text:
From James Samuelson 8 April 1866
6 Montpellier Terrace | New Brighton Cheshire Apl 8/66 Dear Sir.
I am glad you consider the notice of your works an unprejudiced one.1 It seems, strangely enough, to find favor with the rigid orthodox as well as with those who are disposed to consider the subject without reference to theology. & I hope it will give a stimulus to the perusal of your works.
I cannot, for my own part, conceive how any one who considers the past and present of organic life, without theological bias, can find it reasonable to suppose that animals have sprung into existence as it was formerly believed, and a gradual development appears to me the only sensible mode of explaining the matter.
On the other hand, unless you are greatly misunderstood, by me as well as others, I am at a loss to comprehend how you can have arrived at the metaphysical result stated or implied, at p 492 of the 'Origin'. (3d Edn.) "not by means superior to, though analogous with" &c.2
Assuming your theory to be correct; that the metaphysical causes—in other words that the creative or divine power which makes or has made new species is not superior to, though analogous with, human reason; what is the use of continuing your efforts to make a new species? It is only on the assumption that "Nature" acts in a manner superior to though analogous with human reason, that the question of artificial selection can have the slightest weight in determining the natural method of creating species;—I am not going to read you a sermon! You have had enough of those I should think! but judging from some of the explanatory remarks you have introduced into your last edition, referred to by your reviewer at p. 153 (J of S.) I should say that you have, from conviction, not coercion, somewhat changed your views as regards the nature of the metaphysical influences at work; or at least that you have felt yourself to be misunderstood, in the amount of power attributable to "Natural Selection".3
I think you are still misunderstood, in a way which is calculated to impede the progress of your views.
As to 'Natural selection' modifying the Egg—seed &c, "by preserving favorable variations",4 I confess myself totally unable to perceive how you can, in a large majority of cases, ascertain anything whatever as to the influences which are at work in or upon the ovum, germ, or spermatozoon. In the lower & lowest forms of life, these are at present often less than microscopic, & when our powers increase, I do not think we shall be much wiser in that respect. I say this with every deference to your large experience and observation; though as you may perhaps be aware, I have devoted many years to this particular branch of Science. You will probably have seen that I sent a paper last Autumn to the Royal Society in which I have sought to show by experiment that the developmental theory must, in the case of the infusoria, take the place of that of spontaneous generation; but although in the paper (of which an abstract only appeared in the proceedings) I have expressed my conviction that one and the same form of zoospore, monad, or whatever you choose to call it, may become developed into what are now believed to be distinct species of Infusoria, (& I sent an illustration of what I believe to be such a phenomenon) yet I cannot go so far back as the "ovum, germ, or seed" in forming a judgment upon the causes of variation.5
You will consider me very un-Editorial, I know, for entering upon this discussion, but I received your note, & reply to it, not in my official capacity, but as a young fellow-observer—if you will not be scandalised by the comparison.
Believe me, Dear Sir, | With every regard, Yrs truly | James Samuelson
C Darwin Esq
Pleased CD does not consider review of his works prejudiced [Anon., "Darwin and his teachings", Q. J. Sci. 3 (1866): 151–76].
Supports gradual development of species over time.
Confused by the metaphysical view implied in the analogy between a creative power that has made new species and artificial selection governed by human reason (Origin, 3d ed., p. 492).
Doubts natural selection.
Cites his discussion of the origin of Infusoria [Proc. R. Soc. Lond. 14 (1865): 546–7].
Letter details
- Letter no.
- From
- James Samuelson
- To
- Charles Robert Darwin
- Sent from
- New Brighton, Cheshire
- Source of text
- Physical description
- 6pp
Please cite as
Darwin Correspondence Project, "Letter no. 5049," accessed on 19 May 2019, http://www.darwinproject.ac.uk/DCP-LETT-5049
Also published in The Correspondence of Charles Darwin, vol. 14


text:
A great recycling idea from The Goodwill Gal Inbox:
Hello Goodwill Gal,
I thought I would send you a project that I was inspired to do this week.
First, I love following you and your amazing projects. I am beginning to be able to walk through Goodwill and try to look at objects differently and imagine what they can be.
I bought a bookshelf on the day that yellow tags were half off. It was $6.51. I brought it home took the back off and placed it next to my other container garden (which was much more expensive). I filled it with compost and potting soil and marked off the square foot plots. Since I planted seeds, it will be awhile before you can see the final product. In the garden are chamomile, echinechea, fennel, anise, lettuce and spinach.
Last, I also could not pass up these containers that were also at Goodwill (they were purchased with my 20% off coupon). I splurged on the round one (It was 27.99), but I love it! The box with the lid was 11.99. They are going to have some pansies, kale, celery and maybe some daisies (depending on space).

subset: commoncrawl


text:
- Research article
- Open Access
Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase
- James H Keith1Email author,
- Cheryl A Schaeper1Email author,
- Tresa S Fraser1Email author and
- Malcolm J FraserJr1Email author
© Keith et al; licensee BioMed Central Ltd. 2008
- Received: 06 March 2008
- Accepted: 11 August 2008
- Published: 11 August 2008
The piggyBac mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of piggyBac, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for piggyBac at amino acid positions D268, D346, and D447.
This study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the piggyBac transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the piggyBac transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features.
We found all the designated DDD aspartates reside in clusters of amino acids that conserved among piggyBac family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase.
- Donor Plasmid
- Blue Coloni
- Excision Event
- Precise Excision
- Transposition Reaction
With the successful application of the Drosophila melanogaster P element as a tool for the creation of transgenic fruit flies, researchers had high hopes for the stable introduction of transgenes into germlines [1]. Unfortunately, P element mobility was determined to be restricted only to D.melanogaster and very closely related species [2]. The ensuing search for novel mobile elements that are active within a wide range of species led to the classification and characterization of the hAT, Tc1-mariner, and piggyBac families of mobile elements [3]. The piggyBac transposon is a short repeat, class II mobile element originally isolated as the causative agent of the Few Polyhedra plaque morphology mutations of Autographa california nuclear polyhedrosis virus (AcNPV) [4]. It is the archetype of its own family and the only fully active transposable element in that family that is currently in use as a transgenic vector. The wild-type piggyBac element is 2.4 kb long and ends in a 5' CCC...GGG 3' configuration with asymmetric terminal and subterminal repeats. The sub-terminal repeats are 19 bp in length and lie 3 bp and 31 bp inside of the perfect inverted terminal repeats (ITRs) on the 5' and 3' end respectively [4]. The protein catalyzing the movement of piggyBac is encoded within a single open reading frame (ORF) of 1783 bp, with a length of 594 amino acids and a predicated mass of 68 kDa [4, 5]. As a class II mobile element, piggyBac operates via a DNA intermediate in a cut-and-paste mechanism [6]. In this scenario, the transposable element, delineated by transposase-specific ITRs is excised by the transposase and reinserted into a new location. In the case of piggyBac, the bias for individual sites chosen during reinsertion does not follow any clearly defined rules. Although the only sequence requirement for a new insertion event is the presence of a TTAA sequence in the DNA, there is a clear preference for certain TTAA sites over others as shown by inter-plasmid transposition assays [7].
The piggyBac element has been used to transform the species Mus musculus [8], Tribolium castaneum [9], Anopheles gambiae [10], Ceratitis capita [11], Drosophila melanogaster [12], Bactrocera dorsalis [13], Musca domestica [14], Lucilia cuprina [15], Bicyclus anynana [16], Aedes aegypti [17, 18], Anopheles albimanus [19], Anopheles stephensi [20], Bombyx mori [21], Athalia rosae [22], Drosophila willistoni [23], Pectinophora gossypiella [24], Anastrepha suspensa [25], Aedes fluviatilis [26], Harmonia axyridis [27], Schistosoma mansoni [28], and the causative agent of malaria, Plasmodium falciparum [29]. Plasmid-based mobility assays have also shown piggyBac to be active in human and other primate cells [8, 30], in Zea maize cells [31], Spodoptera frugiperda cells [5], and in the embryos of Aedes triseriatus [32], Heliothis virescens [33], and Danio rerio [30], although none of these species have yet been transformed with piggyBac.
