. Lactate concentration was not different between cattle fed ZH in open or shaded pens ( = 0.12). Nonetheless, a tendency for a diet × time interaction was detected for lactate concentration ( = 0.09), in which it was greater in cattle fed the control diet in open pens before being fed ZH. Cortisol concentration was less before and after ZH was fed ( = 0.01). Glucose was greater for cattle fed the control diet than cattle fed ZH for 21 d ( = 0.03). Cattle fed in open vs. shaded pens did not differ in glucose concentration ( = 0.12), whereas glucose concentrations were greater before ZH was fed than after ( = 0.02). In contrast, plasma urea nitrogen (PUN) concentration was not different in response to diet ( = 0.24), housing type ( = 0.65), or before vs. after being fed ZH ( = 0.60). Lactate concentrations were not different across diet or shade treatments before ZH was fed, whereas after ZH, lactate concentrations were greater in control cattle than cattle fed ZH. Additionally, cortisol was less after feeding ZH. Glucose was greater before than after feeding ZH. |
<?xml version="1.0" encoding="UTF-8"?>
<project>
<property name="jnlp.title" value="JST TransformedShape" />
<property name="jnlp.Name" value="TransformedShape" />
<property name="jnlp.name" value="transformedshape" />
<property name="jnlp.codebase" value="https://ateraimemo.com/swing/transformedshape/" />
<property name="jnlp.homepage" value="https://ateraimemo.com/Swing/TransformedShape.html" />
</project>
|
More apps by Miniclip SA
Description
Hit the slopes for some serious shredding in the most fun snowboarding game ever created! To survive you will have to perform impossible stunts, dodge deadly saws and do incredible jumps in a world where gravity laws have been broken!
Combining outstanding physics, amazingly responsive controls and beautiful retina graphics, iStunt 2 is one of the most fun and original games on Windows 8.
With loads of exciting and devilishly tricky levels, iStunt 2 guarantees you many hours of fun.
Free your mind in this mind-blowing action-packed adventure pushing the adrenaline to the limit! |
Q:
AWS KMS Key Policy - How to add Principal
i've been trying to dynamically add Principal into the AWS KMS Policy statement via aws kms cli and I'm dead serious AWS docs are one of the worst docs ever!
anyone knows how to programmatically (using aws kms or any other alternative) to add Principal into the policy statement below ?
{
"Version": "2012-10-17",
"Id": "key-consolepolicy-3",
"Statement": [
{
"Sid": "Allow access for Key Administrators",
"Effect": "Allow",
"Principal": {
"AWS": [
// I need to programatically add the ARN role here
]
},
"Action": [
"kms:Create*",
"kms:Describe*",
"kms:Enable*",
"kms:List*",
"kms:Put*",
"kms:Update*",
"kms:Revoke*",
"kms:Disable*",
"kms:Get*",
"kms:Delete*",
"kms:TagResource",
"kms:UntagResource",
"kms:ScheduleKeyDeletion",
"kms:CancelKeyDeletion"
],
"Resource": "*"
},
{
"Sid": "Allow use of the key",
"Effect": "Allow",
"Principal": {
"AWS": [
// I need to programatically add the ARN role here
]
},
"Action": [
"kms:Encrypt",
"kms:Decrypt",
"kms:ReEncrypt*",
"kms:GenerateDataKey*",
"kms:DescribeKey"
],
"Resource": "*"
},
{
"Sid": "Allow attachment of persistent resources",
"Effect": "Allow",
"Principal": {
"AWS": [
// I need to programatically add the ARN role here
]
},
"Action": [
"kms:CreateGrant",
"kms:ListGrants",
"kms:RevokeGrant"
],
"Resource": "*",
"Condition": {
"Bool": {
"kms:GrantIsForAWSResource": "true"
}
}
}
]
}
A:
It is not possible to add new principals to resource policies using CLI.
You can however replace a key policy with a new version using put-key-policy command.
The best way to add principals dynamically is to generate key policies by leveraging code generation techniques. I usually use Python with Jinja2.
