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Cytoplasmic dynein and its regulatory proteins have been implicated in neuronal and non-neuronal cell migration. A genetic model for analyzing the role of cytoplasmic dynein specifically in these processes has, however, been lacking. The Loa (Legs at odd angles) mouse with a mutation in the dynein heavy chain has been the focus of an increasing number of studies for its role in neuron degeneration. Despite the location of this mutation in the tail domain of the dynein heavy chain, we previously found a striking effect on coordination between the two dynein motor domains, resulting in a defect in dynein run length in vitro and in vivo.
We have now tested for effects of the Loa mutation on neuronal migration in the developing neocortex. Loa homozygotes showed clear defects in neocortical lamination and neuronal migration resulting from a reduction in the rate of radial migration of bipolar neurons.
These results present a new genetic model for understanding the dynein pathway and its functions during neuronal migration. They also provide the first evidence for a link between dynein processivity and somal movement, which is essential for proper development of the brain.
Cytoplasmic dynein is a minus-end directed microtubule motor protein involved in a wide variety of functions. In order to perform such diverse activities in the cell, dynein makes use of numerous regulatory partners, including dynactin, LIS1, NudE and NudEL [1-3]. LIS1 and NudE/L, in particular, have received considerable attention for their role in brain development [4-7]. Sporadic mutations in LIS1 cause type I, or classical, lissencephaly, a severe brain developmental disease characterized by a smooth cortical surface [8]. Patients with lissencephaly exhibit lamination defects of the cerebral cortex, consistent with a defect in neuronal migration. Developmental analysis of LIS1 mutant mouse lines has shown that a reduction of LIS1 protein leads to neocortical organization defects in a dose-dependent manner [5,9-11]. Live analysis of embryonic rat brain subjected to LIS1 RNA interference (RNAi) has directly demonstrated defects in neural progenitor cell division, radial migration, and axon elongation [7,12]. The genes encoding NudE and NudEL, Nde1 and Ndel1, are closely related and interact genetically with LIS1 and cytoplasmic dynein [13]. Nde1 and Ndel1 mutant mice exhibit either microcephalic or lissencephalic brain phenotypes, consistent with a functional relationship to LIS1 [6,14-16].
Several studies have focused on the role of genes in the dynein pathway in mammalian brain development. However, there are no genetic models with which to study the involvement of dynein directly. Mice homozygous null for the cytoplasmic dynein heavy chain gene exhibit early embryonic lethality, whereas heterozygotes show no obvious abnormalities [17]. However, several dynein mutant mouse strains have since been identified in screens for genes involved in neurodegeneration [18,19]. Of these dynein mutants, the Loa mutation has received particular attention [20,21]. The Loa heterozygous mice are viable and develop early onset neurodegenerative disease, but the homozygous animals die perinatally due to an inability to feed [18]. Interestingly, both the Loa/+ and the Loa/Loa mice exhibited defects in retrograde axonal transport [18,22]. The Loa mutation resides in the amino-terminal tail region of the dynein heavy chain. Nonetheless, enzymatic, biochemical, and single molecule analysis revealed a decreased microtubule affinity for the Loa mutant dynein due to a disruption in the coordination of its two motor domains [23]. This defect was manifested as a decrease in processivity for purified mutant dynein in in vitro single molecule assays. Particle tracking of lysosome/late endosome transport in cultured wild-type and Loa hippocampal neurons revealed a marked reduction in vesicular run lengths that could be accounted for quantitatively by the reduced processivity of the mutant dynein [23].
The Loa mouse represents an important model for the study of neurodegeneration. It should also, in principle, be useful in evaluating the role of dynein in other functions, such as neuronal migration. In view of its particular effects on dynein mechanochemical behavior, Loa also affords an opportunity to test whether dynein processivity contributes to this process. The Loa/Loa mice have been reported to display abnormal facial motor neuron organization, suggesting potential cellular migration defects [18]. We report direct evidence that the Loa mutation disrupts this process, causing a delay in neocortical development, a result that confirms a more general value for this mutant mouse strain and has implications for the role of motor processivity in neuronal migration.
To test whether the Loa mutation affects brain development, we first examined the lamination pattern of the cerebral cortex in postnatal day zero (P0) mouse pups. Wild-type and Loa/Loa brains had a similar cortical thickness: 1,198 ± 150 μm for wild type versus 1,220 ± 160 μm for Loa/Loa (P = 0.8705; n = 3 brains per genotype). Six distinct layers could be identified by DAPI staining in both wild-type and Loa/+ brains; however, the boundaries were blurred in the Loa/Loa brains, especially between neuronal layers III and IV (Figure (Figure1A).1A). To identify individual layers, we measured the density of Ctip2 and Foxp2 staining, which serve as markers for layers III and VI, respectively. In the Loa/Loa brains, Ctip2-positive cells were spread over an area roughly twice as broad as the compact layer observed in the wild-type brain (Figure (Figure1B;1B; P < 0.005; n = 3 brains per genotype). In contrast, there were fewer Foxp2-positive cells in layer VI of the Loa/Loa brain compared with wild type (Figure (Figure1C;1C; P < 0.005; n = 3 brains per genotype), a further indication of cortical disorganization.
We also examined the hippocampus of the Loa/Loa mutant brain and noticed that, although overall structure was normal, the dentate gyrus was smaller than in the wild-type brain (Figure (Figure2A).2A). We therefore analyzed the subset of granule neurons (labeled by Prox-1) that are born in the subventricular zone of the neocortex and migrate to form the dentate gyrus [24]. In the Loa/Loa hippocampus, granule cell redistribution to the dentate gyrus was delayed at all time-points examined, and by P0 the dentate gyrus was smaller and more compact than in the wild-type hippocampus (Figure (Figure2B).2B). Together our data revealed lamination defects in both the neocortical and hippocampal regions of the Loa/Loa mouse that were similar to, though less severe than, those reported in the LIS1 compound heterozygous mutant mice [9,10].
To explore the origin of the lamination abnormalities, we first performed a BrdU birthdating study by injection of pregnant Loa/+ mice with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) at either embryonic day (E) 16 or 18. BrdU is incorporated into the DNA of S-phase progenitor cells and serves as a stable marker for cells born around the time of injection. Brains from pups exposed to the label at E16 or E18 were analyzed for the distribution of BrdU-positive cells at P0. In wild-type brains, E16 BrdU-labeled cells formed a compact layer near the pial surface by P0 (Figure (Figure3A).3A). In the Loa/Loa brains, the majority of BrdU-positive cells migrated to the same cortical region of the brain, but formed a broader layer and showed many trailing neurons (Figure (Figure3A;3A; P < 0.005 for cell density within the first two cortical layers; n = 3 brains per genotype). A similar effect was observed with neurons labeled with BrdU at E18: the neurons showed a reduced migration distance, as well as a more scattered distribution in the Loa/Loa mutants relative to wild-type controls (Figure (Figure3B;3B; P < 0.001 for cell density within the first two cortical layers; n = 3 brains per genotype).
We also introduced GFP cDNA into neural progenitor cells in the mouse neocortex by in utero electroporation at E15 [25]. GFP-positive cells in the ventricular zone/subventricular zone, intermediate zone (IZ), and cortical plate (CP) were examined 3, 4, or 5 days later (Figure (Figure4;4; n = 3 brains per genotype, per time-point). In the wild-type brains, 27% of the transfected neurons had reached the CP by day 3 (E18), and 57.5% by day 4 (Figure 4A,C), a time-course consistent with previous observations [26,27]. In Loa/Loa brains, however, the majority of transfected cells remained within the ventricular zone and subventricular zone at both day 3 and day 4, with only a subset having reached the IZ (Figure 4B,C; 45% by day 4; P < 0.01). However, by day 5, the distribution of GFP-positive cells in Loa/Loa brains was almost identical to that at day 3 in the wild-type brains (Figure 4B,C; 22% in the CP of Loa/Loa brains). Thus, the Loa mutation delays the radial redistribution to the CP by approximately 2 days in the mouse neocortex.
To obtain further insight into the basis for the delay in neuronal redistribution, we performed live cell imaging of E16 wild-type and Loa/Loa brain slices labeled with Oregon Green BAPTA AM. Labeled cells within the IZ exhibited the normal bipolar morphology of radially migrating neurons. The rates of neuronal movement towards the pial surface varied with time and from cell to cell as previously reported [12,28,29] (Figure (Figure5).5). The tip of the leading process progressed at a similar average rate for both wild-type and Loa/Loa neurons (0.16 ± 0.10 μm/minute and 0.17 ± 0.11 μm/minute, respectively; P = 0.817). However, the rate of somal translocation was significantly different between genotypes, with an average rate of 0.38 ± .09 μm/minute for wild-type neurons, but only 0.23 ± 0.6 μm/minute for Loa/Loa neurons (Figure 5A-C; P < 0.0001; n = 12 cells from at least three brains per genotype followed for at least 110 minutes).