Recently, Mitra et al. [34] have defined piggyBac mediated transposition as involving several discrete steps. First, the transposase creates 3' nicks at each end of the terminal repeats just internal of the 'TTAA'. The free 3'-OH attacks the complimentary strand, four nucleotides downstream of the initial nick, just outside of the 'TTAA' repeat, forming a transient hairpin structure and releasing the transposon ends. The transposase quickly resolves these hairpin structures into 5' 'TTAA' overhangs on either side of the excision product. For reinsertion, the transposase joins the excised element to a new 'TTAA' target site in a staggered pattern, completing the transposition reaction. This mechanism accounts for both precise excision and target site duplication.
For all transposable elements, there are certain requirements for transposition, namely the recognition and binding of the terminal repeats, double stranded DNA breakage (DSB), and reinsertion of the element into a target, as well as possibly the repair of the excision site, which may or may not involve host factors [3]. DNA manipulating enzymes, such as integrases, recombinases, resolvases, endonucleases, and transposases must meet one or more of these requirements in order to successfully catalyze their respective reactions. With this commonality in function comes some similarity in mechanism and structure. One of the most widespread of these features is the 'DDE/DDD' motif first identified and designated in retroviral and retrotransposon integrases [35]. This motif has since been identified among many different families of DNA manipulating enzymes, from certain nucleases to the enzymes that carry out V(D)J recombination and even the human RNAi enzyme, Ago2 [36–47]. 'DDE/DDD' has been implicated as the active site for such enzymes by providing carboxyl groups used to recruit metal ions as essential cofactors. These residues are required to precisely position the metal ions, usually magnesium or manganese, used to recruit nucleophilic groups to the catalytic center of their respective enzymes.
Sarkar et al. published an extensive computational analysis of the piggyBac element and related sequences [48] that demonstrated the widespread occurrence of the piggyBac family of elements. While no actual experiments were performed by Sarkar et al., they proposed a 'DDD' motif for piggyBac, similar to a paper published in 1996 in which Bigot et al. speculated the existence of a 'DSE' motif in hAT elements based on sequence alignments. Sarkar et al. based their proposal on weak matches for two piggyBac related proteins to the bacterial IS 4/5 transposase family DDE motif. This alignment suggested the first two of these aspartates are D268, and D346. While this similarity with the IS 4/5 transposases did not yield a hit for the third member of the 'DDE' triad, Sarkar et al. speculated that the third member was an aspartate at D447.
In this report, we align very closely related members of the piggyBac family across kingdoms and identify several highly conserved regions. We propose that a direct relationship exists between the degree of conservation of a particular feature, and its necessity to catalyze transposition, as they are favored at the time of horizontal transmission. To this end, we only considered the proteins most closely related to piggyBac in our alignment. However, it should be made clear, when considering the results of our alignment, aside from piggyBac itself, only one other family member has been shown to be a functional element: Xtr-Uribo2_PCR_Iv1b (Genbank Accession: BAF82025) from the Xenopus family, which catalyzes movement in GP293 cells [49]. Therefore, we are careful to judge the relevance of any well conserved feature primarily upon its presence in either piggyBac or Uribo-2.
Finally, we mutate the three aspartates identified by Sarkar et al. as well as D450, another highly conserved residue close to the third proposed aspartate, which may also contribute to the catalytic center of the transposase. We test these site-directed mutant transposases for excision activity in two different donor plasmids and report our results.
Sequence alignment
Proteins used in the alignment
Protein Name
Trichoplusia ni
Daphnia pulicara
Pokey 6.6 kb
Daphnia pulicara
Pokey 5 kb
Helicoverpa armigera
Bombyx mori
Bombyx mori
Bombyx mori
Xenopus laevis
Xenopus tropicalis
Xenopus laevis
Xenopus borealis
Xenopus tropicalis
Tetraodon nigroviridis
Unnamed Product
Homo sapiens
Homo sapiens
Mus musculus
Homo sapiens
Homo sapiens
Homo sapiens
Tetrahymena thermophila
Predicted protein
Ajellomyces capsulatus
Predicted protein
Nasonia vitripennis
Predicted protein
Anopheles Gambiae
Bos taurus
Predicted protein
Danio rero
Predicted protein
Strongylocentrotus purpuratus
Predicted protein
The core of the transposase possesses clusters of conserved amino acids with very well defined borders for each conserved stretch. The first conserved cluster we identified begins shortly downstream of a conserved proline, P131, and may mark the beginning of a functional domain in piggyBac transposase family members. The next clusters of conservation lie roughly from P131 – N152, E175 – K190, and V207 – D228 (Fig. 2, 3). The region between D239 – Y259, while not particularly conserved in piggyBac itself, is conserved in other members of the family. Additionally, the next proline from this cluster, P261, starts another highly conserved region which includes the first member of the proposed 'DDD' triad, D268 (Fig. 4).
The next group of conserved residues contains the second aspartate, D346 and an absolutely conserved glycine at position 369. G369 marks the start of the well conserved motif 'GTVRxNKRxIP' (Fig. 5). This motif ends with P379, possibly delineating the end of a functional domain. This motif is not present in its entirety in Uribo-2. However, the analogous sequence of Uribo-2 'GTINRNRKxLP' does preserve similar features.
Downstream from this region is another conserved group that starts with a highly conserved proline at position 433. P433 is only a short distance from D447 and D450, the last two aspartates tested in our study, and is the start of another motif: 'PxxxxxxYxxxxGGVDxxD.' The aspartates in this motif are D447 and D450, respectively (Fig. 5, 6).
A cysteine-rich domain overlapping with the piggyBac nuclear localization signal (NLS) is positioned at the C-terminus of piggyBac. The spacing between these cysteines is well conserved among piggyBac family members when present, even though the intervening amino acids are not. Additional cysteines also lie within this region, conforming to a novel zinc-finger domain in piggyBac [52, 53]. A basic amino charge at R568 is also well conserved (Fig. 7).
Excision assay
Our mutational analysis demonstrates that all members of the proposed triad, D268, D346, and D447, are required for efficient piggyBac catalyzed excision in a cellular environment. The additional amino acid targeted for mutational analysis, D450, has a primary requirement for a negative charge, since the asparagine replacement significantly inhibited excision, while a glutamate replacement yielded moderate excision, although not to the degree of the wild-type transposase.
Excision frequencies for donor plasmids
REN Screen
Excision Frequency
Excision Frequency
Western blot
Sarkar et al. suggested the presence of a 'DDD' motif for piggyBac, without empirical evidence of the association of the motif with mobility. Their proposal was based on two weak matches returned when two piggyBac related proteins, but not piggyBac itself, were used as a query in an NCBI Conserved Domain Search. These query hits were for the Pfam domain pfam01609 [48]. The conserved D268 and D346 were thus matched to the bacterial IS 4/5 transposase family DDE motif based on their degree of conservation in the transposase core and the presence of a glutamate immediately following D268, and an asparagine following D346. No reason for choosing D447 was cited, but both D447 and D450 are part of a highly conserved motif discussed below.
Bigot and colleagues [54] used a similar approach of transposase alignments to propose the existence of a 'DDE' motif in hAT elements, with the second aspartate replaced by a serine in Ac, hobo, and Hermes. They aligned members of the Tc1-mariner superfamily with members of the hAT family and identified conserved and similar residues common between both groups. The 'DSE' speculation has since been empirically disproven [55]. However, of this 'DSE' triad, both D402 and E572 (with respect to Hermes) were essential for transposition while alteration of S535 to either an alanine or an aspartate had no statistically significant effect on transposition efficiency. This, at least, demonstrated the necessity for D402 and E572, but did not prove if either residue was part of an essential triad.
Starting at the N-terminus of piggyBac there are four acidic amino acids: D32, D38, E45, and D49 that are present in most of the aligned proteins (Fig. 1). While charges are conserved at these positions, the residues themselves do not seem to have any requirement as to whether they are an aspartate or a glutamate. Interestingly, this interchangeability is particularly variable at these positions, even within closely related proteins, such as the piggyBac related proteins in three different species of Xenopus, and among the human piggyBac derived proteins (PGBD). Examination of piggyBac related transposons in Xenopus identified three elements, Uribo-1, Uribo-2, and Kobuta. Xtr-Uribo2_PCR_Iv1b proved to be a functional mobile element complete with transposase able to catalyze movement in GP293 cells [49]. Kobuta, however, lacks excision activity. Xtr-Uribo2_PCR_Iv1b possesses a glutamate at piggyBac's D32 and does not have a match for E45, even though other inactive Xenopus putative transposases, Uribo-1 and Kobuta, do have matches for E45. Additionally, these N-terminal acidic residues are spaced so closely together that they are unlikely to be the DDD/DDE triad in our opinion. Interestingly, both Uribo proteins contained analogs to D268, D346, and D447, while the inactive Kobuta protein contained only D346 as a rule with two divergent Kobuta examples also having the D268 residue. All Xenopus proteins also harbor the highly conserved D450 residue.
