text:
By: Neoriceisgood
Learn to trust me.
The moral of this story is: always trust your teachers, kids!
Gazz should try to save him.
Know what would make it more uncomfortable? Having Eloy in there!
Hey! Kid. Wanna learn how I lost my virginity? Too bad!
I suspect that Garth wants them to break the rule he just set up...
Hmm, is this going to be one of those "loyal enough to help him anyway" sort of things?
Didn't the teacher guy say that he'd explain how Noah made Felix hit himself? He said that Noah used the mask technique but I don't think that explains how Felix could've hit himself because of it.
Don't they have any other classes? Isn't someone at least going to teach them maths?
It'll be interesting to see what Ciel makes of all this.
@Hevel: He's a P.E. teacher, what else did you expect?
@Felix: https://www.youtube.com/watch?v=rX7wtNOkuHo
Of course I trust you, Garth! You are the very personification of trustworthiness, you are. Not batshit insane, no sir. I'll be slowly stepping away, now. With your permission, oh bestest of teachers.
...If it rains, Felix is screwed.
welp, chapter change
Krishnik: I think, felix needs to use juice to lift and throw the ball, but then it homes in on ppl by following their juice. if noah masks himself, felix is then the most juiced up thing nearby so it turns around. idk thats my take
I bet he forces him to get out by himself by using his powers.
Alright, magic speculation time since we've learned all those things, cause I have nothing better to do, apparently.
So, this 'spirit' is said to flow isn't it. Fluid mechanics come to mind. Does it behave like a gas or liquid? Or is it a field in that it doesn't *really* exist and the word flow refers to the things that react to its field lines?
The way it behaves around 'magic potential'(and 'pandemonium') magic users reminds me of the experiment with the magnet and iron fillings, only instead of magnet we have a human that produces a magic field and instead of iron fillings we have spirit.
Instead of how a magnet of a certain shape only produces one kind of magnetic field, though, they can somehow manipulate it. Maybe not 'instead', either. We haven't been told what exactly produces the field. Maybe it's something physical that actually changes shape... Oh shit, midi-chlorians!!:P:P ...So this is the train of thought that created them... I need some brain bleach.:P
Still, the magnet analogy doesn't look unreasonable, given what we know. They do seem to be creating a field, somehow, that is interacting with spirit. This is also consistent with the knowledge that magic appeared suddenly and turned the world upside down.
Either some people always had the ability to create this field but there was nothing there for it to interact with. Then something happened and the world got filled with spirit and suddenly those people are mages.
Or spirit was always there but didn't really interact with the electromagnetic forces, kind of like neutrinos. Then some people got exposed to something that gave them the ability to create a field that interacts with it. That something doesn't seem to have been present in the empire, up till recently. There was Veda saying that it's common for people to have strange features when they are born near magical places or events as an explanation about her green hair. Maybe it's the same with magical abilities.
continued below...
Though, she also said she has no magic... yet something nasty happened to that nationalist who tried to kill her...
None of this explains pandemonium's other abilities and free magic, though, does it?
Let's see...
If they work by the same mechanics and keeping true to the magnet analogy, 'magic potential' is more like a simple magnet while the other two are more like specialized contraptions, whose effects can't be replicated by simple magnets... If that's true, pandemonium is unchanging, though inherent so it comes naturally and without effort, while free magic is more like you have the tools to create what you need, though it requires effort.
If they do not work by the same mechanics I can't really say anything about them... I have an idea about spirit then. It could be the waste/fallout of pandemonium and free magic spells. Like a byproduct of a chemical reaction. Or like heat. Magical Entropy! Speaking of the second law of thermodynamics, Hevel's part of the stolen documents had something about a combustion engine...http://neorice.com/hoh_noah_530
Is the empire preparing to make a magical machine? Spirit fueled, maybe? Then that, along with veda mentioning a chemical test to detect mages, makes my (pseudo)scientific approach not completely uncalled for! Judging by that ball there actually can be spirit sensitive objects, so it's not impossible. (By the way it could be a great weapon against someone who can't use spirit. Light as a feather to you, a ton to them. Or an armor made like that. Armor thicker than steel plate, weighting nothing. Just don't use it against mages...)
I admit none of this explains why pandemonium people can use spirit, while free magic can't... I mean I think they can't. Maybe their magic is too useful to waste time with spirit training but the way I read it, is while they can duplicate the effects of more-or-less anything so they could be using things resembling spirit magic, it's not *really* spirit magic. /speculation
...Am I overthinking this?:P
Hey, it's relaxing.
