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Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)‐based motor. Dynactin binds both dynein and MTs via its p150Glued subunit, but little is known about the ‘pointed‐end complex’ that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin‐dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo‐like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore–MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left‐handed β‐helix domain, with the phosphorylation site located within a disordered, C‐terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.
During interphase, dynein, a microtubule (MT)‐associated, minus end‐directed motor, and dynactin, its ≈1 MDa partner complex, function primarily to move membrane‐bound organelles and other intracellular components. During mitosis, the dynein/dynactin assembly plays a completely different set of roles. It is essential for spindle pole focusing, helps chromosomes engage with and move on spindle MTs, and is believed to facilitate spindle checkpoint silencing by transporting regulators away from kinetochores (Echeverri et al, 1996; Howell et al, 2000; Yang et al, 2007; Bader and Vaughan, 2010).
Dynactin comprises 11 different polypeptide components, each present in one or more copies, which are organized into three structural/functional domains. The structural core of dynactin is a short copolymer of the actin family proteins Arp1, Arp11 and actin, plus the actin capping protein, CP (CapZ). This central structure is flanked by distinct structures at each end. The shoulder/sidearm assembly (p150Glued, dynamitin and p24 subunits), which envelops the ‘barbed’ end of the Arp polymer (Imai et al, manuscript in preparation), binds to dynein and MTs. Located at the opposite, ‘pointed’ end is the p62 subunit and the p27/ p25 heterodimer (Schafer et al, 1994; Eckley et al, 1999). The Arp polymer and the pointed end complex (p27/p25 plus p62 and Arp11) are assumed to participate in cargo binding (Schroer and Cheong, 2012) and specification (Yeh et al, 2012) although the molecular details remain undefined.
Of all dynactin subunits, those that comprise the pointed end complex are most mysterious. The p62, p27 and p25 components appear to be dispensable for minimal dynactin function as they are absent in yeast. The p62 subunit, which is required for dynactin stability, contains a predicted Zn2+ binding, RING/LIM‐like motif near the N‐terminus and, along with its closely associated subunit Arp11, contributes to dynactin targeting to the nuclear envelope (Yeh et al, 2012). The two smallest dynactin subunits, p27 and p25, are important dynactin and dynein binding to endomembranes (Zhang et al, 2011; Yeh et al, 2012). A mitotic role for the p27/p25 heterodimer has not been explored.
In this study, we investigated the function of dynactin's p27 component in mitosis. Using RNA interference (RNAi), we determined that p27 and p25, which are co‐depleted by small‐interfering RNA (siRNAs) to either, are required for normal mitotic timing. Cells depleted of p27 and p25 show defects in kinetochore/MT interactions and chromosome alignment, but unlike cells lacking other dynactin subunits that also exhibit these behaviours, they show accelerated progression through mitosis. The mitotic function of p27 depends upon cyclin‐dependent kinase 1 (Cdk1) phosphorylation at Thr186, which generates a binding site for the important mitotic kinase, polo kinase 1 (Plk1). Depletion of p27 and p25 does not interfere with formation of mitotic spindles, but Plk1 levels are significantly reduced at kinetochores. In conjunction with our functional work, we solved the crystal structure of human recombinant p27 at 2.2 Å resolution. The molecular model revealed a unique left‐handed β‐helix domain, the first such tertiary fold to be characterized in a vertebrate protein, and showed that the Cdk1 phosphorylation site is in the unstructured, C‐terminal segment. Our work reveals that dynactin serves as a binding platform for dynein and Plk1 at kinetochores that governs passage through the spindle assembly checkpoint.
Dynactin p27 and p25 are required for normal mitotic timing
To investigate whether p27 and p25 might play roles in mitosis, we removed them from dynactin using RNAi. Treatment of Cos7 fibroblast cells with p27 siRNAs results in depletion of both p27 and p25, but leaves the rest of the dynactin complex intact (Yeh et al, 2012). To compare the effects of p27/p25 depletion with general impairment of dynactin function, we performed parallel experiments in which p150Glued, the dynein‐binding component of dynactin, was depleted via RNAi. As a final control, we evaluated the effect of depleting Arp11, which leads to the loss of the entire dynactin molecule (Yeh et al, 2012). As expected, depletion of either p150Glued or Arp11 resulted in profound spindle defects (Yeh et al, 2012; Figure 6C, MTs), pseudoprometaphase arrest and an increase in mitotic index (Figure 1A and B). By contrast, depletion of p27 and p25 yielded cells whose spindle structure appeared normal (Yeh et al, 2012; Figure 6C). We were surprised to find that the mitotic index of the p27/p25‐depleted population was reduced (3.1 versus 5.6% for controls; Figure 1B) due to a reduction in the proportion of Cos7 cells at all stages of mitosis (Figure 1A). Closer inspection of mitotic cells revealed lagging chromosomes and misaligned metaphase plates, suggesting chromosome congression defects (Figure 1C and D). Expression of siRNA‐resistant, mouse p27, which rescues both p27 and p25 (Yeh et al, 2012) and yields their full incorporation into dynactin (Supplementary Figure 1A), restored the mitotic index to normal (Figure 1A and B), indicating that the defects were not due to off‐target effects of the p27 siRNAs.
The combination of chromosome alignment defects and a reduced number of cells in mitosis suggests that cells are proceeding through mitosis more rapidly than normal, due to lack of activation of the spindle assembly checkpoint in the face of defects (i.e., checkpoint bypass) (Fava et al, 2011). This behaviour is markedly different from the pseudoprometaphase arrest and prolonged checkpoint activation that is typical of dynein or dynactin perturbation (Echeverri et al, 1996; Howell et al, 2000). Live imaging, either via phase contrast (Supplementary Movies 7 and 8) or of cells expressing mCherry‐tagged histone 2B, revealed that cells depleted of p27 and p25 spend significantly less time in the interval between nuclear envelope breakdown (NEBD) and anaphase (28.1±2.2 min versus 41.7±2.4 min for controls; Figure 1E and 1F; Supplementary Movies 1 and 2). Instead of pausing in metaphase for a few minutes, the cells initiated anaphase as soon as the bulk of chromosomes had converged into a single mass even though uncongressed chromosomes were still observed (Figure 1E). By contrast, cells expressing siRNA‐resistant mouse p27 remained in metaphase for a few minutes and exhibited a normal interval between NEBD and anaphase (42±2.3 min, Figure 1F; Supplementary Movies 3 and 9). The lack of a ‘wait‐anaphase’ period and the presence of uncongressed chromosomes strongly suggest that cells depleted of p27 and p25 do not execute the spindle assembly checkpoint effectively. Treatment of p27/p25‐depleted cells with nocodazole was found to result in mitotic arrest (mitotic index 64.5±4.2 versus 65.4±4.2% for control; Supplementary Figure 2), indicating that the checkpoint was operational, however, it was not activated in cells depleted of p27 and p25. Chromosome alignment in cells depleted of Arp11 or p150Glued was aberrant and mitosis was highly prolonged (Supplementary Movies 5 and 6), in agreement with our analysis of fixed cells (Figure 1A–D).
Efficient and accurate chromosome congression depends upon the presence of both dynein and dynactin at kinetochores (Starr et al, 1998; Yang et al, 2007). Recruitment involves an interaction of dynactin's dynamitin component with the RZZ (Rod/ZW10/Zwilch) complex (Starr et al, 1998) plus a number of other interactions. p27/p25 is required for dynactin association with endosomes and dynein‐based endosome movement (Zhang et al, 2011; Yeh et al, 2012), but whether these two dynactin components also contribute to kinetochore binding and dynein recruitment to kinetochores is unknown. To explore these questions, we quantified the levels of dynactin's p150Glued, Arp1 and p27 components, plus dynein (dynein intermediate chain, DIC) in cells treated with dynactin subunit siRNAs (Figure 2). Cells depleted of either p150Glued or the entire dynactin molecule (via Arp11 depletion) showed a significant reduction in kinetochore‐associated dynactin. Depletion of p27 and p25, by contrast, left kinetochore dynactin in place. Free, exogenous p27 expressed at low levels (via the siRNA‐resistant p27 rescue vector) in cells depleted of p150Glued or Arp11 did not target to kinetochores (Supplementary Figure 3), indicating that p27 does not bind this site unless it is incorporated into dynactin. Depletion of p150Glued or Arp11 also caused significant reduction in dynein at kinetochores, whereas p27 depletion did not (Figure 2A and B). This result agrees with previous reports that dynein binding to kinetochores involves dynactin's p150Glued component (Echeverri et al, 1996; Whyte et al, 2008). Levels of the kinetochore components ZW10, Bub1, BubR1, Bub3, MPS1 and the dynein/dynactin interactor, Spindly (Liu et al, 2006; Griffis et al, 2007; Liang et al, 2007; Whyte et al, 2008; Chan et al, 2009; Gassmann et al, 2010) were unchanged under all three depletion conditions (Figure 2C; Supplementary Figure 4) confirming that none of these proteins depend upon dynein or dynactin for kinetochore binding and verifying that kinetochore structure was not grossly perturbed. This analysis demonstrates that kinetochores in cells depleted of p27 and p25 still contain normal steady‐state levels of both dynein and dynactin, with the only notable difference being the absence of p27 and p25 from dynactin. This system thus permits study of the function of p27 and p25 at kinetochores selectively, without concerns about possible non‐specific or downstream consequences of loss of dynactin or dynein.
Depletion of p27 and p25 from kinetochore dynactin appears to interfere with normal execution of the spindle assembly checkpoint, a phenomenon of great interest in which many molecules have been implicated. Among these are the mitotic checkpoint complex (MCC) and the mitotic regulatory kinase, Plk1. Both dynactin and dynein are reported substrates of Plk1 and two dynactin components, p150Glued and dynamitin, are components of the polo‐binding interactome, suggesting an interaction with Plk1 (Lowery et al, 2007; Li et al, 2010; Bader et al, 2011). Whether the dynactin/Plk1 interaction is direct, and if so, which dynactin component is involved, has not been explored.
Dynactin p27 is phosphorylated by Cdk1 and binds Plk1
Plk1 binding commonly requires priming phosphorylation by Cdk1. In search of regulatory mechanisms that might involve p27 or p25 phosphorylation, we searched their amino‐acid sequences for predicted phosphorylation motifs. The human p27 sequence contains a single consensus Cdk1 phosphorylation site, Thr186, that is conserved among vertebrates (Figure 3A). Phosphorylation at this site creates a consensus Plk1 binding site (Elia et al, 2003). Although p25 is somewhat related to p27, no p25 species we examined contained a Cdk1 consensus sequence or a predicted polo‐binding motif. To verify that p27 could serve as a substrate for Cdk1, we performed in vitro phosphorylation using purified bovine and embryonic chick brain dynactin (Figure 3B). p27 was found to be a major target, along with p150Glued. We then used point mutagenesis to verify that p27 Thr186 was the Cdk1 phosphorylation site. For this work, we utilized purified, recombinant p27/p25 heterodimers to mimic p27 in its native context. Wild‐type p27, along with two variants, T186A and S184A (a MAPKKK consensus site), prepared as dimers with p25, were evaluated as Cdk1 substrates in vitro. Both wild‐type p27 and the S184A variant were phosphorylated, but the T186A variant was not (Figure 3C). The p25 component of the p27/p25 heterodimer was not phosphorylated.
Source Data for Figure 3B
Source Data for Figure 3C
Source Data for Figure 3D‐G1
Source Data for Figure 3D ‐ mitosis
Source Data for Figure 3E
Source Data for Figure 3F
To evaluate p27 phosphorylation in vivo, we performed two‐dimensional immunoblotting on lysates of Cos7 cells synchronized in G1 or M phase (Figure 3D). p27 was seen to be phosphorylated in M phase, but not in G1. When expressed in cells, E‐GFP‐tagged wild‐type p27 also underwent mitotic phosphorylation, whereas the T186A mutant did not (Figure 3E). Taken together, these results verify that p27 is mitotically phosphorylated at Thr186 in vivo.
Phospho‐Thr186 is part of a consensus binding site for the master regulator, Plk1, that acts broadly during the process of cell division, from mitotic entry through cytokinesis. It is found at multiple sites in the spindle, including spindle poles, kinetochores, the spindle midzone and the intracellular cytokinetic bridge, all sites where dynactin and dynein also accumulate. Plk1 is recruited to these sites via a number of different binding partners (Petronczki et al, 2008). Dynactin, more specifically its p27 subunit, may be a previously unidentified Plk1 targeting factor. To demonstrate that p27 can bind Plk1 directly, we performed in vitro experiments using GST‐tagged polo‐binding domain (PBD; AA 365–603 of human Plk1) and purified recombinant p27/p25 heterodimers that had been phosphorylated by purified Cdk1 in vitro (Figure 3F). Phospho‐p27 bound to immobilized wild‐type PBD but not a PBD variant that is attenuated for binding (Elia et al, 2003). This interaction was dependent on Cdk1 phosphorylation of p27 because minimal binding was observed to the non‐phosphorylated control.
The crystal structure of dynactin p27
