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tomekkorbak

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[ "Hughesville, Maryland\n\nHughesville is a census-designated place (CDP) in Charles County, Maryland, United States. ", "The population was 2,197 at the 2010 census. ", "Truman's Place was listed on the National Register of Historic Places in 1988.", "\n\nGeography\n\nHughesville is located at (38.533731, −76.782220).", "\n\nAccording to the United States Census Bureau, the CDP has a total area of , of which is land and , or 0.69%, is water.", "\n\nHughesville was a tobacco market town. ", " The former tobacco warehouses are now used for second-hand thrift stores and boutique gift and craft shops. ", "A multi-lane bypass of the town by Maryland Route 5 opened in February 2007, which has alleviated previous rush-hour traffic backups at the single traffic signal; beside it are two unusual side-by-side traffic roundabouts.", "\n\nBecause Hughesville, though small, is considered the strategic geographic center of the tri-county Southern Maryland region, it houses institutions such as the headquarters for the Southern Maryland Electric Cooperative, an animal shelter, an economic development council, a board of realtors, and a homeless women's shelter, which serve the tri-county area. ", "There is also a Girl Scout camp. ", "The town has no traditional grid-layout streets, but consists of merely businesses along the highway and some very small outlying housing developments. ", "It was announced in 2013 that the College of Southern Maryland is building their fourth campus in the town.", "\n\nA 5.5-MW solar farm near Hughesville generates enough power for 600 homes, and offsets the pollution of 1,600 cars.", "\n\nDemographics\nAs of the census of 2000, there were 1,537 people, 503 households, and 407 families residing in the CDP. ", "The population density was 137.4 people per square mile (53.0/km²). ", "There were 529 housing units at an average density of 47.3/sq mi (18.3/km²). ", "The racial makeup of the CDP was 82.04% White, 13.08% African American, 0.72% Native American, 2.73% Asian, 0.07% Pacific Islander, 0.13% from other races, and 1.24% from two or more races. ", "Hispanic or Latino of any race were 0.65% of the population.", "\n\nThere were 503 households out of which 36.4% had children under the age of 18 living with them, 68.2% were married couples living together, 7.4% had a female householder with no husband present, and 18.9% were non-families. ", "12.7% of all households were made up of individuals and 4.6% had someone living alone who was 65 years of age or older. ", "The average household size was 2.97 and the average family size was 3.25.", "\n\nIn the CDP, the population was spread out with 26.3% under the age of 18, 6.5% from 18 to 24, 30.0% from 25 to 44, 27.3% from 45 to 64, and 10.0% who were 65 years of age or older. ", "The median age was 38 years. ", "For every 100 females, there were 99.4 males. ", "For every 100 females age 18 and over, there were 97.4 males.", "\n\nThe median income for a household in the CDP was $90,697, and the median income for a family was $103,393. ", "Males had a median income of $49,500 versus $36,563 for females. ", "The per capita income for the CDP was $29,884. ", "About 4.8% of families and 7.1% of the population were below the poverty line, including 7.9% of those under age 18 and 5.1% of those age 65 or over.", "\n\nReferences\n\nCategory:Census-designated places in Charles County, Maryland\nCategory:Census-designated places in Maryland" ]

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[ "This invention relates to a three-ring binder having an actuating crank.", "\nMany modem ring binder mechanisms have actuating levers for opening and closing two, three or more rings. ", "In some such devices, the levers also lock the rings closed. ", "The typical arrangement is to attach the bottoms of the ring halves to hinged plates confined between the edges of an arcuate metal housing which provides a toggling action as the plates snap between open and closed positions.", "\nOther devices have been proposed in which the rings are opened and/or closed by a cam-type mechanism. ", "Prior such constructions are seen in U.S. Pat. ", "Nos. ", "778,910, 2,494,898, 2,789,561, and 2,894,513. ", "U.S. Pat. ", "No. ", "778,910 discloses a two-ring binder which is opened by lifting the end of a lever which depresses a crank whose ends are the movable ends of the two rings. ", "It would be advantageous to have a three-ring mechanism of the crank-actuated type.", "\nAn object of the invention is to improve the operation of a crank-operated ring binder having three or more rings.", "\nThese and other objects are attained by a three-ring binder having a support plate, and at least three rings, each comprising a movable segment pivotally attached to the support plate and an immovable segment affixed to said support plate, and a mechanism for moving the rings between an open position and a closed and locked position. ", "The mechanism includes a crank pivotally supported on the support plate for oscillation about a longitudinal axis. ", "The crank has plural throws offset from the longitudinal axis. ", "The movable ring segments are integrally attached to said crank. ", "A leaf spring biases the crank toward a rings-closed position, and a manually operable lever moves the crank toward a rings-open position. ", "The lever is pivotally mounted on said support plate and depresses the throw, moving the crank towards its rings-closed position, as the lever is depressed." ]

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[ "Q:\n\nHigh speed steering & wheel vibration\n\nFirst post and I'm flummoxed. ", "I have an 2009 Lexus RX350 AWD. ", "At speeds of 70-75 one can feel the steering wheel vibrate fast. ", "At 80mph(for testing purposes) it largely disappears. ", "Back down to 65 the vibration in the steering dissapates greatly but one can still feel the front wheels shake. ", "\nI've done the following:\nNew tires. ", "I had them rebalanced three times. ", "A fourth time at an independent shop. ", "No improvement. ", "Went back to the original store and had them put on two new front tires on the off chance it might be a bad set of tires. ", "Had them road force balance them too. ", "No improvements.", "\nReplaced rotors, brakes, entire front strut assembly along with lower ball joints. ", "Also replaced steering column.", "\nAlignment done after that.", "\nTook it for a drive today and no improvement. ", "Full disclosure: My mechanic at the outset said I didn't need new ball joint or struts as the whole suspension looked new almost. ", "\nSo I'm stuck as he is too. ", "Not sure what to do without throwing money at this problem. ", "Did the 12-6 & 9-3 tire test and the wheels don't jiggle or move at all.", "\nCould it be a bad set of rotors? ", "This didn't happen while I had my old tires & rotors on them prior to replacement.", "\nAny ideas? ", "Thanks.", "\n\nA:\n\nHave you tried swapping the front and rear wheels, you could have one slightly bent wheel.", "\n\n" ]

{ "pile_set_name": "StackExchange" }

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[ "Q:\n\nEmacs: Is it possible to expand/collapse single chapters?", "\n\nIn Emacs + org-mode it is possible to collapse the whole text, expand all the subtitles and all the text with SHIFT+TAB.", "\nIs it possible to expand/collapse single chapters?", "\n\nA:\n\nYes, just omit the Shift and use Tab only.", "\n\n" ]

