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LA CROSSE, Wis. - Seven members of the MIT women's track and field team made the trip to the University of Wisconsin-La Crosse for the 2013 NCAA Division III Men's and Women's Outdoor Track and Field Championships, which will run through Saturday, May 25. After the first day of competition, the Engineers are currently in tenth place with six points, scored solely by sophomore Elaine McVay's (San Ramon, Calif.) impressive performance in the 10,000m.
Junior Jaclyn Konopka (Novi, Mich.) opened the Championships with the Heptathlon, finishing the first day in 11th place. She dropped over half a second off her time in the 100m Hurdles from last weekend's ECACs when she ran a 14.83 to start the day. She jumped a 5-03 in the High Jump, threw a 31-01 in the Shot Put and wrapped things up with a time of 26.77 in the 200m. Her 2,826 points is just shy of what earned her a first place finish at ECACs.
The next day, Konopka competed in the final three events of the Heptathlon. She jumped 4.96m in the Long Jump, threw 62-07 in the Javelin Throw and ran a 2:34.67 in the 800m. She ended the day in 20th place with 4,282 points.
In her first showing at nationals, freshman Cimran Virdi (Walnut Creek, Calif.) finished in ninth place in the Pole Vault with an 11-09.75, moving up nine places from where she was ranked going into the Championships.
In prelims of the 1500m, senior Brooke Johnson (Shoreham, N.Y.) stayed true to her eighth place seeding with her time of 4:36.27. Freshman Sarah Quinn (Watchung, N.J.) was not far behind, moving up from 14th to tenth with a 4:32.19, once again improving on her MIT freshman record. Both will get another chance to improve their times at finals on Saturday.
McVay dropped a second off her seed time and moved up five spots to take third place in the 10,000m with her time of 35:53.33, earning six points towards MIT's final score and getting All-America honors. To end the first day, junior Martina de Geus (Los Altos, Calif.) finished in 19th place in the prelims of the 3000m Steeplechase with a time of 11:12.12.
MIT's only athlete competing in the second day of NCAAs is Konopka in the remaining Heptathlon events.


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Computer software designed for "'click fraud'—bogus clicks that do not come from genuinely interested customers," to abuse the 'pay-per-click' model of Internet advertising, "the single largest category within this flourishing industry, accounting for nearly half of all spending". (Nov 23rd 2006 The Economist)
Dick signed on with a company specializing in fraudware, used to increase the number of hits to all advertising banners on his Web site, which ranged in price from $0.10 to as much as $30.
by David R. Gahary January 16, 2007
buy the domain for your pet blog

subset: commoncrawl


text:
Dhole, Asiatic Wild Dog, Indian Wild Dog, Red Dog
(Cuon alpinus)
Facts about this animal
The dhole is a medium-sized dog of light and graceful proportions. The head-body length ranges from 75-110 cm, the tail from 41-50 cm, and the body-weight from 10-20 kg.
The muzzle is shorter than in most other dogs, with a large nose pad. There are 40 teeth, two less than in the lower jaw than above, the 3rd lower molar is absent. The ears are erect and rounded. The sexes look alike. The female has 16 teats instead of the usual 10 of Canis.
The coat is uniform, dorsally red or yellow or rust, the underparts and throat are whitish. The back of the ears, neck and shoulders are slightly duller but the same colour. The dorsal guard hairs are 25-30 mm long. They are shorter in southern animals. The tail is darker than the body and usually black-tipped. The outer parts of the legs are of the same colour as the body, the inner sides are pale and the paws can be white.
Dholes are highly social animals living in packs and hunting cooperatively. A pack consists of usually 5 to 12, sometimes up to 20 members and contains a dominant monogamous pair. After a gestation period of 60-62 days, the female gives birth to a litter of usually eight pups, which are born in a den built near riverbeds or among rocks. Birth season is from November to March. Subordinate pack members help care for the young of the dominant pair. The pups reach sexual maturity at about a year.
The dhole is an opportunistic feeder eating berries, fruit, insects, lizards, and mammals from small rodent to large deer size, including hares, wild boar, various ruminants, and even monkeys.
Did you know?
That the dhole has some extraordinary vocal calls - it can whistle, scream, mew- and even cluck like a chicken, and that it can pee while doing a handstand on its front two legs?
|Name (Scientific)
|Cuon alpinus
|Name (English)
|Dhole, Asiatic Wild Dog, Indian Wild Dog, Red Dog
|Name (French)
|Dhole, Chien sauvage d'Asie, Cuon d'Asie
|Name (German)
|Name (Spanish)
|Cuon asiático, Perro salvaje asiático
|Local names
|Gujarati: Kol kutta
Hindi: Dhole, Son, Ram, Jangli kutta, Ban kutta
Kashmiri: Kadu nai, Chira nai
Tamil: Chen nai, Kattu nai
Tibetan: Sidda ki
|CITES Status
|Appendix II
|CMS Status
|Not listed
Photo Copyright by
Ber' Zophus
|Central and eastern Asia: Bangladesh, Bhutan, Cambodia, China, India, Indonesia, Kazakhstan, Kyrgyzstan, Lao PDR, Malaysia, Mongolia, Myanmar, Nepal, Russian Federation, Tajikistan, Thailand, Turkey, Viet Nam
|The dhole is found in a wide variety of vegetation types, including: primary, secondary and degraded forms of tropical dry and moist deciduous forest; evergreen and semi-evergreen forests; dry thorn forests; grassland–scrub–forest mosaics; and alpine steppe (above 3,000 m). They are not recorded from desert regions (IUCN Red List 2006)
|Wild population
|It is estimated that fewer than 2,500 mature individuals remain in the wild and the declining population trend is expected to continue. Main threats to the species include ongoing habitat loss, depletion of prey base, interspecific comeptition, persecution and possibly disease transfer from domestic and feral dogs.(IUCN Red List 2006)
|Zoo population
|There are at least 110 dholes in captivity, and the sex ratio is approximately even. Except for some captive populations in India heterozygosity appears to be good, but there is little chance of breeding the putative subspecies as animals from diverse geographical origins have been widely interbred (M. Boeer, pers. comm.). 163 reported to ISIS (2007).
In the Zoo
How this animal should be transported
For air transport, Container Note 82 of the IATA Live Animals Regulations should be followed.
Find this animal on ZooLex
Photo Copyright by
Why do zoos keep this animal
The dhole is an endangered species in the wild with a small and declining population. Building up and maintaining an ex situ-reserve population is therefore of conservation interest. But the species is also of educational value because of its complex social life.


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Your Scientology Orgs and What They Do For You
It has been my ambition for a very long time to be able to say to any human being on Earth that they could walk up to any organization of Scientology, whether it was a Center, a City Office or a Central Organization—be able to walk in the front door and at the end of some, well, you could even say a nebulous number of hours of processing, emerge out of that front door Clear. —L. Ron Hubbard
As Scientology expands ever more deeply and broadly across the planet, the purpose remains what it has always been—to Clear every being on Earth. But although the technical procedures have long existed to reach that target, so momentous an undertaking requires something else as well. Specifically, the means to ensure that regardless of whether a session takes place in Los Angeles, London, Johannesburg or Keokuk, what auditors deliver to their preclears is the exact, correct technology, applied down to the last comma of the HCOB.
The need to accomplish that degree of duplication brings into focus the paramount importance of Scientology organizations. For upon the successful execution of their functions depends whether Man goes free. In this definitive lecture on how orgs fulfill their supreme responsibilities, L. Ron Hubbard not only reveals the crucial link between technology and organization but, within that relationship, the accomplishment of World Clearing.


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Eukaryotic mRNAs exist in vivo as ribonucleoprotein particles (mRNPs). The protein components of mRNPs have important functions in mRNA metabolism, including effects on subcellular localization, translational efficiency and mRNA half‐life. There is accumulating evidence that pre‐mRNA splicing can alter mRNP structure and thereby affect downstream mRNA metabolism. Here, we report that the spliceosome stably deposits several proteins on mRNAs, probably as a single complex of ∼335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20–24 nucleotides upstream of exon–exon junctions. Splicing‐dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing‐associated factors SRm160, DEK and RNPS1, the mRNA‐associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense‐mediated mRNA decay pathway, are discussed.
