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text:
Weekend of July 13 – Mid Summer Sportsman Auction
2 Big Sessions
Firearms will be sold live @ 9 am Saturday July 13
Sporting equipment will be online only @ 7 Pm Saturday July 13
Firearms: Long Guns, Pistols, Revolvers, Muzzle Loaders; Ammunition; Archery, Reloading, Mounts, Pocket Knives, Fixed Blade Knives, Fishing Boats, ATV's, Etc.
If it is sporting related just give us a call or send us a picture of it. One item or one hundred items, we will sell it all for you!!! We already have over 100 Firearms, Tree Stands, Gun Cases, Game Calls, Mounts and More!!! Let us add your items to this auction!! Everything sells absolute!!! It's time to get ready for Fall Hunting Season NOW!!!
Firearm Brands already here: Colt, Glock, Sig Sauer, Remington, Winchester, Ruger, Smith & Wesson, Hi-Point, Davis Ind., Raven Arms, Iver Johnson, J.C. Higgins, H&R, Stevens, Savage, Mosin Nagant, Walther, Mossberg, Jennings, Bersa and Lots, Lots More!!!

subset: commoncrawl


text:
Fundraising campaign by ahmed bakhit
by ahmed bakhit
Assiut, eg
Assiut, EG
| Weddings & Honeymoon
My dear brothers,
I'm supposed to get married this month And I've got many debt has to be repaid
so that I can hold my wedding :(
I ask you to help me in any amount you can
The amount that I needed to pay off debts and expenses of about 450$
I do not ask you this amount because many
But I ask you to send me any amount could help me
do and I'll be very thankful to you
And I shall take you to this religion and will refund the money to you someday
Waiting for your reply and your help
Accept greetings to you brothers
ahmed bakhit
Campaign Owner
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Owayis Diabat
Following Since Feb 12, 2017
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PDCD4 inhibits translation initiation by binding to eIF4A using both its MA3 domains
Communicated by Charles C. Richardson, Harvard Medical School, Boston, MA, December 28, 2007 (received for review November 14, 2007)
Programmed Cell Death 4 (PDCD4) is a protein known to bind eukaryotic initiation factor 4A (eIF4A), inhibit translation initiation, and act as a tumor suppressor. PDCD4 contains two C-terminal MA3 domains, which are thought to be responsible for its inhibitory function. Here, we analyze the structures and inhibitory functions of these two PDCD4 MA3 domains by x-ray crystallography, NMR, and surface plasmon resonance. We show that both MA3 domains are structurally and functionally very similar and bind specifically to the eIF4A N-terminal domain (eIF4A-NTD) using similar binding interfaces. We found that the PDCD4 MA3 domains compete with the eIF4G MA3 domain and RNA for eIF4A binding. Our data provide evidence that PDCD4 inhibits translation initiation by displacing eIF4G and RNA from eIF4A. The PDCD4 MA3 domains act synergistically to form a tighter and more stable complex with eIF4A, which explains the need for two tandem MA3 domains.
PDCD4 is a tumor-suppressor protein that is up-regulated on induction of apoptosis (1) and down-regulated in certain aggressive tumors (2). PDCD4 is controlled by protein kinase S6K1 and the ubiquitin ligase SCFβTRCP, and its degradation is necessary for efficient protein translation in vivo, which is a prerequisite for cell growth and, consequently, for cell proliferation (3).
PDCD4 is known to bind two eukaryotic translation initiation factors eIF4A and eIF4G (4–6). eIF4A is an RNA helicase that works as a subunit of eIF4F, a complex composed of eIF4G and eIF4E. The helicase activity of eIF4A itself is weak but is enhanced upon binding to eIF4G (7, 8). eIF4G has two independent binding sites for eIF4A (9), one in the conserved middle domain (eIF4G-m, HEAT1/MIF4G) (Fig. 1), and the other in the adjacent second HEAT domain (eIF4G-MA3, HEAT2/MA3) (reviewed in ref. 10). NMR binding studies have shown that eIF4G-m interacts mainly with the C-terminal domain of eIF4A (eIF4A-CTD) (11), whereas eIF4G-MA3 binds to the N-terminal domain of eIF4A (eIF4A-NTD) and only weakly to eIF4A-CTD (A.M., C.S., K.A.E., and G.W., unpublished work). Mutation and deletion analysis indicates that the interaction of eIF4A with eIF4G-m is necessary for translation, whereas the interaction of eIF4A with eIF4G-c (eIF4G-MA3+HEAT3 domain) plays a modulatory role (12).
PDCD4 contains two MA3 domains after an N-terminal segment of little known function (1). MA3 is a well conserved α-helical motif with typically 3–5 helical hairpins and is a subtype of HEAT domains. A single MA3 domain is found in eIF4G-c (eIF4G-MA3, Fig. 1), which has been reported to also bind eIF4A (10, 13). Recently, crystal and solution structures of mouse PDCD4's C-terminal MA3 domain (MA3-c) were reported (14, 15). MA3-c was shown to bind eIF4A, compete with eIF4G-c, and was sufficient to inhibit translation initiation. However, some questions remained. First, the function of the N-terminal MA3 domain (MA3-m) was unclear. Mutations of conserved amino acid residues in either MA3-c or MA3-m affect eIF4A binding, which implied that both PDCD4 MA3 domains have eIF4A-binding abilities and contribute in translation inhibition (16). NMR experiment revealed that MA3-c binds to eIF4A-NTD through the loop between α5 and α6 and the turn linking α3 and α4 (15), which is well conserved among MA3-c, MA3-m, and the eIF4G-MA3, also supporting a direct role for the MA3-m domain in eIF4A binding and inhibition. Despite these observations, however, the exact function of MA3-m remained unclear. Second, no structure was available for MA3-m. The sequence homology between MA3-m and MA3-c suggests a similar fold but the extent of similarity was not clear. Furthermore, it was open whether the two domains have similar or complementary function, and why PDCD4 needs two MA3 domains.