Distinct clusters of conserved amino acids are present through the rest of the piggyBac family starting at P131 (Fig. 2). The fact that the first constellation of conservation begins with a proline is worth noting, as proline is known to disrupt the periodic structure of α-helices and β-sheets, often demarcating the protein from one functional domain to the next [56]. Together with the adjacent region of conservation, we speculate that the area just downstream of P131 is most likely a functional domain in the transposase. A conserved domain search returns an extremely weak (e = .44) match to pfam02388, Staphylococcus proteins involved in formation of the peptidoglycan layer, a coincidence in our opinion.
The piggyBac family analog to K246 is also a highly conserved proline, but as this amino acid is not present in piggyBac it is not required for a functional transposase (Fig. 3). However, P261, while less conserved than the K246 proline analogs, is present in both piggyBac and Uribo-2. It also lays just N-terminal of the very well conserved residues D268, the first member of the 'DDD' triad, and E269.
The next cluster of conserved amino acids includes D346, the second member of the proposed 'DDD' triad (Fig. 4). Also in this region lies the only residue in the alignment that is absolutely conserved is G369 (Fig. 5). This glycine is the start of a nearly universally conserved motif among piggyBac related proteins: 'GTVRxNKRxIP.' While R372 is limited to arginine, the other two basic amino acids, K375 and R376, seem to be conserved only in charge, as some proteins use either arginine or lysine at these positions. When a basic amino acid occupies sites analogous to both position 375 and 376 in a protein, it is always one of each and never the same residue, except for Strongylocentrotus purpuratus which utilizes arginines in both locations. I378 is also another residue conserved only in properties, in this case hydrophobicity. Methionine, leucine, and isoleucine each are employed at this site with no clear pattern as to which is used outside of immediately related members. Finally, P379 does not seem to indicate the start of any highly conserved clusters, but it does lie at the end of a conserved cluster starting at approximately E331, possibly ending a functional domain in piggyBac. Of note, this motif is not present in its entirety in the functional transposase, Uribo-2. However, the analogous sequence of Uribo-2 'GTINRNRKxLP' does preserve similar features.
The next highly conserved proline, P433, is located only a short distance from P379, just upstream of the third proposed aspartate, D447, and another highly conserved aspartate at D450 (Fig. 5). This proline delineates the start of one of the most highly conserved motifs we found in our alignment: 'PxxxxxxYxxxxGGVDxxD' which contains both D447 and D450. This motif is present in both piggyBac and Uribo-2 as well as most other members of our analysis. Among members where this motif is loosely present, G444 is replaced by an alanine in the silkworm Yabusame proteins, and G445 is replaced with an alanine in the human PGBD-4. P433 could mark the start of another required functional domain in piggyBac, and for this reason we decided to mutate both D447 and D450. In the more divergent proteins, mouse PGBD-5, human PGBD-5, human PGBD-1, Anopheles gambiae PGDB protein XP_312615, and Ajellomyces capsulatus predicted protein XP_001599370, this motif is not fully present.
The C-terminus of most piggyBac related proteins contains a cysteine-rich motif: C559, C562, C574, C577, and C582 (Fig. 7). The spacing between these residues is somewhat conserved even though the intervening residues are not. In piggyBac, this region is also a novel match for a RING-finger motif [52], a type of Zinc-finger which has been implicated in protein-protein interactions [53]. The high degree of conservation in this region suggests a selective pressure for the presence and spacing of these cysteines. Recently, Mitra and colleagues [34] have suggested this Zinc-finger could in fact be a PHD finger, implicated in heterochromatin interactions. The ability of their purified transposase to function in an in vitro environment on free, unwound DNA despite the removal of this C-terminal domain is consistent with this hypothesis. The occurrence of a basic charged residue at R569 is also a commonality.
In piggyBac, we have demonstrated that this area contains a functional NLS (aa 551–571) (in press). In fact, this region is so rich with basic amino acids, that 4 PSORTII [57] predicted NLS matches exist in this short region: 2 monopartite at positions 551 and 563, and 2 bipartite signals at positions 554 and 555, respectively. Interestingly, this NLS cluster is not present in the functional Uribo-2 transposase, however a PSORT analysis of Uribo-2 shows it harbors a NLS matching a monopartite pattern beginning at P276, with respect to the Uribo-2 sequence.
The excision assays reveal that substitution of D268, D346, D447, or D450 with either an asparagine or a glutamate impairs the function of the transposase from the donor plasmid pCR2.1-piggyBac{SV40}. In contrast, while the substitution D450E significantly impaired excision from pCR2.1-piggyBac{SV40}, it did not appear to have a statistically significant effect with regards to pBKOα. The reasons for this apparent difference reflect level of error inherent in the protocols between the two assays. For the REN colony screen, a randomly chosen representative cross section is cultured and digested from the colonies available. This method is prone to a larger amount of error as fewer colonies are screened for excision events. However, the blue/white colony screen permits the analysis of all the colonies on a plate for excision events. This provides a much larger population of donor plasmids to be analyzed and reduces the standard error.
These four residues mutated in our study are far from the only conserved acidic amino acids in the piggyBac family alignment: the short cluster at the amino terminus previously discussed, as well as D141, E175, D249, and D300 are all conserved to a degree, and may be involved in transposition. Each of the mutations tested severely reduced or completely shut down excision activity of the transposase. Previous studies of acidic amino acids in Hermes support the idea that essential amino acids are not limited to just the three members of a proposed triad, as alterations to D180, D248, D402, and E572 in the Hermes transposase all affect transpositional activity [55, 58].
Here, we test only the excision step of the entire transposition reaction in a cell culture system. There are other possibilities that cannot be ruled out by our data, including the necessity of these residues in forming a functional secondary structure of the transposase. We cannot say for certain which specific parts of the excision process these mutations hinder. It is entirely possible that they are required for DNA binding or possibly the recruitment of auxiliary host factors. However, the reports of Mitra et al. [34] examining piggyBac in function an in vitro system suggests that the transposase alone may carry out all steps of transposition without any additional requirement for host factors. Substitution of D268, D346, and D447 with alanine does not inhibit specific binding of the transposase to the terminal repeats of piggyBac [34], but each of these mutations abrogates all steps of transposition, including 3' OH nicking and hairpin formation – both integral steps for excision. Furthermore, unlike wild type piggyBac, these mutants are defective for target joining when synapsed with an artificial excision fragment. Testing these mutants in a yeast system also demonstrated they were functionally defective [34]. The lack of catalytic activity in our D268, D346, and D447 mutant assays confirms the relevance of the observation of Mitra et al. to piggyBac movement in insect cell systems.
In contrast, Mitra et al. demonstrate that D450A is still catalytically active in an in vitro environment, while our results show a definite interfering effect. One explanation for this discrepancy is that D450 is likely not a part of the metal interaction motif, but may be involved in the proper folding of the protein with limited tolerances for certain amino acids. Certainly, our findings support that D450 is the least critical of the four aspartates we tested, but is still necessary to a degree.
Just because an amino acid is acidic and required for transposition does not automatically make it a member of a divalent metal interaction motif, nor does particular mutant interfere with all steps of transposition. An example of this can be found in a mutational study of the V(D)J recombination initiator, RAG1. In this experiment, a great number of acidic amino acids were mutated, some of which impaired cleavage activity. One of these mutations, E811Q, was deficient for DNA binding activity, a loss-of-function that could indirectly lead to decreased cleavage activity. This study also defined two classes of mutants: class I which retained a measure of cleavage activity, and class II which yielded no detectable cleavage products, even though they retained DNA binding activity [42]. It is possible that alteration of D450 in piggyBac may be analogous to such a class I mutant.
Another example of a mutant transposase deficient for specific steps of transposition would be the D248A mutant of Hermes, which was deficient for DSB activity. Upon further investigation, this mutant appeared to only be deficient in the first 5' nicking step of excision. When supplied with a pre-nicked substrate, it was able to facilitate hairpin formation and a measure of target joining in the presence of Mn2+, the second step of DSB and the final step of transposition, respectively [58]. These examples illustrate that a number of steps exist in which a mutant could interfere with transposition, thereby decreasing the formation of the end product.