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[ "Introduction {#s1}\n============\n\nPhagocytosis of apoptotic cells is essential for animal development, tissue homeostasis and regulation of immune responses. ", "Defects in this process contribute to the development of various human diseases including persistent inflammatory diseases and autoimmune disorders [@pgen.1000679-Savill1]. ", "In *C. elegans*, phagocytosis of apoptotic cells is controlled by two partially redundant signaling pathways. ", "In one pathway, three genes, *ced-1*, *ced-7* and *ced-6*, are involved in recognizing and transducing the engulfment signal(s), while *dyn-1* acts downstream of them to promote vesicle delivery for cell corpse internalization [@pgen.1000679-Zhou1]--[@pgen.1000679-Yu1]. ", "In the other pathway, several evolutionarily conserved intracellular signaling molecules, CED-2/CrkII, CED-5/Dock180 and CED-12/ELMO, act downstream of PSR-1, the *C. elegans* homologue of the human phosphatidylserine receptor. ", "These signaling molecules mediate activation of the small GTPase CED-10/Rac, leading to rearrangement of the actin cytoskeleton which is needed for cell corpse engulfment [@pgen.1000679-Fadok1]--[@pgen.1000679-Zhou2]. ", "In addition, CED-10/Rac may also function downstream of the CED-1/6/7 pathway to mediate the engulfment of apoptotic cells [@pgen.1000679-Kinchen1]. ", "CED-2 belongs to the Crk family whose members are widely expressed adaptor proteins that mediate the formation of protein complexes for signal transduction in response to various extracellular stimuli [@pgen.1000679-Feller1]. ", "However, the way in which CED-2 functions to regulate cell corpse engulfment is not well understood. ", "Biochemical studies in mammalian cells indicate that DOCK180/CED-5 and ELMO/CED-12 function as an unconventional bipartite nucleotide exchange factor for Rac/CED-10 activation, which leads to cytoskeleton reorganization during engulfment [@pgen.1000679-Brugnera1]. ", "Moreover, an UNC-73/TRIO-MIG-2/RhoG signaling module regulates CED-10/Rac activation through its interaction with the armadillo repeat of CED-12/ELMO [@pgen.1000679-deBakker1]. ", "However, no defect in cell corpse engulfment was observed in animals which completely lose the activity of either *mig-2* or *unc-73* or both, indicating that more complex regulatory mechanisms are involved in CED-10/Rac activation. ", "In addition, as earlier studies mainly focused only on positive regulation of engulfment, it is less well understood whether any negative regulatory mechanism is involved.", "\n\nMyotubularin phosphatases belong to the tyrosine/dual-specificity phosphatase super-family (PTP/DSP) whose members have been found in almost all eukaryotes. ", "Mutations in myotubularin genes are associated with several human diseases [@pgen.1000679-Laporte1]. ", "For example, mutations in *MTM1*, the founder member of this family, cause X-linked myotubular myopathy (XLMTM), a severe congenital muscular disorder. ", "Mutations in *MTMR2* and *MTMR13* are associated with Charcot-Marie-Tooth disease (CMT4B1) [@pgen.1000679-Azzedine1]--[@pgen.1000679-Bolino1]. ", "The hallmark of the protein tyrosine phosphatase (PTP) super-family is an active site motif (CX~5~R). ", "Intriguingly, nearly half of the known MTM1-related proteins (MTMRs) carry sequence variations in this motif and are predicted to be catalytically inactive [@pgen.1000679-Robinson1]. ", "Nevertheless, these inactive MTMRs are also evolutionarily conserved and have essential biological functions. ", "Recently, several active-inactive pairings of myotubularins have been identified which appear to be important for the function of the active myotubularins, indicating that the inactive MTMRs likely serve as regulatory units for the active ones [@pgen.1000679-Robinson2]--[@pgen.1000679-Berger1].", "\n\nSurprisingly, instead of acting as protein tyrosine phosphatases, MTM1 and its related proteins were later found to function primarily as lipid phosphatases with specificity for phosphatidylinositol 3-phosphate (PtdIns(3)P) and its metabolite phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P~2~) [@pgen.1000679-Parrish1]--[@pgen.1000679-Blondeau1]. ", "PtdIns(3)P is mainly generated by the Class III PI3-kinase VPS34 in vivo, and can be further modified by the phosphatidylinositol 3-phosphate 5-kinase PIKFYVE to generate PtdIns(3,5)P~2~ [@pgen.1000679-Vanhaesebroeck1]. ", "Both PtdIns(3)P and PtdIns(3,5)P~2~ are key regulators of the endocytic pathway [@pgen.1000679-Clague1]. ", "Therefore, the identification of MTM1 and MTMRs as lipid phosphatases that use PtdIns(3)P and PtdIns(3,5)P~2~ as substrates suggests that they may regulate endocytosis and/or other class III PI3-kinase-mediated cellular events. ", "This idea is supported by evidence from a variety of experiments in yeast and *C. elegans*. ", "Overexpression of human MTM1 decreased the level of PtdIns(3)P in *S. pombe* and induced a vacuolar phenotype similar to that of mutants defective in *VPS34* [@pgen.1000679-Blondeau1]. ", "Ymr1p, the sole myotubularin in yeast, cooperates with the synaptojanin-like PI phosphatase Sjl3p to regulate PtdIns(3)P levels and vesicular transport [@pgen.1000679-Parrish1]. ", "Reduction of *C. elegans mtm-6* activity by RNAi rescued the larval lethality of *vps-34(lf)* mutants, while inactivation of *mtm-1* rescued the endocytosis defect in *vps-34(lf)* coelomocytes (the specialized cells that constantly take up fluid and macromolecules from the body cavity), indicating that myotubularins likely modulate VPS-34-mediated cellular processes in worms [@pgen.1000679-Xue1]. ", "In addition, MTM-6, an active MTM, was found to act together with MTM-9, a catalytically inactive MTM, to regulate an ARF-6 and RME-1-mediated endocytic pathway [@pgen.1000679-Dang1]. ", "However, it is not known how *C. elegans* MTM-1 regulates endocytosis or whether it functions in other cellular processes by regulating PtdIns(3)P.\n\nOn the other hand, the cellular function of MTM1 in mammals appears to be more obscure. ", "In one study, it was reported that MTM1 translocates to late endosomes after EGF stimulation and negatively regulates EGFR degradation and vesicular formation at the late stage of endosomal trafficking [@pgen.1000679-Tsujita1]. ", "This process is mediated through interaction between the MTM1 PH-GRAM domain and PtdIns(3,5)P~2~ [@pgen.1000679-Tsujita1]. ", "In another study, however, MTM1 siRNA caused increased PtdIns(3)P levels and accumulation of EGFR in early but not late endosomes, whereas MTMR2 RNAi resulted in elevated PtdIns(3)P levels and EGFR accumulation in late endosomes [@pgen.1000679-Cao1]. ", "This suggested that MTM1 and MTMR2 function sequentially in the endocytic pathway with MTM1 acting in an early step [@pgen.1000679-Cao1]. ", "Therefore, the cellular functions of MTM1 either in endocytic transport or in other cellular events still remain elusive.", "\n\nIn the present study, we have identified *C. elegans* MTM-1 as a negative regulator of cell corpse engulfment. ", "We found that inactivation of MTM-1 by RNAi promotes cell corpse engulfment, whereas overexpression of MTM-1 results in accumulation of cell corpses in a manner dependent on its lipid phosphatase activity. ", "We show that the reduction of cell corpses caused by *mtm-1* RNAi requires the functions of CED-5, CED-12 and CED-10 and the activities of PI3-kinases VPS-34 and PIKI-1. ", "MTM-1 is widely expressed in many cell types, localizes to the plasma membrane and negatively regulates the vesicular accumulation of PtdIns(3)P in *C. elegans*. ", "Our data suggest that MTM-1 acts as a lipid phosphatase to negatively regulate cell corpse engulfment through the CED-5/CED-12/CED-10 module, which is likely achieved by dephosphorylating PtdIns(3)P on the plasma membrane.", "\n\nResults {#s2}\n=======\n\nIsolation of *mtm-1* as a negative regulator of cell corpse engulfment {#s2a}\n----------------------------------------------------------------------\n\nTo identify negative regulators of cell corpse engulfment, we performed an RNAi screen to search for genes which when inactivated cause a reduction in the number of cell corpses in *ced-1(e1735)* mutants ([Materials and Methods](#s4){ref-type=\"sec\"}). *", "mtm-1* was one of the candidate genes identified from this screen because the accumulation of cell corpses in *ced-1(e1735);rrf-3(pk1426)* double mutants was significantly reduced when treated with *mtm-1* RNAi ([Table 1](#pgen-1000679-t001){ref-type=\"table\"}; [Materials and Methods](#s4){ref-type=\"sec\"}). ", "Interestingly, a similar reduction in cell corpses was also observed in strong loss-of-function mutants of *ced-6*, *ced-7*, *ced-2* after *mtm-1* RNAi treatment, but not in animals with mutations in the *ced-5*, *ced-12* or *ced-10* genes ([Table 1](#pgen-1000679-t001){ref-type=\"table\"}). ", "The RNAi treatment likely caused a specific inactivation of the *mtm-1* gene as injection of an in vitro-synthesized dsRNA of *mtm-1* resulted in a similar reduction of cell corpses in *ced-1(e1735)* mutants ([Figure S1A](#pgen.1000679.s001){ref-type=\"supplementary-material\"}; [Materials and Methods](#s4){ref-type=\"sec\"}). ", "Moreover, MTM-1::GFP expression was greatly reduced after *mtm-1* RNAi treatment, either by injecting in vitro-synthesized *mtm-1* dsRNA or by feeding with bacteria expressing *mtm-1* dsRNA ([Figure S1A](#pgen.1000679.s001){ref-type=\"supplementary-material\"}). ", "To further confirm the RNAi results, we also analyzed an *mtm-1* deletion mutant, *ok742*, which contains a 1314 bp deletion that removes the region from intron 4 to exon 7 of the *mtm-1* gene ([Figure S1B](#pgen.1000679.s001){ref-type=\"supplementary-material\"}). *", "ok742* causes embryonic lethality and early larval arrest, indicating that *mtm-1* is essential for embryonic and larval development in *C. elegans*. ", "We examined the appearance of cell corpses in *ok742* homozygous embryos derived from *ok742/+* mothers (*ht2/mtm-1(ok742)*) and found that it was indistinguishable from that in wild-type embryos ([Table 1](#pgen-1000679-t001){ref-type=\"table\"}). ", "However, consistent with the RNAi results, *mtm-1(ok742)* deletion mutants caused significant reduction in cell corpses in *ced-2(n1994)* mutants, but not in *ced-5(n1812)* or *ced-10(n3246)* mutants ([Table 1](#pgen-1000679-t001){ref-type=\"table\"}). ", "Moreover, similar reduction of cell corpses by *mtm-1* RNAi was also observed in *ced-1(e1735);ced-2(n1994)* double mutants, but not in *ced-1(e1735);ced-5(n1812)*, *ced-1(e1735)ced-12(n3261)* or *ced-1(e1735);ced-10(n3246)* double mutants ([Table 1](#pgen-1000679-t001){ref-type=\"table\"}). ", "These data suggest that *mtm-1* likely antagonizes cell corpse engulfment in a way that requires the activity of *ced-5*, *ced-12* and *ced-10*. ", "In agreement with this notion, overexpression of MTM-1 driven by *C. elegans* heat-shock promoters (*qxIs156*: P*~hsp~mtm-1*) led to accumulation of cell corpses at every embryonic stage ([Figure 1A](#pgen-1000679-g001){ref-type=\"fig\"}). ", "The engulfment defect caused by mutations in the *ced-2*, *ced-5*, *ced-12* or *ced-10* genes was not further enhanced by overexpression of MTM-1, whereas significantly more cell corpses were observed in loss-of-function mutants of *ced-1*, *ced-6* and *ced-7* which overexpressed MTM-1, indicating that MTM-1 negatively regulates cell corpse engulfment and likely acts through the CED-5/CED-12/CED-10 module ([Figure 1B](#pgen-1000679-g001){ref-type=\"fig\"}). ", "Since *mig-2* modulates the CED-10/Rac pathway through CED-12/CED-5 GEF and seems to act in parallel to *ced-2* [@pgen.1000679-deBakker1], we tested whether loss of *mtm-1* function could reduce cell corpses when *mig-2* function was lost. ", "We found that *mtm-1* RNAi resulted in reduced numbers of cell corpses in both *ced-1(e1735);mig-2(mu28)* and *ced-2(n1994);mig-2(mu28)* double mutants, suggesting that an additional input of the CED-10/Rac pathway in parallel to *mig-2* and *ced-2* may exist and *mtm-1* probably functions downstream of them to inhibit the CED-5/CED-12/CED-10 module ([Table 1](#pgen-1000679-t001){ref-type=\"table\"}). ", "Consistent with this idea, we observed a significant reduction in cell corpses by *mtm-1* RNAi in a weak loss-of-function mutant of *ced-10*, *ced-10(n1993)*, in which *ced-10* activity is only partially blocked ([Table 1](#pgen-1000679-t001){ref-type=\"table\"}).", "\n\n{ref-type=\"sec\"}.](pgen.1000679.g001){#pgen-1000679-g001}\n\n10.1371/journal.pgen.1000679.t001\n\n###### *mtm-1* negatively regulates cell corpse engulfment through *ced-5/12/10*.", "\n\n![](", "pgen.1000679.t001){#pgen-1000679-t001-1}\n\n Genotype No. ", "of cell corpses[5](#nt106){ref-type=\"table-fn\"} Changes *p*- value[6](#nt107){ref-type=\"table-fn\"}\n ---------------------------------------------------------------------------------------------------- ----------------------------------------------------- --------- --------------------------------------------\n Wild type 0.3±0.1 \n *mtm-1(ok742)* [1](#nt102){ref-type=\"table-fn\"} 0.4±0.1 No 0.7\n *rrf-3(pk1426);control RNAi* 0.6±0.1 \n *rrf-3(pk1426);mtm-1 RNAi* 0.5±0.1 No 0.6\n *ced-1(e1735);control RNAi* [2](#nt103){ref-type=\"table-fn\"} 31.8±0.8 \n *ced-1(e1735);mtm-1 RNAi* [2](#nt103){ref-type=\"table-fn\"} 17.1±0.8 Reduced \\<0.0001\n *ced-6(n2095);control RNAi* [2](#nt103){ref-type=\"table-fn\"} 31.2±0.7 \n *ced-6(n2095);mtm-1 RNAi* [2](#nt103){ref-type=\"table-fn\"} 19.2±1.2 Reduced \\<0.0001\n *ced-6(qx17);control RNAi* 13.7±0.6 \n *ced-6(qx17); mtm-1 RNAi* 5.7±0.6 Reduced \\<0.0001\n *ced-7(n2094);control RNAi* [2](#nt103){ref-type=\"table-fn\"} 29.9±0.8 \n *ced-7(n2094);mtm-1 RNAi* [2](#nt103){ref-type=\"table-fn\"} 23.8±0.8 Reduced \\<0.0001\n *ced-2(n1994);control RNAi* [2](#nt103){ref-type=\"table-fn\"} 29.0±0.8 \n *ced-2(n1994);mtm-1RNAi* [2](#nt103){ref-type=\"table-fn\"} 15.8±0.8 Reduced \\<0.0001\n *ced-2(n1994)* [3](#nt104){ref-type=\"table-fn\"} 29.3±0.6 \n *mtm-1(ok742);ced-2(n1994)* [1](#nt102){ref-type=\"table-fn\"} ^,^ [3](#nt104){ref-type=\"table-fn\"} 17.3±0.7 Reduced \\<0.0001\n *ced-5(n1812);control RNAi* [2](#nt103){ref-type=\"table-fn\"} 33.7±0.7 \n *ced-5(n1812);mtm-1 RNAi* [2](#nt103){ref-type=\"table-fn\"} 33.4±1.2 No 0.8\n *ced-5(n1812)* [3](#nt104){ref-type=\"table-fn\"} 32.3±0.6 \n *mtm-1(ok742); ced-5(n1812)* [1](#nt102){ref-type=\"table-fn\"} ^,^ [3](#nt104){ref-type=\"table-fn\"} 32.6±0.7 No 0.8\n *ced-12(n3261);control RNAi* [2](#nt103){ref-type=\"table-fn\"} 29.9±0.6 \n *ced-12(n3261);mtm-1 RNAi* [2](#nt103){ref-type=\"table-fn\"} 30.2±0.7 No 0.8\n *ced-10(n3246);control RNAi* 29.4±0.5 \n *ced-10(n3246);mtm-1 RNAi* 28.2±0.8 No 0.8\n *ced-10(n1993);control RNAi* 16.6±0.6 \n *ced-10(n1993);mtm-1 RNAi* 11.7±0.9 Reduced \\<0.0001\n *ced-10(n3246)* [3](#nt104){ref-type=\"table-fn\"} 28.4±0.5 \n *mtm-1(ok742);ced-10(n3264)* [1](#nt102){ref-type=\"table-fn\"} ^,^ [3](#nt104){ref-type=\"table-fn\"} 28.2±0.5 No 0.2\n *ced-1(e1735);ced-2(n1994);control RNAi* 44.6±0.9 \n *ced-1(e1735);ced-2(n1994);mtm-1 RNAi* 34.1±1.2 Reduced \\<0.0001\n *ced-1(e1735);ced-5(n1812);control RNAi* 42.7±0.9 \n *ced-1(e1735);ced-5(n1812);mtm-1 RNAi* 42.9±0.8 No 0.8\n *ced-1(e1735) ced-12(n3261);control RNAi* [4](#nt105){ref-type=\"table-fn\"} 42.7±1.2 \n *ced-1(e1735) ced-12(n3261);mtm-1 RNAi* [4](#nt105){ref-type=\"table-fn\"} 43.4±1.1 No 0.6\n *ced-1(e1735);ced-10(n3246);control RNAi* 45.1±0.8 \n *ced-1(e1735);ced-10(n3246);mtm-1 RNAi* 43.7±0.6 No 0.2\n *ced-1(e1735);mig-2(mu28);control RNAi* 37.7±0.4 \n *ced-1(e1735);mig-2(mu28);mtm-1 RNAi* 31.4±0.6 Reduced \\<0.0001\n *ced-2(n1994);mig-2(mu28);control RNAi* 39.6±0.7 \n *ced-2(n1994);mig-2(mu28);mtm-1 RNAi* 30.2±0.6 Reduced \\<0.0001\n\nRNAi experiments were performed as described in [Materials and Methods](#s4){ref-type=\"sec\"}.", "\n\n*mtm-1(ok742)* is balanced by hT2 and non-green progeny were scored as *mtm-1(ok742)* homozygotes.", "\n\nStrains also carry *rrf-3(pk1426)*.", "\n\nStrains were kept on NGM plates seeded with OP50.", "\n\nStrains also carry *unc-101(m1)*.", "\n\nCell corpses were scored in the head region of 4-fold stage embryos and are shown as mean±s.e.m. ", "At least 15 embryos were scored for each strain.", "\n\nUnpaired *t* tests were performed to compare the average number of cell corpses in *mtm-1* RNAi-treated animals with that in control animals, or to compare the number of cell corpses in double mutants with that in the respective single mutants.", "\n\nLoss of *mtm-1* function promotes the internalization of cell corpses {#s2b}\n---------------------------------------------------------------------\n\nTo further investigate whether the reduction in cell corpses caused by inactivation of *mtm-1* is due to an accelerated clearance of cell corpses, we performed a time-lapse analysis to follow cell corpse duration in *qx17*, a weak loss-of-function allele of the *ced-6* gene which showed a mild engulfment defect on its own and a reduced number of cell corpses after *mtm-1* RNAi treatment ([Table 1](#pgen-1000679-t001){ref-type=\"table\"}; [Materials and Methods](#s4){ref-type=\"sec\"}). ", "In *ced-6(qx17)* animals treated with control RNAi, most cell corpses persisted from 30 to 70 min, with an average duration of 63 min ([Figure 2A](#pgen-1000679-g002){ref-type=\"fig\"}). ", "In contrast, in *ced-6(qx17)* embryos treated with *mtm-1* RNAi, most cell corpses persisted from 20 to 40 min, with an average duration of 34 min, which is 46% shorter than in control animals ([Figure 2A](#pgen-1000679-g002){ref-type=\"fig\"}). ", "This suggests that *mtm-1* RNAi may promote cell corpse clearance. ", "We next examined the embryonic cell deaths that occurred during a period of 200--400 min past the first cleavage. ", "We observed similar numbers of cell death events in *qx17* animals treated with either control or *mtm-1* RNAi, indicating that *mtm-1* RNAi does not obviously affect the occurrence of cell death ([Figure 2B](#pgen-1000679-g002){ref-type=\"fig\"}). ", "Consistent with this, no missing or extra cells were observed in the anterior pharynx of *ced-6(qx17)* animals treated with either control or *mtm-1* RNAi (data not shown). ", "Therefore, inactivation of *mtm-1* accelerates the clearance of cell corpses.", "\n\n{#pgen-1000679-g002}\n\n*ced-1* encodes a transmembrane phagocytic receptor that acts specifically in engulfing cells to mediate the recognition and internalization of cell corpses [@pgen.1000679-Zhou1]. ", "In wild-type animals, CED-1::GFP was detected along the surface of apoptotic cells, which are distinguishable using Nomarski optics [@pgen.1000679-Zhou1],[@pgen.1000679-Yu1] ([Figure 3A](#pgen-1000679-g003){ref-type=\"fig\"}; [Figure S2A](#pgen.1000679.s002){ref-type=\"supplementary-material\"}). ", "Moreover, we found that clustering of CED-1::GFP around cell corpses completely overlapped with a secreted Annexin V::mRFP fusion protein expressed under the control of heat-shock promoters (P*~hsp~annexin v::mrfp*) ([Figure S2A](#pgen.1000679.s002){ref-type=\"supplementary-material\"}). ", "Annexin V specifically