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Padmanabhan Ananth
Lovelocks Bridge - Poem by Padmanabhan Ananth
Walking from The Louvre to Musée d' Orsay
In Paris, winter afternoon of a relaxed Sunday
When I decided to spend the day on arts
Fancied as I took a journey of 'love locks'!
Over River Seine, that majestically flowed
Pont de l'Archeveche* so serenely stood
Built a quarter after start of 19th century
Proudly linking the left bank to Île de la Cité
Narrow bridge welcomed loving pedestrians
From the city to where all art remains
Under cloudy sky, the painted fences glowed
On the fences were love locks a thousand
In the shadow of this old French span
Gene Kelly danced with Leslie Caron
In 'An American in Paris', the 50s musical
Memorable love waltz of the epic couple
Fascinated I was by sheer number-n-variety
Padlocks many from every known country
Brass, steel, branded, polished, painted
Each had names of a boy-n-girl printed
To these love padlocks, no long history
Just a romantic expression of lovers, truly
They'd meet on bridge, on lock engrave names
They'd hug, kiss and in river below drop keys
They would also take back home a key
As a souvenir over bridge of their intimacy
In the City of Love, great way to express passion
Naïve padlock truly turns article of adulation!
- - -
*Archbishop Bridge
Poet's Notes about The Poem
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WikiLeaks has released what it says is an annotated transcript of a documentary that takes a critical look at the anti-secrecy group ahead of the US film's premiere.
WikiLeaks said it had not participated in the making of We Steal Secrets: The Story of WikiLeaks, a film by Alex Gibney that focuses on the website's founder Julian Assange and its main source of leaks, Bradley Manning.
Manning, a US Army intelligence analyst who admitted to leaking hundreds of thousands of secret military logs and confidential embassy cables to WikiLeaks, faces possible life imprisonment in a military trial to resume on June 3.
Assange, the Australian hacker who founded the site, has been holed up in Ecuador's London embassy for nearly a year, seeking to avoid extradition to Sweden, where he is wanted for questioning in a sexual assault case.
WikiLeaks said late on Thursday that the film "portrays Manning's alleged acts as a failure of character rather than a triumph of conscience", and said the film's portrayal of his relationship with Assange was "grossly irresponsible".
The portrayal "suggests - erroneously and when evidence is to the contrary - that Assange may be guilty of conspiring with Bradley Manning to commit espionage or similar offences."
The WikiLeaks statement added "neither Julian Assange nor anyone associated with WikiLeaks over the past two-and-a-half years agreed to participate in the film".
It then posted what appeared to be a full transcript of the documentary with copious notes alleging factual errors and misrepresentations.
Gibney could not immediately be reached for comment.
But in a recent interview with CBS news, the director said the film, which premieres on Friday, is concerned with "both the abuse of power and hubris on the part of the US government, but certainly also on the part of Julian Assange".
"At his moment of greatest fame, (Assange) ends up becoming all too much like the enemies he sought to take out or expose," Gibney said.
Gibney told CBS he engaged in "endless negotiations" with Assange, but his requests to interview the WikiLeaks founder were ultimately denied.
He was also unable to interview Manning, who has been in military detention since his arrest in 2010.

subset: commoncrawl


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Cameron Tool and Supply Company, Cameron, WV in 1915.
Master Blacksmith, Verne Monroe (far right), and his crew. The 8" diameter round stock is being heated in the forge prior to forge welding. Source of photo is unknown and was sent to our chapter.
Cameron Tool is now CamForge, a maker of large crane hooks.
The ABA toured the plant in 2004 -- see the slideshow.


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by Staff Writers
Valverde, Spain (AFP) Sept 28, 2011
Police evacuated residents and tourists from houses at the foot of a volcano on Spain's Canary Islands after a growing series of earthquakes raised fears of an eruption, officials and locals said Wednesday.
With the Pico de Malpaso mountain spitting rocks intermittently and a growing rumbling underground, authorities made emergency preparations in case the volcano blows its top on the Atlantic island of El Hierro.
"I have never felt shaking like it," said Herminio Barrera, 25, a mechanic in the town of La Frontera. "I notice it especially at night. We can also hear a rumbling and sounds from deep down."
A municipal official who asked not to be named told AFP that 53 residents and tourists had been evacuated to protect them from the occasional flying volcanic rock and the defence ministry said it was preparing emergency shelter for 2,000 people.
"I am staying calm but there are people who are more worried, particularly those with children," Barrera told AFP. "We are very close to the mountain. My father-in-law left yesterday."
The Canary Islands regional government said it was in a state of pre-alert and was stocking drinking water and medical supplies, but officials played down any risk of a disaster.
"We will not have to evacuate the island," population 11,000, said the head of the island's local council, Alpidio Armas. "The number of tremors has increased, but most of them are in the sea."
The municipal official said the authorities did not expect an "imminent eruption" and the island's official risk alert for the 1,500-metre (4,9200-foot) peak remained on yellow, or intermediate.
The national defence ministry said it had sent 31 military personnel to the island to help with the evacuation and Defence Minister Carme Chacon was heading there to inspect the emergency preparations.
The Spanish National Geographic Institute has recorded 8,000 tremors since July 19, most of them too small to be felt, but they have been growing in intensity. One recorded overnight on Wednesday reached 3.4 magnitude.
"We have not seen this kind of movement with such frequency on El Hierro since records began" more than a century ago, said a spokeswoman for the Canary Islands government, Ana Vidal.
The last volcanic eruption on the Canary Islands was on nearby La Palma in 1971, she said.
The evacuation disrupted the holidays of a number of tourists, including Tuengen Maier, 62, from Germany, who said he and his wife would spend the rest of their holiday on nearby Tenerife.
"We were having some wine yesterday evening when the Civil Guard told us to leave the house because we were too close to the mountain," he told AFP by telephone on Wednesday.
"We are just going to pick up our luggage this morning. This is too dangerous. We cannot stay."
Related Links
Bringing Order To A World Of Disasters
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Scientists find eruption at undersea volcano after forecasting the event
Newport OR (SPX) Aug 11, 2011
A team of scientists just discovered a new eruption of Axial Seamount, an undersea volcano located about 250 miles off the Oregon coast - and one of the most active and intensely studied seamounts in the world. What makes the event so intriguing is that the scientists had forecast the eruption starting five years ago - the first successful forecast of an undersea volcano. Bill Chadwi ... read more
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text: 075 NOUS44 KSHV 231343 PNSSHV ARZ050-051-059>061-070>073-LAZ001>006-010>014-017>022-OKZ077- TXZ096-097-108>112-124>126-136>138-149>153-165>167-240145- Public Information Statement National Weather Service Shreveport La 843 AM CDT Fri Jun 23 2017 ...Public Information Statement... A damage survey team from the National Weather Service in Shreveport will be investigating storm-related damage near Campti and Powhatan in Natchitoches Parish this afternoon, from severe thunderstorms which moved through this area earlier this morning. Results from this survey will be made available later this evening. $$ 29


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Note to flyers: If you've got a beef with flight attendants, try not to cuss them out, because losing your temper could lead to your detention and arrest.
Man Arrested At Airport After Swearing At Flight Crew
Carl's Jr. Guy Tells Me He Doesn't Give A F*ck
Frequent commenter Jook checks in with his tale of confronting an alarmingly indifferent and potty-mouthed Carl's Jr. drive-thru dude who took his time getting some fries through the window, then explained he didn't "give a f*ck" how long he took.
The Pizza Delivery Guy Cussed Me Out
James says a Papa John's delivery dude called him a "f*gg*t m*th*rf*ck*r" as part of a blowup in a dispute about whether or not the driver rang the doorbell during an unsuccessful delivery attempt.
Texting While Driving So Distracting That Meredith Vieira Yells "Oh Shit" On TV
Texting while driving is pretty damn distracting. How distracting? Well, it can make a seasoned professional TV anchor yell "Oh shit!" on the air.
Mother Thinks Hannah Montana Doll Is Swearing
We're going to go ahead and suggest that if you are the type to hear swear words when confronted with awful audio recordings, you not buy dolls that sing. One Florida mom is such a person, and is convinced that her daughter's Hannah Montana doll has taught her kid to say the "F" word.
Shopper Arrested For Saying "The F Word" At Walmart
Well, here's a truly weird story, disorderly conduct charges have been dropped against a Texas woman who dropped an f-bomb at her local Walmart.
Parents Think Bratz Baby Doll Says "Fuck You"
Is there no end to the foul things parents hear their children's toys say? Already this season Ariel the Mermaid has been accused of calling people "sluts." Now the Bratz baby is saying fuck you to people. Could it be that the doll is singing "So Cute?" That's what the lyrics posted on the Bratz website say, but that didn't stop parents from alerting the media. In the news reports they actually beeped out "So Cute" when they played the song, as if that was going to help parents make an informed decision about whether or not "so cute" sounds too much like "fuck you" for their tastes.