Iron-induced hydroxyl radical protein cleavage is one direct test for metal binding activity [59]. This assay takes advantage of the ability of Fe2+, in the presence of ascorbate or H2O2, to generate hydroxyl radicals. If the protein in question has the ability to bind Fe2+, then hydroxyl radicals will be generated at the sites where the ion is located. These radicals subsequently cleave the peptide backbone of the protein which bound the Fe2+, yielding cleavage products with sizes consistent with the location of the metal binding pockets. Regarding recombinases, this approach was used to narrow down the location of the metal binding residues of RAG1 prior to the generation of site-specific mutants [43].
Another common test for metal interaction is the replacement of either the aspartate or the glutamate with a cysteine [60]. This test is based on the ability of glutamate and aspartate metal ligand residues to supply a carboxyl group for metal interaction. These amino acids use the oxygen on their R-chain as the interacting atom via O-Mg2+ or O-Mn2+ formation. However, interactions with Mn2+, but not Mg2+, are also able to occur using a cysteine via a S-Mn2+ bond [61]. Such an interaction can be analogous to the metal binding activities of glutamate and aspartate. For example, substitutions D180C, D248A, and D248C in Hermes all showed the ability to catalyze hairpin formation of substrate DNA in the presence of Mn2+ during the excision step of transposition, but only D180C and D248A were able to complete the joining of the transpososome to target DNA. E572A and E572C left Hermes unable to facilitate any step of the transposition reaction [58].
Finally, much work has been done with X-ray crystallography on integrases, resolvases, and transposases at different stages of catalysis, including multimers, free floating proteins, and enzymes complexed with their target DNA. Direct resolution of these structures has been invaluable to the understanding of structure-function relationships. Indeed, the various studies have found structural similarities in the active sites of the recombinase family and nucleases, including the DDD/DDE metal binding motif for both one and two metal binding proteins [62–69]. In light of what we and others have found [34] it would not be surprising to find piggyBac shared many features common to the recombinase family.
The piggyBac family alignment revealed a number of interesting features in the transposase. Regions of high conservation in the catalytic core were sometimes demarcated by proline residues, possibility separating functional domains. Furthermore, we found four conserved acidic residues at the N-terminus of the transposase, and four more throughout the rest of the transposase. A cysteine-rich region with somewhat conserved spacing exists at the C-terminus and is a novel match for the RING finger protein-interaction motif. Our data indicate which of the four tested residues are essential for transposase-mediated excision from a donor plasmid in Drosophila cultured cells. Our donor plasmids yielded similar results, however a charge-preserving substitution from aspartate to glutamate was at least moderately tolerated at positions D450. The existence of an acidic charge was necessary at all four positions tested for both of our donor plasmids with a specific requirement for an aspartate at D268, D346, and D447. We conclude that the four amino acids tested are indeed vital for efficient excision of the piggyBac element from a donor plasmid in cell culture.
Sequence retrieval and alignment
The predicted piggyBac translated protein was used to scan GenBank [50] in a protein-BLAST search of the non-redundant database. Hits that were not from Trichoplusia ni were chosen for a multiple sequence alignment in ClustalW [70]. The resulting .msf alignment was input into the BoxShade server in order to more easily identify the conserved regions.
Plasmid Construction
pXL-BacII-SV40 was constructed by amplifying the SV40 terminator sequence from pMT/V5-HisA (Invitrogen, Carlsbad, CA) with the primers: Sense: 5' TGTGCGGCCGCAGTCTAGAAAAGGATCCTAGATCATAATCAGCCATACCACATTTGTAGAGG 3' and antisense: 5' TGGGAGCTCATAAGCCGTATCGATAAGCTTTAAGATACATTGATG 3' with Pfx high-fidelity polymerase (Invitrogen) according to manufacturer protocol. The PCR reaction was separated on .9% agarose gel stained with 10 μg/ml ethidium bromide and the band corresponding to the SV40 fragment was isolated with the Wizard SV Gel Purification System (Invitrogen). The fragment was then cut with Bam HI (New England Biolabs (NEB), Ipswitch, MA) and Cla I (NEB) and ligated into the vector pXL-BacII [71] at the same sites to form pXL-BacII-SV40. The plasmid was sequenced to verify accuracy.
To create pCR2.1-piggyBac{SV40}, the piggyBac{SV40} fragment was amplified from pXL-BacII-SV40 with the primers: Sense: 5' AAACCCAAAGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGGGG TT AA CCCTAGAAAGATAATCATATTGTGACG 3' and Antisense: 5' CCCAAACCCGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGGGG TT AA CCCTAGAAAGATAGTCTGCGTAAAATTG 3' using Pfx high-fidelity polymerase. The 'TTAA' sites delineating the ends of the piggyBac donor fragment are in bold while the sequences remaining behind after precise excision are underlined. The fragment corresponding to piggyBac{SV40} was band-isolated as described above and cut with the restriction enzymes Acc 65I (NEB) and Not I (NEB) This fragment was then ligated into the vector pCR2.1-Topo (Invitrogen) at the same sites. The plasmid was sequenced to verify accuracy.
The various mutants of the piggyBac open reading frame were prepared by Bio Basic Inc. (Biobasic Inc., Ontario, Canada) in the vector backbone phsp-pBac [12] according to the specifications listed in Table 1.
Blue/White colony screen
The blue/white colony screen was developed to enable us to determine, by visual inspection, which colonies had undergone precise excision. The piggyBac{SV40} cassette was inserted at a 'TTAA' site in the pCR2.1 backbone, in the midst of the lacZ ORF. The piggyBac expression plasmid and the pCR2.1-piggyBac{SV40} are co-transfected into D. melanogaster Schneider2 (S2) cells and incubated. The plasmids are recovered by Hirt extraction and transformed into E. coli. In the presence of the cassette, the lacZ ORF is interrupted, yielding a white colony phenotype on kanamycin containing media with IPTG and 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal). However, when precise piggyBac-catalyzed excision occurs in this vector, the lacZ ORF is restored and the subsequent E. coli colony will possess a blue phenotype (Fig. 10). Plating on kanamycin containing media ensures that only donor plasmids will develop into colonies, and helper expression plasmids will not lead to false white colonies. By comparing the number of blue colonies to the number of total (blue+white) colonies, we can extrapolate the excision frequency. That is, the number of excision events occurring out of the total number of potential excision events.
106 S2 cells were allowed to adhere to the bottom of a 9.6 cm2 cell culture well and were transfected with .4 μg of phsp-pBac helper plasmid and .4 μg of pCR2.1-piggyBac{SV40} donor plasmid with Transfectin liposomal transfection reagent (Bio-Rad Laboratories, Hercules, CA) according to manufacturer protocol in minimal S2 media (Gibco, Carlsbad, CA). The cell/transfection media mixture was allowed to sit at room temperature overnight. The next morning, the transfection media was aspirated from the cells and replaced with complete S2 media (Minimal S2 supplemented with 10% FBS, .1 mg/mL streptomycin, and .25 μg/mL amphotericin). The cells were heat-shocked to induce phsp-pBac promoter activation at 37°C for 60 minutes. The cells were then moved to a 28°C incubator for 48 hours prior to harvesting.
Plasmid DNA was isolated from the S2 cells according to the standard Hirt LMW DNA extraction protocol [72]. Additional steps taken were as follows: Following the centrifugation of the lysate, the DNA-containing supernatant was removed, extracted once in phenol:chloroform:isoamyl alcohol (25:24:1) and washed once with chloroform:isoamyl alcohol (24:1). DNA was precipitated with 2 volumes 100% ethanol and 1/10 volume 3 M sodium acetate (pH 4.5) and stored at -20°C overnight. DNA was pelleted by centrifugation at 14,000 rpm at 4°C and the supernatant aspirated. The pellet was washed 3 times with 70% ethanol to remove the excess salt from the Hirt extraction buffers, dried, and resuspended in 40 μl of nuclease-free water. 1 μl of the Hirt extraction DNA was mixed in 21 μl nuclease-free water and transformed by electroporation into 3 μl E coli. DH10B cells (Invitrogen) in a 1 mm gap electroporation cuvette according to manufacturer protocol. 150 μl of S.O.C. medium was pipetted directly into the electroporation cuvette and mixed well by pipetting. Cells were allowed to recover at 37°C for 30 minutes prior to plating. 10 μl and 100 μl portions of this cell-media mixture were spread onto LB-Kanamycin plates prepared with 4 μl 100 μg/μl IPTG and 70 μl 20 mg/ml X-gal. The plates were incubated overnight at 37°C and scored by counting the number of blue colonies on the plate spread with 100 μl cell-media mixture and the total number of colonies on the plate spread with 10 μl of the cell-media mixture.
