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Florida Manatee
Collisions with Watercraft
Slow-moving manatees often cannot avoid the speed boats and other watercraft that frequent the Florida waterways they call home. As a result, propellers and boat hulls inflict serious or mortal wounds, and most manatees have a pattern of scars on their backs or tails after surviving collisions with boats. Defenders is working to reduce this cause of death and make waterways safer for these unique marine mammals.
The Problem
Over the past five years alone, more than 80 manatees have died from watercraft-related incidents each year. The highest year on record was 2009, when a devastating 97 manatees were killed in collisions with boats over the course of the year, not to mention those that were seriously injured but went unrecorded. Population scientists believe that unless this cause of death is controlled, the manatee population will not recover.
How We're Helping
Defenders has been advocating for slow speed zones to protect manatees since 1997, and many efforts have been successful. We have spoken with city, county, state and federal officials, participated in working groups, submitted written comments and testified at public meetings.
Since 2004, the Florida Fish and Wildlife Conservation Commission has approved new manatee protection rules for three counties in Tampa Bay. The agency also reviewed and updated speed zones in Sarasota, Broward, Charlotte, Lee and Duval counties. In October 2005, the Hillsborough County Commission adopted mandatory manatee protection slow-speed zones in the Cockroach Bay Aquatic Preserve that previously had been voluntary. In 2012, speed zones were established in the Intracoastal Waterway in Flagler County. Defenders of Wildlife worked to see these rules through, providing comments on how to make them effective devices to save manatee lives.
Thanks in part to Defenders' advocacy efforts—the National Park Service designated slow-speed zones to protect manatees in Everglades National Park. The U.S. Fish and Wildlife Service also improved protections for manatees at Crystal River National Wildlife Refuge by establishing the Kings Bay Manatee Refuge, designating most of Kings Bay as a slow speed area and improving refuge managers' abilities to protect manatees from boaters and swimmers wanting to interact with them during cold weather.
Today, through public outreach and education programs, we continue trying to make Florida's waters safer for manatees. We talk to boaters about ways they can minimize the danger of hitting these often hard-to-see mammals. We also advocate for additional slow speed zones in areas that remain dangerous to manatees, such as western Pinellas County.
You may also be interested in:
Length: 10-12 feet
Weight: 1,500-1,800 lbs
Lifespan: 50-60 years in the wild


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Hello all,
I found a picture of the Carradale school class of 1920 posted on your forum back in 2010. As normal the image was very small (55kb) and I would really like to find a better quality copy. It contains the ONLY known photograph of the mother of someone close to me. The original message used by user: chuckiebay is below:
If anyone has a high quality scan or can contact chuckiebay I'd be really grateful.
Carradale School circa 1920
Postby chuckiebay » Thu Nov 18, 2010 3:42 pm
Back row L/R: John McBride; Archie Brown; Angus Cook; John Campbell 9Nona); Donald Morrison; Alister Galbraith; Hector McMillan; Johnny McMillan; Donald Paterson(Fergie)
2nd row: Peggy Galbraith; Margt McDougal; Lizzie Campbell; Annie McBride; Jeanie Girgen; Annie W Paterson; Mairi McDougal; Peggy McKinnon;
3rd row: Marion McDougal; Chrissie McKechnie; Flora Blue; Lizzie McLean; Flora Downie; Agnes Brown; Alice Thomson; Betty Galbraith; BuntyMcPherson.
4th row: Walter McDougal; Davey Mitchel; Gibby Nicholson; Duncan Fisher; Alister McMillan.
The teacher was Miss Fettes.
http://i1119.photobucket.com/albums/k62 ... os/001.jpg

subset: commoncrawl


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Friday, September 14, 2012
An alternative view of the NHL lockout
On Saturday night at 11:59 p.m. EDT, the National Hockey League plans to lockout the players if no new Collective Bargaining Agreement is reached before the deadline.
As it currently stands, no meetings are planned and the sides are miles apart. In 2004, the last time that the CBA was up for grabs, there was a three month delay before further negotiations were attempted and eventually the entire season was lost.
Hockey fans lived through the ordeal when the 2004-05 season was the first season completely lost due to labor strife in the history of North American professional sports. The NHL returned after the layoff with new rules that quickened the pace of the game and fans quickly returned to the sport that they loved.
The NHL and the NHL Players Association are again banking on the fan's patience and their assumption that fans will put another lockout behind them and return in the same manner.
NHL Commissioner Gary Bettman offended many fans last month when he arrogantly stated "We recovered last time because we have the world's greatest fans."
That may have worked in 2004 but will it fly once again?
I was probably one of the few followers of the game that actually enjoyed the time away from the rink during the last lockout. I had two son's playing hockey and a mother living out her last few months of life. I had plenty to do and made lasting memories with my boys and mother instead of going to NHL games.
When hockey returned, I did too, and was somewhat refreshed from a year where I didn't drive 100 miles fifty times in a six month period to attend games.
Now as we await the lockout keys turning, instead of being up in arms that the owners and players will give fans the middle finger once again, fans should start planning how to live life to its fullest.
I really like Jiff peanut butter. If it suddenly became unavailable I would not be happy. I would begrudgingly buy Skippy or Peter Pan and move on.
Hockey fans should do the same. Go to minor or junior games or if you are really Jonesing for a cold building, go watch a bunch of kids play at the local house league. Football, pro or college, the baseball postseason, or many other sports are not affected by the lockout if you savor the stadium experience.
Better yet, check out other things that you have never been able to do in the fall because of your dying allegiance to the sport of hockey. With all the money saved on tickets, parking and food and beverages, there are plenty of options out there.
Each dollar you don't spend on "hockey related revenue" is one dollar less for the millionaires to fight over. At some point, they may miss a few of your dollars and decide to play hockey again.
My suggestion is that fans tune out all the negotiations (or lack of them) and try to live life to its fullest in the extra time you are being given. Time is one of the few things that you can't make more of or increase. Value every minute and don't waste it worrying about something that is completely out of your control.
Personally, I am enjoying the extra time I have been given and have not used one ounce of emotional energy being upset about the lack of hockey. I'm not the least bit concerned about the players or owners since, to an extent, they control their own destiny.
The folks that we should be concerned about in this whole mess are all the folks who depend on NHL games to put food on the family table by working part-time jobs at the rink or are in businesses that depend on 41 home games to make payroll and keep the doors open. Our prayers go out to them.
In summary, I'm sure everyone is hopeful for a quick resolution to the situation but instead of being upset, consider it a vacation and enjoy the extra time you have with your family and friends.
More Later...
Buddy Oakes for PredsOnTheGlass
2 comments:
- Paul Nicholson said...
Agreed Buddy. I think my only Preds related spending will be making good on my promise to get my wife a ladies cut home gold jersey as a belated mother's day present (they were out of stock). otherwise I'll enjoy the extra time with my family and maybe give playing the game myself another shot
- September 14, 2012 at 3:29 PM
- Dirk Hoag said...
I'm really bummed that I'm not on a rec league team this fall (gotta shed this spare tire first), but hey, maybe I'll actually get my yard looking somewhat respectable!
- September 14, 2012 at 9:08 PM
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The many meanings of "evolution" - no wonder we get confused!
One thing that confuses people who are trying to follow the intelligent design controversy is that the word "evolution" is used by different people to mean strikingly different things. Embryologist Jonathan Wells notes,
"Evolution" has many meanings. It can mean simply "change over time." The present is different from the past. The cosmos evolves. Technology evolves. No sane person denies evolution in this sense.
In biology, "evolution" can also refer to minor changes within existing species. Nobody denies evolution in this sense, either. People were observing such changes for centuries before Darwin came long; they were even producing them in plants and animals by artificial selection. Darwin (and Wallace) pointed out that something analogous to artificial selection operates in natural populations, but there is nothing "Darwinian" about artificial selection.
Lest some people mistakenly conclude that I accept Darwinian evolution broadly defined, I will make two other distinctions. The first is between "microevolution" and "macroevolution," and the second is between "macroevolution" and "Darwinism."
Regarding the first: Almost eighty years after Darwin published The Origin of Species, neo-Darwinist Theodosius Dobzhansky noted that there was still no hard evidence to connect small-scale changes within existing species (which he called "microevolution") to the origin of new species, organs and body plans (which he called "macroevolution"). Dobzhansky wrote: "We are compelled at the present level of knowledge reluctantly to put a sign of equality between the mechanisms of macro- and microevolution, and proceeding on this assumption, to push our
investigations as far ahead as this working hypothesis will permit."
1. Unfortunately for Darwin, there has never been a confirmed case of natural selection producing a new species, much less new organs or body plans
2. So in 2008, seventy years after Dobzhansky and a century and a half after Darwin, the extrapolation from microevolution to macroevolution is still just an assumption.
Concerning the second and final distinction: Darwin didn't just claim that natural selection could produce new species, organs and body plans. He went much further and argued that (a) all species are biologically descended from a common ancestor, and (b) their features were produced entirely by unguided natural processes. He wrote: "There seems to be no more design in the variability of organic beings, and in the action of natural selection, than in the course which the winds blow."
3 It is the claim of universal common ancestry coupled with the exclusion of design that I call "Darwinism."
So "Darwinian evolution" can mean changes in existing species ("microevolution") due to natural selection. This is what Egnor referred to as "obviously true," and I agree. But for some people "Darwinian evolution" also includes the extrapolation from microevolution to macroevolution. This is far from obviously true; indeed, the evidence for it is underwhelming, at best.
Actually, natural selection tends to conserve, not create.
See, for example, Thoroughbred by Design? Mustang by Chance?
Natural Selection: Tracking the Primitive Dog
to see how natural selection works in horses and dogs.
Labels: natural selection


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Every now and then we have to tip our hats to the guys at Wall Street Oasis— they've really created a place where bloggers feel like they can let loose.
That's what one female banker (with the handle, bankarella) did in a post called "What every banker girl needs."
As she correctly points out, man or woman, Wall Streeters don't have a lot of time, so they need to know what they want (in terms of the opposite sex) and how/where to get it quickly.
That's why she ranked men from all over the financial services industry (from private wealth management to trading) in terms of their dateability — this is all based on research, of course.
So lets go down the list of Wall Streeters she's evaluated, starting with her fellow bankers (from Wall Street Oasis).
Bankers: "I give the banker guy 1 star out of 3. Pros: not clingy, spends money. Cons: Frequently not available. Work is always top-of-mind. Things stall when work gets heavy. Unfortunate tendency to end phone calls with "Thanks. Bye." Also, there's just too much room for comparison. There's just no way to not compare status, deals, money. You have to be okay with that going in."
Private Equity guys:2 stars (of 3). Same pros as the banker guy with fewer cons, more confidence, and more free time.
Private Wealth Management guys:One star (of three). Pros: Knows all the good restaurants, expenses everything. Cons: Too fake, too fratty, too status-oriented, seems to put girls into two buckets: marriage material and date rape territory. Talks endlessly. Can't be squeezed into a two-hour time slot.
Hedge Fund guys: my sample is weak here; would benchmark him to PE guy with potential upside based on role, experience, and personality.