To not go into specifics, the reason "Free Magic" users can't use "spirit" is more akin to why people generally don't use dynamite to light cigarettes.
It's not so much that they absolutely can't use free magic, but rather that free magic and spirit are closer related to eachother in how they work v.s. pandemonium so it's very difficult for free magic users to execute the more "subtle" spirit magic techniques without involuntarily going overkill.
So basically, Free Magic users are better off using their free magic to replicate the same 'effects' as spirit, rather than attempting to use it in the same way.
For example, creating a sparkly rainbow armor around yourself, instead of simply trying to defend yourself with spirit. Same result, but free magic is better off continuing to be "free", instead of trying to restrict itself.
More or less, yep!
Is Gazz smirking in the first panel or does the shading just make it look that way?
Wouldn't being overkill sometimes benefit you better than free magic? For example, making overprotective spirit armor could be a better defense than magical potato shields, right?
I can't be possibly the first one to have noticed this, but searching on the site turns up nothing.
Is Felix a reference to a certain character from the Yu-gi-oh? The way he talks and his deal with his cards.
@Krishnik Mask makes spirit literally flow around the person. Noah's Mask made the spirit sensitive ball flow around him and hit Felix.
@Neoriceisgood "generally"? You must know some interesting people :P.
So it's pandemonium that is more of an outlier, not free magic...
Anyway, I like the magic system and I could see it working very well in a video game (which I mean as a compliment)


text:
The discovery reveals important links in the molecular pathway by which complex genetic alterations arm the immune system to target myriad potential bacterial and viral invaders with swiftness and precision. The discovery may also provide welcome new information about lymphoma, a form of leukemia in which certain cells of the immune system proliferate uncontrollably.
Howard Hughes Medical Institute investigator Frederick W. Alt led the research team that published its findings August 26, 2004, in the journal Nature. Alt and lead author Jayanta Chaudhuri are at Children's Hospital, Boston, and Harvard Medical School.
The studies focus on B cells, specialized immune cells responsible for producing antibodies, and how an enzyme in those cells known as activation-induced cytidine deaminase (AID) triggers mutations of antibody gene segments to produce an assortment of antibody proteins. This process enables the immune system to produce antibodies that will recognize billions of different antigens the fragments of foreign invaders that are used to call the immune system to arms.
The presence of an antigen on the surface of a B cell stimulates it to produce antibodies. An important step in this process is the activation of AID, which causes largely random mutations in the genes for the antibody segments that recognize antigens. These mutations occur about a million times more frequently than spontaneous mutations in other genes. In this process, known as somatic hypermutation, AID selectively "damages" the DNA strand, prompting the DNA repair system to create the mutations.
AID also triggers class switch recombination, a highly specific process that involves re
Contact: Jim Keeley
Howard Hughes Medical Institute

subset: commoncrawl


text:
Abraham Bowman Sweet XVI Bourbon
Abraham Bowman // Virginia, USA
Abraham Bowman represents the experimental line released by the A. Smith Bowman Distillery and this bottling marks the 17th release in the line. Sweet XVI Bourbon is made from a selection of bourbons all aged for 16 years in new, charred American oak. The distillery experimented with different barrel entry proofs and for this bourbon they included 125, 114, 105, and 90 proof. The bottle includes the distillation date (April 16, 2002) and bottling date (April 26, 2018) along with the batch number and bottle number. Availability is limited with Virginia getting the bulk of it, but it will expand to a few other states in summer 2018.
BourbonProduced anywhere in USA; Mash bill of at least 51% corn; aged in new, charred oak containers.
Cask Typenew, charred American oak
Tasting Notes
"The nose is syrupy and rich, with vanilla, caramel, barrel char, and hot cornbread with melting maple butter, as well as deep, leathery aromas. The palate is big, yet not hot given the proof, showcasing spicy oak, with molasses, sour cherry, and cloves, and a sweetness reminiscent of glazed donuts. The finish is long, with astringent oak balanced by spearmint and a thread of rich vanilla sweetness. An eminently sippable bourbon."


text:
Membrane targeting of the human immunodeficiency virus Gag proteins is dependent on phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] located in the plasma membrane. In order to determine if evolutionarily distant retroviral Gag proteins are targeted by a similar mechanism, we generated mutants of the matrix (MA) domain of murine leukemia virus (MuLV) Gag, examined their binding to membrane models in vitro, and analyzed their phenotypes in cell culture. In vitro, we showed that MA bound all the phosphatidylinositol phosphates with significant affinity but displayed a strong specificity for PI(4,5)P2 only if enhanced by phosphatidylserine. Mutations in the polybasic region in MA dramatically reduced this affinity. In cells, virus production was strongly impaired by PI(4,5)P2 depletion under conditions of 5ptaseIV overexpression, and mutations in the MA polybasic region altered Gag localization, membrane binding, and virion production. Our results suggest that the N-terminal polybasic cluster of MA is essential for Gag targeting to the plasma membrane. The binding of the MA domain to PI(4,5)P2 appears to be a conserved feature among retroviruses despite the fact that the MuLV-MA domain is structurally different from that of human immunodeficiency virus types 1 and 2 and lacks a readily identifiable PI(4,5)P2 binding cleft.