The generated policy can be attached to a CMK by using the following command:
aws kms put-key-policy \
--policy-name default \
--key-id <kms-key-id> \
--policy file://new_key_policy.json
|
import * as React from "react";
import PropTypes from "prop-types";
import FormGroupContainer from "#SRC/js/components/form/FormGroupContainer";
import FormRow from "#SRC/js/components/form/FormRow";
import SecretFormInput from "./SecretFormInput";
import SecretExposureTypeSelect from "./SecretExposureTypeSelect";
import EnvironmentVariableInput from "./EnvironmentVariableInput";
import SingleContainerFileInput from "./SingleContainerFileInput";
function valueRow(props) {
const { secretIndex, variableIndex, variable } = props;
let exposureValue;
exposureValue = null;
const valueName = `secrets.${secretIndex}.exposures.${variableIndex}.value`;
switch (variable.type) {
case "file":
exposureValue = (
<SingleContainerFileInput
name={valueName}
value={variable.value || ""}
showErrors={props.showErrors}
errors={props.errors}
/>
);
break;
case "envVar":
exposureValue = (
<EnvironmentVariableInput
name={valueName}
value={variable.value || ""}
showErrors={props.showErrors}
errors={props.errors}
/>
);
break;
}
return (
<FormRow>
<SecretExposureTypeSelect
secretIndex={secretIndex}
variableIndex={variableIndex}
type={variable.type}
/>
{exposureValue}
</FormRow>
);
}
const SingleContainerSecret = (props) => {
const {
secretIndex,
variableIndex,
secret,
secrets,
variable,
onRemoveItem,
} = props;
const secretKey = secret.key || `secret${secretIndex}`;
const removeTransaction =
secret.exposures.length > 1
? { value: variableIndex, path: `secrets.${secretIndex}.exposures` }
: { value: secretIndex, path: "secrets" };
const showValue =
(secret.value && secret.value.length > 0) ||
(variable.type &&
variable.type.length > 0 &&
variable.value &&
variable.value.length > 0);
return (
<FormGroupContainer
key={`${secretIndex}-${variableIndex}`}
onRemove={() => onRemoveItem(removeTransaction)}
>
<FormRow>
<SecretFormInput
secretKey={secretKey}
index={secretIndex}
value={secret.value}
secrets={secrets}
showErrors={props.showErrors}
errors={props.errors}
/>
</FormRow>
{showValue ? valueRow(props) : null}
</FormGroupContainer>
);
};
SingleContainerSecret.propTypes = {
secretIndex: PropTypes.number.isRequired,
variableIndex: PropTypes.number.isRequired,
secret: PropTypes.shape({
exposures: PropTypes.arrayOf(
PropTypes.shape({
type: PropTypes.oneOf(["", "file", "envVar"]),
value: PropTypes.string,
})
),
key: PropTypes.string,
value: PropTypes.string,
}),
secrets: PropTypes.arrayOf(PropTypes.string),
variable: PropTypes.shape({
type: PropTypes.oneOf(["", "file", "envVar"]),
value: PropTypes.string,
}),
showErrors: PropTypes.bool.isRequired,
errors: PropTypes.object,
onRemoveItem: PropTypes.func.isRequired,
};
export default SingleContainerSecret;
|
[[AC:FridgeBrilliance]]* I took a while to realize the meaning of the line "Rangers at the core" in the ''Power Rangers Zeo'' theme song - I thought it might have something to do with the Rangers in the cockpit of the Megazord, or perhaps them being inside their suits, but eventually I realized that what it actually meant was that even though the Rangers had different powers and equipment, they were, at the core, still the same Rangers continuing their fight against evil in the tradition they'd followed in the ''Mighty Morphin''' series.* "Mondo's Last Stand" featured Rocky displaying an inferiority complex. Whilst it never got brought up again in ''Zeo'', this would eventually lead to Rocky's GameBreakingInjury in ''Film/TurboAPowerRangersMovie''.* Prince Gasket attempts to brainwash Tommy into becoming the ruler of the Machine Empire in "King for a Day". In the episode immediately following it, "A Brief Mystery of Time", side effects from the brainwashing allows Tommy to recognize that the exact same day is looping repeatedly. It may be possible that Tommy's sudden boost in intelligence and memory skills (as seen in [[Series/PowerRangersDinoThunder Dino Thunder]]) are also due to these side effects.* Rita being pregnant with [[EnfantTerrible their child]] by the events of the season finale as per WordOfGod explains quite nicely why she and Zedd left at the end of the season, rather than resume conquering -- they were taking time off to raise their son.** In addition they may have gone off to another planet that wasn't as well defended to build their forces up so they'd have a better footing when they came back.* If one really thinks about it, the Zeo Rangers as described in the series canon are, literally, the most powerful rangers ''of all time''. The Zeo Crystal was an infinitely self-strengthening power source that would never stop growing more potent. The Zeo Ranger arsenal of weapons and zords was growing more complex and varied with every major episode. But this leads to a serious question that was never answered in any way. Why did they stop using the Zeo powers and move to the Turbo powers? Tommy at the least clearly retains the Red Zeonizer all the way up to Forever Red and it's unlikely he got rid of it afterword, unless that was the base he used to develop the Dino Gems. But that also begs the question of "Why mess with the Zeo Crystal and turn it into something clearly less powerful?" The only answer that logically comes to mind is that the Zeo Crystal is not as much of a penalty free power source as first believed. As we see with season team-ups and was Lampshaded in ''Series/PowerRangersRPM'', morphing tends to cause explosions. [[EarthShatteringKaboom Imagine what Zeo morphing would eventually do]]. That's right, the Zeo powers were pocket supernovas waiting to happen; since they were self-strengthening and ever-growing, it's quite possible that they could eventually reach a stage where the Rangers' bodies are no longer able to tolerate them, much like with Jason and the Gold Ranger powers.