Traders:Three stars. Pros: transactional awareness, speed, bandwidth, value/time ratio, ability to close out the trade and walk away without drama. Typically feel they have something to prove, like to throw money around. Cons: Slightly scruffier than the guys above. Thinks that traders (rather than bankers) are masters of the universe. Keeps comparing his best month's earnings to your average month; sometimes "forgets" to add in your bonus. (It's not discretionary! It's a sure thing!) Most importantly: when he has a bad day, it's a really bad day.
We appreciate her honesty (especially about the hedge fund sample size, very scientific) and we wish her luck in her quest not to waste her time. It is, after all, so precious.


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Kenny Chesney 'Shocked' by Riley Cooper Racial Slur Video
Kenny Chesney has reacted to a video showing Philadelphia Eagles wide receiver Riley Cooper making threats and using a racial slur at the country singer's June 9 concert.
Chesney says he was shocked by the video, which recently surfaced on YouTube. In a lengthy statement, the star goes on to admonish Cooper's use of the N-word.
"I don't believe in discrimination in any form, and I think using language like that is not only unacceptable, it is hateful beyond words," Chesney remarked to ESPN this week.
He also tells writer LZ Granderson that he finds NFL players to be an inspiration, as they're very charitable as a group. His statement reads:
"To judge an entire audience by one loud mouth isn't fair ... not to the NFL, not to the city of Philadelphia and that awesome crowd, not to my band and crew and certainly not to me, who believes music is about bringing people together for friendship and forgetting about the things in life that bring you down. The music I make is about living life, loving life and loving everybody -- no matter who they are. That's how I was raised, and what someone else does or says doesn't reflect who I am or what my fans stand for."
The statement comes one day after Cooper apologized for his actions. The Eagles fined him for the incident.
This is the second time a Chesney show in Pennsylvania has led to bad press, although it'd be unfair to blame the singer for either incident. On June 22, he played Pittsburgh's Heinz Field. Afterward, video and pictures of multiple fights and a mountain of trash surfaced, leading to several complaints from city officials and residents.


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- Research
- Open Access
Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum
- Sophie C Oehring†1, 2,
- Ben J Woodcroft†3,
- Suzette Moes4,
- Johanna Wetzel1, 2,
- Olivier Dietz1, 2,
- Andreas Pulfer1, 2,
- Chaitali Dekiwadia3,
- Pascal Maeser1, 2,
- Christian Flueck1, 2,
- Kathrin Witmer1, 2,
- Nicolas MB Brancucci1, 2,
- Igor Niederwieser1, 2,
- Paul Jenoe4,
- Stuart A Ralph3 and
- Till S Voss1, 2Email author
© Oehring et al.; licensee BioMed Central Ltd. 2013
- Received: 24 May 2012
- Accepted: 26 November 2012
- Published: 26 November 2012
The post-genomic era of malaria research provided unprecedented insights into the biology of Plasmodium parasites. Due to the large evolutionary distance to model eukaryotes, however, we lack a profound understanding of many processes in Plasmodium biology. One example is the cell nucleus, which controls the parasite genome in a development- and cell cycle-specific manner through mostly unknown mechanisms. To study this important organelle in detail, we conducted an integrative analysis of the P. falciparum nuclear proteome.
We combined high accuracy mass spectrometry and bioinformatic approaches to present for the first time an experimentally determined core nuclear proteome for P. falciparum. Besides a large number of factors implicated in known nuclear processes, one-third of all detected proteins carry no functional annotation, including many phylum- or genus-specific factors. Importantly, extensive experimental validation using 30 transgenic cell lines confirmed the high specificity of this inventory, and revealed distinct nuclear localization patterns of hitherto uncharacterized proteins. Further, our detailed analysis identified novel protein domains potentially implicated in gene transcription pathways, and sheds important new light on nuclear compartments and processes including regulatory complexes, the nucleolus, nuclear pores, and nuclear import pathways.
Our study provides comprehensive new insight into the biology of the Plasmodium nucleus and will serve as an important platform for dissecting general and parasite-specific nuclear processes in malaria parasites. Moreover, as the first nuclear proteome characterized in any protist organism, it will provide an important resource for studying evolutionary aspects of nuclear biology.
- Malaria
- Plasmodium falciparum
- Nucleus
- Proteomics
- Bioinformatics
- Transcription
- Nucleolus
- Nuclear pore
- Transfection
As one of the most deadly infectious diseases in the world, malaria causes close to 500 million clinical cases and 1 million deaths every year [1, 2]. Most of this burden is due to infections with Plasmodium falciparum, one of six Plasmodium species known to elicit malaria in humans [3, 4]. Malaria-related morbidity and mortality is exclusively associated with the erythrocytic stage of infection where repeated rounds of intracellular parasite development and re-invasion into red blood cells (RBCs) lead to exponential parasite proliferation. The entire parasite life cycle is much more complex involving several morphologically and functionally distinct extra- and intracellular stages, and obligate transmission between two hosts, female Anopheles spp. and humans.
The key to this amazing biological complexity lies within the parasite nucleus that, in the case of P. falciparum, encloses and regulates a 23Mb genome encoding 5,400 genes on 14 linear chromosomes [5]. However, albeit many nuclear processes such as transcription, splicing, DNA replication/repair, mitosis, and the temporal and spatial organization of the nucleus have been studied in detail in model eukaryotes our understanding of nuclear biology in P. falciparum is very limited. This is not surprising given that >50% of all genes code for proteins with no known or even inferred function [5–7]. While many seminal studies in the post-genomic era of malaria research provided unprecedented insights into the biology of P. falciparum, they also highlighted our profound lack of understanding of basic biological processes in this parasite. In light of spreading drug resistance and the eager expectation for an effective vaccine, acquisition of such knowledge is urgently needed.
During the pre-replicative phase of the intra-erythrocytic developmental cycle (IDC), parasites develop into morphologically distinct ring and trophozoite stages. Schizogony is characterized by multiple rounds of genome replication and closed mitosis before cytokinesis produces new daughter merozoites from multinucleated schizonts [8, 9]. At the ultrastructural level, the parasite nucleus appears spherical and contains a mixture of electron-sparse and electron-dense material probably reflecting euchromatic and heterochromatic zones, respectively. The distribution of this material appears to be sensitive to its precise fixation and preparation [10–12].
Several high-throughput transcriptome and proteome profiling studies revealed that in addition to a core set of genes expressed in multiple/all life cycle stages, a large number of genes are specifically expressed in only a single stage, many of which are involved in highly specialized processes and pathways [13–19]. Differential gene expression is also strikingly observed during the 48-h IDC. Detailed microarray experiments performed at high temporal resolution identified a surprisingly structured cascade of gene transcription during this stage [20–22]. About 80% of the genes expressed during the IDC display temporal variation in transcript abundance where genes appear to be activated only when their encoded protein functions are required [21]. Notably, the timely expression of variant protein families involved in immune evasion and RBC invasion is directly related to the pronounced virulence of P. falciparum [23]. However, despite the fact that many of these processes are likely governed by transcriptional control little detail on the underlying mechanisms has yet been elucidated.
General transcription factors (TFs) and chromatin remodelling activities are well conserved in the P. falciparum proteome [24–27]. Several factors of the latter class have recently been localized to different subcompartments within the parasite nucleus [28]. Functional studies identified important roles for the histone deacetylase PfGCN5, silent information regulator 2 (PfSIR2) and heterochromatin protein 1 (PfHP1) in parasite development, heterochromatin formation, and virulence gene expression [29–37]. In contrast, however, the striking under-representation of identified sequence-specific TFs in the P. falciparum proteome compared to those of fungi, plants, and metazoans has hampered targeted research to understand gene-specific control [24, 38]. Until recently, only a single TF, PfMYB1, had been analyzed to any extent in vivo [39]. Fortunately, the discovery of the apicomplexan-specific ApiAP2 family of DNA-binding factors and functional analysis of some members sparked new interest in this field [40–46].
Most proteomic studies on Plasmodium parasites have focused on elucidating whole cell proteomes, which generated valuable insight into the overall structure of, and differences between, the active proteomes in different parasite life cycle stages [13, 14, 16, 17, 47], or in response to perturbations such as drug treatment [48–51]. However, while these approaches typically detect large numbers of different proteins they fail to provide information on subcellular protein localization. Organellar proteomics is an important tool to gain new insight into cellular structures and functions since proteins localizing to distinct subcellular compartments are usually associated with the function of these compartments. To date, only a few mass spectrometry-based studies aimed at identifying protein compositions of Plasmodium cellular compartments. These include the analysis of fractions enriched for P. falciparum food vacuoles (116 proteins) [52], Maurer's clefts (78 proteins) [53], and the infected RBC membrane (36 proteins) [54]. Rodent Plasmodium spp. have also been analyzed, searching for rhoptry (36 proteins) [55] and micronemal proteins (345 proteins) [56].
Here, we performed a detailed proteomic analysis of P. falciparum nuclei during intra-erythrocytic development. Our approach combined multidimensional protein identification technology (MudPIT) of purified and fractionated nuclei with validation by both bioinformatic appraisal and in-vivo localization experiments. We present a robust core nuclear proteome consisting of 802 proteins. Our comprehensive analysis of this inventory provides unprecedented insight into the parasite nucleus and will be of great benefit to future studies investigating nuclear biology in this important pathogen.
Isolation and fractionation of parasite nuclei
We next performed protein fractionation to reduce sample complexity. Nuclei were serially extracted with 0.1 M KCl (fraction 2), DNAseI (fraction 3), 1 M KCl (fraction 4), and 2% SDS (fraction 5). Each fraction displayed a distinct 1D-SDS-PAGE protein pattern indicative of differential protein extraction (Figure 1dand Additional file 1). The cytosolic enzyme glyeraldehyde-3-phosphate dehydrogenase (GAPDH) and the mitochondrial heat shock protein 60 (HSP60) were exclusively detected in the NP40-soluble cytoplasmic extract (fraction 1) demonstrating efficient lysis of the parasite plasma membrane and the double membrane-bound mitochondrion (Figure 1e). In contrast, H4 and PfHP1 were soluble only after extraction of nuclear pellets with high salt and SDS, consistent with their intimate association with chromatin. In summary, the microscopy-, SDS-PAGE-, and immunodetection-based assessments show that the protocol applied here efficiently separated the cytoplasmic and nuclear compartments and yielded distinct subnuclear protein fractions suitable for mass spectrometry analysis.
Nuclear proteome determination by MudPIT
On average, 69.7% (+/- 14.7 SD) of proteins measured in a given sample were also detected in its matching replicate. Furthermore, calculation of Spearman rank coefficients showed that each sample correlated best with its matching replicate (Additional file 2). This clearly underlines the repeatability of the fractionation and the mass spectrometric methods applied. The absolute correlations between replicates were moderate (0.50 +/- 0.09 SD; Spearman rank coefficients). This can be explained by the stochastic sampling of peptides in the mass spectrometer, a limitation inherently associated with shotgun proteomics of complex samples, and by biological and experimental sample-to-sample variation. Undersampling is a consequence of the limited speed with which MS/MS spectra can be acquired and is specific for the instrument used. Both factors contribute to the differential detection/identification of proteins, particularly for low abundance proteins.