Throughout their lifetimes, pre‐mRNAs and their product mRNAs exist in vivo as RNA–protein particles (RNPs; Dreyfuss et al., 1993). The complement of proteins bound to these RNAs evolves significantly as nuclear pre‐mRNA is processed into mRNA and nuclear mRNA is exported to the cytoplasm. For example, Pol II transcripts acquire CBC20/80, the nuclear cap‐binding proteins, after extensive modifications to their 5′ ends, and these proteins are exchanged for the translation initiation factor eIF4E upon export to the cytoplasm (Izaurralde et al., 1995; Visa et al., 1996; Fortes et al., 2000). As another example, Pol II transcripts acquire PABP2, the nuclear poly(A) binding protein, as part of a series of protein–RNA interactions that direct 3′ end cleavage and polyadenylation (Wahle, 1991; Krause et al., 1994). In the cytoplasm, mRNA poly(A) tails are bound by PABP1 (Gorlach et al., 1994), which functions in the initiation of protein synthesis (reviewed by Sachs and Varani, 2000). Some heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins, which coat pre‐mRNAs as they are synthesized, accompany mRNAs into the cytoplasm. Such hnRNP proteins can regulate cytoplasmic events, including mRNA localization, translation and mRNA turnover (reviewed by Shyu and Wilkinson, 2000). Therefore, the nuclear history of an mRNA can have substantial effects on its downstream metabolic fate.
Like capping and polyadenylation, it has recently become apparent that pre‐mRNA splicing can significantly influence the downstream metabolism of product mRNAs. There are several examples in the literature indicating that the expression of specific genes in metazoans is enhanced by the presence of an intron (Luo and Reed, 1999 and references therein). It has also been shown that metazoan mRNAs containing an exon–exon junction >50 nucleotides (nt) downstream of the first in‐frame termination codon are targeted for nonsense‐mediated mRNA decay (NMD; reviewed by Maquat, 1995, 2000; Li and Wilkinson, 1998; Hentze and Kulozik, 1999). Thus, mRNAs must somehow carry a memory of the introns contained in their precursors.
The most likely mechanism mediating the downstream effects of pre‐mRNA splicing on mRNA metabolism is a splicing‐dependent modification of mRNP protein composition (Le Hir et al., 2000). To date, several proteins have been shown to specifically associate with spliced mRNA. Antibodies against SRm160, a co‐activator of splicing (Blencowe et al., 1998), and RNPS1, a general splicing activator (Mayeda et al., 1999), specifically precipitate mRNAs produced by splicing in vitro. SRm160 and hPrp8p, a core component of the spliceosome (Luo et al., 1999), could be cross‐linked to one mRNA in vitro in a splicing‐dependent manner (Le Hir et al., 2000). Other proteins that are not demonstrable splicing factors have also been found associated with spliced mRNA. Antibodies against the oncoprotein DEK, which co‐purifies with SRm160 (McGarvey et al., 2000), and Y14, a novel RNA‐binding protein that shuttles in and out of the nucleus (Kataoka et al., 2000), preferentially precipitate spliced mRNAs. Additionally, the protein REF (also called Aly) was found to be present in spliced mRNP complexes, but absent from an analogous mRNP complex containing unspliced mRNA (Zhou et al., 2000). However, whether these proteins bind along the length of spliced mRNA or only at particular positions remained to be determined.
In a previous study, we described a novel in vitro cross‐linking approach for detecting proteins that associate with mRNAs only as a consequence of splicing (Le Hir et al., 2000). While that study provided direct evidence that splicing can alter the proteins associated with mRNA, only selected positions could be assayed for an interaction with splicing‐dependent proteins. Furthermore, it was not possible to determine the proportion of spliced mRNA molecules carrying these proteins. Here, we took advantage of a targeted RNase H analysis (Gunzl and Bindereif, 1999) to explore in more detail where and how the splicing machinery alters spliced mRNP structure in vitro and in vivo. Remarkably, this approach revealed that spliceosomes deposit multiple proteins, probably as a single complex, at a conserved position 20–24 nt upstream of spliced mRNA exon–exon junctions. This −20/24 complex contains SRm160, DEK, RNPS1, Y14 and REF.
RNase H footprinting reveals a splicing‐dependent protection of mRNA
If pre‐mRNA splicing stably alters mRNP structure, such alterations might be detectable as splicing‐dependent protections of mRNA to RNase digestion. We initially tested this possibility by incubating a uniformly labeled pre‐mRNA that derived from the human triose phosphate isomerase (TPI) gene under splicing conditions in HeLa cell nuclear extract to generate mRNA (spliced mRNA). Subsequently, the reaction was divided into aliquots and individual cDNA oligonucleotides (oligos; 12 nt each) were added to each aliquot to activate targeted RNase H cleavage (Figure 1A, right panel). Each oligo was named according to the position of its center relative to the exon–exon junction (Figure 1A, top). A series of 13 such oligos allowed for analysis across the entire TPI mRNA. To reveal any splicing‐dependent changes in the mRNA cleavage pattern, an identical mRNA that had been transcribed from cDNA (control mRNA) was analyzed in parallel (Figure 1A, left panel).
We found that all regions of control mRNA were accessible to RNase H cleavage. This was shown by the appearance of two cleavage products in all digestion reactions (Figure 1A, left). Although a significant amount of uncleaved mRNA remained after digestion at some positions, this amount was not consistent in every experiment (data not shown). In reactions containing spliced mRNA, only one cleavage product specifically reflected RNase H digestion of mRNA; the other product could also be generated by digestion of pre‐mRNA and/or splicing intermediates (except at the exon–exon junction). Compared with the control mRNA digestions, this mRNA‐specific cleavage product was either absent or dramatically reduced at two positions in spliced mRNA: 24 nt upstream of the exon–exon junction (oligo centered at position −24; Figure 1A, lane 24), and at the junction itself (oligo centered at the exon–exon junction, position 0; Figure 1A, lane 26). This indicated that two positions in TPI mRNA were almost completely protected from RNase H cleavage after splicing.
To test the generality of the RNase H resistances observed for spliced TPI mRNA, we performed the same analysis with three other splicing substrates and their corresponding control mRNAs: AdML, β‐globin and PIP85.B (Figure 1B–D). No splicing‐dependent protection at the exon–exon junction was observed with any of these mRNAs (Figure 1B, lane 14; Figure 1C, lane 11; Figure 1D, lanes 11 and 25). Remarkably, however, splicing‐dependent protection from cleavage by each of the oligos centered at position −24 was characteristic of all mRNAs tested (Figure 1B, lane 12; Figure 1C, lane 9; Figure 1D, lanes 9 and 23).
Since no conserved sequence element was apparent in the vicinity of position −24 (data not shown), it seemed likely that the location of this splicing‐dependent protection was defined mainly by its distance from the exon– exon junction rather than by RNA sequence. To test this hypothesis, we inserted 10 nt at the 3′ end of PIP85.B exon 1 (Figure 1D). Although this insertion moved the sequence surrounding position −24 in the PIP85.B construct to the vicinity of position −34 in the PIP85.B+10 construct, the protected region in the new construct remained 24 nt upstream of the exon–exon junction. Thus, a general consequence of pre‐mRNA splicing is the deposition of some stably associated species a fixed distance 24 nt upstream of mRNA exon–exon junctions.
Splicing‐dependent protection of mRNA is conserved in Xenopus oocytes
To test whether the splicing‐dependent protection of mRNA observed in HeLa cell nuclear extract could be reproduced in vivo, we performed a similar RNase H analysis in Xenopus laevis oocytes (Figure 2). The same AdML (Figure 2A) and β‐globin (Figure 2B) pre‐mRNAs and control mRNAs as above were injected into oocyte nuclei. After 90 min, more than half of each pre‐mRNA had been converted to spliced products. Following oocyte dissection, isolated nuclei were further incubated in the presence of the indicated cDNA oligos to activate endogenous RNase H.