Many cell bodies of the migrating wild-type and Loa/Loa neurons were distorted into an elongated shape during migration. However, the wild-type cell bodies remained in this stretched conformation for 20.0 ± 11.5 minutes before returning to their normal rounded morphology after advancing, whereas Loa/Loa cell bodies remained in the stretched state for 38.0 ± 13.0 minutes (Figure 5A,B; P < 0.05; n = 12). These results are indicative of a prolonged somal translocation state due to the reduced transport by the Loa mutant motors.
Cytoplasmic dynein is involved in numerous aspects of mitosis, including mitotic spindle organization and orientation, interactions of kinetochores with microtubules, and mitotic checkpoint regulation [30-34]. To test for accumulation of dynein mutant cells in mitosis, we stained wild-type and Loa/Loa E16 brain slices with an antibody to phospho-histone 3 (PH3). Although we did find a slight increase in the number of PH3-positive cells in the Loa/Loa brain slices compared with wild type, this difference was not statistically significant (Figure (Figure6A;6A; P = 0.054; n = 5 brain slices per genotype). Similarly, counts of PH3-positive cells located in the ventricular or abventricular (>100 μm from the ventricle) regions were not significantly different between wild-type and Loa/Loa brains (Figure (Figure6A;6A; P = 0.068; n = 5 brain slices per genotype). As an additional test for impaired cell cycle progression, we performed a 2-hour BrdU pulse in E16 mice and found a similar number of BrdU-positive cells in wild-type versus mutant brain (Figure (Figure6B).6B). We also observed a comparable fraction of Pax6-positive cells between wild-type and Loa/Loa brains at both E16 and E18 (data not shown). Wild-type and Loa/Loa brains are of comparable size and have a similar cortical thickness (Figure (Figure1);1); thus, together, these results argue against the gross mitotic defects that have been reported for a NudE null mouse [6].
Dynein and its regulatory factors, dynactin and LIS1, have been implicated in axon elongation in rat brain slices subjected to LIS1 RNAi and in cultured neurons [7,35,36]. Oregon Green BAPTA staining could detect clear trailing axons in a majority of radially migrating neurons in wild-type mice (Figure 7A,C; 72.3%; n = 32 cells from three brain slices); however, axons were detectable in fewer neurons in Loa/Loa slices (Figure 7B,C; 50.6%; n = 30 cells from three brain slices; P < 0.0001). Of the visible axons, 62.5% of the wild-type axons could be followed for longer than 40 μm compared with only 40.0% of the Loa/Loa axons (Figure (Figure7D).7D). We also observed a decrease in axon extension for Loa/Loa neurons in vitro. We cultured neurons on laminin for 2 or 3 days in vitro (DIV), then examined axonal length. At DIV2, the average axon length of wild-type neurons was 111 ± 23 μm compared with 54 ± 19 μm for the Loa/Loa neurons (n = 57 and 53 neurons, respectively; P < 0.0001), and by DIV3, the average axon length of wild-type neurons was 125 ± 27 μm compared with 89 ± 28 μm for the Loa/Loa neurons (n = 60 and 55 neurons, respectively; P < 0.0001) (Figure 8A,B). Together with our earlier results [23], these results reveal that dynein processivity is necessary for axon elongation.
We previously reported that the Loa mutation specifically affects the processivity of dynein due to apparent miscoordination of the motor domains. We observed no change in the association of the mutant dynein with isolated neuronal membranes, further indicating that the physiological effects of Loa could be accounted for by altered mechanochemical activity alone [23]. However, because the cytoplasmic dynein regulators NudE, LIS1, and dynactin are also associated with brain development, we tested the mutant dynein further for its ability to interact with these factors. We performed pull-downs with purified proteins rather than immunoprecipitations with a dynein intermediate chain antibody, which might interfere with these interactions. Pull-downs from brain cytosol with recombinant glutathione S-transferase (GST NudE revealed no difference in the associated dynein or LIS1 between wild-type and Loa/Loa mutants (Figure 9A,C; 66.2 ± 8.8% and 57.7 ± 9.1% of heavy chain in the NudE pellet, respectively; P = 0.310). We similarly tested the GST-tagged CC1 fragment from the dynactin subunit p150Glued, which interacts with the dynein intermediate chain [37,38]. Comparable levels of wild-type and Loa/Loa dynein were again found in the CC1 pull-downs (Figure 9B,C; 45.3 ± 2.2% and 40.7 ± 10.7% of heavy chain in the pellet, respectively; P = 0.506). Therefore, the developmental defects we observe in the Loa/Loa mice likely reflect intrinsic abnormalities in the mutant dynein.
We find that Loa mutant mice exhibit defects in cortical and hippocampal development. Using live cell imaging techniques, we found direct evidence for a migration delay of bipolar neurons. Our results further indicate that this effect is not associated with defects in dynein interactions with LIS, NudE, or dynactin, and is therefore likely to result directly from the inherent mechanochemical defects caused by the Loa mutation [23].
The abnormal cortical lamination we observe in the Loa/Loa mouse brain is similar to that seen in LIS1 compound heterozygous mutant mice [5,9]. LIS1 mice exhibit severe disorganization of pyramidal cells in the CA1, CA2, and CA3 regions, as well as a reduced density of granule cells in the dentate gyrus [9,39]. We observe a clear delay in the migration of granule neurons in the Loa/Loa mouse to form the dentate gyrus. Although the formation of the dentate gyrus has not been examined in the LIS1 mice, a delay in granule cell migration could provide a potential explanation for the diminished size of the dentate gyrus in these mice.
We detected a decrease in axon extension in Loa/Loa neurons both in vivo and in vitro, as well as a decrease in axon length. Dynein as well as LIS1 are necessary for the reorganization and extension of the growth cone during axonogenesis [7,35,36]. These proteins are required to help microtubules extending into the peripheral zone of the growth cone resist retrograde actin flow [36]. A Loa mutant dynein detaches from microtubules about twice as frequently as the wild-type motor protein [23]. Therefore, microtubule penetration into the peripheral zone could be compromised, providing an explanation for altered axon elongation.
Altered expression of LIS, cytoplasmic dynein, and NudE have each been found to affect mitotic index in the developing brain [6,7,40]. These effects reflect direct roles for these proteins in mitosis [31,41,42]. Furthermore, LIS1 and cytoplasmic dynein RNAi interfere with apical migration of radial glial nuclei to the ventricular surface, which has been found to be essential for mitotic entry [7,43]. The similarity in mitotic index between Loa/Loa and wild-type mouse brains suggests that any effect on mitotic entry or progression must be relatively minor, though further live analysis of mitosis might still reveal altered mitotic behavior.
The current study indicates that the Loa mutation affects neuronal migration in a similar manner as reduced LIS1 expression [5,7,9]. However, the Loa mutation affects dynein processivity [23], whereas LIS1 contributes to dynein force production [44]. Our results indicate, therefore, that different defects in dynein function can lead to a common developmental outcome. The mechanical basis for this result is uncertain, but is likely a consequence of the coordinate behavior of multiple dynein molecules during high load functions. In in vitro studies, LIS1 was found to convert cytoplasmic dynein to a persistent force-producing state, though the level of force generation by individual dynein molecules was unaffected. However, force generation by multiple motors is summated, and this effect was further enhanced by LIS1 as revealed in in vitro laser trap bead assays and in computational simulations [44]. Thus, LIS1 appears to be ideally suited for a role in high load functions such as nuclear transport [12,44]. The Loa mutation, in contrast, had a clear effect on motor processivity [23]. How the Loa mutation might affect transport of high load structures is uncertain, but could also involve coordination of multiple dynein molecules. The Loa mutation results not only in decreased single molecule processivity in vitro, but also in decreased run-lengths for lysosomes/late endosomes in vivo, which are thought to be driven by an average of approximately five to seven dyneins [23,45,46]. Computational modeling indicates that, although cargo run length increases with motor number, the Loa processivity defect extends to the multimotor condition [23]. Thus, defects in neuronal migration could conceivably be explained by a reduction in run length during nuclear transport. We reason, however, that Loa should also affect aggregate force production by multiple motors, which is a reflection of the number of individual motors simultaneously 'engaged' with microtubules at any given time. This value should be reduced for Loa as a simple consequence of its increased microtubule detachment rate. Thus, the defect in neuronal migration we observe in the Loa/Loa mouse could result from shorter nuclear run lengths, decreased force, or both. The prolonged state of somal distortion we observe during neuronal migration in the Loa/Loa neurons could be indicative, in particular, of a reduction in nuclear translocation forces, but further work will be needed to directly determine the effects of the Loa mutation at the multiple motor level.