Here, we report the crystal structure of the PDCD4 MA3-m domain and its NMR-derived eIF4A-binding face. We show that the PDCD4 MA3 domains compete with the eIF4G-MA3 and with RNA for binding to eIF4A. Both the structures of the two MA3 domains and their eIF4A-binding faces are very similar. However, the two PDCD4 MA3 domains act synergistically to form a tighter and more stable complex with eIF4A, which explains the need for two tandem MA3 domains.
MA3-m Is Structurally Similar to MA3-c but Contains an Additional C-Terminal Helix.
A series of truncation mutations were carried out to identify the domain boundaries of human PDCD4 MA3-m. The region 157–302 (Fig. 1) exhibited the best NMR spectra and was thus assumed to contain the intact MA3-m domain. Backbone resonances for 142 of 158 nonprolines could be assigned by using triple-resonance NMR experiments (reviewed in ref. 17). Similarly, 114 of 127 nonproline residues of human PDCD4 MA3-c were assigned. A few short segments are invisible in the NMR spectra presumably because of line broadening due to conformational exchange. Chemical shift analysis indicates that MA3-m contains eight helices [supporting information (SI) Fig. 6A ].
Both native and Se-Met MA3-m were crystallized. MA3-m (Se-Met) crystals belonged to space group P212121 with cell axes a = 37.68 Å, b = 70.06 Å, c = 110.88 Å, α = β = γ = 90° with two monomers per asymmetric unit (SI Table 1). The structure was solved at 1.7 Å resolution and is shown in Fig. 1. The domain consists of a stack of four α-helical hairpins, displaying a similar conformation as PDCD4 MA3-c (14) and eIF4G-MA3 (18). The crystal structure reveals that the folded part of MA3-m extends between residues 161 (α1) and 302 (α8) and is ≈20 residues longer than previously predicted by analogy to MA3-c (14). The C-terminal 18 residues of the MA3-m fragment (after α8) have high amino acid sequence homology to helix α9 in eIF4G-MA3. However, this region was not visible in the electron density and also appears unstructured in the NMR data (SI Fig. 6A ).
Both PDCD4 MA3 Domains Bind to eIF4A-NTD but Not to eIF4A-CTD.
To test for the interaction between the two PDCD4 MA3 domains (MA3-m and MA3-c) and eIF4A domains (eIF4A-NTD and eIF4A-CTD), four separate NMR titration assays were performed. [15N,1H]TROSY-HSQC spectra were recorded for uniformly 15N-labeled MA3-m or MA3-c. Increasing amounts of nonlabeled eIF4A-NTD or eIF4A-CTD were added to each MA3 domain, and the backbone chemical shift changes were monitored. Distinct changes were observed for both MA3-m and MA3-c when eIF4A-NTD was added (Fig. 2 A and B). No significant changes in the peak positions were observed when eIF4A-CTD was added to either MA3-m or MA3-c. (Fig. 2 C and D). This indicates that both PDCD4 MA3 domains bind specifically to eIF4A-NTD but not to eIF4A-CTD.
Both MA3-m and MA3-c Bind to eIF4A-NTD with a Similar Surface.
Chemical shift changes of backbone amide signals of PDCD4 MA3 domains upon adding eIF4A-NTD are summarized in Fig. 3 A. The final spectra obtained in the NMR titration assays (MA3-m/-c–eIF4A-NTD ratio 1:2) were used for data analysis. To visualize the eIF4A-binding surface on both MA3 domains, we mapped the residues with distinct chemical shift changes onto the respective crystal structures. Affected residues are localized primarily in two adjacent regions, forming a contiguous surface (Fig. 3 B and C). It is located in the bent portion of helix α5 and the following loop between α5 and α6. The second region is located in the end of α3 and the connection to α4. Thus, MA3-m and MA3-c have similar binding surfaces for eIF4A-NTD. A few of the residues that showed significant chemical shift changes are located in the middle portion of α5 and α6 (L243, F264, R267 for MA3-m, I412, E431, E432 for MA3-c), which presumably reflects slight conformational changes in α5 and α6 upon eIF4A-NTD binding. MA3-m has an additional small group of residues in the loop between α7 and α8 that were affected by eIF4A-NTD binding (K283, G284). This will be further analyzed in Discussion.
PDCD4 MA3 Domains Efficiently Compete with eIF4G-c and RNA for Binding to eIF4A.
Several reports have shown that PDCD4 inhibits the eIF4A-eIF4G interaction, and recently MA3-c alone was shown to compete with eIF4G-c (14, 16). To confirm this MA3-c inhibition, and to investigate whether MA3-m also competes with eIF4G-c for eIF4A binding, NMR competition assays were used. [15N,1H]TROSY-HSQC spectra of 15N-labeled eIF4A-NTD (≈26 kDa) were recorded (SI Fig. 7A ), whereas unlabeled GB1-tagged eIF4G-c and/or unlabeled PDCD4 MA3 domains were added (SI Fig. 7 B–F ). When a stoichiometric amount of eIF4G-c (≈54 kDa) was added to eIF4A-NTD, a significant decrease in the signal intensities was observed, due to formation of the high molecular mass eIF4A-NTD–eIF4G-c complex (≈80 kDa, SI Fig. 7B ; red, remaining peaks). Addition of an equivalent amount of PDCD4 MA3-m (SI Fig. 7C ) or MA3-c (SI Fig. 7D ) to the eIF4A-NTD-eIF4G-c mixture resulted in the reappearance of some peaks. When fourfold excess of MA3-m or MA3-c was added to the mixture, most of the signals recovered with a spectrum pattern virtually identical to that of the eIF4A-NTD–MA3-m/MA3-c complexes (SI Fig. 7 E–F ). The recovery of the signals indicated that MA3-m and MA-c competed with eIF4G-c and formed the eIF4A-NTD:MA3-m and eIF4A-NTD–MA3-c complexes (≈43.7 kDa for MA3-m and ≈40 kDa for MA3-c).