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The PRAME gene family belongs to the group of cancer/testis genes whose expression is restricted primarily to the testis and a variety of cancers. The expansion of this gene family as a result of gene duplication has been observed in primates and rodents. We analyzed the PRAME gene family in Eutheria and discovered a novel Y-linked PRAME gene family in bovine, PRAMEY, which underwent amplification after a lineage-specific, autosome-to-Y transposition. Phylogenetic analyses revealed two major evolutionary clades. Clade I containing the amplified PRAMEYs and the unamplified autosomal homologs in cattle and other eutherians is under stronger functional constraints; whereas, Clade II containing the amplified autosomal PRAMEs is under positive selection. Deep-sequencing analysis indicated that eight of the identified 16 PRAMEY loci are active transcriptionally. Compared to the bovine autosomal PRAME that is expressed predominantly in testis, the PRAMEY gene family is expressed exclusively in testis and is up-regulated during testicular maturation. Furthermore, the sense RNA of PRAMEY is expressed specifically whereas the antisense RNA is expressed predominantly in spermatids. This study revealed that the expansion of the PRAME family occurred in both autosomes and sex chromosomes in a lineage-dependent manner. Differential selection forces have shaped the evolution and function of the PRAME family. The positive selection observed on the autosomal PRAMEs (Clade II) may result in their functional diversification in immunity and reproduction. Conversely, selective constraints have operated on the expanded PRAMEYs to preserve their essential function in spermatogenesis.
Citation: Chang T-C, Yang Y, Yasue H, Bharti AK, Retzel EF, Liu W-S (2011) The Expansion of the PRAME Gene Family in Eutheria. PLoS ONE 6(2): e16867. https://doi.org/10.1371/journal.pone.0016867
Editor: David Liberles, University of Wyoming, United States of America
Received: September 21, 2010; Accepted: January 16, 2011; Published: February 10, 2011
Copyright: © 2011 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from USDA-CSREES (No. 2005-35205-18653 and No. 2010-65205-20362) and start-up funds from the Pennsylvania State University to Liu, W-S. http://www.csrees.usda.gov/fo/funding.cfm, http://www.psu.edu/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cancer/testis (CT) genes comprise a group of genes involved primarily in immunity and reproduction. They are expressed in various types of cancers when abnormally activated, whereas, the normal expression of CT genes is restricted mainly to the testis, but it has been detected also in other tissues such as fetal ovary [1]–[3]. CT genes have more than 240 members from 70 families. Twenty-four of these families are located on the human X-chromosome (CT-X) and two families, the TSPY (testis-specific protein Y-linked) gene family and the Y-linked TPTE (transmembrane phosphatase with tensin homology) pseudogene family, are on the Y-chromosome (Y-chr) [4]. Interestingly, many amplified CT gene families are located within direct or inverted repeats on the sex chromosomes (chrs) [1], [4]. The autosomal CT genes were conserved during evolution and play roles in spermatogenesis, fertilization, and apoptosis in malignant cells [5]–[7]. However, knowledge about the CT genes on the sex chrs is still limited. A comparative study suggested that the CT-X genes were subject to positive selection and evolved faster than the autosomal CT genes [4]. The Y-linked TSPY gene family is conserved among most mammalian species, and has 30–60 copies on the human Y-chr [8] and 50–200 copies on the bovine Y-chr (BTAY) [8], [9]. This family has a typical CT tissue-restricted expression pattern with functions in immunity and spermatogenesis [10]. In this study, we identified a novel Y-linked CT gene family, preferentially expressed antigen in melanoma, Y-linked (PRAMEY), and examined its evolution in Eutheria.
PRAME, as one of the CT genes, first identified as an antigen-encoding gene related to immunity in a melanoma cell line [2], is expressed predominantly in normal testis and melanoma, lung squamous cell carcinoma, and acute leukemia, and at much lower levels in the ovary and other tissues [2], [11]. The human PRAME gene, located on chromosome 22 (HSA22), encodes a protein with seven leucine-rich (LXXLL) motifs through which PRAME interferes with the retinoic acid receptor (RAR) pathway, and leads to the inhibition of RA-induced differentiation, growth arrest, and apoptosis [12]. Thus, PRAME functions as a transcriptional repressor in the signaling cascade, and the over-expression of PRAME results in tumorigenesis [12]. Similar to the other multi-copy CT genes, PRAME went through expansion and constituted a large gene family in most mammalian species [13], [14]. A previous phylogenetic analysis of the primate PRAME family has revealed that the expansion of the human paralogs is hominin-specific and occurred within the past three million years [13]. Several potential surface-accessible sites of the human PRAME protein have been identified under positive selection during evolution [13]. Even though the evolutionary pattern and oncogenic roles of the PRAME family have been studied in the human and rodent [2], [11]–[13], [15]–[17], the phylogeny of the PRAME orthologs in other mammalian species and the function of PRAME in normal tissues, such as testis, remain unclear.
To delineate the macro-evolution of PRAME, we analyzed the PRAME gene family in Eutheria. We discovered a bovine Y-linked PRAME family, namely PRAMEY, which was derived from an autosome-to-Y transposition and underwent amplification after the transposition. A phylogenetic analysis of PRAME/PRAMEY orthologs in Eutheria identified two major clades, which were subject to diverse selection pressures. The origination of the PRAMEY family and its unique expression patterns in spermatids suggest that it plays an important role in spermatogenesis.
Discovery of the PRAMEY Family
Two PRAMEY transcripts (PRAMEY1 and PRAMEY2) were identified through a large-scale direct testis cDNA selection using a micro-dissected, PCR amplified BTAY probe. PRAMEY1 is 99% identical to a predicted mRNA (GenBank acc. no. XM_001253165.1) located in a non-annotated bovine bacterial artificial chromosome (BAC) (GenBank acc. no. AC234911.1). This clone was validated as a Y-linked BAC by a male-specific PCR (Fig. 1). PRAMEY2 is 99% identical to an mRNA (GenBank acc. no. NM_001077979) located in a bovine Y-BAC (GenBank acc. no. AC234853.4). Full-length mRNAs of both transcripts were obtained by RACE (rapid amplification of cDNA ends) (Fig. 2). The mRNA of PRAMEY1 (GenBank acc. no. GU144301) is 2747 bp, with an open reading frame (ORF) from nucleotide (nt) 895 to 2436, and it encodes a peptide of 513 amino acids (aa). The mRNA of PRAMEY2 (GenBank acc. no. GU144302) is shorter (1888 bp), with an ORF from nt 104 to 1639, encoding a peptide of 511 aa (Fig. 2). The similarity between the coding regions of PRAMEY1 and PRAMEY2 is 88% at the nucleotide level and 90% at the protein level.
A. RT-PCR results (lanes 2-10). PRAMEY is expressed specifically in the testis, whereas the autosomal PRAME is expressed in the testis (predominantly), kidney, brain and the muscle tissues. Bovine male genomic DNA-specific PCR (lanes 11–12) confirmed that PRAMEY is Y-specific. Te, testis; Li, liver; Ki, kidney; Sp, spleen; Br, brain (cerebrum); Ad, adrenal gland; Mu, muscle; Ly, lymph node; Ov, ovary; ♂, bovine male genomic DNA control; ♀, bovine female genomic DNA control; -, negative control (water); M, 1 kb DNA ladder. B. The expression of the PRAMEY loci by deep-sequencing analysis. The alignment of reads derived from deep-sequencing of selected cDNAs against coding regions of the PRAMEY loci (Table S2) reveals that seven of the 10 active PRAMEY genes are expressed differentially, six of which have significant numbers of both read-pairs matching exactly to the specific loci.
Schematic representations of PRAMEY1 and PRAMEY2. Compared to the PRAMEY2 (GenBank acc. no. GU144302) that contains five exons, the first exon of the PRAMEY1 (GenBank acc. no. GU144301) reads through to the second exon and forms a larger exon. The introns are drawn to scale. The open boxes represent UTR regions and the filled black boxes are coding segments (CDS). The numbers denote the length of exons, introns and CDS in bp. The polyA [(A)n] sites are indicated.
To address the question whether more loci of PRAMEY are present on BTAY, we searched PRAMEY1/2 against the bovine Y-BACs (available in NCBI) and identified a total of 10 potentially active PRAMEY paralogs (named PRAMEY1-10, Table S1) and 6 pseudogenes. The active- and pseudo-genes were mapped to a total of 11 Y-BACs, each containing one or two copies (Table S1). The pairwise similarity of the 10 active PRAMEY loci was >86%, with a 100% similarity between PRAMEY2 and PRAMEY3 in AC234853.4 (Table S2). PRAMEY1 contains 4 exons whereas PRAMEY2 contains 5 exons because the first exon of PRAMEY1 reads through the second exon, resulting in a single, larger exon (Fig. 2). The first two introns in the coding regions are conserved across all the PRAMEY loci, with a slight difference in length (1289–1371 bp and 274–284 bp) (Fig. 2). A major difference is present in the last intron (Fig. 2): the size is 758 bp in PRAMEY2/3/8, compared to 1161–1212 bp in the remaining PRAMEYs. This difference is the result of an indel of 403–454 bp that is specific to BTAY.