The defects we observe in the mutant neurons may explain the perilethality of the Loa/Loa mice. The Loa homozygotes die within 24 hours of birth due to an inability to move their mouths, and therefore feed. A 2-day delay in the migration of facial motor neurons to their destination may be responsible for this facial paralysis. Correspondingly, a delay in axon extension could also contribute to facial paralysis due to a lack of muscle innervation.
Our results reveal a clear effect of the Loa mutation on neuronal migration, in addition to its previously reported effects on retrograde vesicular transport and on motor and sensory neuron viability. Our observations establish the utility of the Loa mouse as a hypomorphic dynein mutant model for the investigation of not only neurodegeneration but also general cytoplasmic dynein function.
Mice were housed in standard cages on a 12:12 light:dark schedule at 25°C. All experimental research was performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC). All experimental analyses were performed on mutant animals with littermate-matched controls.
Brains were fixed in 4% paraformaldehyde for 24 hours after extraction, then transferred to phosphate-buffered saline (PBS) until sectioning. Brains were sectioned on a Vibrotome (Leica VT 1200S) into 50-μm thick slices. For staining with primary antibodies, slices were blocked at room temperature for 1 hour in PBS with 10% donkey serum and 1% Triton X-100. For antibodies requiring antigen retrieval (such as Foxp2), slices were boiled in 10 mM sodium citrate buffer (pH 6), then cooled back to room temperature before blocking. Primary antibodies were applied overnight at 4°C in PBS with 1% donkey serum and 0.1% Triton-X-100 at the following dilutions: anti-ctip2, 1:500 (rat; Abcam, Cambridge, MA, USA); anti-foxp2, 1:200 (rabbit; Abcam); PH3, 1:500 (rabbit; Millipore, Billerica, MA, USA); Prox1, 1:500 (rabbit; Abcam); Pax6, 1:200 (rabbit; Covance, Princeton, NJ, USA); and TujI, 1:500 (mouse; Covance). Slices were then washed in PBS and incubated with Cy2, Cy3, or Cy5 secondary antibodies (1:1,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Sections were mounted onto slides using VECTASHIELD HardSet Mounting Medium with DAPI (VectorLabs, Burlingame, CA, USA), then imaged on an Olympus IX81 inverted microscope. At least three brains per genotype were analyzed for each experiment.
For the BrdU birthdating assay in Figure Figure2,2, pregnant mice were injected intraperitoneally with BrdU at 100 μg/g of body weight at either E16 or E18. The BrdU-labeled females were left to give birth and their offspring were sacrificed on P0. BrdU-positive cells in the cerebral cortex were detected by immunostaining with an anti-BrdU antibody (1:200, rat; Abcam). This antibody required pre-treatment with 2N HCl for 15 minutes at 37°C prior to blocking. Staining was performed as described above. To analyze the BrdU-positive neurons in Loa/Loa mutants and their wild-type littermates, each brain section was divided into six equal regions, and the relative density and distribution of the BrdU-stained neurons in each region was plotted.
For the BrdU pulse-chase experiment in Figure Figure5B,5B, pregnant mice were injected intraperitoneally with BrdU at 100 μg/g of body weight at E16. The mice were then sacrificed 2 hours after the injection to analyze the number of BrdU pulse-labeled cells at the ventricular surface in Loa/Loa and wild-type embryonic brains.
Plasmids were transfected using intraventricular injection followed by in utero electroporation as previously described [25]. Briefly, 1 to 2 μl of GFP cDNA (concentration of 1 to 3 μg/μl) were injected into the ventricle of embryonic brains at E15. A pair of copper alloy oval plates attached to the electroporation generator (Harvard Apparatus, Holliston, MA, USA) transmitted five electric pulses at 40 V for 50 milliseconds at 1 second intervals through the uterine wall. The embryos were then sacrificed 3, 4, or 5 days later and their brains were prepared as described above. The number of GFP-labeled neurons at the ventricular zone, subventricular/intermediate zone, and cortical plate was plotted for Loa/Loa and wild-type littermates to assess the progression of neuronal migration from the ventricular surface.
These experiments were performed using a modified procedure of previously described methods [28,47]. Briefly, E16 brains were collected and sectioned coronally on a vibratome (Leica), then placed in a 50-mm MatTek dish (MatTek Corporation, Ashland, MA, USA) containing imaging medium (F12/MEM medium with 10% fetal bovine serum and 1 × penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) with 10 μg/ml of Oregon Green BAPTA-1 488 AM (Invitrogen). Slices were then incubated for 1 hour at 37°C with 5% CO2. After the incubation, the slices were washed in warmed PBS, then covered with a thin layer of Matrigel (BD Biosciences, San Diego, CA, USA) diluted 70 × in imaging medium, then incubated again for 30 minutes. The dish was then imaged in 1 ml of additional imaging media with 10 mM Hepes at 37°C with 5% CO2 on an Olympus IX81 inverted microscope. Images were captured every 10 minutes for 3 to 5 hours using an Hamamatsu ORCA-R2 CCD camera and Metamorph software (Molecular Devices, Sunnyvale, CA, USA. The speed of migration and length of axon were analyzed with Metamorph software. The position of the soma center was measured in each image and displacement of the soma was defined by the difference in location of the center between consecutive frames. Therefore, the somal speed is described here as the displacement per unit time. The length of the axon was defined as the distance from the basal end of the soma to the tip of the process. Live cell imaging experiments and axon length measurements were performed on three brains per genotype.
Whole brain lysates from P0 wild-type and Loa/Loa mouse pups were used for the NudE and CC1 pull-downs. For each experiment, the lysate was first pre-cleared with glutathione agarose 4B (USB Corporation, Cleveland, OH, USA) in order to prevent non-specific binding in the real assay. The beads were then spun down, and the pre-cleared lysate was used for the pull-downs. The pull-down solution contained the brain lysate, phosphate-glutamate buffer (10 mM sodium phosphate, 100 mM sodium glutamate, pH 7.0 with 1 mM MgSO4, 1 mM EDTA, 1 mM DTT, and 0.2% NP-40) up to 350 μl and either beads alone, 1 μg/μl GST-NudE [44] or 1 μg/μl GST-CC1 [37,38]. The solutions rotated at 4°C for 2 hours, then the beads were spun down to separate the supernatants and pellets. The supernatants and pellets from each pull-down (beads alone, NudE, or CC1) were collected and analyzed by SDS-PAGE and western blot probing for dynein heavy chain (rabbit, 1:500) [48] and dynein intermediate chain (mouse, 1:1500; clone 74.1 provided by K Pfister, University of Virginia, Charlottesville, VA, USA). In the case of the NudE pull-down, we also probed for LIS1 (mouse, 1:1500; clone 338, Sigma, St. Louis, MO, USA).
BrdU: 5-bromo-2'-deoxyuridine; CP: cortical plate; DIV: days in vitro; E: embryonic day; GFP: green fluorescent protein; GST: glutathione S-transferase; IZ: intermediate zone; Loa: Legs at odd angles; P: postnatal day; PBS: phosphate-buffered saline; PH3: phospho-histone 3; RNAi: RNA interference.
The authors declare that they have no competing interests.
KMOM and RBV designed experiments, KMOM performed experiments and analyzed data, and KMOM and RBV wrote the manuscript. Both authors read and approved the final manuscript.
We thank Richard McKenney for providing the GST-NudE and GST-CC1 constructs, as well as for useful discussion. This work was supported by NIH grants GM47434 and HD40182 to RBV and GM008798-09 to KMOM, and the Columbia University Motor Neuron Center.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Tradition Encounter Brief Global History ItemsMore than 100 items were found
Outlines & Highlights for Traditions and Encounters, Brief Global History, Volume I by Jerry Bentley, Herbert Ziegler, Heather Streets
Studyguide for Traditions and Encounters, Brief Global History, Volume 2 by Jerry H. Bentley, ISBN 9780073207032 (Cram101 Textbook Outlines)
Traditions & Encounters + AP World History Practice Test Booklet: A Global Perspective on the Past, AP EditionRentList Price:$176.56
List Price:$92.67
RentList Price:$16.13
RentList Price:$16.99
Emperors in the Jungle: The Hidden History of the U.S. in Panama (American Encounters/Global Interactions)RentList Price:$25.95
List Price:$7.89
List Price:$201.33
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Vertigo Photography Photographer
JK Digital DesignerRetoucher Male York, Pennsylvania, US
My Website: JK DesignsMy MM URL: http://www.modelmayhem.com/jkdigitaldesigner
Mayhem # 683475
About Me
Print work I can do is business cards, logos, flyers, posters, book jackets, album covers and almost anything else.
I'm also a part of Vertigo Photography so any photo work needed is okay to do and i have extensive knowledge of Photoshop. I can retouch portraits, add a creative flare to modeling portfolios or take pictures for album covers or anything where pictures are needed!
I can use flash for 2D animations or for web use or I can use HTML for web use as well.