To maximally cover the nuclear proteome, we accepted protein identifications based on single peptide matches. For reliable peptide identifications we used the decoy search strategy querying a reversed sequence database [60]. This strategy resulted in very low mean false discovery rates of 0.0028 +/- 0.0024 SD per sample, reflecting the stringent search criteria applied for peptide identification and acceptance of single peptide hits (Additional files 1 and 2). Furthermore, random inspection of single peptide-based protein identifications confirmed the automated protein identification of the search engine. In light of these high confidence identifications, proteins represented by single peptides were included in all downstream analyses. The SEQUEST output data for all stages and fractions (Additional files 3, 4, and 5) and a comprehensive summary table (Additional file 6) are provided as supplementary information.
The preparation of isolated nuclei is enriched in nuclear proteins
Distribution of peptides derived from a representative selection of known nuclear proteins across the three IDC stages.
Protein ID
Product description
Fraction 1
Fraction 2
Fraction 3
Fraction 4
Fraction 5
histone H2A
histone H2A.Z
histone H2B
histone H2B variant
histone H3
histone H3 variant
histone H3, CENP-A variant
histone H4
DNA-directed RNA polymerase II complex
DNA-directed RNA pol II, putative
DNA-directed RNA pol II, putative
DNA-directed RNA pol II, putative
DNA-directed RNA pol II 2nd largest subunit, putative
RNA polymerase subunit 8c, putative
DNA-directed RNA pol II 16 kDa subunit, putative
DNA-directed RNA pol II, putative
DNA-directed RNA pol II, putative
DNA-directed RNA pol II, putative
DNA-directed RNA pol II 8.2 kDa polypeptide, putative
ApiAP2 transcription factors
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
transcription factor with AP2 domain(s), putative
Next, we applied two measures, positive predictive value (PPV) and the number of proteins remaining in each set, to assess differences in content between the nuclear fractions. The pool of detected proteins annotated as nuclear in the ApiLoc database [64] and/or by literature review was used as reference set (159 proteins). As expected, the sets of proteins found in individual fractions showed varying PPVs with the least and most predictive observed in the cytoplasmic lysate and high salt nuclear extract, respectively (Figure 3b). The sum of proteins found in the combined nuclear fractions gave the largest set at a high PPV. Considering only those proteins in this set that were not also detected in the cytoplasm increased the PPV by 8% but came at too steep a cost by removing 49% of all proteins including many of nuclear and unknown localization. This shows that the cytoplasmic fraction is not especially deficient in nuclear proteins, consistent with the presence of many nuclear proteins in both compartments. In contrast, proteins found exclusively in the cytoplasmic fraction were markedly deficient in predicted nuclear proteins.
Application of bioinformatic reduction techniques to generate a high-confidence core nuclear proteome dataset
Applying the five filters resulted in a set of 841 proteins with a PPV of 70% (Figure 4b). After removal of 55 proteins experimentally localized to non-nuclear compartments, re-addition of 16 known nuclear proteins, and adjustments due to altered SP annotations (Additional file 9) we present a set of 802 proteins, whereof we estimate that 76% represent true nuclear proteins (compared to 46% in the unfiltered dataset) (Figure 4b). This set is henceforth referred to as the 'core nuclear proteome' and all further analyses were carried out on this set (Additional file 10).
An overview of the contents of the core nuclear proteome is shown in Figure 4c where all proteins are categorized into broad generic classes based on functional annotation. Apart from proteins engaged in typical nuclear processes such as transcription, chromatin remodeling, DNA replication/repair, splicing, and nucleolar functions, the core nuclear proteome contains many putative RNA-binding proteins and factors involved in RNA processing and metabolism, protein folding, modification and degradation, and cytoskeletal organization. Notably, one-third of the core nuclear proteome consists of proteins with no annotated function, and only 5% of all proteins are reliably allocated to non-nuclear compartments.
Experimental validation by subcellular localization of selected nuclear candidates
To test if the core nuclear proteome will be useful in identifying true nuclear proteins we experimentally validated the subcellular localization of 28 proteins by indirect immuno-fluorescence assays (IFA). We generated transgenic parasite lines expressing full-length C-terminally tagged proteins from episomally maintained plasmids. We chose 22 nuclear protein candidates (NuProCs1-22) that were included in the core nuclear proteome, and six non-nuclear protein candidates (n-NuProCs1-6) that were excluded from the list based on the presence of predicted TM domains and/or SPs (Additional file 11). Of the 22 NuProCs, seven carried annotations indicative for nuclear localization and 15 were annotated as hypothetical proteins at the time of selection on PlasmoDB [68]. Amongst the negative control set, n-NuProC1 (PF11_0099) is annotated as heat shock protein DnaJ homolog Pfj2 whereas all other n-NuProCs are hypothetical proteins with unknown function.
Ten candidates annotated as hypothetical proteins (NuProC9, 10, 11, 12, 14, 15, 17-20) were also unambiguously localized within the parasite nucleus. A diffuse pattern was evident for NuProC12, NuProC15, NuProC17, and NuProC20. NuProC18 showed a cytosolic staining in late schizont and ring stages but was clearly nuclear in trophozoites. NuProC9 changed from a rather ubiquitous to a more condensed nuclear localization upon transition from the early to late schizont stage. Both NuProc10 and NuProC11 are localized to unknown nuclear subcompartments. NuProC14 and NuProC19 were preferentially associated with the nuclear periphery in ring stages but showed a diffuse pattern in trophozoite nuclei.
Two additional proteins, NuProC4 (putative multiprotein bridging factor type 1 MBF1) and NuProC13 localized outside but in close proximity to the DAPI signal (Figure 5b). These patterns were clearly distinct from the cytosolic GAPDH signal and reminiscent of that obtained for the nuclear porin PfNUP100 [28] suggesting that these two factors may be associated with the nuclear periphery. In addition, NuProC4 appeared to accumulate in the cytosol in mature trophozoites.
None of the six non-nuclear protein candidates localized to the nucleus. Five co-localized with PfBIP throughout the IDC, and n-NuProC3 was associated with the ER in early stages and exported into the host cell in trophozoites and schizonts (Figure 6b) (detailed IFA localization results throughout the IDC for all n-NuProC cell lines are available in Additional file 13). The strong representation of ER-localized proteins in the negative control set indicates that the crude nuclear preparation was enriched in ER-associated proteins. However, we cannot exclude the possibility that ER association of some of these proteins may be artefactual due to over-expression of the tagged proteins.
In summary, our experimental validation of the predicted core nuclear proteome tested 16 out of 22 nuclear protein candidates (72.7%) positive for true nuclear localization, plus another two that are associated with the nuclear periphery. This figure is consistent with the estimated precision of the bioinformatically reduced core set of nuclear proteins and validates this inventory as an important platform for the identification and characterization of novel parasite nuclear proteins.
Two novel nucleolar proteins in P. falciparum
Identification of novel domains associated with P. falciparum nuclear proteins
Using the core nuclear proteome as an input collection, we sought to detect novel functional domains enriched in nuclear proteins using an all-versus-all BLAST search followed by manual inspection (Additional file 1). Six novel domains comprised of 17 proteins warranted further inspection, all of which were conserved across Apicomplexa providing further evidence that these modules are functional.
Classical nuclear localization signals are only marginally over-represented in P. falciparum nuclear proteins
Classical nuclear localization signals (cNLSs) have been the subject of several bioinformatic prediction algorithms, and our core nuclear proteome provided an opportunity to test their utility in identifying P. falciparum nuclear proteins. We used three different bioinformatic tools to predict cNLSs: NLStradamus [76], predictNLS [77], and cNLS mapper [78]. All three algorithms attempt to predict nuclear localization based solely on the presence of cNLSs and not other protein domains.
NLStradamus, PredictNLS, and cNLS mapper suggest 51%, 37%, and 22% of proteins found in the core nuclear proteome contain a cNLS, respectively (Additional file 10), compared to 45%, 31%, and 17% of the entire P. falciparum proteome (P values 0.008, 0.006, and 0.006, respectively; Fisher's exact test). In contrast, only 47%, 32%, and 18% of proteins found in the whole cell trophozoite proteome [17] were predicted by these algorithms (P values 0.17, 0.4, and 0.18, respectively, relative to all P. falciparum proteins; Fisher's exact test). While this result adds some evidence to the hypothesis that cNLS-mediated nuclear import may operate in P. falciparum similarly to model eukaryotes, the difference in the percentage of cNLS-containing proteins in the core nuclear proteome compared to the set of all P. falciparum proteins is only marginal. Likewise, the same three predictors identify only 50% (NLStradamus), 37% (PredictNLS), and 28% (cNLS mapper) of proteins in the combined ApiLoc and literature review reference set of 317 P. falciparum nuclear proteins to contain a cNLS (P values 0.08, 0.07, and 0.0004, respectively, relative to all P. falciparum proteins; Fisher's exact test). Using more stringent bioinformatic filters on the core nuclear proteome resulted only in a slightly higher percentage of proteins with predicted cNLS (Additional file 22). Together, the low percentage of nuclear proteins being predicted suggests that either P. falciparum cNLSs may be too divergent for current bioinformatic predictors, or that the major mode of nuclear import may not occur through classical import signals, or a combination of both.
To deepen our insight into nuclear biology in P. falciparum we performed a comprehensive proteomic analysis of the parasite nucleus and detected a total of 1,273 proteins in sequential extracts of crude nuclei. The proportion of predicted nuclear proteins in this set was estimated at 46%, which compares well with two similar studies in S. cerevisiae. Mosley et al. detected 2,674 yeast proteins in crude nuclei, 46% of which are annotated as nuclear proteins in the Saccharomyces genome database SGD [79], and a second study based on sucrose gradient purification of nuclei detected 1,889 proteins, 55% of which were annotated as nuclear in SGD [80].
The major sources of contamination in nuclear preparations are membrane fractions of non-nuclear organelles, particularly the ER. To eliminate such likely contaminants we applied an informed bioinformatic filtering approach that resulted in a markedly improved positive predictive value (76%) for true nuclear proteins in the core nuclear proteome. Importantly, we confirmed the specificity of this set by in-vivo experimental validation. Out of 22 candidates 18 localized in different patterns to the nucleus. Some proteins displayed a rather ubiquitous distribution within the nucleus, whereas others localized to more restricted, undefined subnuclear regions, to the nucleolus, or to the nuclear periphery. These alternative localizations are a likely consequence of the different functions these factors carry out in the nucleus. Four candidates localized primarily to the cytoplasm, however, some of them may also be nuclear. First, we determined localizations using episomally expressed epitope-tagged proteins, which may cause over-expression and cytosolic accumulation of some candidates. Second, proteins primarily located in the cytosol may still be present in the nucleus in lower concentrations. For instance, PfNAPL (NuProC21), which localized to the cytoplasm here and in a previous study [81], is a putative orthologue of ScNAP1 that shuttles between the nucleus and cytosol [82]. Another example is PFL0450c (NuProc22), a protein with unknown function that localized to the cytoplasm in ring and trophozoite stages but was found at the nuclear periphery in late schizonts.
Functional classification of the core nuclear proteome
We detected 299 proteins (37.3%) implicated in nuclear processes such as transcription, chromatin remodeling, DNA replication/repair, RNA binding and processing, ribosome biogenesis, but also in more general processes such as protein folding and modification, protein degradation, translation, cytoskeleton organization, and metabolism. This classification is based on direct experimental evidence, literature review, and/or sequence similarity to known S. cerevisiae nuclear proteins (Additional file 10). A total of 118 proteins (14.7%), for which such evidence is missing, carry annotations similarly consistent with roles in DNA/chromatin interactions, cell cycle control, mitosis, RNA binding and processing, protein folding and modification, protein degradation, cytoskeleton organization, and metabolic processes. Ninety-five proteins (11.8%) represent ribosomal subunits or translation-associated factors, and 35 proteins (4.4%) are predicted to localize to other compartments such as the mitochondrion, ER/Golgi, protein sorting vesicles, or the vacuole. Finally, the largest fraction of the core nuclear proteome consists of 255 proteins of unknown function (31.8%).