PCR verification of excision events
A portion of the blue colonies were screened for excision events to verify accuracy of the test. Briefly, a 20 μl total volume PCR was prepared with a final concentration of 50 mM KCl, 10 mM Tris pH 8.3, 1.5 mM MgCl2, 5 units of Taq polymerase, and 10 pmol of each standard M13 primers: sense 5' GTAAAACGACGGCCAGTG 3' and antisense: 5' GGAAACAGCTATGACCATG 3'. The colony was taken directly from the plate and resuspended into the 20 μl PCR reaction by pipetting. 10 μl of the thermocycled PCR reaction was separated on .9% agarose stained with 10 μg/ml ethidium bromide.
Restriction analysis colony screen
The REN digestion excision assay follows pre-established methods, using the plasmid pBKOα [32] (Fig. 9). pBKOα and a piggyBac expression helper plasmid are cotransfected into S2 cells and incubated. Plasmid DNA is recovered by modified Hirt extraction as detailed in the blue/white colony screen section of methods and transformed into E. coli cells. A portion of this recovered plasmid is digested with Bgl II to remove any helper and non-excised donor plasmids and plated on LB-Amp/X-gal/IPTG plates. Because excision removes the lacZ gene, white colonies are scored as excision events. A portion of the undigested Hirt extraction is also transformed and plated on LB-Amp/X-gal/IPTG plates and the number of blue colonies is scored as the total number of potential donor plasmids.
106 S2 cells were allowed to adhere to the bottom of a 9.6 cm2 cell culture well and transfected with 0.5 μg of phsp-pBac helper plasmid and 0.5 μg of pBKOα donor plasmid with Transfectin liposomal transfection reagent (Bio-Rad) according to manufacturer protocol in minimal S2 media (Gibco). Two wells were transfected per helper-donor pair. The transfection media remained on the cells overnight and was replaced with Schneider's complete media. At 24 hours post transfection the cells were heat shocked for 1 hour at 37°C. At 24 hours post heat shock the cells were harvested by Hirt extraction as detailed above and resuspended in 10 μl water. At this time the two individual samples were pooled and 2 μl of the DNA sample was digested with high activity Bgl II restriction enzyme (NEB) for 4 hours at 37°C, ethanol precipitated as above and resuspended in 22 μl water. The entire sample was transformed by electroporation into 3 μl E. coli DH10B (Invitrogen) in a 1 mm gap cuvette and recovered in 75 μl S.O.C. media (Invitrogen). 50 μl of sample was immediately plated onto two LB-Ampicillin plates prepared with IPTG and X-gal and incubated at 37°C for 16 hours. White colonies were picked and cultured overnight in 3 ml LB-Ampicillin broth. The DNA was extracted by crude boiling mini-prep and digested with Hind III restriction enzyme (NEB) and run on a 0.8% agarose gel to screen for positive excision events.
For the background control 1 μl of the pooled Hirt extracted DNA was resuspended in 21 μl of water and transformed into 3 μl of DH10B E. coli and recovered in 75 μl of S.O.C. media. 5 μl of this preparation was immediately plated on two LB-Ampicillin agar plates containing IPTG and X-gal and incubated at 37°C for 16 hours and the blue colonies were counted.
piggyBac antibody
5 ml of rabbit anti-serum was obtained from Proteintech Group, Inc. after injection with truncated piggyBac protein. The antiserum was subjected to 50% ammonium sulfate precipitation for 60 minutes at 4°C and centrifuged at 10,000 g for 60 minutes. After resuspending the ammonium sulfate pellet in 20 mM K2HPO4 pH 6.8, 50% ammonium sulfate precipitation was performed again. After centrifugation, the resulting pellet was resuspended again and underwent buffer exchange in 20 mM K2HPO4 using a Centriprep YM-50 (Fisher Scientific, St. Louis, MO) according to manufacturer's protocol. This was followed by concentration of the IgG protein fraction using a Centricon YM-50 (Fisher Scientific). The concentrated IgG fraction was further purified using a 15.5 mL DEAE Affi-Gel Blue Gel Column (Bio-Rad Laboratories, Hercules, CA) according to manufacturer's protocol. The elution buffer was 20 mM K2HPO4, 0.02% sodium azide pH 6.8. The eluted IgG anti-piggyBac antibody was concentrated using a Centricon YM-50 to a final concentration of 16.57 mg/mL as determined by optical density spectrophotometry at 280 nm on a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE). Anti-piggyBac antibody function was verified through western blotting of known positive and negative protein samples (data not shown).
Western Blot
S2 Cells were co-transfected as described above along with two additional samples containing 1 μg of either a mutant helper, phsp-pBac, as a positive control or a non-piggyBac expression plasmid as a negative control. 48 hours post-induction, the cells were collected and pelleted by centrifugation at 3,000 rpm for 2 minutes. This was followed by two 1 ml washes with 1× PBS. The resulting cell pellets were lysed by resuspending in 100 μl 2× Laemmli Sample Buffer containing 10 mM benzamidine, 10 mM sodium fluoride, 100 mM sodium vanadate, 1 mM phenylmethanesulphonylfluoride (PMSF), 25 μg/ml leupeptin, 25 μg/ml aprotinin, and 25 μg/mL pepstatin. The whole cell lysates were then sonicated on ice for 15 seconds. The protein concentration was measured by optical density spectrophotometry at 280 nm on a Nanodrop ND-1000 spectrophotometer.
The whole cell lysates were prepared for electrophoresis on a 1.0 mm thick 7.5% acrylamide SDS-PAGE gel by diluting 125 μg of total protein to a total volume of 25 μl using 2× Laemmli sample buffer. This was followed by heating for 5 minutes at 95°C with 1.25 μl 2-mercaptoethanol to completely denature the protein. The samples were loaded onto two separate gels with 5 samples per gel and electrophoresed at 100V using 1× Tris-glycine plus 0.1% SDS as the running buffer. The protein standards used were Invitrogen's Benchmark Prestained protein ladder. After electrophoresis was complete, the separated proteins were transferred to a Hybond-ECL nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) using the Bio-Rad Mini Trans-Blot Cell and Module for 1 hour at 350 mAmps. 1× Tris-glycine with 20% methanol was the transfer buffer.
Following transfer, the membranes were stained with Ponceau stain and subsequently destained with 5% acetic acid and 1× PBS washes (images not shown). The blots were blocked using 1× PBS plus 5% non fat dry milk for 2 hours while shaking at room temperature. The blots were subsequently probed for piggyBac protein expression using a 1/1000 dilution of anti-piggyBac, purification described above, or for actin using anti-actin I-19 (Santa Cruz Biotechnology, Santa Cruz, CA) in a 1/500 dilution with 1× PBS plus 0.1% Tween-20 (PBS-T) and 5% non-fat milk for one hour while shaking at room temperature. After the primary incubation, the blots were washed four times for five minutes each with 1× PBS-T. The secondary antibody, ECL rabbit IgG, HRP-linked whole antibody (Amersham Biosciences) or donkey anti-goat IgG-HRP (Santa Cruz Biotechnology), respectively, was added in a 1/5000 dilution with 1XPBS-T and 5% non-fat dry milk for one hour at room temperature while shaking. This was followed by washing with 1× PBS-T four times for five minutes each. The blots were developed with Pierce's SuperSignal West Dura Extended Duration Substrate kit (Pierce, Rockford, IL) according to manufacture protocol except 0.5 ml of each substrate was used per blot.
Statistical analysis
Data are expressed as a mean of three replicates +/- standard. ANOVA test was performed using GraphPad Prism 3.0 software. All values were adjusted to a positive control setting of 100%. Means were considered statistically significant when p-values less than 0.05 were obtained with the Dunnett's post-test. Statistical significance is indicated on all data figures as asterisks above bars.
Many thanks to Meggan Keith for her help with the western blot. This research was funded by NIH grant RO1 AI48561 to Malcolm J. Fraser Jr.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Here we are going to see, How to Export PUTTY Sessions list to Another Computer,
Run the below command on a source system ( to taking backup / export existing session list)
regedit /e "%userprofile%\desktop\putty-sessions.reg" HKEY_CURRENT_USER\Software\SimonTatham\PuTTY\Sessions
Do the below taskson a remote system ( Importing from reg file)
to copy the putty-sessions.reg file on remote system.
then right click on a file & merge it.