PDCD4 is known to inhibit cap-dependent translation initiation by interacting with eIF4A. To investigate the mechanism of inhibition of eIF4A helicase activity by PDCD4, we tested whether PDCD4 competes with RNA for binding to eIF4A or forms an inactive PDCD4–RNA–eIF4A ternary complex. Because a single MA3 domain is sufficient to inhibit cap-dependent translation, and the MA3 domains bind specifically to eIF4A-NTD, we used MA3-m, eIF4A-NTD, and U6-RNA in this experiment. Again, a [15N,1H]TROSY-HSQC spectrum of 15N-labeled eIF4A-NTD was recorded as a control (SI Fig. 8A ). When twofold excess U6-RNA was added, significant changes in the signals were observed, indicating the formation of eIF4A-NTD–U6-RNA complex (SI Fig. 8B , red). When fourfold excess PDCD4 MA3-m was added to the mixture, the spectrum changed again (SI Fig. 8D , red) and became virtually indistinguishable from that of the eIF4A-NTD–MA3-m complex in the absence of U6-RNA (SI Fig. 8C , red). These peak pattern changes indicate that MA3-m competed with U6-RNA and displaced it from its complex with eIF4A-NTD. As a control, we verified that neither MA3-m nor MA3-c binds to U6-RNA by NMR and fluorescent polarization, using the same concentration in both experiments (data not shown).
Intramolecular Interaction of PDCD4 MA3 Domains.
To test whether there is intramolecular interaction between MA3-m and MA3-c, we performed binding experiments with one of the MA3 domains 15N-labeled and the other unlabeled. No changes in the spectra were observed upon addition of the counterpart for both combinations (data not shown), indicating that MA3-m and MA3-c do not interact in trans. We then expressed a MA3-dual domain construct (157–449), which contains MA3-m and MA3-c. The [15N,1H]TROSY-HSQC spectrum of MA3-dual showed overall line broadening, presumably because of the large molecular size (>30 kDa), but overlays of the spectra of MA3-m, MA3-c, and MA3-dual showed some distinct chemical shift changes in peaks from both domains (SI Fig. 9A , marked by circle). The changes in the signals are mainly located at the N termini of α4, α6, and the middle of α8 for MA3-m and α1 and α2 for MA3-c (SI Fig. 9 B and C ). Analysis of the electrostatic surface potential of the domains shows a broad positively charged area in MA3-m (Fig. 1 D) and a broad negatively charged surface in the N-terminal region of MA3-c (14). These areas overlap with the regions where distinct chemical shift changes were observed in MA3-dual, which suggests electrostatic interaction between the two MA3 domains in cis.
Tandem MA3 Domains Bind Synergistically to eIF4A.
PDCD4 MA3-m, MA3-c, and MA3-dual were immobilized onto a sensor chip to ≈2,000 response units (RU), and various eIF4A fragments were passed over. First, binding of eIF4A fragments to immobilized MA3-dual was confirmed by flowing 100 μM eIF4A-NTD or eIF4A-CTD and 50 μM eIF4A-FL (Fig. 4 A Lower). The concentration of eIF4A-FL was chosen (50 μM) to keep the total concentration of eIF4A domains constant for a fair comparison. eIF4A-FL showed the highest increase in RU for all PDCD4 MA3 domains, whereas moderate RU increase were observed for eIF4A-NTD, and no apparent binding was observed for eIF4A-CTD.
The binding affinity between MA3-dual and eIF4A-FL was strikingly higher than all other combinations. MA3-dual showed 5- to 6-fold greater increase in RU for 50 μM eIF4A-FL compared with MA3-m or MA3-c, both of which have a similar moderate affinity (Fig. 4 A Upper). Among the three eIF4A fragments tested, eIF4A-FL showed higher binding affinity to the single MA3 domains, but the binding affinity between eIF4A-FL and MA3-dual stands out among all (Fig. 4 A Lower).
To analyze the effect of tandem MA3 domains versus single MA3 domain, a more precise binding analysis was performed by using MA3-dual and MA3-m. eIF4A-NTD and eIF4A-FL were passed over the sensor chip with concentrations ranging from 0 to 50 μM and from 0 to 20 μM, respectively (Fig. 4 C and D).
When MA3-dual and MA3-m were compared for their eIF4A-NTD-binding affinity, MA3-dual binds somewhat stronger than MA3-m (Fig. 4 D). When compared for their eIF4A-FL-binding affinity, the difference was much more significant, and MA3-dual showed ≈8-fold greater RU increase at 20 μM eIF4A-FL, compared with that of MA3-m (Fig. 4 C). Because relative numbers of immobilized MA3 domains are set to be equal between MA3-dual and MA3-m, the RU increase caused by binding directly corresponds to the affinity differences between the two fragments. Fig. 4 D clearly shows that each MA3 domain of MA3-dual has a capacity to bind individually to different eIF4A-NTD molecules, because the RU increases for MA3-m and MA3-dual were comparable. When MA3-m is compared for its binding to eIF4A-FL and eIF4A-NTD, >3-fold increase in RU was observed for eIF4A-FL compared with eIF4A-NTD. Considering the molecular mass difference between eIF4A-FL (46.2 kDa) and eIF4A-NTD (26.6 kDa), the actual increase in the binding is ≈1.5-fold. This indicates that binding to eIF4A-FL is somewhat stronger than binding to eIF4A-NTD.
PDCD4 has been known to bind eIF4A and inhibit eIF4A-mediated translation initiation (4, 6). Based on the findings that PDCD4 prevents eIF4A binding to eIF4G-c and that PDCD4 binds to eIF4G-m in the absence of eIF4A, a possible inhibition model has been proposed, which implies two alternative inhibitory functions for PDCD4 (6): (i) PDCD4 prevents eIF4G-c from binding to eIF4A and blocks stimulation of eIF4F activity and (ii) PDCD4 binds to eIF4A and traps the inactivated eIF4A on the eIF4G-m surface, which inactivates the eIF4F complex.
Further studies revealed that PDCD4 MA3 domains play important roles in eIF4A binding and inhibition of translation (16), and the latest report showed MA3-c alone is sufficient for inhibition of eIF4A translation (14). However, the detailed functions of MA3-c and the rest of PDCD4 are still unclear. The structure of the MA3-c domain of mouse PDCD4 that has >90% homology to that of human has been solved by both x-ray crystallography and NMR (14, 15), but a structure of MA3-m has not been reported so far, and the specific functions of the two MA3 domains have been unresolved. Whether the two MA3 domains bind to eIF4A simultaneously, and how these two MA3 domains function in eIF4A binding and inhibition of translation is also not clear. In addition, PDCD4 was reported to bind eIF4G-m, but whether the PDCD4 MA3 domains are involved in the binding remains to be answered.