To date, only three proteins have been localized to the parasite nucleolus (RNA polI, PFE0465c; fibrillarin/PfNOP1, Pf14_0068; PfNOP5, PF10_0085) [83, 84]. Further, only six P. falciparum proteins are annotated with the GO term 'nucleolus', and 36 are annotated by GeneDB [85] as putative nucleolar proteins. Here, we detected 12 of these proteins including fibrillarin, PfNOP5, RNA methyltransferase (PF11_0305), putative ribonucleoprotein (RNP) components such as LSM homologs (PFL0460w, PF08_0049), and several predicted pre-ribosomal assembly proteins, as well as another 19 putative homologs of S. cerevisiae nucleolar proteins (Additional file 10). Notably, two of these candidates, NuProC2 (PF10_0278) and NuProC3 (PF11_0250), co-localized with fibrillarin to the parasite nucleolus. These novel identifications expand our current knowledge of the P. falciparum nucleolus and provide a basis for detailed analyses of this essential nuclear compartment.
Plasmodium protein kinases (PK) play central roles in growth, development, and differentiation throughout the life cycle, and are intensely studied as a class of promising antimalarial targets [86, 87]. We identified kinases or their accessory factors of seven PK systems, six of which have been implicated in nuclear roles in Plasmodium or other eukaryotes: PfMAT1 (PFE0610c) [88]; the α- and β-subunits of casein kinase 2 (PF11_0096, PF11_0048) [89–91]; casein kinase 1 (PF11_0377) [92, 93]; the catalytic and regulatory subunits of cAMP-dependent protein kinase (PFI1685w, PFL1110c) [94]; NIMA-related protein kinase PfNEK-1 (PFL1370w) [95, 96]; and mitogen-activated protein kinase 2 PfMAP2 (PF11_0147) [97, 98]. PfMAP2 is a substrate of PfNEK-1 [96] and both kinases are essential for completion of the IDC in P. falciparum [95, 99], whereas in P. berghei PbMAP2 is implicated specifically in the development of male gametes [97]. Interestingly, while the targets of these and other nuclear kinases remain largely unknown, we find that 70% of proteins in the core nuclear proteome (562/802) were recently shown to be phosphorylated [100] (Additional file 10) suggesting an important role for kinase signalling in the regulation of nuclear processes in P. falciparum.
We detected 32 of the 33 annotated P. falciparum proteasome components and all, except two of the 19S regulatory particle (RP) subunits, were represented in the cytoplasmic fraction. Intriguingly, all subunits of the RP were also associated with the insoluble nuclear fraction in ring stage parasites (Additional file 10). In other species, the RP interacts with chromatin and is involved in transcriptional regulation [101]. For example, the S. cerevisiae RP participates in SAGA histone acetyltransferase complex recruitment [102], interacts with the FACT (facilitates chromatin transcription) complex [103], and influences H3 methylation and gene silencing [104]. Hence, we speculate that the 19S RP may have similar non-proteolytic roles in P. falciparum transcriptional regulation.
The nuclear proteome also contained several orthologues of the mRNP complex implicated in translational repression during P. berghei gametocytogenesis [105, 106], such as the RNA helicase PfDOZI (PFC0915w), PfCITH (PF14_0717), the RNA-binding proteins PfHOBO (PF14_0096) and PfHOMU (PFI0820c), and a homolog of a yeast poly(A)-binding protein (PFL1170w). Although DOZI and CITH were described as cytoplasmic proteins in P. berghei, their localizations do not appear to exclude the nucleus. Furthermore, homologs of PfHOMU and PFL1170w shuttle between the cytosol and nucleus in mammalian cells and S. cerevisiae, respectively [107, 108]. The detection of these proteins in the asexual nuclear proteome suggests the presence of translational repression machinery during the IDC, although P. berghei DOZI and CITH loss-of-function mutants have no apparent phenotype in asexual parasites [105, 106].
We also discovered novel domains that are likely linked to previously unrecognized nuclear functions of parasite proteins, and we envisage their roles will be more thoroughly recognized in future studies. Of particular interest is the identification of the ACDC domain, which we identified exclusively in members of the ApiAP2 family. Moreover, we experimentally validated the identification of PF14_0442 as a novel subunit of P. falciparum nuclear pores. Testing this in more detail will be an important future task given that Plasmodium parasites lack identifiable orthologues of most nuclear pore components [109].
Stage- and fraction-specific aspects of the core nuclear proteome
We made several interesting observations regarding stage- and fraction-specific protein profiles that allow us to speculate about potential temporal and spatial expression patterns of nuclear proteins. In light of the stochastic undersampling of complex proteomes in shotgun proteomics approaches and possible sample-to-sample variation, however, these data have to be interpreted with caution.
A total of 58, 90, and 105 proteins were found exclusively in ring, trophozoite, and schizont stages, respectively. In trophozoites and schizonts, expression of these proteins occurred roughly in line with transcription of the encoding genes (Additional files 1 and 23). mRNA expression of ring stage-specific nuclear proteins was somewhat surprising, with a collective profile similar to that observed in schizonts, and an additional smaller peak at 15 to 20 hpi. This suggests that some proteins were newly synthesized in the ring stage while some remained from the preceding schizont stage. A total of 159 of the 253 stage-specifically detected proteins carry no functional annotation and several others carry annotated domains that indicate little about their function. Hence, a large number of novel proteins have been assigned a potential stage-specific role in nuclear biology where previously no informative annotation was available. Further, 13 predicted TFs [63] were detected specifically in a single IDC stage only, indicating a role for these factors as cell cycle-specific regulators of transcription and genome regulation (Additional file 24).
Of 145 proteins predicted to interact with DNA and/or chromatin, 137 (94.5%) were identified in the chromatin-containing fractions (DNAseI-, high salt-, and/or SDS-soluble fractions) (Additional file 10). Some of these factors, such as histones, SNF2 helicase (PFF1185w), chromodomain-helicase-DNA-binding protein 1 (CHD1) (PF10_0232), putative chromosome assembly factor 1 (PFE0090w), and the nuclear peroxiredoxin PfnPRX (PF10_0268) [110] were detected in all three chromatin-associated fractions. In contrast, nine out of the 10 RNA pol II subunits identified were exclusively detected in the high salt nuclear extract. Both FACT components (PFE0870w, PF14_0393) extracted almost identically only after high salt and SDS extraction. The two recently described high mobility group box proteins (PFL0145c, MAL8P1.72) [111] were identified in DNAse1- and high salt-soluble extracts but not in the SDS fraction. These examples suggest that at least some regulatory complexes were extracted as interacting entities. Interestingly, all ApiAP2 factors showed a noticeable association with the insoluble nuclear fraction. While the reason for this remains unknown our observation hints at possible functions of these DNA-binding proteins. Most ApiAP2 proteins are large and, apart from the short and well-defined DNA-binding AP2 domains, consist of extensive uncharacterized regions. It is possible that these non-AP2 regions may mediate the formation of regulatory complexes involved in diverse processes such as DNA replication, transcriptional regulation, or functional organization of the genome. Such complexes are often resistant to extraction with DNAseI and high salt buffers, as observed for many RNA- or DNA-binding proteins associated with the nuclear matrix [112]. In case of PfSIP2, the only P. falciparum ApiAP2 characterized in vivo, the detection of PfSIP2-derived peptides in the insoluble nuclear fraction is consistent with the association of this factor with condensed heterochromatin [46].
A large number of proteins in the core nuclear proteome (379) were also detected in the cytoplasmic fraction. This pool of proteins contained members of all functional classes but was clearly enriched in distinct pathways associated with the various functions of the nucleolus in other eukaryotes [113–115]. These include the majority of ribosomal subunits (90.5%), RNA-binding proteins (78.9%), factors involved in protein degradation (69.0%), and protein folding and modification (65.1%). Furthermore, 84.4% of translation-related factors were identified in the cytoplasmic and nuclear fractions, a finding consistent with the existence of nuclear translation [116, 117]. We also noticed that 42% (62 proteins) of confirmed or likely nuclear proteins in the core nuclear proteome were detected in the cytoplasmic fraction. Moreover, six proteins experimentally localized to the nucleus in this study had peptides detected in the cytoplasmic fraction. Hence, while some of the dually detected proteins may represent cytoplasmic contaminants such as abundant cytosolic proteins or macromolecular complexes, our results show that many nuclear proteins in P. falciparum shuttle between the nucleus and cytosol and/or perform their tasks at multiple destinations.
Nuclear import in P. falciparum
Transport of proteins into the nucleus remains poorly understood in apicomplexan parasites [118]. In yeast and mammalian model systems short, arginine-, and lysine-rich cNLSs are thought to be the major mediators of nuclear import, though alternative and redundant mechanisms have been described [119]. The poor enrichments of cNLSs in both the core nuclear proteome and the set of 317 curated nuclear proteins show that bioinformatic discrimination of nuclear vs. non-nuclear P. falciparum proteins via prediction of cNLSs remains impractical. Notably, however, cNLS predictors are also problematic in the reliable identification of nuclear proteins in the model systems they were designed for [120]. Nevertheless, differences between P. falciparum nuclear proteins and these current computational models of cNLSs appear prevalent but remain unclear. It has not been determined whether the majority of P. falciparum nuclear proteins are imported independently of importin α, the protein that binds cNLSs [118], or alternately by similar but unrecognized cNLSs that do mediate importin α-dependent translocation. The fact that only 22% to 51% of nuclear proteins are predicted to contain cNLSs (depending on the predictor used) suggests that in its current definitional form the cNLS is not the major mode of nuclear import in P. falciparum. Notably, the important insight that most verifiable Plasmodium nuclear proteins lack a recognizable nuclear localization sequence, and need thus be identified through empirical strategies, reinforces the value of an experimentally robust nuclear proteome for understanding Plasmodium nuclear biology.
Lineage-specific nuclear proteins
Using a combination of OrthoMCL and synteny analyses (Additional files 1 and 25) only around 10 proteins are genuinely falciparum-specific. Unsurprisingly, nearly all of them have no functional annotation, though one is the gametocyte-specific protein Pfg27 (PF13_0011). Pfg27 binds RNA and has previously been localized to the cytoplasm and nucleus of gametocytes [121]. Interestingly, we detected another nuclear P. falciparum-specific protein of unknown function (PFB0115w) that is expressed during the IDC and in gametocytes and contains C-terminal homology to the Pfg27-specific fold [122] suggesting a functional relation between these two proteins.
The pool of around 100 genus-specific proteins represents a promising group from which to characterize features of nuclear biology peculiar to Plasmodium. Several are potentially worthy of prioritized treatment; these include some of the previously characterized ApiAP2s [40], as well as a large number of uncharacterized proteins with nucleic acid-binding and chromatin-interacting domains. Several others in this group have kinase or phosphatase domains and may be involved in expression regulation cascades. One of the Plasmodium-specific nuclear proteins is a putative metacaspase (PF14_0363). Interestingly, unrelated trypanosomatid parasites also possess nuclear localized metacaspases that are required for proliferation, possibly through modulation of cell cycle progression [123].