Retroviruses are a large family of enveloped viruses that contain two copies of a positive-strand RNA genome. The retroviral replication cycle is canonically divided into early and late stages. The early stage corresponds to virus infection and entry into the target cell, reverse transcription of the genome, and proviral DNA integration into the host cell genome. During the late stage, the integrated provirus is transcribed, generating unspliced and spliced viral RNAs that are translated by the host ribosome machinery. All replicating retroviruses encode three major structural proteins and enzymes, namely, Gag, Pol, and Env, that assemble to form new virions. Much of the current understanding of retroviral replication has been derived from studies of murine leukemia virus (MuLV), a simple gammaretrovirus with a full-length primary transcript that encodes four polyproteins: Pr65Gag, Pr180Gag-Pol, Pr75glyco-Gag, and Pr190glyco-Gag-Pol (13) (the Pol domain contains protease [PR], reverse transcriptase [RT] proteins, and integrase [IN]). Gag, the main retroviral structural protein, consists of several domains, with three of them being functionally conserved among retroviruses: the nucleocapsid (NC) domain, which selects the genomic RNA (17, 34); the capsid (CA) domain, which forms the internal capsid structure of the virus; and the myristoylated matrix (MA) domain, which targets Gag to cellular membranes (24, 44). MuLV Gag also contains a long polypeptide called p12 that connects the MA and CA domains and is involved in recruiting cellular cofactors important for budding (59). The Gag polyprotein multimerizes, the assembly process occurs, and Gag is then cleaved into these individual domains by the viral PR during the particle maturation process concomitant with budding (43).
A number of recent studies have focused on identifying the mechanism by which retroviral Gag proteins are targeted to specific virus assembly sites. Although a large fraction of Gag assembles and buds from the plasma membrane, there is not uniform agreement regarding the path(s) that Gag follows to reach the assembly sites. Newly synthesized Gag proteins may be targeted directly to the plasma membrane (54), or they may indirectly traffic via endosomes (3). MuLV budding can occur in intracellular compartments similar or identical in nature to late endosomes/multivesicular bodies (27, 51), but the importance of this pathway in virus egress was recently questioned (9), as MuLV spreading was not significantly impaired in transgenic mice with defects in multivesicular body biogenesis and trafficking.
Gag trafficking may be driven, at least in part, by the ability of the MA domain to interact with host protein and membranes. Some studies indicated that the MuLV MA domain can be engaged in interactions with cellular proteins directly involved in vesicular trafficking, such as AP-1 (6) or Cav-1 (58), or in cytoskeleton regulation, such as IQGAP-1 (31). In addition, a recent study suggests that there is a preferential budding of MuLV at sites of cell-to-cell contact due to the long cytoplasmic tail of Env (28). However, another element important for Gag trafficking and virus assembly is the ability of MA to bind cellular membranes of specific compositions.
In fact, the conserved myristoylation of MA is necessary for Gag anchoring into membranes and infectious particle production for different retroviruses such as MuLV (44) and human immunodeficiency virus type 1 (HIV-1) (20, 56). As shown previously for HIV-1 (60, 62) or Rous sarcoma virus (RSV) (16), membrane binding also requires a polybasic region, which is highly conserved among retroviruses. This region is thought to interact with the negatively charged heads of the cellular membrane phospholipids (36, 62). A previous study (52) showed that mutating this region of Moloney MuLV (MoMuLV) MA impairs viral production and Gag localization, but there is as yet no further functional study. Basic residues could reinforce MA interactions with membranes via electrostatic interactions with the acidic lipids of the membrane, as in the case of RSV (16). For several retroviruses, specific membrane binding has been shown to be mediated by cellular lipids. In particular, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] was recently shown to mediate plasma membrane binding by HIV-1 and -2 (12, 39, 48) and equine infectious anemia virus (EIAV) (11). PI(4,5)P2 plays an important role in cellular trafficking, similarly to all members of the phosphatidylinositol phosphate (PIP) family (see references 19, 29, and 30 for reviews). The PIPs are membrane phospholipids, phosphorylated derivatives of phosphatidylinositol. Their inositol ring can be phosphorylated at three positions (positions 3, 4, and 5), and all combinations of mono-, bi-, or triphosphates are present in cells. They are involved in membrane dynamics and trafficking and play essential roles in major cellular signalization pathways. The synthesis, localization, and degradation of PIPs are highly regulated (30): the location of specific kinases and phosphatases can modify the number and the position of the inositol phosphorylations. Therefore, potential interactions of the PIPs with retroviral MA might determine on which cell membrane Gag is targeted for viral assembly.