Structural Features of PDCD4 MA3-m and Comparison with MA3-c.
We have solved the crystal structure of the human PDCD4 MA3-m domain. MA3-m was found to consist of four helical hairpins and is longer than MA3-c. Thus, its size is intermediate between PDCD4 MA3-c and eIF4G-MA3, which have three and five helical hairpins, respectively. Superimposition of MA3-m and MA3-c clearly shows that they adopt similar secondary and tertiary structures. (SI Fig. 6C ) It had been predicted in a previous report (14) that a proline residue in the middle of helix α3 (P200) might cause disruption or a bend in the helix conformation, resulting in overall structural change. Looking at MA3-m crystal structure, a slight bend in the helix α3 with the angle ≈20° was observed at the position of P200. No overall distortion of the structure was caused by P200, and the orientation of the helices is conserved.
Computation analysis of MA3 domain boundaries of human PDCD4 predicts MA3-m to be 164–275, and MA3-c to be 327–440 (SMART, simple modular architecture research tool). The region between the two domains is predicted to be a linker containing another α-helix. Upon inspection of the actual 3D structures of MA3-m and MA3-c, the observable last helix in MA3-m ends at the position L302, and the first helix in MA3-c begins at H326. There are ≈23 aa residues between the two MA3 domains, and no additional α-helix was found for this region both in the crystal structure and NMR data. This region might purely act as a flexible linker or might form an additional α-helix when MA3-m and MA3-c are tethered.
eIF4A Interaction with PDCD4 MA3 Domains.
NMR titration assays showed that both MA3 domains bind specifically to eIF4A-NTD and use homologous epitopes. (Fig. 3 B and C). This is consistent with existing mutation data: Mutations that have been reported to inhibit the interaction with eIF4A (E249 and D253 for MA3-m, D414 and D418 for MA3-c) are located within these surface areas. However, a second set of residues, whose mutation also affected eIF4A binding are located in α2 (D180 and L191 in MA3-m and D343 and L354 in MA3-c) and were not in the regions affected by eIF4A-NTD binding (16). This is consistent with the finding that the corresponding region of the human eIF4G-MA3 contacts eIF4A-CTD and not eIF4A-NTD (A.M., C.S., K.A.E., and G.W., unpublished work).
K283 and G284, which are located in the loop between α7 and α8 of MA3-m, were also affected by eIF4A-NTD binding (Fig. 3 A). This loop does not exist in MA3-c and is somewhat separated from the main interaction surface. The backbone signals were significantly broadened for the C-terminal half of the loop (V286–C288) and the N-termini of α8 (A293–A295), which implies that this region is in conformational exchange. Upon eIF4A-NTD binding, the α7-α8 loop may move closer to the main interaction surface and form an additional binding interface adjacent to the main surface. This additional interface may lead to a tighter binding between MA3-m and eIF4A-NTD.
Recently, mutations in eIF4AII were reported that affect PDCD4 binding (19). As described, most of the mutated residues are buried inside of the protein and are likely to affect the structure of eIF4AII but are unlikely to be part of the binding epitope.
PDCD4 Displaces eIF4G-c and RNA from eIF4A.
A recent study has shown that MA3-c alone can efficiently compete with eIF4G-c (MA3 + HEAT3) for binding to eIF4A (14). Considering the remarkable similarity between MA3-m and MA3-c, we were intrigued by the possibility that MA3-m might also compete with eIF4G-c, and indeed it does efficiently so as revealed with NMR competition assays.
Because eIF4A-NTD has an RNA binding site, there is a possibility that PDCD4 MA3 domains occupy the same binding site and inhibit RNA binding. Using an NMR competition assay, we found that MA3-m efficiently competed with RNA, and the RNA binding to eIF4A-NTD was abolished when twofold excess MA3-m was added to eIF4A. This means that PDCD4- and RNA-binding interfaces on eIF4A-NTD are at least partially overlapped. Because PDCD4 MA3 domains and eIF4G-MA3 share the same binding interface on eIF4A-NTD, this could indicate that eIF4G-MA3 also competes with RNA. However, when eIF4G-MA3 was used instead of MA3-m, no competition was observed under the same experimental conditions (K.A.E., A.M., C.S., and G.W., unpublished data). Thus, eIF4G-MA3 has either a binding interface that overlaps with PDCD4 but does not compete with RNA, or it has a weaker affinity to eIF4A and was not potent enough to compete with RNA under the same conditions as MA3-m.
Whereas eIF4G-MA3 is dispensable for stimulation of eIF4A helicase activity and is thought to have only a modulatory function, point mutations in this domain were reported to result in 76% decrease in translation, a much more severe defect than when the entire C-terminal region of eIF4G is deleted (12). Although the reason for this phenomenon is unclear, it suggests two possible inhibitory mechanisms for the PDCD4 MA3 domains: (i) interference with the modulatory function of eIF4G-MA3 and (ii) blocking RNA from binding to eIF4A-NTD. These two possible inhibitory mechanisms are not mutually exclusive (Fig. 5 C).
Regions from both domains of eIF4A are thought to form a contiguous RNA-binding surface in the helicase's closed (active) conformation, and RNA and ATP bind cooperatively (20). Because the intracellular concentrations of ATP and ADP are very high (≈500 and ≈50 μM, respectively), it is likely that the predominant states of eIF4A in the cell are the two different versions of the closed conformation: ATP-bound and ADP-bound. We have previously proposed that eIF4G-m binding to eIF4A promotes the closed active conformation, and stimulates ATP and RNA binding (11). Because both PDCD4 MA3 domains compete with RNA and eIF4G-MA3, PDCD4 is presumably able to inhibit eIF4A activity by preventing RNA binding and thus formation of the active RNA-bound conformation. A previous report showed that full-length PDCD4 binds to eIF4G-m and eIF4A at the same time, which implicates an additional inhibitory function by PDCD4 (6). In this context, the interaction of PDCD4 with eIF4G-m likely serves a dual role. First, it directs PDCD4 to the eIF4E/4G/4A complex (eIF4F), which is much less abundant than free eIF4A (21) and, thus, a better target for inhibition of translation initiation. Second, it anchors PDCD4 to eIF4F and increases its effective concentration in the vicinity of eIF4G-bound eIF4A. We have performed binding assays for both PDCD4 MA3 domains with eIF4G-m, but neither MA3-m nor MA3-c showed any detectable chemical shift changes (data not shown). The N-terminal region of PDCD4 preceding MA3-m (1–157) may be responsible for binding to eIF4G-m (Fig. 5 C).