labels apoptotic cells by binding to phosphatidylserine (PS), an \"eat me\" signal which appears only on the surface of dying cells [@pgen.1000679-Fadok2]--[@pgen.1000679-Wang2]. ", "In fact, over 96% of cell corpses were found to be labeled by the secreted Annexin V::mRFP in both wild type and *ced-6(qx17)* mutants (at least 15 embryos were scored in each strain). ", "We therefore used CED-1::GFP as a marker to examine whether the acceleration of cell corpse clearance caused by *mtm-1* RNAi is achieved by facilitating the internalization of apoptotic cells. ", "We found that more cell corpses were surrounded by CED-1::GFP in *ced-6(qx17)* animals treated with *mtm-1* RNAi, suggesting that more apoptotic cells might be internalized ([Figure 3B](#pgen-1000679-g003){ref-type=\"fig\"}). ", "A similar increase in CED-1::GFP clustering by *mtm-1* RNAi was also observed in the strong loss-of-function mutants of *ced-6* and *ced-2*, but not *ced-5* ([Figure S3A](#pgen.1000679.s003){ref-type=\"supplementary-material\"}). ", "Since CED-1 is a phagocytic receptor that localizes to extending pseudopods and only transiently associates with nascent phagosomes after engulfment [@pgen.1000679-Zhou1],[@pgen.1000679-Yu1], we further examined the internalization of cell corpses by monitoring both the formation and duration of the CED-1::GFP ring around cell corpses. ", "We found that in *ced-6(qx17)* animals treated with *mtm-1* RNAi, the CED-1::GFP ring formed rapidly around dying cells, in an average of 4.5 min ([Figure 4A and 4B](#pgen-1000679-g004){ref-type=\"fig\"}; [Video S1](#pgen.1000679.s010){ref-type=\"supplementary-material\"}). ", "In control RNAi-treated *ced-6(qx17)* embryos, however, many cell corpses were not fully surrounded by CED-1::GFP even after 10 min and the average formation time of a full CED-1::GFP ring was 7.4 min, which is 64% longer than that in *mtm-1* RNAi-treated embryos ([Figure 4A and 4B](#pgen-1000679-g004){ref-type=\"fig\"}; [Video S2](#pgen.1000679.s011){ref-type=\"supplementary-material\"}). ", "Moreover, we observed that CED-1::GFP associated with extending pseudopods or nascent phagosomes for an average duration of 29 min in control animals, compared to an average of just 15 min in *ced-6(qx17);mtm-1(RNAi)* embryos ([Figure 4C and 4D](#pgen-1000679-g004){ref-type=\"fig\"}; [Video S3](#pgen.1000679.s012){ref-type=\"supplementary-material\"}, [Video S4](#pgen.1000679.s013){ref-type=\"supplementary-material\"}). ", "To exclude the possibility that the observed effect in cell corpse engulfment by *mtm-1* RNAi is caused by the individual variability of different apoptotic cells or different developmental locations, we also monitored the clearance of a specific apoptotic cell C3, which undergoes apoptosis at a mid-embryonic stage and is engulfed by a ventral hypodermal cell [@pgen.1000679-Yu1],[@pgen.1000679-Zhou3] ([Figure S2A](#pgen.1000679.s002){ref-type=\"supplementary-material\"}). ", "We found that both formation and duration of the CED-1::GFP ring around C3 were significantly shortened after *mtm-1* RNAi treatment, indicating that the internalization of C3 was accelerated similar to other apoptotic cells ([Figure S2B, S2C](#pgen.1000679.s002){ref-type=\"supplementary-material\"}). ", "Collectively, these data suggest that inactivation of MTM-1 promotes cell corpse internalization in *ced-6(qx17)* mutants.", "\n\n{#pgen-1000679-g003}\n\n{#pgen-1000679-g004}\n\nTo further prove that *mtm-1* RNAi promotes the engulfment of cell corpses, we examined the internalization of apoptotic cells by using a GFP fusion of a cytosolic actin isoform, ACT-5, which clusters around cell corpses during early stages of engulfment and disappears after they are fully engulfed [@pgen.1000679-Kinchen1]. ", "We found that more cell corpses were labeled by ACT-5::GFP in *ced-6(qx17)* embryos treated with *mtm-1* RNAi than in control animals ([Figure 3](#pgen-1000679-g003){ref-type=\"fig\"}). ", "A similar increase in the labeling of cell corpses was also observed in *ced-6(qx17);mtm-1(RNAi)* embryos when two different phagosomal markers, GFP::RAB-7 and LMP-1::GFP, were monitored ([Figure 3](#pgen-1000679-g003){ref-type=\"fig\"}). ", "RAB-7 associates with phagosomal membranes and controls late steps of phagosome maturation, while LMP-1, a lysosome-associated membrane protein, is recruited to phagosomes during late stages of phagosome maturation [@pgen.1000679-Yu2]--[@pgen.1000679-Dowling1]. ", "Therefore, our data suggest that more cell corpses are internalized and enclosed in phagosomes when *mtm-1* function is inhibited. ", "As expected, the clustering of both GFP::RAB-7 and LMP-1::GFP around cell corpses was significantly enhanced in *ced-1(e1735)*, *ced-6(n2095)* and *ced-2(n1994)* mutants but not *ced-5(n1812)* mutants after *mtm-1* RNAi treatment ([Figure S3B, S3C](#pgen.1000679.s003){ref-type=\"supplementary-material\"}). ", "This supports the hypothesis that *mtm-1* negatively regulates cell corpse engulfment through *ced-5*/*12/10*.", "\n\nMTM-1 does not play a similar role in the migration of distal tip cells {#s2c}\n-----------------------------------------------------------------------\n\nMutations in *ced-2*, *ced-5*, *ced-12* and *ced-10*, but not *ced-1*, *ced-6* or *ced-7*, affect the migration of the two distal tip cells (DTCs), which are located at the tips of the two gonad arms and guide the formation of gonads during larval development [@pgen.1000679-Reddien1],[@pgen.1000679-Wu2],[@pgen.1000679-Wu3],[@pgen.1000679-Hedgecock1],[@pgen.1000679-Kimble1]. ", "In *ced-2*, *ced-5*, *ced-12* and *ced-10* mutants, the DTCs often make extra turns, which causes abnormally shaped gonads [@pgen.1000679-Reddien1],[@pgen.1000679-Wu2],[@pgen.1000679-Wu3]. ", "Since *mtm-1* negatively regulates cell corpse engulfment through the CED-5/CED-12/CED-10 complex and both MTM-1 and CED-10 are highly expressed in DTCs ([@pgen.1000679-Lundquist1] and see below), we tested whether *mtm-1* could also modify the DTC migration defect in *ced-2*, *ced-5*, *ced-12* or *ced-10* mutants. ", "We found that *mtm-1* RNAi did not obviously affect the DTC migration defect in strong loss-of-function mutants of *ced-2*, *mig-2*, *ced-5*, *ced-12* or *ced-10*, nor did it significantly suppress or enhance the abnormal DTC migration phenotype in *ced-2;mig-2* or *ced-1;ced-2* double mutants ([Table S1](#pgen.1000679.s006){ref-type=\"supplementary-material\"}). ", "However, we observed that *mtm-1* RNAi resulted in a weak DTC migration defect in both wild type and *rrf-3(pk1426)* mutants (which are hypersensitive to RNAi treatment). ", "Moreover, inactivation of *mtm-1* by RNAi significantly enhanced the defect of DTC migration in *ced-10(n1993)* weak loss-of-function mutants ([Table S1](#pgen.1000679.s006){ref-type=\"supplementary-material\"}). ", "This suggests that *mtm-1* may play a positive role in the migration of distal tip cells and may act in the same genetic pathway as *ced-10*.", "\n\nOther MTMs do not play redundant roles with MTM-1 in cell corpse engulfment {#s2d}\n---------------------------------------------------------------------------\n\nMTM-1 belongs to the myotubularin family, which constitutes a large group within the tyrosine/dual-specificity phosphatase (PTP/DSP) super-family and which are evolutionarily conserved in yeast, worms and humans [@pgen.1000679-Laporte1] ([Figure S4](#pgen.1000679.s004){ref-type=\"supplementary-material\"}). ", "To examine whether the function of MTM-1 in cell corpse engulfment is conserved, we overexpressed human MTM1 under the control of the *C. elegans* heat-shock promoters (P*~hsp~*hMTM1) and found that it efficiently rescued the reduced cell corpse phenotype in *mtm-1(ok742);ced-2(n1994)* mutants ([Table S2](#pgen.1000679.s007){ref-type=\"supplementary-material\"}). ", "This suggests that human MTM1 can substitute for the function of worm MTM-1 in regulating cell corpse engulfment.", "\n\nIn *C. elegans*, 5 MTMs have been identified based on sequence homology [@pgen.1000679-Xue1]. ", "Like mammalian MTMs, they may have non-redundant functions. ", "To determine whether other *C. elegans* MTMs are also involved in cell corpse clearance, we analyzed the cell corpse phenotype of *mtm(lf)* in the background of *ced-1(e1735)*, which has a cell corpse phenotype that can be attenuated by *mtm-1* RNAi. ", "We found that the persistent cell corpse phenotype of *ced-1(e1735)* mutants was not affected when *mtm-9*, *mtm-6*, or *mtm-5* was inactivated either individually or in combination ([Table S3](#pgen.1000679.s008){ref-type=\"supplementary-material\"}). ", "Interestingly, when *mtm-3* was inactivated by RNAi in *ced-1(e1735)* mutants, we observed a slight enhancement of cell corpse numbers which was further enhanced by *mtm-6(ok330)* but not *mtm-5(ok469)* mutants ([Table S3](#pgen.1000679.s008){ref-type=\"supplementary-material\"}). ", "In wild-type animals, *mtm-3* RNAi also caused increased cell corpse numbers, which were significantly enhanced by *mtm-6(ok330)* but not *mtm-5(ok469)* mutants ([Table S3](#pgen.1000679.s008){ref-type=\"supplementary-material\"}). ", "This suggests that *mtm-3* may play a redundant role with *mtm-6* to promote cell corpse clearance or to affect cell death activation. ", "However, since *mtm-1* RNAi treatment resulted in an opposite phenotype in *ced-1(e1735)* mutants, these MTMs appear not to act redundantly with MTM-1 in cell corpse engulfment.", "\n\nMTM-1 acts as a lipid phosphatase to regulate cell corpse engulfment {#s2e}\n--------------------------------------------------------------------\n\nAlthough myotubularins contain CX~5~R active site motifs characteristic of the protein tyrosine phosphatase super-family, they primarily function as lipid phosphatases to dephosphorylate phosphatidylinositol 3-phosphate (PtdIns(3)P) or phosphatidylinositiol 3, 5-bisphosphate (PtdIns(3,5)P~2~) [@pgen.1000679-Taylor1],[@pgen.1000679-Kim2],[@pgen.1000679-Schaletzky1]. ", "To determine whether MTM-1 acts as a lipid phosphatase to regulate cell corpse engulfment, we generated a catalytically inactive mutant of MTM-1, MTM-1(C378S) [@pgen.1000679-Chaussade1]. ", "In contrast to overexpression of MTM-1, overexpression of MTM-1(C378S) driven by the *C. elegans* heat-shock promoters (P*~hsp~*MTM-1(C378S)) failed to cause accumulation of cell corpses and was unable to rescue the reduced cell corpse phenotype in *mtm-1(ok742);ced-2(n1994)* double mutants ([Figure 1A](#pgen-1000679-g001){ref-type=\"fig\"}; [Table S2](#pgen.1000679.s007){ref-type=\"supplementary-material\"}). ", "This indicates that the lipid phosphatase activity of MTM-1 is required for its function in cell corpse engulfment.", "\n\nSince human MTM1 mainly uses PtdIns(3)P as a substrate both in vitro and in vivo [@pgen.1000679-Taylor1],[@pgen.1000679-Kim2],[@pgen.1000679-Blondeau1], we next examined whether phosphatidylinositol 3-kinase (PI3K) activity is required for the reduction of cell corpses caused by *mtm-1* RNAi. ", "Three classes of phosphatidylinositol 3-kinases (PI3K) are responsible for generating 3-phosphoinositides, among which class I PI3Ks are mainly responsible for generating PtdIns(3,4,5)P~3~, while the synthesis of PtdIns(3)P in vivo is mostly carried out by class III PI3Ks [@pgen.1000679-Vanhaesebroeck1]. ", "In addition, class II PI3Ks produce PtdIns(3)P both in vitro and in vivo [@pgen.1000679-Maffucci1],[@pgen.1000679-Brown1]. *", "C. elegans* contains a single homolog of each class: AGE-1, a class I PI3K essential for the *C. elegans* insulin-like signaling pathway [@pgen.1000679-Tissenbaum1]; VPS-34, a class III PI3K that regulates larval development, endocytosis and cell corpse degradation by generating PtdIns(3)P [@pgen.1000679-Xue1],[@pgen.1000679-Kinchen2],[@pgen.1000679-Roggo1]; and a class II PI3-kinase encoded by the open reading frame F39B1.1 which can compensate for the loss of *vps-34* function when *mtm-6* is inactivated by RNAi [@pgen.1000679-Xue1]. ", "We found that the deletion mutant *ok2346* of the class II PI3-kinase F39B1.1, which we named *piki-1* ([p]{.ul}hosphatidyl[i]{.ul}nositol 3 -[ki]{.ul}nase), resulted in accumulation of cell corpses at several embryonic stages similar to *vps-34(h797)*, a strong loss-of-function mutant of *vps-34* ([Figure 5A](#pgen-1000679-g005){ref-type=\"fig\"}; [Figure S1C](#pgen.1000679.s001){ref-type=\"supplementary-material\"}). ", "Interestingly, significantly more cell corpses were observed in *vps-34(h797);piki-1(ok2346)* double mutants than in either single mutant alone ([Figure 5A](#pgen-1000679-g005){ref-type=\"fig\"}). ", "The increase in cell corpses observed in *vps-34(h797);piki-1(ok2346)* double mutants is likely due to a defect in apoptotic cell clearance because cell corpses persist on average 1.6 times longer in embryos lacking both *vps-34* and *piki-1* activities than in wild type ([Figure 5B](#pgen-1000679-g005){ref-type=\"fig\"}). ", "This indicates that *vps-34* and *piki-1* act redundantly to regulate the removal of apoptotic cells. ", "In contrast, neither an obvious cell corpse phenotype nor an enhancement of the cell corpse clearance defect was observed when the class I PI3K AGE-1 was inactivated by RNAi in either wild-type animals or *vps-34;piki-1* double mutants, indicating that the class I PI3K is not involved in this process (data not shown).", "\n\n{ref-type=\"fig\"}. ", "Data from wild type and mutant animals were compared as described in [Figure 1](#pgen-1000679-g001){ref-type=\"fig\"}. ", "\\*\\**P*\\<0.0001; all other points had *P* value\\>0.05. (", "B) Four-dimensional microscopy analysis of cell corpse duration in *vps-34(h797);piki-1(ok2346)* mutants. ", "The durations of 30 cell corpses from wild type (n = 3; black bars) and 34 cell corpses from *vps-34(h797);piki-1(ok2346)* double mutants (n = 3; gray bars) were monitored as described in [Figure 2](#pgen-1000679-g002){ref-type=\"fig\"} and [Materials and Methods](#s4){ref-type=\"sec\"}. ", "The numbers in parenthesis indicate the average duration of cell corpses (±s.e.m).](pgen.1000679.g005){#pgen-1000679-g005}\n\nWe next examined whether *mtm-1* RNAi is able to reduce cell corpse numbers in *ced-1(e1735)* mutants when PI3K activity is blocked. ", "Although a reduction in cell corpses was still observed in *vps-34(h797)ced-1(e1735)* or *ced-1(e1735);piki-1(ok2346)* double mutants treated with *mtm-1* RNAi, no obvious difference in the number of cell corpses was seen in *vps-34ced-1;piki-1* triple mutants after *mtm-1* RNAi treatment ([Table 2](#pgen-1000679-t002){ref-type=\"table\"}). ", "This indicates that the reduction of cell corpses in *ced-1(e1735)* mutants by *mtm-1* RNAi requires the activity of both class III and class II PI3Ks. ", "Similar effects were also observed when *mtm-1* was inactivated in *vps-34;piki-1* double mutants or in *ced-6* or *ced-7* mutants lacking both *vps-34* and *piki-1* functions ([Table 2](#pgen-1000679-t002){ref-type=\"table\"}). ", "Therefore, MTM-1 may coordinate with the PI3Ks VPS-34 and PIKI-1 to regulate the level of PtdIns(3)P for cell corpse engulfment.", "\n\n10.1371/journal.pgen.1000679.t002\n\n###### *vps-34* and *piki-1* are required for the reduction in cell corpses by *mtm-1* RNAi.", "\n\n![](", "pgen.1000679.t002){#pgen-1000679-t002-2}\n\n Genotype No. ", "of cell corpses[1](#nt109){ref-type=\"table-fn\"} Changes *p*- value[2](#nt110){ref-type=\"table-fn\"}\n --------------------------------------------------------- ----------------------------------------------------- ----------- --------------------------------------------\n *vps-34(h797);piki-1(ok2346);control RNAi* 16.3±0.9 \n *vps-34(h797);piki-1(ok2346);mtm-1 RNAi* 14.7±1.2 No 0.3\n *vps-34(h797)ced-1(e1735);control RNAi* 33.6±0.8 \n *vps-34(h797)ced-1(e1735);mtm-1 RNAi* 22.6±1.0 Reduced \\<0.0001\n *ced-1(e1735);piki-1(ok2346);control RNAi* 36.9±0.5 \n *ced-1(e1735);piki-1(ok2346);mtm-1 RNAi* 25.9±1.0 Reduced \\<0.0001\n *vps-34(h797)ced-1(e1735);piki-1(ok2346);control RNAi* 46.7±0.5 \n *vps-34(h797)ced-1(e1735);piki-1(ok2346);mtm-1 RNAi* 47.5±0.8 No 0.4\n *vps-34(h797);ced-6(n2095);control RNAi* 38.4±0.6 \n *vps-34(h797)ced-6(n2095);mtm-1 RNAi* 31.6±0.7 Reduced \\<0.0001\n *ced-6(n2095);piki-1(ok2346);control RNAi* 37.0±0.7 \n *ced-6(n2095);piki-1(ok2346);mtm-1 RNAi* 27.3±0.9 Reduced \\<0.0001\n *vps-34(h797);ced-6(n2095);piki-1(ok2346);control RNAi* 43.5±0.8 \n *vps-34(h797);ced-6(n2095);piki-1(ok2346);mtm-1 RNAi* 44.0±0.5 No 0.6\n *vps-34(h797);ced-7(n2094);control RNAi* 32.3±0.5 \n *vps-34(h797)ced-7(n2094);mtm-1 RNAi* 23.2±0.8 Reduced \\<0.0001\n *ced-7(n2094);piki-1(ok2346);control RNAi* 39.1±1.0 \n *ced-7(n2094);piki-1(ok2346);mtm-1 RNAi* 32.9±1.6 Reduced 0.004\n *vps-34(h797);ced-7(n2094);piki-1(ok2346);control RNAi* 43.5±0.6 \n *vps-34(h797);ced-7(n2094);piki-1(ok2346);mtm-1 RNAi* 46.0±0.7 increased 0.009\n\nRNAi experiments were performed as described in [Materials and Methods](#s4){ref-type=\"sec\"}. *", "vps-34(h797)* mutants were maintained and scored as described in [Materials and Methods](#s4){ref-type=\"sec\"}.", "\n\nCell corpses were scored in the head region of 4-fold stage embryos as described in [Materials and Methods](#s4){ref-type=\"sec\"} and are shown as mean±s.e.m. ", "At least 15 embryos were scored for each strain.", "\n\nUnpaired *t* tests were performed to compare control animals with *mtm-1* RNAi-treated worms.", "\n\nMTM-1 functions in engulfing cells and localizes to the plasma membrane {#s2f}\n-----------------------------------------------------------------------\n\nTo determine the subcellular localization of MTM-1, we generated a MTM-1::GFP fusion driven by the *mtm-1* promoter (P*~mtm-1~mtm-1::gfp*), which fully rescued the cell corpse phenotype of *mtm-1(ok742);ced-2(n1994)* mutants ([Table S2](#pgen.1000679.s007){ref-type=\"supplementary-material\"}). ", "The expression of MTM-1::GFP was seen from embryogenesis throughout larval and adult stages in many known engulfing cell types including hypodermal cells, body wall muscle cells, pharyngeal muscle cells and sheath cells ([Figure 6A](#pgen-1000679-g006){ref-type=\"fig\"}). ", "MTM-1::GFP was also observed in vulva cells, distal tip cells and coelomocytes, which is consistent with previous findings that *mtm-1* RNAi rescues the coelomocyte uptake defect in *vps-34(lf)* mutants ([Figure S5A](#pgen.1000679.s005){ref-type=\"supplementary-material\"}) [@pgen.1000679-Xue1]. ", "In agreement with this expression pattern, we found that overexpression of MTM-1 controlled by the *ced-1* promoter (P*~ced-1~mtm-1*), which drives gene expression specifically in engulfing cells, fully rescued the reduced cell corpse phenotype in *mtm-1(ok742);ced-2(n1994)* mutants. ", "This rescuing activity was not observed when MTM-1 expression was controlled by the *egl-1* promoter (P*~egl-1~mtm-1*), which drives gene expression specifically in dying cells ([Table S2](#pgen.1000679.s007){ref-type=\"supplementary-material\"}) [@pgen.1000679-Zhou1],[@pgen.1000679-Conradt1]. ", "This indicates that *mtm-1* needs to function in engulfing cells to regulate cell corpse engulfment.", "\n\n{#pgen-1000679-g006}\n\nWe found that MTM-1::GFP is