Thats it.


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About Element Games
Element Games officially opened its virtual doors in May 2012 with the aim of offering low prices, excellent service and fast delivery time, without compromise.
We also have both a store and a gaming centre (Element Games North West Gaming Centre) in Stockport, Manchester, UK. You can find more information (address, opening times and facilities) here.
Element Games at Salute 2016
Element Games Store
Click here for more information on our Store, including opening times and facilities.
Element Games North West Gaming Centre
Click here for more information on our Gaming Centre, including opening times and facilities.


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Update on Alphablox in WebSphere Business Monitor
October 31, 2009
Earlier this year I blogged about installing Alphablox as part of WebSphere Business Monitor. Real production environments require clustering, and it was difficult to accomplish. Using DB2 on z/OS for data repository was particularly daunting task. Mike Killelea commented on my original post, noting that IBM disclaimed support for this scenario.
This time of the year, IBM is preparing version 7 of its BPM stack for release. From what I saw, Alphablox is much better integrated into Business Monitor (BAM). Remember, until now you had to run a separate Alphablox installer (with the exception of non-production-grade standalone profile). Now, ABX install is fully integrated. I specifically inquired about z/OS database support and was told that it is ON. I'll post an update when I learn more.


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Hendrick Motorsports contributors from the 2000s share their thoughts and memories of working with Hendrick Motorsports owner Rick Hendrick:
"He's just one of those people when you walk in a room, which I've never met the president of countries, but he's just one of those people where when he walks in you feel the power coming out of him and he expresses a genuineness and realness. Whether it's the CEO of Lowe's or a grandmother who is standing in the shop hoping to get a glimpse of one of the drivers walking by, he'll take her from the lobby and walk her all the way through the shop and you'll think she's the CEO of Lowe's getting a tour."Robbie Loomis, champion crew chief
"I tell people all the time, anything that I do I always think of Mr. Hendrick. The lessons of dealing with people, whether it's a waitress at a restaurant or a CEO of a company or just a friend, just how to get along with people and help everybody be their best and realize that we are all in this world together. That's the thing I always admired of him."Robbie Loomis, champion crew chief
"My impression always was that he was an absolutely great guy that had a very strong organization with really good people. But he was always first-class. He was a serious competitor and I was around from his very beginning days. I was in the sport when he was first getting in and that's one thing – I don't think I've ever heard anyone say something bad about Rick. He's a fierce competitor but he's a really first-class man."Mark Martin, driver of the No. 5 Chevrolet from 2009-11
"There were just so many good times that we had but I think the one thing was the pride that I had just being one of his four drivers. We had a great team with (Dale Earnhardt) Jr., Jeff (Gordon) and Jimmie (Johnson), great teammates and great crew chiefs and great people. At that time I felt like it was the highlight of my career, to be able to step into an organization that was so strong and have so much support from people that I thought the world of. It was a real special time in my career. He made that happen."Mark Martin, driver of the No. 5 Chevrolet from 2009-11
"I was very excited for sure when I first got the opportunity at such a great organization, and that's where I got my first and only win at this point, so it's definitely a special place for me. Knowing Rick beforehand, obviously I knew him as a car owner but I knew him probably more as a father to Ricky. Ricky and I were buddies and that's how I first got to know Rick before I ever got an opportunity there so it was cool to drive for a guy that you respected as a successful car owner but also as a successful father. That was really cool."Casey Mears, driver of the No. 25 Chevrolet from 2007-08
"Obviously the record speaks for itself – all the wins, the guys that have driven for him in the past. It just goes without saying that he's definitely one of the top guys in NASCAR's history, period."Casey Mears, driver of the No. 25 Chevrolet from 2007-08
"(Joining Hendrick Motorsports) was a huge event. The only event that was probably more significant in my racing career was my dad putting me in a go kart. It certainly changed the course of my career and there's no way to look backwards in time and say what would have happened otherwise."Brian Vickers, driver of the No. 25 Chevrolet from 2003-06
"He was a lot of things through those years. He was a car owner, a friend, a boss, a mentor, a coach and got through very difficult times. Even though he was going through so much himself, a lot of people leaned on him and he leaned on them at the same time but a lot of people were leaning on him when it should have been probably the other way around. That's something I'll never forget."Brian Vickers, driver of the No. 25 Chevrolet from 2003-06


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And of course, because it's New Year's, it's time for me to sit down and evaluate how I did.
My writing goals? Knocked that puppy out of the park! I completed each project I listed, and then a few more. There are two books here on my list that I did not get done, but those were held up by other people and aren't yet ready. I did my part on them, though, so I consider them a success. This was made possible in large part by the fact that I spent a lot of 2012 in bed, recovering from the December 2011 car accident and the March 2012 broken foot.
If you're interested, the books I produced in 2012 are:
Taking Out the Trash
Million Dollar Diva
Turning Pages
Virtual Book Tours
Women of Strength
Targets in Ties
The Writer's Workbook
Word Count Tracker
Dialogue Dynamics
Creating Characters
Taking Care of BusinessI also worked on a few more that will come out next year.
My personal goals? I did pretty well. I didn't have "breaking my foot" down in my plans, so a few things did go a bit awry. Nothing like having a cast for a month and then recovering strength in the foot for two months to put a bit of a damper on things. But I did accomplish three of my goals and made good progress on two, leaving just one to wonder why it got left out in the cold.
My business goals? I did well here too. I met two of my goals and am still struggling to accomplish the third. But that third one will be vanquished come 2013. Mark my words! (I'll wait while you find a marker.)
My family goals? Ah ... this is where the wake-up call comes in. I did not accomplish one thing on this list. Granted, some things were just plain out of my control. But the things that were in my control ... didn't get done either.
What did I learn? I learned that when I put my mind to it, I can get a lot of writing done. I learned that car accidents and broken bones are not helpful to goals that require physical movement. I learned that laptops are wonderful things and that WiFi is like a miracle from on high. I also learned that we can't let less-than-ideal circumstances keep us from striving to improve in every aspect and not just some.
My motto for 2012 was: "2012 is my year - not because everything is going to go my way, but because I will face everything that does come with dignity and faith." Was this tested? Oh, holy cow, yes. A whole lot of stuff came our way. Did I face it all with dignity and faith? I have to say that in most respects, I impressed myself. :) There were definitely times when I dropped the ball and didn't react how I would have liked. By and large, though, I see real growth in myself.
And there you have it. Goals keep me moving forward, and they also serve to show me where I'm missing my mark. 2013 will be a year of no excuses - I know where I'm weak, I know where I need to improve, I know how I need to improve, and so now it's a matter of getting 'er done.
Happy New Year ... and may we all realize our dreams, accomplish our goals, and take huge steps toward becoming the people we want to be.


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5 Physiotherapy Tools - Down Syndrome/
Here is the latest PT happenings in our house (from 7-9 months).
All of the activities shown here have been approved by our PT and OT. Also, these activities benefit Hazel and you should consult your PT and OT if you are thinking about trying any of these with your children.
Another important thing to mention, is that when Hazel is not happy doing any of these activities, we stop. We try something new and if she's not into it, we drop it for a while. I never pressure her or force her to do any PT or OT.
Our Tools:
1. The Foam
When using the foam in this position, it encourages Hazel to weight bare on both her knees as well as her arms. These pics are from a while ago.
If I lean her up against the side like this, she leans and rests her arm on the foam. It gives her practice weight baring on one side at a time. It, also, helps her to support herself better because her arms are a little shorter than most.
We cut the foam so that Hazel's legs will stay together. This ensures that the weight is on her knees and that she is not flat on her hips.
Sometimes I put the foam out in front of her so that she can reach to put weight on her hands as well as her knees. Trying to get her into the 4-point crawling position.
We are doing some PT outside (this pic is old). I think it's nice to change the scenery so that Hazel always has something new to look at.
In this position, Hazel is learning to put weight on her hands and elbows.
I often will hold a toy up for her to reach for so that she learns to shift her weight from one side to the other.
Sometimes she doesn't like to do PT, so we stop.
We have her sit on the foam and pull herself up. She gets a great bend in her knees and it forces her to use all the muscles in her legs to stand.
As you can see, she is pretty proud of herself (I know the pic is blurry but look at that face).