The putative PRAMEY protein isoforms share an identity of ≥82%. Seven important leucine-rich motifs have been identified in the human PRAME protein [12]. The alignment of the bovine PRAMEY/PRAME with the human PRAME on HSA22 revealed that these motifs are highly conserved (Fig. S1).
In addition, we found a predicted gene (GenBank acc. no. XR_082974.1) located on BTA17. This gene shares ∼87% similarity with the identified Y-linked PRAMEYs (Table S2). Gene-specific PCR and sequencing (Table S3) confirmed the predicted PRAME on BTA17. This autosomal gene encodes a putative peptide of 410 aa and is located at 74.35 Mb close to two zinc-finger genes, ZNF280A (also known as SUHW1) and ZNF280B (SUHW2).
Expression analysis of the bovine PRAMEY
Expression of the putative PRAMEY loci was investigated by deep-sequencing of the selected testis cDNAs using the Illumina GAIIx (see methods) and aligning the short sequence reads (pair-ends, 2×36 bp) against unique coding regions of the PRAMEY genes (Table S2). Seven of the 10 PRAMEY loci are active at the transcription level (PRAMEY2/3/6-10), and six of the seven loci have exactly matched read-pairs (Fig. 1B); in contrast, PRAMEY1/4/5 have no matched reads. Further, PRAMEY2/3/6 have more uniquely matched reads (>20), suggesting a higher expression level at these loci. Taken together with the RACE result, at least eight of the 10 loci on BTAY have been confirmed to be active at the transcription level.
RT-PCR analysis (Table S3) across nine tissues revealed that PRAMEY2 was expressed specifically in the testis. In contrast, the autosomal PRAME gene on BTA17 was expressed highly in the testis, and low in the kidney, brain and muscle (Fig. 1A). In situ hybridization (ISH) of PRAMEY2 cRNA probes (Table S4) revealed that both sense and antisense transcripts of PRAMEY2 were expressed in adult testis (Fig. 3). The sense RNA of PRAMEY2 was expressed specifically in spermatids (Fig. 3A), whereas the antisense RNA was expressed in all cell types in the seminiferous tubules, with the highest expression occurring in spermatids (Fig. 3B). Quantitative (q) RT-PCR analysis of PRAMEY2 indicated that the expression of the sense RNA was low in 5-11-day and 3-month-old testes, but up-regulated in 8-month- and 24-month-old testes (Fig. 3E); the expression of antisense PRAMEY2 RNA increased slightly with age.
A. The sense RNA of PRAMEY2 is expressed specifically in spermatids. B. The antisense RNA of PRAMEY2 is expressed broadly across seminiferous tubules with a predominant expression in spermatids. Sense and antisense RNAs of PRAMEY2 were detected by DIG-labeled cRNA probes. C. The bovine PRM1gene was used as positive control, and there is no antisense mRNA of PRM1 detected in the bovine testis [38]. D. Haematoxylin and Eosin (H&E) staining is shown. Scale: bar = 200 µm. E. Temporal expression pattern of PRAMEY2. The relative expression levels of the PRAMEY2 sense and antisense transcripts at different ages (X-axis), measured by the strand-specific qPCR, were normalized by the 18S rRNA (Y-axis). The PRAMEY2 sense RNA is expressed very low in earlier stage, but up-regulated in the 8 months and 2 years-old testis. Similarly, antisense RNA of PRAMEY2 is detected in the 8 months and 2 years-old testis. Values are means ± SD of the three biological replicates.
Phylogenetic tree of the PRAME/PRAMEY family
To investigate the evolution of PRAME/PRAMEY, the sequences of multiple PRAME loci in the human, chimpanzee, orangutan, mouse, rat and cattle were retrieved from NCBI (Table S2) [18]. A single autosomal ortholog was found in dog and horse. Multiple PRAME loci were detected on the pig chr 6 (SSC6), similar to the expansions observed in primates (HSA1, PTR1 and MMUL1) and rodents (MMU4 and RNO5/14) [13]. Since SSC6 has not been well-annotated, the corresponding matched regions were collected and aligned with the HSA22 ortholog by Splign [19] to confirm gene structures and splicing signals/sites, which gave rise to 10 swine orthologs containing long ORFs (ranging from 470 to 528 aa) with corresponding splicing sites (Table S2). In addition to the autosomal copies, we found X-linked PRAME (PRAMEX) in rodents and horses. However, we did not identify any ortholog of PRAME in the non-eutherian lineages examined, including opossum, platypus, chicken, frog and zebrafish, all of which have a genome sequence coverage of ≥6X, implying that the PRAME gene family is present in eutherian mammals only.
The coding regions of the retrieved PRAME sequences were used to establish phylogenetic trees using Maximum-likelihood (ML), Bayesian-inference (BI) and Neighbor-joining (NJ) methods. All the tree topologies were consistent and contained two major clades (Fig. 4). The first clade (Clade I) included the syntenic orthologs of the BTA17 PRAME on human (HSA22), macaque (MMUL10), chimpanzee (PRT22), dog (CFA26), horse (ECA8) and pig (SSC14). Interestingly, all the active bovine PRAMEY loci and PRAME on BTA17 were clustered on the same branch with a strong bootstrap support value (100%) (Fig. 4). This clade also included the orthologs on the horse and mouse X-chrs (ECAX and MMUX), which have a closer evolutionary distance to Clade I (0.713) than Clade II (0.814) (Maximum-Composite-Likelihood method) [20]. In Clade I, only the PRAMEY gene contains multiple copies, whereas the other homologs are all single-copy genes. Since no Y-linked ortholog was identified among the available Y-chrs of the other eutherian mammals, we propose that the bovine PRAMEY was derived by a lineage-specific, autosome-to-Y transposition event.
Two major PRAME/PRAMEY clades are shown in this tree. The PRAME locus on HSA22 and its syntenic orthologs in other species are clustered with the bovine PRAME and PRAMEY loci in Clade I (branches in red). The orthologs on the X-chrs of horse and mouse are also clustered with Clade I. The PRAME orthologs syntenic to HSA1 are clustered in Clade II (branches in light blue), which contains three sub-clusters, IIa (Artiodactyla), IIb (Primates) and IIc (Rodentia). The tree was built based on the ML method and bootstrap values (1000 replicates) are shown above the branches. The branches corresponding to partitions reproduced in less than 80% bootstrap replicates are collapsed.
Clade II included the remaining orthologs with three internal clusters (Fig. 4). The first cluster (IIa) comprised the orthologs in Artiodactyla, including those on BTA16 and SSC6. The second cluster (IIb) included all the orthologs on chr 1 in primates, where the human orthologs were intermingled with chimpanzee and orangutan orthologs as demonstrated previously [13]. The autosomal orthologs in Rodentia constituted the third cluster (IIc) and the mouse and rat orthologs were intermingled within the cluster. The X-linked orthologs in rats were also nested within this cluster. The orthologs in Clade II were all located in a chromosomal region syntenic to HSA1 except for the rat X-orthologs. The PRAME gene tree was reconciled with a species tree to reveal potential duplication and speciation events (Fig. 5) [21], [22].
Two branches in Clade I and 14 branches in Clade II are under positive selection (red) based on the branch-site model tests (Model A versus Model A null). The branches under positive selection are numbered and the selected sites along each foreground lineage are detailed in Table S5. The nodes underwent duplication are marked with a yellow circle and speciation with a blue circle.
Selection forces acting on the PRAME genes
The lineage-specific selection test using the PAML (Phylogenetic Analysis by Maximum Likelihood) package revealed that the dN/dS ratios varied significantly among different lineages (p<0.001, fixed ratio/free ratio branch model) [23]. We applied the branch-site models (model A null/model A) to examine whether any lineage is under positive selection [24]. In Clade I, we observed two branches, leading to the primate homologs and the bovine PRAME on BTA17, which were subject to positive selection (Fig. 5). Three positively selected sites were found along these two branches (probability >0.8, Table S5) [24]. We also tested different pairs of site-specific models (see methods) in a dataset containing only the homologs in Clade I (Table S2 and S6) [23], and the results were all negative (p>0.1). It is noteworthy that the homologs in Clade I had a significant lower median dN/dS ratio when compared to the three clusters in Clade II (p<0.001, Fig. 6A). Taken together, these data suggest that Clade I was under stronger functional constraints.