Structural Model for the Mechanism of Inhibition of eIF4A Activity by PDCD4.
Although no apparent interaction was observed between the two MA3 domains in trans, distinct chemical shift differences were observed in both domains between the spectra of the free domains and MA3-dual (SI Fig. 9A ). Mapping of these residues onto the structures implies a rough orientation of the two MA3 domains as shown in SI Fig. 9 B and C : There is a linker composed of ≈23 aa residues between the two MA3 domains, and this may allow MA3-c to rotate further. The oppositely charged surfaces that exist in the putative interface between MA3-m and MA3-c also indicate that the two MA3 domains actually function as a pair.
SPR data showed that MA3-m binds to eIF4A-FL ≈1.5-fold tighter than eIF4A-NTD (Fig. 4 C and D, filled squares) even though no obvious binding was observed between MA3-m and eIF4A-CTD (data not shown). We recently found that the eIF4G-MA3 binds not only to eIF4A-NTD but also weakly to eIF4A-CTD and that a mutation in α2 (D1259N) affects binding to eIF4A-CTD but not eIF4A-NTD (A.M., C.S., K.A.E., and G.W., unpublished work). The corresponding mutations in MA3-m (D180N) and in MA3-c (D343N) were reported previously to affect PDCD4 binding to eIF4A (16). Therefore, it is likely that the binding of the PDCD4 MA3 domains with eIF4A-FL is stabilized by additional interaction with eIF4A-CTD (Fig. 5 A), which is too weak to be observed with the isolated eIF4A-CTD. The necessity of the two MA3 domains was shown by SPR analysis. MA3-dual displayed significantly tighter binding to eIF4A-FL compared with the single-domain MA3-m. This synergistic effect could be explained if simultaneous binding of eIF4A-NTD to one MA3 domain and of eIF4A-CTD to the other (Fig. 9B) is more favorable than both eIF4A domains binding to the same MA3 domain (Fig. 5 A). Because the 1:1 complex shown on Fig. 5 B Upper leaves the eIF4A-NTD-binding site on one of the MA3 domains unoccupied (as well as one putative eIF4A-CTD-binding site on the other MA3 domain), it is likely that PDCD4 binds two eIF4A molecules simultaneously. Both MA3 domains of MA3-dual have a capacity to bind an individual eIF4A-NTD (Fig. 4 D), and, judging from the proposed orientation of MA3-m and MA3-c (SI Fig. 9 B and C ), the eIF4A-NTD-binding site and, presumably, the eIF4A-CTD-binding site from each MA3 domains are separated enough to bind two eIF4A-FL molecules without steric hindrance.
A model for the inhibition mechanism of PDCD4 is proposed in Fig. 5 C. In the absence of PDCD4, eIF4G-m and eIF4G-MA3 associate with eIF4A and RNA. PDCD4 displaces RNA and eIF4G-MA3 from eIF4A. Whether RNA remains bound to the inactive eIF4A/eIF4G/PDCD4 complex is still unresolved because both domains of eIF4A have been shown to interact with RNA, and eIF4A-CTD does not measurably bind PDCD4; it is likely that RNA remains weakly associated to the complex through eIF4A-CTD as indicated in Fig. 5 C iii.
In summary, we determined the x-ray crystal structure of PDCD4 MA3-m. We found that MA3-m and MA3-c have similar structure, and NMR data revealed that both domains have similar eIF4A-binding sites. PDCD4 competes with both RNA and the eIF4G-MA3 for binding to eIF4A-NTD, thus inhibiting eIF4A activity. We also found that both PDCD4 MA3 domains bind to eIF4A-NTD, and the presence of the two MA3 domains together is essential for maximum eIF4A binding, which helps explain the presence of pairs of tandem MA3 domains in all PDCD4 homologues. We provide evidence for at least transient interactions between the two MA3 domains, but the exact interdomain orientation remains to be determined.
Materials and Methods
Vector constructions, protein expression, and purification protocols and additional experimental details are described in SI Text .
Crystallization, Data Collection, and Structure Determination.
MA3-m was crystallized by vapor diffusion against 100 mM Tris·Cl (pH 8.5), 200 mM sodium acetate, 28–30% PEG 4000, and 8% Jeffamine M-600. All datasets were collected at the Advanced Photon Source beamline 24C (NE-CAT) by using a Q4 area detector. A single-wavelength anomalous dispersion (SAD) dataset was obtained from a Se-Met MA3-m crystal at λ = 0.9793 Å; for higher resolution datasets, further datasets were obtained at λremote = 0.8551 Å. Data were processed by using HKL2000 (HKL Research). Structure determination and refinement procedures are mentioned in SI Text .
NMR Resonance Assignments, Titration, and Competition Assays.
NMR spectra were recorded at 298 K on either Bruker Avance 500-MHz, 600-MHz, or 750-MHz spectrometers, all of them equipped with cryogenic triple-resonance probes. Samples for NMR measurements typically contained 0.5–1 mM protein in buffer containing 20 mM Tris·Cl (pH 7.0), 150 mM NaCl, 2 mM DTT, 1 mM EDTA, and 10% D2O. Chemical-shift changes in titration experiments were reported as normalized values by using . Details of the NMR experiments are described in SI Text .
Binding Analysis by Surface Plasmon Resonance (SPR).
SPR binding analysis was carried out by using a BIAcore 3000 instrument (Biacore). PDCD4 MA3-m, MA3-c, and MA3-dual were immobilized on a research-grade CM5 sensor chip by using the amino coupling kit supplied by the manufacturer in 10 mM sodium acetate (pH 5.5). A control flow cell was subjected to activation and blocking in the same procedure but without proteins. The surface densities of immobilized proteins were ≈2,000 RU for MA3-m, MA3-c, and MA-dual. The signal generated in the control flow cell was subtracted from the experimental signals to correct for refractive index changes and nonspecific binding.