Remarkably, we observed exactly the same cleavage and protection patterns for control and spliced mRNAs in Xenopus oocytes in vivo as were observed in HeLa cell nuclear extract in vitro. These results indicate that the splicing‐dependent protection at −24 (Figure 2A, lane 14; Figure 2B, lane 11) is conserved from amphibians to humans.
Identification of proteins associated with the exon–exon junction region of spliced mRNA
To identify factors that associate with the region around the exon–exon junction in a splicing‐dependent manner, we undertook a selective co‐immunoprecipitation (co‐IP) strategy (Figure 3A). Briefly, uniformly labeled AdML pre‐mRNA was spliced in HeLa cell nuclear extract. To ensure that only released mRNPs were analyzed, spliced mRNPs were separated from spliceosomes by glycerol gradient fractionation (Le Hir et al., 2000). The mRNA in these isolated mRNPs was then subjected to RNase H cleavage by a pair of oligos, one annealing upstream and the other annealing downstream of the exon–exon junction, to generate three separate mRNA fragments: 5′ (83 nt), middle (m; 64 nt) and 3′ (36 nt), where the −24 region and the exon–exon junction were contained in the middle fragment. Reactions were then subjected to IP with different antibodies, and the co‐IP of mRNA fragments was analyzed by denaturing PAGE. In parallel, control mRNA was incubated under splicing conditions, cleaved with the identical oligos, and subjected to IP directly (Figure 3B).
As expected from previous studies (see Introduction), antibodies against SRm160, DEK, RNPS1, Y14 and REF all precipitated full‐length spliced mRNA to a much greater extent than control mRNA (Figure 3B and data not shown). Remarkably, all of these antibodies also selectively co‐immunoprecipitated the middle (m) fragment from spliced mRNA, but not the same fragment from control mRNA (Figure 3B and C). All other fragments, whether arising from the spliced or control mRNA, were either precipitated with greatly reduced efficiency (e.g. the 5′ fragment from spliced mRNA) or were observed at background levels only. These differences in IP efficiencies were readily quantifiable, as the histograms shown in Figure 3C represent averages of 2–5 independent experiments. The generality of the splicing‐dependent association of SRm160, DEK, RNPS1, Y14 and REF with the region around the exon–exon junction of spliced mRNA was assessed by performing similar analyses with TPI, β‐globin and PIP85.B mRNAs. In those experiments, antisera against these proteins invariably co‐immunoprecipitated RNA fragments containing the −24 region of spliced mRNA more efficiently than any other fragment (data not shown). Thus, the association of SRm160, DEK, RNPS1, Y14 and REF with this region is a general feature of spliced mRNA.
Selective co‐IP of the middle fragment from spliced mRNA was not a general property of all antibodies tested. Antibodies against the mRNA export factor TAP (Gruter et al., 1998; Braun et al., 1999; Katahira et al., 1999) did not co‐immunoprecipitate any RNA we tested (Figure 3B and data not shown). Anti‐trimethyl cap antibodies, which recognize U1, U2, U4 and U5 spliceosomal snRNAs, and Y12 antibodies, which recognize the core Sm snRNP proteins, failed to co‐immunoprecipitate full‐length spliced mRNA, control mRNA, or any RNase H‐generated fragment thereof. However, both antibodies did effectively co‐immunoprecipitate RNAs contained in spliceosomes (S) purified from the same glycerol gradients as spliced mRNP (Figure 3B and C). Antisera against the hnRNP A1 protein, which actively shuttles between the nucleus and cytoplasm and is thought to be associated with mRNAs in vivo (Nakielny and Dreyfuss, 1999), co‐immunoprecipitated both spliced and control mRNAs (and their respective RNase H digestion fragments) with comparable efficiencies (Figure 3B and C). In this case, co‐IP efficiency correlated more with RNA length than any other attribute, consistent with the minimal RNA recognition requirements of hnRNP A1 (see below). Antisera recognizing hnRNP C1/C2, a non‐shuttling protein (Nakielny and Dreyfuss, 1996), co‐immunoprecipitated RNAs contained in spliceosomes, but neither spliced nor control mRNA or their fragments. Similar results were obtained with an antiserum directed against the C‐terminus of hPrp8p (Le Hir et al., 2000). Thus, the epitopes recognized by these antisera are either not present or are inaccessible in spliced mRNP.
Taken together, the above results indicate that the SRm160, DEK, RNPS1, Y14 and REF proteins all become associated with the exon–exon junction region of mRNA as a consequence of splicing. Furthermore, their association with spliced mRNA is sufficiently stable to withstand glycerol gradient fractionation followed by IP. Finally, in contrast to hnRNP A1, which apparently binds at multiple points along the length of mRNA, selective co‐IP of the exon–exon junction region by antibodies against SRm160, DEK, RNPS1, Y14 and REF suggests that this region is their primary point of association with spliced mRNA.
The splicing‐dependent protection spans 8 nt, including positions −20 and −24
The above experiments indicated that antibodies against the SRm160, DEK, RNPS1, Y14 and REF proteins all selectively co‐immunoprecipitate a 64 nt fragment containing position −24 and the exon–exon junction of spliced mRNA. We next investigated whether the association point of these proteins corresponded exactly to the region protected by splicing from RNase H cleavage (i.e. the nucleotides immediately flanking position −24). To do so, we first determined whether the splicing‐dependent protection of the −24 region could withstand more stringent RNase digestion. AdML pre‐mRNA containing a single labeled phosphate located 20 nt upstream of the 5′ splice site was incubated under splicing conditions for various times and then each reaction was divided in half. One half was analyzed directly by denaturing PAGE to follow the evolution of splicing intermediates and products (Figure 4A, upper panels). The other half was treated with RNase A or RNases A and T1 before PAGE to detect any protected fragments (Figure 4A, lower panels).
Prior to the formation of splicing products, no major protected fragments were observed (Figure 4A, lanes 1, 2, 6 and 7). However, as spliced mRNA appeared, a 19 nt protected fragment appeared with identical kinetics in the RNase A‐digested samples (lanes 3–5). Similarly, an 8 nt protected fragment appeared in the RNase A‐ and T1‐digested samples (lanes 8–10). An 8 nt protected fragment was also observed when the single label was incorporated into pre‐mRNA at position −24 (Figure 4B). In contrast, no protected fragments were observed when control mRNA containing a single labeled phosphate at position −20 was incubated under splicing conditions and then digested with RNases A and T1 (Figure 4C, lane 6). Neither were any protected fragments observed when a sample containing spliced mRNA was treated by SDS prior to RNase addition (Figure 4C, lane 8). Thus, the splicing‐dependent protection of AdML mRNA spans 8 nt, including positions −24 and −20 relative to the exon–exon junction. Furthermore, whereas the species responsible for this protection is resistant to extensive RNase degradation, its sensitivity to SDS treatment suggests that it is not covalently attached to mRNA.
All five spliced mRNA‐specific proteins are associated with the 8 nt protected fragment
To examine which proteins remained associated with the protected region of spliced mRNA after stringent RNase digestion, we next performed IPs from digestion reactions similar to those above with antibodies against SRm160, DEK, RNPS1, Y14, REF, hnRNP A1 and hnRNP C1/C2 (Figure 5A). Notably, antibodies against all five of the spliced mRNA‐specific proteins (SRm160, DEK, RNPS1, Y14 and REF) precipitated both the 19 and 8 nt protected fragments from spliced mRNA. Each of these antibodies precipitated both fragments with equal efficiencies, and these efficiencies were similar to those observed for the larger RNase H fragments in Figure 3. In contrast, antibodies against hnRNP A1 precipitated the 19 nt fragment, but not the 8 nt fragment. This latter result may be explained by the existence of a nearly consensus binding site for hnRNP A1 spanning nt −16 to −13 [AGGG (Figure 5A, bottom); the hnRNP A1 consensus is uAGGGa/u, where upper case letters indicate the most important positions for efficient binding (Burd and Dreyfuss, 1994)]. As expected, antibodies against hnRNP C1/C2 failed to precipitate either fragment. These results indicate that SRm160, DEK, RNPS1, Y14 and REF are all part of the species responsible for the splicing‐dependent RNase protection of 8 nt. However, consistent with the results from Figure 3, neither hnRNP A1 nor C1/C2 is specifically associated with this region.