2. The Mirror
One of my favourite PT toys to use is this mirror. Hazel will sit up for the longest time talking to herself, and trying to grab at her face and hands. It is great for encouraging her to remain in the sitting position, rather than grab a toy and then fall over and just lay down and play.
Plus, it has the added feature of being the most entertaining thing for me to watch. She yells, dances, makes silly faces. It's great!!!
Oh yes...and tries to give herself a kiss.
3. The Medicine Ball
The medicine ball is the BEST!!!
Hazel loves it and it is so effective for core strengthening. It was a little nerve racking at first but we took it slow and now I really love it. We use it everyday. I bounce her really slowly and move the ball from side to side and front to back. Her natural reaction is to stay up so she is constantly flexing her abs trying to balance. She thinks it is pretty funny.
4. The Play Mat
These pics are pretty old but I still put her on her play mat sitting so that she can reach up and play with her toys.
Hazel has been really working on core control. Her posture is getting very straight. She practices sitting often and enjoys the freedom she has with her hands.
5. The Exersaucer
She spends time in here everyday and is really getting the hang of standing and sitting. She thinks she's hilarious when she stands (again...blurry but the faces kill me).


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Not sure if you've heard yet, but, apparently white potato is now considered paleo in paleo circles. There has been a debate over white potato for some time. Many claim that due to its high starch content, high glycemic index, and ,of course, the fact that they did not exist in Paleolithic times, regular white potatoes should be taboo on paleo diet. Others argue that the nutritious benefits of white potatoes outweigh the drawbacks, and that the mere fact that our paleo ancestors didn't eat potatoes shouldn't stop modern, educated, healthy lifestyle seekers from incorporating this controversial vegetable into their diets. Personally, I'm undecided on this one. And until I make up my mind, I'm sticking to good old sweet potatoes! The following recipe is for baked sweet potato canapes with roasted red pepper sauce. Its a twist on chips and dip—baked sweet potato chips topped with a zesty cream cheese—to start off your informal get together at home or a cottage dinner in style. It makes a great low carb appetizer and is pretty straightforward to make.
Now, remember dairy is also a major grey area in paleo. With the paleo camp divided into two: those who allow high quality dairy and those who are against any. The overall consensus seems to be such that one needs to test and decide for oneself. Eliminate all dairy from you diet for a month and see if you notice a difference. If you don't see any difference, and enjoy good quality milk products, then why suffer? I myself am very tolerant to dairy and love cheese, so for me, going non-dairy would be a torture. So I do incorporate some dairy into my diet as an occasional treat.
Make sure to use only high quality organic cream cheese for the sauce. My local grocery store carries a brand that has only the following ingredients in contents: Pasteurized organic milk, salt, bacterial culture, microbial enzymes. Looks pretty natural to me.
In winter, when fresh red peppers are at a premium price, use prepared roasted red peppers from a jar or the deli, or ones that you froze or canned in summer. Some are more acidic than others, so have a taste to decide if you wish to rinse them to remove some of the brine or marinade before puréeing them. Choose sweet potatoes that are a fairly even thickness the length of the potato for even cooking and equal-size canapés. Extra red pepper cream cheese can be stored in the refrigerator and spread on sandwiches or tossed with hot pasta.
Last time I made these canapes was for a get together with a girlfriend who came over for some vino and tasty gossip. We attacked these canapes and I swear the whole batch was gone in a few minutes. They are the best when fresh out of the oven. But don't worry, when you serve them to your guests, they are guaranteed to be gone before they get to cool down;)
As always, let me know how this recipe worked out for you. Go to the Review section below and share your experience.


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Have you recently been inspired and blessed by Uncle Marvin and Auntie Hiroko? Do you feel a call to be of support to their mission? Below is a list of needs, immediate and long term, to guide your calling to help.
Please email us at email@example.com for more details.
If you would like to make a monetary donation please do so via PAYPAL( firstname.lastname@example.com )
100% of your donation will go directly to Marvin & Hiroko.
• Horse & garden caretaker
• Plumbing in 3 buildings
• Deck/stairs for main house entrance
• Roofing - main house
• Kitchen sink - main house
• Remodel bathroom - main house
• Drywall straw bale house
• Build community laundry & storage rooms
• Build quarters for visitors / volunteers
• Build outdoor area for gatherings
• Expand living room in main house
• Build kids' playground
• Build secure gate
• Outdoor dish washing station
• Outhouses
• Outdoor showers
• Shade - outside main kitchen
• Build dog kennels
• Build adobe house for Marvin & Hiroko
• Build large community building for events
• Dig stream pond / sitting area
• Efficient vehicle in good condition, such as a mini van or station wagon
• 2 Large on-demand water heaters
• Stove
• Refrigerator
• Wind generator
• Solar panels
• Large outdoor tables
• Gravel for living grounds
• Drywall for straw bale house
• Gardening tools
• Organic soil
• Window screens
• Blankets
• Utensils, bowls, plates, serving utensils, etc...
• Tipi canvas
• Goose neck horse trailer
• Tiny houses / living structures
4x4 Vehicle - Teeki
Car - Martinez Family
Tipi poles - Dominic & Nina
Industrial washer and dryer - Maurer Family
Fence around garden - Andy Molnar & Cid Little Elk
Shade area near Sweat Lodge - Val & relatives
Constant support: bringing wood, food, etc... - Val


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The good thing is that I lost track of what I have accomplished for this semester. Once I finish a task, I take a big breathe of relief and move on. I can't wait to do that December 16th.
What I do like is how I get li'l breaks to be with my cute li'l nieces and nephews. Like this li'l guy. Baby Ethan and I took a li'l break from the composing, the memorizing, the theory and the practicing to hang out on a Friday night.
Ethan had fun watching himself on the computer


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Some clarity on David Stern's advice and Hamady Ndiaye's contract situation
I wanted to clarify a few things that I've reported over the past few days. First off, David Stern's advice to Gilbert Arenas and the Wizards to remain silent on his gun incident is not a "gag order" but rather a strong suggestion. No one would get fined for making comments, however, it would be frowned upon with the franchise looking to move in a new direction, with a new owner and some fresh new faces.
Wizards owner Ted Leonsis told me that he wasn't going to go against Stern's recommendation for fear of getting fined, but team spokesman Kurt Kehl told the Associated Press on Thursday that the organization was never issued the threat of a financial penalty. "The idea of being fined was never intimated to him at all," Kehl said.
As I wrote in my blog post on Thursday, they have been advised not to talk about the situation. Arenas will assuredly be asked about it, either Monday at media day or some time throughout the season, but Stern supports him should the three-time all-star decide to issue no comment on the matter. Since he has been given permission to avoid the subject, Arenas is expected to follow those orders.
Secondly, while Hamady Ndiaye is working out at Verizon Center and expected to participate in training camp with the Wizards beginning next week, he has yet to accept the non-qualifying tender. Ndiaye most likely will take it, with only a few days left before camp and him unable to get offers from elsewhere.
Ndiaye and the Wizards remain hopeful that he finds employment overseas before the regular season begins, because the longer he sticks around, the more problems it could possibly present for both sides. Unless Ndiaye completely blows away the Wizards in training camp and the preseason, the team does not plan on having him in uniform in the upcoming season.
The Wizards don't want to use a roster spot and send him down to the NBA Developmental League, either. They would like to maintain some roster flexibility by going into the season with either 13 or 14 players. Having that 15th roster spot available could help the Wizards if they feel the need to add some veteran help in an area of need or to take back another player in a possible trade down the road.
The dilemma for both sides is that the Wizards would lose Ndiaye's rights if they have to cut him in training camp. Ndiaye is seen as an extra asset for the organization, since the Wizards only asked for the 57th pick from Minnesota after they had already agreed to move up to get Trevor Booker. They would like to have Ndiaye in a Wizards uniform at some point, just not before he has developed and received some playing time and experience elsewhere. The way the Wizards are currently constructed, Ndiaye is unlikely to gain sufficient playing time this season.
I hope that clears up any confusion for you guys.
Michael Lee
| September 24, 2010; 9:19 AM ET
Categories: Gilbert Arenas, Hamady Ndiaye
Save & Share: Previous: Grunfeld says Wizards are changing to red, white and blue
Next: Ernie Grunfeld: "This freshness is exciting to me"
I don't see Namady in the active list if he stays here and sitting on the bench will hinder his development.