All of the binding experiments were carried out in Hepes-EP buffer (Biacore) at 25°C and with the flow rate of 20 μl/min.
We thank Drs. D. Frueh, H. Arthanari, and K. Takeuchi for help with NMR spectroscopy. This work was supported by National Institutes of Health (NIH) Grants CA68262 and GM47467 (to G.W.) and National Cancer Institute Grant K01 CA119107 (to A.M.). The crystallographic study was supported by NIH Grant RR-15301 and Department of Energy contract DE-AC02-06CH11357.
- *To whom correspondence may be addressed at: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. E-mail: gerhard_wagner{at}hms.harvard.edu or assen_marintchev{at}hms.harvard.edu
Author contributions: C.S., A.M., and G.W. designed research; C.S., R.G.G., and S.H. performed research; C.S., R.G.G., K.A.E., S.H., S.G.H., and A.M. analyzed data; and C.S., R.G.G., A.M., and G.W. wrote the paper.
The authors declare no conflict of interest.
The atomic coordinates reported in this paper have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, www.pdb.org (PDB ID code 2RG8).
This article contains supporting information online at www.pnas.org/cgi/content/full/0712235105/DC1.
- © 2008 by The National Academy of Sciences of the USA
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Diabetes medicine from ramdev baba news,homeopathic treatment for type 1 diabetes nieuws,gc gents sport chic collection watch x72005g2s,diabetes log books free shipping zazzle - PDF 2016
Very obese patients whose diabetes is not well managed with diet and medicine may 5) Converting to hose use is neither quick nor intuititive and once done it is not even very practical.
Once-daily basal insulin glargine versus thricedaily prandial insulin lispro in people with type 2 diabetes on oral hypoglycaemic agents (APOLLO): an open randomised controlled trial R G Bretzel et al. Individualize metformin treatment with diabetes forecast phone number diabetes mellitus research questions Each time we eat insulin is released into the bloodstream. Pill box: This machine is used to manufacture the insulin pills being tested by Novo Nordisk.
Three of the thirteen eye patients who underwent catract surgery at a hospital in Hyderabad. Mogens Lykketoft, President of the seventieth session of the United Nations General Assembly and others performing Yoga exercises on 2nd International Yoga day at UN in New York.
People at a mass yoga session on 2nd International Yoga Day at Belur temple near Chikmagalur in Karnataka. Officers and sailors of Indian Navy participate in a yoga session on the deck of INS Viraat on the occasion of 2nd 'World Yoga Day', in Mumbai.
School children perform yoga at an event to celebrate International Yoga Day near Victoria Memorial, the iconic heritage landmark in Kolkata. Monsoon clouds loom over as people perform yoga at an event to celebrate International Yoga Day in Guwahati.
The participants in the mass performance of Common Yoga Protocol, organised by the Ministry of AYUSH and NDMC with support of Patanjali Yog Samiti, on the occasion of the International Day of Yoga 2016, at Lodhi Garden, in New Delhi. People participate in a yoga session on International Day of Yoga 2016, at Lodhi Garden, in New Delhi. Members of Madhya Pradesh Backward Class Commission Laxmi Narayan Yadav with his nephew Yash and niece Tanvi performs Jal Yoga on the eve of International Day of Yoga, at a swimming pool. A rainbow appears in the sky as NCC cadets perform yoga at a ground in Kolkata on the eve of International Yoga day. Dulce the dog sits next to its owner during an outdoor yoga session in Mexico City, on Sunday, June 19, 2016.
Yoga guru Baba Ram Dev, leads a yoga session at a rehearsal ahead of International Yoga Day at Rajpath, in New Delhi.
Diabetes which is also sometimes referred to as diabetes mellitus or blood sugar is a health issue related to the endocrine system of human body. As a result of this, the concerned person suffers from low levels of energy and complains of constant fatigue and tiredness.
Type 1 diabetes is an autoimmune disease under which the beta cells of the pancreas are affected adversely by the immune system.
Contrary to this, second type of diabetes is a condition under which the pancreatic cells produce insulin hormone but it is insufficient for sugar or glucose oxidation.
Since diabetes is a health condition that becomes sometimes incurable therefore it is always best to use some safe and herbal products so that the patient may get long lasting relief from this problem. The herbal formulas as mentioned above contained in this wonderful health pack offer multiple benefits to the diabetic patients as mentioned below. Pancreatic cells are stimulated to secrete required amount of insulin so that it may help in oxidation of sugar or glucose present in the blood. Feeling of exhaustion and tiredness is also eradicated from the body so that the patient may be able to perform routine activities well. All these herbal remedies are totally safe and can be used in long run without the fear of any side-effects or health hazards. And they should havetheir feet checked regularly for nerve damage circulation problems and infections. The other Baba Ramdev Yoga Asanas For Diabetes will diabetes educator jobs au have a heartbeat they ill always know.
The model estimates total diabetes prevalence by local authority and by clinical commissioning group (CCG). Seven patients are in danger of losing vision as a solution used during surgeries was found contaminated. Under this condition, apt or required amount of insulin is not produced by the beta cells of the pancreas. Baba Ramdev Health pack for Diabetes is apt in this task and can help in offering effective and permanent relief from this condition. Baba Ramdev Yoga Asanas For Baba Ramdev Yoga Asanas For Diabetes Diabetes natural Baba Ramdev Yoga Asanas For Diabetes food cures for diabetes homeopathic medicine cure diabetes Insulin resistance has been known to occur. However there are several major limitations – cost the potential for hypoglycemic episodes catheter problems and at the time of writing no approvable means Depending on the size of your doses a cartridge may give you enough insulin to last for several days of injections. We have spent multiple years doing the scientific research on marijuana and we have gathered the definitive facts. Experts explain how losing weight and journal of diabetes technology and science maintaining the weight loss will significantly reduce your risk of heart disease high blood pressure diabetes and Weight loss is associated with improvements in cardiometabolic risk factors including serum glucose insulin C?reactive protein and blood lipids. O diabetes pode ter origem gentica e hereditria na qual Baba Ramdev Yoga Asanas For Diabetes chamado de diabetes mellitus insulino dependente (DMID) ou seja tipo I The Sound+Sleep blows the competition away.