mainly localized to the plasma membrane and coincides with myri::mCHERRY, which specifically labels cell membranes ([Figure 6](#pgen-1000679-g006){ref-type=\"fig\"}; personal communication with Dr. David Sherwood and Dr. Guangshuo Ou; [Materials and Methods](#s4){ref-type=\"sec\"}). ", "GFP::MTM-1 expressed from engulfing cells (P*~ced-1~gfp::mtm-1*) also localizes to plasma membranes and overlaps with CED-1::mCHERRY, a cell surface phagocytic receptor ([Figure 7A](#pgen-1000679-g007){ref-type=\"fig\"}) [@pgen.1000679-Zhou1]. ", "Interestingly, we found that GFP::MTM-1 and CED-1::mCHERRY not only co-localized to the plasma membrane, but clustered around the same apoptotic cell, suggesting that MTM-1 may associate with extending pseudopods or nascent phagosomes at a similar stage to CED-1 ([Figure 7A](#pgen-1000679-g007){ref-type=\"fig\"}). ", "Indeed, by time-lapse analysis, we observed that GFP::MTM-1 and CED-1::mCHERRY appeared on the surface of dying cells simultaneously during the early stage of engulfment ([Figure 7B](#pgen-1000679-g007){ref-type=\"fig\"}). ", "However, GFP::MTM-1 disappeared more quickly than CED-1::mCHERRY from the phagosome after internalization, suggesting that MTM-1 only transiently associates with apoptotic cells during engulfment ([Figure 7B](#pgen-1000679-g007){ref-type=\"fig\"}).", "\n\n{#pgen-1000679-g007}\n\nSimilar to the mammalian MTM1 protein, *C. elegans* MTM-1 contains several conserved motifs including an N-terminal PH-GRAM domain which may have the capacity to bind phosphoinositides, a central myotubularin-related domain, a protein tyrosine phosphatase domain (PTP) that contains the catalytic activity, and a C-terminal coiled-coil domain (CC) which might be involved in protein-protein interactions [@pgen.1000679-Laporte1] ([Figure S4](#pgen.1000679.s004){ref-type=\"supplementary-material\"}). ", "To find out which domain is important for the membrane localization of MTM-1, we generated several truncated GFP::MTM-1 fusions driven by the *ced-1* promoter and examined their localizations. ", "All of the MTM-1 truncations were expressed at the expected size in *C. elegans* ([Figure S5B](#pgen.1000679.s005){ref-type=\"supplementary-material\"}). ", "We found that MTM-1 lacking either the PH-GRAM domain (GFP::MTM-1(ΔGRAM)) or the PTP domain (GFP::MTM-1(ΔPTP)) completely lost its membrane localization and instead displayed a punctate vesicular localization pattern. ", "This indicates that both the PH-GRAM and PTP domains are required for the plasma membrane localization of MTM-1 ([Figure 6C](#pgen-1000679-g006){ref-type=\"fig\"}). ", "In contrast, GFP::MTM-1(ΔCC), in which the C-terminal coiled-coil motif is deleted, still localized to the plasma membrane ([Figure 6C](#pgen-1000679-g006){ref-type=\"fig\"}). ", "Moreover, we found that this membrane-localized GFP::MTM-1(ΔCC) was able to rescue the reduced cell corpse phenotype of *mtm-1(ok742);ced-2(n1994)* mutants, suggesting that the coiled-coil motif is dispensable for MTM-1 function in cell corpse engulfment ([Table S2](#pgen.1000679.s007){ref-type=\"supplementary-material\"}). ", "Conversely, neither ΔPH-GRAM nor ΔPTP truncations could rescue the cell corpse phenotype of *mtm-1* deletion mutants ([Table S2](#pgen.1000679.s007){ref-type=\"supplementary-material\"}).", "\n\nSince the PH-GRAM domain likely mediates the binding of myotubularins to phosphoinositides, and the PTP domain contains catalytic activity, we further determined whether PtdIns(3)P is required for locating MTM-1 to the cell membrane by examining the MTM-1::GFP expression pattern in *vps-34(lf)* mutants in which the production of PtdIns(3)P is largely blocked [@pgen.1000679-Roggo1]. ", "We found that the plasma membrane localization of MTM-1 was not obviously affected in *vps-34(h797)* mutants ([Figure S5C](#pgen.1000679.s005){ref-type=\"supplementary-material\"}). ", "Moreover, no significant difference in the localization of MTM-1::GFP was observed in either *piki-1(ok2346)* mutants or *vps-34(h797);piki-1(ok2346)* double mutants ([Figure S5C](#pgen.1000679.s005){ref-type=\"supplementary-material\"}). ", "Therefore, neither PI3Ks nor substrates of MTM-1 seem to be required for its plasma membrane localization.", "\n\nInactivation of MTM-1 increases the accumulation of PtdIns(3)P on intracellular vesicles {#s2g}\n----------------------------------------------------------------------------------------\n\nBecause MTM-1 is a lipid phosphatase, its negative role in cell corpse engulfment might be achieved by regulating the level of PtdIns(3)P. To test this hypothesis and assess whether *mtm-1* can negatively regulate PtdIns(3)P levels in *C. elegans*, we monitored the localization and accumulation of PtdIns(3)P in both wild type and *mtm-1(ok742)* mutants using a YFP::2xFYVE probe which specifically binds PtdIns(3)P on both endosomes and phagosomes [@pgen.1000679-Kinchen2],[@pgen.1000679-Roggo1]. ", "Compared to wild-type animals, there were significantly more YFP::2xFYVE-positive vesicles in *mtm-1(ok742)* larvae, and the number of positive vesicles was greatly reduced when *vps-34* activity was inhibited ([Figure 8A](#pgen-1000679-g008){ref-type=\"fig\"}). ", "This indicates that MTM-1 may modulate the level of PtdIns(3)P on intracellular vesicles. ", "We then quantified YFP::2xFYVE labeling in hypodermal cells which highly express MTM-1 and can act as engulfing cells. ", "In wild-type animals, we observed an average of 79 YFP::2xFYVE-positive vesicles, which was significantly reduced to 27 by treatment with *vps-34* RNAi ([Figure 8B](#pgen-1000679-g008){ref-type=\"fig\"}). ", "This indicates that *vps-34* is responsible for generating PtdIns(3)P on these vesicles. ", "In contrast, over 66% of *mtm-1(ok742)* worms contained more than 110 vesicles positive for YFP::2xFYVE. ", "The average number of YFP-2xFYVE-positive vesicles in *mtm-1(ok742)* animals was 128, which is 62% more than in wild type, suggesting that MTM-1 negatively regulates the vesicular accumulation of PtdIns(3)P ([Figure 8B](#pgen-1000679-g008){ref-type=\"fig\"}).", "\n\n{ref-type=\"sec\"}. ", "The *y*-axis shows the number of animals that contain YFP::2xFYVE-positive vesicles within a specific range as shown on the *x*-axis. ", "The numbers in parenthesis indicate the average number of vesicles positive for YFP::2xFYVE (±s.e.m).](pgen.1000679.g008){#pgen-1000679-g008}\n\nDiscussion {#s3}\n==========\n\nWhy is a negative regulation of engulfment required? {#", "s3a}\n----------------------------------------------------\n\nThe rapid and efficient phagocytosis of apoptotic cells is crucial for maintaining homeostasis as well as regulating the immune responses. ", "Therefore, it seems to be counterintuitive to think that engulfment needs to be down-regulated. ", "In fact, phagocytosis of apoptotic cell not only clears dead cells but promotes cell killing. ", "For example, macrophages taking up apoptotic cells will release FasL and promote Fas-mediated apoptosis of monocytes and neutrophils for quick resolution of inflammation [@pgen.1000679-Brown1]. ", "It has also been reported that macrophages are involved in tissue remodeling by promoting apoptosis in vertebrates [@pgen.1000679-DiezRoux1]--[@pgen.1000679-Little1]. ", "Furthermore, genetic studies in *C. elegans* showed that blocking engulfment enhances the survival of cells triggered to initiate programmed cell death [@pgen.1000679-Reddien2],[@pgen.1000679-Hoeppner1]. ", "Hence, it is conceivable that uncontrolled engulfment may lead to unexpected cell deaths under certain circumstances and an inhibitory mechanism may protect cells from inappropriate death. ", "In addition, negative regulation of engulfment may also contribute to the correct targeting of apoptotic cells by phagocytes. ", "In contrast to apoptotic cells that expose \"eat me\" flags on the surface, living cells exhibit \"don\\'t eat me\" signals, such as CD47 and CD31, in order to be discriminated from dead cells [@pgen.1000679-Brown1],[@pgen.1000679-Gardai1]. ", "Although intracellular pathways transducing this type of signal have not been identified, it is possible that negative regulators are involved in mediating \"don\\'t eat me\" signals within engulfing cells and in inhibiting the engulfment of normal healthy cells.", "\n\nMTM-1 negatively regulates cell corpse engulfment through CED-5/12/10 {#s3b}\n---------------------------------------------------------------------\n\nAlthough many genes are known be involved in apoptotic cell clearance, very few of them play a negative role. ", "In mammalian macrophages, the small GTPase RhoA and its effector Rho-kinase were shown to negatively regulate the engulfment of apoptotic cells [@pgen.1000679-ToselloTrampont1],[@pgen.1000679-Nakaya1]. ", "Recently, *C. elegans* ABL-1 kinase was reported to inhibit cell corpse engulfment and DTC migration through its interacting protein ABI-1 [@pgen.1000679-Hurwitz1]. ", "However, the underlying mechanisms whereby these genes negatively regulate engulfment are not clear.", "\n\nIn order to gain more insight into the negative regulation of phagocytosis, we performed an RNAi screen to search for negative regulators of cell corpse engulfment and identified *C. elegans* myotubularin MTM-1, which was previously reported to play a role in endocytosis. ", "Our genetic and cell biological analyses indicate that inactivation of MTM-1 promotes cell corpse engulfment, while overexpression of MTM-1 results in accumulation of cell corpses, suggesting that MTM-1 negatively regulates the engulfment of apoptotic cells. ", "Given that inactivation of *mtm-1* reduced the number of cell corpses in strong loss-of-function mutants of *ced-1*, *ced-6*, *ced-7* and *ced-2* but not *ced-5*, *ced-12* or *ced-10*, *mtm-1* likely functions through the CED-5/CED-12/CED-10 complex. ", "We still observed a significant reduction in cell corpses when *mtm-1* was inhibited in *ced-2(n1994);mig-2(mu28)* double mutants, which suggests that MIG-2 and CED-2 do not mediate all the inputs into the CED-10 pathway and an additional branch may exist in parallel to both of them. ", "Since overexpression of MTM-1 failed to enhance the engulfment phenotype in *ced-2*, *ced-5*, *ced-12* or *ced-10* mutants, we prefer a model in which *mtm-1* functions downstream of *mig-2* and *ced-2* to negatively regulate the CED-5/CED-12/CED-10 complex rather than a model where *mtm-1* acts in a parallel pathway which requires CED-10 function for engulfment. ", "Recently, ABL-1 kinase was reported to inhibit both cell corpse engulfment and DTC migration by acting in a parallel pathway to the two known engulfment pathways. ", "Moreover, the functions of CED-5, CED-12 and CED-10 were required for its inhibition of engulfment but not DTC migration [@pgen.1000679-Hurwitz1]. ", "As both ABL-1 and MTM-1 negatively regulate cell corpse engulfment through CED-5, CED-12 and CED-10, it will be interesting to test the genetic interaction between them.", "\n\nMTMs play non-redundant roles in the engulfment of apoptotic cells {#s3c}\n------------------------------------------------------------------\n\nIn *C. elegans*, 5 MTMs (MTM-1, 3, 6, 9 and 5) have been identified based on sequence homology. ", "They belong to 5 different subgroups with *mtm-5* and *mtm*-*9* encoding catalytically inactive phosphatases and probably perform non-redundant functions like mammalian MTMs [@pgen.1000679-Xue1]. ", "In agreement with this notion, we found that MTM-1, but not other MTMs, plays a negative role in cell corpse engulfment. ", "Interestingly, *mtm-3(lf);mtm-6(lf)* double mutants contain significantly higher numbers of cell corpses than wild type and they also enhance the cell corpse phenotype of *ced-1(e1735)* mutants, suggesting that *mtm-3* and *mtm-6* may play a redundant role to either promote cell corpse clearance or affect cell death activation. ", "Since *mtm-6* has only been implicated in regulating an ARF-6- and RME-1-dependent endocytic pathway together with *mtm-9*, whereas the functions of *mtm-3* are mostly unknown, it will be important to determine whether these MTMs are directly involved in cell death processes and if so, which specific steps they regulate and how their activities are coordinated.", "\n\nMTM-1 may down-regulate cell corpse engulfment by modulating plasma membrane PtdIns(3)P levels {#s3d}\n----------------------------------------------------------------------------------------------\n\nMyotubularins are lipid phosphatases that specifically dephosphorylate PtdIns(3)P or its metabolite PtdIns(3,5)P~2~ [@pgen.1000679-Clague1]. ", "Our findings that MTM-1 acts as a negative regulator of cell corpse engulfment dependent on its lipid phosphatase activity suggest that 3-phosphoinositides may act as signaling molecules during engulfment. ", "Given that both the class III PI3K *vps-34* and the class II PI3K *piki-1* are required for reducing cell corpses by *mtm-1* RNAi and that loss of *mtm-1* function increases the vesicular accumulation of PtdIns(3)P in vivo, PtdIns(3)P likely serves as a substrate of MTM-1 during engulfment. ", "Although PtdIns(3)P is enriched in endocytic compartments [@pgen.1000679-Roggo1], we found that MTM-1 is mainly localized to plasma membrane. ", "Although both the phosphoinositide-binding domain PH-GRAM and the catalytic domain PTP are required for membrane association of MTM-1, PtdIns(3)P seems to be dispensable for its membrane localization because MTM-1 still localizes to the plasma membrane in *vps-34(lf)*, *piki-1(lf)* or *vps-34(lf);piki-1(lf)* double mutants, in which PtdIns(3)P generation is probably blocked [@pgen.1000679-Roggo1]. ", "This observation also excludes the possibility that MTM-1 is recruited to plasma membranes through direct interaction with VPS-34 or PIKI-1. ", "Therefore, MTM-1 may associate with plasma membranes through its interactions with other phosphinositides or protein partners such as VPS34 adaptor protein which binds hMTM1 on endosomes [@pgen.1000679-Cao2]. ", "Consistent with our observations, several human MTMs were also reported to localize to plasma membranes although their substrates PtdIns(3)P and PtdIns(3,5)P~2~ are concentrated on early and late endocytic compartments, respectively, which leads to the hypothesis that myotubularins may act to prevent accumulation of PtdIns(3)P or PtdIns(3,5)P~2~ at inappropriate compartments [@pgen.1000679-Robinson1],[@pgen.1000679-Clague1]. ", "In the case of MTM-1, it is possible that MTM-1 coordinates with PI3Ks VPS-34 and PIKI-1 to maintain an appropriate level of PtdIns(3)P on the plasma membrane for the internalization of cell corpses. ", "In agreement with this hypothesis, we found that MTM-1 transiently associates with extending pseduopods and nascent phagosomes at a similar stage to the phagocytic receptor CED-1 during engulfment.", "\n\nAlthough PtdIns(3)P is a proven critical regulator of early endosomal traffic and phagosome maturation, its involvement in cell corpse engulfment has not been reported. ", "We propose that PtdIns(3)P acts as a positive signal for engulfment based on our findings that (i) *mtm-1* antagonizes cell corpse internalization, which requires both its lipid phosphatase activity and the functions of PI3Ks VPS-34 and PIKI-1, (ii) MTM-1 localizes to the plasma membrane, functions in engulfing cells and clusters around cell corpses during engulfment, and (iii) MTM-1 negatively regulates the vesicular accumulation of PtdIns(3)P in vivo. ", "Interestingly, DOCK180, the mammalian homolog of CED-5, binds directly to PtdIns(3,4,5)P~3~ through its DHR1 domain in vitro and translocates to the cell membrane in response to PtdIns(3,4,5)P~3~ production in NIH3T3 cells [@pgen.1000679-Cote1]. ", "Since the Class I PI3K AGE-1 which generates PtdIns(3,4,5)P~3~ appears not to be required for cell corpse engulfment in *C. elegans*, it is possible that CED-5/CED-12 GEF is recruited to the plasma membrane by PtdIns(3)P to activate CED-10/Rac, and that MTM-1 acts antagonistically to terminate the signal and release the complex after engulfment. ", "Alternatively, PtdIns(3)P may bind and facilitate the nucleotide exchange of CED-10/Rac during engulfment, similar to the way in which PtdIns(4,5)P~2~ promotes Rho and Rac activation under certain conditions [@pgen.1000679-Ren1].", "\n\nThe function of MTM-1 in cell corpse engulfment may be conserved from *C. elegans* to mammals {#s3e}\n---------------------------------------------------------------------------------------------\n\nMyotubularin family phosphatases are conserved amongst all eukaryotic organisms, but their cellular functions are not well understood. ", "Our finding that overexpression of human MTM1 can efficiently rescue the cell corpse phenotype of *mtm-1(lf)* mutants suggests that the role of MTM-1 in cell corpse engulfment is likely conserved from worms to humans and that similar mechanisms may also be used in mammals to regulate the removal of apoptotic cells.", "\n\nWe found that MTM-1 acts as a lipid phosphatase to negatively regulate the engulfment of apoptotic cells through CED-10/Rac, which raises the interesting question of whether MTM-1 is similarly involved in other CED-10/Rac-mediated cellular processes by regulating PtdIns(3)P. Intriguingly, we found that *mtm-1* seems to play a positive instead of a negative role in the migration of DTCs, a process which also requires the activity of CED-5, CED-12 and CED-10. ", "MTM-1 may therefore play distinct roles in different CED-10/Rac-mediated processes. ", "However, we currently do not know whether the function of MTM-1 in DTC migration is also mediated by PtdIns(3)P and, if so, whether the opposite roles of MTM-1 in cell corpse engulfment and DTC migration are caused by distinct effects of PtdIns(3)P on CED-10/Rac in these two processes. ", "On the other hand, loss of myotubularin has been found to cause muscle defects in human, mouse and zebrafish [@pgen.1000679-Laporte1],[@pgen.1000679-Dowling1],[@pgen.1000679-BujBello1], but the underlying mechanism is not understood. ", "Since we have identified a genetic link between MTM-1 and CED-10/Rac in cell corpse engulfment, it will be interesting to test whether misregulation of Rac GTPase might be relevant to the muscle defect caused by lack of myotubularin.", "\n\nMaterials and Methods {#s4}\n=====================\n\n*C. elegans* strains {#s4a}\n--------------------\n\nStrains of *C. elegans* were cultured at 20°C using standard procedures [@pgen.1000679-Brenner1]. ", "The N2 Bristol strain was used as the wild-type strain. ", "Mutations used are described in *C. elegans* II [@pgen.1000679-Riddle1] unless otherwise indicated. ", "Linkage group I (LGI): *ced-1(e1735)*, *vps-34(h797)*, *dpy-5(e61)*, *unc-13(e450am)*, *mtm-1(ok742)* (this study), *ced-12(n3261)* [@pgen.1000679-Zhou2], *hT2(bli-4(e937);let-?