A. The dN/dS ratio distributions in different clades. Clade I has the lowest mean and median dN/dS ratios. The vertical axis represents the dN/dS ratio. The asterisk (*) represents the outliers of the data. B. Map of the positively selected sites detected in Clade IIa to the PRAME protein model. The selected sites derived from PAML analyses are mapped to the protein homology model. Eight of the 12 selected sites (red) are clustered in the inner concave region of the protein model. The model was built based on the PRAME gene (GenBank acc. no. XM_001256020.1) on BTA16. The predicted DNA binding site is highlighted in orange. The LXXLL motifs are highlighted in pink.
In Clade II, we detected a total of 17 sites from 14 different branches under positive selection (Fig. 5). Four sites and three branches were observed in Artiodactyla (Clade IIa), five sites/five branches in Primate (Clade IIb), and nine sites/six branches in Rodentia (Clade IIc) (Fig. 5). Our findings support a previous report that the primate and rodent PRAME homologs were subject to positive selection [13]. In this study, we further examined the potential selected sites in the homologs in Artiodactyla (Clade IIa) using the site-specific models (see methods) and detected eight more positively selected sites (model M8, probability >0.8, Table S6 and S7). Therefore, 12 sites in total were found under positive selection in Clade IIa. We built a PRAME protein homology model using the PRAME gene on BTA16 (GenBank acc. no. XM_001256020) as the template, and mapped the positively selected sites on the model (Fig. 5). In contrast to the primate and rodent PRAME, in which the positively selected sites were clustered in the outer surface of the protein [13], the majority (8/12) of the positively selected sites in the bovine PRAME were located in the inner concave region (Fig. 6B). Furthermore, a DNA binding site was predicted in this protein model. This could be important as one of the positively selected sites (329M) and two of the leucine-rich motifs were located in this region. In addition, we also investigated whether or not the bovine paralogs, including the pseudogenes, were subject to gene conversion during evolution using the GENECONV program [25]. The results did not indicate any gene conversion events.
Lineage-specific amplification of PRAMEs
PRAME is one of the most amplified gene families in mammals and is considered the third largest gene family in the mouse genome [26]. In the present study, we found that the PRAME gene family is present only in Eutheria, indicating that this family may have originated de novo in the eutherian lineages [27]. The birth-and-death model of gene duplication, instead of concerted evolution, has been suggested to be the major evolutionary mechanism accounting for the expansion of autosomal PRAME and the resemblance between each copy [13]. Our analysis revealed that: 1) during eutherian evolution, the expansion of PRAME genes was not limited to autosomes, but also occurred in sex chrs; 2) the expansion of PRAMEs is lineage-dependent. This conclusion was based upon the finding that the PRAME gene was transposed to and amplified on BTAY, but not on the other mammalian Y-chrs; 3) the intra- (cis-) and inter- (trans-) chromosomal duplications occurred during the expansion of the PRAME gene family. The cis-duplications occurred mainly for the syntenic PRAMEs in Clade II and the bovine PRAMEYs in Clade I (Fig.4, Table S2). The rat X-orthologs may be derived from the trans-duplication of the autosomal paralogs on RNO14, but the origin of the mouse X-ortholog is unclear (Fig. 4). It is noteworthy that the PRAME genes appear to (cis-) duplicate largely only on those chromosomal regions syntenic to HSA1 in Clade II. In contrast, the orthologs clustered with the PRAME gene on HSA22 tend to be maintained as single-copy genes in the respective genome, except for the bovine PRAMEY family, which could be a consequence of abundant reorganization and duplication events that occurred during the evolution of the Y-chr [28]. We observed five BACs, each containing two PRAMEY loci (Table S1), suggesting that the expansion of PRAMEY occurred in tandem on BTAY and gene duplication was the predominant process during the expansion. However, we cannot exclude the possibility that concerted evolution may also have contributed to the similarity between the PRAMEY genes because of potential Y-Y gene conversions [29], [30]. The mechanism behind the frequent cis-duplications and limited trans-duplications of the PRAME gene family in Eutheria may be related to genomic contexts on each chromosome, including local gene density, repeat density, GC content and recombination rate [31].
Selective pressures on PRAME(Y)
Positive selection tends to increase the frequency of advantageous mutations; negative selection eliminates the deleterious mutations resulting in less genetic variation. A previous study found a large number of positively selected sites in both human and mouse PRAME orthologs on HSA1 and MMU4 [13]. In the present study, we found several branches leading to the orthologs in primates, rodents and artiodactyls in Clade II under positive selection (Fig. 5), which supports the previous report [13]. The selection test for the homologs on BTA16 and SSC6 detected 12 sites that were subject to positive selection (Fig. 6B, Table S5 and S7). Unlike the primate [13], the positively selected sites in Artiodactyla were clustered in the inner concave region, suggesting that the functional accommodations of PRAMEs are lineage-dependent. The protein structure of the bovine PRAME model (Fig. 6B) is close to the ribonuclease inhibitor (PDB: 1DFJ), which interacts with its substrate through a similar concave region [32]. Thus, the modifications of PRAME in Artiodactyla appear to occur along the regions essential for protein interaction during evolution. Further, the difference in the median dN/dS ratios between Clade I and Clade II (Fig. 6A) suggested differential selection pressures acting on the PRAME gene family.
Origin of PRAMEY in cattle
Our recent study [29] in cattle has shown that a gene block containing ZNF280BY and ZNF280AY was transposed from BTA17 and duplicated on the Y-chr after the transposition. In the present study, we found a PRAME on BTA17, which is linked to ZNF280B/ZNF280A within a 60 kb region (74.30–74.36 Mb). Meanwhile, the same gene order (ZNF280BY -ZNF280AY-PRAMEY) was observed in two non-overlapping Y-BACs (GenBank acc. no. AC234853.4 and AC233215.5), leading us to hypothesize that the PRAMEYs were derived from the transposition of the block on BTA17. Unlike the human DAZ and feline TETY1 and FLJ36031 genes, in which the translocation was involved in a single autosomal gene, the bovine ZNF280B-ZNF280A-PRAME was transposed to the Y-chr as a block. However, the established phylogenetic tree of PRAME/PRAMEY in this study was not clear because the BTA17 locus was nested within the PRAMEY cluster (Fig. 3), raising an alternative but unlikely hypothesis that the PRAME on BTA17 was derived from the loci on BTAY. If we assume a "Y-to-autosome" transposition occurred during evolution, we would expect this gene block to be conserved on the Y-chr of most, if not all, eutherians, but not conserved on autosomes. However, this block is highly conserved on autosomes (Fig. S2) instead of the Y, which apparently conflicts with the alternative hypothesis. Thus, we proposed that the PRAMEY genes in cattle were derived from the transposition of the ZNF280B/ZNF280A/PRAME on BTA17 and duplicated separately thereafter.
Furthermore, based on the tree topology (Fig. 3), it appears that PRAMEYs were clustered into two subgroups and could be derived from two transposition events. However, several observations led us to postulate that PRAMEYs were derived from a single transposition of the BTA17 gene block. First, all PRAMEYs are highly similar (>86%) and amplify tandemly in a narrow genomic region just like the PRAME expansion within 740 kb on HSA1 [13]. Several Y-BACs contain two copies of PRAMEY, such as PRAMEY2 and PRAMEY3, which are identical and located in a BAC with a distance of 22 kb. More importantly, PRAMEY6 and PRAMEY7, falling into different subgroups, are also located in one Y-BAC with a distance of 97 kb (Table S1). The narrow distance and high similarity of each copy indicated that the gene duplication is the major evolutionary mechanism of PRAMEYs after transposition. Two separate transpositions occurring within a narrow genomic region are implausible. Thus, we propose that the distinct clusters of PRAMEYs are the synergic consequence of a higher mutation rate on the non-recombining Y-chr [33] and Y-Y gene conversions [29], [30]. The diversity of the duplicated PRAMEY sequences reflects a response of Y-chr to diverse selection pressures.
Potential roles of PRAME/PRAMEY
Several lines of evidence have indicated a close relationship between PRAME and tumorigenesis [12], [13], [15], [16], [34]. PRAME acts as a ligand-dependent co-repressor in the important retinoic acids receptor (RAR) pathway [12], [34]. When PRAME is absent, the activation of the RAR pathway by retinoids will lead to proliferation arrest, cell differentiation and apoptosis [12]. Conversely, the RAR pathway is inhibited when PRAME is abnormally present, resulting in incessant cell proliferation and tumorigenesis.