The −20/24 complex has an apparent molecular mass of 335 kDa
SRm160, DEK, RNPS1, Y14 and REF have a combined molecular mass of at least 222 kDa [individual molecular masses (in kDa) based on sequence calculations are: SRm160, 93.5; DEK, 42.7; RNPS1, 34.2; REF, 32; and Y14, 20; although some of these proteins are likely to be subject to post‐translational modification]. To determine whether the −20/24 complex was large enough to accommodate all of these proteins, we measured its approximate mass by gel filtration. To do so, AdML pre‐mRNA containing a single labeled phosphate at position −20 was incubated under splicing conditions to generate mRNA; the reaction was treated with RNase A and then loaded onto a Superose 6 gel filtration column. Compared with molecular weight standards (data not shown), the 19 nt protected fragment eluted as a single peak with an apparent molecular mass of 335 kDa (Figure 5B). Even when hnRNP A1 (38.5 kDa), which is associated with this particular RNA fragment (see above), and the RNA fragment itself (6 kDa) are taken into account, the measured mass is large enough to accommodate all of the factors we have described here. Taken together with the IP results, the observation of a single gel filtration peak suggests that SRm160, DEK, RNPS1, Y14 and REF associate with this region of spliced mRNA as a single multi‐protein complex.
In this study, we demonstrate that pre‐mRNA splicing stably alters mRNP structure at a conserved position, 20–24 nt upstream of mRNA exon–exon junctions, both in vitro and in vivo. At least five proteins become tightly associated with this region, probably as part of a single complex (Figure 6). The diverse attributes of these proteins may explain previously reported effects of pre‐mRNA splicing on downstream mRNA metabolism (see below).
Pre‐mRNA splicing deposits multiple proteins at a conserved position upstream of exon–exon junctions independent of mRNA sequence
The RNase H analysis performed here is a very useful and straightforward assay for detecting stable structural changes in RNA–protein complexes (Gunzl and Bindereif, 1999). In this study, splicing‐dependent changes in mRNP structure were readily detected by comparing the cleavage patterns of spliced and control mRNAs (Figure 1). When the entire length of TPI mRNA was evaluated, two regions were sufficiently altered by splicing to produce significant resistance to RNase H: the exon–exon junction and a region centered 24 nt upstream of the junction. Surprisingly, the latter protection proved characteristic of every mRNA tested. This protection exists in vivo and is conserved between amphibians and humans (Figure 2). The protection can be attributed to the splicing‐dependent deposition of at least five proteins (Figure 3), probably as a single complex (Figure 5). The binding site for this complex spans 8 nt, including positions −20 and −24 relative to the exon–exon junction (Figure 4).
That pre‐mRNA splicing can alter mRNP structure was expected from several earlier studies (Thermann et al., 1998; Zhang et al., 1998a,b; Luo and Reed, 1999; Le Hir et al., 2000; Zhou et al., 2000). However, the exact location(s) on mRNA where such changes occur had not been previously mapped in any systematic way. Our finding that the spliceosome deposits a multi‐protein complex 20‐‐24 nt upstream of exon–exon junctions on multiple mRNAs was unexpected. Not only is there is no consensus sequence in this region, but exons as short as 12 nt can support both steps of splicing in vitro (Duchene et al., 1988). In fact, previous studies analyzing protein–RNA interactions in splicing complexes have almost entirely ignored this region. In one study, Chiara et al. (1996) did report that the SR protein splicing factors SRp20 and SRp30 could be cross‐linked in the early (E) splicing complex at positions −31 and −26 relative to the 3′ end of exon 1 of an AdML derivative. However, neither the generality of those cross‐links nor whether they were detectable in later splicing complexes was examined. Our results indicate that contact between the spliceosome and the −20 to −24 region of the 5′ exon is a general occurrence, at least late in the splicing reaction.
The −20/24 proteins remain stably bound to mRNA without apparent preference for a particular RNA sequence. In this regard, the −20/24 proteins show some similarity to the protein–RNA interactions observed at a site in the intron that helps anchor U2 snRNP during assembly of splicing complex A. This 'anchoring site' corresponds to the 20 nt region immediately 5′ of the branch site. Although components of U2 snRNP make stable interactions with the anchoring site, this region contains no obvious conserved sequence elements (Gozani et al., 1996). Consistent with there being no consensus sequence in the −24 region of the 5′ exon, we demonstrated that the position of −20/24 protection is determined exclusively by its distance from the exon–exon junction and not by exon sequence (Figure 1D). Furthermore, −20/24 protein association with spliced mRNA is stable enough to survive complete RNase digestion or glycerol gradient fractionation followed by IP (Figures 3,4,5). It will be of interest to determine which of the protein(s) in this region interacts directly with mRNA and how these proteins maintain their grip without substantial base‐specific contacts.
The −20/24 proteins include both pre‐mRNA splicing and mRNA export factors
Three identified components of the splicing‐dependent −20/24 complex are directly or indirectly related to the splicing process. SRm160, originally defined as a nuclear matrix component (Blencowe et al., 1994), is a member of the SR splicing factor family (Blencowe et al., 1998). A complex formed by SRm160 and SRm300, another nuclear matrix component, activates splicing of specific pre‐mRNAs by interacting with SR proteins and U1 snRNP (Eldridge et al., 1999; Blencowe et al., 2000). Like SRm160, the phosphoprotein DEK associates with some pre‐mRNAs and splicing complexes in vitro through interactions mediated by SR proteins (McGarvey et al., 2000). Interestingly, DEK also has chromatin remodeling activity (Alexiadis et al., 2000), but the relationship of this function to pre‐mRNA splicing, if any, is unknown. RNPS1 contains an RNA recognition motif (RRM) and has been shown to generally activate splicing of both constitutively and alternatively spliced pre‐mRNAs in synergy with SR proteins (Mayeda et al., 1999). Thus, the association of SRm160, DEK and RNPS1 with spliced mRNA may result from their associations with pre‐mRNA during the early stages of spliceosome assembly.
The Y14 and REF proteins have no known functions in the splicing process per se, but they have both been linked to mRNA export (Kataoka et al., 2000; Stutz et al., 2000; Zhou et al., 2000; E.Izaurralde, unpublished results). Like RNPS1, both proteins contain RRMs, and REF can bind RNA in vitro (E.Izaurralde, unpublished results). Thus, RNPS1, Y14 and REF are all good candidates for being the −20/24 complex components that interact directly with spliced mRNA.
Another splicing factor known to interact directly with the 5′ exon during splicing is hPrp8p, a core component of U5 snRNP (Teigelkamp et al., 1995; Collins and Guthrie, 1999; Luo et al., 1999; Siatecka et al., 1999). We have previously shown that, like SRm160, hPrp8p can be cross‐linked to the PIP85.B exon–exon junction specifically as a consequence of splicing (Le Hir et al., 2000). However, our current data suggest that hPrp8p is unlikely to be a general mRNP component. Not only is the mass of the −20/24 mRNP complex too small to accommodate a 270 kDa protein, but anti‐hPrp8p antibodies fail to co‐immunoprecipitate any mRNA that has been purified away from spliceosomes (data not shown). Furthermore, although hPrp8p could also be cross‐linked to the exon– exon junction of AdML and TPI mRNAs in a splicing‐dependent manner, not all such cross‐linked species were released from spliceosomes (data not shown). This suggests that, while hPrp8p does generally interact with mRNA exon–exon junctions prior to their release from spliceosomes, this interaction does not persist in most mRNP complexes.
Functional implications of the −20/24 complex for mRNA export
It was recently demonstrated that some mRNAs produced by splicing are exported from the nucleus more efficiently than those not produced by splicing, suggesting that spliceosomes alter the mRNP so as to enhance its productive interaction with the nucleocytoplasmic transport machinery (Luo and Reed, 1999; Zhou et al., 2000). Therefore, it is notable that two of the −20/24 complex components defined here have been directly or indirectly linked to mRNA export.