To gain further insight into structure–function relationships in the p27/p25 heterodimer and to elucidate the structural context of the p27 phosphorylation site, we embarked on a crystallographic study of these proteins. Amino‐acid sequence‐based analysis of p27 and p25 using 3D‐Jury (Ginalski et al, 2003; Kajan and Rychlewski, 2007) suggested that both proteins contain an unusual left‐handed parallel β‐helix (LβH) domain, in agreement with earlier hypotheses (Parisi et al, 2004). Co‐expression of the two proteins in E. coli using a bi‐cistronic vector resulted in a stable, soluble complex that was used in the in vitro phosphorylation and PBD binding experiments described above. Unfortunately, the complex did not yield crystals suitable for structural characterization despite extensive effort. Interestingly, recombinant isolated human p27, but not p25 (which is unstable in the absence of p27), could also be expressed in soluble form in E. coli. Starting with this protein, we obtained a good‐quality atomic model using diffraction data to 2.2 Å resolution (see Supplementary data, Materials and methods; Table I). The asymmetric unit contains two independent monomers, each with interpretable electron density for residues Ser8 to Thr70 and Lys80 to Gln159, accounting for 74% of the polypeptide chain.
The ordered domain of p27 visualized by the crystal structure (Figure 4) shows the predicted LβH tertiary fold, making it the first vertebrate protein experimentally shown to display this architecture. Like its right‐handed counterpart, this fold contains a β‐helical coil formed by repeating units of three β‐strands (‘rungs’) separated by turns or loops where the polypeptide chain takes a 300° turn (Choi et al, 2008). The rungs pack against each other by main‐chain to main‐chain hydrogen bonds parallel to the helical axis. The p27 LβH domain is a type I β‐helix, containing seven rungs, with six residues per strand (i.e., 18 residues per rung). The first four amino acids in each rung display the canonical β‐sheet secondary structure, with the side chains of the first and third pointing into the interior of the protein, and the side chains of the second and fourth pointing into the solvent. The fifth and sixth residues in each rung make up the turn. Thus, the molecule has a distinctly triangular cross section, with three faces (A, B and C) separated by edges (Figure 4A, referred to as ‘Turns’ in Figure 4C).
The three faces of the p27 LβH domain are characterized by different types of exposed amino acids. In each rung, the second residue is positioned in the centre and thus contributes significantly to the physicochemical properties of that respective face. Faces A and C show a variety of amino acids in position 2, but face B has exclusively hydrophobic amino acids at this position (Figure 4C). This is likely to have functional significance, as discussed below. The edge between the A and B faces (Turn 1) is made up of regular, two‐residue long turns, as is the edge between the B and C faces (Turn 2). The turns that make up the edge between the C and A faces (Turn 3) are irregular and include two longer loops, one comprising residues 43–47 and a longer one comprising residues 64–81, most of which are not resolved in the electron density map.
Beyond Gln159, the last residue visible in the electron density, secondary structure prediction suggests the presence of a short disordered sequence, a region with high propensity to form an α‐helix (residues 170–185; a similar α‐helix is predicted in p25), and a disordered C‐terminus. The Cdk1 phosphorylation site, Thr186, is the first residue in the disordered segment, suggesting high accessibility to kinase.
A model for p25 structure and mechanism of heterodimerization
The shared predicted LβH tertiary fold of p27 and p25 allowed us to propose a model of the p25 LβH domain based on the p27 crystal structure. The model (Figure 5) shows that the C face of the p25 LβH domain also contains a ridge of exposed hydrophobic residues, making this face structurally similar to the B face of p27. This raises the possibility that the p27 and p25 proteins utilize their respective B and C faces to interact within the heterodimer. This hypothesis is corroborated by the fact that within the asymmetric unit of the p27 crystal, two independent monomers form an intimate homotypic interface between the two B faces, burying ∼1300 Å2 of solvent exposed surface (Figure 5A). In this geometry (irrespective of orientation), the loops in Turn 3 of both proteins are near each other, raising the possibility that this part of the structure contributes to interactions with other dynactin components or extrinsic binding partners.
Cdk1‐phosphorylated p27 targets Plk1 to kinetochores
Our imaging analysis indicates that loss of the p27/p25 heterodimer from kinetochore‐associated dynactin allows cells to enter anaphase more quickly without altering steady‐state levels of many kinetochore‐associated molecules. To explore the possibility that the accelerated mitotic progression might be due to impaired Plk1 targeting to kinetochores, we examined Plk1 localization in mitotic cells. The amount of Plk1 present at spindle poles and in intracellular cytokinetic bridges was unchanged in cells depleted of any of the dynactin subunits (Figure 6A–C) indicating that Plk1 recruitment to these sites depends primarily upon binding partners other than the dynactin p27 component. However, cells from which kinetochore dynactin was removed completely (by p150Glued or Arp11 depletion) or stripped of p27/p25 (by p27 depletion) showed a significant reduction in Plk1 at prometaphase kinetochores (Figure 6D and F). These results indicate that dynactin, specifically its p27 and p25 subunits, is required for full Plk1 targeting to kinetochores. The residual Plk1 present at kinetochores in p27/p25‐depleted cells is most likely recruited via other known polo‐binding proteins such as Bub1, BubR1, PBIP1 and/or NudC, none of which are reduced as a result of p27/p25 depletion (Figure 2C; Supplementary Figure 4).
To verify that dynactin‐mediated recruitment of Plk1 to kinetochores was functionally relevant, we used the 3F3/2 antibody to assess the levels of phosphorylated Plk1 substrates. This antibody recognizes a phosphoepitope generated by Plk1 phosphorylation on a number of spindle‐associated proteins (Ahonen et al, 2005; Wong and Fang, 2005). Kinetochore labelling with 3F3/2 has been shown to be reduced when Plk1 is depleted or inhibited (Ahonen et al, 2005; Wong and Fang, 2005). 3F3/2 labelling was significantly reduced at kinetochores of cells treated with dynactin subunit siRNAs, verifying that Plk1 activity is attenuated (Figure 6D and G). Depletion of Spindly, which causes both dynein and dynactin to be lost from kinetochores (Griffis et al, 2007; Chan et al, 2009; Barisic et al, 2010; Gassmann et al, 2010) and thus removes p27, also resulted in dramatically reduced Plk1 binding to prometaphase kinetochores (Supplementary Figure 5). The reduction in Plk1 and 3F3/2 staining seen in cells depleted of dynactin subunits reflected the intrinsic composition of the kinetochore because similar results were observed in nocodazole‐treated cells in which MT‐dependent tension has been eliminated (Figure 6E, H and I).
These findings provide strong evidence that dynactin targets Plk1 to mitotic kinetochores via phospho‐Thr186 in the p27/p25 heterodimer. As a final test of this hypothesis, we evaluated whether the non‐phosphorylatable p27 variant, T186A, expressed at roughly the same level as endogenous p27 so it incorporates fully into dynactin‐like wild‐type p27 (Supplementary Figure 1), could rescue the mitotic phenotypes seen in cells depleted of p27 and p25. Wild‐type p27, but not the T186A variant, fully rescued the mitotic timing defects (Figure 1A, B and F; Supplementary Movies 4 and 10). It also restored Plk1 and 3F3/2 at kinetochores to normal levels (Figure 6F and G), whereas the T186A variant did not (Figure 6J and K; Supplementary Figure 6). In cells depleted of Arp11 or p150Glued, expression of wild‐type p27 did not restore kinetochore Plk1 or 3F3/2 to normal levels (Figure 6F and G), as expected given that p27/p25 is not recruited to kinetochores unless they are incorporated into dynactin (Supplementary Figure 3). Expression of neither wild‐type p27 nor the T186A variant had a discernable impact on mitotic timing or Plk1 targeting in cells transfected with control siRNAs (Supplementary Figures 6 and 7), verifying that low level expression of these proteins in cells containing endogenous dynactin has no effect.
Kinetochore–MT attachment, but not kinetochore spacing, is perturbed when p27 and p25 are removed from dynactin
Kinetochore‐associated Plk1 has been implicated in the establishment of MT–kinetochore attachments (Sumara et al, 2004; Lenart et al, 2007; Petronczki et al, 2008). To determine whether MT–kinetochore interactions were perturbed in cells depleted of dynactin subunits, we treated cells with monastrol to generate monopolar spindles. These structures allow intimate end‐on attachments to be distinguished from lateral interactions and unattached kinetochores (Kapoor et al, 2000; Lenart et al, 2007). Under normal conditions, this assay reveals that >90% of kinetochores sit at the ends of bundles of MTs that emanate from the pole in an orientation where one sister faces the pole the other sister faces away and no MTs appear to run between them (i.e., mono‐oriented k‐fibres; Figure 7A and B). Mono‐oriented k‐fibres are seen in only about 60% of cells depleted of p150Glued and we see an increased number of cases where tubulin bundles run between sisters (lateral k‐fibres), consistent with an increase in merotelic attachments. In cells depleted of p27/p25 we also observed fewer mono‐oriented attachments and more lateral associations. This is as expected given that these cells show an increased number of uncongressed and lagging chromosomes (Figure 1E) and supports the idea that p27/p25‐based recruitment of Plk1 contributes to proper establishment of kinetochore–MT interactions.
The spindle assembly checkpoint has been proposed to monitor tension between and within kinetochores to ensure that chromosomes are properly bi‐oriented before anaphase is initiated. Kinetochores from which dynactin or dynein has been removed exhibit decreased tension between sister kinetochore pairs, as evidenced by closer interkinetochore spacing (Varma et al, 2008; Maresca and Salmon, 2009). Depletion of dynactin from kinetochores via Arp11 or p150Glued siRNA treatment also reduced interkinetochore spacing in bi‐oriented pairs (Figure 7C). This is most likely due to the absence of dynein, which is believed to exert poleward pulling forces at kinetochores (Varma et al, 2008). Depletion of p27/p25, by contrast, does not result in a loss of tension, as expected given that kinetochores in these cells still have normal steady‐state levels of dynein. This suggests that inhibition of Plk1 binding to dynactin at kinetochores does not alter the tension generating mechanism, but instead interferes with the generation of and/or prematurely terminates the ‘wait‐anaphase’ signal.
p27/p25 contributes to recruitment of Mad1 to kinetochores
Kinetochore‐associated Plk1 has also been implicated in the recruitment of spindle checkpoint proteins to kinetochores and checkpoint signalling (Ahonen et al, 2005; Kang et al, 2006; Elowe et al, 2007). A key governor of the checkpoint is the MCC. This ‘wait‐anaphase’ inhibitory complex, comprising the proteins Mad2, BubR1, Bub3 and Cdc20, is assembled at kinetochores of unaligned chromosomes and is disassembled prior to anaphase in a process that involves component removal (‘stripping’) by the dynein/dynactin motor. Recruitment of the essential MCC component, Mad2, depends upon the presence of a binding partner, Mad1, at kinetochores. Like the MCC, Mad1 is present on the kinetochores of unaligned chromosomes but is transported away by dynein/dynactin once chromosomes are properly aligned and the checkpoint is satisfied (DeLuca et al, 2003; Figure 7E, Control, right). Unlike what was observed in controls, Mad1 levels were high on aligned chromosomes in cells treated with either Arp11 or p150Glued siRNAs. This is expected given that their kinetochores contain less dynein/dynactin that can remove Mad1. Aligned chromosomes in cells depleted of p27, by contrast, showed very low levels of Mad1, just like controls, consistent with the fact that their kinetochores bear a normal amount of dynein. Mad1 levels on the kinetochores of unaligned chromosomes in cells lacking the dynactin‐bound pool of Plk1 (i.e., cells treated with p27, Arp11 or p150Glued siRNAs) were significantly lower than controls (Figure 7D and E). A similar result was seen in cells treated with nocodazole (Figure 7F and G) or depleted of Spindly (Supplementary Figure 5). Although it has been reported that Spindly depletion has no effect on Mad1 (or Mad2) localization (Chan et al, 2009; Barisic et al, 2010; Gassmann et al, 2010), efficiency of knockdown or sensitivity of Mad1 recruitment to Spindly levels in the different cell lines used in these studies may explain the discrepancy. In any case, our results indicate that the dynactin p27 component is necessary for binding or retention of Mad1 at kinetochores. This might be due to phosphorylation of Mad1, a known Plk1 substrate (Grosstessner‐Hain et al, 2011; Kettenbach et al, 2011; Santamaria et al, 2011), or phosphorylation of a molecule such as dynein that governs Mad1 retention at kinetochores. Reduced levels of Mad1 at unaligned prometaphase kinetochores would interfere with the assembly of the MCC and might contribute to the accelerated mitotic progression we observe.
In this study, we show that dynactin's p27 component contributes to cell‐cycle progression in a completely unexpected way. Rather than helping target the dynein/dynactin motor to motile cargoes as it does in interphase, mitotically phosphorylated p27 recruits an extrinsic protein, Plk1, to the dynein motor complex at kinetochores. Our crystallographic study reveals that the Plk1 docking site is located near the C‐terminus of p27, in a region outside the well‐folded, type I left‐handed β‐helix domain.
All but one structurally characterized type I LβH family members are bacterial enzymes that typically exhibit acyltransferase activity, but eukaryotic p27 lacks the residues required for catalysis. The only other proteins in the human genome predicted to share the type I LβH fold are the ε and γ subunits of the translation initiation factor eIF2B and GDP‐mannose pyrophosphorylase A and B (Choi et al, 2008). The evolutionary origins of these rare eukaryotic LβH proteins are not known and it is unclear how their unusual structure contributes to their physiological functions.