In addition to tumor development, PRAME is implicated in germ cell development. In the mouse, an autosomal Prame-like gene, Oogenesin, is expressed in oocytes and early cleavage-stage embryos with a role in oogenesis [13], [35], suggesting that the duplicated PRAME genes on autosomes are related to rapid cell mitosis. The mouse X-linked Prame-like 3 (Pramel3) is expressed specifically in spermatogonia and may function in early stage of spermatogenesis [36]. Since maintaining and amplifying male fertility factors on the Y-chr may provide selection advantages during evolution [37], the origin and retention of these Y-linked copies are expected to be crucial for spermatogenesis. The exclusive expression of PRAMEY (Fig. 1 and 3) in spermatids provides a strong support for this hypothesis. We validated that at least eight of the 10 predicted PRAMEY loci are active at the transcription level, and differentially expressed in the testis (Fig. 1B). Future research is needed to investigate the biological meanings behind this differential expression. It is worth noting that the predominant expression of the bovine PRAMEY2 antisense transcript in spermatid may be essential biologically (Fig. 3E). Our previous works demonstrated that the antisense RNAs of three other Y-related and testis-expressed genes (ZNF280BY, DDX3Y and CDYL) in cattle all appear to be expressed in late stage spermatocytes and/or spermatids, indicating that antisense RNA is crucial in the regulation of bovine spermiogenesis [29], [38], [39].
Recent and extensive duplications of PRAME and other CT genes in human are consistently involved in adaptive functions including reproduction and immunity [13], [40]. PRAME and neighboring ZNF280BY/ZNF280AY on HSA22 are reportedly associated with immune responsiveness [41], [42]. Thus, the PRAME/PRAMEY gene family may also participate in auto-immunity to sperm, which is prevented by the blood-testis barrier in normal males [43]. Anti-sperm immunity is considered as one of the causes of infertility in humans [44] and it is thus important to clarify the immunological roles of PRAME in male-related functions.
In conclusion, we have identified a lineage-specific PRAMEY gene family in bovine, which was derived from the transposition of a gene block, ZNF280B-ZNF280A-PRAME, on BTA17, and duplicated afterwards. The expansion of PRAME genes occurred not only in Primates and Rodentia, but also in Artiodactyla. The phylogenetic analysis revealed two distinct clades of PRAME that evolved under different selection forces. The largely amplified autosomal PRAMEs are under positive selection, whereas the PRAMEYs are under stronger functional constraints. The PRAMEY gene family is expected to be important in spermatogenesis. We anticipate that future research on the roles of PRAME and PRAMEY in the crosstalk between the spermatogenesis and immunoresponse will facilitate understanding of both spermatogenesis and tumor developments.
Materials and Methods
RNA extraction and cDNA synthesis
Total RNA was extracted from bovine testicular tissue at 4 days, 20 days, 3–4 months, 8 months, and 2 years of age with Trizol® reagent (Invitrogen, Carlsbad, CA, USA). Equal amounts of total RNA from different ages of testes were pooled and treated with DNase I twice (before and after mRNA purification) (Ambion, Austin, Texas, USA). Messenger RNAs were purified from the pooled total RNA (Oligotex; Qiagen, Valencia, CA, USA). First strand cDNAs were synthesized with random hexamers and oligo-T primers using SuperscriptIII reverse transcriptase (Invitrogen, Carlsbad, CA, USA); blunt-ended double-stranded cDNAs were generated as described [45]. Adaptors [phosphorylated oligonucleotides 1 (5′-CTGAGCGGAATTCGTGAGACC-3′) and 2 (5′-CCAGAGTGCTTAAGGCGAGTCAA-3′)] were attached to cDNAs using T4 polynucleotide kinase (NEB, Ipswich, MA, USA)[46]. Adaptor-ligated cDNA products were used for direct testis cDNA selection.
Direct testis cDNA selection and sequencing
The entire BTAY DNA was isolated by a micro-dissection approach [47]. The DNA fragments were PCR amplified and labeled with biotin-16-dUTP (Roche, Indianapolis, IN, USA) by nick translation (Roche, Indianapolis, IN, USA). Direct testis cDNA selection was detailed in Yang et al. (2011) [29] and Del Mastro and Lovett (1997) [46]. The selected cDNAs were PCR-amplified using the adaptor oligo 1 as the primer. Selection efficiency was assessed by qPCR with Y-linked genes, SRY and DDX3Y, as positive controls and β-Actin and CDYL as negative controls. PCR products were cloned using a TOPO-TA cloning kit (Invitrogen, Carlsbad, CA, USA). Randomly selected clones (n = 2208) were grown overnight at 37°C in 2 ml, 96-deep-well culture plates. All clones were dot-blotted on nylon transfer membranes and hybridized with 32P-dCTP labeled BTAY fragments and PCR fragments of four genes (HSFY, UBE2D3, RPL23A, and ZNF280B) that were highly redundant in our test sequencing result. After dot-blot and elimination of the most likely repetitive clones, 753 clones were selected for sequencing. Plasmid DNA was purified by alkaline lysis (Qiagen, Valencia, CA, USA), and sequenced on an ABI-3730XL DNA analyzer at the Pennsylvania State University Genomics Core Facility.
Total RNAs were extracted from nine different tissues (testis, liver, kidney, spleen, cerebellum, adrenal gland, longissimus muscle, lymph node, and spinal cord) of a 2-years old bull and ovarian tissue from a mature cow, then treated with DNase I (Ambion, Austin, TX, USA) and reverse transcribed using Superscript™ III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed in 20 µl containing 10 ng cDNA, 200 µM dNTPs, 1.5 mM MgCl2, 2.5 µM of each primer, 1 unit Taq DNA polymerase (Bioline, Taunton, MA, USA). The PCR conditions were: 94°C for 7 min followed by 35 cycles each of 95°C for 40 sec, 55°C–65°C for 40 sec, 72°C for 40 sec, with a final extension at 72°C for 7 min. Products were resolved on 1.5% agarose gels with ethidium bromide in 1× TAE buffer.
Total RNAs from bovine testis (5–11 days, 3 months, 8 months and 2 years of age) were used for 5′ and 3′ rapid amplification of cDNA ends (RACE). The RACE experiment was conducted essentially as described in Yang et al. [29].
Short-read sequencing for locus-specific expression
The selected cDNAs were sequenced at the National Center for Genome Resources using an Illumina GAIIx. Library construction and sequence methods were described previously [29]. A total of 6,710,574 high-quality paired end reads of 2×36 bp were generated. These reads were aligned to nine unique PRAMEY sequences identified through BlastClust [48] with 100% similarity and 100% coverage as the criteria. For aligning the short-reads, the software GSNAP [49] was used as part of the Alpheus pipeline [50]. Two mismatches were allowed during the alignment step and only the reads that hit the reference uniquely were considered for counting towards locus-specific expression. Since the reads were paired end, only the reads where both ends hit the same reference were considered. These counts were further sub-grouped under two categories: (A) both reads are unique hits with at least one of them being exact match and (B) both reads are unique hits & both are exact matches. The read counts in these two categories were considered a measure of expression pertaining to the specific locus.
Testis tissue section in situ hybridization (ISH)
The bovine testis was fixed [51], embedded in paraffin and sectioned (4 µm). Sense and antisense RNA probes of PRAMEY were selected (Table S4) using G-PROBE (Genetyx Co., Tokyo, Japan) and the 120-bp probes were subjected to in vitro transcription to produce digoxigenin (DIG)-labeled cRNA with the AmpliScribe T7-Flash Transcription Kit (Epicentre, Madison, WI, USA). Uniform labeling of DIG-labeling was confirmed using the NBT/BCIP detection system (Roche Diagnostics, Indianapolis, IN, USA). ISH was performed as described previously [38], [39]. Serial tissue sections were used for sense and antisense probe hybridizations. The spermatid-specific gene Protamine 1 (PRM1) served as the positive control, while LNE120 staining was used as the negative control.
Strand-specific qPCR
First strand sense and antisense cDNAs were developed with strand-specific reverse transcript primers (Table S3) (SuperScript™ III First-Strand Synthesis System, Invitrogen, Carlsbad, CA, USA) from 5–11 day, 3 month, 8 month and >24-month bovine testis total RNA and used as templates for qPCR with gene specific primer sets (Table S3). All qPCRs were performed in the Power SYBR Green PCR Master Mix (Applied Biosystems, CA, USA) and Applied Biosystems 7500 real-time PCR system following the manufacturer's instructions. Amplification conditions were 2 min at 50°C; 10 min at 95°C; followed by 40 cycles of 20 sec at 95°C, 20 sec at 57°C and 30 sec at 72°C. Cycle threshold acquisition used default parameters with CT values for PRAMEY sense/antisense RNAs normalized to 18S rRNA in each sample. RNA samples without a reverse transcript served as the negative control. Each qPCR was conducted in duplicate on three independent RNA samples (biological replicates). Significance was evaluated by one-way ANOVA using SAS (SAS Institute Inc., NC, USA).
Sequence alignment, gene prediction and phylogenetic tree construction
For the identification of bovine PRAME paralogs, we used the two identified transcripts (GenBank acc. no. GU144301 and GU144302) to blast against ∼600 bovine Y-BACs that are available in GenBank to retrieve all potential paralogous regions on BTAY. The redundant regions were removed by detecting the overlaps between Y-BACs using purpose-designed scripts. The paralogs with inferred splicing sites/signals and comparable coding regions were considered as active PRAMEY genes; in contrast, the others were pseudogenes due to either frameshift mutations or premature stop codons.