To our knowledge, this is the most detailed organellar proteome analysis in apicomplexan parasites and the first study describing a large scale nuclear proteome of any protist species. During the past decade several intriguing features of nuclear biology in P. falciparum have been discovered, including the periodical behavior of gene transcription during the IDC [21, 22], the discovery of the ApiAP2 family [40], the epigenetic control of genes related to virulence [29, 30, 33–35, 124], or the fascinating dynamic surface distribution of nuclear pores throughout the IDC [12]. These examples highlight the existence of nuclear processes distinct from those in the host and underscore the importance of understanding the underlying mechanisms. For the first time, our study provides a comprehensive and unprecedented insight into the biology of the P. falciparum nucleus, and the nuclear proteome presented herein will assist greatly in dissecting conserved as well as evolutionary specialized nuclear processes in Plasmodium and related parasites.
Materials and methods
Parasite culture and transfection
P. falciparum 3D7 parasites were cultured as described previously [125]. Growth synchronization was achieved by repeated sorbitol lysis [126]. Transfections were performed as described [127] and selected on 5 μg/mL blasticidin-S-HCl and/or 4 nM WR99210. Parasites transfected with pH_0442-GFP were cycled on/off WR three times to select for integration of the plasmid into the endogenous locus.
Isolation of nuclei and nuclear fractionation
Nuclei from ring stages, trophozoites, and schizonts were isolated and fractionated as previously described with minor modifications [35]. A detailed protocol is provided in Additional file 1. For western blot analysis protein fractions were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Schleicher&Schuell). Primary antibody dilutions were: anti-H4 (Abcam, ab10158) 1:10,000; anti-HSP60 (kind gift from Geoff McFadden, University of Melbourne) 1:2,000; mAb anti-PfGAPDH 1:2,000 [128]; rabbit anti-PfHP1 (kind gift from Mike Duffy, University of Melbourne) 1:2,000; and anti-GFP (Roche Diagnostics, 11814460001) 1:1000.
Microscopic assessment of isolated nuclei
Nuclei were incubated with DAPI (10 μg/mL) in CLB for 10 min on ice and directly observed by light and fluorescence microscopy. Images were obtained using a Leica DM 5000B microscope with a Leica DFC 300 FX camera and acquired via the Leica IM 1000 software and processed and overlayed using Adobe Photoshop CS2. For TEM analysis isolated nuclei or whole asynchronous 3D7 cultures were fixed, prepared for microscopy and imaged as described [129]. For immunolabelling, nuclei were incubated with rabbit anti-H3K4Me3 (Abcam ab8580) diluted 1:300, then detected with 18 nm colloidal gold-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, Baltimore, MD, USA) diluted 1:20.
Sample preparation, MudPIT liquid chromatography tandem mass spectrometry, and protein identification
Detailed protocols for sample preparation, MudPIT analysis, and protein identification are provided in Additional file 1. All proteomics data are publicly available on PlasmoDB [68] and in Additional files 3, 4, and 5.
Bioinformatic data analysis
Bioinformatic methods used to assess and analyze the nuclear proteome are presented in detail in Additional file 1.
Transfection constructs
To express epitope-tagged candidate proteins in P. falciparum, full-length genes were amplified from gDNA or cDNA and cloned into pBcam-3xHA [35] using BamHI (or BglII) and NcoI (or NheI) restriction sites. The PFL0635c gene (NuProC6) was cloned into BamHI/NheI-digested pHcam-2xTy [46]. To generate the double transfectant co-expressing NuProC2-3xHA/NuProC3-2xTy the PF11_0250 gene (NuProC3) was cloned into BamHI/NcoI-digested pHcam-2xTy and transfected into BSD-resistant 3D7/NuProC2-3xHA parasites. All forward primers included five wild-type nucleotides directly upstream of the natural ATG start codon. pH_0442-GFP was obtained by cloning the 3' end of PF14_0442 (551 bp) into PstI/NotI-digested pHcam-GFP [130]. Successful 3' replacement integration of pH_0442-GFP into the endogenous locus was verified by PCR and western blot (Additional file 21). Primer details are listed in Additional file 26.
Indirect immunofluorescence assays
IFAs were performed with iRBCs fixed in 4% formaldehyde/0.01% glutaraldehyde as described elsewhere [131]. Primary antibody dilutions were: rat mAb anti-HA 3F10 (Roche Diagnostics) 1:100; mouse mAb anti-Ty BB2 1:2,000 (kind gift from Keith Gull); mouse mAb anti-GAPDH 1:500 [128]; rabbit anti-PfBIP 1:500 [132, 133] (kind gift from Tim Gilberger); and mouse mAb anti-fibrillarin 1:500 [71] (kind gift from Michael Terns, University of Georgia). Secondary antibody dilutions were: Alexa-Fluor® 568-conjugated anti-rat IgG (Molecular Probes) 1:500; Alexa-Fluor® 488-conjugated anti-rabbit IgG (Molecular Probes) 1:500; and FITC-conjugated anti-mouse IgG (Kirkegaard Perry Laboratories) 1:250. Images were taken on a Leica DM 5000B microscope with a Leica DFC 300 FX camera and acquired via the Leica IM 1000 software and processed and overlayed using Adobe Photoshop CS2. Observed protein localizations have been deposited in ApiLoc [64].
The authors would like to thank Terry Speed and James Bailey for helpful comments. We are grateful to Tim-Wolf Gilberger, Mike Duffy, Michael Terns, and Geoff McFadden for providing antibodies. BJW is funded by a University of Melbourne MRS scholarship. SAR is funded by an Australian Research Council Future Fellowship (FT0990350). NMBB received a Boehringer Ingelheim PhD fellowship. This work was supported by the Swiss National Science Foundation (PP00A-110835; PP00P3_130203), the Novartis Foundation for Medicine and Biology (08C46), the Emilia-Guggenheim-Schnurr Foundation, and the Rudolf Geigy Foundation. The funders had no role in study design, data collection, analysis, and interpretation, the decision to publish, or preparation of the manuscript.
Authors' Affiliations
- Hay SI, Okiro EA, Gething PW, Patil AP, Tatem AJ, Guerra CA, Snow RW: Estimating the global clinical burden of Plasmodium falciparum Malaria in 2007. PLoS Med. 2010, 7: e1000290-PubMedPubMed CentralGoogle Scholar
- World Health Organization: World Malaria Report 2010. 2010, Geneva: WHO PressGoogle Scholar
- White NJ: Plasmodium knowlesi: the fifth human malaria parasite. Clin Infect Dis. 2008, 46: 172-173.PubMedGoogle Scholar
- Sutherland CJ, Tanomsing N, Nolder D, Oguike M, Jennison C, Pukrittayakamee S, Dolecek C, Hien TT, Do R, Arez AP, Pinto J, Michon P, Escalante AA, Nosten F, Burke M, Lee R, Blaze M, Otto TD, Barnwell JW, Pain A, Williams J, White NJ, Day NP, Snounou G, Lockhart PJ, Chiodini PL, Imwong M, Polley SD: Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally. J Infect Dis. 2010, 201: 1544-1550.PubMedGoogle Scholar
- Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan MS, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DM, et al: Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002, 419: 498-511.PubMedGoogle Scholar
- Hall N, Karras M, Raine JD, Carlton JM, Kooij TW, Berriman M, Florens L, Janssen CS, Pain A, Christophides GK, James K, Rutherford K, Harris B, Harris D, Churcher C, Quail MA, Ormond D, Doggett J, Trueman HE, Mendoza J, Bidwell SL, Rajandream MA, Carucci DJ, Yates JR, Kafatos FC, Janse CJ, Barrell B, Turner CM, Waters AP, Sinden RE: A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses. Science. 2005, 307: 82-86.PubMedGoogle Scholar
- Waters AP: Genome-informed contributions to malaria therapies: feeding somewhere down the (pipe)line. Cell Host Microbe. 2008, 3: 280-283.PubMedGoogle Scholar
- Bannister LH, Hopkins JM, Fowler RE, Krishna S, Mitchell GH: A brief illustrated guide to the ultrastructure of Plasmodium falciparum asexual blood stages. Parasitol Today. 2000, 16: 427-433.PubMedGoogle Scholar
- Striepen B, Jordan CN, Reiff S, van Dooren GG: Building the perfect parasite: cell division in apicomplexa. PLoS Pathog. 2007, 3: e78-PubMedPubMed CentralGoogle Scholar
- Aikawa M: The fine structure of the erythrocytic stages of three avian malarial parasites, Plasmodium fallax, P. lophurae, and P. cathemerium. Am J Trop Med Hyg. 1966, 15: 449-471.PubMedGoogle Scholar
- Ralph SA, Scheidig-Benatar C, Scherf A: Antigenic variation in Plasmodium falciparum is associated with movement of var loci between subnuclear locations. Proc Natl Acad Sci USA. 2005, 102: 5414-5419.PubMedPubMed CentralGoogle Scholar
- Weiner A, han-Pasternak N, Shimoni E, Shinder V, von HP, Elbaum M, Dzikowski R: 3D nuclear architecture reveals coupled cell cycle dynamics of chromatin and nuclear pores in the malaria parasite Plasmodium falciparum. Cell Microbiol. 2011, 13: 967-977.PubMedGoogle Scholar
- Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ: A proteomic view of the Plasmodium falciparum life cycle. Nature. 2002, 419: 520-526.PubMedGoogle Scholar
- Silvestrini F, Lasonder E, Olivieri A, Camarda G, van SB, Sanchez M, Younis YS, Sauerwein R, Alano P: Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum. Mol Cell Proteomics. 2010, 9: 1437-1448.PubMedPubMed CentralGoogle Scholar
- Lasonder E, Janse CJ, van Gemert GJ, Mair GR, Vermunt AM, Douradinha BG, van N, Huynen MA, Luty AJ, Kroeze H, Khan SM, Sauerwein RW, Waters AP, Mann M, Stunnenberg HG: Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity. PLoS Pathog. 2008, 4: e1000195-PubMedPubMed CentralGoogle Scholar
- Khan SM, Franke-Fayard B, Mair GR, Lasonder E, Janse CJ, Mann M, Waters AP: Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology. Cell. 2005, 121: 675-687.PubMedGoogle Scholar
- Lasonder E, Ishihama Y, Andersen JS, Vermunt AM, Pain A, Sauerwein RW, Eling WM, Hall N, Waters AP, Stunnenberg HG, Mann M: Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature. 2002, 419: 537-542.PubMedGoogle Scholar