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Vertigo Photography
YC Underground
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Cosenza has a steep, stair-filled centro storico that truly hails from another age. Wrought-iron balconies overlook narrow alleyways with old-fashioned storefronts and bars that have barely been touched by centuries of development. Flung haphazardly—and beautifully—across the top and side of a steep hill ringed by mountains, and watched over by a great, crumbling medieval castle, Cosenza also provides the best gateway for the Sila, whose steep walls rear up to the town's eastern side. Though Cosenza's outskirts are largely modern and ugly, culinary gems and picturesque views await in the rolling farmland nearby and the mountains to the east.
Don't go to these destinations in 2017—try our suggestions instead.More


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http://bit.ly/kKdcVi I am posting this link because the rules to get a free copy of a book were to link this on a blog or something. I am
Also, I have been doing Couch 2 5k . Next week will be my 3rd week and I am doing well with it. I actually really like it. I am still unsure if I see myself being a 5k runner, but I am going to try like hell
Everything & Nothing
Exactly what the title says
Wednesday, April 27, 2011
Wednesday, March 30, 2011
today I had the MUNCHIES~ I made the girls Fried French Fries and chicken nuggets and I could not keep my hands off of the french fries. I was going to go over on calories tonight, but the BFF talked me into my senses. I so need that girl in my life. I really was in a I DON'T give a shit kinda mood, but she reminded me that come Monday when the scales not saying good things that I only would have myself to blame. I love that girl!
It really sucks that some days this comes so easy and other days I just want to give in to every temptation. I guess I will just have these struggles for the rest of my life so I will have to get used to it.
It really sucks that some days this comes so easy and other days I just want to give in to every temptation. I guess I will just have these struggles for the rest of my life so I will have to get used to it.
Tuesday, March 29, 2011
Nothing exciting really has been going on. I did enjoy 2 pieces of PAPA JOHNS pizza today. One for lunch and one for dinner. It was soooooo good.
Monday, March 28, 2011
Quick post
Today I weigh 138.4!!!!!
Sunday, March 27, 2011
Weigh day
In the morning. I hope to be 138. I also did 3 miles today. It was cold, but I made it through and felt good when I was done.
Saturday, March 26, 2011
Back in track
Cheated yesterday and back on track today. Usually if I would have a cheat day it would lead to more cheating, but today I'm right back where I should be. It feels good to know I can do that on occasion and not have to worry. I will feel confident with it as long as the scale stays nice on Monday.
Friday, March 25, 2011
cheater cheater
I cheated on calories today. I shouldn't really let it bother me, but I FEEL SO GUILTY~ I went over by about 700 calories. The food was really good, but I wish I would have had more will power to not do it. Oh well, tomorrow is a new day and back on track I must be!~
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Category: Inspiration
5 Best Websites to send gifts online
Lets face it. Some of us aren't really that enthusiastic when it comes down to going out to shop for others. Like most of our problems, the internet has also provided a solution for this dilemma. There are numerous websites online that let you buy gifts for you loved ones and also to send them […]
30+ Inspirational 3D Illustrations
Designers seek ideas that might help locate a stupendous overflow of creativity, with a load full of imagination. Some seek inspiration while some hunt rapture & originality. And hence, they all prefer to jump-off to a kick start when a hard slogged design is on the line. This provides an obvious ground to majors like, […]
50 Tumblr Blogs for Inspiration
Want to blog? Use Tumblr, which says: "Tumblr celebrates creativity. We want you to express yourself freely and use Tumblr to reflect who you are, and what you love, think, witness, and believe." A popular social service, Tumblr is a great place to create your own blog, customize it and fill it with your own content […]
85+ Redesigns of Famous Logos
An established fact amongst marketeers worldwide is that the logo speaks in a way no other thing can, for a brand. Logos of some of the world's leading companies are household pictures for a reason. The simplicity of some, the high-end design of some or simply the millions of memories consumers attach to them make […]
15+ Kickass Free Web Fonts For Web Designers
Days of hard work doesn't pay off until it meets utter perfection. Same way how designing is left unfinished, without the arrangement of letters in style. Designing a website maybe crucial but what gleams it up is the, smart and wise use of typography. A font plays a significant character in any design. It can […]
25 Best Responsive Metro WordPress Themes
Metro Designs are becoming epidemic after the arrival of Windows 8. Although, Metro user interface was first introduced by Windows 7 phone, but it did not much influenced the market. Websites were being designed in the same old overused patterns until the release of Windows 8. Since then, websites are experiencing a rife of metro […]
6 Professional Music Recording Audio Processors
It is a hard fact that a software cannot enjoy the monopoly for a long time. Rivals keep on launching softwares as soon as first of its kind is pioneered and released into the market. Same had been the case with Music Recording Audio Processors. Since 1997 many music recording softwares have consistently been developed and […]
Trending Hot Popular
30 Useful HTML5 and CSS3 Form Designs
Forms are widely used as an essential component of the websites. It does not matter what sort of website you possess, it must be having at least one type of a form. There are different form types: contact forms, sign up/log in forms and questionnaires are some of its most used types. A nicely designed. […]
21+ Kickass Fonts For Designers (Newer)
Logos play an important role in contributing to the value of a brand or product. An overview of logo redesigns of popular companies of the world and new ventures alike will tell that simplicity and an educated font selection is the mantra. Reiterating the ideas expressed in previous posts, fonts have indeed become a lifeline […]
20 HTML5 & CSS3 Templates for Inspiration
The arrival of CSS3 and HTML5 in the purview of designers have immensely increased their productivity. In other word, the span of creative designing have been dramatically expanded by the arrival of this dual pack. Designers have now been able to inspire their imaginations to design more abstract designs that previously were not possible or […]


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Freak Me - Silk MP3
"Freak Me!" Written and produced by Keith Sweat, Freak Me was a Billboard Hot 100 #1 single in 1993 dominating the chart for 14 consecutive days. By the end ...
Silk - Freak Me MP3
Lyrics video.
Silk - Freak me MP3
lyrics---- Freak me baby aawww yea (4x) (Chorus) let me lick you up and down till you say stop let me play with you baby make you real hot let me do all the ...
Silk - Freak Me MP3
Album " Lose Control " - 1992 -
Silk - Freak Me Interlude MP3
Sex Video Here : http://goo.gl/h0wK8A.
Silk - Freak Me live at the sweat hotel MP3
the sweat hotel.
keith sweat ft silk - Freak me MP3
lyrics---- Freak me baby aawww yea (4x) (Chorus) let me lick you up and down till you say stop let me play with you baby make you real hot let me do all the ...
Silk - Meeting In My Bedroom (Video) MP3
2006 WMG Meeting In My Bedroom (Video)
Freak Me(Remix)- Silk MP3
1993. I like the remix much better. Lose Control was THAT album.
Freak Me Lyrics - Silk MP3
Silk - Freak Me (REMIX) MP3
Silk - Freak Me (Remix) Video.
SILK - Girl U For Me MP3
"Girl U For Me" was a released on the "B side" of our single, "Lose Control". Written by Keith Sweat, "Girl U For Me" helped push our debut LP into ...
Another Level - Freak Me MP3
Another Level's official music video for 'Freak Me'. Click to listen to Another Level on Spotify: http://smarturl.it/AnotherLSpotify?IQid=AnotherLFM As featured on ...
Freak Me
Another LevelFreak me baby (ah yeah) Freak me baby (yeah just like that) Freak me baby (c'mon.....c'mon) Freak me baby Let me lick you up and down Til' you say stop Let me play with your body baby Make you real hot Let me do all the things you want me to do Cuz tonight baby I wanna get freaky with you Baby don't you understand I wanna be your nasty man I wanna make[...]
Silk - Freak Me MP3
{DISCLAIMER} I Don't Own This Song No Copy Right Intended This Song Belongs To It Respective Owners Please Support The Artist By Buying Their ...
Freak Me Baby ~ Silk + Lyrics Below MP3
"Copyright Disclaimer Under Section 107 of the Copyright Act 1976, allowance is made for "fair use" for purposes such as criticism, comment, news reporting, ...
Silk ft. Teno West - Freak Me MP3
Freak Me / Silk Featuring Teno West [album version] Written by Roy Murray, Keith Sweat Produced by Keith Sweat Co-Produced by T.H. Executive Produced by ...
Nick Young's Here I Go Again vs. Iggy Azalea's Freak Me | Lip Sync Battle MP3
The battle between this power couple continues! Iggy Azalea breaks out a sultry performance of "Freak Me" by Silk. Nick Young retaliates with a rocking ...
Iggy Azalea performs Freak Me on Lip Sync Battle MP3
This week's battle is Iggy Azalea vs. Nick Young! We have a Sneak Peak of Iggy's sexy performance of "Freak Me" by Silk. Watch the full performance this week.