** Keep in mind that these crystals were split from one single crystal... Imagine the power ''that'' would cause if they were still united.** It's also possible that when Katherine's morph failed in the Turbo movie, the whole Zeo powerset was damaged or incomplete, and it just couldn't be repaired before the old team left.** Another possible explanation is the Zeo Cyrstals are the power source for the Turbo Powers. This would answer the question of why the Turbo Powers are supposed to be more powerful then the Zeo powers. They're not, they're just more efficient, using less power for the same effect.** One thing to consider: It's stated that the morphing grid gets its overall power from the balance between good and evil. Perhaps they had to stop using the Zeo powers or risk destabilizing the whole morphing grid due to a shift in the balance. This also explains why each season of rangers stops being rangers when their evil bad guys get defeated, and when the monsters get bigger and badder, so do the rangers. A subtle, justified example of Mid-season upgrades, available for every season ever.* It didn't make much sense why, after the Command Centre blew up, the rangers found the Zeo Crystal abandoned, and why in the first half of Zeo, Goldar and Rito had lost their memories. But with some further analysis, it makes sense that it was because of the forcefield around the Zeo Crystal.** It had been established in Season 3 of MMPR that a forcefield had been placed around the Zeo Crystal by the inhabitants of M51, to destroy anything evil that comes near it. It had been believed that it would destroy anyone who was evil, or who was ONCE evil, but when Tommy, who had been the evil Green Ranger, took the crystal, it was clear that the forcefield did have an effect on him, but merely removed any remaining evil that was in his system.** It was also made clear that this forcefield remained around the Zeo Crystal even after it was removed by Tommy from underneath Zedd and Rita's palace, because Tommy used it to cleanse all the evil from Kat.** So when Goldar and Rito broke into the Command Centre and stole the Zeo Crystal, the forcefield acted on them, removing all the evil from them, including their memories of ever having done any evil. This explains why Goldar lost his wings (they were given as a present by Zedd, presumably made of pure evil), and also explains why Goldar and Rito never had any desire to do anything evil while they were in this state, as all the evil had been cleansed from them.** Unfortunately, Zedd and Rita caught up with them, and in an even more prominent form of Fridge Brilliance, it makes sense why Zordon's Wave turned Goldar to sand when he wasn't destroyed by the Zeo Crystal's forcefield. It's because when Zedd and Rita found Goldar and Rito, Zedd agreed to restore Goldar's memory, wings and sword, on the condition that Goldar never do anything good again. So, in making this agreement, Goldar literally stripped himself of any remaining good that was in his system, which is why when Zordon's Wave hit, there was nothing to save.* There wasn't really a sufficient explanation for Billy's mysterious absences in some episodes (before the character officially left). However, there is a very popular fan theory which posits that during those mysterious absences, he was actually developing the Turbo powers, weapons, morphers, and Zords. ** As to why the Turbo powers didn't really match up to the superiority of the Zeo Crystals -- maybe Billy ran out of time and therefore couldn't finish them or fix them due to his rapid ageing and having no choice but to go to Aquitar to get treatment. But again, it seemed like they were more of backups as opposed to true replacements to the Zeo Crystals.*** The last sentence here makes perfect sense: Billy has seen the Power Coins and Ninja Coins destroyed and suffered with his teammates to get replacement powers. Billy was smart enough to consider that the Zeo Powers could suffer the same fate.*** There actually WAS one line given as to why Billy was frequently missing during the Gold Ranger build up: He was building new vehicles for the team. That were never used or shown, or even mentioned after that line. The line was given by Old Billy.* It took me a while to understand Tommy's Tearjerker narration at the beginning of the first episode: "Little did we know how soon our world as we knew it would come crashing down. And our lives as Power Rangers were about to an end.", when in the next episode, they got new Zeo powers to fight King Mondo. I now realize that Tommy was right: Their world (the Command Centre, which like a second home to them) ''did'' come crashing down (via implosion device), and their lives as ''Mighty Morphin'' Power Rangers ''did'' come to an end (the Zeo Crystal gave them new powers instead of restoring their old powers). Even though they got a new power base and stronger powers, they are going to miss their old ones greatly. |
Greed, in my view, is like a sickness. It is like an addiction.