Dynein and dynactin have numerous, well‐characterized functions in the mitotic spindle. They are recruited to kinetochores via multiple binding partners, including NudE, NudEL, Spindly and the RZZ complex, which provides for a complicated protein–protein interaction network (Bader and Vaughan, 2010). Loss of any component impairs dynein/dynactin binding (Starr et al, 1998; Griffis et al, 2007; Yang et al, 2007; Chan et al, 2009) and leads to improper kinetochore–MT attachments in prometaphase (Sharp et al, 2000; Gassmann et al, 2008; Varma et al, 2008) and loss of Mad1 and Mad2 (Buffin et al, 2005; Kops et al, 2005). In metaphase, impairment of dynein/dynactin function leads to prolonged checkpoint activation and retention of kinetochore‐associated checkpoint proteins, which has led to the hypothesis that dynein‐based ‘stripping’ of key regulators from kinetochores drives checkpoint silencing (Howell et al, 2000; Sivaram et al, 2009). Dynein/dynactin perturbation also typically results in significant spindle derangement owing to the focusing activities they provide at spindle poles, which makes it all the more difficult to tease out their roles at kinetochores. Because p27/p25 depletion does not affect spindle formation or dynein/dynactin integrity and recruitment to kinetochores, but does result in selective loss of Plk1 from this site, it represents a powerful new tool for analysing the functions of kinetochore‐associated Plk1 and its downstream targets in the processes of chromosome congression and execution of the spindle assembly checkpoint.
Kinetochore‐associated Plk1 has been implicated in kinetochore–MT engagement and checkpoint protein recruitment (Sumara et al, 2004; Ahonen et al, 2005; Wong and Fang, 2005; Elowe et al, 2007; Lenart et al, 2007; Hood et al, 2012; Liu et al, 2012; Maia et al, 2012). Our data suggest that full Plk1 activity is needed for establishment of proper kinetochore–MT attachments. However, unlike what is usually observed when kinetochore–MT interactions are impaired, cells depleted of p27 do not exhibit prolonged spindle assembly checkpoint activation, delay of anaphase onset and mitotic arrest. Instead, the checkpoint, if it is activated at all, appears to be prematurely satisfied, resulting in accelerated mitotic progression. Our findings suggest the following scenarios, which are not mutually exclusive. p27‐based recruitment of Plk1 to kinetochores may contribute to recognition or resolution of merotelic attachments, or it may influence recruitment, activation and/or retention of checkpoint proteins.
Impaired resolution of merotelic attachments in cells lacking p27 and p25 could lead to accelerated mitotic progression if this is a predominant mode of attachment, because such a situation would not activate the spindle assembly checkpoint. Selective pharmacological perturbation of kinetochore Plk1 has been shown to yield an increase in merotelic attachments (Lera and Burkard, 2012). Three major contributors to resolution of such attachments, Aurora B, MCAK and Kif2B, are known Pkl1 substrates (Grosstessner‐Hain et al, 2011; Kettenbach et al, 2011; Santamaria et al, 2011; Hood et al, 2012).
Accelerated mitotic progression might also be the result of aberrant checkpoint protein turnover. Unaligned chromosomes in cells depleted of p27/p25 show greatly reduced levels of Mad1, a protein that plays a key role in assembly of the MCC. Plk1 has been reported to phosphorylate the MCC component BubR1, as well as Mad1, which binds the MCC component Mad2 and facilitates its incorporation into the complex (Elowe et al, 2007; Grosstessner‐Hain et al, 2011; Kettenbach et al, 2011; Santamaria et al, 2011). Phosphorylation might alter the affinity of these proteins for their binding partners, enhancing MCC formation and/or retention at the kinetochore. Plk1 has also been linked to phosphorylation of dynein on its intermediate and light intermediate chains (Bader et al, 2011; Grosstessner‐Hain et al, 2011; Santamaria et al, 2011). Because dynactin binds dynein directly, it provides an ideal mechanism for localizing Plk1 activity nearby to regulate dynein activity. Plk1 phosphorylation is thought to reduce dynein's affinity for dynactin. This has been proposed to prevent the motor from switching from a state in which it is inactive for motility and tightly bound to the RZZ complex to a fully active form that can remove checkpoint proteins (Bader et al, 2011). Attenuation of a Plk1‐dependent switch might yield constitutively or prematurely motile dynein, which has the potential to impact the checkpoint in multiple ways. Dynein‐based removal of Mad1 would prevent conversion of Mad2 from its autoinhibited form to the form that is incorporated into the MCC (Fava et al, 2011). Alternatively, constitutively active dynein might prematurely remove the fully assembled MCC, triggering anaphase. Finally, enhanced dynein activity would be expected to generate more tension at the kinetochore and increase intrakinetochore stretch which has been correlated with checkpoint satisfaction (Maresca and Salmon, 2009).
Materials and methods
Cell culture, RNAi, 2D immunoblotting, immunofluorescence and image analysis
See Supplementary data for details.
Commercial antibodies are α‐tubulin (DM1A), phospho‐histone 3 (serine 10): Sigma; p150Glued (clone 1): BD, DIC clone 74.1: Millipore; p25 and p27: ProteinTech Group; Plk1: Santa Cruz; GFP: Invitrogen; PBIP1: Rockland; NudC: Abcam; GST: GE, Alexa488‐, Alexa568‐, and Alexa633‐conjugated secondary antibodies: Invitrogen; alkaline phosphatase‐conjugated secondary antibodies: Tropix. Monoclonal antibodies against p27 (27A) and Arp1 (45A) were produced and purified as described previously (Schafer et al, 1994; Melkonian et al, 2007). Other antibodies were obtained from our colleagues: mouse 3F3/2 ascites (Campbell and Gorbsky, 1995): Gary Gorbsky; rat anti‐ZW10 3.2 (Chan et al, 2000): Gordon Chan, sheep anti‐Bub1 (SB1.3), BubR1 (SBR1.1), Bub3 (SB3.2), MPS1 (SMP1.1) (Taylor et al, 2001; Tighe et al, 2008; Gurden et al, 2010): Stephen Taylor; rabbit anti‐Spindly (Griffis et al, 2007): Eric Griffis; mouse anti‐Mad1 (BB3–8) (De Antoni et al, 2005): Andrea Musacchio; human anti‐centromere serum (ACA; Martin et al, 1990): JB Rattner.
Expression and purification of p25/p27 heterodimers
Full‐length human p27 and full‐length mouse p25 cDNA (ATCC) were amplified and cloned into pDEST15 to create the bi‐cistronic expression vector pDEST‐GST‐p27/p25‐His6, with an rTEV cleavage site downstream of GST and a ribosomal binding site (RBS) between the p27 and p25 coding sequences. The non‐cleavable C‐terminal His6 tag was inserted for purification purposes. The T186A (ACT to GCT) and S184A (AGC to GCC) p27 variants were created in pDEST‐GST‐p27/p25‐His6 via QuikChange. BL21‐CodonPlus (DE3)‐RIL cells (Agilent) were transformed with the plasmid and grown to an OD600 of 0.6 in 0.5 l LB at 37°C, then induced with 0.1 mM IPTG for 16 h at 18°C. For purification of GST‐tagged heterodimers, the bacterial pellets were resuspended in 40 ml PBS (pH 7.4) containing 1 mM DTT and 1 mg/ml lysozyme, rotated for 20 min at 25°C and sonicated. The clarified bacterial supernatants were applied to a GSTtrap FPLC column (GE) and eluted with 50 mM Tris–HCl, 100 mM NaCl, 10 mM l‐glutathione and 1 mM DTT, pH 8.0.
GST‐PBD binding assays
Preparation of GST‐PBD beads: The human Plk1 PBD (AA 365–603; accession #BC01486) from a pCMV‐SPORT6 clone (ATCC) was cloned into pGEX4T‐2. The PBD binding‐null variant, GST‐PBD H538A/K540M (Elia et al, 2003), was generated using engineered PCR primers that also contained a unique restriction site (BsaBI) for insertion into the parent vector. The constructs were transformed into BL21‐CodonPlus (DE3)‐RIL, grown to an OD600 of 0.6 at 37°C in 0.5 l LB and induced with 0.1 mM IPTG for 3 h at 30°C. The bacterial pellet was resuspended in 40 ml binding buffer (20 mM Tris–HCl, 250 mM NaCl, 1 mM EDTA, 1 mM NaVO4, 20 mM NaF, 5 mM β‐ME, pH 8.0) containing 1 mg/ml lysozyme, 5 U/ml Benzonase and protease inhibitors, subjected to a freeze/thaw cycle, and sonicated. Two milliliters of the post‐sonication supernatant was incubated with Glutathione‐Sepharose (for each 10 μl aliquot; GE) pre‐blocked with 5% non‐fat milk in binding buffer, washed 5 × with 500 μl binding buffer, then test proteins were added.
To prepare p27/p25 heterodimers for the binding assay, the GST tag was cleaved via TEV protease (Invitrogen) for 16 h at 4°C from samples that had been phosphorylated in vitro (see below). The p27/p25 heterodimers were separated from GST and TEV protease using a Superose12 FPLC column attached to a GSTrap column (GE) in 20 mM Tris–HCl, 250 mM NaCl, 1 mM EDTA, 1 mM NaVO4, 20 mM NaF, 5 mM β‐ME, pH 8.0. The peak fractions containing p27/p25 heterodimers were identified by Coomassie staining. The purified p27/p25 heterodimers (25 μg) were incubated with 10 μl Glutathione‐Sepharose carrying GST‐PBD for 1 h at 4°C. The beads were washed with 3 × 500 μl binding buffer and proteins were eluted in 500 μl binding buffer plus 30 mM l‐glutathione for 1 h at 4°C prior to analysis of 10 μl by immunoblotting.
In vitro phosphorylation
To detect Cdk1 phosphorylation, purified dynactin (27 nM) or recombinant GST‐p27/p25‐His6 heterodimers (0.57 μM) were incubated with 10 units of Cdk1 (0.01 μg, NEB), 100 μM ATP and 3.75 μCi γ‐32P ATP in Cdk1 buffer in a 15‐μl reaction volume for 30 min at 30°C. The entire sample was subjected to SDS–PAGE and autoradiography.
To generate phosphorylated proteins for GST‐PBD binding, 350 μg purified GST‐p27/p25‐His6 heterodimers were incubated with 20 units of Cdk1 (NEB) plus 500 μM ATP in a 1‐ml volume for 16 h at 30°C. A parallel sample incubated without Cdk1 was used as the non‐phosphorylated control. Phosphorylation was verified using Pro‐Q Diamond Stain (Invitrogen).
To image mitosis in cells expressing histone 2B‐mCherry, cells were harvested by trypsinization 24 h after transfection with siRNAs, histone 2B‐mCherry and rescue plasmids, then 1.5 × 105 cells were plated into glass bottom dishes (MatTek) and cultured in HEPES‐buffered DMEM for 24 h prior to synchronization using a double thymidine block. Live‐cell recordings were initiated at 9–10 h after release from the block (G1/S boundary). Cells were imaged on a Zeiss Axiovert 200M inverted microscope (40 × /0.85 NA Plan‐Neofluor air objective) heated to 37°C using an In Vivo Scientific incubator. Images were collected using a Cooke SensiCam camera. Samples were illuminated at 25% intensity via a 120W X‐Cite 120PC Xenon arc lamp (EXFO Life Science). To reduce intensity and prevent radiation‐induced changes in the cell cycle (Khodjakov and Rieder, 1999), the light beam was passed through GG400 (UV) and KG5 (IR) filters (Newport Corporation) as well as a 12% neutral density filter (Zeiss). This filter combination allowed cells to proceed through mitosis without discernable delays. Images (100 ms/frame) were collected at 120‐s intervals. Three z planes (0.5 μm step size) were collected at each time point and at the end of the imaging interval the best optical plane for each cell was chosen and used to generate movies in SlideBook.
For phase contrast imaging, RNAi‐treated cells synchronized as above were illuminated using a 12V/100W halogen lamp at 30–35% intensity with heat absorbing (KG1) and green interference filters (546±20 nm bandpass, Zeiss) (Wadsworth et al, 2005). Time‐lapse movies were acquired at 90‐s intervals on a Zeiss Axiovert 200 Microscope (LD Plan‐Neuofluor 40 × , 0.6 NA Phase2 objective). Three z planes (0.5 μm step size) were collected at each time point, and the best optical plane for each cell was used to generate movies.
Analysis of interkinetochore spacing and kinetochore–MT interactions
Cells transfected with siRNAs were methanol fixed, stained for tubulin and ACA, and 0.15 μm image planes were collected as for immunofluorescence. Image stacks were processed using SlideBook Constrained Iterative deconvolution software and deconvolved images were exported to Image J. Only bi‐oriented kinetochore pairs were used for measurement of the interkinetochore distance.
Kinetochore–MT interactions were analysed in cells treated with 100 μM monastrol (Sigma) for 2 h followed by tubulin and ACA immunostaining, and deconvolution imaging as described (Lenart et al, 2007).
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary Movie 1
Supplementary Movie 2
Supplementary Movie 3
Supplementary Movie 4
Supplementary Movie 5
Supplementary Movie 6
Supplementary Movie 7
Supplementary Movie 8
Supplementary Movie 9
Supplementary Movie 10
We thank Ms Natalya Olekhnovich for technical assistance. We are grateful to Drs G Chan, G Gorbsky, E Griffis, JB Rattner, A Musacchio and S Taylor for generously providing antibodies, to Dr Q Vong and Y Zheng for the histone 2B‐mCherry plasmid, and the two anonymous reviewers plus Dr T Stukenberg for input and comments on the manuscript. The use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract No. W‐31‐109‐Eng‐38. We thank the SER‐CAT staff for assistance with data collection. Supporting institutions of SER‐CAT may be found at http://www.ser-cat.org/members.html. This research was supported by grants from the NIH to TAS (GM044589) and ZSD (NS036267) and training grant T32 GM07231 (Johns Hopkins University CMDB Program). Microscopy was performed at the Johns Hopkins Integrated Imaging Center.
Author contributions: TAS and ZSD supervised the experimental design and wrote the paper. TY performed the experiments in Figures 1, 2, 3D, E, 6 and 7, Supplementary Movies, and Supplementary Figures. BRS performed the experiments in Figure 3C and F. FKC performed the experiments in Figure 3B and C. AK, MYZ and UD performed the experiments in Figures 4 and 5. All the authors understand their responsibilities connected to authorship.
↵§ On leave from: Institute of Technical Biochemistry, Technical University of Łódź, Stefanowskiego 4/10, 90‐924 Łódź, Poland
- Copyright © 2013 European Molecular Biology Organization