HIV-1 Gag membrane binding requires a "myristyl switch," i.e., a conformational change during which the N-terminal myristoylation of Gag, initially sequestered in the MA, becomes exposed and is able to interact with membrane phospholipids (25, 40, 45, 47, 48, 53, 55, 61). Recent structural studies of HIV-1 and -2 suggest that the binding of PI(4,5)P2 to a conserved cleft triggers this allosteric conformational change that exposes the myristyl group (48, 49). The MuLV MA protein does not contain an obvious PIP binding site, and poor solubility has thus far precluded high-resolution structural studies of the myristoylated MuLV MA protein. It is possible, however, to replace HIV MA with MuLV MA with limited effects on viral replication, suggesting a conserved function for this domain among retroviruses (10, 42). To determine if this evolutionarily distant retrovirus uses a similar PIP-dependent mechanism for membrane targeting of the Gag polyprotein, we prepared and characterized native and mutant forms of MuLV MA mutants. In doing so, we discovered an unusual lipid binding mode that appears to depend on both PI(4,5)P2 and phosphatidylserine.
Plasmids and site-directed mutagenesis.Plasmids used were pRR88 expressing MoMuLV (35) and pFMLV-Y1 (pY1), which contains the gag-pol gene of Friend MuLV (FrMuLV) (57). Substitution mutations were made in the MA coding region in pY1 by using the QuikChange mutagenesis kit (Stratagene), according to the manufacturer's protocol, as shown in Fig. 4. The first mutant series, m1 to m3, are substitutions in the polybasic region of MA. Mutant m1 harbors a replacement of basic arginines 31, 33, and 34 by glycines, mutant m2 harbors a replacement of basic residues R31 to R33 by alanines, and mutant m3 harbors a replacement of basic arginines R31 and R33 by glutamic acids to neutralize charges of the basic K32 and R34 residues. The second mutant series, m4 to m6, are replacements of single basic arginines in MA by glycines. The primers used for mutagenesis are as follows (5′ to 3′): CTGTCGGTAGAGGTTGGAAAAGGGGGCTGGGTTACATTC for m1, CTGTCGGTAGAGGTTGGAGGAGGGCGCTGGGTTACATTCTGC for m2, CTGTCGGTAGAGGTTGAAAAAGAGCGCTGGGTTACATTCTGC for m3, TGGAGGGATGTCGAAGGGACAGCCCACAACCTG for m4, AACGTCGGATGGCCAGGAGACGGCACTTTTA for m5, GACCCCCCTCCCTGGGTCGGACCCTTCGTGCAC for m6, and the corresponding reverse primers. Plasmid pMyr(−) (2) codes for the unmyristylated protein G2A-Gag-Pol. Plasmid phCMV-MLV-EnvA (50) codes for the envelope glycoprotein derived from amphotropic MuLV strain 4070A. Plasmid pMLV-VL30-GFP (Transgene SA) allows the expression of the MLV-VL30-Psi RNA coding for the green fluorescent protein (GFP) reporter. Plasmid PCS2-Venus, a kind gift from A. Miyawaki, encodes yellow fluorescent protein (YFP) under the cytomegalovirus promoter. The His6-tagged MuLV unmyristoylated MA [myr(−)MA] plasmid (pET16b-MA) was constructed by ligating the MA coding sequence, which was PCR amplified from the pNCA isolate (NCBI accession number NC_001501), into Escherichia coli expression vector pET16b at BamHI and NcoI sites in frame with the C-terminal His6 tag and stop codon of the plasmid. The plasmid was verified by sequencing at the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University (New Haven, CT). Substitution mutations were made in the MA coding region in pET-16b-MA using the QuikChange mutagenesis kit (Stratagene) according to the manufacturer's protocol, as shown in Fig. 2. The mutations are the same as the first group of mutants described above except that the primers used for the mutagenesis are adapted to an MoMuLV strain and are as follows (5′ to 3′): CAGTCGGTAGATGTTGGAAAAGGGGGCTGGGTTACCTTC for m1, CAGTCGGTAGATGTTGGAGGAGGGAGATGGGTTACCTTCTGC for m2, CAGTCGGTTGATGTTGAAAAAGAGCGCTGGGTTACCTTCTGC for m3, and the corresponding reverse primers.