(q782)qIs48/sep-1(e2406)* (Wormbase:[www.wormbase.org](http://www.wormbase.org)). ", "LGII: *rrf-3(pk1426)* [@pgen.1000679-Simmer1]. ", "LGIII: *ced-6(n2095), ced-6(qx17)* (this study and see below), *ced-7(n2094)*, *mtm-6(ok330)* (this study, Wormbase: [www.wormbase.org](http://www.wormbase.org)). ", "LGIV: *ced-2(n1994), ced-5(n1812), ced-10(n3246), ced-10(n1993)*. ", "LGX: *mig-2(mu28)* [@pgen.1000679-Zipkin1], *piki-1(ok2346)* and *mtm-5(ok469)* (this study, Wormbase: [www.wormbase.org](http://www.wormbase.org)). *", "smIs34* (P*~ced-1~ced-1::gfp*) and *smIs95* (P*~hsp~annexin v::mrfp*) were provided by Dr. Ding Xue (University of Colorado, CO). *", "opIs334* (P*~ced-1~*YFP::2xFYVE) was a gift from Dr. K. S. Ravichandran (Univ. ", "of Virginia, Charlottesville, VA) and Dr. M. O. Hengartner (Univ. ", "of Zurich, Zurich, Switzerland) [@pgen.1000679-Kinchen2].", "\n\nOther strains carrying integrated arrays used in this study are listed below:\n\n*qxIs156* (P*~hsp~*MTM-1), *qxIs210* (P*~hsp~*MTM-1(C378S)), *qxIs197* (P*~mtm-1~*MTM-1::GFP), *qxIs66* (P*~ced-1~*GFP::RAB-7), *qxIs40* (P*~ced-1~*ACT-5::GFP), *qxIs60* (P*~ced-1~*LMP-1::GFP). ", "The *vps-34*-deficient strain, *vps-34(h797)*, is maintained as *dpy-5(e61)vps-34(h797)*; *qxEx* \\[*vps-34*(+); P*~sur-5~sur-5::gfp*\\]. ", "Non-green embryos (*dpy-5(e61)vps-34(h797))* were scored as *vps-34(lf)* and green embryos that carry the *vps-34(+)* transgene were scored as wild type for *vps-34*.", "\n\n*qx17* is a recessive mutation isolated from a forward genetic screen for additional regulators of cell corpse engulfment and was mapped to linkage group III very close to *ced-6*. ", "A complementation test between *qx17* and *ced-6(n2095)* was performed and a similar engulfment phenotype was observed in the double heterozygote, indicating that *qx17* is an allele of *ced-6*. ", "We determined the sequence of *ced-6* in *qx17* mutants and identified a T to A transition that results in a premature stop codon after Glu 475 and generates a truncated CED-6 protein lacking the last 17 amino acids at the C-terminus.", "\n\nQuantification of cell corpses, cell death events, and cell corpse duration {#s4b}\n---------------------------------------------------------------------------\n\nSomatic cell corpses were directly visualized by differential interference contrast (DIC) microscopy as highly refractile button-like objects distinct from normal living cells [@pgen.1000679-Sulston1],[@pgen.1000679-Sulston2]. *", "C. elegans* embryos were mounted on agar pads in M9 and viewed using a 100× Plan-Neofluar DIC objective on an Axioimager M1 microscope (Carl Zeiss, Inc.). ", "The number of cell corpses was quantified in the head region of living embryos either at the six different embryonic stages (bean/comma, 1.5-fold, 2-fold, 2.5-fold, 3-fold and 4-fold) for a time-course analysis or at the 4-fold embryonic stage as described before [@pgen.1000679-Stanfield1]. ", "To measure the duration of cell corpses, four-dimensional microscopy (4D) analysis was performed at 20°C as described before with some modifications for *vps-34(h797);piki-1(ok2346)* mutants [@pgen.1000679-Wang1]. ", "Since *vps-34(lf)* causes embryonic lethality and most *vps-34(h797)* mutant embryos die before the 4-fold embryonic stage, it is maintained as *dpy-5(e61)vps-34(h797)*; *qxEx* \\[*vps-34*(+); P*~sur-5~sur-5::gfp*\\]. ", "To monitor the cell corpse duration in *vps-34(h797);piki-1(ok2346)* mutants, only non-green embryos which developed normally until the 2.5- or 3-fold stage were followed and quantified. ", "To examine the internalization of cell corpses, apoptotic cells were first identified by their refractile disk-like morphology using Nomarski DIC microscopy and then different fluorescent markers that associate with cell corpses at different stages of engulfment were examined. ", "To monitor the occurrence of embryonic cell death, embryos at the 2--4 cell stage were mounted on agar pads and images in a 30 µm z series (0.75 µm/section) were captured every 1.5 minutes for 8 h using a Zeiss Axioimager M1 microscope (Carl Zeiss, Inc.). ", "Images were processed and viewed using Axiovision Rel 4.5 software (Carl Zeiss, Inc.).", "\n\nRNAi and genome-wide RNAi screen {#s4c}\n--------------------------------\n\nThe genome-wide RNAi screen was performed as described before with some modifications [@pgen.1000679-Kamath1]. ", "Briefly, adult hermaphrodites of *ced-1(e1735);rrf-3(pk1426)* were bleached on NGM plates without OP50. ", "The hatched larvae were washed off with M9 and added to individual RNAi plates with 30 larvae per plate (*C. elegans* RNAi library, Geneservice, UK). ", "Cell corpses were scored in the progeny at the 4-fold embryonic stage. ", "For *mtm-1* RNAi by feeding, gravid adults of the indicated strains (P0) were picked and bleached on either control (pPD129.36-gfp or pPD129.36 for examining MTM-1::GFP expression) or *mtm-1* RNAi plates (I-6C09) and the number of embryonic cell corpses or the expression of MTM-1::GFP was scored in the F2 generation. ", "To quantify the DTC migration defect, L4 larvae of the F2 generation were aged 24 h before examination. *", "mtm-1* RNAi caused 14% and 40% embryonic lethality in wild type and the RNAi hypersensitive mutant *rrf-3(pk1426)*, respectively, while 7% of wild-type and 29% of *rrf-3(pk1426)* embryos died when treated with *gfp* RNAi. ", "For *mtm-1* RNAi by injection, a double-stranded RNA (dsRNA) of *mtm-1* (436--2489 bp of Y110A7A.5) was synthesized in vitro and injected into *ced-1(e1735);qxIs197* animals, which carry an integrated array of MTM-1::GFP controlled by the *mtm-1* promoter (P*~mtm-1~*MTM-1::GFP). ", "Embryonic cell corpses and expression of MTM-1::GFP were both examined 24 h post injection. ", "For *mtm-3* and *mtm-9* RNAi, double-stranded RNAs (dsRNA) were synthesized (*mtm-3*: 5812--6435 bp of T24A11.1a; *mtm-9*: 1583--2031 bp of Y39J10A.3a) and injected individually or in combination. ", "Embryonic cell corpses were quantified 24 h post injection.", "\n\nHeat-shock experiments {#s4d}\n----------------------\n\nYoung adults were moved to fresh nematode growth medium (NGM) plates and cultured at 20°C for 12 h before they were incubated at 33°C for 1 h (+HS) or left at 20°C without heat-shock treatment (−HS), followed by recovery at 20°C for 1.5 h. Adult worms were removed and embryos were incubated at 20°C and scored for the number of cell corpses 5 to 10 h after treatment.", "\n\nConfocal microscopy {#s4e}\n-------------------\n\nA Zeiss LSM 510 Pascal inverted confocal microscope with 488, 514, 633 lasers (Carl Zeiss Inc.) was used to capture fluorescent images which were processed and viewed using LSM Image Browser software. ", "Time-lapse imaging of CED-1::GFP, GFP::MTM-1 and CED-1::mCHERRY was performed as described [@pgen.1000679-Laporte3]. ", "Briefly, *C. elegans* embryos at the pre-comma or comma stage were mounted on agar pads and images in a 20--25 z series (1.0 µm/section) were captured every 1 min, 1.5 min or 2 min for 120 min using a Zeiss LSM 510 Pascal inverted confocal microscope (Carl Zeiss, Inc.). ", "Images were processed and viewed using LSM Image Browser software.", "\n\nExamination of PtdIns(3)P level by YFP::2xFYVE probe {#s4f}\n----------------------------------------------------\n\nTo examine the level of PtdIns(3)P using the YFP::2xFYVE probe, L2 larvae of wild type, surviving *mtm-1(ok742)* mutants derived from *hT2/mtm-1(ok742)* worms, and *vps-34* RNAi-treated wild type or *mtm-1(ok742)* mutants were mounted on agar pads. ", "Nomarski and fluorescent images of the midbody region of L2 larvae in a 20 z series (1.0 µm/section) were captured using a fixed exposure time with a Zeiss Axioimager A1 equipped with epifluorescence and an Axiocam monochrome digital camera. ", "The exposure time was 550 ms for wild type and *mtm-1(ok742)* mutants, but 1500 ms for animals treated with *vps-34* RNAi because only very faint YFP::2xFYVE signal can be observed at 550 ms in these worms due to significant reduction of PtdIns(3)P accumulation caused by loss of *vps-34* function. ", "Serial optical sections were analyzed and the numbers of YFP::2xFYVE-positive vesicles were quantified in hypodermal cells in a region of approximately 88 µm×18 µm. ", "At least 30 animals were quantified in each strain.", "\n\nPlasmid construction {#s4g}\n--------------------\n\nPCR primer sequences are shown in [Table S4](#pgen.1000679.s009){ref-type=\"supplementary-material\"}. ", "To generate P*~mtm-1~mtm-1::gfp* , a 3.9 kb genomic fragment of *mtm-1* including a 0.8 kb promoter region was amplified by PCR from WRM0617dG02 using primers PQL121/120 and cloned into the pPD95.77 vector through its Bam HI and Sma I sites. ", "A 3.1 kb fragment containing the full-length genomic sequence of the *mtm-1* gene was PCR-amplified from WRM0617dG02 using primers PWDL108/109 and cloned into both pPD49.78 and pPD49.83 through the Nhe I-Kpn I sites to generate P*~hsp~mtm-1*. ", "The same *mtm-1* genomic fragment was also amplified by primers PWZ215/PWDL109 and cloned into P*~ced-1~gfp* vector through the Kpn I site to generate P*~ced-1~gfp::mtm-1*. ", "The C378S mutation was introduced into pPD49.83-mtm-1 by site-directed mutagenesis using primers PQL148/149 (QuickChange; Stratagene, USA) and re-cloned into both pPD49.78 and pPD49.83 via their Nhe I and Kpn I sites. ", "To construct P*~ced-1~mtm-1* and P*~egl-1~mtm-1*, the full-length cDNA of *mtm-1* was amplified from a *C. elegans* cDNA library (Invitrogen, USA) by PWZ215/PWDL109 and cloned into both P*~ced-1~* and P*~egl-1~* vector through the Kpn I site. ", "To generate P*~ced-1~*GFP::MTM-1(ΔGRAM), P*~ced-1~*GFP::MTM-1(ΔPTP) and P*~ced-1~*GFP::MTM-1(Δcoiled-coil), a 2.1 kb genomic fragment of *mtm-1* (ΔGRAM: 975--3072 bp) or a 1.7 kb *mtm-1* genomic sequence (1--1739 bp) (ΔPTP) or a 3 kb *mtm-1* fragment (Δcoiled-coil: 1--3000 bp) were PCR-amplified from WRM0617dG02 using primers PWZ291/PWDL109, PWZ215/PWZ151 and PWZ215/PWZ322, respectively, and cloned into the P*~ced-1~gfp* vector via its Kpn I site. ", "To construct P*~hsp~*hMTM1, the full-length cDNA of human *MTM1* was amplified from a human cDNA library (Clontech, USA ) with primers PWZ384/385 and cloned into both pPD48.78 and pPD49.83 through the Nhe I-Kpn I sites. ", "To generate P*~hsp~myri::mcherry*, mcherry was amplified from pAA65 [@pgen.1000679-Green1] using primers PWZ421 (which contains a myristoylation signal) and PWZ427 and cloned into both pPD49.78 and pPD49.83 through the Kpn I site.", "\n\nSupporting Information {#s5}\n======================\n\n###### \n\n*mtm-1* RNAi treatments specifically inhibit the expression of *mtm-1*. (", "A) *mtm-1* RNAi treatments (either feeding with bacteria expressing *mtm-1* dsRNA or injecting in vitro-synthesized *mtm-1* dsRNA) result in reduction of cell corpse numbers and inhibition of MTM-1::GFP expression. ", "RNAi experiments were performed as described in [Materials and Methods](#s4){ref-type=\"sec\"}. ", "Cell corpses were scored at the 4-fold embryonic stage and are shown as mean±s.e.m. ", "At least 15 embryos were scored for cell corpses and 40 embryos at the 4-fold embryonic stage were examined for expression of MTM-1::GFP. ", "Representative pictures of MTM-1::GFP expression before and after *mtm-1* RNAi treatment are also shown. ", "The exposure time of both pictures was 2000 ms. (", "B,C) The gene structures of *mtm-1* and *piki-1* are shown, with filled boxes representing the exons and thin lines indicating the introns. ", "The arrows show the direction of the transcript. ", "The gray bars below the genes indicate the position and size of the deletions in the *ok742* and *ok2346* mutant.", "\n\n(1.05 MB TIF)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\n*mtm-1* RNAi accelerates internalization of the apoptotic cell C3. (", "A) DIC and fluorescence images of a wild-type embryo co-expressing CED-1::GFP (P*~ced-1~ced-1::gfp*) and a secreted Annexin V::mRFP under the control of heat-shock promoters (P*~hsp~annexin v::mrfp*) are shown. ", "The apoptotic cell C3 (white arrow) and a posterior apoptotic cell (blue arrow) were labeled by both CED-1::GFP and Annexin V::mRFP. ", "The ventral hypodermal cell that engulfs C3 is indicated by the arrowhead. ", "Bars: 5 µm. (", "B,C) The formation and duration of the CED-1::GFP ring around C3 (arrowed) were followed in *ced-6(qx17)* mutants treated with either control (i--iv in B and i--v in C) or *mtm-1* RNAi (v--viii in B and vi--x in C). ", "To monitor the formation of CED-1 rings, the \"0 min\" time point was set immediately prior to the appearance of trace amounts of CED-1::GFP around C3. ", "To monitoring the duration of CED-1 rings, the \"0 min\" time point was set when a full CED-1::GFP ring was just visible. ", "13 C3 corpses were monitored and quantified for either formation or duration of CED-1::GFP rings (ix in B and xi in C). ", "The numbers in parenthesis indicate average formation or duration times of CED-1::GFP rings (±s.e.m). ", "Bars: 5 µm.", "\n\n(2.95 MB TIF)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nInactivation of MTM-1 promotes cell corpse internalization. ", "Clustering of CED-1::GFP around cell corpses (A) or the phagosomal association of GFP::RAB-7 (B) and LMP-1::GFP (C) was quantified in 1.5-fold stage embryos in the indicated strains. ", "At least 15 embryos were scored in each strain. ", "Error bars indicate s.e.m. ", "Unpaired *t* tests were performed to compare the data derived from *mtm-1* RNAi-treated embryos with that from control animals. ", "\\*\\**P*\\<0.0001, \\**P*\\<0.01; all other points had *P* value\\>0.01.", "\n\n(0.13 MB TIF)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nMyotubularin is conserved in yeast, worms and humans. ", "Protein sequence alignments of *C. elegans* MTM-1 (c.eMTM-1), human myotubularin (hMTM1) and yeast myotubularin (Ymr1p) are shown. ", "Identical residues are in black and similar ones are in gray. ", "Conserved motifs are boxed. ", "The signature CX5R active site motif for the protein tyrosine phosphatase super-family is circled in red. ", "The critical cysteine residue, which is changed to serine in the *C. elegans* MTM-1(C378S) mutant, is marked by a red arrowhead.", "\n\n(0.50 MB TIF)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nPlasma membrane localization of MTM-1 does not require the activities of PI3-kinases. (", "A) MTM-1::GFP is expressed in many different cell types. ", "DIC and fluorescence images of wild-type animals expressing P*~mtm-1~mtm-1::gfp* are shown. ", "MTM-1::GFP was seen in distal tip cells (i, ii), coelomocytes (iii, iv) and vulva cells (v, vi). ", "Bars: 10 µm. (", "B) Both full-length and truncated GFP::MTM-1 were stably expressed in *C. elegans*. ", "Lysates were prepared from 200 adult transgenic worms carrying P*~ced-1~*GFP::MTM-1, P*~ced-1~*GFP::MTM-1(δGRAM), P*~ced-1~*GFP::MTM-1(δPTP) or P*~ced-1~*GFP::MTM-1(δCC) and western blot analysis was performed using an anti-GFP antibody. ", "Full-length GFP::MTM-1 (93 Kd) and the three GFP::MTM-1 truncations (δCC: 91 Kd, δGRAM: 76 Kd, δPTP: 65 Kd) were all expressed at the expected size. (", "C) DIC and fluorescence images of MTM-1::GFP in wild-type (i, ii), *vps-34(h797)* (iii, iv), *piki-1(ok2346)* (v, vi) and *vps-34(h797);piki-1(ok2346)* (vii, viii) embryos are shown. ", "The plasma membrane localization of MTM-1::GFP is not affected in the loss-of-function mutants of PI3-kinases. ", "Bars: 5 µm.", "\n\n(2.85 MB TIF)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nInactivation of MTM-1 affects the migration of DTCs.", "\n\n(0.04 MB DOC)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nThe lipid phosphatase activity and conserved domains of MTM-1 are important for its function in cell corpse engulfment.", "\n\n(0.04 MB DOC)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nOther MTMs do not play redundant roles with MTM-1 in cell corpse engulfment.", "\n\n(0.04 MB DOC)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nPrimers used for plasmid construction.", "\n\n(0.04 MB DOC)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nThe clustering of CED-1::GFP in a *ced-6(qx17)* embryo treated with *mtm-1* RNAi. ", "The formation of a CED-1::GFP ring around a dying cell in a *mtm-1* RNAi-treated *ced-6(qx17)* embryo is shown. ", "The cell corpse followed is indicated by an arrow. ", "The frames were collected every 1 min and displayed every 1 sec. ", "Selected images are shown in [Figure 4A](#pgen-1000679-g004){ref-type=\"fig\"}.", "\n\n(0.35 MB AVI)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nThe clustering of CED-1::GFP in a *ced-6(qx17)* embryo treated with control RNAi.", "The formation of a CED-1::GFP ring around a dying cell in a control RNAi-treated *ced-6(qx17)* embryo is shown. ", "The cell corpse followed is indicated by an arrow. ", "The frames were collected every 1.5 min and displayed every 1 sec. ", "Selected images are shown in [Figure 4A](#pgen-1000679-g004){ref-type=\"fig\"}.", "\n\n(0.33 MB AVI)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nThe duration of CED-1::GFP around dying cell in a *ced-6(qx17)* embryo treated with control RNAi.", "The duration of the CED-1::GFP ring around a cell corpse in a control RNAi-treated *ced-6(qx17)* embryo is shown. ", "The cell corpse followed is indicated by an arrow. ", "The frames were collected every 1 min and displayed every 1 sec. ", "Selected images are shown in [Figure 4C](#pgen-1000679-g004){ref-type=\"fig\"}.", "\n\n(0.81 MB AVI)\n\n###### \n\nClick here for additional data file.", "\n\n###### \n\nThe duration of CED-1::GFP around dying cell in a *ced-6(qx17)* embryo treated with *mtm-1* RNAi.", "The duration of CED-1::GFP around a cell corpse in a *mtm-1* RNAi-treated *ced-6(qx17)* embryo is shown. ", "The cell corpse followed is indicated by an arrow. ", "The frames were collected every 1 min and displayed every 1 sec. ", "Selected images are shown in [Figure 4C](#pgen-1000679-g004){ref-type=\"fig\"}.", "\n\n(0.52 MB AVI)\n\n###### \n\nClick here for additional data file.", "\n\nWe thank Drs. ", "Ding Xue for providing the *smIs34* and *smIs95* strains, K. S. Ravichandran, and M.O. Hengartner for providing the *opIs334* strain and A. Fire for vectors. ", "We thank Dr. David Sherwood and Dr. Guangshuo Ou for sharing information about myri::mcherry and Dr. Karen Oegema for the pAA65 vector. ", "We thank the *C. elegans* gene knockout consortium for generating deletion mutants of *piki-1, mtm-1, mtm-5* and *mtm-6* and the *Caenorhabditis* Genetic Center (CGC) for strains. ", "We thank C. Zhan and L. Wang for the SLM 510 confocal microscope, Dr. C.L. Yang for his critical reading of the manuscript, Dr. Isabel Hanson for editing services, and members of our laboratory for helpful discussion and suggestion.", "\n\nThe authors have declared that no competing interests exist.", "\n\nThis work was supported by the National High Technology Project 863 from the Ministry of Science and Technology, grant number: 2007AA02Z1A0, [www.most.gov.cn](http://www.most.gov.cn). ", "The funders has no role in study design, data, collection and analysis, decision to publish, or preparation of the manuscript.", "\n\n[^1]: Conceived and designed the experiments: WZ QL XW. ", "Performed the experiments: WZ QL. ", "Analyzed the data: WZ QL XW. ", "Contributed reagents/materials/analysis tools: JM. ", "Wrote the paper: XW. ", "Contributed to some of the experiments: DZ, WL, YX.", "\n" ]