REF, like its yeast ortholog Yra1p, is functionally required for mRNA export (E.Izaurralde, unpublished results; Strasser and Hurt, 2000; Stutz et al., 2000; Zhou et al., 2000). REF/Yra1p can bind RNA and simultaneously contact the mRNA export factor TAP (Mex67p in yeast; E.Izaurralde, unpublished results; Strasser and Hurt, 2000; Stutz et al., 2000). TAP/Mex67p is a shuttling protein that interacts with components of the nuclear pore complex, and Mex67p is essential for viability in yeast (Katahira et al., 1999; Bachi et al., 2000). Although TAP can bind directly to certain viral RNA export elements (Gruter et al., 1998; Bear et al., 1999), it does not have high affinity for RNA in general (Braun et al., 1999; Stutz et al., 2000). It has been suggested that the principal means of TAP association with cellular mRNA is through protein–protein interactions (Bachi et al., 2000; Strasser and Hurt, 2000; Stutz et al., 2000; E.Izaurralde, unpublished results). In addition to REF, TAP can associate with other RNA binding proteins in vitro and in vivo. One of these is Y14, which is found complexed in vivo with TAP, spliced mRNA and hnRNP proteins (Kataoka et al., 2000). Our findings that both REF and Y14 are contained in the −20/24 complex suggest that these proteins may mediate the preferential export of spliced mRNA by recruiting export factors, such as TAP, to spliced mRNPs. Since we could not detect TAP in the −20/24 complex (Figure 3B), it is possible that TAP is recruited at an intermediate stage between splicing and export that is not replicated under our in vitro conditions.
The conserved position of the −20/24 complex suggests a role in NMD
In addition to a role in mRNA export, it is possible that components of the −20/24 complex may 'mark' the positions of exon–exon junctions for the process of NMD (reviewed in Li and Wilkinson, 1998; Nagy and Maquat, 1998; Hentze and Kulozik, 1999; Maquat, 2000). In particular, the location of this complex at a fixed position upstream of the exon–exon junction could explain the previous observations that in‐frame termination codons are generally not recognized as premature unless they are located >50–55 nt upstream of the 3′‐most exon–exon junction of an mRNA. It is now known that introns and, by implication, a splicing‐dependent modification of mRNP, are required for both nucleus‐associated and cytoplasmic NMD (Sun et al., 2000). While it is debatable whether nucleus‐associated NMD involves cytoplasmic ribosomes, the involvement of cytoplasmic ribosomes in cytoplasmic NMD indicates that at least some components of the splicing‐dependent modification to mRNP accompany mRNA into the cytoplasm. Since REF and Y14 are known shuttling proteins (Kataoka et al., 2000; Zhou et al., 2000; E.Izaurralde, unpublished results), they are candidates for such a role. Future studies will explore possible functional interactions between components of the −20/24 complex and components of the translational machinery required for NMD.
Materials and methods
In vitro splicing
Uniformly labeled splicing substrates with a GpppG dinucleotide cap were synthesized by standard run‐off transcription. TPI pre‐mRNA corresponded to a portion of exon 6 through exon 7 of the human TPI gene. DNA templates for AdML (HMS81) (Gozani et al., 1994), β‐globin (Reed and Maniatis, 1988) and PIP85.B (Query et al., 1994) were described previously. DNA templates for corresponding intronless control mRNAs were constructed by PCR. AdML pre‐mRNAs and control mRNA containing a single labeled phosphate were synthesized by splinted ligation (Moore and Query, 1998). In vitro splicing reactions, analysis of splicing products and glycerol gradients were performed as previously described (Le Hir et al., 2000). Glycerol gradients (10–30% glycerol) contained 8 mM Tris–HCl pH 7.6, 40 mM KCl, 20 mM potassium glutamate, 2 mM magnesium acetate.
RNase digestions
Activation of endogenous RNase H activity was accomplished by adding each cDNA oligo (1 μM) directly to splicing reactions and incubating the reaction for an additional 10 min at 30°C. When mRNAs were purified by glycerol gradient fractionation, they were supplemented with exogenous RNase H (0.1 U/μl; USB). For complete RNase digestion, splicing reactions were supplemented with 0.5 mg/ml heparin, RNase T1 (1 U/μl; Sigma) and/or RNase A (0.02 μg/μl; Sigma) and incubated for an additional 10 min at 30°C. For gel filtration analysis, a 10 μl splicing reaction containing 4 nM singly labeled AdML pre‐mRNA was incubated for 90 min at 30°C, supplemented with heparin, treated with RNase A, diluted with 300 μl of IP‐150 buffer [150 mM NaCl, 50 mM Tris–HCl pH 7.6, 2 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.05% NP‐40] and loaded onto a Superose 6 FPLC column (Pharmacia) equilibrated with the same buffer. Molecular weight standards were analyzed in parallel under the same conditions.
Xenopus laevis oocyte microinjections
Uniformly labeled AdML and β‐globin pre‐mRNAs and control mRNAs were synthesized as above, except with a 7mGpppG dinucleotide cap. Oocyte injections and analysis of microinjected RNA by denaturing gel electrophoresis and autoradiography were performed as previously described (Jarmolowski et al., 1994). For RNase H cleavage, 10 nuclei were collected 90 min after injection in 35 μl of RNase H buffer (100 mM Tris–HCl pH 7.5, 50 mM MgCl2, 250 mM KCl, 0.5 mM DTT) containing the corresponding cDNA oligo at 5 μM and incubated for 15 min at 30°C. RNase H cleavage was stopped by the addition of 200 μl of SDS–proteinase K buffer (300 mM NaCl, 50 mM Tris–HCl pH 7.5, 5 mM EDTA, 1.5% SDS, 2 mg/ml proteinase K). RNAs were extracted with phenol, precipitated and separated by 15% (AdML) and 10% (β‐globin) denaturing PAGE, respectively.
Antibodies and IPs
The following antibodies were kindly provided by the person indicated: anti‐SRm160 [monoclonal antibody (mAb) B1C8; B.J.Blencowe]; anti‐DEK (G.Grosveld); anti‐RNPS1 (A.Mayeda); anti‐REF and anti‐TAP‐C (raised again the C‐terminal domain of TAP; E.Izaurralde); anti‐Y14 (mAb‐4C4; G.Dreyfuss); anti‐hnRNP A1 (mAb‐4B10), anti‐hnRNP C1/C2 (mAb‐4F4) and anti‐Sm (Ab‐Y12; S.Piñol‐Roma). Anti‐3mCAP antibodies were purchased from Roche. All antibodies (except anti‐3mCAP) were independently confirmed by western blotting; each detected a major band with the expected mobility in HeLa cell nuclear extract (data not shown). mAbs B1C8 and 4C4 were bound to protein A–Sepharose (PAS) beads (Pharmacia) via rabbit anti‐mouse IgG + IgM (Pierce). All other antibodies were bound directly to PAS beads. IPs were performed as previously described (Le Hir et al., 2000) in IP‐100 buffer (the same as IP‐150, except 100 mM NaCl). IP efficiencies were calculated by dividing the intensity of a given precipitated fragment (measured by PhosphorImager; Molecular Dynamics) by the intensity of the same fragment present in the input reaction, taking into account the fact that only one‐fifteenth of each input reaction was loaded on every gel.
We are grateful to the following people for providing antibodies: Ben Blencowe, Gideon Dreyfuss, Gerard Grosveld, Akila Mayeda and Serafin Piñol‐Roma. We thank Natasha Levin, Cecilia Lu and Maidung Nguyen for technical support; members of the Moore and Maquat laboratories for helpful discussions; Torben Heick‐Jensen, Charles Query and Michael Rosbash for critical comments on the manuscript; and Ben Blencowe and Gideon Dreyfuss for communicating results before publication. M.J.M. is an HHMI assistant investigator and H.L.H. was partially supported by a fellowship from Philippe Foundation. L.E.M. was funded by NIH grant DK33938 and E.I. was supported by the European Molecular Biology Organization (EMBO).