Say what you will, this presidential race has been, at the very least, an intensely interesting affair. Of course, there is the debacle of the Republican primaries; but those are mainly interesting in the same way that a car accident is interesting—you can’t help but rubberneck, even if you’re a bit disgusted with yourself for doing so. Much more engrossing, for me, has been the rise of Bernie Sanders, something which seems to have surprised everybody, even Sanders himself.I should admit right off the bat that I like Sanders; but I’m going to try my best in this review, however ineffectual that may be, to maintain some skeptical distance. I suggest you do the same for me.This book was first released in 2011. As its back cover will tell you, it is a transcription of Sanders’s long filibuster speech, delivered on December 10, 2010, on the eve of a deal, brokered by Obama and the Republicans that extended the Bush tax cuts on the super-rich, among other things. The whole speech is on YouTube, if you’re interested, all eight-and-a-half hours. This book is just a transcription of the speech.As Sanders warns in the beginning, this speech is quite repetitive, deliberately so; he expected viewers to turn in for only a few minutes on CSPAN, and not to stick through the whole thing. This redundancy is probably the worst aspect of this book. I don’t see why it couldn’t have been edited and neatened up. Even so, despite the recurring sections there is just enough new material scattered throughout the speech to keep the reader’s interest—or at least to keep mine.The subject of Sanders’s speech is most immediately the financial legislation in question and its shortcomings; but Sanders uses this as a jumping-off point to discuss what he sees as the pressing and dire problems facing the United States. Sanders is a remarkably consistent politician, and you will see him focused on the same issues, often using the same language that he’s employed during his presidential bid this year.The core of Sanders’s speech (like his campaign) is income inequality. Truly, the level of income inequality in the United States is staggering and hard to wrap one’s head around. Sanders does his best by hammering his listeners with statistic after statistic, numbers so big and so stark that they baffle the mind. After about five repetitions, they start to sink in; and after ten, your own moral outrage begins to simmer along with Sanders’s.It’s worthwhile to compare Sanders’s speaking style with that of Obama. Obama is, I think, certainly the stronger and more versatile speaker. He is capable of sharp wit, of passionate outrage, of good-natured jocularity. But where I think he most excels, and what was his biggest asset when he ran for president, was his ability to inspire. He does this mainly through the use of anecdotes. He makes his speeches very personal; the way he speaks of nurses and teachers and firefighters is not at all condescending or pandering, but really makes you feel he knows them, knows them personally and intimately.Sanders’s approach is quite different. For one, he is certainly more narrow in ability and focus. What Sanders conveys with his voice, with his words, with his thrashing body language and unkempt appearance is moral outrage. Indeed, I find something Biblical about Sanders’s speeches. He shouts until his voice cracks, until he is absolutely hoarse, detailing in a long, grotesque list how unfair and unequal our society has become. You don’t so much feel inspired as galvanized, jolted with a mixture of desperation and indignation.To create these feelings, he does not tell stories, but recites facts. It’s astonishingly simple, really; he just has to read off a long list of ways that America is doing poorly—our shamefully huge prison population, our crumbling infrastructure, our soaring college tuition and health costs, and of course the absurd level of wealth and income inequality.To speak personally for a moment, I remember the moment when his message really hit me. First I have to tell you that among my friends it’s almost a cliché to talk about how much better life in Europe is than in America. In fact, one of my friends, after a long vacation in Europe, said to me: “It’s honest really depressing how much better life is over there.” And it’s not just us; a lot of people have these thoughts. You get used to thinking of the United States as poorer, less prosperous, more benighted than places like Germany and Denmark.Anyhow, one day when I was listening to a Sanders speech, he said: “Some of you may not know this, it’s easy to forget it sometimes, but the United States is the wealthiest country in the history of the world.” This really made something click within me. I’d gotten so used to thinking of the United States as poor and inferior—a place where you can’t afford to go to school or to get sick—that I was shocked to be reminded that we have more wealth in this country than anywhere else. This, I think, is what’s so effective and compelling about Sanders: you feel you’re being snapped back into reality.So this is what I like about Sanders. What I dislike is his tendency to demonize the rich. He speaks of the