{ "pile_set_name": "PubMed Central" }

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[ "In an earlier thread, as a Rams fan, I expressed concern with the Rams selection of Brockers because LSU players seem to underwhelm in the NFL. ", "Below is a list of LSU players who are on, or will be on, NFL rosters.", "\n\nMcClure has been a good center for the Falcons; Peterson has loads of potential as he is already one of the best return men; Landry has been solid, but only 4 career ints????; ", "Henderson has been a consistent deep threat for the Saints; while Bowe is a stud. ", "But overall, LSU players have not exactly set the NFL on fire. ", "I think the only player who receives consistent pro bowl recognition is Bowe, while Peterson may warrant that type of attention sooner rather than later.", "\n\nNow let's take a school such as Boston College, who has spent the last few years near the bottom of the weak ACC.", "\n\nI don't think it's just LSU, it's alot of big college programs. ", "USC, Texas, Florida, they seem to produce lots of busts. ", "Talented teams mask alot of individual flaws that prospects might have. ", "For example Brockers, it didn't seem to matter if he didn't push the pocket or have any pass rush moves because he's playing next to two stud pass-rushers. ", "I'm not saying he's going to bust although he wasn't my favorite prospect but at the end of the day his weakness doesn't hurt LSU as much as it would a team that didn't also have Mingo and Montgomery, therefore making everyone look better. ", "It's not that teams should just avoid these guys from powerhouse programs because that would be stupid, but I think you need to take a harder look at their flaws than you would a kid from a smaller school.", "\n\nI don't think it looks that out of the ordinary. ", "And a perfectly reasonable explanation is that high-profile schools may be more complete teams in the first place, and have great coaching to develop their players, and are more heavily scouted by NFL teams, and thus have more mediocre-to-decent roster-filler types that make the NFL, where similar guys from smaller schools may not have the kind of experience, development, coaching, and exposure that those from the big schools do.", "\n\nI don't think it's just LSU, it's alot of big college programs. ", "USC, Texas, Florida, they seem to produce lots of busts. ", "Talented teams mask alot of individual flaws that prospects might have. ", "For example Brockers, it didn't seem to matter if he didn't push the pocket or have any pass rush moves because he's playing next to two stud pass-rushers. ", "I'm not saying he's going to bust although he wasn't my favorite prospect but at the end of the day his weakness doesn't hurt LSU as much as it would a team that didn't also have Mingo and Montgomery, therefore making everyone look better. ", "It's not that teams should just avoid these guys from powerhouse programs because that would be stupid, but I think you need to take a harder look at their flaws than you would a kid from a smaller school.", "\n\nValid point. ", "But USC, Texas and Florida have produced some studs as well. ", "Troy Palomala (sp?), ", "Ryan Khalil, Clay Matthews, Brian Cushing for USC; Casey Hampton, Jamaal Charles, Jermicheal Finley, Brian Orakpo, and Earl Thomas for Texas; the Pouncy's, Dunlap, Hernandez, and Percy Harvin for Florida. ", "Granted, these schools had their share of disappointments and busts, they also appear to have more success at the pro level than LSU (though one could make a argument that Florida players are somewhat disappointing).", "\n\nTwo future Hall of Famers (Lewis and Reed), another who is well on his way (Andre Johnson), one who has Hall of Fame numbers (Reggie Wayne), one of the best running backs in the league (Frank Gore), and Pro Bowlers at one time or another such as McKinnie, Vilma, Graham, Wilfork, Rolle, Myers, Moss, Hester, and McGahee - as well as a plethora of others who are very good NFL players.", "\n\nI don't want to get too much into it, but LSU wasn't a powerhouse program in the 90s, and Miami was in the 90s and early 00s.", "\n\nLSU has, really in just the past decade or so became a national powerhouse. ", "Give LSU some time and I'm sure there list will look almost as impressive as Miami.", "\n\nMiami was producing stars at nearly every level (although it's amazing QB and O-Line never seemed to develop much) while LSU really just seems to produce DT's, DB's, a few WR's and some odd ones here and there/\n\nI don't want to get too much into it, but LSU wasn't a powerhouse program in the 90s, and Miami was in the 90s and early 00s.", "\n\nLSU has, really in just the past decade or so became a national powerhouse. ", "Give LSU some time and I'm sure there list will look almost as impressive as Miami.", "\n\nMiami was producing stars at nearly every level (although it's amazing QB and O-Line never seemed to develop much) while LSU really just seems to produce DT's, DB's, a few WR's and some odd ones here and there/\n\nGood point regarding quarterbacks. ", "Other than Kelly and perhaps Kosar, Miami never really produced top flight qbs. ", "Yeah, Testeverde played for decades, but never was the player many thought he would be.", "\n\nEven as of late, Miami has produced good players. ", "Olsen, Graham, Beason, Winston, Phillips, Shields, Franklin, and Campbell.", "\n\nLSU has not really produced too many DTs, though they have had a few good db's (Peterson and Landry being two of the better ones).", "\n\nYou may be onto something as far as LSU lacking in the pro department due to their lack of past success....look at Alabama. ", "They are not exactly supplying the NFL with Pro Bowlers. ", "Just five or six years ago, they were dismal.", "\n\nGood point regarding quarterbacks. ", "Other than Kelly and perhaps Kosar, Miami never really produced top flight qbs. ", "Yeah, Testeverde played for decades, but never was the player many thought he would be.", "\n\nLSU has not really produced too many DTs, though they have had a few good db's (Peterson and Landry being two of the better ones).", "\n\nYeah I was talking the second life of Miami football, from the 90s-2000s they havent produced any QBs.", "\n\nit's true. ", "I hate this program actually, and am still fairly bitter about buster davis and jacob hester being not just awful, but really pretty pathetic to the extent that you never thought they were even trying hard. ", "Hester actually is still on the Bolt roster despite being a fullback that takes away from the running game's potency, although will be a special teamer/backup to McClain this year.", "\n\nWe also have Scafe and Lee in camp, AJ Smith loves LSU like i loved that girl in high school who did meth, until she literally got a facial scar from a car crash. ", "Man that was insaaaane. ", "waht an apt metaphor.", "\n\nMiami has obviously produced some great pros (or at least they used to) but half the players on that Miami list absolutely suck, or are barely rosterable players.", "\n\nIt's hard to give Miami credit for \"producing\" solid pros lately because even though some of them turned out to be solid pros, they it's only because of the coaching they got once in the NFL. ", "They were raw as a turnip leaving Miami and the program did absolutely nothing to develope these players.", "\n\nA lot of the best pros that Bama produced over the years are recently retired like Chris Samuels, Shaun Alexander, etc... or were tragically killed in car accidents like Derrick Thomas was.", "\n\nAlabama has produced some of the best pros that the NFL has ever had if you go back a little... guys like Ozzie Newsome, John Hannah, Dwight Stephenson, Joe Namath, Bart Starr, Kenny Stabler, etc.", "\n\nThe more draft picks a program produces, the higher the bust percentage increases. ", "Likewise, so does the odds of produces average pros.", "\n\nAs great as Miami was in the 90's and 2000's, look at all the 1st round busts... Yatil Green, Mike Rumph, Phillip Buchanon, Jerome McDougle, Damione Lewis, William Joseph, etc. ", "Dan Morgan was a nice player but made of glass.", "\n\nRemember Randall Hill?", "\n\nYou have to produce a lot of average pros in order to produce a lot of great ones.", "\n\nTwo future Hall of Famers (Lewis and Reed), another who is well on his way (Andre Johnson), one who has Hall of Fame numbers (Reggie Wayne), one of the best running backs in the league (Frank Gore), and Pro Bowlers at one time or another such as McKinnie, Vilma, Graham, Wilfork, Rolle, Myers, Moss, Hester, and McGahee - as well as a plethora of others who are very good NFL players.", "\n\nThose great Miami teams from the late 90s to the start of the last decade also featured Clinton Portis, Edge James, Jeremy Shockey and the late great Sean Taylor.", "\n\nGo back a bit further and you'll find Warren Sapp, Michael Irvin, Cortez Kennedy, Russell Maryland, the Blades brothers and Jerome Brown (RIP). ", "Even long before the Hurricanes became a powerhouse in the 80s they still produced two HOF Raiders legends in Ted Hendricks and Jim Otto.", "\n\nI hate the LSU program for this reason. ", "There is just a lack of good-great players that come out of it. ", "Someone mentioned that it is a problem with many of the bigger schools, and that may be true. ", "A program like LSU gets a lot of hype because they have been so good the past decade, and I feel that causes people to \"fall in love\" with certain players even if they aren't that good.", "\n\nI'm going to throw out a 'correlation does not imply causation' worry here. ", "There are only so many productive NFL players at a given time. ", "Right now, LSU hasn't produced as many as they could have. ", "I think guys like Brockers, Claiborne and Randle will help change this trend.", "\n\nJust glancing over the list those guys right there were/are pretty darn good players in the NFL - most of them pro bowl or at least pro bowl quality. ", "There are a bunch of other guys on that list who are established starters in the league even if they aren't pro bowlers or stars. ", "I'm pretty positive Morris Claiborne is going to be really good as well.", "\n\nSure LSU has had a fair share of high pick busts, but so does every other school who produces that large of a volume of top picks (look at USC, Ohio State, Oklahoma, Georgia, Florida State, etc.)", "\n\nIt's been covered pretty well but when you're on a top team you get more exposure, hence guys at LSU that would've been 2nd-3rd rounders elsewhere going in round 1, same for 4th-5th rounders getting pushed up, etc\n\nBoston College is in the dumpster now but they were a very consistent, if unexciting, 8-9 win team for a very long time (save the one year with Matt Ryan where they peaked at #2 overall). ", "They get no exposure being in a pro sports town with a small fanbase\n\nMiami has obviously produced some great pros (or at least they used to) but half the players on that Miami list absolutely suck, or are barely rosterable players.", "\n\nIt's hard to give Miami credit for \"producing\" solid pros lately because even though some of them turned out to be solid pros, they it's only because of the coaching they got once in the NFL. ", "They were raw as a turnip leaving Miami and the program did absolutely nothing to develope these players." ]

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[ "Brodersby, Rendsburg-Eckernförde\n\nBrodersby is a municipality in the district of Rendsburg-Eckernförde, in Schleswig-Holstein, Germany.", "\n\nReferences\n\nCategory:Municipalities in Schleswig-Holstein\nCategory:Rendsburg-Eckernförde" ]

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[ "Guibemantis punctatus\n\nGuibemantis punctatus is a species of frog in the family Mantellidae.", "\nIt is endemic to Madagascar.", "\nIts natural habitats are subtropical or tropical moist lowland forests, subtropical or tropical moist montane forests, and heavily degraded former forest.", "\nIt is threatened by habitat loss.", "\n\nSources\n Nussbaum, R., Vallan, D. & Raxworthy, C. 2004. ", " Mantidactylus punctatus. ", " 2006 IUCN Red List of Threatened Species. ", " Downloaded on 23 July 2007.", "\n\nCategory:Mantellidae\nCategory:Endemic frogs of Madagascar\nCategory:Taxonomy articles created by Polbot\nCategory:Amphibians described in 1979" ]

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[ "Aptech launches month-long career guidance initiative for students\n\nMumbai: Aptech Computer Education has launched ‘Career Next’ - a month-long career guidance initiative for students interested in making a career in IT.", "\n\nThrough this initiative, Aptech aims to provide essential ‘first step’ guidance to students, while helping them make informed career decisions in the IT industry.", "\n\nTalking on the rationale behind launching Career Next, Anuj Kacker, Senior Vice President & Global Head - Retail Education, Aptech, said, “Career Next is aimed to provide students with the initial step in the pathway to future employment in the IT sector. ", "We will provide students with free career counselling & aptitude tests to help them become confident professionals.”", "\nThe program started off in the first week of April 2009. ", "Aptech kiosks have been installed outside colleges, malls, multiplexes, popular eating joints, cafés, etc. ", "across the country. ", "Students & parents visiting the kiosks are guided to attend free career guidance session and aptitude test at the nearest Aptech centre and are being given the Microsoft DreamSpark software free of cost.", "\nWhile at the centre, students get the opportunity to have a face-to-face interaction with Aptech career counsellors on selecting the next generation of IT courses. ", "They also undergo a free aptitude test which helps them match their aptitude with the right career." ]

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[ "Authorities say an Alabama woman who asked deputies to test her methamphetamine for purity has been arrested on drug charges.", "\n\nThe News Courier reports 48-year-old Jennifer Colyne Hall was taken into custody Wednesday after calling law enforcement dispatch.", "\n\nLimestone County Sheriff's Office deputy and spokesman Stephen Young told the newspaper the woman pulled a bag containing methamphetamine from a baby wipes container when deputies arrived and said, “I want this dope tested.”", "\n\nAccording to authorities, she said she believed it had been tainted with another drug.", "\n\nYoung says the woman acknowledged taking the drug, but couldn’t remember when. ", "The newspaper reports Hall was charged with possession of a controlled substance and remained jailed early Thursday with bail set at $2,500.", "\n\nIt wasn’t immediately known if she had a lawyer." ]

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[ "Hollywood makes a lot of World War II movies — but the vast majority of them are told from the American perspective, in English. ", "If you want to learn more about WWII in a more comprehensive manner, sticking to Hollywood isn’t enough.", "\n\nThat’s why we’ve compiled this list of five best WWII movies from the German perspective, which will allow you to see the war from the other side. ", "We tried to find films that are fairly recent, actually made by Germans, and won’t bore you to death — check them out!", "\n\n• • •\n\n5. ", "Sophie Scholl – The Final Days (2002)\n\nIf you’re not into combat footage, then Sophie Scholl – The Final Days might be more your cup of tea. ", "This 2002 movie centers on Sophie Scholl, a 21 year-old student in Munich who was guillotined in 1943 for distributing anti-Hitler propaganda. ", "Today, Scholl is commemorated alongside other members of the White Rose student movement for standing up to Nazism at a time when few dared.", "\n\nFind Sophie Scholl – The Final Days on Amazon\n\n4. ", "13 Minutes (2015)\n\nMaybe you’ve heard of Valkyire, the 2008 movie starring Tom Cruise as Colonel Claus von Stauffenberg, a Wehrmacht officer who tried to assassinate Hitler. ", "As it turns out, Stauffenberg wasn’t the only aspiring Hitler assassin in Nazi Germany. ", "In fact, five years before Stauffenberg’s 1944 attempt, a German worker named George Elser planned and executed an elaborate (but unsuccessful) attempt to kill Hitler. ", "13 Minutes is a 2015 film based on Elser’s true story, its name inspired by the fact that Hitler missed Elser’s bomb by just 13 minutes.", "\n\nFind 13 Minutes on Amazon\n\n3. ", "Stalingrad (1993)\n\nYou could say that Nazi Germany’s defeat at Stalingrad in 1942 was the most important turning point of WWII… so it’s not surprising that quite a few films have been made about the battle. ", "One of these is 1993’s Stalingrad, which depicts the battle from a German perspective. ", "Stalingrad follows a platoon of Wehrmacht soldiers as they’re transferred from sunny North Africa to the Eastern Front, and shows the platoon’s descent into despair and death as the tide of war turns against them.", "\n\nFind Stalingrad on Amazon\n\n2. ", "Das Boot (1981)\n\nThis is the oldest film on our list, but it’s on here because it’s one of the most well-known WWII movies from the German perspective. ", "Das Boot — translated as “The Boat” in English — depicts the trials and tribulations of a German submarine crew as their vessel, U-96, cruises the Atlantic and Mediterranean. ", "Besides exhibiting the diverse personalities and backgrounds of U-96’s crew, the movie contains numerous tension-packed scenes of submarine warfare.", "\n\nFind Das Boot on Amazon\n\n1. ", "Downfall (2004)\n\nIf you’ve spent enough time on the internet, you’ve probably seen one of the many “Hitler reacts” clips where various parody subtitles accompany a scene of the Fuhrer getting mad. ", "As it turns out, the original footage for that clip comes from one of the most renowned WWII movies from the German perspective — Downfall.", "\n\nThrough the lens of his personal secretary, the film focuses on the last days of Hitler’s life during the Battle of Berlin. ", "Besides showing the desperate machinations of Nazi leadership, it includes gritty combat footage, stirring depictions of the battle’s toll on Berlin’s civilians, and, overall, a historically rigorous yet entertaining look at Nazi Germany’s final days.", "\n\nFind Downfall on Amazon\n\nBonus: Generation War (2013 TV miniseries)\n\nGeneration War is a TV miniseries rather than a movie, but we have to put it on here because it’s probably the most comprehensive cinematic depiction of WWII from the German perspective. ", "You can think of Generation War as Band of Brothers or The Pacific, but from a German point of view.", "\n\nSet between 1941-1945, the series traces the lives of five friends in their twenties from Berlin. ", "It intimately explores how the war shapes their lives across multiple dimensions. ", "Two of the friends are brothers who are sent off to the Eastern Front with the Wehrmacht, giving us a view into combat between Nazi Germany and the Soviet Union. ", "Another is a Jew who escapes into the forests, linking up with Polish partisans who fight Nazis. ", "The two female characters end up as a nurse and a mistress to a high-ranking SS officer; they become portals through which we see how the violence of total war touched women as much as men.", "\n\nThough the series is not without controversy, it was one of the most popular German shows in recent memory, and is well worth your time if you’re interested in WWII from the German perspective.", "\n\nFind Generation War on Amazon\n\n• • •\n\nDo you agree with our list? ", "Let us know in the comments below!", "\n\nFor more war movies, check out our list of WWII movies from the Japanese perspective, or list of the Best Korean War Movies." ]

{ "pile_set_name": "OpenWebText2" }

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[ "Vibratory stimulation for alleviation of chronic pain\n\nThe pain relieving effect of vibratory stimulation was studied in 731 patients suffering from acute pain (135 patients) or chronic pain (596 patients). ", "Most of the patients had previously undergone treatments of various kinds without sufficient pain relief. ", "The effect of vibratory stimulation was assessed before, during and after stimulation using different rating scales.", "\n\nThe majority of patients reported reduction of pain during vibratory stimulation. ", "In most patients suffering from musculoskeletal pain the best pain reducing effect was obtained when the vibratory stimulation was applied with moderate pressure at a frequency of 50-150 Hz. ", "To obtain a maximal duration of pain relief the stimulation had to be applied for 30-45 minutes.", "\n\nMany of the patients experienced pain relief lasting for more than 3 hours. ", "In many patients the pain relief lasted for 12 hours or more. ", "82% of the patients experienced a relief of pain; 47% of the patients experienced a reduction of pain." ]

{ "pile_set_name": "Pile-CC" }

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[ "Gigahertz analog modulation and differential delay of GaAIAs lasers: temperature and current behavior.", "\nGigahertz analog modulation characteristics of broad-area commercially available GaAlAs lasers have been investigated as a function of temperature and current in the vicinity of the upper frequency limit, where the resonance phenomena occur. ", "The optimum temperature for small-signal amplitude modulation was found to be around -15 degrees C for our particular broad-stripe geometry double-heterostructure laser. ", "The Q was found to increase by a factor of 2 and the bandwidth by about 2%; the external quantum efficiency was maximized in this range. ", "The optimum dc current bias was about 2% above the threshold current. ", "Differential delays have also been measured down to a few picosecond accuracy by a unique phase-angle measurement method using a vector voltmeter. ", "Some of the temperature effects observed may be related to mode changes and multimode and superradiance behavior." ]