Using the human PRAME sequences on HSA22 (GenBank acc. no. NM_206956.1) and HSA1 (GenBank acc. no. NM_023013.1) to blast [48] against the nucleotide databases in NCBI [18], we were able to retrieve the annotated PRAME homologs in humans, chimpanzees, orangutans, horses, cats and cattle (e-value <1E-50 and coverage >40%, Table S1 and S2). For the swine orthologs, the blast search was against the swine HTGS database as the swine genome sequence was not well annotated. The retrieved porcine BAC sequences were annotated for PRAME in this study using Splign [19] and the getorf program in EMBOSS [52]. The redundant porcine paralogs were removed. The identified homologs were used to construct the phylogenetic trees using the ML, BI and NJ methods (substitution model: TrNef + I + G) implemented in the TOPALi program [53]. The alignment gaps were trimmed using Gblocks [54], [55]. The branches with a bootstrap value <80% were collapsed (Fig. 4). We further investigated the duplication and speciation events by reconciling the PRAME gene tree with a species tree obtained from NCBI taxonomy database [56] using Notung 2.6 [22] (Fig. 5). No PRAME ortholog was identified from the lineages beyond Eutheria, including opossum (6.8x genome coverage), platypus (6x), chicken (6.6x), frog (7.5x) and zebrafish (6.5x).
Lineage- and site-specific selection test
We conducted a pairwise dN and dS analysis (Dnasp version 5.0) [57] for the orthologs located on the same chromosome across species studied. The sequences with a pairwise dS value of <0.02 were removed, and the resulted 78 sequences were used for lineage-specific positive selection test [23] (Table S1). The median dN/dS ratio was calculated for different clades and compared by the Mann-Whitney test [58]. The 78 sequences were aligned by ClustalW [59] and the gaps were trimmed by Gblocks. The aligned segments included 912 positions of the original 2677 positions. We used the codeml program implemented in PAML package for the selection test. A simple model assuming a single dN/dS ratio for branches was compared with another model assuming free dN/dS ratio for all the branches (branch models). The likelihood ratio test (LRT) indicates that the dN/dS ratios are significantly varied among lineages (p<0.001, ). We conducted LRT for each branch using the branch-site models, model A null and model A [24]. The sites under positive selection detected by Bayes Empirical Bayes (BEB) analyses were retrieved when the LRTs were significant.
For the site-specific positive selection test [23], [60], we focused on investigating the Clade I and Clade IIa, which were newly identified in this study. We established two datasets, one with the 12 sequences in Clade I and the other with the 12 sequences in Clade IIa (Table S1). The Clade I dataset included 1290 aligned positions of the original 1677 positions; The Clade IIa dataset included 1065 bases of the original 1902 positions. PAML [23] and HyPhy [60] packages were used to detect the selection. We compared four different model pairs, M0 (one-ratio)/M3 (discrete), M1a (nearly neutral)/M2a (positive selection), M7 (beta)/M8 (beta and ω>1), and M8a (beta and ωs = 1)/M8 in PAML. Three methods, SLAC (Single Likelihood Ancestor Counting), FEL (Fixed Effects Likelihood) and REL (Random Effects Likelihood), implemented in HyPhy (Hypothesis Testing Using Phylogenies) package [60] were also used to detect the positive selection sites (Table S7). The protein model of the PRAME gene on BTA16 (GenBank acc. no. XM_001256020) was built by I-TASSER [61].
Supporting Information
Figure S1.
Motif alignment between the bovine PRAMEY and the human PRAME on HSA22. The aliphatic sites of LXXLL motifs observed on the human PRAME on HSA22 [16], [12] are conserved in the bovine PRAME(Y). These motif modifications are restricted to the aliphatic group, including the leucine to valine in the third and seventh motifs and leucine to isoleucine in the fourth motif. An exception is that the first leucine in the fifth motif was modified to the non-aliphatic phenylalanine. The colors in the alignment indicated different types of amino acids (White: Aliphatic sites; Red: Acidic sites; Cyan: Basic sites; Purple: Aromatic sites; Yellow: Cystenine). * The aliphatic site positions were annotated based on the PRAME on HSA22.
Figure S2.
Alignment of the ZNF280B/ZNF280A/PRAME gene block across 17 species. The ZNF280B/ZNF280A/PRAME gene blocks are conserved in the syntenic regions in most mammals except the rodents where the block was rearranged in two different chromosomes (MMU4/10 and RNO5/20). This plot was generated based on the HSA22 assembly (hg19, Feb. 2009). The boxes represent ungapped alignments; the lines represent gaps. This plot was generated using blastz alignment from the UCSC genome browser (http://genome.ucsc.edu/).
Table S1.
A list of BACs containing homologous PRAMEY.
Table S2.
PRAME/PRAMEY homologs in the phylogenetic tree.
Table S3.
Primers for (RT-) PCR and strand-specific qRT-PCR.
Table S4.
Probes for in situ hybridization.
Table S5.
Positively selected sites detected from branch-site model tests.
Table S6.
Site-specific selection tests on the homologs in Clade I and Clade IIa.
Table S7.
The integrative analysis of positively selected sites in Clade IIa.
We thank Dr. Abel Ponce de León at the University of Minnesota for providing the micro-dissected bovine Y-chr DNA fragments, and Dr. Jon Oatley, Ms. Melissa Oatley and Dr. Daniel Kniffen at the Pennsylvania State University for providing the bovine testis tissues and partial RNA samples of the bovine testes for this study. We thank the Human Genome Sequencing Center (HGSC) at Baylor College of Medicine for sequencing the BTAY BACs. We are grateful to Dr. Daniel Hagen, Dr. Craig Beattie, Dr. Kateryna Makova, Dr. Cooduvalli Shashikant, and Dr. Melissa Wilson for their comments on the manuscript. We also want to thank the three anonymous reviewers for their critical comments and suggestions.
Author Contributions
Conceived and designed the experiments: TCC YY WSL. Performed the experiments: TCC YY HY EFR. Analyzed the data: TCC YY WSL. Contributed reagents/materials/analysis tools: TCC YY HY AKB EFR WSL. Wrote the paper: TCC YY WSL.
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During the 1890s, the successful British pharmacist Henry Wellcome established a pair of labs in London to conduct physiological and chemical research. Then he gave his team of researchers a mandate that most scientists today can only dream of: Follow your noses, he told them. And they did. In the face of skepticism from academics, the Wellcome labs began pumping out peer-reviewed scholarship that made an important contribution to science while putting Wellcome - a company with a promising future - on the map.
When Willard Boyle, Canada's newest Nobel laureate, spoke disapprovingly last week about how scientists have to ante up a "business plan" in order to get funding for curiosity-driven research, his words resonated with researchers across the country. In accepting the prestigious prize - for discovering an electronic device that would lay the groundwork for the development of digital imaging - Prof. Boyle said governments are increasingly compelling scientists to focus on applied research that pays off quickly, in the form of a patent, a marketable product or a spin-off business. Cast in the language of investment portfolios, public research funders are leaning too heavily toward short-term returns at the expense of long-term growth.
Neuroscientist Bruce McNaughton has a more trenchant analogy that most politicians will recognize: "Curiosity-driven research is a bit like passenger railroads," says the renowned University of Lethbridge researcher, who recently returned to Canada after a long stint in the United States. "If you let the knowledge base, skills and infrastructure deteriorate, they are not going to be there when you need them, like we desperately do today in Canada."
It's not a new accusation. Twenty-three years ago, University of Toronto chemist John Polanyi lobbed out a similar criticism when he went to Stockholm to accept his own Nobel Prize. At the time, Brian Mulroney's Tory government was slashing funding for research while touting a "matching" grant program intended to accelerate the commercialization of university research.
Musing that ambitious young researchers would be well advised to go abroad, Prof. Polanyi warned that scientists become risk-averse if there are too many strings attached. "If they were asked three years later, 'Did you actually make some progress toward the sort of technology which you said might flow from your work?' and you would admit you didn't - because you usually don't - the sponsor of the research … simply takes the research money to someone else and asks the same impossible requirement."
Nobel winners, of course, receive a big soapbox along with their prize money and the medal. But are these lions of science simply expressing the self-importance of academe? Or have they put their fingers on a chronic source of short-sightedness embedded in Canada's overall philosophy about research and development?
About 15 years after the Wellcome labs were established, a pair of scientists working there - George Barger and James Ewens - synthesized a chemical called dopamine, a compound that one of their colleagues, Henry Dale, found to behave like the hormone adrenaline. At the time, it was little more than an interesting observation. And so the story about the recognition of dopamine's potential - which would eventually lead to dramatic advances in the treatment of neurological disorders like Parkinson's disease - ground to a halt.