The highest magnetic field is up to 15000GS. The mass and grade of both concentrate and tailings can be controlled through stepless speed regulation of roller and adjustment of the separator.
It's suitable for processing hematite, limonite, mirror iron ore, chromite, manganese ore, wolframite and other weak magnetic minerals in the tailing discarding stage; Remove harmful magnetic impurities from different products.
The materials convey into belt by vibrating feeder with uniformly layering, when materials pass through the strength magnetic roller, the magnetic particles are adsorbed to the strength magnetic roller due to the magnetic force, with rotation of the belt, the magnetic particles break away from magnetic filed and fall into mags collect hopper, nonmagnetic materials are discharged over the roller in a normal trajectory., then fall into non-mags collect hopper, the working principle shown as figure 2. The equipment can be configured into a multi-layer separation structure, the principle is the same as above.
Being the leader in the industry, SLon's magnetic separator equipment has been widely applied in the field of metallic and non-metallic minerals. Its tracks cover more than 20 countries and regions, including America, Canada, Brazil, Peru, Russia, Korea, India, Iran, Thailand, Philippines, Australia, Sierra Leone and so on. With market holdings of over 4000 machines, SLon has been the largest supplier of magnetic separation equipment in the industry.
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text:
My daughter got a new puzzle for her birthday that you get to put together and then color in. I can’t think of a person this would fit better! The puzzle was quite the challenge to figure out, but she enjoyed even the tricky bits. Within minutes of it being completed, she got to work giving it some beautiful color.
My daughter has been wanting to do henna here for a long time. We decided that her birthday party with her friends would be a good time to go ahead and do it. She also picked out some other activities and added to the ideas until a bunch of girls slept over. It was a huge success!
13 is not too old for running through the sprinklers:
Piñata in the basement (to keep the candy away from all the cicada exoskeletons littering our backyard):
Fun with plastic bubbles:
Dessert and candy cigarettes (her favorite candy):
Ready for sleep:
We celebrated my daughter’s birthday with some family before her friends came over today.
She got handmade presents from me- a mermaid tail blanket and a Dharma shark zippered pouch. (LOST is her favorite show and the shark is her favorite “character”) We also got her a ukulele bag for her uke.
She received lots of fantastic clothes, some candy, and a puzzle from family.