The plasmids expressing 5PhosphataseIV (pcDNA4TOMyc5ptaseIV and the Δ1 mutant) were a kind gift of Eric Freed (HIVDRP, NCI—Frederick) (as described in reference 39). The plasmid expressing the plextrin homology (PH) domain of phospholipase C fused to cyan fluorescent protein (CFP) (pPH-PLC-CFP) was a kind gift of Walter Mothes (Section of Microbial Pathogenesis, Yale University School of Medicine).
Production of recombinant MA.Wild-type MuLV-MA protein, purified under native conditions, was overexpressed in E. coli cells (BL21 Codon Plus RIL) by the transformation of plasmid pET16b-MA. Cells were grown at 37°C in LB medium until the optical density at 600 nm reached 0.6 to 0.7 and were then induced with 1 mM isopropyl-d-thiogalactoside (IPTG) for 4 h. Cells were then spun down, and the pellet was lysed using a microfluidizer in a buffer containing 50 mM sodium phosphate, 500 mM sodium chloride, 5 mM β-mercaptoethanol, and 5 mM methionine. The MuLV myr(−)MA protein was purified by cobalt affinity chromatography (Clontech, Mountain View, CA) and ion-exchange column chromatography (SP column). Molecular weight was confirmed by electrospray ionization mass spectrometry. The MuLV myr(−)MA protein was then stored in a buffer containing 50 mM sodium phosphate, 100 mM NaCl, and 5 mM dithiothreitol.
Mutants and wild-type MA proteins, purified under denaturing conditions, were overexpressed in E. coli cells (BL21 Codon Plus RIL). Cells were grown at 30°C in LB medium until the optical density at 600 nm reached 0.6 to 0.7 and were the induced with 0.5 mM IPTG overnight. Cells were then spun down, and the pellet was lysed by sonication in a buffer containing 6 M guanidinium, 0.1 M NaH2PO4, 0.01 M Tris, 10% ethyl alcohol, 10 mM imidazole, 500 mM NaCl, and 10 mM β-mercaptoethanol (pH 7). The unmyristylated wild-type and mutant MA proteins were purified by nickel affinity chromatography (Ni-nitrilotriacetic acid spin columns; Invitrogen). Elution was done by using a buffer containing 6 M guanidinium, 0.1 M NaH2PO4, and 0.01 M Tris (pH 4.5). A 48-h dialysis was performed against KCl-HEPES buffer (100 mM KCl, 0.5 mM EGTA, 20 mM HEPES [pH 7.4]). Aggregated proteins were removed by centrifugation (20 min at 13,000 rpm).
Reagents.1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (PC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (PS) were obtained from Avanti Polar Lipids (Alabaster, AL). The ammonium salt of PI(4,5)P2 was purchased from Lipid Products (Surrey, Great Britain). d-myo-Phosphatidylinositol-3-phosphate [PI(3)P], d-myo-phosphatidylinositol-4-phosphate [PI(4)P], d-myo-phosphatidylinositol-5-phosphate [PI(5)P], d-myo-phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2], d-myo-phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2], and d-myo-phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3] were purchased from Echelon Bioscience (Tebu-Bio, France).
Quantitative cosedimentation assay with LUVs.MA affinity for phospholipids was determined by sedimentation assays with sucrose-loaded large unilamellar vesicles (LUVs) as previously described (4). We incubated a constant amount (1 μM) of recombinant MA protein (see the purification protocol described above) with different concentrations of LUVs, from 1.5 mg/ml to 15.625 μg/ml of total accessible lipids in KCl-HEPES buffer (100 mM KCl, 0.5 mM EGTA, 20 mM HEPES [pH 7.4]) for 10 min at room temperature. The 100-μl samples were then centrifuged (30 min at 42,200 rpm in a Beckman 42.2 Ti rotor). The top 90 μl of each sample was removed and considered the supernatant, and the pellet was resuspended by the addition of 80 μl of KCl-HEPES buffer. The supernatant and pellet were analyzed on a 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel, transferred onto Immobilon-P (Millipore) membranes, and stained with Coomassie blue for quantification (4). Since 10 μl of the supernatant was counted as pellet, and since the supernatant and pellet resuspension volumes were identical, the true pellet intensity was calculated using the following formula: IPellet = IP − 0.1 ISN, where IP and ISN are the experimental intensity values of the pellet and supernatant, respectively. The corresponding percentage of MA bound was deduced [the percentage of MA bound is IPellet/(IP + ISN)]. The binding of the protein to lipid bilayers can be described by defining an apparent association constant, K, as described previously (7). K is the proportionality constant between the fraction of proteins bound to the lipids, [MA]B, and the total protein concentration in the aqueous phase, [MA]F.