On a per-capita basis, Canada doesn't do a lot of research compared with innovation-driven nations such as Finland. But since Prof. Polanyi won his Nobel in 1986, successive governments have rolled out a series of lucrative research programs dominated by conditions and specific policy goals: reversing the brain drain, improving living standards, accelerating technology transfer.
These days, the Harper government pumps hundreds of millions into energy-industry technologies such as carbon capture and storage. Yet the Tories last winter picked a fight with the scientific community by tabling a stimulus budget that proposed cutting $150-million in funding to Canada's three national research funding agencies.Report Typo/Error


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Lacrosse news straight to your inbox.
Tuesday July 24th, 2012 7:39pm
Watched a ton of lacrosse today at Richard Stockton College, covering four grass fields and one turf field at the ComLax.com Powerball Lacrosse Tournament near Atlantic City, N.J.
The top four teams emerged in the Money Division and will advance to compete Sunday for the $25,000 grand prize. Reebok Blacks Out Cancer took the Red Division No. 1 seed and will play Black No. 2 HEADstrong at 9:30 Sunday, while Motive Pure won the Black Division and will take on Red No. 2 Flow Society at 11 a.m.
Before we get to the playoffs, Liquid Flow put together a solid squad, but just missed out making the final four. Cortland alum Brian Krol put up five goals and an assist in the first half of an 11-3 win over Bass Massters, and the LF squad dropped a key game to Flow Society 6-2.
Gotta say this facility is excellent, with a host of hotels, shops and restaurants nearby right out the entrance, and the glitz of AC about 15 minutes away. Seems like most of the people I talked to are staying all over the place: the beach, the AC strip and some other hotels in between.
Also caught some of the Fruit Loops game in the High Rollers (Open) Division. Host of MIAA players, including Deemer Class, Pat Powderly, Adam Cohen and Connor Daulton. Saw Powderly score a goal and dish three assists in the less than a half that I watched. Anxious to check those guys out Sunday in the championship game.
Since the theme of the Powerball Tournament centers on nearby Atlantic City, we're going to take a shot at some odds for Sunday's Money Division Final and the ultimate $25K grand prize.
So, here's the scoop on the four Money Division playoff teams, who I saw play in 1-2 games on Saturday.
Reebok Blacks Out Cancer 3:1
As was expected, the mostly Canadian squad went 3-0, beating Flow Society 8-5, Liquid Flow 8-5 and Bass Massters 17-4. Kevin Huntley, Dan Dawson and Zack Greer form a dangerous attack. Huntley scored a hat trick versus Liquid Flow. They also got scoring from Peter Milliman and Dan Deckelbaum, who had big games against Bass Massters. David Mitchell, Mark Steenhuis and Dave Brock were also all over the field in the midfield.
The defense of Tom Hajek, Matt Vinc, Pat Merrill and Shawn Nadelen and d-middies George Castle and Billy Dee Smith in front of goalie Rich D'Andrea looked pretty formidable.
Not surprisingly, this is definitely the most physical team at the tournament. And with Andrew Hennessey taking face-offs, they're going to get plenty of possessions.
This is the most complete team at Powerball, and thanks to nice corporate sponsorship from RBK and Route1, they are doing it in style, staying on the strip in AC and wheeling around in a bus.
If this team wins, they're donating the money to the families of Chris Sanderson and Kyle Miller. Sanderson recently passed away after a four-year battle with brain cancer; his father-in-law made it out Saturday. Miller is another former Team Canada goalie, who is suffering from Osteogenic Sarcoma; he's here this weekend with the squad.
Motive Pure 4:1
Though RBK certainly has a great attack, the all-Virginia attack of Tim Whiteley, Mike Watson and Doug Knight goes down in history as one of the all-time best. Sure enough, two minutes into MP's first game of the tournament, Knight scored one of his patented diving goals; and later on Whiteley hooked up Watson with a sweet pass for another dive. Knight and Watson, for those who don't know, are the reason they outlawed the dive. Fun watching those guys play, and fun to hear 2012 UVa grad John Haldy talk in awe of playing with those Cavalier legends at Powerball.
Another player to watch for MP is Billy Glading. The athletic middie who walked on to the UVa hoops team his final year there and also played MLL for a while, had four goals in the morning game against HEADstrong, a narrow 9-8 win that came with 17 seconds left on a Drew Thompson goal, his second of the game.
Trey Whitty, Matt Passagno and Mike Timms looked tough on D, and goalie Frank Maguire made some huge saves late in the HEADstrong game to keep them at bay.
Maryland's David Tamberrino, one of just a handful of non-UVa players on the squad, ran the show at face-offs and, if they advance, should have a nice battle against Hennessey. He was rocking a Portside helmet Saturday, saying "I decided to leave all the failed MLL helmets at home." There were a host of MLL helmets out Saturday, including Bayhawks, Nationals, Lizards and Outlaws.
MP beat Team 110% Play Harder 15-4, Beerwolves 13-4 and HEADstrong 9-8.
Motive Pure also decided to donate their winnings to benefit Sanderson, who led the Cavaliers to two final fours before moving on to team Canada.
HEADstrong 6:1
The team from nearby Philadelphia looked solid on Saturday. Pretty well-rounded roster that had a bunch of chances late against Motive Pure in the morning game. Ohio State's Jimmy Green scored two big second-half goals in that one, while Ryan Cranston, Neal Hicks and Mike Collelouri also scored big goals for HEADstrong. Cranston even ran some longstick; the big middie from Lynchburg says he enjoys that more sometimes.
HEADstrong was missing defenseman Casey Connor and face-off man Tim Treubig, who should be coming in for the playoffs Sunday. Chris and Mike Creighton helped hold down the D for HEADstrong, and Julian Watts looked very athletic playing mostly shortstick while rocking a Nationals helmet and Lizards gloves. Also great to see Kevin Finneran back on the field. The former pro ironman who is down in Delray Beach, Fla., won four NLL titles in Philadelphia. After the first game he said he was initially planning on playing more player-coach but the competitive juices got him going and fired him up.
They beat Team 110% Play Harder 11-6 and Beerwolves 14-4, while losing to Motive Pure 9-8.
HEADstrong is playing to benefit the families of cancer patients via their HEADstrong Foundation.
Flow Society 7:1
Flow Society landed UMass alum Tim McCormack in goal, and he helped them limit Liquid Flow in a 6-2 win and Bass Massters in an 11-4 win. RBK only won 8-5 versus Flow Society, and considering how much offensive talent they have that's a huge accomplishment for the Flow D. Flow will be playing for the Jimmy Regan Fund.
Regis McDermott (Notre Dame) is the most recognizable name on the defense; he made a great check in the game against Liquid Flow that would have resulted in a turnover if LF hadn't called a timeout. He had a great line after: "Come on, I hustled on that play."
Their attack of Edison Parzanese (Notre Dame), Brendan Gibson (Yale and Tom Perini (Lafayette) leads the offense for Flow. Each scored nice goals in the game I saw against Bass Massters. Middies Colin Serling (Lafayette), Mike Pratt (Yale), Shaun Dunn (Siena) and Kevin Young (Duke) also helped fuel the offense.


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==== Poor Law Unions ====
==== Poor Law Unions ====
Revision as of 12:02, 29 September 2012
England Lancashire Lancashire Parishes
Chapelry History
Tonge cum Alkrington St Michael was created a district chapelry in 1842 from, and lying within the boundaries of Prestwich St Mary, Lancashire Ancient Parish.
Other places in the parish include: Alkrington.
TONGE, a township, in the parish of Prestwich-cum-Oldham, union of Oldham, hundred of Salford, S. division of Lancashire, 5½ miles (N. N. E.) from Manchester; containing 2423 inhabitants. It is probable that the family of Tonge gave name to this place, which in the 43rd of Elizabeth was possessed by a member of it named Christopher Tonge. The township adjoins Middleton, and forms a populous part of the environs of that town. The soil here is various, chiefly sand, gravel, and clay: a coal-mine is in operation. The population is mostly employed in silk hand-loom weaving, and in cotton-mills and print-works. Tonge, with Alkrington, forms an ecclesiastical district: the church, dedicated to St. Michael, was built in 1839, by Her Majesty's Commissioners, at a cost of £1773, and is a plain brick edifice. The living is a perpetual curacy, in the patronage of the Rector of Prestwich; net income, £150, of which £137 are granted by the Ecclesiastical Commissioners. The tithes of Tonge have been commuted for £20. There are national schools.
From: A Topographical Dictionary of England by Samuel Lewis (1848), pp. 372-377. URL: http://www.british-history.ac.uk/report.aspx?compid=51349 Date accessed: 31 July 2010.
Civil Registration
Birth, marriages and deaths were kept by the government, from July 1837 to the present day. The civil registration article tells more about these records. There are several Internet sites with name lists or indexes. A popular site is FreeBMD.