{ "pile_set_name": "PubMed Abstracts" }

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[ "<?", "php\n\nnamespace Oro\\Bundle\\WorkflowBundle\\Tests\\Unit\\Model;\n\nuse Oro\\Bundle\\ActionBundle\\Button\\ButtonSearchContext;\nuse Oro\\Bundle\\ActionBundle\\Model\\Attribute;\nuse Oro\\Bundle\\ActionBundle\\Model\\AttributeGuesser;\nuse Oro\\Bundle\\WorkflowBundle\\Entity\\WorkflowDefinition;\nuse Oro\\Bundle\\WorkflowBundle\\Model\\AttributeAssembler;\nuse Oro\\Component\\Action\\Exception\\AssemblerException;\nuse Symfony\\Contracts\\Translation\\TranslatorInterface;\n\nclass AttributeAssemblerTest extends \\PHPUnit\\Framework\\TestCase\n{\n /** @var TranslatorInterface */\n protected $translator;\n\n protected function setUp(): void\n {\n $this->translator = $this->createMock(TranslatorInterface::class);\n $this->translator->expects($this->any())\n ->method('trans')\n ->willReturnCallback(\n static function (string $key) {\n return sprintf('[trans]%s[/trans]', $key);\n }\n );\n }\n\n /**\n * @dataProvider invalidOptionsDataProvider\n *\n * @param array $configuration\n * @param string $exception\n * @param string $message\n */\n public function testAssembleRequiredOptionException($configuration, $exception, $message)\n {\n $this->expectException($exception);\n $this->expectExceptionMessage($message);\n\n $assembler = new AttributeAssembler($this->getAttributeGuesser(), $this->translator);\n $definition = $this->getWorkflowDefinition();\n $assembler->assemble($definition, $configuration);\n }\n\n /**\n * @return WorkflowDefinition|\\PHPUnit\\Framework\\MockObject\\MockObject\n */\n protected function getWorkflowDefinition()\n {\n $definition = $this->createMock(WorkflowDefinition::class);\n $definition->expects($this->any())->method('getLabel')->willReturn('test_workflow_label');\n\n return $definition;\n }\n\n /**\n * @return AttributeGuesser|\\PHPUnit\\Framework\\MockObject\\MockObject\n */\n protected function getAttributeGuesser()\n {\n $guesser = $this->getMockBuilder('Oro\\Bundle\\ActionBundle\\Model\\AttributeGuesser')\n ->disableOriginalConstructor()\n ->getMock();\n return $guesser;\n }\n\n /**\n * @return array\n */\n public function invalidOptionsDataProvider()\n {\n return array(\n 'no_options' => array(\n array('name' => array('property_path' => null)),\n 'Oro\\Component\\Action\\Exception\\AssemblerException',\n 'Option \"label\" is required'\n ),\n 'no_type' => array(\n array('name' => array('label' => 'test', 'property_path' => null)),\n 'Oro\\Component\\Action\\Exception\\AssemblerException',\n 'Option \"type\" is required'\n ),\n 'no_label' => array(\n array('name' => array('type' => 'test', 'property_path' => null)),\n 'Oro\\Component\\Action\\Exception\\AssemblerException',\n 'Option \"label\" is required'\n ),\n 'invalid_type' => array(\n array('name' => array('label' => 'Label', 'type' => 'text', 'property_path' => null)),\n 'Oro\\Component\\Action\\Exception\\AssemblerException',\n 'Invalid attribute type \"text\", allowed types are \"bool\", \"boolean\", \"int\", \"integer\", ' .", "\n '\"float\", \"string\", \"array\", \"object\", \"entity\"'\n ),\n 'can_not_guess_type' => [\n ['test_name' => ['property_path' => 'test.property']],\n AssemblerException::class,\n 'Workflow \"[trans]test_workflow_label[/trans]\": Option \"type\" for attribute \"test_name\" ' .", "\n 'with property path \"test.property\" can not be guessed'\n ],\n 'invalid_type_class' => array(\n array(\n 'name' => array(\n 'label' => 'Label', 'type' => 'string', 'options' => array('class' => 'stdClass'),\n 'property_path' => null\n )\n ),\n 'Oro\\Component\\Action\\Exception\\AssemblerException',\n 'Option \"class\" cannot be used in attribute \"name\"'\n ),\n 'missing_object_class' => array(\n array('name' => array('label' => 'Label', 'type' => 'object', 'property_path' => null)),\n 'Oro\\Component\\Action\\Exception\\AssemblerException',\n 'Option \"class\" is required in attribute \"name\"'\n ),\n 'missing_entity_class' => array(\n array('name' => array('label' => 'Label', 'type' => 'entity', 'property_path' => null)),\n 'Oro\\Component\\Action\\Exception\\AssemblerException',\n 'Option \"class\" is required in attribute \"name\"'\n ),\n 'invalid_class' => array(\n array(\n 'name' => array(\n 'label' => 'Label', 'type' => 'object', 'options' => array('class' => 'InvalidClass'),\n 'property_path' => null\n )\n ),\n 'Oro\\Component\\Action\\Exception\\AssemblerException',\n 'Class \"InvalidClass\" referenced by \"class\" option in attribute \"name\" not found'\n ),\n 'not_allowed_entity_acl' => array(\n array(\n 'name' => array(\n 'label' => 'Label', 'type' => 'object', 'options' => array('class' => 'stdClass'),\n 'entity_acl' => array('update' => false),\n )\n ),\n 'Oro\\Component\\Action\\Exception\\AssemblerException',\n 'Attribute \"Label\" with type \"object\" can\\'t have entity ACL'\n ),\n );\n }\n\n /**\n * @dataProvider configurationDataProvider\n * @param array $configuration\n * @param Attribute $expectedAttribute\n * @param array $guessedParameters\n * @param array $transitionConfigurations\n */\n public function testAssemble(\n $configuration,\n $expectedAttribute,\n array $guessedParameters = [],\n array $transitionConfigurations = []\n ) {\n $relatedEntity = '\\stdClass';\n\n $attributeGuesser = $this->getAttributeGuesser();\n $attributeConfiguration = current($configuration);\n if ($guessedParameters && array_key_exists('property_path', $attributeConfiguration)) {\n $attributeGuesser->expects($this->any())\n ->method('guessParameters')\n ->with($relatedEntity, $attributeConfiguration['property_path'])\n ->will($this->returnValue($guessedParameters));\n }\n\n $assembler = new AttributeAssembler($attributeGuesser, $this->translator);\n $definition = $this->getWorkflowDefinition();\n $definition->expects($this->once())\n ->method('getEntityAttributeName')\n ->will($this->returnValue('entity_attribute'));\n $definition->expects($this->any())\n ->method('getRelatedEntity')\n ->will($this->returnValue($relatedEntity));\n\n $expectedAttributesCount = count($configuration) + count($transitionConfigurations);\n if (!", "array_key_exists('entity_attribute', $configuration)) {\n $expectedAttributesCount++;\n }\n\n $attributes = $assembler->assemble($definition, $configuration, $transitionConfigurations);\n $this->assertInstanceOf('Doctrine\\Common\\Collections\\ArrayCollection', $attributes);\n $this->assertCount($expectedAttributesCount, $attributes);\n $this->assertTrue($attributes->containsKey($expectedAttribute->getName()));\n\n $this->assertEquals($expectedAttribute, $attributes->get($expectedAttribute->getName()));\n }\n\n /**\n * @SuppressWarnings(PHPMD.ExcessiveMethodLength)\n * @return array\n */\n public function configurationDataProvider()\n {\n return array(\n 'string' => array(\n array(\n 'attribute_one' => array('label' => 'label', 'type' => 'string', 'property_path' => null)),\n $this->getAttribute('attribute_one', 'label', 'string')\n ),\n 'bool' => array(\n array('attribute_one' => array('label' => 'label', 'type' => 'bool', 'property_path' => null)),\n $this->getAttribute('attribute_one', 'label', 'bool')\n ),\n 'boolean' => array(\n array('attribute_one' => array('label' => 'label', 'type' => 'boolean', 'property_path' => null)),\n $this->getAttribute('attribute_one', 'label', 'boolean')\n ),\n 'int' => array(\n array('attribute_one' => array('label' => 'label', 'type' => 'int', 'property_path' => null)),\n $this->getAttribute('attribute_one', 'label', 'int')\n ),\n 'integer' => array(\n array('attribute_one' => array('label' => 'label', 'type' => 'integer', 'property_path' => null)),\n $this->getAttribute('attribute_one', 'label', 'integer')\n ),\n 'float' => array(\n array('attribute_one' => array('label' => 'label', 'type' => 'float', 'property_path' => null)),\n $this->getAttribute('attribute_one', 'label', 'float')\n ),\n 'array' => array(\n array('attribute_one' => array('label' => 'label', 'type' => 'array', 'property_path' => null)\n ),\n $this->getAttribute('attribute_one', 'label', 'array')\n ),\n 'object' => array(\n array(\n 'attribute_one' => array(\n 'label' => 'label',\n 'type' => 'object',\n 'options' => array('class' => 'stdClass'),\n 'property_path' => null\n )\n ),\n $this->getAttribute('attribute_one', 'label', 'object', array('class' => 'stdClass'))\n ),\n 'entity_minimal' => array(\n array(\n 'attribute_one' => array(\n 'label' => 'label',\n 'type' => 'entity',\n 'options' => array('class' => 'stdClass'),\n 'property_path' => null\n )\n ),\n $this->getAttribute(\n 'attribute_one',\n 'label',\n 'entity',\n array('class' => 'stdClass')\n )\n ),\n 'with_related_entity' => array(\n array(\n 'entity_attribute' => array(\n 'label' => 'label',\n 'type' => 'entity',\n 'options' => array('class' => 'stdClass'),\n 'property_path' => null\n )\n ),\n $this->getAttribute(\n 'entity_attribute',\n 'label',\n 'entity',\n array('class' => 'stdClass')\n )\n ),\n 'with_entity_acl' => array(\n array(\n 'attribute_one' => array(\n 'label' => 'label',\n 'type' => 'entity',\n 'options' => array('class' => 'stdClass'),\n 'property_path' => null,\n 'entity_acl' => array('update' => false),\n )\n ),\n $this->getAttribute(\n 'attribute_one',\n 'label',\n 'entity',\n array('class' => 'stdClass'),\n null,\n array('update' => false)\n )\n ),\n 'entity_minimal_guessed_parameters' => array(\n array(\n 'attribute_one' => array(\n 'property_path' => 'entity.field'\n )\n ),\n $this->getAttribute(\n 'attribute_one',\n 'label',\n 'entity',\n array('class' => 'stdClass'),\n 'entity.field'\n ),\n 'guessedParameters' => array(\n 'label' => 'label',\n 'type' => 'entity',\n 'options' => array('class' => 'stdClass'),\n ),\n ),\n 'entity_full_guessed_parameters' => array(\n array(\n 'attribute_one' => array(\n 'label' => 'label',\n 'type' => 'entity',\n 'options' => array('class' => 'stdClass'),\n 'property_path' => 'entity.field'\n )\n ),\n $this->getAttribute(\n 'attribute_one',\n 'label',\n 'entity',\n array('class' => 'stdClass'),\n 'entity.field'\n ),\n 'guessedParameters' => array(\n 'label' => 'guessed label',\n 'type' => 'object',\n 'options' => array('class' => 'GuessedClass'),\n ),\n ),\n 'add_init_context_attribute' => array(\n array(),\n $this->getAttribute(\n 'attribute_one',\n 'attribute_one',\n 'object',\n array('class' => ButtonSearchContext::class)\n ),\n array(),\n array(\n 'transition1' => array('init_context_attribute' => 'attribute_one'),\n 'transition2' => array('init_context_attribute' => 'source')\n )\n ),\n );\n }\n\n /**\n * @param string $name\n * @param string $label\n * @param string $type\n * @param array $options\n * @param string $propertyPath\n * @param array $entityAcl\n * @return Attribute\n */\n protected function getAttribute(\n $name,\n $label,\n $type,\n array $options = array(),\n $propertyPath = null,\n array $entityAcl = array()\n ) {\n $attribute = new Attribute();\n $attribute->setName($name);\n $attribute->setLabel($label);\n $attribute->setType($type);\n $attribute->setOptions($options);\n $attribute->setPropertyPath($propertyPath);\n $attribute->setEntityAcl($entityAcl);\n return $attribute;\n }\n}\n" ]

{ "pile_set_name": "Github" }

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[ "In the Asian\ncurrency markets, 2015 has been a year of surprising volatility.", "\n\nIn particular, the RMB devaluations in China and the political\nscandals in Malaysia have had an impact on currency values, and an\neven bigger impact on sentiment.", "\n\nFor many larger institutional businesses, this is no great\ninconvenience. ", "They have hedging strategies in place and may also have\na trading desk where they may be able to take advantage of the\nfluctuations.", "\n\nFor small and medium sized businesses, however, it is often not the\ncase, and their day to day activities and bottom line can receive a\nmauling if they are on the wrong side of the currency movements.", "\n\nFor most SMEs, foreign exchange means the spot market, and they are\nhappy to take whatever conversion rate applies at the time of the\ntransaction.", "\n\nWith the recent volatility, however, come indications that Asian SMEs\nare more inclined to engage with hedging products such as FX forwards\nand options.", "\n\nThe increased engagement signals a more sophisticated approach from\nregional SMEs, and an opportunity for FX providers used to the\none-dimensional approach of this market segment.", "\n\nIn the most recent iteration of the bi-annual report, 1861 SME\nbusinesses with an annual turnover of between US$1-20 million were\ninterviewed about their FX use.", "\n\nEvery one of the 1861 businesses were engaging with Spot FX, which was\na pre-condition to participating in the research.", "\n\nThe most recent report, from August 2015, shows significantly higher\nlevels of engagement with Options and Forwards products.", "\n\nWhere 25.6 percent of SMEs were using options in May, this had grown\nto 27.0 percent by August.", "\n\nEngagement rates for Forwards were higher, moving from 29.5 to 31.5\npercent over the same period.", "\n\nSingapore has consistently been the most sophisticated, in addition to\nthe most competitive, of the markets.", "\n\nIn Singapore,\n35.0 percent of SMEs use options, and the percentage using options\nmoved through the 40 percent barrier for the first time in August, and\nhit 41.7 percent.", "\n\nWhen East began the ABFX research in August 2013, only 30 percent of\nSingapore SMEs were using FX Forwards, so the product penetration in\nthat market has effectively moved from three in ten to four in ten in\ntwo years.", "\n\nPart of this can be attributed to the increased sophistication of the\nSMEs, but currency volatility must also have played a part.", "\n\nIn Malaysia, for example, where the ringgit has lost just over 15\npercent of its value so far this year, 22.9 percent of SME businesses\ncurrently use FX Forwards. ", "In August 2013, this figure was 18.0\npercent.", "\n\nMalaysian SMEs are also turning away from using their own currency to\nconduct international business.", "\nAs a percentage of FX volumes, Malaysian SMEs report that the ringgit\nhas fallen from comprising 13.9 percent of FX volumes in May to 13.0\npercent in August, with a forecast to fall to 12.4 percent in six\nmonths’ time.", "\n\nAll this is occurring in the context of a highly competitive market,\nwhere SMEs are choosing to spread their FX wallets among multiple\nproviders.", "\n\nFX continues to be the most consistently multi-banked financial\nproduct, with SMEs – who are usually averse to sharing their wallets,\nusing up to four or five providers for Spot FX.", "\n\nIn Singapore, which is the most fragmented and competitive of the four\nFX markets, the average wallet share for primary providers in the Spot\nmarket has fallen to a region-wide low of 18.6 percent. ", "In August\n2013, this was at 21.9 percent.", "\n\nFor this reason, the increased engagement with Forwards and Options is\na welcome opportunity for providers.", "\nWhile they might be facing declining wallet share, this can be\ncompensated in part if they can provider hedging products to their\nclients.", "\n\nVolatility might pose some difficulties for some, but for others it\ncan be an opportunity to increase the cross-sell." ]

{ "pile_set_name": "Pile-CC" }

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[ "gardening without weeds\n\nEvery spring I imagine my ideal garden: weed free, self-tilling, self-watering, disease-resistant, a garden safe from hungry, pesky bugs and critters. ", "Every spring for the past seven years I set out to my little strip of trucked-in topsoil behind the garage of our Lanesboro farm house, determined to coax the ideal garden out of the ground this time, at last! ", "And every midsummer about the time a good dry spell sets in, I am humbled by the many troubles that have cropped up in my perfect little garden. ", "The weeds have somehow managed to out-strip everything – many as tall as I am (granted, I’m short). ", "Still, it’s sobering to realize how entirely I’ve lost my focus on weeding and watering, how compacted the soil has become, how cabbage moths and tomato blight have taken a harsh toll." ]