The fraction of MA bound is given by equation 1: $$mathtex$$\[\frac{[\mathrm{MA}]_{\mathrm{B}}}{[\mathrm{MA}]_{\mathrm{T}}}{=}\frac{K[\mathrm{lipid}]_{\mathrm{ACC}}}{1{+}K[\mathrm{lipid}]_{\mathrm{ACC}}}\]$$mathtex$$(1) where [MA]T is total MA, and [lipid]ACC is the accessible lipid concentration. It is assumed here that all the lipids of the outer leaflet of the LUVs are accessible. The association constant, K, deduced from the above-described equation is the reciprocal of the apparent dissociation constant, which is often called the affinity constant, Kd. All the calculated Kd values are given in Table 1.
The composition of the LUVs indicated in Results is expressed in weight percentages. LUVs were composed of PC (neutral) and PIPs (acid) and/or PS (acidic), as indicated in Results.
Cell culture.Human 293T cells and mouse NIH 3T3 cells used in this study were maintained, as described previously (34), in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and antibiotics (penicillin-streptomycin).
Antibodies.The polyclonal antibody against MuLV CAp30 used is a purified rabbit antiserum raised against the capsid domain of the MuLV-Gag protein (kind gift from A. Rein, NCI), used at a dilution of 1/5,000. Another anti-CAp30 rabbit antibody was produced by Invitrogen and was used at a dilution of 1/700. A goat antiserum against MuLV-Env gp70 (Quality Biotech) was used at a dilution of 1/2,000. The following monoclonal antibodies were purchased from Santa Cruz: anti-Lamp2 (mouse) (dilution of 1/400) anti-human S6 (mouse) (1/1,000 dilution), and anti-human alpha/beta actin (mouse) (1/200 dilution).
Anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase (Dako) were used at a dilution of 1/2,000. Anti-mouse antibody coupled to Alexa546 and anti-rabbit antibody coupled to Alexa488 (Molecular Probes, Invitrogen) were used at a dilution of 1/2,000.
Viral protein expression in cells.Viral protein expression was obtained by the transfection of MuLV Gag-Pol expression vectors in 293T and NIH 3T3 cell lines. Transfection was performed by use of the calcium phosphate precipitate method on 293T cells (as described in reference 35) and by use of Lipofectamine coupled to Plus reagent (Invitrogen) on NIH 3T3 cells, according to the manufacturer's instructions. Briefly, cells were plated at a density of 40,000 cells per cm2 and transfected the day after with 100 ng of DNA per cm2. This protocol was used for viral production characterization, electron microscopy analysis, and membrane flotation assays. For nonreplicative retroviral vector production, 100 ng of Gag-Pol expression vector, 33 ng of phCMV-MLV-A (Env) vector, and 33 ng of p-Psi-GFP per cm2 were transfected in 293T cells. For confocal microscopy, 16,000 cells per cm2 were plated and transfected the day after with 70 ng per cm2 of Gag-Pol expression vector. For viral expression in the presence of 5PtaseIV, 50 ng of pY1 together with 40 ng of phosphatase or pCS2-Venus vector per cm2 were cotransfected.
Viral particle purification and immunoblotting.To monitor viral particle production, culture medium containing virus-like particles or retroviral vectors was harvested 24 or 48 h posttransfection. After filtration (0.45 μm), the culture medium was centrifuged on a cushion of 25% sucrose in TNE buffer (10 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 mM EDTA), at 28,000 rpm for 1 h 15 min in a Beckman SW 32 rotor. The pellet was resuspended overnight at 4°C in TNE buffer and stored at −80°C. To analyze intracellular viral protein content, cells were lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 20 mM Tris-HCl [pH 8], 1% NP-40, 0.1% SDS, 0.2 mM EDTA) and sonicated. The cell lysate was then clarified for 10 min at 13,000 rpm and tested by the Bradford assay (4a). For Western blot analysis, the purified culture medium (equivalent of 430 μl) and cell lysate (50 μg of total proteins) or 28 μl from membrane flotation assay fractions was mixed into the loading buffer. The proteins were separated onto an SDS-10% PAGE gel and transferred onto a polyvinylidene difluoride membrane, and immunoblotting was performed by using the corresponding antibodies. Secondary antibodies were revealed by using the SuperSignal West Pico substrate (Thermo Scientific).