- Le Roch KG, Johnson JR, Florens L, Zhou Y, Santrosyan A, Grainger M, Yan SF, Williamson KC, Holder AA, Carucci DJ, Yates JR, Winzeler EA: Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res. 2004, 14: 2308-2318.PubMedPubMed CentralGoogle Scholar
- Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD, de la Vega P, Holder AA, Batalov S, Carucci DJ, Winzeler EA: Discovery of gene function by expression profiling of the malaria parasite life cycle. Science. 2003, 301: 1503-1508.PubMedGoogle Scholar
- Otto TD, Wilinski D, Assefa S, Keane TM, Sarry LR, Bohme U, Lemieux J, Barrell B, Pain A, Berriman M, Newbold C, Llinas M: New insights into the blood-stage transcriptome of Plasmodium falciparum using RNA-Seq. Mol Microbiol. 2010, 76: 12-24.PubMedPubMed CentralGoogle Scholar
- Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol. 2003, 1: E5-PubMedPubMed CentralGoogle Scholar
- Llinas M, Bozdech Z, Wong ED, Adai AT, DeRisi JL: Comparative whole genome transcriptome analysis of three Plasmodium falciparum strains. Nucleic Acids Res. 2006, 34: 1166-1173.PubMedPubMed CentralGoogle Scholar
- Coleman BI, Duraisingh MT: Transcriptional control and gene silencing in Plasmodium falciparum. Cell Microbiol. 2008, 10: 1935-1946.PubMedGoogle Scholar
- Aravind L, Iyer LM, Wellems TE, Miller LH: Plasmodium biology: genomic gleanings. Cell. 2003, 115: 771-785.PubMedGoogle Scholar
- Meissner M, Soldati D: The transcription machinery and the molecular toolbox to control gene expression in Toxoplasma gondii and other protozoan parasites. Microbes Infect. 2005, 7: 1376-1384.PubMedGoogle Scholar
- Horrocks P, Wong E, Russell K, Emes RD: Control of gene expression in Plasmodium falciparum - Ten years on. Mol Biochem Parasitol. 2009, 164: 9-25.PubMedGoogle Scholar
- Cui L, Miao J: Chromatin-mediated epigenetic regulation in the malaria parasite Plasmodium falciparum. Eukaryot Cell. 2010, 9: 1138-1149.PubMedPubMed CentralGoogle Scholar
- Volz J, Carvalho TG, Ralph SA, Gilson P, Thompson J, Tonkin CJ, Langer C, Crabb BS, Cowman AF: Potential epigenetic regulatory proteins localise to distinct nuclear sub-compartments in Plasmodium falciparum. Int J Parasitol. 2010, 40: 109-121.PubMedGoogle Scholar
- Lopez-Rubio JJ, Mancio-Silva L, Scherf A: Genome-wide analysis of heterochromatin associates clonally variant gene regulation with perinuclear repressive centers in malaria parasites. Cell Host Microbe. 2009, 5: 179-190.PubMedGoogle Scholar
- Tonkin CJ, Carret CK, Duraisingh MT, Voss TS, Ralph SA, Hommel M, Duffy MF, Silva LM, Scherf A, Ivens A, Speed TP, Beeson JG, Cowman AF: Sir2 paralogues cooperate to regulate virulence genes and antigenic variation in Plasmodium falciparum. PLoS Biol. 2009, 7: e84-PubMedGoogle Scholar
- Perez-Toledo K, Rojas-Meza AP, Mancio-Silva L, Hernandez-Cuevas NA, Delgadillo DM, Vargas M, Martinez-Calvillo S, Scherf A, Hernandez-Rivas R: Plasmodium falciparum heterochromatin protein 1 binds to tri-methylated histone 3 lysine 9 and is linked to mutually exclusive expression of var genes. Nucleic Acids Res. 2009, 37: 2596-2606.PubMedPubMed CentralGoogle Scholar
- Freitas-Junior LH, Hernandez-Rivas R, Ralph SA, Montiel-Condado D, Ruvalcaba-Salazar OK, Rojas-Meza AP, Mancio-Silva L, Leal-Silvestre RJ, Gontijo AM, Shorte S, Scherf A: Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites. Cell. 2005, 121: 25-36.PubMedGoogle Scholar
- Duraisingh MT, Voss TS, Marty AJ, Duffy MF, Good RT, Thompson JK, Freitas-Junior LH, Scherf A, Crabb BS, Cowman AF: Heterochromatin silencing and locus repositioning linked to regulation of virulence genes in Plasmodium falciparum. Cell. 2005, 121: 13-24.PubMedGoogle Scholar
- Salcedo-Amaya AM, van Driel MA, Alako BT, Trelle MB, van den Elzen AM, Cohen AM, Janssen-Megens EM, van d, Selzer RR, Iniguez AL, Green RD, Sauerwein RW, Jensen ON, Stunnenberg HG: Dynamic histone H3 epigenome marking during the intraerythrocytic cycle of Plasmodium falciparum. Proc Natl Acad Sci USA. 2009, 106: 9655-9660.PubMedPubMed CentralGoogle Scholar
- Flueck C, Bartfai R, Volz J, Niederwieser I, Salcedo-Amaya AM, Alako BT, Ehlgen F, Ralph SA, Cowman AF, Bozdech Z, Stunnenberg HG, Voss TS: Plasmodium falciparum heterochromatin protein 1 marks genomic loci linked to phenotypic variation of exported virulence factors. PLoS Pathog. 2009, 5: e1000569-PubMedPubMed CentralGoogle Scholar
- Cui L, Miao J, Furuya T, Fan Q, Li X, Rathod PK, Su XZ, Cui L: Histone acetyltransferase inhibitor anacardic acid causes changes in global gene expression during in vitro Plasmodium falciparum development. Eukaryot Cell. 2008, 7: 1200-1210.PubMedPubMed CentralGoogle Scholar
- Cui L, Miao J, Furuya T, Li X, Su XZ, Cui L: PfGCN5-mediated histone H3 acetylation plays a key role in gene expression in Plasmodium falciparum. Eukaryot Cell. 2007, 6: 1219-1227.PubMedPubMed CentralGoogle Scholar
- Coulson RM, Hall N, Ouzounis CA: Comparative genomics of transcriptional control in the human malaria parasite Plasmodium falciparum. Genome Res. 2004, 14: 1548-1554.PubMedPubMed CentralGoogle Scholar
- Gissot M, Briquet S, Refour P, Boschet C, Vaquero C: PfMyb1, a Plasmodium falciparum transcription factor, is required for intra-erythrocytic growth and controls key genes for cell cycle regulation. J Mol Biol. 2005, 346: 29-42.PubMedGoogle Scholar
- Balaji S, Babu MM, Iyer LM, Aravind L: Discovery of the principal specific transcription factors of Apicomplexa and their implication for the evolution of the AP2-integrase DNA binding domains. Nucleic Acids Res. 2005, 33: 3994-4006.PubMedPubMed CentralGoogle Scholar
- Yuda M, Iwanaga S, Shigenobu S, Mair GR, Janse CJ, Waters AP, Kato T, Kaneko I: Identification of a transcription factor in the mosquito-invasive stage of malaria parasites. Mol Microbiol. 2009, 71: 1402-1414.PubMedGoogle Scholar
- Yuda M, Iwanaga S, Shigenobu S, Kato T, Kaneko I: Transcription factor AP2-Sp and its target genes in malarial sporozoites. Mol Microbiol. 2010, 75: 854-863.PubMedGoogle Scholar
- Lindner SE, De Silva EK, Keck JL, Llinas M: Structural determinants of DNA binding by a P. falciparum ApiAP2 transcriptional regulator. J Mol Biol. 2010, 395: 558-567.PubMedPubMed CentralGoogle Scholar
- Campbell TL, De Silva EK, Olszewski KL, Elemento O, Llinas M: Identification and genome-wide prediction of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite. PLoS Pathog. 2010, 6: e1001165-PubMedPubMed CentralGoogle Scholar
- De Silva EK, Gehrke AR, Olszewski K, Leon I, Chahal JS, Bulyk ML, Llinas M: Specific DNA-binding by apicomplexan AP2 transcription factors. Proc Natl Acad Sci USA. 2008, 105: 8393-8398.PubMedPubMed CentralGoogle Scholar
- Flueck C, Bartfai R, Niederwieser I, Witmer K, Alako BT, Moes S, Bozdech Z, Jenoe P, Stunnenberg HG, Voss TS: A major role for the Plasmodium falciparum ApiAP2 protein PfSIP2 in chromosome end biology. PLoS Pathog. 2010, 6: e1000784-PubMedPubMed CentralGoogle Scholar
- Roobsoong W, Roytrakul S, Sattabongkot J, Li J, Udomsangpetch R, Cui L: Determination of the Plasmodium vivax schizont stage proteome. J Proteomics. 2011, 74: 1701-1710.PubMedPubMed CentralGoogle Scholar
- Radfar A, Diez A, Bautista JM: Chloroquine mediates specific proteome oxidative damage across the erythrocytic cycle of resistant Plasmodium falciparum. Free Radic Biol Med. 2008, 44: 2034-2042.PubMedGoogle Scholar
- van Brummelen AC, Olszewski KL, Wilinski D, Llinas M, Louw AI, Birkholtz LM: Co-inhibition of Plasmodium falciparum S-adenosylmethionine decarboxylase/ornithine decarboxylase reveals perturbation-specific compensatory mechanisms by transcriptome, proteome, and metabolome analyses. J Biol Chem. 2009, 284: 4635-4646.PubMedPubMed CentralGoogle Scholar
- Aly NS, Hiramoto A, Sanai H, Hiraoka O, Hiramoto K, Kataoka H, Wu JM, Masuyama A, Nojima M, Kawai S, Kim HS, Wataya Y: Proteome analysis of new antimalarial endoperoxide against Plasmodium falciparum. Parasitol Res. 2007, 100: 1119-1124.PubMedGoogle Scholar
- Briolant S, Almeras L, Belghazi M, Boucomont-Chapeaublanc E, Wurtz N, Fontaine A, Granjeaud S, Fusai T, Rogier C, Pradines B: Plasmodium falciparum proteome changes in response to doxycycline treatment. Malar J. 2010, 9: 141-PubMedPubMed CentralGoogle Scholar
- Lamarque M, Tastet C, Poncet J, Demettre E, Jouin P, Vial H, Dubremetz JF: Food vacuole proteome of the malarial parasite Plasmodium falciparum. Proteomics Clin Appl. 2008, 2: 1361-1374.PubMedGoogle Scholar
- Vincensini L, Richert S, Blisnick T, Van DA, Leize-Wagner E, Rabilloud T, Braun BC: Proteomic analysis identifies novel proteins of the Maurer's clefts, a secretory compartment delivering Plasmodium falciparum proteins to the surface of its host cell. Mol Cell Proteomics. 2005, 4: 582-593.PubMedGoogle Scholar
- Florens L, Liu X, Wang Y, Yang S, Schwartz O, Peglar M, Carucci DJ, Yates JR, Wub Y: Proteomics approach reveals novel proteins on the surface of malaria-infected erythrocytes. Mol Biochem Parasitol. 2004, 135: 1-11.PubMedGoogle Scholar
- Sam-Yellowe TY, Florens L, Wang T, Raine JD, Carucci DJ, Sinden R, Yates JR: Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites. J Proteome Res. 2004, 3: 995-1001.PubMedGoogle Scholar
- Lal K, Prieto JH, Bromley E, Sanderson SJ, Yates JR, Wastling JM, Tomley FM, Sinden RE: Characterisation of Plasmodium invasive organelles; an ookinete microneme proteome. Proteomics. 2009, 9: 1142-1151.PubMedPubMed CentralGoogle Scholar
- Cohn ZA, Hirsch JG: The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. J Exp Med. 1960, 112: 983-1004.PubMedPubMed CentralGoogle Scholar
- Crawley JC, Harris H: The fine structure of isolated hela cell nuclei. Exp Cell Res. 1963, 31: 70-81.PubMedGoogle Scholar
- Gatlin CL, Eng JK, Cross ST, Detter JC, Yates JR: Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry. Anal Chem. 2000, 72: 757-763.PubMedGoogle Scholar
- Kall L, Storey JD, MacCoss MJ, Noble WS: Assigning significance to peptides identified by tandem mass spectrometry using decoy databases. J Proteome Res. 2008, 7: 29-34.PubMedGoogle Scholar