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We have no intention to hurt anyone, please use this information at your own understanding. Since no insulin is produced under the first type of diabetes therefore it requires administration of artificial insulin. What is important in this case is early or timely diagnosis of this health condition by considering the various signs and symptoms so that necessary curative or preventive measures may be taken so as to protect body against further health issues or complications. It consists of different types of herbal formulas or products that help in relieving all the signs and symptoms of this health issue. The newest research has found that vitamin D sufficiency is important for preventing type 2 diabetes cognitive decline asthma and cardiovascular disease.
It is owing to the reason that sugar present in the blood is oxidized or converted into energy required for various physical and mental functions only with the help of insulin hormone. If your blood sugar level becomes low during exercise, eat a snack according to the guidelines below.
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Due to dependence upon artificial insulin, it is also called as insulin dependent or juvenile diabetes. No matter how hungry I am, one of everything in the snack aisle never seems like a good idea.
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On Wednesday, July 10, 2019, Peoria Magazines will host a reception at the Country Club of Peoria to honor "Local Legends"—remarkable individuals who have had a lifetime of impact on the Peoria-area community.
This year, we celebrate the lifelong accomplishments of:
G. Raymond Becker
Dr. Patrick Elwood
Hon. Joe Billy McDade
Doug & Diane Oberhelman.
The event kicks off at 5:00pm, and the program will begin promptly at 5:30pm. Your ticket includes drinks and hors d'oeuvres.
Available seating is limited.
For more information, visit peoriamagazines.com/local-legends.
Thanks to our sponsors for making this special event possible!


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JACKSONVILLE, Fla. - The Jacksonville Sheriff's Office said it is investigating after a person was shot in the 400 block of Nixon Street.
Police are expected to hold a briefing on the situation later tonight.
This is a developing story. Refresh this page, follow @ActionNewsJax on Twitter and watch FOX30 Action News Jax at 10 for updates.
© 2019 Cox Media Group.


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Rolling Hill Drive Homes For Sale in Sebastian
Sebastian Highlands homes for sale on Rolling Hill
We found 1 Rolling Hill Homes For Sale in Sebastian.
Single Family Home
Sebastian Highlands
761 Rolling Hill
Sebastian, FL 32958
3 Beds / 2 Baths
Courtesy of The Keyes Company
|More Info...
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|Vero Beach, FL The Laurel spec home in The Willows community features a corner lake view, upgraded kitchen with white cabinets and quartz countertops, ceramic tile and impact windows throughout! Also comes with open floor plans designer color packages - wood cabinetry, GE appliances, hurricane protection, irrigated homesites, a 10-yr warranty come standard and energy efficient easy living. The Willows is a gated community of 92 homes featuring pool and cabana. Lawn maintenance included in HOA. 2 series of home designs to select from. Great location near beaches, golf courses, museums, and nature tours - Vero Beach is the ideal spot for your new home. Lot 73 Visit http://www.livinginvero.com/ for more info...
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|Sebastian, FL Drastically Reduced, this Boater's Paradise awaits you! This 3BD/2BA sits on the Sebastian River and backs up to an island preserve. Watch the Dolphins & Manatees from your back porch w/no homes blocking your view. Your private dock has a 12,000 lb boat Visit http://www.livinginsebastian.com/ for more info...
© 2019 Starfish Real Estate. The data relating to real estate for sale on this Website come in part from the Broker Reciprocity Program (BR Program) of Regional Multiple Listing Service, Inc.. Properties listed with brokerage firms other than Starfish Real Estate are marked with the MLS Logo and detailed information about them includes the name of the Listing Brokers. The properties displayed may not be all the properties available through the BR Program. BR data on this website is provided by Starfish Real Estate on behalf of Paul Kitchen. The source of this real property information is the copyrighted and proprietary database compilation of the Regional Multiple Listing Service, Inc.. Copyright 2019 Regional Multiple Listing Service, Inc.. All rights reserved. The accuracy of this information is not warranted or guaranteed. This information should be independently verified if any person intends to engage in a transaction in reliance upon it.
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Symphonic Broadway
Presented by CFCArts Symphony Orchestra
Join the 160-member CFCArts Symphony Orchestra as they present their fall concert, A Hero's Journey. A hero's journey involves a hero who goes on an adventure, and in a decisive crisis wins a victory, and then comes home changed or transformed. Be transformed by the power and passion of Florida's largest symphony orchestra combined with sweeping scores and heroic music. This one-of-a-kind concert is one you and the entire family will not want to miss!
October 25 at 8pm
October 26 at 2pm & 7pm
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WABASHA, Minn. — A human leg — from femur to foot with the shoe still on — was found Saturday afternoon in the Mississippi River.
According to a press release from the Wabasha County Sheriff's Office, deputies responded to a call at about 4:50 p.m. that human remains were found while a resident was cleaning out a debris pile about 20 feet from shore south of Lock and Dam No. 4 in West Newton near Kellogg.
UTICA — A Utica man was arrested Sunday night after opening fire with a semi-automatic rifle and attacking a vehicle that was parked outside his house, leaving the driver with non-life-threatening injuries.
No other signs of human remains were located.
The area was searched again on Sunday. No other items of interest were located.
Chief deputy Jim Warren said an investigation into the matter is ongoing. More information will be available after a report is issued by the medical examiner's office.
A patron at Steiny's Bar foiled an attempted robbery early Thursday.


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Recreation / Outdoor in Georgetown
Browse 34 attractions, meet 16 travelers, 12 tour guides and discover 27 photos
Travelers are voting Hickman Creek, Keeneland and The Red Mile as the best of 5 recreation / outdoor in Georgetown. There are 6 recreation / outdoor in Danville a city just 89 km from Georgetown and 2 recreation / outdoor in Owingsville which is 92 km distant. The most popular of these are Chaplin River in Danville and Cave Run Lake in Owingsville. You can find 212 recreation / outdoor in Kentucky and 23466 recreation / outdoor in the country of United States. Our members favorite in Kentucky is Mammoth Cave National Park and Lake Superior is the top pick in United States. Want more? Check out our map of recreation / outdoor in Georgetown.