- Copyright © 2000 European Molecular Biology Organization


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The Red Sox are in some trouble. Like the race just started and the boat is on fire trouble. The team many if not most picked to win the AL East (guilty) just snapped a 10-game losing streak, which pushed them from second to last place in the division. If it weren't for the Astros, Boston would be last in the American League.
Dear Astros, thanks! Love, Boston.
That might not be news or would certainly be less newsworthy were it another team, but we media types love irony, and the bigger the dose the better. We also love it when the big guys fall, so this works on lots of levels. You see, just a few months ago the Boston Red Sox were baseball's model organization. A dominating and inspiring World Series win coupled with a top five minor league system bursting with talent is a desirable combination and made the Red Sox the team to emulate, likely along side or maybe just behind St. Louis, depending on who you asked. Entire articles were written advancing this idea. And now, 10 straight losses later, the Red Sox are terrible. They can't hit, they can't pitch, they can't play defense and most importantly they can't win a baseball game. The jamming of brakes by baseball's punditry is enough to give readers whiplash.
It's easy to look at a team on a 10-game losing streak and say they're (insert your expletive of choice here). But no major league team is that bad. So you won't find charities to donate to in lieu of flowers here. There are 113 games left in the season and the trade deadline is still months away, meaning there is ample opportunity to turn things around, to the extent that things can be turned around.
Before looking at some fixes, I thought it would be instructive to see where the Red Sox are and how they got here. So how did the Red Sox get here? Before the streak, Boston was chugging along, a game over .500 and, thanks to the generally crappy turn the AL East has taken so far this season, just a half game out of first place. Everything wasn't perfect, but nothing was inexorably broken.
Over the next 10 games Boston was out-scored by 27 runs, while the Red Sox themselves scored 26 runs. There are, of course, other stats, but the team has just come off losing 10 straight games so you'd expect they're not playing well in any phase of the game and you'd be correct in that expectation. The shocking part, as has been mentioned, is that this is the team fresh off a World Series win. Mostly. There are some differences.
The Red Sox made the following changes to their World Series winning lineup:
• Will Middlebrooks to start at third base
• Xander Bogaerts to start at shortstop
• A.J. Pierzynski to start at catcher
• Jackie Bradley to start in center field
That seems like a lot (there are only eight non-pitching starters after all), but when you remember that Will Middlebrooks was the starter at third most of last season, and Xander Bogaerts was the starter at third at the end of the season and into the playoffs, things don't look so different. What's more, Bogaerts has been quite good with the bat, so it can't really be that bad, can it? Well, yes.
First, Middlebrooks. He's been on the DL twice, which has almost been a blessing for the Red Sox considering he's hit .197/.305/.324 when healthy. That's from a guy whose primary skill is hitting for power. So that hasn't worked. Xander Bogaerts moved from third to shortstop, the position the Red Sox were hoping (and probably are still hoping) he could occupy for the next decade. But Bogaerts' defense at shortstop has been bad. There have been errors and there have been mental gaffes, and there have been mistakes that shortstops still learning the position tend to make. It has been a large downgrade from the steady and occasionally spectacular Stephen Drew to Bogaerts at a vital defensive position.
Beyond the left side of the infield, the Red Sox declined to match Jarrod Saltalamacchia's free agent offer from Miami and instead spent $8.25 million to bring in A.J. Pierzynski on a one-year deal. At 37 years old, Pierzynski is a known quantity. He has some power and can hit for some average, and works well with a pitching staff. But he doesn't get on base really at all, isn't much of a pitch framer, and as for throwing out runners, well, not so much. That's pretty much what the Red Sox have received minus the batting average and it's a big downgrade on Saltalamacchia's 2013 performance.
Moving to the outfield, Jacoby Ellsbury's indentured servitude was up and he signed a seven-year contract with the Yankees. The Red Sox had, they thought, two players to replace him. The first is Jackie Bradley, a first round draft pick whose defense has been described as gold-glove ready. What's more, Bradley has hit and gotten on base as well or better than Ellsbury did in the minor leagues. But, while Ellsbury set the world afire when he arrived on the scene in 2007, Bradley has done the opposite. His defense has been as advertised, but to date, he's hit a horrendous .193/.280/.283. That might be more palatable if the second option hadn't failed as well, but Grady Sizemore has managed a similarly awful .221/.291/.353 slash line, but with defense that suggests his time as a center fielder passed several seasons ago.
So the Red Sox exchanged a borderline MVP candidate in center field for replacement-level production, a good hitting catcher for a bad hitting one, a good fielding shortstop for a bad fielding one, and a good hitting third baseman for a bad hitting one. That's four downgrades, and two at key up-the-middle defensive positions. This helps explain why the Red Sox turn balls in play into outs worse than any team in baseball except Texas. Last season Boston was 17 (out of 30) so it wasn't like they were wonderful then either, but a good hitting and pitching team can thrive with an average defense at its back. It's much harder to win with a bad defense, offense and pitching be damned.
Beyond those four, the Red Sox have had other steps back. Daniel Nava, one of the Red Sox best hitters last season, couldn't buy a hit (at least part of this was just bad luck) at the start of the season and was demoted to Triple-A, partly so the team could keep Sizemore on the roster. Nava was just recalled when Shane Victorino was put on the DL for the second time with a hamstring strain. And that's the other thing. The team that stayed relatively healthy last season has had to deal with some injuries. As mentioned above, Middlebrooks and Victorino have seen the DL twice apiece, and first baseman Mike Napoli and starter Felix Doubront were introduced to it this past week as well. It's hardly a cavalcade of stars like the Texas Rangers or Atlanta Braves (at least at the beginning of the season) have lost, and it's not the sole reason for the 10 straight losses, but it's not nothing.
All of the above would put a hurt on the bottom line, but the team would still be fine if the rest of the club were producing. After Lester and John Lackey, the starting rotation has varied from mediocre results with the potential to be worse (Jake Peavy) to hot garbage (Clay Buchholz). Buchholz in particular is interesting as he went from Cy Young contender last season to injured, to, now, a barely major league-quality pitcher. Buchholz's changeup, the pitch that set up his fast but straight fastball, and kept lefties from teeing off on him, has disappeared. [insert milk carton joke] The end result has been a five run jump in ERA and the results to match. As for the bottom line, the Red Sox are 4-6 in games started by Buchholz. Compare that to last season when they went 14-2 in Buchholz's starts and you can start to see how a huge strength became a huge weakness.
It's not all flaming bags of poo and sadness. Jon Lester has had a terrific start to the season, though he got shelled in his last start and will be a free agent at the end of the season, so … yeah.
So that's how the Red Sox got where they are. Now, how do they get out of it? That's a tougher article to write. Like most teams at this point in the season, there are only so many moves available. I can suggest trading for Giancarlo Stanton and Jeff Samardzija (if for no other reason then I'd learn how to spell it), but that's not realistic. Most teams aren't looking to make moves this time of the season. There are some things the team can do though.
My first suggestion has already been taken, and that's recall Daniel Nava as the team did that last Friday. Nava isn't a panacea but he can hit right-handed pitching. The Red Sox have a .683 OPS from their left fielders so far this season (it should be pointed out that some small part of that is Daniel Nava), and Nava figures to be an improvement on that against righties (he had an .894 OPS against right-handed pitchers last season). Against lefties, well, that's why they have Jonny Gomes. Between those two players, left field should be a productive spot in the lineup. Second take!
As for the left side of the infield, they've already (seemingly) solved that as well with the signing of Stephen Drew last week. Drew will start at shortstop and Bogaerts will slide back to third base. Assuming Drew is some variation on his 2013 performance, this improves two positions simultaneously. Bogaerts and his 15 percent above average bat (to date) remain in the lineup but he moves from short to third base. This should improve the defense at both positions (or at least isn't a downgrade at third). The addition of Drew's bat is also an upgrade over what Middlebrooks was providing, so even better.