- Huang da W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009, 4: 44-57.PubMedGoogle Scholar
- Beissbarth T, Speed TP: GOstat: find statistically overrepresented Gene Ontologies within a group of genes. Bioinformatics. 2004, 20: 1464-1465.PubMedGoogle Scholar
- Bischoff E, Vaquero C: In silico and biological survey of transcription-associated proteins implicated in the transcriptional machinery during the erythrocytic development of Plasmodium falciparum. BMC Genomics. 2010, 15: 34-Google Scholar
- ApiLoc - A database of published protein sub-cellular localization in Apicomplexa. [http://apiloc.biochem.unimelb.edu.au/apiloc/apiloc]
- Marti M, Good RT, Rug M, Knuepfer E, Cowman AF: Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science. 2004, 306: 1930-1933.PubMedGoogle Scholar
- Hiller NL, Bhattacharjee S, van OC, Liolios K, Harrison T, Lopez-Estrano C, Haldar K: A host-targeting signal in virulence proteins reveals a secretome in malarial infection. Science. 2004, 306: 1934-1937.PubMedGoogle Scholar
- Bender A, van Dooren GG, Ralph SA, McFadden GI, Schneider G: Properties and prediction of mitochondrial transit peptides from Plasmodium falciparum. Mol Biochem Parasitol. 2003, 132: 59-66.PubMedGoogle Scholar
- Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS, Heiges M, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Miller JA, Nayak V, Pennington C, Pinney DF, Roos DS, Ross C, Stoeckert CJ, Treatman C, Wang H: PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Res. 2009, 37: D539-D543.PubMedPubMed CentralGoogle Scholar
- Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR, Bateman A: The Pfam protein families database. Nucleic Acids Res. 2010, 38: D211-D222.PubMedPubMed CentralGoogle Scholar
- Wiley SE, Murphy AN, Ross SA, van der GP, Dixon JE: MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity. Proc Natl Acad Sci USA. 2007, 104: 5318-5323.PubMedPubMed CentralGoogle Scholar
- Yang JM, Baserga SJ, Turley SJ, Pollard KM: Fibrillarin and other snoRNP proteins are targets of autoantibodies in xenobiotic-induced autoimmunity. Clin Immunol. 2001, 101: 38-50.PubMedGoogle Scholar
- Gubbels MJ, Wieffer M, Striepen B: Fluorescent protein tagging in Toxoplasma gondii: identification of a novel inner membrane complex component conserved among Apicomplexa. Mol Biochem Parasitol. 2004, 137: 99-110.PubMedGoogle Scholar
- Altschul SF, Wootton JC, Zaslavsky E, Yu YK: The construction and use of log-odds substitution scores for multiple sequence alignment. PLoS Comput Biol. 2010, 6: e1000852-PubMedPubMed CentralGoogle Scholar
- Iyer LM, Anantharaman V, Wolf MY, Aravind L: Comparative genomics of transcription factors and chromatin proteins in parasitic protists and other eukaryotes. Int J Parasitol. 2008, 38: 1-31.PubMedGoogle Scholar
- Peleg O, Lim RY: Converging on the function of intrinsically disordered nucleoporins in the nuclear pore complex. Biol Chem. 2010, 391: 719-730.PubMedGoogle Scholar
- Nguyen Ba AN, Pogoutse A, Provart N, Moses AM: NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction. BMC Bioinformatics. 2009, 10: 202-PubMedPubMed CentralGoogle Scholar
- Cokol M, Nair R, Rost B: Finding nuclear localization signals. EMBO Rep. 2000, 1: 411-415.PubMedPubMed CentralGoogle Scholar
- Kosugi S, Hasebe M, Tomita M, Yanagawa H: Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc Natl Acad Sci USA. 2009, 106: 10171-10176.PubMedPubMed CentralGoogle Scholar
- Mosley AL, Pattenden SG, Carey M, Venkatesh S, Gilmore JM, Florens L, Workman JL, Washburn MP: Rtr1 is a CTD phosphatase that regulates RNA polymerase II during the transition from serine 5 to serine 2 phosphorylation. Mol Cell. 2009, 34: 168-178.PubMedPubMed CentralGoogle Scholar
- Gauci S, Veenhoff LM, Heck AJ, Krijgsveld J: Orthogonal separation techniques for the characterization of the yeast nuclear proteome. J Proteome Res. 2009, 8: 3451-3463.PubMedGoogle Scholar
- Chandra BR, Olivieri A, Silvestrini F, Alano P, Sharma A: Biochemical characterization of the two nucleosome assembly proteins from Plasmodium falciparum. Mol Biochem Parasitol. 2005, 142: 237-247.PubMedGoogle Scholar
- Calvert ME, Keck KM, Ptak C, Shabanowitz J, Hunt DF, Pemberton LF: Phosphorylation by casein kinase 2 regulates Nap1 localization and function. Mol Cell Biol. 2008, 28: 1313-1325.PubMedPubMed CentralGoogle Scholar
- Mancio-Silva L, Zhang Q, Scheidig-Benatar C, Scherf A: Clustering of dispersed ribosomal DNA and its role in gene regulation and chromosome-end associations in malaria parasites. Proc Natl Acad Sci USA. 2010, 107: 15117-15122.PubMedPubMed CentralGoogle Scholar
- Mancio-Silva L, Rojas-Meza AP, Vargas M, Scherf A, Hernandez-Rivas R: Differential association of Orc1 and Sir2 proteins to telomeric domains in Plasmodium falciparum. J Cell Sci. 2008, 121: 2046-2053.PubMedGoogle Scholar
- Logan-Klumpler FJ, De SN, Boehme U, Rogers MB, Velarde G, McQuillan JA, Carver T, Aslett M, Olsen C, Subramanian S, Phan I, Farris C, Mitra S, Ramasamy G, Wang H, Tivey A, Jackson A, Houston R, Parkhill J, Holden M, Harb OS, Brunk BP, Myler PJ, Roos D, Carrington M, Smith DF, Hertz-Fowler C, Berriman M: GeneDB--an annotation database for pathogens. Nucleic Acids Res. 2012, 40: D98-108.PubMedPubMed CentralGoogle Scholar
- Doerig C, Meijer L: Antimalarial drug discovery: targeting protein kinases. Expert Opin Ther Targets. 2007, 11: 279-290.PubMedGoogle Scholar
- Jirage D, Keenan SM, Waters NC: Exploring novel targets for antimalarial drug discovery: plasmodial protein kinases. Infect Disord Drug Targets. 2010, 10: 134-146.PubMedGoogle Scholar
- Jirage D, Chen Y, Caridha D, O'Neil MT, Eyase F, Witola WH, Mamoun CB, Waters NC: The malarial CDK Pfmrk and its effector PfMAT1 phosphorylate DNA replication proteins and co-localize in the nucleus. Mol Biochem Parasitol. 2010, 172: 9-18.PubMedGoogle Scholar
- Dastidar EG, Dayer G, Holland ZM, Dorin-Semblat D, Claes A, Chene A, Sharma A, Hamelin R, Moniatte M, Lopez-Rubio JJ, Scherf A, Doerig C: Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway. BMC Biol. 2012, 10: 5-PubMedPubMed CentralGoogle Scholar
- Holland Z, Prudent R, Reiser JB, Cochet C, Doerig C: Functional analysis of protein kinase CK2 of the human malaria parasite Plasmodium falciparum. Eukaryot Cell. 2009, 8: 388-397.PubMedPubMed CentralGoogle Scholar
- Filhol O, Cochet C: Protein kinase CK2 in health and disease: Cellular functions of protein kinase CK2: a dynamic affair. Cell Mol Life Sci. 2009, 66: 1830-1839.PubMedGoogle Scholar
- Milne DM, Looby P, Meek DW: Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization. Exp Cell Res. 2001, 263: 43-54.PubMedGoogle Scholar
- Cheong JK, Virshup DM: Casein kinase 1: Complexity in the family. Int J Biochem Cell Biol. 2011, 43: 465-469.PubMedGoogle Scholar
- Tudisca V, Recouvreux V, Moreno S, Boy-Marcotte E, Jacquet M, Portela P: Differential localization to cytoplasm, nucleus or P-bodies of yeast PKA subunits under different growth conditions. Eur J Cell Biol. 2010, 89: 339-348.PubMedGoogle Scholar
- Dorin-Semblat D, Schmitt S, Semblat JP, Sicard A, Reininger L, Goldring D, Patterson S, Quashie N, Chakrabarti D, Meijer L, Doerig C: Plasmodium falciparum NIMA-related kinase Pfnek-1: sex-specificity and assessment of essentiality for the erythrocytic asexual cycle. Microbiology. 2011, 157: 2785-2794.PubMedPubMed CentralGoogle Scholar
- Dorin D, Le RK, Sallicandro P, Alano P, Parzy D, Poullet P, Meijer L, Doerig C: Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum Biochemical properties and possible involvement in MAPK regulation. Eur J Biochem. 2001, 268: 2600-2608.PubMedGoogle Scholar
- Rangarajan R, Bei AK, Jethwaney D, Maldonado P, Dorin D, Sultan AA, Doerig C: A mitogen-activated protein kinase regulates male gametogenesis and transmission of the malaria parasite Plasmodium berghei. EMBO Rep. 2005, 6: 464-469.PubMedPubMed CentralGoogle Scholar
- Dorin D, Alano P, Boccaccio I, Ciceron L, Doerig C, Sulpice R, Parzy D, Doerig C: An atypical mitogen-activated protein kinase (MAPK) homologue expressed in gametocytes of the human malaria parasite Plasmodium falciparum. Identification of a MAPK signature. J Biol Chem. 1999, 274: 29912-29920.PubMedGoogle Scholar
- Dorin-Semblat D, Quashie N, Halbert J, Sicard A, Doerig C, Peat E, Ranford-Cartwright L, Doerig C: Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics. Mol Microbiol. 2007, 65: 1170-1180.PubMedGoogle Scholar
- Treeck M, Sanders JL, Elias JE, Boothroyd JC: The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries. Cell Host Microbe. 2011, 10: 410-419.PubMedPubMed CentralGoogle Scholar
- Collins GA, Tansey WP: The proteasome: a utility tool for transcription?. Curr Opin Genet Dev. 2006, 16: 197-202.PubMedGoogle Scholar
- Lee D, Ezhkova E, Li B, Pattenden SG, Tansey WP, Workman JL: The proteasome regulatory particle alters the SAGA coactivator to enhance its interactions with transcriptional activators. Cell. 2005, 123: 423-436.PubMedGoogle Scholar
- Ferdous A, Gonzalez F, Sun L, Kodadek T, Johnston SA: The 19S regulatory particle of the proteasome is required for efficient transcription elongation by RNA polymerase II. Mol Cell. 2001, 7: 981-991.PubMedGoogle Scholar
- Ezhkova E, Tansey WP: Proteasomal ATPases link ubiquitylation of histone H2B to methylation of histone H3. Mol Cell. 2004, 13: 435-442.PubMedGoogle Scholar
- Mair GR, Lasonder E, Garver LS, Franke-Fayard BM, Carret CK, Wiegant JC, Dirks RW, Dimopoulos G, Janse CJ, Waters AP: Universal features of post-transcriptional gene regulation are critical for Plasmodium zygote development. PLoS Pathog. 2010, 6: e1000767-PubMedPubMed CentralGoogle Scholar
- Mair GR, Braks JA, Garver LS, Wiegant JC, Hall N, Dirks RW, Khan SM, Dimopoulos G, Janse CJ, Waters AP: Regulation of sexual development of Plasmodium by translational repression. Science. 2006, 313: 667-669.PubMedPubMed CentralGoogle Scholar