super-wealthy as if they’re a bunch of nefarious, mustache-twirling, conscience-less devils trying to enslave the rest of the world. I just don’t see this rhetoric as necessary. First, everybody pursues their own interests—the poor, the middle-class, and the wealthy—so I don’t see any reason to act morally superior. And second, I simply don’t think it’s true, strictly speaking, that the economy is hurting solely because of the greed of the wealthy. Yes, I am sure that a lot of stupid, selfish greed contributed to our economic situation today; but the economy is bloody complicated; it’s not a moral playground, but a vast system that even the best minds have failed to understand.The cynical side of me sees this finger-wagging as just the sort of us-versus-them rhetoric that politicians use to gain power. But I do think, to be honest, that Sanders is not capable of something so underhanded. He’s been ragging on the rich for his whole career; it’s only recently that this strategy has started to pay off. And besides, I do think his larger point is not only valid, but vitally important—namely, that the influence of the wealthy class on politics, with campaign contributions and corporate lobbyists, has to be curtailed in order to preserve a working democracy.As for Sanders’s political vision, I can’t deny that it appeals to me deeply. In a nutshell, Sanders’s vision is to make the United States more like Europe, with cheap college education, with free healthcare, with a strong social safety net, with higher taxes on the rich, with stronger infrastructure, and with a great deal more economic regulation. For the truth is, life in European countries often sounds too good to be true to young Americans.Let me give you some concrete examples. Just the other day, I was in a car with a Spaniard. We got on the topic of vacation. She said she has a friend in the States who only gets 8 vacation days per year. "Is that typical?" she asks. Yes, we tell her. In my last job I got 15, but my girlfriend only had 5. Our driver is aghast. “I get thirty,” she says, “and I think that’s too few!"Here’s another example, with regards to infrastructure. A monthly subway card in New York City costs $117; the equivalent here in Madrid costs 55€, and only 20€ if you’re 25 or under. What’s more, the subways in New York are overcrowded and dirty, with constant delays due to lines being shut down for repair; whereas the metro here is clean and always has good service. I’ve even seen a video—here’s the link —which shows some of the machines being used today in the NYC subway system. They were built in the 1930s, if you can believe that.And this is not to mention the looks of shocked disbelief on the faces of Europeans when I tell them just how expensive college and healthcare are in the United States. So really, when you’re reminded that your country—the place with the slow and expensive and obsolescent trains, where every young person is several thousand dollars in debt from college, and where we still have high levels of unemployment and child poverty—is the richest country in all of history, it hits a nerve.And while I don’t like demonizing the rich, I do agree that the rich in the U.S. live in a world apart. This was illustrated for me last year when, by chance, I found myself looking through a yacht magazine. Have you ever seen one? It was unbelievable, and I mean I honestly couldn’t believe what I saw. These ships were just huge. Inside they had bowling alleys, movie theaters; they had personal gyms and helicopter landing pads; they had living rooms created by world-famous interior designers. The boats were, I admit, super cool. But what does it say about our society that there are people who can afford things like this when on every corner is somebody on the street?This review has already dragged on too long, and still there is so much to be said about Sanders and what his campaign means. The pundits dismissed him before he began, and even now, even in some liberal publications, he’s discussed—discussed all too rarely—with a kind of guarded skepticism. Ironically enough, the only thing that seems to get the journalists' attention is how much money Sanders is managing to make without accepting donations from corporations—which says quite a lot about the American media. Some have said that the media is ignoring him because of their corporate overlords. But in general I don’t think conspiracy theories are necessary. The news media in the U.S. is not evil, it’s just shamefully bad.Almost every prediction I’ve heard about this election cycle has been shown to be foolish, so perhaps I should demure. But let me give it a go. Even if he doesn’t quite win, I think Sanders will surprise everyone on election day by how close he gets. And even if he loses, I predict that his presidential run will serve a similar function as Barry Goldwater’s did, back in the 60s, giving impetus and direction to a new political movement in the country. In other words, even if he loses the political battle, I think he’s already won the battle of ideas. And who knows? Maybe he’ll win the political battle, too. |
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