domain: hobby


President Donald Trump arrives to speak at a campaign rally, Thursday, Aug. 15, 2019, in Manchester, N.H. (AP Photo/Patrick Semansky)
On Friday morning, Trump’s campaign manager Brad Parscale released some very surprising date from the Dallas, TX rally held the night before. First, their data showed that 53,985 voters had attended. The stadium had a maximum capacity of 20,000. The overflow crowd remained outside watching on a giant television screen.
Next, 12% of those who attended indicated they had not voted in the last four elections. Can you imagine if this group were to actually go to the polls next year and cast their ballot for Trump?
The third data point showed that 21.4% of attendees were Democrats. The significance of this is monumental. I wonder what the percentage of Republicans attending Democratic rallies is. I imagine it would be low to nil.
This tells us that a certain percentage of Democrats are looking at the weak field of candidates and are searching for an alternative. I would guess many of them are turned off by the party’s sharp left turn. It might surprise them to hear that not every Democrat shares their enthusiasm for a socialist America.
Rush Limbaugh discussed this data on his radio show on Friday. He said, “Folks, if this is right — if this is even close — the Democrats can’t win anything with that amount of defection, if that’s really percolating.”
Finally, Parscale finds that 11% of them were Latino. The implications of such a strong Hispanic presence lays to waste the Democratic talking point that Trump’s insistence on building a border wall and taking a tougher stand against illegal immigration is resonating. Admittedly, close to 40% of the state’s population is Hispanic compared to about 18% for the general U.S. population. Still, an 11% Hispanic turnout to a Trump rally is impressive. The Hispanic vote will have a big impact on the results in 2020.
Parscale’s comment on this data was encouraging. He wrote, “These are winning numbers that will help win #FourMoreYears for @realDonaldTrump! We continue to outperform 2016.”
Incredible data from last night’s Dallas rally:
✅ 53,985 voters identified
✅ 12% have not voted in the last 4 elections
✅ 21.4% Democrat
✅ 11% Latino
These are winning numbers that will help win #FourMoreYears for @realDonaldTrump!
We continue to outperform 2016.
— Brad Parscale (@parscale) October 18, 2019
I’ve watched the last two Trump rallies on television and I’ve concluded:
1. There is no way that any of the 2020 Democratic candidates could ever attract the crowd sizes that Trump regularly receives at his rallies.
2. Nor would supporters line up 24-48 hours to hear any of them speak.
3. No one in the current field could generate the level of excitement Trump creates routinely at these events.
4. President Trump connects with attendees on an emotional level.
5. Trump himself loves holding rallies and the crowd feels it, knows it.
Limbaugh can’t get over the energy of these rallies. He told listeners:
It’s just a guy at a podium and a microphone. It’s not like you’ve got Mick Jagger in there strumming and running around singing with the guitars and drums being played.
I mean, it’s just one guy, and yet this kind of charismatic excitement… I’m telling you that there isn’t anybody anywhere in the Democrat Party who could muster anything like that, and yet we are told to believe that the country hates Donald Trump. We’re told to believe the country wants Trump gone. We’re told to believe the country’s embarrassed as hell and regret really regretful of the decision they made back in 2016 — and this is a giant disconnect.
He also thinks Trump will win Texas. He said:
Now, this is Texas. I’ll tell you something about Texas. The Democrats will tell anybody who will listen that they’re gonna win Texas. They’re not gonna win Texas, but they’ve got the Drive-Bys putting it out there as a possibility. They’re doing it on the basis of a lot of people having left — a lot of liberals leaving California, a lot of liberals leaving the Northeast. And then of course the illegal aliens crossing the border. They will find a way to get them to vote. The counties all along the Texas-Mexico border are turning blue. So the Democrats in the media are out there saying, “Hey, Texas is in play.” Well, it’s not in play.
Aside from excitement, Trump offers hope, a strong economic record, a far more conservative and fair judiciary, strength against the unrelenting attacks from the left, transparency (like his tweeting or not, we know where he stands), and he acts as a bulwark against socialism.
The legacy of the current Democrats in Congress will be one of hatred, injustice, secrecy, lies, anger, bending the rules and norms to advance their toxic agenda and trying to gaslight American voters.
When asked what specifically Trump has done that rises to the level of impeachment, Democrats have little of substance to offer. The 2020 candidates were questioned about their positions on this issue at CNN’s presidential nomination debate held on Tuesday night. Sen. Kamala Harris answered, “The reality of it is that I don’t really think this impeachment process is going to take very long, because, as a former prosecutor, I know a confession when I see it! And he did it in plain sight. He has given us the evidence, and he tried to cover it up, putting it in that special server. This will not take very long.”
Harris knows a confession when she sees it? He tried to cover it up by putting it on a special server? After two of Trump’s early conversations with world leaders were leaked to the press, he was forced to take extra precautions. She knows this, but since she can’t point to any valid reason for his impeachment, she was forced to use a debunked talking point.
Later in the debate, Harris said, “When I think about where we are right now, in 2020 I do believe justice is on the ballot.”
If justice is truly on the ballot in 2020, then Trump will win in a landslide.


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Search results 1-20 of 107 for pee pee dance 6
The Burger King pee dance (1:39)
Macho's excitement at Burger King led him to creat the official Burger King pee dance!!! Lol.
Pee Wee Herman Dancing to Tequila. For more great clips visit me at http://www.shaunsayre.com
... fox Michael tomorrow's is heating up on the Fox Network with the return of season ten. Of the smash So You Think You Can Dance our next guest is…
http://www.themoralliberal.com/2011/11/23/u-s-seals-court-records-of-border-patrol%E2%80%99s-murder/ U.S. Seals Court Records Of Border…
Kayla's Pee Pee Dance (0:08)
We were in 6th hour and kayla was showing us her dance she does when she has to pee!!!!!!!!! (lmao)
Girl is WEIRD.
Because it's funny enough watching a child do it
I'm Lori Rothman has your back is history in the midst of a rising stock market American optimism over the US economy. This is sentiment going nearly…
A performance by the 6 piece version of the band with caller Pee Rees calling Jack's Alive.
Guitar - Brian Heywood
Fiddle - Mark Knight…
Ring Bearer Pee Dance (4:02)
My son doing the pee pee dance while he is the ring bearer at his grandma's wedding. Watch as he tries to hold it all in.
http://bit.ly/OSxSbE - Watch the videos!http://Bit.ly/SubClevverTV - Click to Subscribe!http://Facebook.com/ClevverTV - Become a Fan!http://Twitter…
this is my pee pee!
Getting down in the dance box
this video is about 6 min and really only about 15 secs of absolute funniness, when it gets to the part where miya is gonna chase the bubbles watch…
Hysterical dance performance that will make you crack up and put a smile on your face no matter what.
Pee Pee Dance (5:00)
Nervous business man gives Boss presentation that will make or break his career. trouble is he drinks too much water in the waiting room. Can he make…
guy stops to pee
... head to Las Vegas where there will be paired up each week. And dance for the that come up with judge Mary Murphy and find out what ..... brings…

domain: entertainment


text:
Муниципальная политика и местное самоуправление в России
The Routledge Handbook of International Local Government conducts a rigorous, innovative and distinctive analysis of local government within a comparative, international context.
Examining the subject matter with unrivalled breadth and depth, this handbook shows how different cultures and countries develop different institutions, structures and processes over time, yet that all have some features in common – the most obvious of which is the recognition that some decisions are better made, some services better delivered, and some engagement with the state better organised if there is structured organisational expression of the importance of the local dimension of all these factors .
Thematically organised, it includes contributions from international experts with reference to the wider context in terms of geographies, local government modes, recent developments and possible further lines of research. It has a wide academic appeal internationally and will steer a course between the two dimensions of mono-jurisdictional studies and ‘cataloguing’ forms of comparison.
The Routledge Handbook of International Local Government will be essential reading and an authoritative reference for scholars, students, researchers and practitioners involved in, and actively concerned about, research on local government.
The article is based on the empirical research done during 2008-2010 within the framework of research projects of the Project-academic laboratory of the municipal management at the National Research University High School of Economics on municipal territories of Krasnodar, Tver, Kostroma, Kaliningrad, Rostov, Novosibirsk, Sverdlovsk. Th e research found that municipal territorial structure is unbalanced especially in city districts. Th e article considers such a phenomenon of the territorial structure as territorial forms independent of geographical regions and the subjects of the Russian Federation. Th e existing forms of city districts are characterized and a forecast of possible prospects of their development is made in the article.
The paper examines the structure, governance, and balance sheets of state-controlled banks in Russia, which accounted for over 55 percent of the total assets in the country's banking system in early 2012. The author offers a credible estimate of the size of the country's state banking sector by including banks that are indirectly owned by public organizations. Contrary to some predictions based on the theoretical literature on economic transition, he explains the relatively high profitability and efficiency of Russian state-controlled banks by pointing to their competitive position in such functions as acquisition and disposal of assets on behalf of the government. Also suggested in the paper is a different way of looking at market concentration in Russia (by consolidating the market shares of core state-controlled banks), which produces a picture of a more concentrated market than officially reported. Lastly, one of the author's interesting conclusions is that China provides a better benchmark than the formerly centrally planned economies of Central and Eastern Europe by which to assess the viability of state ownership of banks in Russia and to evaluate the country's banking sector.
The paper examines the principles for the supervision of financial conglomerates proposed by BCBS in the consultative document published in December 2011. Moreover, the article proposes a number of suggestions worked out by the authors within the HSE research team.
The article is devoted to the study of the authoritarianism prevalent in the mass consciousness of Russians. The article describes a new approach to the consideration of the authoritarian syndrome as the effects of the cultural trauma as a result of political and socio-cultural transformation of society. The article shows the dynamics of the symptoms of the authoritarianism, which appear in the mass consciousness of Russians from 1993 to 2011. This paper proposes a package of measures aimed at reducing the level of the authoritarianism in Russian society.
This work looks at a model of spatial election competition with two candidates who can spend effort in order to increase their popularity through advertisement. It is shown that under certain condition the political programs of the candidates will be different. The work derives the comparative statics of equilibrium policy platform and campaign spending with respect the distribution of voter policy preferences and the proportionality of the electoral system. In particular, it is whown that the equilibrium does not exist if the policy preferences are distributed over too narrow an interval.
The article examines "regulatory requirements" as a subject of state control over business in Russia. The author deliberately does not use the term "the rule of law". The article states that a set of requirements for business is wider than the legislative regulation.
First, the article analyzes the regulatory nature of the requirements, especially in the technical field. The requirements are considered in relation to the rule of law. The article explores approaches to the definition of regulatory requirements in Russian legal science. The author analyzes legislation definitions for a set of requirements for business. The author concludes that regulatory requirements are not always identical to the rule of law. Regulatory requirements are a set of obligatory requirements for entrepreneurs’ economic activity. Validation failure leads to negative consequences.
Second, the article analyzes the problems of the regulatory requirements in practice. Lack of information about the requirements, their irrelevance and inconsistency are problems of the regulatory requirements in Russia.
Many requirements regulating economic activity are not compatible with the current development level of science and technology. The problems are analyzed on the basis of the Russian judicial practice and annual monitoring reports by Higher School of Economics.
Finally, the author provides an approach to the possible solution of the regulatory requirements’ problem. The author proposes to create a nationwide Internet portal about regulatory requirements. The portal should contain full information about all regulatory requirements. The author recommends extending moratorium on the use of the requirements adopted by the bodies and organizations of the former USSR government.