Silk - Freak Me (12 Remix) MP3
Album " The best of Silk " - 2004 -


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2035 Coit Avenue NE, Grand Rapids MI, 49505
Sold Price:
3 beds , 1 Full/1 Half baths
2,000 sqft
Status: Closed
Last Updated: 7/26/2017 5:43 PM
Description for 2035 Coit Avenue NE Grand Rapids, MI 49505
Welcome home to this nice ranch style home with all the charm of the past. This home features a nice kitchen, dining area, living room, Two nice size bedrooms 1-1/2 Baths. There is also a flex room along the back of the home that could be used as a 3rd bedroom/family room with a 1/2 bath. The lower level has a really nice size family room, non-conforming bedroom and a nice size storage area.
Sold Price:
Sold Date:07/29/2015
City Of Grand Rapids
1 Full/1 Half
2,000 (approx)
Listing Information
City Of Grand Rapids
Public Sewer
Natural Gas
School Information
Room Information
Eat-In Kitchen, Butler Pantry
Interior Features
Dishwasher, Dryer, Microwave, Range, Refrigerator, Washer
Ceiling Fan(s)
Gas Heat, Forced Air
Basement, Full
Exterior / Lot Features
Detached Garage
50 x 129
Sidewalks, Paved Road, Acreage
Driving Directions
West on Knapp, N on Coit.
Financial Considerations
Listing Price History
Grand Rapids Real Estate
Grand Rapids is located in Michigan. Grand Rapids, Michigan 49505 has a population of 195,097.
The median household income in Grand Rapids, Michigan 49505 is $39,913. The median household income for the surrounding county is $52,716 compared to the national median of $53,482. The median age of people living in Grand Rapids 49505 is 30.9 years.
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Our Journey
For over 80 years and three generations in manufacturing in various industries, the Tahincioglu family has experience in confectionery, real estate development including interiors and they are recognized as world-wide brand.
The family enjoys extending their industrial experience in the yacht sector, by providing one of the best services in building, high-quality custom and serial production yachts.
Beside new building projects, ICT Shipyard is also servicing the marine industry in refitting, restoring and remodelling yachts.


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Choose Between Two Options
- $25 for four group training classes ($60 value)
- $48 for four 25-minute personal-training sessions ($114 value)
See the schedule.
Fitnessology's crew of professional fitness consultants, nutrition counselors, and pain relievers works to help patrons meet their health and fitness goals with personal training, nutrition counseling, and small-group classes. The staff focuses on aiding clients in their quests for total-body wellness by teaching them how to integrate fitness, healthy habits, and thumb wars into their everyday lives. They are so committed to total wellness that they've created The Clinic—a subset of the Fitnessology facility that treats muscular pain and is staffed by a chiropractor, neuromuscular therapist, and corrective exercise specialist. Their thorough assessments help them understand pain before treating conditions that include lower-back pain, headaches, tennis elbow, and motion restrictions.


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Foreclosure Alternatives - Avoid Foreclosure, Short Sale, Deed-in-Lieu of Foreclosure
Foreclosure Alternatives So You Can Avoid a Foreclosure
Last Updated: March 15, 2016
A press release issued by MarketWatch in January of 2016 stated there were nearly 1.1 million U.S. properties with foreclosure filings in 2015, which was down 3 percent from 2014 to a new 9-year low. Since its peak of 2.9 million properties with foreclosure filings in 2010, it seems there has been a slow downward trend. But foreclosure activity was up in Boston, St. Louis, Dallas, Detroit, New York City, and Houston. States where foreclosure rates increased in 2015 were Massachusetts (up 55 percent), Missouri (up 50 percent), Oklahoma (up 36 percent), New York (up 24 percent), and Texas (up 16 percent). Foreclosure is financially and psychologically devastating to families and affects the stability of their lives but there are ways you can avoid going through a foreclosure.
Possible Reasons for Foreclosure
Many homeowners who cannot afford their house payments simply walk away because they don't realize that banks and mortgage companies may be willing to work with them in an effort to avoid foreclosure. Foreclosures are costly and time-consuming for financial institutions, and depending on the state in which you live, the process can take months. Besides valuable time, foreclosure costs a considerable amount of money, and lenders would much rather keep the lines of communication open and work with those who can't pay their house payments to find a mutually beneficial solution. They may not be aware of the alternatives, but there are some alternatives available for those who qualify.
Alternatives to Foreclosure
FORBEARANCE PLAN: Forbearance means you are allowed to delay or reduce mortgage payments on a temporary basis with the agreement that another option will be used at the end of that time to bring your account to current status. Most lenders have a forbearance program available, but you need to be diligent and immediately contact the lender to report a temporary loss, hardship and/or reduction in income and to request a forbearance agreement. Your lender, if in agreement, will then temporarily cease legal actions, or not proceed initially, whichever the case may be.
Lenders may agree to combine your forbearance with a reinstatement or a repayment plan if you can provide adequate assurance that you can raise the needed funds to bring your account current by a specified date. Be sure you are provided this agreement in writing. Forbearance programs are best for people who have experienced sudden unexpected income loss or debt, and simply need some extra time to readjust their spending and recover financially.
SHORT SALE: Those who can't afford to pay their house payments and know they can't afford to keep their home can request a short sale. A short sale is when a lender accepts a discount or shortage on a mortgage balance to avoid a possible foreclosure auction or bankruptcy. Depending on your particular situation, this could be one of the best alternatives to foreclosure. Mortgage companies and banks that offer a short sale as a option are sometimes willing to take less than the amount owed on the home.
Depending on circumstances, lenders are often willing to completely forgive the homeowner of the debt. In many cases, the homeowner and lender have certain provisions that must be adhered to when transacting a short sale. For instance, many lenders require the home to be on the market for a minimum of 90 days, have established maximum commission levels for any real estate agents involved, and may require the broker be a member of the Multiple Listing Service (MLS). A hardship letter explaining the reasons for financial problems as well as a list of debts and income are required, as well as any other bank-specific paperwork and forms. If you can't pay your house payments and can prove financial hardship, a short sale could be one of the most viable alternatives to your financial problems.
DEED IN LIEU OF FORECLOSURE: As a last resort, you may be able to voluntarily "give back" your property to the lender. This won't save your house, but may increase your chances of qualifying for a mortgage loan in the future. A deed in lieu of foreclosure may be a viable option for individuals who can verify they are incapable of paying their mortgage payments due to financial hardship. This option may save lenders the time and expense caused by homeowners who abandon their homes, and it can be one of the best long-term solutions for all concerned.
Before most lenders will offer a deed in lieu of foreclosure, a typical requirement is that the house must be listed for sale at fair market value. The homeowner needs to demonstrate that they are trying to find a solution, therefore lending credibility to the homeowner through the action of actively trying to sell the home. If the market is stagnant and the home doesn't sell, and you qualify for a deed in lieu of foreclosure, the lender may allow you to sign off on the house and walk away without owing on the mortgage.
The deed in lieu of foreclosure offers several advantages to both the borrower and the lender. The principle advantage to the borrower is that it immediately releases him/her from most or all of the personal indebtedness associated with the defaulted loan. The borrower also avoids the public notoriety of a foreclosure proceeding and may receive more generous terms that he or she would in a formal foreclosure.
The alternatives above are the three most common situations utilized to avoid foreclosure. Contingent on the type of mortgage loan you have, there may be others. Specifically: if you have a Federal Housing Insured (FHA) loan, you will want to contact HUD/FHA for guidance. The Department of Housing and Urban Development (HUD) has been instrumental in establishing guidelines to assist homeowners experiencing financial hardships. The goal is to offer financial alternatives to foreclosure, while allowing lenders to make determinations based on certain risk criteria.
Additionally, here is a link to a website which provides guidance applicable to homeowners with FHA Insured loans.
How Will These Alternatives Affect My Credit?
We went right to the myfico.com "Credit Education Center" to ask that question. Here is the answer verbatim:
"The common alternatives to foreclosure, such as short sales, and deeds-in-lieu of foreclosure are all "not paid as agreed" accounts, and considered the same by your FICO score. This is not to say that these may not be better options for you from a financial perspective, just that they will be considered no better or worse for your FICO score."
If you are considering bankruptcy as an alternative to foreclosure, that may have a greater impact to your FICO score. While a foreclosure is a single account that you default on, declaring bankruptcy has the opportunity to affect multiple accounts and therefore has potential to have a greater negative impact on your FICO score.


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Description: For the first time, a leadership expert has been able to quantify the bottom-line value of keeping your word: Companies with high measures of integrity outperform all companies by at least 2.5%., and exhibit: Deeper employee committment Lower turnover Superior customer service Higher profitability Tony Simon reverses the usual maxim: "Talk the Walk"-it is better to be honest about what you can and can't do rather than over promise and fall short. Beginning with scholarly research into "word-action alignment," Simons interviewed hundreds of employees in dozens of workplaces-and bases this book on focused work with 100 senior managers. Simons moves from individual integrity at the beginning of the book to creating an organizational culture of integrity in later chapters. Throughout, he uses real-world insight and feedback drawn from both line managers and CEOs. His model for organizational integrity is completely new and will reach the market at a critical time. Author plans to expand his model into a resource for training and assessment for all businesses.