Online index of Lancashire Births, Marriages and Deaths Lancashire BMD
Church records
Include here information for parish registers, Bishop's Transcripts and other types of church records, such as parish chest records. Add the contact information for the office holding the original records. Add links to the Family History Library Catalog showing the film numbers in their collection
Parish registers for St. Michael's Church, Tonge-cum-Alkrington, 1839-1947 Microfilm copy of originals formerly held at the Manchester Central Library, Manchester, England.
Tonge is a township and a chapelry in Prestwich parish. The chapelry includes Alkrington township.
Manchester Central Library call nos.: L148/1/1/1-7, L148/2/1-4, L148/1/3/1-3.
| Content
| Film
| Baptisms, 1839-March 1906.
| FHL BRITISH Film
| Baptisms, March 1906-1923. Marriages, 1840-1922.
| FHL BRITISH Film
| Marriages, June 1922-1943.
| FHL BRITISH Film
2356369 Items 5 - 6
| Burials, 1839-1925.
| FHL BRITISH Film
| Banns, 1917-1947.
| FHL BRITISH Film
Census records
Census records from 1841 to 1911 are available online. For access, see England Census Records and Indexes Online. Census records from 1841 to 1891 are also available on film through a Family History Center or at the Family History Library.
Poor Law Unions
Probate records
Records of wills, administrations, inventories, indexes, etc. were filed by the court with jurisdiction over this parish. Go to Lancashire Probate Records to find the name of the court having primary jurisdiction. Scroll down in the article to the section Court Jurisdictions by Parish.
Maps and Gazetteers
Maps are a visual look at the locations in England. Gazetteers contain brief summaries about a place.
Web sites
| This section requires expansion with:
any relevant sites that aren't mentioned above..
http://www.british-history.ac.uk/report.aspx?compid=53004 British History Online Alkrington
http://www.british-history.ac.uk/report.aspx?compid=53005 Tonge


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Sewing Design Class
An eight week Sewing Design class is being offered at the Career-Tech (formerly Vo-Tech) Center. Tentatively the class will meet on Mondays from 6-9:15 p.m. beginning March 18. 2.5 CEUs will be given for this course. The cost is $75 due at registration. A minimum number of people must register for the class to make. For more information on registering, you may contact us Monday through Thursday from 7:30 a.m. until 4 p.m. or Friday from 7:30 a.m. until 3 p.m. at 601-656-8544.
Neshoba Central High School Class of '73
Our class reunion is coming up May 4. Organizers need a head count for the caterers. Please send in your money and profile sheets before March 15. If you are unable to attend, we would still like to have your profile sheet. Everyone, please help us make this the best reunion yet. The next meeting will be April 1 at Ronnie's Steak and Grill at 6 p.m.
Junior Auxillary
Junior Auxiliary will be hosting annual Easter Egg Hunt on Sunday March 24 from 2 p.m. to 4 p.m. at Northside Park. We are asking for a $2 donation per child. All proceeds will be going to the Philadelphia Boys and Girls Club. Bring cameras as the Easter Bunny will make special appearance.
Registration for Preschool
First United Methodist Church Weekday Children's Ministry will hold registration in March for toddler and Preschool classes. So stop on by and fill out an application. Registration fee is $50. If you have any questions, please call 601-656-1417.
NCHS Class of 1988
Attention Neshoba Central class of 1988. It's time for your 25th class reunion! The date chosen is June 22 starting at 5 at the Senior Citizen's building at Northside park. Please r.s.v.p. with your address to email@example.com, or send your $50 check or money order to NCHS class of '88, 10430 rd 404, Philadelphia, MS 39350. Cut off date is May 15th. Any questions? Call 601-504-1337. We look forward to seeing you!
Easter Bunny Coming to Library
The Easter Bunny is coming to the Neshoba County Library on March 28. Stop by the library from 9 to 11 a.m. or 3 to 5 p.m. and bring your our camera for a free picture with the Easter Bunny. Call 601-656-4911 for more details.
Longino Dance
Longino Senior Citizens Center Dance, every Friday and Saturday night from 8 - 12 p.m.
Smoke free family environment. $8 cover charge. Music by Sam Holding and the Sun Downers. County music and old time rock. For more information call 601-728-0313.
The Recovery First NA meeting will be held at God's Tabernacle Church every Thursday at 6 p.m.
For more information call Ruthie Nash at 601-656-3565.
Mt. Olive Baptist Church
Anyone with family members buried at the cemetery is ask to help with its upkeep. Contributions can be mailed to: Mt. Olive Baptist Church, 11881 Road 347, Philadelphia, MS 39350.
Donations Requested
The Philadelphia Police Department and the Community Policing Neighborhood Association are asking for your assistance in donations for school supplies for the children of Philadelphia Elementary School. Any donation would be greatly appreciated.
Bingo Night
Calling all senior citizens ages 60 and up. Come join us every Tuesday at 5 p.m. for a fun night of bingo and a chance to win great prizes.
This event will be held at the Senior Citizens building located at Northside Park. There will be a $2 entry fee and refreshments will be served. So come out and have some bingo fun.
Benefit for Ricky Boyles
A benefit is planned for Ricky Boyles. His family will be holding a garage sale but needs donations as well. Items can be delivered to 214 Hospital Road west. If items need to be picked up please call 601-562-3915 or 601-562-6416. Sale is scheduled for March 22 and 23.
Concert at Prospect Church
The Spoken 4 Quartet from Branson, Missouri, will be featured in concert at Prospect Church, Collinsville, for Friend Day on Sunday, April 7 at 10 a.m. The public is invited. Prospect Church is located on the left side of Highway 19, 6 miles north of Collinsville. For further information, call 601-986-8392.
Heath Fair
There will be a health fair on Thursday, March 21 from 10 a.m. - 12 p.m. at the Mississippi Action for Progress, Inc. Exhibit Hall Headstart Center (234 Carver Avenue, Philadelphia, MS 39350.
NCHS Class of 1963
The Neshoba Central High School Class of 1963 will celebrate their 50th anniversary with a class reunion on May 11 at the Senior Citizens Building at Northside Park. All former classmates (whether in our class or another class), spouses of deceased classmates, friends and former teachers are invited to attend. We need contact information for Betty Hardy Kelly and Thomas Kilpatrick. If you have information on these two, please contact one of the committee. For further details on the reunion, please call Ruth Whitehead 601-389-1946, Ann Ethredge 601-562-4851, Judy Elwood 601-562-2509, Sarah Waddell 601-201-4085, Barbara Dunn 601-416-4584, or Voncile Breazeale 601-506-1922.
Center Hill Baptist Church Revival
Center Hill Baptist Church will have revival on March 17-20. Sunday services will be 11 a.m. and 6 p.m. Monday through Wednesday services will be at 7 p.m. Speaker will be Rev. Tom McCurley. The public is invited.
Goodway M.B. Church
Join us for worship at Goodway M.B. Church Youth Revival on March 11-13 at 7 p.m. The speaker for Monday night is Rev. Harold Coburn, Tuesday night will be Rev. Darsett Buckley and Wednesday night will be Rev. Jarvis Rush. Rev. W. C. Rush is pastor.
Laurel Hill Baptist Church
Laurel Hill Baptist Church will have new times for worship services, Sunday school and discipleship training beginning March 10. Sunday morning worship services will begin at 9:30 a.m. each Sunday with Sunday School to follow. Sunday Night Discipleship will begin at 6 p.m. each Sunday with worship services beginning at 6:45 p.m.
Please come and worship with us. Bro. Jason Willis is pastor.
Food Giveaway
The Mars Hill Outreach Ministry will be open Saturday, March 16 from 10 a.m. until 12 p.m. First time applicants must bring I.D. of income (stubs for all household members) and proof of residence (utility bill).
We are a 501 (C)(3) non profit organization. To make donations or get information. Please contact Patricia Wilson at 601-656-3549 or Latonya Smith at 601-416-2319.
Friends of the Library
Friends of the Library will meet on March 21 at 4 p.m. The meeting is to get ready for National Library week in April.
Miss Neshoba County Fair 2013
Miss Neshoba County Fair 2013 first interest meeting is set for Sunday, March 24 at the CDP at 2 p.m. Contact Gail Breazeale for information call (601)416-5529.
Women's Ministry
The Women's Ministry of First Baptist Church is having a spring fling called "Make Time for Joy" on Saturday, March 23 from 8:30 a.m. until 2:30 p.m. Special guests are Heaven Bound Clowns from Pickensville, Alabama and Sidney Covington from Meridian. The event is free, including lunch and child care. You must register by March 18 to attend. For more information call FBC at 601-656-1507.
NC School Board
Work Session
The Neshoba County School Board will hold a School Board work session on Wednesday, March 20 from 1-4 p.m. in the data room of the Neshoba Elementary School. Parking will be across from the Middle School/Gym.
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439 E Beacon ST, Philadelphia, MS 39350 | 601.656.4000
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