Viral titer measurement and reverse transcription assay.To measure viral titers, serial dilutions of clarified culture supernatants containing the nonreplicative retroviral vectors (made of MLV Gag, Env, and Psi RNA-GFP) were applied onto NIH 3T3 cells, and the number of cells expressing GFP was analyzed by fluorescence-activated cell sorter (FACS) analysis after 48 h. In each case, the limit dilution was reached to assess the amount of transductant retroviral vector particles. To assess the amount of viral particles produced, the RT activity (RT assay) was tested on the same serial dilutions. Ten microliters of culture supernatant was added to 50 μl of a reverse transcription mix {60 mM Tris-Cl (pH 8), 75 mM NaCl, 0.06% Nonidet P-40 substitute (Sigma), 0.7 mM MnCl2, 6 mM dithiothreitol, 6 μg·ml−1 oligo(dT), 12 μg·ml−1 poly(rA), and 2 μCi·ml−1 [α-32P]dTTP (specific activity, 3,000 Ci/mmol)}. After 1 h of incubation at 37°C, 5 μl was loaded onto DEAE paper (DE-81; Whatman) and then rinsed with 2× SSC (0.3 M NaCl, 0.03 M sodium citrate [pH 5]). The radioactivity (β-rays) of the sample was recorded by a storage phosphorscreen (Molecular Dynamics), measured with a phosophorimager (Fuji), and quantified by using MultiGauge software (Fuji).
Immunofluorescence staining and confocal microscopy.Immunofluorescence staining and confocal microscopy imaging were performed as previously described (23). Briefly, 293T cells (grown on polylysine-coated coverslips) or NIH 3T3 cells (grown directly on glass coverslips) were transfected and, 24 h to 48 h hours later (as indicated in Results), fixed (3% paraformaldehyde in phosphate-buffered saline [PBS]), permeabilized (0.2% Triton), and stained (see above for the antibodies used). In the case of CFP and Myc detection (see Fig. 3), images were acquired by use of a TCS SP5 AOBS spectral confocal microscope with 405-nm and 561-nm laser diodes and an HCX Plan Apo 63× 1.2 W Corr oil objective, supplied with Leica LAS AF SP5 software. In the case of capsid detection (see Fig. 6), images were acquired on an Axioplan 2 Zeiss CLSM 510 confocal microscope with an Argon 488/458 Plan Apochromat 63× 1.4-numerical-aperture oil objective (supplied with LSM 510 software) in the case of capsid detection.
Electron microscopy.Transfected 293T cells expressing wild-type and mutant F-MuLV Gag-Pol were washed in PBS and then fixed in a buffer containing 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate (pH 7.2) for 48 h. Cells were washed again, harvested, and postfixed with 1% osmium tetroxide for 1 h. Cells were dehydrated in a graded series of ethanol solutions, and the final cell pellets were embedded in Epon resin, which was allowed to polymerize for 48 h at 60°C. Ultrathin sections were cut with a Reichert (Heidelberg, Germany) ultramicrotome, collected on copper grids, and stained with 5% uranyl acetate and 5% lead citrate. The grids were then observed with a Jeol (Tokyo, Japan) 1010 XC electron microscope.
Membrane flotation assay.The membrane flotation assay protocol used was adapted from one described previously (38). For each experiment, 110 cm2 of 293T cells was transfected (see above). Twenty-two hours after transfection, cells were kept at 20°C for 2 h to slow down intracellular trafficking. The supernatant was then harvested and treated as described above. Cells were washed in ice-cold PBS and then resuspended in 10 mM Tris-HCl containing 1 mM EDTA and Complete protease inhibitor cocktail (Roche). All subsequent stages were performed in a cold room. Postnuclear supernatants (PNSs) were obtained after Dounce homogenization of cell suspensions and low-speed centrifugation and were adjusted at 150 nM NaCl. One hundred eighty microliters of this PNS was then mixed with 820 μl of 75% (wt/vol) sucrose in TNE (25 mM Tris-HCl [pH 7.4], 4 mM EDTA, 150 mM NaCl) and placed into the bottom of a centrifuge tube. On top of this PNS-containing 62% (wt/vol) sucrose mixture, 2.3 ml of 50% (wt/vol) sucrose in TNE buffer and 0.9 ml of 10% (wt/vol) sucrose in TNE buffer were layered. The gradients were centrifuged at 35,000 rpm for 16 h at 4°C in a Beckman SW60Ti rotor. Eight fractions of 500 μl were collected from the top of the centrifuge tube for immunoblotting as described above. The sucrose percentage after centrifugation was verified by refractometry to confirm that the discontinuity of the gradient was conserved.