- Brune C, Munchel SE, Fischer N, Podtelejnikov AV, Weis K: Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export. RNA. 2005, 11: 517-531.PubMedPubMed CentralGoogle Scholar
- MacNicol AM, Wilczynska A, MacNicol MC: Function and regulation of the mammalian Musashi mRNA translational regulator. Biochem Soc Trans. 2008, 36: 528-530.PubMedPubMed CentralGoogle Scholar
- Neumann N, Lundin D, Poole AM: Comparative genomic evidence for a complete nuclear pore complex in the last eukaryotic common ancestor. PLoS One. 2010, 5: e13241-PubMedPubMed CentralGoogle Scholar
- Richard D, Bartfai R, Volz J, Ralph SA, Muller S, Stunnenberg HG, Cowman AF: A genome-wide chromatin-associated nuclear peroxiredoxin from the malaria parasite Plasmodium falciparum. J Biol Chem. 2011, 286: 11746-11755.PubMedPubMed CentralGoogle Scholar
- Briquet S, Boschet C, Gissot M, Tissandie E, Sevilla E, Franetich JF, Thiery I, Hamid Z, Bourgouin C, Vaquero C: High-mobility-group box nuclear factors of Plasmodium falciparum. Eukaryot Cell. 2006, 5: 672-682.PubMedPubMed CentralGoogle Scholar
- Albrethsen J, Knol JC, Jimenez CR: Unravelling the nuclear matrix proteome. J Proteomics. 2009, 72: 71-81.PubMedGoogle Scholar
- Boisvert FM, van KS, Navascues J, Lamond AI: The multifunctional nucleolus. Nat Rev Mol Cell Biol. 2007, 8: 574-585.PubMedGoogle Scholar
- Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, Mann M: Nucleolar proteome dynamics. Nature. 2005, 433: 77-83.PubMedGoogle Scholar
- Shaw P, Doonan J: The nucleolus. Playing by different rules?. Cell Cycle. 2005, 4: 102-105.PubMedGoogle Scholar
- Iborra FJ, Jackson DA, Cook PR: Coupled transcription and translation within nuclei of mammalian cells. Science. 2001, 293: 1139-1142.PubMedGoogle Scholar
- Iborra FJ, Jackson DA, Cook PR: The case for nuclear translation. J Cell Sci. 2004, 117: 5713-5720.PubMedGoogle Scholar
- Frankel MB, Knoll LJ: The ins and outs of nuclear trafficking: unusual aspects in apicomplexan parasites. DNA Cell Biol. 2009, 28: 277-284.PubMedPubMed CentralGoogle Scholar
- Chook YM, Suel KE: Nuclear import by karyopherin-betas: recognition and inhibition. Biochim Biophys Acta. 2011, 1813: 1593-1606.PubMedPubMed CentralGoogle Scholar
- Marfori M, Mynott A, Ellis JJ, Mehdi AM, Saunders NF, Curmi PM, Forwood JK, Boden M, Kobe B: Molecular basis for specificity of nuclear import and prediction of nuclear localization. Biochim Biophys Acta. 2011, 1813: 1562-1577.PubMedGoogle Scholar
- Camarda G, Bertuccini L, Singh SK, Salzano AM, Lanfrancotti A, Olivieri A, Scaloni A, Sharma A, Alano P: Regulated oligomerisation and molecular interactions of the early gametocyte protein Pfg27 in Plasmodium falciparum sexual differentiation. Int J Parasitol. 2010, 40: 663-673.PubMedGoogle Scholar
- Sharma A, Sharma I, Kogkasuriyachai D, Kumar N: Structure of a gametocyte protein essential for sexual development in Plasmodium falciparum. Nat Struct Biol. 2003, 10: 197-203.PubMedGoogle Scholar
- Szallies A, Kubata BK, Duszenko M: A metacaspase of Trypanosoma brucei causes loss of respiration competence and clonal death in the yeast Saccharomyces cerevisiae. FEBS Lett. 2002, 517: 144-150.PubMedGoogle Scholar
- Cortes A, Carret C, Kaneko O, Yim Lim BY, Ivens A, Holder AA: Epigenetic silencing of Plasmodium falciparum genes linked to erythrocyte invasion189. PLoS Pathog. 2007, 3: e107-PubMedPubMed CentralGoogle Scholar
- Trager W, Jenson JB: Cultivation of malarial parasites. Nature. 1978, 273: 621-622.PubMedGoogle Scholar
- Lambros C, Vanderberg JP: Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol. 1979, 65: 418-420.PubMedGoogle Scholar
- Voss TS, Healer J, Marty AJ, Duffy MF, Thompson JK, Beeson JG, Reeder JC, Crabb BS, Cowman AF: A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria. Nature. 2006, 439: 1004-1008.PubMedGoogle Scholar
- Daubenberger CA, Tisdale EJ, Curcic M, Diaz D, Silvie O, Mazier D, Eling W, Bohrmann B, Matile H, Pluschke G: The N'-terminal domain of glyceraldehyde-3-phosphate dehydrogenase of the apicomplexan Plasmodium falciparum mediates GTPase Rab2-dependent recruitment to membranes. Biol Chem. 2003, 384: 1227-1237.PubMedGoogle Scholar
- Riglar DT, Richard D, Wilson DW, Boyle MJ, Dekiwadia C, Turnbull L, Angrisano F, Marapana DS, Rogers KL, Whitchurch CB, Beeson JG, Cowman AF, Ralph SA, Baum J: Super-resolution dissection of coordinated events during malaria parasite invasion of the human erythrocyte. Cell Host Microbe. 2011, 9: 9-20.PubMedGoogle Scholar
- Witmer K, Schmid CD, Brancucci NM, Luah YH, Preiser PR, Bozdech Z, Voss TS: Analysis of subtelomeric virulence gene families in Plasmodium falciparum by comparative transcriptional profiling. Mol Microbiol. 2012, 84: 243-59.PubMedPubMed CentralGoogle Scholar
- Tonkin CJ, van Dooren GG, Spurck TP, Struck NS, Good RT, Handman E, Cowman AF, McFadden GI: Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method. Mol Biochem Parasitol. 2004, 137: 13-21.PubMedGoogle Scholar
- Kumar N, Koski G, Harada M, Aikawa M, Zheng H: Induction and localization of Plasmodium falciparum stress proteins related to the heat shock protein 70 family. Mol Biochem Parasitol. 1991, 48: 47-58.PubMedGoogle Scholar
- Struck NS, Herrmann S, Schmuck-Barkmann I, de Souza DS, Haase S, Cabrera AL, Treeck M, Bruns C, Langer C, Cowman AF, Marti M, Spielmann T, Gilberger TW: Spatial dissection of the cis- and trans-Golgi compartments in the malaria parasite Plasmodium falciparum. Mol Microbiol. 2008, 67: 1320-1330.PubMedGoogle Scholar
- Fink JL, Hamilton N: DomainDraw: a macromolecular feature drawing program. In Silico Biol. 2007, 7: 145-150.PubMedGoogle Scholar
- Jalview. [http://www.jalview.org]
- LogoMat-P. [http://www.sanger.ac.uk/resources/software/logomat-p/]
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|Copying 18th-century letters
Fredericksburgh Meeting members considered themselves remote and independent, so they reported directly to London. I never thought that was strange until I happened to mention to the librarian that most of these pioneers were Irish. His implied question was immediate: wouldn't they report to Ireland? But no, the letters are there. Maybe it was because the Religious Society of Friends started in England; London was considered the mother ship. The letters show great loyalty and attachment, even though the people writing and reading them probably never met.
With London's insistence, Fredericksburgh eventually joined New Garden Quarter, based in North Carolina. They really didn't want to. They wrote that they were "isolate" and "remote." The nearest monthly meeting, Bush River, was about 75 miles away, and New Garden a couple hundred miles. The funny thing is that during those days of walking or traveling by horse, Friends "in this wilderness" felt like New Garden was as out of reach as Mars; they preferred to answer to people on the other side of the ocean.
I found a clue. The helpful librarian brought me an article about Fredericksburgh. They turned over their minutes to Bush River Meeting about 1783. So, at some point, over 200 years ago, the minutes existed. Somebody did write things down.
|The library at Quaker House. Old prohibition posters adorn the balcony.


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Tenugui Japanese Towel - Fan & Flying Crane
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Availability: In Stock
Item# M-7105
Overall size: 35" (L) x 13" (W)
Finish: Plain cut, edge not hemmed
A tenugui is a thin Japanese hand towel made of cotton with over 1,000 years of history. Originally, tenugui were used in Shinto rituals and by samurai to keep the sweat out of their eyes when wearing helmets. In the Edo period, the tenugui became valued not only for its practical function but also for its artistic possibilities. To this day the tenugui retains some traces of its samurai origins. Lacking a sewn in hem enabled samurai to tear long strips from their tenugui to bind wounds sustained in battle.
Tenugui can be used as hand towels, bandanas, handkerchiefs, cloth for wrapping small gifts and an elegant towel to clean and polish tools. Cut them in half and use as sustainable cloth napkins!
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The most disturbingly long absence in the history of Plenty of Colour! Well, to be honest, 2014 didn't have the robust number of posts I was shooting for overall. Thank you to those who kindly emailed me. Sometimes life gets in the way and one ends up with a pile of half-done posts. But, I am still as colour-obsessed as ever so upward and onward in 2015! And perhaps a blog overhaul too? Regardless, I think a great way to start the new year is with some beautiful packaging. Project Charisma is a a promotion project for Polytrade Paper designed by design studio BLOW. The showcase includes striking, simple cosmetic packaging — blocks of colour, clean type and downright beautiful metallic foils. A happy and colour-filled 2015 to you all!
(photographs via ken lo / blow)


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UNIVERSITY PARK, Pa. – New York University professor Pedro Noguera discusses the U.S. education system's most pressing issues on the next installment of WPSU-TV's "Conversations from Penn State."
The episode will air at 8 p.m. Thursday, Nov. 14 on WPSU-TV. The show can also be viewed at http://conversations.psu.edu.
Noguera joins host Patty Satalia to examine strategies for addressing achievement gaps in gender and race, as well as the issues with federal education programs like No Child Left Behind and Race to the Top.
Noguera is the Peter L. Agnew Professor of Education at NYU. Also an affiliate faculty member of NYU's Department of Sociology, Noguera's research focuses on how social and economic conditions impact the education system.
"Conversations from Penn State," with Satalia at the helm, offers thoughtful, in-depth exchanges with a broad range of people. In many cases, their trailblazing work has advanced their field, drawing national and worldwide attention.