PG: Wall, Hinrich
SG: Arenas, Young
SF: Thornton, Booker, Howard
PF: Blatch, Yi, Seraphin
C: McGee, Armstrong
Thats already 12 players on the active list
Posted by: jefferu | September 24, 2010 9:55 AM | Report abuse
I understand the flexibility issue very well, and Wiz flexibility helped them get Yi and Hinrich for this year. However, when EG talked about (yesterday) "wanting to develop young players throughout the season," and liking the D-League, but "we couldn't really use it the past few years," I am a little confused.
A player like Ndiaye would seem like a perfect example of how to use the D-League. Yes, he takes up a roster spot, but considering that spots 13-15 are seldom used, why not give one to Ndiaye if you think he has a future with the team, but needs more playing time to improve his skills. I could also see a situation where Seraphin or even possibly Booker struggle or are not getting enough playing time, and the Wiz may want them to play more. Then, bring up Ndiaye to give him practice time with the big team and send down one of the other rookies. Also, when the injury bug hits (pray that it doesn't), we always go to the D-League for help. With N'diaye, we already have one of our own players that we can bring up in the front court. I am not understanding the downside.
Worst case, let's say this "incredible deal" comes before the trade deadline. Ndiaye is on a rookie contract for a 2nd rounder, not much money. They spent a lot more to get Hinrich and move up to get Seraphin, so if the deal is attractive enough, cut Ndiaye, make the deal and bring him back later. I believe they can do that, but they may need to eat the rest of his salary, but that won't be much, at most the remainder of the year for a 2nd round rookie contract??
I just believe this could be another sign, especially if they cut Ndiaye, of the Wiz saying they believe in the D-league and wanting to develop players for the future and then turning around and doing something else.
Posted by: faninAlex | September 24, 2010 10:45 AM | Report abuse
"I don't see Namady in the active list if he stays here and sitting on the bench will hinder his development."
Very little "development" happens in games. That comes with work put in during practice, offseason, tape study, etc. Game time is when they put into practice what they've learned. But they have to learn it first. A season spent working with coaches, learning the system, getting familiar with teammates, and going head to head with NBA caliber players will likely do more good than a season spend competing against D-League scrubs.
Besides, even if they keep Ndiaye, they'll still only have 13 players. That leaves two spots. Fill one with someone (a SF?) out of camp (Martin? Morrison?) and they still have one left.
Posted by: kalo_rama | September 24, 2010 11:20 AM | Report abuse
In other NBA news:
Looks like the inevitable has finally happend and Don Nelson is out in Golden State. That writing has been on the wall for a while.
And in Miami's ongoing quest to corner the market on decrepit big men, it looks like Erick Dampier is leaning towards signing with the Heat.
Posted by: kalo_rama | September 24, 2010 11:22 AM | Report abuse
N'Diaye is a dleague scrub, summer league showed that. It's not bad luck that no team on earth (apparently) has made him an offer. I dont know what they should do with him, but it's also ok to concede they missed with the 57th pick in the draft and just move on. This guy is older than Mcgee but looks years away from being able to contribute....no harm in parting ways if he shows nothing in camp.
Posted by: divi3 | September 24, 2010 11:28 AM | Report abuse
What's a "non-qualifying tender?" How does it work?
If Ndiaye signs such a tender, does that mean Wiz will have to either cut him and loose his right, or keep him on the roster?
Posted by: sagaliba | September 24, 2010 11:50 AM | Report abuse
I believe you can sign a player to a non-guaranteed contract. The contract would only become guaranteed if the player stays past certain date. So if you cut the player (due to roster spot) before that date, you won't even loose any money other than the salary that you've already paid him.
Posted by: sagaliba | September 24, 2010 11:56 AM | Report abuse
With a scoring 2, 3 and 4 all then need is a sound point guard and some bangers down low and a coach with some championship experience. I somehow dont see young Erik outcoaching, Doc, Phil or Pop in a series. I think they need Pat to guide the egos and coach in a competitive series. Dampier is a banger with six fouls to give.
Posted by: ged0386 | September 24, 2010 12:22 PM | Report abuse
"With a scoring 2, 3 and 4 all then need is a sound point guard and some bangers down low and a coach with some championship experience. I somehow dont see young Erik outcoaching, Doc, Phil or Pop in a series."
That's nice. Why are you telling me?
Posted by: kalo_rama | September 24, 2010 2:10 PM | Report abuse
As for N'Diaye . . . he may or may not be a bust, but that determination can't be made with any substance based on summer league because (A) he barely played and (B) summer league proves a grand total of bupkis. Every year there's a handful of players who look great in summer league and end up nothing in the NBA (if they even make it that far) and vice versa.
Posted by: kalo_rama | September 24, 2010 2:12 PM | Report abuse
With the team that we have; we don't need All-Star centers.
All we need is a collection of Big-Bodies (prefferably 7-footers) to play Hack-a-Shaq, Ala "Da Bull's".
During their 6 Championship seasons, name one all-star?
Give the guy (Namady) a chance at training camp, I will be there. See ya!
Peace-out, I love ya'll!, Chef John.
Posted by: upscalechef | September 24, 2010 2:26 PM | Report abuse
I think the Wiz should switch to all-Black uniforms and Gilbert should be nicknamed "The Silencer" and John Wall "The Sheriff!" Yeah! lmao
Posted by: terptek | September 24, 2010 2:31 PM | Report abuse
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Broward County
Ex-exec at Hollywood facility admits to Medicare fraud
A former longtime executive at Hollywood Pavilion, a psychiatric facility, pleaded guilty Tuesday to Medicare fraud charges stemming from $67 million in bogus billings to the taxpayer-funded program.
Christopher Gabel, 61, of Davie, the former chief operating officer, pleaded guilty to conspiring to commit healthcare fraud and pay kickbacks to patient recruiters.
In court papers, Gabel admitted that Hollywood Pavilion submitted false claims for mental health treatment that was not necessary or provided to patients between 2003 and 2012. Medicare paid out $40 million for those claims.
Gabel was the supervisor of both inpatient and outpatient facilities, where Medicare beneficiaries were admitted regardless of whether they qualified for treatment or even saw a doctor.
Gabel, represented by defense attorneys Bruce Lehr and Andrew Levi, is scheduled for sentencing Jan. 26 before U.S. District Judge Cecilia Altonaga. Last year, Hollywood Pavilion's chief executive and others were convicted at trial and given lengthy sentences.


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Funny Veterans Day Meme 2017 – Happy Veterans Day Memes & Trolls – In the United States of America, Veterans Day is celebrated as an official public holiday, which observed on November, 11th every year. This day coincides with other two holidays, including Remembrance Day and Armistice Day, which celebrated in other countries of the world that mark the anniversary of the end of the World war I. On this extra special day, Americans honor their military veterans who served in the armed force of the United States of America. The occasion of Veterans Day is celebrated throughout the United States with much enthusiasm and passion. Veterans Day is not to be confused with Memorial Day.
Funny Veterans Day Meme 2017
Veterans Day Meme Funny – Veterans Day is one of the grateful and precious events of the United States of America. It is a special day where Americans take a time to reflect and honor military men and women for what they have done for their great country. In times of peace and war, from the Army to the Airforce, to the Marines to the Navy, you have all these brave soldiers who have selflessly sacrifice their own lives for preserving freedom and safety of your nation. So, here we have collected a huge collection of Veterans Day funny meme which you can send to your veterans. These hilarious memes celebrate this day with both humor and solemness.
Veterans day vs Memorial Day Funny MEMES
Happy Veterans Day Memes
Veterans Day Memes – Each year 11th of November is grandly celebrated as Veterans Day in the United States. On this day, the people of US nation, honor and be thankful for the flawless service of their military personnel. They celebrate this day with immense respect and zeal by arranging several parades, attending church services and many other awesome activities. As this year's Veterans Day is on the way, so here we have gathered some inspirational and wonderful Veterans Day memes. You can greet your brave heroes by sharing these fabulous memes.
Veterans Day Trolls Funny Pictures
Veterans Day Funny Pictures Trolls – Veterans Day is one of the most prideful occasions of the year when Americans honor all those veterans who have served in the US military. With Veterans Day is approaching fast, so here we have assembled a variety of Veterans Day trolls funny pictures. You can download these hilarious pictures for free and showcase your heartfelt gratitude towards your veterans and spread some laughter by sending these gatherings to them on this precious event of Veterans Day. Many peoples have confusion between veterans day and memorial day so they are keep confusing other with their stupidity. So after lots of frustration from that kind of people i have created some memorial day vs veterans day memes this year.