In center, well, Jacoby Ellsbury isn't walking through that door. Jackie Bradley is though. As noted, Bradley has been awful so far at the plate, but the fact that he's hit at every level of the minor leagues should give his critics pause. He just turned 24, so there is reason to believe the young guy can make some improvements. Not everyone is Mike Trout, and if you go back and look at Trout's 2011 season, even Mike Trout wasn't Mike Trout. Prospects, even the best of prospects, need time to adjust and teams that don't provide them with that time don't reap the benefits just the drawbacks of playing young players. Bradley does bring good defense at a premium defensive position, so the Red Sox need to ride that, install him at the back of the lineup, and make the young guy comfortable*
* Unless…well, keep reading.
It would be nice if the Red Sox could bring up a young prospect to fill in at a position of need, but despite the pedigrees, the Triple-A roster is devoid of anyone pushing for promotion. There might come a time when Christian Vasquez (at least for now, the Triple-A catcher version of Jackie Bradley [all defense, little offense]) makes a case for playing time over either David Ross or A.J. Pierzynski, but that time isn't now unless the Red Sox are willing to play two players solely based on defense.
The Red Sox will need a starter to step in and possibly stay for a while depending on the nature of Doubront's injury and they might need a second starter if Clay Buchholz doesn't figure out which way is up. There is now room for people who can pitch effectively in the rotation. Boston is rich in Triple-A pitching prospects, but none are distinguishing themselves at the moment. They could bring up Allen Webster or Anthony Ranuado and hope, but neither of those guys appear ready to successfully do battle with major league hitters. Looking one rung down the ladder though presents some interesting options. Henry Owens is both highly regarded and is shutting down Double-A hitters for the Portland (Maine) Sea Dogs. Owens has had some issues with walks, but he does just about everything else wonderfully. In 57.1 innings Owens has 63 strikeouts to 28 walks. What's more, he's given up just 38 hits in those innings. Over his last four starts, Owens has given up seven hits and three runs in 22.2 innings while striking out 28 and walking 15. The Red Sox are typically conservative when it comes to promoting from Double-A and Owens isn't on the 40 man roster yet (though he'll have to be added after the season) but adding Owens could be an electrifying jolt to the team's rotation.
The other player in Double-A to consider is Mookie Betts. Betts is hitting .366/.452/.575 on the season with 22 stolen bases (three caught-stealings) and six homers. Betts is a second baseman by trade, but recently the Red Sox have been experimenting with him in center field. This could be a precursor to moving him up to Triple-A, but if he's comfortable enough, why not skip Pawtucket altogether and move him up to the majors? Well, again, this would require a 40 man roster spot, so that's certainly a strong argument against because it would necessitate cutting someone. But in this particular case, there is someone who can be cut: Grady Sizemore. Sizemore has been a wonderful story in that he's stayed healthy after returning to the majors leagues from more knee surgeries than one should be able to count, but the wonderfulness starts to fall away when you look at how Sizemore has hit and the fact that he can no longer play an effective center field. With Gomes and Nava in left and Victorino in right, there isn't a spot for Sizemore unless his production warrants it, and as of now his production warrants being sent to Triple-A. Meanwhile Betts continues to tear up Double-A. In such a move, Betts would displace Jackie Bradley who would move to right field, or if Shane Victorino is healthy, back to Triple-A.
This all leaves us with the following lineup:
1. Dustin Pedroia, 2B
2. Xander Bogaerts, 3B
3. David Ortiz, DH
4. Mike Napoli, 1B
5. Daniel Nava, LF
6. Stephen Drew, SS
7. A.J. Pierzynski, C
8. Jackie Bradley/Shane Victorino, RF
9. Mookie Betts, CF
…and starting rotation
1. Jon Lester
2. John Lackey
3. Jake Peavy
4. Brandon Workman/Clay Buchholz
5. Henry Owens
I don't like using the word 'spark.' The Red Sox have more problems than a spark can solve, but eliminating Sizemore and potentially Bradley for one of the hottest hitters in the minor leagues should help improve the offense. The Drew signing will improve the production from the left side of the infield both offensively and defensively, and adding Owens gives the rotation some actual upside beyond waiting for Clay Buchholz to stop being awful.
Does raiding the Double-A roster after a bad two-week stretch qualify as panicking? Maybe. But the season is slipping away. Ten losses is one thing, but some serious cracks showing in the armor is another. It's time to bring the full force of the organization to bear. Even so, it's likely none of this will save the Red Sox. Dustin Pedroia, Shane Victorino, and David Ortiz are all going to have to remember how to hit, and Buchholz will have to fix his changeup or go somewhere else. But, like their team, their track records say the chances they improve are pretty good. Remember, this team won the World Series last year. It's getting harder and harder to remember.


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There's something about Jennifer Lopez's love life that never gets old. Despite three divorces and three major flings, she seems to be a romantic idealist at heart. And, seriously, it's super cute.
The "I Luh Ya Papi" singer stopped by Chelsea Handler's E! talk show "Chelsea Lately" on Thursday to chat about who she's dated, what kind of men she prefers and what the future holds -- you know, all that good stuff.
Handler began by asking J.Lo about her romantic game plan, specifically if marriage was on the horizon. "I would get married again," the


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PowerColor Radeon HD 6970 LCS Review
Of all the cooling solutions on the market one of the most appealing and yet most feared is water cooling. Who in their right mind would put water in with electronics and hope that the inevitable does not happen (sheepishly raising my hand)? Water cooling is one of the cooling solutions that offer tremendous upsides when done correctly and that most feared of things when done incorrectly. The last factory released water cooled card I looked at was made by PowerColor, the HD 5970 LCS. What I found was that by liquid cooling the trouble spots with a full cover water block, the core clock speeds were increased by another 120Mhz over a non-water cooled card. Peformance was increased significantly as well with that card so the expectation is that the HD 6970 LCS from PowerColor will offer up some nice gains in performance with the enhanced cooling. PowerColor have again gone with EK Waterblocks as their supplier of choice to equip this card. The Acetal and Nickel plated copper is an enticing look that is all the rage right now.
So what do you get with a card of this caliber when you shell out a cool 5 bills for it? You get a card equipped with a high-end cooling solution that is already installed (saving you the trouble and potential heartaches), comes with a warranty and gets a generous increase in the base clock speeds to 925MHz on the Cayman XT core and 1425MHz on the 2GB of GDDR5 memory. Seeing as how just about every one of the HD 6970 cards I have tested have delivered a clock speed of just over 1000MHz I have to wonder if the cooling will allow for some significant increases above that number or will the main benefit be just reduced operating temperatures? Let's see what the HD 6970 LCS from PowerColor has to offer and see if it can live up to the hype.
Closer Look:
The packaging used for the HD 6970 LCS is traditional PowerColor and is on the smallish side. The front panel of the shell shows the card with the model numbers and series listed under the card. To the bottom right is a picture of four monitors in an Eyefinity setup. The total amount of the GDDR5 frame buffer is listed at the top right and is 2GB in size. Just under this listing are the type of outputs this card is capable of using. The back side of the box talks about AMD specific technologies that are employed by the HD 6900 series including Accelerated Parallel Processing, AMD HD3D, HDMI 1.4, CrossfireX and DirectX 11. Also discussed on the right hand rear panel are the benefits of water cooling as PowerColor sees them. Chief among those reasons are the immense cooling potential of a liquid cooled solution.
Inside the shell is the box that holds the HD 6970 LCS. Inside are the driver disk and a generic installation manual. Digging further you find the video card and hidden in a compartment up at the front side of the box is the large selection of accessories.
The bundle of accessories that come with this card include just about everything you need to get the HD 6970 LCS installed in your system save the liquid loop. That you need to supply on your own! The balance of the bundle includes 3/8 and 1/2 inch barbed fittings, a pair of extensions in case your fittings are to long to fit the EK block, clamps for both sizes of tubing, a Crossfire bridge connection, Mini HDMI to HDMI adapter and the 6 Pin PCIe power to 8 pin adapter.
You can see from the packaging and contents that PowerColor has something special here. It will be interesting to find out just how special it is for the premium price.


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The Ravens' scouting system, envied around the league, has remained remarkably consistent under General Manager Ozzie Newsome.
There have been a few tweaks along the way to adapt to technology and the times, but the organization still doesn't belong to the two main national scouting services, and still does put the onus on their own area scouts to do the bulk of the heavy lifting.