domain: politics


text:
Discussion in 'Games, Gaming & Game-demos' started by IcE, Jun 30, 2011.
terraria has no options for pad in menus. but havent tryd using pad though
it wouldnt really work, you have to be able to place and destroy blocks not near your main character
idk COD7 Blackops 59.99€ on steam vs 50€ retail, same with other newer titles.. :3eyes:
If its 100% the game companies setting the prices, and not Steam, then does that mean this isn't Steam discounting these games lol.
Valve are too much of big player in the DD market these days to not get a say in prices, and the more expensive the game, the higher their cut will be.
On the other hand, since most publishers also have deals with big retail outlets worldwide, digital downlaod services obviously can't price them out the market altogether, which means that for now retail will more often than not be the cheaper choice, especially in Europe.
Usually steam prices are on par with physical copies or even less because you get to save that money of making the disk and all that crap.
And computer games that cost $60 instead of $50 just means the publisher is greedy. The only reason console games cost that is they have to pay $10 to the consoles. Computer games don't have that fee.
Maybe in the US, but thats not the case in Europe, especially the UK.
Here, the average price of a new release retail PC game is £25 ($40) compared to £30-35 on Steam or other DD services, and most of the time that includes shipping.
Even Black Ops which is on sale on Steam at £30 ($48) (34€), can be found on GAME for £20 ($32) (23€) and as far as i know it uses Steamworks, so you should still get all the "benefits" of Steam anyway.
Craaaaaaaaaaaap. Lost all my chances to buy any game on steam ..... Daaaaaaaaaammmmmn it :bang:
yeah,....someone just gifted me Killing floor Full Bundle
It had to be a gift so its UNCUT..hehe...
for 12 euro thats totaly ok...
is that zombie/monster slayer game good?
Killing Floor is fun with a full team! In single player it is good to practice on but the game shines with good players. Thank God it is not another deathmatch game.
The id Software's super pack is superb at that price. If for one own more than half of the games in there but if I didn't I'd buy it in a heart beat.
Does anyone have an extra copy of Half Life: Source (first one?) I never bought that since I had the original Half Life Platinum Pack but I found out a mod requires me to have this, even though I have all of HL2 + Garry's mod. And it seems I can't even buy it!
I can paypal you a couple of bux or gift you a cheapy game or DLC in return. PM me if you can.
@Valken - I managed to download Half-Life: Source and the two expansion packs just by entering the CD codes from my retail game into Steam around the time I also got Half-Life 2 on disc several years ago so you could try entering that and seeing if that allows you to download it.
Yeah this explains it, was also looking to buy at less 75%
@ Darren, thats how I registered HL Platinum - with the CD codes off the back of the case but did not ever let me get HL:S. I guess I'll wait for it to go on sale again but its weird a mod would require a few files from that game.
I really wish Black Mesa Source would hurry up and release!
@ Blesner - thanks for the info but I think STEAM should have popped out a message saying something like "Woops! We goofed! The price for Terrania should be 50% off and not 75% off! Sorry about that. If you still would like to purchase Terrania, click CONTINUE. Otherwise, click CANCEL to tell us to eff off." j/k about the eff off part.

domain: game


text:
A drug dealer was given the shortest sentence possible after a court heard a grim account of coronavirus lockdown conditions at HMP Liverpool.
Daniel McCann, 32, of Waterloo Street, St Helens, faced a sentence of anywhere between 42 months and seven years after admitting two counts of possession with intent to supply Class A drugs heroin and crack cocaine plus criminal damage.
At Liverpool Crown Court on Friday, McCann received the minimum of three-and-a-half years behind bars after defence counsel John Rowan told Judge Garrett Byrne that prisoners have "no toilet roll" and "no hand sanitiser".
Confined to his cell 23 hours, 45 minutes a day, McCann is also only allowed out of his cell for 15 minutes exercise a day, Mr Rowan said, with no access to courses, prison jobs or family visits.
A prison spokesperson said they have 'robust plans' in place to keep prisoners safe based in advice from Public Health England.
But Mr Rowan's description of the conditions played a part in convincing Judge Byrne to limit the sentence to 42 months - the shortest sentence available, with no separate penalty for the criminal damage.
McCann had been caught by police earlier this year heading to Widnes to sell Class A drugs.
Chris Hopkins, prosecuting via videolink, told Liverpool Crown Court today McCann was driving a VW Golf on Speke Road on January 28 when Cheshire police officers spotted the vehicle and began to follow behind, having received "intelligence" indicating the car was involved in drug supply.
McCann realised he was being tailed, stopped the car and ran, dropping two wraps of drugs as he went.
He then clambered to the roof of Widnes Spiritualist Church, on Lacey Street on the edge of Widnes town centre, where he caused £60 worth of damage.
Mr Hopkins said: “He dropped two wraps of heroin and climbed up the roof of Widnes Spiritualist Church and had to be brought down by the fire service.”
Back at the VW Golf, officers found 26 wraps of crack cocaine and 26 wraps of heroin in the footwell of the passenger seat, where a co-defendant - to be sentenced at a future date after a disputed issue is resolved - was sitting.
Police found two mobile phones on the passenger, and another phone in the driver’s side.
Officers searched McCann’s home address and found a "golf-ball-sized bag of powder", namely paracetamol and caffeine, suspected to be for mixing with drugs.
At the co-defendant’s home address, they found 24 wraps of crack, scales and what the prosecutors say was a "tick list".
The phone in the driving seat had sent bulk messages with the word "on", meaning drugs were available to buy.
McCann gave no comment in interview and admitted criminal damage at the earliest opportunity, but denied dealing drugs at first only to later change his pleas to guilty to two counts of possession with intent to supply Class A drugs, heroin and cocaine.
The 32-year-old had previous convictions including for the production of cannabis and harassment - the community order for which was still in place when police arrested him over the crack and heroin.
John Rowan, defending, cited a reference from McCann’s wife regarding their relationship and three children, and he requested credit for the guilty pleas.
He added that McCann's involvement in selling drugs stemmed from his own use of them, but was now clean and said that although McCann had believed using Class A drugs had been useful to him for dealing with "financial and emotional pressure", he was now realising "they are causing more problems than they are solving".
Mr Rowan conceded that custody was inevitable but he argued to Judge Garrett Byrne that it should be kept as short as possible due to the conditions faced by inmates in HMP Liverpool during the coronavirus lockdown.
He said that as well as having "no toilet roll" and "no hand sanitiser" or other hygiene products, McCann like other prisoners was being kept in his cell 23 hours and 45 minutes a day with only 15 minutes out for exercise, and with no family visitors or being allowed out to do their prison jobs.
Mr Rowan said: “All courses have stopped, all visits have stopped.
“He has a partner, that’s difficult.
“Of course the defendant is aware these procedures and policies are being put in place to protect him and to protect other prisoners and staff as well.
“It doesn’t make his time in custody any less difficult.”
The two Class A possession with intent counts carried a sentencing starting point of four-and-a-half years.
Judge Byrne slashed a year off for McCann’s guilty pleas and due to the conditions in prison, making a total sentence of 42 months. - the least he could receive.
He also ordered a victim surcharge and forfeiture and destruction order for the drugs and phone.
During his summing-up he said: “I’ve considered a letter from your wife.
“I accept you have a young family and I accept it will affect them.
“I will keep this to a minimum.
“I accept you’re not at the top of the chain and I accept conditions in prison are not easy for anybody.”
There was no separate penalty for the criminal damage charge in connection with the church roof.
A Ministry of Justice spokesman said: "We have robust and flexible plans in place keep prisoners, staff and the wider public safe based on the latest advice from Public Health England.”