Hickman CreekRecreation / Outdoor RiversKentucky, United States
Hickman Creek is a 25.5-mile-long (41.0 km) tributary of the Kentucky River, flowing through Jessamof the Mississippi River watershed.
KeenelandRecreation / Outdoor Sporting AreasLexington, Kentucky, United States
Keeneland is a Thoroughbred horse racing facility and sales complex in Lexington, Kentucky, USA. Operated by the Keeneland Association, Inc., it is…
The Red MileRecreation / Outdoor Sporting AreasLexington, Kentucky, United States
The Red Mile is a horse racing track located in Lexington, KY. The track hosts harness racing, a type of horse racing in which the horses must pull…
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Kentucky Horse Park is a working horse farm and an educational theme park opened in 1978 in Lexington, Kentucky. It is located off Kentucky…0
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Samangan launches women's Shuras and starts women's rights awareness campaign with German help
Women all around Samangan celebrated the launch of the province's first women's shura councils on Sunday. Backed by the German Government through Afghan-German Cooperation, the shuras offer women the chance to meet in the capital city of Aybak and throughout districts in the northern Afghan province of Samangan. The councils are led by women and deal specifically with women's affairs and representing women's rights in local government. To coincide with the launch, a six-day workshop also began in Aybak today on raising awareness of women's rights.
The shuras represent women's interests and can allow greater participation in the administrative level. The aim is to boost the profile of women in politics, both at the grass-roots level and in high-ranking governmental positions in ministries, governors' offices, and in district governors' and mayors' posts, for example. The shuras' long-term goal is to empower women to take on more responsibility and a greater presence both in political roles and in the public and private sectors, for example in schools, hospitals, business and the media.
The focus of the six-day workshop for women is on women's rights, specifically in the context of forced marriages. Around 200 women registered for the six days of training, which also acts as the start of an awareness-raising campaign on women's rights and privileges. Participants include female members of development committees in districts and villages, principals and headmasters of female high schools in Aybak, widows and mothers from martyrs' families and representatives of the Department of Women Affairs and other governmental departments. Through their work and through the shuras, they will pass on their knowledge to other women in their communities.
The aim of the course is to help improve women's lives through greater awareness of their rights. Violence against women, family issues and forced marriages are the main topics the course will be dealing with. With the help of a variety of workshop teaching methods, the two trainers will also be teaching the participants about the right to healthcare and the right to education for women.
The cost of the training is AFN 512,200, and the project was approved by the Samangan Provincial Development Committee (PDC). Support for conducting the training has come from the Regional Capacity Development Fund (RCDF) based in Samangan, which is financed by the German Federal Ministry for Economic Cooperation and Development (BMZ).
As part of Afghan-German Cooperation, the RCDF is implemented by Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH. The RCDF provides support for its Afghan partners' development priorities both on a provincial and district level in six of Afghanistan's northern provinces. The RCDF also provides training and coaching programmes for civil servants. With its broad range of activities, the RCDF aims to increase Afghan ownership of the development process, strengthen Afghan state institutions on the sub-national level and promote transparency and inclusiveness.
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Pakistan's Contribution to Afghanistan's Development Reaches $1bn
Pakistan has made USD 1 billion in contribution to Afghanistan's development, said Pakistan's Ambassador to China Masood Khalid in an
Australian Government to help Afghanistan hundred million dollars after 2014
Julia Glared Prime Minister and Stephen Smith Defense Minister of Australia announced a support of 300 Australian Dollars in joint


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This Property Is Located On A Quiet Street In W...
- 3 Beds
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Single Family
Eagleswood, New Jersey
This Property Is Located On A Quiet Street In West Creek. There Is A House Located On The Property That Could Be Renovated Back To Its 1930 Charm. However, This Property Is Being Sold As Is With All Due Diligence Placed On The Buyer. All Items In The Residence Will Be Removed By The Owner Prior To Closing. The Shed/garage Will Also Be Taken Down Before Closing. This Property Is…


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A pioneer when it comes to scalp concerns, Christophe Robin launched his cleansing Purifying Scrub with Sea Salt in 2013. An instant success in the industry, the award-winning scrub has become a haircare saviour for both women and men. The best-selling scrub features a hybrid formula, combining two potent ingredients to soothe even the most sensitive scalps.
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Where reviews refer to foods or cosmetic products, results may vary from person to person. Customer reviews are independent and do not represent the views of The Hut Group.
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For the little ones, there seems to be a lot of buzz about bunnies, eggs, and candy during Easter time. Yet, the true meaning of the season sometimes gets buried in the fun fluff. There's no reason why we can't celebrate cuteness and the cross ALL at the same time! Bring the reason for the season into homeschool with this free Resurrection Day writing prompts for Kids!
Resurrection Day Writing Prompts
These writing prompt printables are perfect for igniting questions about what the Resurrection truly means. It gives parents and Sunday School teachers an outlet to communicate why we celebrate Easter. While these Easter Writing Prompts are geared towards upper elementary, there's no reason to use it for preschoolers as well!
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This free resurrection printables set includes the following writing prompts worksheets:
- Describe the Resurrection
- Resurrection in Nature
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- Words to describe the church
- Nouns/Adjectives related to the Resurrection
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Click here and complete the checkout process to get your free Resurrection Day Writing Prompts pack!
Sarah is a wife, daughter of the King and Mama to 4 children (one who is a homeschool graduate)! She is a an eclectic, Charlotte Mason style homeschooler that has been homeschooling for over 16 years now. She is still trying to find the balance between work and keeping a home and says she can only do it by the Grace of God, and Coffee!
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Are you tired of the same old tie-dye t-shirt project for the summer? I surely am and so are my kidd
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If you're looking for ways to reduce waste and teach your children about their impact on the environ
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Summers are for being together with your family and friends, relaxing and enjoying the warmer weathe
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Have you desired to see a change in your homeschool, but don't know what to do? Are you beginning