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List price: $27.95Copyright year: 2008Publisher: John Wiley & Sons, IncorporatedPublication date: 10/6/2008Binding: HardcoverPages: 256Size: 6.50" wide x 9.25" long x 1.00" tall Weight: 0.990Language: English
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Giveaway is now over. What do you do with leftover wine (leftover what??!!) Put it in a cupcake! I can't think of a better happy hour treat that a Manischewitz Red Wine Cupcake. Plus, scroll down ...
Dark Chocolate and Sea Salt Matzah Truffles with Manischewitz Glaze
The ultimate Passover dessert. Dark Chocolate and Sea Salt Matzah Truffles with Manischewitz Glaze. Let that sink in for a moment. This is the second installment in my series for The Jewish Week ...
Matzah Bark
Looking for an easy Passover dessert? Matzah bark is here! I even covered it with glitter. Word is still out if glitter is kosher for Passover... I've mentioned before, but Passover desserts aren't ...
40+ Flourless Gluten Free Desserts for Passover
Over 40 Gluten Free Desserts perfect for Passover! And not even one recipe tastes like cardboard! This is beyond macaroons and jelly fruits, people. Tasty Passover desserts almost sounds like ...
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The six-storey hotel in Terre Haute houses 109 guest rooms, including four suites, each furnished with a convenience centre, featuring a fridge, microwave and coffeemaker. The spacious work desks at this hotel in Terre Haute, Indiana feature an ergonomic chair, phone, data port and voicemail. Guests will also enjoy complimentary high-speed Internet access, conveniently located at desk-level, as well as remote printing from each guest room to the business centre. Guests can enjoy evening in-room dining, a complimentary 24-hour business centre and a fitness centre. Meeting rooms, conference facilities and catering are also available.
Located in historic Downtown Terre Haute, immediately adjacent to Indiana State University and the IU School of Medicine, this Indiana hotel is only minutes away from the Rose Hulman Institute of Technology and St. Mary-of-the-Woods College. Major corporations in the area include Clabber Girl, Sony/DADC, Bemis, AET, Columbia House, the Eli Lilly-Clinton Lab, Thyssen Krupp and Presta, Pfizer, and Aisin Brake and Chassis. This Terre Haute, Indiana hotel is within walking distance or a short drive from many restaurants in the area. Guests can even spend the day shopping at the nearby Honey Creek Mall.
The hotel provides convenient guest rooms.
The hotel offers guest an indoor swimming pool, whirlpool and fitness center.
A freshly prepared breakfast is offered daily in the Great American Grill, also open for lunch and dinner. Room service is available.
The hotel accepts all major credit cards.
From Indianapolis, take I-70 west, take IN 46 north. At US 40, go west (left). US 40 turns into Wabash. The hotel is at corner of Wabash and Seventh Ave. Turn right on 7th to hotel entrance.
Room Facilities:
Direct dial telephone
Satellite cable TV
Internet access
Mini fridge
Tea Coffee
Iron and board
Double bed
King size bed
Individual airconditioned
Individual heating
Wheelchair accessible


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Wendy – Female Casting Agents
Released: February 1, 2013
Well known agent Wendy is interviewing a new male actor today. She explains that if he wants her to represent him she must see him naked in case that's what the role requires. She letches over his naked, toned body then calls in her two female colleagues. The women won't leave him alone, stroking his dick and sucking his cock until he explodes all over their faces.


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Many fans only dream of catching a foul ball, yet most never get their hands on one.
One lucky Tampa Bay Rays fan came away with a foul ball Sunday, but he didn't have the ball for long.
In the eighth inning of the Rays 3-0 win against the Miami Marlins, Drew Sutton fouled a ball into the stands, where it was caught by a fan. He gave the ball to his son, who proceeded to test out his throwing arm while the cameras were rolling.
That's one way to say happy Father's Day.
Daily Pitch is a baseball fan's destination for breaking news, emerging trends and enlightening banter. With a veteran group of reporters positioned in three time zones, we provide a balanced, comprehensive glimpse at our national pastime, with a nod to both the passion and complexities that make the sport irresistible to many. More about our reporters


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Benefits of sweating during the exercise
Sweating is the method that the body has to cool off your skin and organs that otherwise would overheat. For many people this turns out to be something nasty because the sticky feeling may remain on the skin after sweating, but it is not a process that should be avoided, since the skin is our largest organ and it must stay fresh.
Through sweat toxins and chemicals that may be harmful are eliminated, such as sodium, ammonia, urea, salt, sugar and potassium. Therefore its salty taste and when it comes to fall in your eyes, burns or itches a little.
During the day, a person can lose up to 10% of water in the form of sweat. This in warmer climates, such as the ones experienced in tropical countries or warmer states like Ecuador, Venezuela or Colombia, you might experience abundant sweating, especially during midday hours until the afternoon when the sun is most imposing moment and warms everything around it.
The amount of sweat that a man and a woman release throughout the day is very different, because of a very simple cause; men tend to be bigger than females. This also applies to overweight people, so Dr. Craig Crandall explains "... your body is larger, resulting in greater heat generation during the activity".
Why is good to sweat during physical exercise?
When people practice some sort of high-impact activity that requires physical effort or that is practiced at high temperatures as hot yoga, then your body temperature increases to the point that you need to freshen and go back to normality. As Dr. Crandall explains, "A high physical level allows a greater exercise load, which generates more heat and in turn results in more sweat"
This brings many additional health benefits because the skin needs to be cleaned from impurities that may penetrate during the day. When your heart activates, it sends more blood to organs such as your lungs and skin causing sweat production that permits contamination to go out from your body, and you can enjoy a healthy and smooth skin.
If we add the fact it also activates your immune system, producing white blood cells that protect and defend your body. At the same time, the risk of suffering from kidney stones is decreased since the salt accumulated in your body is discarded in the form of sweat. That being so, the value of exercise increases.
To enhance the production of heat in your middle zone, an Ann Chery girdle has the advantage of a latex coating that helps generating more heat, and facilitates the production of constant sweat while you wear it. But that's not all, its internal bones make this compression garment an effective and comfortable posture corrector that relieves back and lower back pain that may appear from keeping your back bent in unfavorable positions, either sitting or standing. They also have several levels of compression to suit all body types, and maintain their position throughout training sessions if you practice a sport, or during your household chores.
However, they shouldn't be used for more than four hours a day due to its high level of compression, and can eventually cause damage to your skin and internal organs when if your exceed the recommended hours.
Therefore the girdle turns out to be an ally for sweat production and fat. If you perspire when you exercise you will also lose fat all over your entire body such as your thighs. It's often noticed anti-cosmetic cellulite throughout this area and can be fought in many ways, for example continuously exercising since the more complete your routine is, the more effective it's going to be.
So sweat and don't worry. Just remember to clean the remnants after you refresh yourself since leaving dried sweat on your skin it's not recommended at all, because like makeup when much time left on your skin, it clogs your pores and can cause uncomfortable rashes.
Keep yourself hydrated
A very important fact which cannot be overlooked is that the body needs to continue to be hydrated even if you lose weight in fluids through urine, and sweat.
You can see that a pot of boiling water as long as it has enough liquid the fire will have something to heat, but when it's left too long it begins to evaporate. Unless water is added again, it will come to a point where there isn't any more left to be warmed and the pot might burn.
The same thing occurs to your body, even when it discards a certain amount of liquid through sweat you need to fill these reservoirs, either with energy sports drinks or fresh water so that you can have fuel to continue producing sweat, and clean the pores that have opened due to high body temperature.
What happens if I don't sweat?
If you are doing an activity that will require you much physically or you're in a very humid environment that causes sweating in your body, and this does not occur then you should seek medical help. As a consequence of this situation, you run a high risk of a heatstroke since the skin cannot cool to return to normal temperature.
Since externally you feel heat internally as well, organs feel rising temperatures and may be affected by them.
On the other hand, your skin cannot get rid of harmful toxins found in the environment, food or water that we daily deal with.
What happens if I sweat too much?
This condition is called hyperhidrosis, where excessive sweating occurs for no apparent reason when you feel extreme heat or a strong emotion. It usually occurs more expansively in your armpits, face and hands.
It is advisable that you speak to an expert on this subject, but one the best treatments proven to be quite effective is injecting Botox especially in the armpits to decrease the amount of sweat produced. While it is not a permanent solution, it is a way to attack the disorder.