domain: law


text:
Building Better Wine
Interview with Anabelle Sielecki of Mendel Wines
Conducted by Kate Soto
We’re thrilled to welcome Anabelle to Chicago!
Architect, mother, wife of a diplomat: Anabelle Sielecki wears many hats, but for the last 12 years she’s been occupied as proprietor of Mendel Wines, a small-scale winery in Mendoza, Argentina, focused on producing high-quality wines in a limited volume. Sielecki founded the vineyard and brought Roberto de la Mota on board, son of Argentina’s “Winemaker of the Century,” Raul de la Mota. Together, they’ve set out to carefully cultivate their grapes and craft their wines into reflections of Mendoza’s unique terroir.
Much of the work begins in the vineyard. Their 80-year-old Malbec and Cabernet Sauvignon vines are located on high-altitude (900-1100 meters), alluvial soils in Lujan de Cuyo, Mendoza. They practice artisan flood irrigation, tightly controlling it based on each individual block’s stress levels and yields. The vines are on their original rootstocks, so flood irrigation is an important control against phylloxera. Everything from harvesting to sorting to maceration is done by hand. Aging takes place in the finest French oak barrels.
All this attention to detail has produced a suite of Malbecs, Semillon, and blends that express intensity and complexity, and regularly receive scores over 90 from Wine Spectator and the Wine Advocate. We’re lucky to have Anabelle visit us in Chicago this week, so we sat down with her to pick her brain about her interests, vinous and otherwise.
Cream: You bring such a unique background to wine. How did you go from architecture to wine? Aside from a concern with place do you find that there is any crossover between your professional interests? Do you still practice architecture?
AS: With architecture, I am building abstract designs in a determined location. The architect plus program create a building made with diverse materials, and almost always clash with the environment. Viticulture, however, involves growing roots into the soil, and creating something that amazingly goes hand in hand with the nature of the place. I practiced architecture for a few years, developing real estate. Because of an economic crisis in the sector over 12 years ago, I started looking at other projects, and along came some old vineyards in Mendoza. I still practice architecture as a hobby, doing very small renovations and concepts.
Cream: I’ve read that you split your time between Washington, D.C., and Mendoza. I’m curious of your take on how wine culture varies on the two continents. Especially as the wife of an ambassador, I’m guessing you have a good sense of cultural identities.
AS: I was splitting my time between D.C. and Argentina until a couple of years ago. My husband was stationed in D.C., so I took advantage and stayed in order to get to know this beautiful city and many other areas of the country, and to get to meet wine people and talk to them about wine. Wine is now a very globalized drink in the western world. Obviously in some regions there are more women drinking rose wines, or white wines, and different varietals or blends go better given the climate of the cities, and also there are traditions of drinking at play. In Central America, for example, there was historically a more embedded tradition of drinking harder liquors, and almost no wine. Slowly, trends have changed and the world has become a bit more homogenized. Of course, countries in the Baltic, Russia, etc., have a long tradition of drinking white liquors, and even if some people now drink wines, it is still a very small percent of the population. The climate is a factor in people’s habits of drinking harder alcohols. You also have many countries that do not consume alcohol, some for religious reasons, and protocol calls for not serving them these drinks.
But having said this, I must confess I was very surprised at the huge amount of people who know about Malbec and other wines from Argentina. Almost everyone I’ve encountered has tasted a Malbec wine and liked it.
Cream: Mendel was named after your father. How did he inspire the personality of the company?
AS: My dad was an immigrant, a self-made man who came to Argentina in 1926, from what was considered one day Russia and Poland the next. He managed to establish a pharmaceutical business and be successful. He appreciated the best quality in things, be it a work of art, a building, or a glass of Champagne. My brothers and I were taught early in life that we did not have to settle until we got the best possible products.
Cream: Do you work closely with Roberto de la Mota in the cellar? Has he brought any techniques or ideas to winemaking that have surprised you?
AS: I work very closely with Roberto. He is our partner, our winemaker, our friend and our inspiration for this project. Any technique he brings along is never questioned--we assume he is always right. Roberto has a rare gift of combining best technique with the finest sensibility.
Cream: We have a lot of wine geeks in our readership--any fun or particularly geeky technology/techniques that you guys employ in the winemaking process?
AS: Roberto has tried numerous techniques in the last ten vintages. Some have been successful, some not quite (some of his experiments we’ll never taste). The one I like most is the way he discovered the perfect aging technique for our Semillon. When he made the first vintage of Semillon, it was quite a small amount, about 3,000 bottles. He wanted to use some wood, and ended up using about 15 percent American oak that time. The wine came out fantastic. When we asked why he used that exact percentage, he responded quite frankly..... because that was what was on hand at the time. Needless to say, we still use the same percentage of American oak.
Cream: What’s your own passion in wine? Is your heart in Argentinian wines or do you have favorites in other regions as well?
AS: I like wines from different countries in addition to Mendoza Malbecs and Cabernet Sauvignon blends. I like Burgundy, Pomerol, Priorat, Amarone, and Super Tuscans. I enjoy tasting the wines in their contexts, and if I travel to a country I try to concentrate my wine drinking to only that made in the specific region.
Cream: Chicagoans love Malbec. It seems to be the perfect wine to reach for during our polar vortex winter. Anything you’re looking forward to doing or drinking while in town?
AS: I am delighted to hear Chicagons like Malbecs. I hope they are also open to other varietals--Malbec is just one example of a fine Argentinian wine. While in Chicago, I’m looking forward to enjoy a typical meal from the region.

domain: artistic


text:
Nov 7, 2017
Regardless of our situation, we make conscious and unconscious money choices. The allure of alchemy, making gold out of other stuff, is something that attracts us … and has for centuries. What we do with money, how we react to it, what we want to do to acquire – all directly relates to being open and willing to dig into how economies, and our role in them, work. Jess Dewell talks with James Adams, author of the book Waffle Street (which has become a feature film).

domain: psychology


text:
Dimensions: D 33 x W 42 x H 17cm
Made from 100% recycled household plastics.
Solid rectangular shaped design, raised feet for easy sink drainage, comfortable easy grip handles and pouring, a perfect addition to any household or business.
This extra large washing up bowl, ideal for larger Belfast or butler style sinks or cleaning utility room uses;Ê Maintaining the same hard wearing quality finish with the Addis 10 year guarantee. Nutritional Value: Country of Origin: UK

domain: design


The best solution is actually sending a low voltage AC or DC coupled with a "Bias T". A Bias T is an inductor with a capacitor that allows for mixing AC+DC signal in a wire pair. Bias T are simple, and very cheap to make or buy. Low frequency Bias T are the solution to add power through the tether cable and being able to transmit the Ethernet commands to the unit.
Gaston T., PhD Physic (with patents applications in Multimode Comunication and plenty of articles of fiber optics communications). Will send by request.


text:
29 May 2019
If you could not make it to the ESCIF Congress 2019 we have a great news. On 29 May at 18:00 you can watch a live webinar with 2 of our keynote speakers, Lisa Harvey and John Chernesky.
The two will discuss what we can do to form strong bond between consumers, clinicians and researchers - something they, together, have nearly 50 years of experience...
John Chernesky is the Consumer Engagement Lead at the Rick Hansen Institute and the Vice-president of the North American Spinal Cord Injury Consortium.
With nearly 25 years of lived experience of spinal cord injury since being paralyzed in 1993, John has participated in dozens of research studies in addition to being a co-investigator on a number of research projects. He works closely with spinal cord injury consumers to ensure research is addressing their priorities and will have an impact on their lives.
Lisa Harvey is a Physiotherapist and a Professor at the University of Sydney School of Medicine.
She is primarily interested in clinical trials designed to determine the effectiveness of different physiotherapy interventions for people with spinal cord injury. She has research experience in a diverse range of topics including contracture and hand management of people with tetraplegia, orthotic gait along with exercise and respiratory physiology.

domain: health


text:
Basic CPR/AED First Aid $75.00
MEDIC First Aid PediatricPlus CPR, AED and First Aid for Children, Infants & Adults (EMSA APPROVED) Perfect For: Individuals who desire or are required to be certified in Pediatric First Aid, CPR and AED, such as:
- School bus drivers
- Child care workers
Occupational first aid providers who are not healthcare providers or professional rescuers.
HEART SAVER CPR/AED FIRST AID $110.00
PROVIDER: American Heart Association
Heart Saver CPR, AED, and Basic First Aid combination training allows instructors an easy option for students seeking a comprehensive training class that builds the confidence to respond. This first aid, AED, and CPR certification course option is an excellent choice for both the community and workplace setting, and conforms to the 2015 AHA Guidelines Update for CPR and ECC, and the 2015 AHA and ARC Guidelines Update for First Aid.
Individuals who require or desire first aid, AED, and CPR classes that deliver comprehensive emergency care knowledge and skills:
- Emergency response teams in business and industry
- Adult residential care personnel
- Adult and Child
- Adult, Child and Infant
Required for Successful Completion (Certification)
Optional except when required by organizational, local or state regulation.
Students must perform required skills competently without assistance.

domain: medical


text:
Got a craving to show off your "Monster Mash" or bust a move to "Thriller?"
Even if your dancing is monstrous, you can still have a frightening amount of fun during a pair of dances put on by local Carnival krewes.
Local band "Good Feelings" will entertain at the Krewe of Ambrosia’s second-annual Halloween dance, set for 9 p.m.-1 a.m. Oct. 27 at the Thibodaux Civic Center, 310 N. Canal Blvd., Thibodaux.
The dance, for ages 18 and up, is open to the public. The event is BYOL, and tickets are $15 at the door.
Costumes are optional, but welcome and encouraged. A costume contest will determine the funniest, scariest and most-original attire. The dance will include door-prize giveaways throughout the night.
The Thibodaux-based krewe hopes to draw a larger crowd for this year’s dance, said Sharon Daigle, Ambrosia treasurer and dance organizer. Last year’s dance drew about 200 people, she said. Organizers want the dance to grow into a large annual event, she said.
Proceeds help the 400-member krewe hire local marching bands to entertain during the Krewe of Ambrosia’s annual Mardi Gras parade, Daigle said.
In Terrebonne Parish, the Krewe de Bonne Terre plans to hold its first Halloween dance Oct. 27 in the Montegut Gym, 107 Recreation Drive. Doors open at 7 p.m. The dance begins at 9 p.m. The dance is open to anyone age 18 and up. Admission is $10 each.
BYOL. Only ice chests without plugs will be allowed.
Christina LeBlanc, president of the krewe, said guests will enjoy hearing from the evening’s musical act, local band Southern Boyz, a group that includes former members of the band LA 56. The homegrown band plays swamp pop, Zydeco and "a little bit of everything."
LeBlanc said krewe members are eager to see the interesting costumes worn by guests. The carnival club, which includes between 100 and 150 members, will use proceeds to help fund its annual Mardi Gras day parade.
Carnival krewes putting the 'boogie’ in boogey man