{ "pile_set_name": "Pile-CC" }

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[ "using Consul;\nusing Microsoft.", "Extensions.", "Logging;\nusing Surging.", "Core.", "Consul.", "Configurations;\nusing Surging.", "Core.", "Consul.", "Internal;\nusing Surging.", "Core.", "Consul.", "Utilitys;\nusing Surging.", "Core.", "Consul.", "WatcherProvider;\nusing Surging.", "Core.", "Consul.", "WatcherProvider.", "Implementation;\nusing Surging.", "Core.", "CPlatform.", "Address;\nusing Surging.", "Core.", "CPlatform.", "Routing;\nusing Surging.", "Core.", "CPlatform.", "Routing.", "Implementation;\nusing Surging.", "Core.", "CPlatform.", "Runtime.", "Client;\nusing Surging.", "Core.", "CPlatform.", "Serialization;\nusing Surging.", "Core.", "CPlatform.", "Utilities;\nusing System;\nusing System.", "Collections.", "Generic;\nusing System.", "Linq;\nusing System.", "Text;\nusing System.", "Threading;\nusing System.", "Threading.", "Tasks;\n\nnamespace Surging.", "Core.", "Consul\n{\n /// <summary>\n /// consul服务路由管理器 \n /// </summary>\n public class ConsulServiceRouteManager : ServiceRouteManagerBase, IDisposable\n {\n private readonly ConfigInfo _configInfo;\n private readonly ISerializer<byte[]> _serializer;\n private readonly IServiceRouteFactory _serviceRouteFactory;\n private readonly ILogger<ConsulServiceRouteManager> _logger;\n private readonly ISerializer<string> _stringSerializer;\n private readonly IClientWatchManager _manager;\n private ServiceRoute[] _routes;\n private readonly IConsulClientProvider _consulClientProvider;\n private readonly IServiceHeartbeatManager _serviceHeartbeatManager;\n\n public ConsulServiceRouteManager(ConfigInfo configInfo, ISerializer<byte[]> serializer,\n ISerializer<string> stringSerializer, IClientWatchManager manager, IServiceRouteFactory serviceRouteFactory,\n ILogger<ConsulServiceRouteManager> logger,\n IServiceHeartbeatManager serviceHeartbeatManager, IConsulClientProvider consulClientProvider) : base(stringSerializer)\n {\n _configInfo = configInfo;\n _serializer = serializer;\n _stringSerializer = stringSerializer;\n _serviceRouteFactory = serviceRouteFactory;\n _logger = logger;\n _consulClientProvider = consulClientProvider;\n _manager = manager;\n _serviceHeartbeatManager = serviceHeartbeatManager;\n EnterRoutes().Wait();\n }\n\n /// <summary>\n /// 清空服务路由\n /// </summary>\n /// <returns></returns>\n public override async Task ClearAsync()\n {\n var clients = await _consulClientProvider.", "GetClients();\n foreach (var client in clients)\n {\n //根据前缀获取consul结果\n var queryResult = await client.", "KV.List(_configInfo.", "RoutePath);\n var response = queryResult.", "Response;\n if (response !", "= null)\n {\n //删除操作\n foreach (var result in response)\n {\n await client.", "KV.DeleteCAS(result);\n }\n }\n }\n }\n\n public void Dispose()\n {\n }\n\n /// <summary>\n /// 获取所有可用的服务路由信息。", "\n /// </summary>\n /// <returns>服务路由集合。</returns>\n public override async Task<IEnumerable<ServiceRoute>> GetRoutesAsync()\n {\n await EnterRoutes();\n return _routes;\n }\n\n public override async Task SetRoutesAsync(IEnumerable<ServiceRoute> routes)\n {\n var locks = await CreateLock();\n try\n {\n await _consulClientProvider.", "Check();\n var hostAddr = NetUtils.", "GetHostAddress();\n var serviceRoutes = await GetRoutes(routes.", "Select(p => $\"{ _configInfo.", "RoutePath}{p.", "ServiceDescriptor.", "Id}\"));\n foreach (var route in routes)\n {\n var serviceRoute = serviceRoutes.", "Where(p => p.ServiceDescriptor.", "Id == route.", "ServiceDescriptor.", "Id).FirstOrDefault();\n\n if (serviceRoute !", "= null)\n {\n var addresses = serviceRoute.", "Address.", "Concat(\n route.", "Address.", "Except(serviceRoute.", "Address)).ToList();\n\n foreach (var address in route.", "Address)\n {\n addresses.", "Remove(addresses.", "Where(p => p.ToString() == address.", "ToString()).FirstOrDefault());\n addresses.", "Add(address);\n }\n route.", "Address = addresses;\n }\n }\n await RemoveExceptRoutesAsync(routes, hostAddr);\n await base.", "SetRoutesAsync(routes);\n }\n finally\n {\n locks.", "ForEach(p => p.Release());\n }\n }\n\n public override async Task RemveAddressAsync(IEnumerable<AddressModel> Address)\n {\n var routes = await GetRoutesAsync();\n try\n {\n foreach (var route in routes)\n {\n route.", "Address = route.", "Address.", "Except(Address);\n }\n }\n catch (Exception ex)\n {\n throw ex;\n }\n await base.", "SetRoutesAsync(routes);\n }\n\n protected override async Task SetRoutesAsync(IEnumerable<ServiceRouteDescriptor> routes)\n {\n var clients = await _consulClientProvider.", "GetClients();\n foreach (var client in clients)\n {\n foreach (var serviceRoute in routes)\n {\n var nodeData = _serializer.", "Serialize(serviceRoute);\n var keyValuePair = new KVPair($\"{_configInfo.", "RoutePath}{serviceRoute.", "ServiceDescriptor.", "Id}\") { Value = nodeData };\n await client.", "KV.Put(keyValuePair);\n }\n }\n }\n\n #region 私有方法\n\n private async Task RemoveExceptRoutesAsync(IEnumerable<ServiceRoute> routes, AddressModel hostAddr)\n {\n routes = routes.", "ToArray();\n var clients = await _consulClientProvider.", "GetClients();\n foreach (var client in clients)\n {\n if (_routes !", "= null)\n {\n var oldRouteIds = _routes.", "Select(i => i.ServiceDescriptor.", "Id).ToArray();\n var newRouteIds = routes.", "Select(i => i.ServiceDescriptor.", "Id).ToArray();\n var deletedRouteIds = oldRouteIds.", "Except(newRouteIds).ToArray();\n foreach (var deletedRouteId in deletedRouteIds)\n {\n var addresses = _routes.", "Where(p => p.ServiceDescriptor.", "Id == deletedRouteId).Select(p => p.Address).FirstOrDefault();\n if (addresses.", "Contains(hostAddr))\n await client.", "KV.Delete($\"{_configInfo.", "RoutePath}{deletedRouteId}\");\n }\n }\n }\n }\n\n private async Task<List<IDistributedLock>> CreateLock()\n {\n var result = new List<IDistributedLock>();\n var clients = await _consulClientProvider.", "GetClients();\n foreach (var client in clients)\n {\n var key = $\"lock_{_configInfo.", "RoutePath}\";\n var writeResult = await client.", "KV.Get(key);\n if (writeResult.", "Response !", "= null)\n {\n var distributedLock = await client.", "AcquireLock(key);\n result.", "Add(distributedLock);\n }\n else\n {\n var distributedLock = await client.", "AcquireLock(new LockOptions($\"lock_{_configInfo.", "RoutePath}\")\n {\n SessionTTL = TimeSpan.", "FromSeconds(_configInfo.", "LockDelay),\n LockTryOnce = true,\n LockWaitTime = TimeSpan.", "FromSeconds(_configInfo.", "LockDelay)\n }, _configInfo.", "LockDelay == 0 ?", "\n default :\n new CancellationTokenSource(TimeSpan.", "FromSeconds(_configInfo.", "LockDelay)).Token);\n result.", "Add(distributedLock);\n }\n\n }\n return result;\n }\n private async Task<ServiceRoute> GetRoute(byte[] data)\n {\n if (_logger.", "IsEnabled(Microsoft.", "Extensions.", "Logging.", "LogLevel.", "Debug))\n _logger.", "LogDebug($\"准备转换服务路由，配置内容：{Encoding.", "UTF8.GetString(data)}。\");", "\n\n if (data == null)\n return null;\n\n var descriptor = _serializer.", "Deserialize<byte[], ServiceRouteDescriptor>(data);\n return (await _serviceRouteFactory.", "CreateServiceRoutesAsync(new[] { descriptor })).First();\n }\n\n private async Task<ServiceRoute[]> GetRouteDatas(string[] routes)\n {\n List<ServiceRoute> serviceRoutes = new List<ServiceRoute>();\n foreach (var route in routes)\n {\n var serviceRoute = await GetRouteData(route);\n serviceRoutes.", "Add(serviceRoute);\n }\n return serviceRoutes.", "ToArray();\n }\n\n private async Task<ServiceRoute> GetRouteData(string data)\n {\n if (data == null)\n return null;\n\n var descriptor = _stringSerializer.", "Deserialize(data, typeof(ServiceRouteDescriptor)) as ServiceRouteDescriptor;\n return (await _serviceRouteFactory.", "CreateServiceRoutesAsync(new[] { descriptor })).First();\n }\n\n private async Task<ServiceRoute[]> GetRoutes(IEnumerable<string> childrens)\n {\n\n childrens = childrens.", "ToArray();\n var routes = new List<ServiceRoute>(childrens.", "Count());\n\n foreach (var children in childrens)\n {\n if (_logger.", "IsEnabled(Microsoft.", "Extensions.", "Logging.", "LogLevel.", "Debug))\n _logger.", "LogDebug($\"准备从节点：{children}中获取路由信息。\");", "\n\n var route = await GetRoute(children);\n if (route !", "= null)\n routes.", "Add(route);\n }\n\n return routes.", "ToArray();\n }\n\n private async Task<ServiceRoute> GetRoute(string path)\n {\n ServiceRoute result = null;\n var client = await GetConsulClient();\n var watcher = new NodeMonitorWatcher(GetConsulClient, _manager, path,\n async (oldData, newData) => await NodeChange(oldData, newData), tmpPath =>\n {\n var index = tmpPath.", "LastIndexOf(\"/\");\n return _serviceHeartbeatManager.", "ExistsWhitelist(tmpPath.", "Substring(index + 1));\n });\n\n var queryResult = await client.", "KV.Keys(path);\n if (queryResult.", "Response !", "= null)\n {\n var data = (await client.", "GetDataAsync(path));\n if (data !", "= null)\n {\n watcher.", "SetCurrentData(data);\n result = await GetRoute(data);\n }\n }\n return result;\n }\n\n private async ValueTask<ConsulClient> GetConsulClient()\n {\n var client = await _consulClientProvider.", "GetClient();\n return client;\n }\n\n private async Task EnterRoutes()\n {\n if (_routes !", "= null && _routes.", "Length > 0)\n return;\n Action<string[]> action = null;\n var client = await GetConsulClient();\n //判断是否启用子监视器\n if (_configInfo.", "EnableChildrenMonitor)\n {\n //创建子监控类\n var watcher = new ChildrenMonitorWatcher(GetConsulClient, _manager, _configInfo.", "RoutePath,\n async (oldChildrens, newChildrens) => await ChildrenChange(oldChildrens, newChildrens),\n (result) => ConvertPaths(result).Result);\n //对委托绑定方法\n action = currentData => watcher.", "SetCurrentData(currentData);\n }\n if (client.", "KV.Keys(_configInfo.", "RoutePath).Result.", "Response?.Count() > 0)\n {\n var result = await client.", "GetChildrenAsync(_configInfo.", "RoutePath);\n var keys = await client.", "KV.Keys(_configInfo.", "RoutePath);\n var childrens = result;\n //传参数到方法中\n action?.Invoke(ConvertPaths(childrens).Result.", "Select(key => $\"{_configInfo.", "RoutePath}{key}\").ToArray());\n //重新赋值到routes中\n _routes = await GetRoutes(keys.", "Response);\n }\n else\n {\n if (_logger.", "IsEnabled(Microsoft.", "Extensions.", "Logging.", "LogLevel.", "Warning))\n _logger.", "LogWarning($\"无法获取路由信息，因为节点：{_configInfo.", "RoutePath}，不存在。\");", "\n _routes = new ServiceRoute[0];\n }\n }\n\n private static bool DataEquals(IReadOnlyList<byte> data1, IReadOnlyList<byte> data2)\n {\n if (data1.Count !", "= data2.Count)\n return false;\n for (var i = 0; i < data1.Count; i++)\n {\n var b1 = data1[i];\n var b2 = data2[i];\n if (b1 !", "= b2)\n return false;\n }\n return true;\n }\n\n /// <summary>\n /// 转化路径集合\n /// </summary>\n /// <param name=\"datas\">信息数据集合</param>\n /// <returns>返回路径集合</returns>\n private async Task<string[]> ConvertPaths(string[] datas)\n {\n List<string> paths = new List<string>();\n foreach (var data in datas)\n {\n var result = await GetRouteData(data);\n var serviceId = result?.ServiceDescriptor.", "Id;\n if (!", "string.", "IsNullOrEmpty(serviceId))\n paths.", "Add(serviceId);\n }\n return paths.", "ToArray();\n }\n\n private async Task NodeChange(byte[] oldData, byte[] newData)\n {\n if (DataEquals(oldData, newData))\n return;\n\n var newRoute = await GetRoute(newData);\n //得到旧的路由。", "\n var oldRoute = _routes.", "FirstOrDefault(i => i.ServiceDescriptor.", "Id == newRoute.", "ServiceDescriptor.", "Id);\n\n lock (_routes)\n {\n //删除旧路由，并添加上新的路由。", "\n _routes =\n _routes\n .Where(i => i.ServiceDescriptor.", "Id !", "= newRoute.", "ServiceDescriptor.", "Id)\n .Concat(new[] { newRoute }).ToArray();\n }\n\n //触发路由变更事件。", "\n OnChanged(new ServiceRouteChangedEventArgs(newRoute, oldRoute));\n }\n\n /// <summary>\n /// 数据更新\n /// </summary>\n /// <param name=\"oldChildrens\">旧的节点信息</param>\n /// <param name=\"newChildrens\">最新的节点信息</param>\n /// <returns></returns>\n private async Task ChildrenChange(string[] oldChildrens, string[] newChildrens)\n {\n if (_logger.", "IsEnabled(Microsoft.", "Extensions.", "Logging.", "LogLevel.", "Debug))\n _logger.", "LogDebug($\"最新的节点信息：{string.", "Join(\",\", newChildrens)}\");\n\n if (_logger.", "IsEnabled(Microsoft.", "Extensions.", "Logging.", "LogLevel.", "Debug))\n _logger.", "LogDebug($\"旧的节点信息：{string.", "Join(\",\", oldChildrens)}\");\n\n //计算出已被删除的节点。", "\n var deletedChildrens = oldChildrens.", "Except(newChildrens).ToArray();\n //计算出新增的节点。", "\n var createdChildrens = newChildrens.", "Except(oldChildrens).ToArray();\n\n if (_logger.", "IsEnabled(Microsoft.", "Extensions.", "Logging.", "LogLevel.", "Debug))\n _logger.", "LogDebug($\"需要被删除的路由节点：{string.", "Join(\",\", deletedChildrens)}\");\n if (_logger.", "IsEnabled(Microsoft.", "Extensions.", "Logging.", "LogLevel.", "Debug))\n _logger.", "LogDebug($\"需要被添加的路由节点：{string.", "Join(\",\", createdChildrens)}\");\n\n //获取新增的路由信息。", "\n var newRoutes = (await GetRoutes(createdChildrens)).ToArray();\n\n var routes = _routes.", "ToArray();\n lock (_routes)\n {\n #region 节点变更操作\n _routes = _routes\n //删除无效的节点路由。", "\n .Where(i => !", "deletedChildrens.", "Contains($\"{_configInfo.", "RoutePath}{i.", "ServiceDescriptor.", "Id}\"))\n //连接上新的路由。", "\n .Concat(newRoutes)\n .ToArray();\n #endregion\n }\n //需要删除的路由集合。", "\n var deletedRoutes = routes.", "Where(i => deletedChildrens.", "Contains($\"{_configInfo.", "RoutePath}{i.", "ServiceDescriptor.", "Id}\")).ToArray();\n //触发删除事件。", "\n OnRemoved(deletedRoutes.", "Select(route => new ServiceRouteEventArgs(route)).ToArray());\n\n //触发路由被创建事件。", "\n OnCreated(newRoutes.", "Select(route => new ServiceRouteEventArgs(route)).ToArray());\n\n if (_logger.", "IsEnabled(Microsoft.", "Extensions.", "Logging.", "LogLevel.", "Information))\n _logger.", "LogInformation(\"路由数据更新成功。\");", "\n }\n #endregion\n }\n}" ]

{ "pile_set_name": "Github" }

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[ "/*\n * This file is part of Cleanflight.", "\n *\n * Cleanflight is free software. ", "You can redistribute\n * this software and/or modify this software under the terms of the\n * GNU General Public License as published by the Free Software\n * Foundation, either version 3 of the License, or (at your option)\n * any later version.", "\n *\n * Cleanflight is distributed in the hope that it\n * will be useful, but WITHOUT ANY WARRANTY; without even the implied\n * warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.", "\n * See the GNU General Public License for more details.", "\n *\n * You should have received a copy of the GNU General Public License\n * along with this software.", "\n *\n * If not, see <http://www.gnu.org/licenses/>.", "\n */\n\n#include <stdbool.h>\n#include <stdint.h>\n\n#include \"platform.h\"\n\n#ifdef USE_RX_MSP\n\n#include \"common/utils.h\"\n\n#include \"drivers/io.h\"\n#include \"pg/rx.h\"\n#include \"rx/rx.h\"\n#include \"rx/msp.h\"\n\n\nstatic uint16_t mspFrame[MAX_SUPPORTED_RC_CHANNEL_COUNT];\nstatic bool rxMspFrameDone = false;\n\nstatic uint16_t rxMspReadRawRC(const rxRuntimeState_t *rxRuntimeState, uint8_t chan)\n{\n UNUSED(rxRuntimeState);\n return mspFrame[chan];\n}\n\n/*\n * Called from MSP command handler - mspFcProcessCommand\n */\nvoid rxMspFrameReceive(uint16_t *frame, int channelCount)\n{\n for (int i = 0; i < channelCount; i++) {\n mspFrame[i] = frame[i];\n }\n\n // Any channels not provided will be reset to zero\n for (int i = channelCount; i < MAX_SUPPORTED_RC_CHANNEL_COUNT; i++) {\n mspFrame[i] = 0;\n }\n\n rxMspFrameDone = true;\n}\n\nstatic uint8_t rxMspFrameStatus(rxRuntimeState_t *rxRuntimeState)\n{\n UNUSED(rxRuntimeState);\n\n if (!", "rxMspFrameDone) {\n return RX_FRAME_PENDING;\n }\n\n rxMspFrameDone = false;\n return RX_FRAME_COMPLETE;\n}\n\nvoid rxMspInit(const rxConfig_t *rxConfig, rxRuntimeState_t *rxRuntimeState)\n{\n UNUSED(rxConfig);\n\n rxRuntimeState->channelCount = MAX_SUPPORTED_RC_CHANNEL_COUNT;\n rxRuntimeState->rxRefreshRate = 20000;\n\n rxRuntimeState->rcReadRawFn = rxMspReadRawRC;\n rxRuntimeState->rcFrameStatusFn = rxMspFrameStatus;\n}\n#endif\n" ]

{ "pile_set_name": "Github" }

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[ "Diabetes mellitus increases the risk of bladder cancer: an updated meta-analysis.", "\nStudies have indicated that diabetes mellitus (DM) is a risk factor for bladder cancer; however, not all evidence supports this conclusion. ", "The aim of this meta-analysis was to collate and evaluate all primary observational studies investigating the risk of bladder cancer associated with DM. ", "The PubMed and Google Scholar databases were searched to identify studies that estimated the association of DM and bladder cancer. ", "Summary effect estimates were derived using a random-effects meta-analysis model. ", "A total of 23 studies (8 case-control studies, 15 cohort studies) including 643,683 DM and 4,819,656 non-DM cases were identified. ", "Analysis of all studies showed that DM was associated with an increased risk of bladder cancer compared with non-DM overall (OR=1.68, 95% CI 1.32-2.13). ", "Analysis of subgroups demonstrated this to be the case in both case-control studies (OR=1.59, 95% CI 1.28-1.97, I2=58%) and cohort studies (RR=1.70, 95% CI 1.23-2.33, I2=96%). ", "There was no gender difference in DM-associated bladder cancer risk. ", "Bladder cancer risk was increased in Asia and the North America region, but not in Europe. ", "Furthermore, DM-associated bladder cancer risk was obviously higher in Asia than North America and Europe or in those with Caucasian ethnicity. ", "With extension of follow-up time, the bladder cancer risk was not increased for the patients with DM. ", "This meta-analysis provided further evidence supporting the DM association with a significantly higher risk of bladder cancer obtained from observational studies." ]

{ "pile_set_name": "PubMed Abstracts" }

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[ "[Diagnostic strategies in cases of suspected periprosthetic infection of the knee. ", "A review of the literature and current recommendations].", "\nReliable confirmation of periprosthetic infection after total knee arthroplasty is a diagnostic challenge. ", "The present work reviews published data evaluating the available diagnostic tools. ", "Erythrocyte sedimentation rate and C-reactive protein serum levels are relatively sensitive methods with rather low specificity towards periprosthetic infection and are mainly applied to exclude infection. ", "Studies evaluating scintigraphic methods--especially white cell scans--provide inconsistent data with varying accuracy. ", "Consequently, white cell scans cannot be recommended as standard methods. ", "Immunoscintigraphy with antigranulocyte antibodies and FDG-PET scans demonstrated promising results with particularly high sensitivities, but have to be validated in larger studies. ", "Microbiological evaluation of joint aspirates proved high specificity for periprosthetic infection. ", "However, an average of 20% of infected cases remained undetected. ", "Nevertheless, aspiration is widely recommended for preoperative isolation of the infecting organism. ", "Intraoperative frozen sections demonstrated excellent specificity with good sensitivity. ", "The real accuracy of intraoperative culture and permanent histology cannot be determined due to the missing golden standard; however, a combination of both methods is recommended to define the final diagnosis. ", "Large studies validating both methods and criteria for the final diagnosis of periprosthetic infection are necessary to optimize the diagnostic algorithm." ]

{ "pile_set_name": "PubMed Abstracts" }

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[ "Calcium-lead interactions involving earthworms. ", "Part 2: the effect of accumulated lead on endogenous calcium in Lumbricus rubellus.", "\nThe calcium and lead burdens of tissue fractions of Lumbricus rubellus were quantified in 'native' animals from acidic and calcareous disused lead mines, and from control ('naive') animals exposed to lead-polluted soils under laboratory conditions. ", "Most of the body burden of lead was accumulated within the posterior alimentary canal, and significant positive correlations were generally found between the calcium and lead burdens in this tissue fraction, which were evident in both the naturally and laboratory lead-exposed animals. ", "The calcium:lead correlation is probably due to a proliferation of the calcium-rich, lead-sequestering chloragosome granules, and may thus be regarded as a specific tissue response to cellular lead incursion. ", "No calcium-lead relationship was recorded in the rest (largely composed of the body wall) fraction of earthworms inhabiting the lead-polluted sites. ", "However, a concomitant increase in calcium and lead in this tissue fraction of the laboratory lead-exposed control animals was noted. ", "It is concluded that in naturally lead-exposed earthworms, the cells of this tissue fraction may be relatively resistant to the toxic effects of the metal. ", "By contrast, it is apparent that a non-specific cytotoxic response by the cells of the rest of 'naive' animals occurs, as demonstrated by the concurrent increase in its calcium and lead burdens. ", "These results suggest that a tolerance mechanism to lead, perhaps with a genetic basis, may exist in earthworms naturally exposed to lead." ]

{ "pile_set_name": "PubMed Abstracts" }

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[ "A novel and cost effective method of removing excess albumin from plasma/serum samples and its impacts on LC-MS/MS bioanalysis of therapeutic proteins.", "\nWe have developed an innovative method to remove albumin from plasma/serum samples for the LC-MS/MS quantitation of therapeutic proteins. ", "Different combinations of organic solvents and acids were screened for their ability to remove albumin from plasma and serum samples. ", "Removal efficiency was monitored by two signature peptides (QTALVELVK and LVNEVTEFAK) from albumin. ", "Isopropanol with 1.0% trichloroacetic acid was found to be the most effective combination to remove albumin while retaining the protein of interest. ", "Our approach was compared with a commercial albumin depletion kit on both efficiency of albumin removal and recovery of target proteins. ", "We have demonstrated that our approach can remove 95% of the total albumin in human plasma samples while retaining close to 100% for two of three therapeutic proteins tested, with the third one at 60-80%. ", "The commercial kit removed 98% of albumin but suffered at least 50% recovery loss for all therapeutic proteins when compared to our approach. ", "Using BMS-C as a probe compound, the incorporation of the albumin removal approach has improved both assay sensitivity and ruggedness, compared to the whole plasma protein digestion approach alone. ", "An LC-MS/MS method was developed and validated based on this new approach for the analysis of BMS-C in monkey serum. ", "This assay was successfully applied to a toxicological study. ", "When the albumin removal method was used in another clinical LC-MS/MS method, the sensitivity improved 10-fold to 50 ng/mL LLOQ comparing to a typical pellet digestion method." ]

{ "pile_set_name": "PubMed Abstracts" }

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