Boost Sweat Production
A relaxing way to increase production of sweat is saunas; both traditional and infrared warm your body causing sweat generation that cools your skin and therefore clean pores and skin.
Infrared saunas are said accelerate the expulsion of toxins by 20% because they warm your body from within; unlike traditional ones that help release 3% of toxins because they heat from the outside in, making the process of releasing slower.
This type of treatment can then be used to train your muscles to relax and unwind in more comfortable surroundings. But before you try this technique, you must have the approval of your doctor since people who suffer from a heart condition should not come into saunas of any kind, because it is contraindicated.


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How to Survive in the woods
If you were lost in the woods tomorrow, would you know how to survive? In this video, Reggie Bennett from the Mountain Shepherd Survival School teaches us the skills for surviving in the wild. From how to start a fire to knowing which kinds of berries are safe to eat, this is the sort of info that it's vital to know in case of emergencies.


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It's been nearly three weeks since Joey Feek passed away after a courageous battle with cervical cancer. While her husband and family adjust to life without her, the blessings keep rolling in. It was announced yesterday that Joey+Rory's final album, Hymns That Are Important To Us, tops the Billboard Top Country Albums chart for the second week in a row.
Joey, prior to receiving her terminal cancer diagnosis in October, set out to fulfill her long-time dream of recording a hymns album. Joey fought through the side effects of surgery and cancer treatments to record the album, truly making it a labor of love.
"We recorded it in a studio in Nashville early in the summer – just after recovering from her first big surgery in Chicago. And then she did her vocals where she could… in hotel rooms, our house, wherever and whenever she felt up to singing," Rory wrote in November. "In early October, we even did a tv taping of the songs in the concert hall at our farm in front of a live audience. Joey was weak and it was difficult for her, but she was determined. This record means so much to her. It is the songs that are the most important to her."
Hymns That Are Important To Us, was released in early February just two days before the couple celebrated their first Grammy nomination. The album was an instant success with Cracker Barrel, who was selling exclusive deluxe editions, unable to keep copies on the shelf. So, it's no surprise that for the second week in a row, Joey+Rory's Hymns That Are Important To Us tops the Billboard Top Country Albums chart. The duo's album surpassed Chris Stapleton's Traveller, Sam Hunt's Montevallo, and the much-anticipated new release from Loretta Lynn, Full Circle.
We're sure that Joey never anticipated the success of her final album, but it has turned into a sweet gift and unexpected blessing for her loved ones who want her song to live on.
On April 3, Joey+Rory are up for the Academy of Country Music's Vocal Duo of the Year award. They are nominated alongside Brothers Osborne, Dan+Shay, Florida Georgia Line and Maddie & Tae. The 2016 ACM Awards will broadcast live on April 3 from Las Vegas on CBS at 8PM ET.
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Subject: Re: What version of tapestry-security and tynamo-federated accounts should I use with Tapestry 5.3?
Newsgroups: gmane.comp.java.tynamo.user
Date: Monday 3rd October 2011 23:42:34 UTC (over 5 years ago)
On Mon, Oct 3, 2011 at 2:41 PM, Greg Pagendam-Turner <[email protected]> wrote: > Well 5.3 will be released soon. How long do you think it will take to > update tapestry-security to work with it? AFAIK, tapestry-security works with 5.3 and we are running some integration tests against T5.3 continuously. However, I suspect you are hitting an issue with a combination of libraries and configuration you have in use. I'm planning on releasing a new version of tapestry-security shortly after a public version of T5.3 is available (note that the current alpha, beta releases are only available from Apache's staging repo which complicates things for CI systems). I'm also holding on to hopefully be able to integrate Shiro 1.2 to the the same release. Kalle > On 04/10/11 01:16, Kalle Korhonen wrote: >> I'd generally steer away from using snapshots. While T5 is still a >> beta without generally available releases, your mileage may vary. The >> stack trace below might be related to colliding exception handlers. >> >> Kalle >> >> >> On Fri, Sep 30, 2011 at 9:27 PM, Greg Pagendam-Turner >> <[email protected]> wrote: >>> Hi, >>> >>> I'm upgrading to tapestry beta 15 >>> >>> What version of tapestry-security and tynamo-federated accounts should I use >>> with Tapestry 5.3? >>> >>> I'm currently using: >>>


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Light Rail Collision Nets Bus Driver a Ticket for Running Red Light
Contrary to speculation in several media outlets, including ABC-15 News (KNXV) and the Arizona Republic, the driver of a bus that hit a light rail train yesterday did not have a green light, police said.
The Orbit bus driver received a citation today for running a red light at Lemon and Terrace streets in Tempe, writes Tempe police spokeswoman in a news release. That seems to jibe with an account by a student quoted in the Arizona State University Web Devil, who alleged the driver was making a call at the time of the accident.
That's bad news for the driver, but good news for motorists who wondered -- after reading media reports of this collision -- whether to trust green lights near the light rail trains.


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In the first part of this two-part review of Google Wifi, we kicked off proceedings with a look at creating a mesh network using ASUS and TP-Link's Google OnHub routers. Today, we'll take a look at the new Google Wifi points themselves.
Priced at $129 for one, with a three pack available for $299, Google is selling their Home Wi-Fi system at a considerably cheaper price than their competitors. Compare pricing to NETGEAR Orbi ($399 for two devices, $249 for the base router – see our review) and $499 eero (three pack – review) and, on paper, Google Wifi looks like great value. Ubiquiti Networks' AmpliFi HD (my review) is closer on price – $149 for the base router (which can be meshed with additional devices) and $349 for a three-point pack.
Google WiFi is designed around a dual-band 2×2 AC1200 router offering speeds up to 867 Mbps on the 5 GHz band and 300 Mbps at 2.4 GHz. The specs are broadly equivalent to the likes of eero and Luma, while AmpliFi HD – on paper – offers a slightly higher class AC1750 router.
NETGEAR Orbi is perhaps the most innovative design we've seen to date. It's a tri-band AC3000 device that dedicates on of its two 5 GHz bands to transmit data between the WiFi points. While that means that the network itself (as far as your clients are concerned) is AC1200, that dedicated link (known as a backhaul) has proven itself to offer faster satellite connections than Orbi's competitors.
Google Wifi is powered by a quad-core ARM CPU (of unspecified origin) with each core suporting speeds up to 710 MHz. There's 512 GB RAM in support alongside 4 GB eMMC flash RAM. Each WiFi point is equipped with two Gigabit Ethernet ports. While that's meagre compared to a traditional router (probably meaning that advanced users will require a network switch like the trusty NETGEAR GS108 I use with my OnHub) there's an important distinction to be made. Unlike several competitors, Google has enabled the Ethernet ports for use as backhaul connections. Those that have Ethernet wiring (or coaxial cables for MoCA 2.0 adapters) can take advantage of high-speed connections between their WiFi points, ensuring that speeds are optimised when connecting to remote WiFi points. Not all mesh WiFi systems offer this feature today and we'll be testing performance later with a wired and wireless backhaul to see just how big an advantage it is,
Like many of this new crop of home WiFi systems the hardware is small, compact and cute – a far cry from the traditional router designs with extensive arrays of external antennas. Like its close cousin, Google Home, users are encouraged to place Google Wifi points out on show, helping to ensure strong connectivity with other Wi-fi points and wireless devices around the home.
Google's recommendation is that a single Google Wifi point (the first point will replace your router and will connect to your Internet modem) will support smaller apartments and homes between 500 and 1500 sq. ft. A medium home (1500-3000 square feet) should need two Wifi points for strong coverage while the three pack is designed to serve homes between 3000-4500 square feet.
Completing the specifications are Beamforming support (helping direct wireless signals to your devices) and Bluetooth Smart. Unlike Google OnHub, the company has made no mention of 802.15.4 (Zigbee) smart home support (note that this radio hasn't been enabled on OnHub to date) but investigations at SmallNetBuilder have revealed that it's included. An integrated Infineon SLB9615 TPM (trusted platform module, the same chip fitted to Google OnHub) strengthens security – the device will refuse to boot if any unauthorised changes are made to its software.
Those comparing specifications with Google OnHub (handy rundown here) will note that Google Wifi is equipped with a slightly lower specification on paper (2×2 AC1200 vs 3×3 AC1900) but we'll see whether that translates into a different in real world performance. As discussed in Part 1 of the review, Google WiFi and Google OnHub are compatible and can be combined in a mesh network.
What's in the Box?
One benefit of the slightly humbler specification is that Google Wifi is certainly smaller than the already diminutive Google OnHub. Resembling a over-tall hockey puck, each device measures just 4.17 in (106.12 mm) in diameter and 2.70 in (68.75 mm) in height.
Google Wifi's packaging is as premium as the other devices recently launched under the "Made by Google" banner, Google Home and the Google Pixel/Pixel XL smartphone. The crisp white carton doesn't quite hit the heights of the TP-Link Google OnHub pack, but you're left in no doubt as to the quality you've purchased.