domain: news


text:
When John Crowl sets foot in the wagon sheds at the Carroll County Farm Museum in Westminster, Md., he embarks on a journey down Memory Lane.
John, 80, was born (and still lives) on a farm first developed by his great-grandfather in Westminster in 1884. He stopped active farming in 1986, but maintains a keen interest in preserving the memories and artifacts that relate to a way of life now fading into the past.
In one of the two wagon sheds are a pair of huckster’s wagons. One is fitted out with runners for winter use and the other, which resembles a Conestoga wagon, is prepared for summer service. The wagons were built for William Henry Segafoose of Uniontown, Md., between 1870 and 1900, by Charles Sittig, also of Uniontown. They were owned by three generations of the family before being donated to the museum in 1966.
“I remember the huckster’s wagon coming to the farm,” John says. “It usually came once a week. They would pick up butter, eggs, chickens that were no longer producing, geese, ducks, turkeys … whatever they had. In season, it was fresh fruit and vegetables. And pigeons. The children used to climb up in the barn at night with a flashlight and pull the birds from the rafters. Pigeons got to be a nuisance. They went for 10 cents a pair, and were used to make pigeon pot pie.”
John recalls hucksters as an integral part of the rural community. They had different routes and schedules, depending on the weather and the season. They took the produce from the farms to the city, a journey that could take three days.
“They’d go back and forth to Baltimore,” he says. “They’d bring back fresh fruit, molasses and sugar.”
The summer huckster wagon, which is covered by a canvas top and fitted out with wooden cages for live poultry, carried a cargo that averaged 1,500 pounds and was drawn by two (or occasionally four) horses. On the return journey from Baltimore, Maryland’s major port city, the cargo often included bolts of fabric for women, tools and parts needed by fanners, and other items used in everyday life on the farm.
A 19th century “universal wagon” (also called a hay carriage wagon or a stone bed wagon) was popular with local farmers between 1850 and 1900.
“It was used for all sorts of hauling,” John says. “Stones and rocks from the fields, fodder for the animals, ear corn, sheaves of wheat, oats and rye at harvest time.”
The wagon’s design allows for boards to be added to the frame to make a container for whatever was to be transported. Because of its size, it needed four animals – horses or mules – to pull it.
The sheds also house an exhibit focusing on the ice industry of the 19th century. The exhibit includes an ice wagon, also used to haul wood, and a variety of ice cutting tools. There’s even a 19th century horse-drawn ice plow used to cut ice blocks. For John Crowl, there are more memories.
“My grandfather had an ice cream business for 50 years,” he says. “He always said you should fill the ice house before Christmas because the ice was best before it snowed. We don’t have the ice or the snow ’round here like we used to.”
A community ice cutting was usually held in January or February each year. Farmers met at a pond or lake with their sleighs and ice-cutting equipment. A hole was cut in the ice, and a wooden chute placed in the hole. Crosscut saws were used to cut ice blocks, some of which weighed up to 300 pounds. The blocks were pulled up the chute with ice picks or a line drawn by a horse, loaded into sleighs and delivered to the ice house. There the blocks were packed in sawdust layers a foot thick for insulation, providing a year’s supply of ice. Sleighs and bobsleds are romanticized today, but a century ago, they played a critical role in winter transport. Necessity, as always, was the mother of invention: When the snow piled up, wagons were simply refitted.
“Bobs were used to convert wheeled vehicles to winter vehicles,” John says. “They were made by people who had little education but a lot of intuition. This was when farmers didn’t have the means. They just switched the running gears. The runners were broad so they didn’t cut into the snow.”
Another regular visitor to farms was the dead stock wagon that picked up dead farm animals for disposal. The one displayed at the museum is painted a soft blue-gray and fitted with mechanisms to winch the carcass into the back end of the wagon. Along the side is painted in black capitals “Leidy Dead Stock. Westminster Md. Phone 259.” The wagon was custom built in Doylestown, Penn., about 1915, and was used by the Leidys until World War II.
“I remember that very buggy coming to our farm,” John says. “I knew the Leidys real well. They had a rendering plant. They turned fat into tallow. They sold the hides for leather. The flesh solids would be cooked, ground into scraps called ‘tankage’, and used for chicken feed or hog feed.”
A rural mail delivery wagon dating to the turn of the 19th century is a symbol of the end of rural isolation. Farms once cut off from the mainstream of life suddenly had regular access to news and goods. Once rural free delivery was implemented in 1899, the rural mail carrier quickly became an important part of farm life.
“I remember mail delivered by horse and buggy once a day, six days a week,” John says. “They brought mail, newspapers, packages, baby chicks. Things moved along, and then it was a Model T. My brother carried mail for 40 years; in those days, mail carriers furnished their own vehicles.”
A Dayton spring wagon built in Westminster in the 1890s by John Eckenrode provides an example of how farm families went visiting 100 years ago. Dayton wagons, sometimes referred to as “the poor man’s surrey,” were very popular because of their versatility. With the rear seat removed, they could be used to haul cargo. With the rear seat in place, they served as family transportation.
Eckenrode was born in Carroll County in 1847. He developed a carriage factory in Westminster. By the end of the century, he was producing more than 100 vehicles annually, which were sold to customers in Maryland, Pennsylvania and Virginia. Some of his vehicles won prizes at the Maryland State Fair in Timonium.
For John Crowl, the Carroll County Farm Museum represents a community’s commitment to preservation of the area’s farm heritage.
“We rely on volunteers,” he says. “Without them, this museum could not operate. We do have personnel here who take care of restoration; we do as much as we can to save money. Things we can’t handle we send out. Our goal is to preserve the history of the culture and heritage of this great country.”
The museum hosts a variety of shows each year. One near and dear to John’s heart is Fall Harvest Days, held the first weekend in October. The event includes demonstrations of skills and arts used commonly in rural areas a century ago.
“We do everything they did back then,” he says. “We do threshing and baling, make apple butter and scrapple, make shingles, all those long-forgotten arts. It fascinates those who don’t know about them. I’m deeply embedded in carrying that on. When it’s gone, it’s gone forever.”
Living History Center Focuses on Rural Life
The Carroll County Farm Museum is a center for living history with emphasis on rural life in the 19th century.
The 142-acre complex (operated by the Carroll County Department of Recreation, Parks and Facilities) features original farm buildings dating to the 1850s. The site includes a blacksmith’s shop, broom shop, spring house, one-room schoolhouse, and living history center. Historic artifacts, antique tools and machinery enhance the 19th century atmosphere.
The facility was originally built in 1852 as an almshouse to provide shelter for the homeless. Residents farmed to generate their own food.
John Crowl has been involved in development of the museum from its inception, and has served several terms on the board of directors. He credits longtime county Extension agent Landon Burns as the museum’s driving force.
“Landon Burns dreamed of turning it into a farm museum and had a lot to do with getting it going,” John says. “He was a great man. The idea was very well received, and people donated by the truckload. They wanted what they once used to be preserved for future generations.”
In addition to permanent exhibits, the museum hosts a variety of events year-round, including a week-long 19th century living history summer camp for elementary age children, and the annual “Steam Show Days” in September. FC
For more information: Carroll County Farm Museum, 500 South Center Street, Westminster, Md., 21157; phone (410) 386-3880; online at http://ccgovernment.carr.org/ccg/farmmus/
Jill Teunis is a freelance writer living in Damascus, Md. She is interested in writing about communities, their people and history.

domain: history


text:
26 Nov, 2009
Get Your Mellow Zing On With The “Live-Green” V-8 Smoothie… A Savory Smoothie/Blended Salad
The challenge today: to create a V-8-esque Live Green Smoothie from simple, non-exotic ingredients available at any grocery story, and possibly already in someone’s fridge and cupboard.
Another day, I’ll put up a recipe using fresh horseradish root and a bit of Nama Shoyu unpasteurized soy sauce, and maybe some fresh chili. But for now, we’ve got a great base for a Savory Smoothie/Blended Salad anyone can make using fresh tomatoes, celery, red cabbage, and celery as a base.
This smoothie is a wee bit spicy… but not too much. It’s also a bit creamy, with a mellow, mild taste and a nice zingifyingly zesty finish.
The fresh garlic in the smoothie is an immune system revitalizer, and hey… we all want a bit more of that these days. Perfect for if you’re feeling a little “wan” of late! And you can always take the zest factor up a few notches with the addition of cayenne, fresh chili, horseradish, and a splash of Nama Shoyu, if you have any of that around!
And of course, you could use this as a base for a nice “Bloody Mary” raw cocktail!
Recipe for: The “Live-Green” V-8 Smoothie
A Blended Salad/Savoury Salad
- 1 cup chopped fresh tomatoes
- 1 cup chopped celery
- 1 cup chopped kale
- 1 cup chopped red cabbage
- 1/2 cup red grapes
- 1 tablespoon chopped red onion
- 2 teaspoons chopped garlic
- 1 tablespoon cold-pressed olive oil
- 1 teaspoon raw, unpasteurized apple cider vinegar
- 1 tablespoon fresh lime juice
- 1/2 teaspoon quality natural salt, such as Himalayan Crystal salt
- 3 cups spring water
- 1 cup ice
Blend everything together until smooth and pour out into nice glasses, with celery or fresh lime as a garnish.[/private]

domain: food


Spinner Harmeet Singh, who represented India at the Under-19 World Cup in 2010 and 2012, has been included in the USA’s 15-member squad for the five-match T20I series against Canada, starting April 7.
USA, the co-hosts of the 2024 ICC T20 World Cup, will play all their matches at the Prairie View Cricket Complex (PVCC), in Houston, Texas.
Singh, a former Mumbai and Rajasthan Royals player, was the first-ever draft pick in MLC (Major League Cricket) history—snapped by the Seattle Orcas—and went on to lead the Minor League Cricket (MiLC) team Seattle Thunderbolts to the title in 2022.
He played two Under-19 World Cups for India— 2010 and 2012 – and was instrumental in India’s title win in 2012. In 2013, during the IPL spot-fixing controversy, Singh was investigated by the BCCI for allegedly being in contact with a bookie, but was subsequently cleared of any wrongdoings.
Interestingly, a notable omission from the squad is India’s U19 World Cup winning captain Unmukt Chand. Chand had a good run with the bat in the three MiLC seasons scoring over 1500 runs across 45 innings yet was left out of the squad.
Another notable inclusion in the squad is former New Zealand international Corey Anderson, who last played for New Zealand in 2018. He played in the MiLC, scoring 900 runs in 28 innings at a strike rate of 146. Former Delhi and Royal Champions Bengaluru player Milind Kumar also makes it to the squad alongside former Canada skipper Nitish Kumar and former South African domestic stars Andries Gous and Shadley Van Schalkwyk.


text:
GATINEAU, Que. - The Hydropothecary Corp.'s share price closed up 13 per cent Wednesday after the company said it had struck a cannabis supply deal with the Societe des alcools du Quebec.
The Gatineau-based company says the deal has it set to supply upwards of 200 tonnes of cannabis products over five years to the provincial Crown corporation.
The company says the deal, which sent its share price up 17 per cent earlier in the day on the TSX Venture Exchange, is the largest supply agreement between a producer and a provincial supplier for recreational cannabis.
The deal sees Hydropothecary supplying 20,000 kilograms in the first year, then ramping up to 35,000 kilograms in the second and 45,000 kilograms in the third with the final two years yet to be determined. The contract could be extended for another year.
Chief executive Sebastien St-Louis says the deal is an important step in Hydropothecary's growth strategy of focusing on Quebec before expanding capacity to serve other Canadian markets.
Hydropothecary says it's the only cannabis producer in Quebec with a licence to sell from Health Canada.

domain: finance


text:
Spent two nights here and we're warmly welcomed. The unit had a separate bedroom and a large lounge/ dining area. This also contained a single bed for an extra person. The kitchen was well equipped and around the corner from the lounge which gave it a separate feel. Wifi was free and unlimited for a change. A very comfortable stay.
- Reservation Options:
- TripAdvisor is proud to partner with Agoda, Booking.com, Priceline and Odigeo so you can book your Aorangi Motel reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.

domain: travel


text:
Por Oscar aims to document the movement to free Oscar López Rivera, one of the longest-serving political prisoners in the world. Rivera is a Puerto Rican activist who has been incarcerated since 1981 for his activities in the struggle for Puerto Rican independence. Many attribute Rivera to being the “Nelson Mandela of Puerto Rico.” Local and international activists and groups have been organizing for his release from prison and their demands have recently come into fruition.
On January 17, 2017, U.S. President Barack Obama conceded to the community's demand to commute Rivera's sentence and he is now finishing his 35th year of imprisonment under house arrest in Puerto Rico. One of those groups is the 35 Mujeres Por Oscar (35 Women for Oscar), who have been organizing a protest demanding Rivera’s release on the last Sunday of every month for the last two years. Each woman in the group represents each year that Rivera has been imprisoned. Por Oscar aims to document these women’s stories about how and why they got involved in the movement to free Oscar López Rivera and to win independence for Puerto Rico.

domain: sociology