"We still have our area scouts responsible for every player that's being drafted in their area. So, a lot of that has not changed," Newsome said Wednesday at the team's annual draft luncheon. "There's a lot more information, but I think there's a lot more misinformation, and our scouts are responsible for getting as good information as they can for us and bringing it back to this group here so that we can make decisions. We would much prefer our scouts talking to strength coaches, secretaries at the school and people like that then living off the Internet."
One area, however, that the Ravens, like the rest of the sports world, are now paying particular attention to are past head injuries. The Ravens' scouting reports on draft prospects now include mention of how many concussions a player sustained in college.
"That information is out there," Newsome said. "As far as what we do, and most teams do, is once we get guys here, we'll get a baseline test done with those players, so that if a concussion occurs, then we have something that we can refer back to. But, it is something now that throughout the league – and in all professional sports and on the collegiate level – that everybody is spending more time looking at."
Ravens Director of Player Personnel Eric DeCosta said that the team has gotten the medical information back from the participants in the NFL Scouting Combine last month and will spend next week studying specific players. Prospects that visit the team's Owings Mills facility before the draft are also put through a physical and various medical tests.
DeCosta said that typically, the Ravens drop as many as 10 players off their draft board because of failed physicals and other medical issues. Then, there are other players who are eliminated because of character questions or behavioral issues.
"Any kind of character things are major red flags," DeCosta said. "There is a slotting scale of things that would be major character concerns because society has changed. Some issues that would be major flags 10 or 15 years ago may not be as major of a flag now and there will be some new things that we'll look at character-wise that we really didn't have to deal with 10 or 15 years ago as much. Obviously, any sort of mental concern would be a red flag because football is a game where you have to think quickly. We'll investigate any sort of mental/learning issue with every player that we like because we feel that is critical to a guy's success or a lack of success.
"And then the third thing would be durability issues. One of the things that we've seen with players is when players fail, oftentimes, it's because they just can't stay healthy, and they can't play, they can't practice, they can't get out there. We've tried to do a better job, I think, and we've spent more time trying to figure out what a player's durability is going to be, projecting that forward and also looking at the issues that he's had. I think our doctors and trainers do a great job of that and we've put a burden on our scouts as well to do a better job getting that kind of information."


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Lincoln County Prosecuting Attorney Leah Askey announced on Tuesday, Oct. 2, Charles Neubauer, 39, of Foley, was sentenced in the Circuit Court of Lincoln County by the Honorable Dan Dildine for two counts of Statutory Sodomy in the First Degree and one count of Statutory Sodomy in the Second Degree.
Neubauer received a 15-year sentence on each of the Statutory Sodomy First charges, to run concurrently, and seven years on the Second Degree Sodomy charge, also to run concurrently.
The charges arose out of events occurring as far back as 2007 and as recently as 2011. The victim and his family traveled from the State of Indiana to be present for the defendant's earlier plea of guilty and for sentencing.
"The courage and perseverance of the victim led to the successful prosecution of this case. He saw it through and traveled to ensure justice was served. Hopefully, he will be able to move on with his life and put this traumatic experience behind him," said Case Attorney, First Assistant PA, George Gundy.
Askey said she was pleased with the sentence Judge Dildine imposed in this case, despite the Defendant's plea for probation.
"It is difficult to say what "justice" should be in a case like this. Anytime a child is involved, I am always hopeful for as much time as the Court will allow." Askey said.
This case was prosecuted by First Assistant Prosecuting Attorney George Gundy with the assistance of the Lincoln County Sheriff's Department, including Detective Patrick Harney, who was the lead investigator.
Askey also announced that David Knudsen, 39, of Moscow Mills, has plead guilty and was sentenced for an assault on a Lincoln County woman and her 14-year-old daughter in 2010.
Dildine sentenced Knudsen to 15-years in the Missouri Department of Corrections for Assault in the First Degree and 15-years in the Missouri Department of Corrections for Domestic Assault in the Second Degree.
The charges arose from a Dec. 31, 2010, incident in which Knudsen severely assaulted his then-girlfriend and threatened to kill the victim's 14-year-old daughter while holding her at knife point.
"I am glad to see this case come to end. We strive to gain closure for the victims as quickly as possible and these victims have been diligent and involved in the process," said Assistant PA Therese Marchlewski.
Askey went on to say that it was a good week for sentencing in Lincoln County and hopefully a message is being sent to offenders that Lincoln County takes prosecution of these crimes seriously.
This case was prosecuted by Assistant Prosecuting Attorney Therese Marchlewski. It was investigated by the Lincoln County Sheriff's Office. Deputy Kevin Hunt was the lead investigator.


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Nixon Vows to Lead Efforts for Contribution Limits
JEFFERSON CITY - Missouri Governor Jay Nixon is vowing to back a ballot initiative to restore limits on campaign contribution if lawmakers refuse to approve them.
Nixon argued in his State of the State address Monday that Missouri must reinstate strict limits on how much money candidates can receive from individual donors.
If lawmakers don't approve contribution limits, the Democratic governor said he will do everything within his power to ensure that a citizens' initiative makes the ballot and is approved by a statewide vote.
Missouri's Republican-led legislature repealed the state's campaign contribution limits in 2008. They argued that the limits led big donors to funnel money to politicians through a variety of obscure committees. Republicans said unlimited donations made it easier for people to track the money.


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The Minnesota Commission on Judicial Selection is created for the purpose of soliciting judicial candidates, evaluating applicants and recommending nominees to the Governor for appointment. It will fill vacancies that occur within district courts and any vacancies that occur during the term of a judge for the Workers Compensation Court of Appeals. The Commission has 49 members. 27 are appointed by the Governor and 22 are appointed by the Minnesota Supreme Court. 9 of these members are appointed at large to consider vacancies within every judicial district. Of these 9 members, at least 3 must be non-attorneys. 4 members are appointed to represent each of Minnesota's 10 judicial districts. Of these 4 members, there must be at least 2 non-attorneys. Each member of the Commission is appointed to a 4 year term that ends at the conclusion of the Governor's term.
The Commission is governed by 2016 Minnesota Statutes 480B.
The Commission on Judicial Selection is committed to providing equal access to the judicial selection process for all interested individuals. If you need alternative formats or services because of a disability, please contact Megan Jablonski Johnson at (651) 201-3451 or via email email@example.com with your request.
For a list of members on the Minnesota Commission on Judicial Selection, click here.
Outline of Application Procedures for District Court Judgeship.
To receive judicial announcements via email, please contact Megan Jablonski Johnson at firstname.lastname@example.com
For a list of Judicial Resources, click here.


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Tabac Original Cologne
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Reviews (44)
1 - 5 (of 44 Reviews)
Tabac Original (1959) by Mäurer & Wirtz I was searching for a soapy-clean fragrance, I read all the reviews and decided to purchase Tabac to try. This is a warm, sweet, soapy fragrance that I use for any occasion. After a shower, going to work, or just to shop at the local stores. After dry down, It is not over powering, subtle but still noticeable. I will keep it as one of my standard scents, now that I have tried it and liked it. As with any cologne scent........you either like it, or not. I LIKED IT. And would recommend it for anyone to try and wear.
Written by Keith..... on October 11, 2014
I have been using Tabac cologne for more than 20 years, but it is nearly impossible to find in stores. I was pleased to find it on your website. I just love its clean manly scent. I put it on every morning along with my deodorant.
Written by Jazz on July 07, 2014
My husband was introduced to Tabac in his early 20s when his parents brought it back from Germany. More than 20 years later we still find the scent comforting (no place like home). It's a clean, masculine scent. Only problem is that we could only purchase this in a German Butcher. Unfortunately, once we moved it was hard to locate. I'm so happy for the internet and finding TABAC here at this site.
Written by Livluvlaf on June 16, 2014
I bought this for my brother and he loves it he is always looking out for fresh clean smell in a cologne. I highly recommend this since my brother really likes it and he is one that is very picky when it comes to his colognes. I will be ordering again for him in the future.
Written by Anne on June 04, 2014
1 - 5 (of 44 Reviews)