Each Wi-fi point ships with a 15W power adapter, 6 foot flat Ethernet cable (which is great) and a short quickstart guide (which basically says, "Plug it in. Download the app.")
This year, Google has designed a range of new hardware. Google Wifi follows the Pixel and Pixel XL smartphones, the Google Daydream VR headset and Google Home smart speaker. If you've been tracking these products then you'll know what a great job the company has done in their industrial design. Google Wifi is no exception.
The compact, ice-white puck is perhaps my favourite design from the crop of new Home WiFi systems hitting the market this year. It's certainly taller than eero (if a little narrower) but much more diminutive than the vase-shaped NETGEAR Orbi. Like Google Home, the device is designed to blend into your home environment. It's not entirely invisible, but the design is subtle enough to stand out. It's a shame it's not available with replacement covers, like Google Home (and Google OnHub).
The device is intersected by a thin, horizontal groove which serves as a status light. It glows subtly when the device is powered on and can change colour from blue and white to orange and on to red to announce any issues.
Flip the device over and you'll find the twin Gigabit Ethernet ports (one WAN, one LAN for the first Wi-fi point – two LAN for subsequent points) tucked away neatly with a USB-C port used for power. The reset button is located on the rear of the device.
Two Ethernet ports is an improvement on this device's predecessor, Google OnHub, but behind the four ports equipped on Amplifi HD (see our review). It's likely you'll need a network switch at some point if you're using smart home hubs for devices like Sonos, SmartThings or Philips Hue, so be sure to build that into your thinking when positioning the device. Importantly, as we discussed in the first part of this review, those Ethernet ports are enabled for backhaul duties, which should enhance network speeds across the mesh.
Overall it's a Google Wifi offers a simple design, but care has been taken to ensure the devices remain as neat and unobtrusive as possible.
28 comments
At LONG last, my ideal home Wi-Fi system is in sight. I DO have Ethernet throughout the house, but that has left the problem of connectivity with wireless-only devices — namely, phones and tablets — but also with laptops which could, conceivably, be wired, but are much more convenient when used wirelessly. I've tried Ethernet-wired access points and followed instructions for setup from the manufacturers as well as authorities listing cookbook approaches in forums, but I have always ended up with my printers (wired) and some other connected devices, like my Windows Home Server, on "different sides" of the network from tablets and phones, inaccessible. I'm not a computer pro, let alone a networking professional, so I've made little headway with my problems.
I think that a Google On-Hub router with wired Google Wi-Fi mesh extenders will finally, after years of fretting and finagling, give me the system for which I've been searching for what feels like eons.
Thanks, Terry, for this fabulous review!
Happy to have helped, Ron – best of luck with the new network!
Hi Terry, best review I've seen on Google WiFi, a lot of great info.
I currently have a Asus OnHub as the main router connected to three Google Wifi units. Works quite well. After seeing your review about wired backhaul from the Google WiFi units I'd like to give it a try.
My dilemma is that Ethernet cables or Moca in my house is not an option and was wondering if I could use some gigabit powerline adapters for the wired backhaul?
Hey Paul – really glad that you enjoyed the read.
You *could* try Powerline adapters, but to be honest, I think you'll probably get better performance from wireless backhaul. Powerline can be pretty flaky depending on your home wiring.
Thanks for the reply. I think I'll keep it the way it is, no reason to fix something that isn't broken in the first place. Happy Holidays!
Hi Terry, I enjoyed the review. I have a question for you. Will the Google Wifi or OnHub still function without internet? So say one day my ISP goes out during the day, will I still have local access? I was reading about Eero mesh routers and how if you don't have internet connectivity that they will not function. Well let me correct myself, if your eero is up and running and had connectivity before you lost internet, local access will still function. I was told by eero support that if you reboot the routers after you lost connectivity that when the routers come back online you will not have local network access functionality. Eero support mentioned they are working on "persistent" networking capabilities? have you done any of that kind of testing on the Google Wifi or Onhub devices?
I get pretty good results when connected to the Ethernet port. http://beta.speedtest.net/result/5895400602 http://beta.speedtest.net/result/5895413962
I deeply appreciate your research and testing. I have a problem in that I must have multiple SSID's (at least two, but three is better) on my networks. We have a renter in our basement, and have two duplexes we rent and want their traffic separate and private though sharing the internet connection.
We use one SSID for each "home" in the duplex, and one SSID for the smart home gear (nest thermostats/protects and Wink equipment (managing schlage smart locks, light switches, etc) with Kuna light/cameras. I love having one SSID across all three physical homes for smart home gear as it makes it easy to move gear around or set things up at my house and drop them in the other homes. Also makes it easy for my wife and our cleaners to have their internet "just work" at all three homes.
We have MoCA in two of the three houses. Currently using either two routers or gear from Open-Mesh (Cloudtrax for management) in the homes. I would love to switch to Google WiFi for performance/simplicity but the multiple SSID's is the hold up. With my need for multiple SSIDs what would you recommend? Does the Orbi support that? I would love to stick with ethernet for backhaul, but Google won't work for me. I've been patiently waiting for multiple SSIDs in vain… 🙁
Hi Aaron – all of these home WiFi systems are built with simplicity in mind. None of them are really going to be able to deliver what you're looking for. They're all single SSID (+ guest network) but you really need a device that allows you to manage multiple SSIDs and subnets. Really, your needs are more typical of a small business setup!
Terry, it seems like a number of the other mesh systems — Luma, Eero, and Plume as well as Google Wifi — also allow wired backhaul. Do recommend Google over the others for this application?
I am weighing one of these mesh systems with wired backhaul versus POE WAPs. I live in a recently renovated home with ethernet jacks, and, though I have almost no networking knowledge, I chanced it a couple of years ago and installed Ubiquiti Unifi APs with a Ubiquiti POE router. It was a little scary getting it going, but then it ran without a thought or hiccup for two years straight, until I turned on the automatic firmware updates on the APs, and they all died. My efforts to troubleshoot with Ubiquiti about this were pointless and infuriating, and since they were out of warranty, Ubiquiti didn't bother to respond to my emails. It's a little hard to buy more Unifi's after that experience, and with young kids at home I would like some of the parental control features of the consumer mesh systems, but I wonder if you think I should swallow my pride and stick with Unifi APs.
FYI, I posted a similar question on thewirecutter's review of mesh products. One responder reported that switching from a Ubiquity Unifi setup with 3 AC Pro access points to 3 Google Wifi units with wired backhaul yielded improved range and speed. I'm surprised but excited to give it a try whenever GWifi starts shipping again.
So, for a two story home with the modem coming in to the lower level, would you recommend the Google WiFi (3 positions) or two Onhub units (1 up and 1 down)? Sounds like it would be pretty much a wash. House is not wired throughout.
Whichever is cheaper, TBH. Two OnHubs would provide ample coverage and would be slightly faster with faster wireless clients.
But, you may find a single device covers you in a two storey home. I'd start with an Onhub and then add a second device if needed.
Thanks Terry, Happy Holidays!
Great review Terry.
I'm running a mesh of 3 ASUS OnHubs and things are pretty great. It's just as fast as the ASUS RT-AC5300 for me (500mbit synchronous Ethernet).
My only issue is finicky band steering where devices get pushed onto 2.4 Ghz for no reason and get stuck there. I have to toggle the wifi off/on on the device and then I get 5 Ghz again.
I think this is a firmware bug, but I'm not sure.
Pricing aside, would you recommend google wifi over eero? Also, were your tests conducted with eero 2.0 firmware? Great review by the way!
Thanks! The eero review I did was firmware 1.x. Performance was similar, but the way you frame the question highlights the issue – price.
I found eero to be less stable than Google WiFi, so there are a couple of reasons I'd take GW over eero.
thanks for the awesome review, this indeed is exactly what i am looking for, but i have a single question
router is connected to WAN and chained to port1 of node1, node1 port2 is connected to node2 port1
will that work ? having node one connecting the router with the furthest node2
and i noticed that the radio speeds are 1200, how much is that effect compared to the 1900, 2600 that regular routers offers ?
i have wired connections, but would love the hassle free setup/managment for all the nodes in my network
did you connect the mesh Onhub to ethernet via LAN or WAN? WAN has been shown to slow down ethernet backhaul speed.
Is it possible to connect a switch to the Orbi satellites or Google's Wifi secondary points to connect more than four wired devices?
Hi I was WO daring which would you recommend? Getting rid of current ISP router and then getting three google WiFi nodes. Or getting rid of ISP router and getting an onhub and two google WiFi nodes or getting two more on hubs instead? And BTW I agree with you on the fact that google severely failed on the smart home aspect.
Thanks for this thorough review. Glad you checked the wired backhaul numbers. Netgear says they plan to add that to their devices. It could be awesome if do it right.


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