The specific interactions of MuLV MA with the PIPs are enhanced for PI(4,5)P2 in the presence of PS in vitro.We determined the Kd, expressed in terms of accessible acidic phospholipid, for the interaction of MoMuLV MA and LUVs. To this end, we performed quantitative cosedimentation assays with LUVs composed of PC and the phosphoinositides either in the presence or in the absence of PS. After ultracentrifugation, the supernatant and pellets were analyzed by SDS-PAGE followed by transfer onto membranes, staining, and subsequent image analysis. Typical images of membranes representing the pellets and the supernatants for LUVs of different compositions that had been cosedimented with MA (1 μM) are shown in Fig. 1A for increasing accessible lipid concentrations (from 15.6 to 1.5 mg/ml).
From these images, we determined the fraction of bound protein, which is represented in Fig. 1B as a function of the acidic lipid concentration. Fitting these curves according to equation 1 allowed us to obtain the Kd value between MA and the different LUVs. The Kd value corresponds to the lipid concentration at which one-half of the protein is bound to the LUVs.
In these experiments, the unspecific sedimentation of MuLV MA was very low (less than 20%), which indicates that no MuLV MA aggregates were formed. Of note, no affinity was detected between MA and PC, as expected (data not shown). Also, there was no interaction between MA and PC/PS LUV containing PS at 20% (data not shown) or even at 40% (Fig. 1A and B). This was rather unexpected, since it was previously reported that under the same conditions, unmyristoylated HIV-1 MA showed a strong affinity for PS (15, 62), which was confirmed in our study (Fig. 1B). In one study, no clear interaction was observed between PIPs and HIV-1 MA under equivalent experimental conditions (15). However, under different experimental conditions, i.e., with myristoylated HIV-1 Gag in the presence of reticulocyte lysate factors, another study reported an in vitro interaction between HIV-1 MA and PI(4,5)P2 (14).
From Fig. 1B, it can be seen that the binding of MuLV MA to PI(4,5)P2 LUVs increases when the concentration of LUVs is increased. Of note, PI(4,5)P2 has a higher negative charge per molecule than PS (about −3 for PI(4,5)P2 versus −1 for PS [30]), and thus, 5% of PI(4,5)P2 is approximately equivalent to 15% of PS. As MuLV MA does not interact with PC or PS (even at 40%) but interacts with the phosphoinositides, we conclude that the interaction is more likely to be specific rather than exclusively electrostatic.
The fit of the experimental data using equation 1 leads to a Kd of 21.7 ± 1.37 μM for LUVs containing 5% PI(4,5)P2 (Table 1 and Fig. 1D).
Similarly, we performed the same experiments with all PIPs, and we observed a clear interaction between MuLV MA and all of them (images not shown), which allowed us to determine the Kd for each phosphoinositide (Fig. 1C and Table 1). Kd values were calculated by considering only the concentration of accessible PIPs, as there was no interaction with PC/PS (60:40) LUVs. The Kd ranged from 15.1 ± 0.8 μM for PI(3,4)P2 to 42.1 ± 13.7 μM for PI(3)P (lowest affinity). Some members of the PIP family showed higher affinities than others. For the monophosphorylated PIPs, the highest affinity (lower Kd) was observed for PI(4)P. In the case of polyphosphorylated PIPs, this tendency was conserved, as the only PIP lacking a phosphate at position 4 [PI(3,5)P2] had the lowest affinity.
We also investigated whether the addition of both PS and PIPs potentiated the interaction with MA (Table 1 and Fig. 1D). The interaction of MA and PC/PS/PI(4,5)P2 LUVs is higher than that for PC/PI(4,5)P2 LUVs without PS, as the curve shown in Fig. 1B is shifted toward the left in the presence of PS. The fit of the experimental data using equation 1 leads to a Kd of 5.4 ± 1.7 μM for LUVs containing 20% PS and 5% PI(4,5)P2, which represents approximately a fourfold-lower affinity than that without PS (Table 1 and Fig. 1D). In contrast, there was no significant decrease in the Kd upon the addition of PS together with the other PIPs (Fig. 1D and Table 1). Thus, the addition of PS to the LUV provided stereospecificity to the MA/PI(4,5)P2 interactions.
Our results suggest that there is a difference between HIV-1 MA and MuLV MA in the way in which they bind PIPs. In our test, unmyristoylated MuLV MA binds to PI(4,5)P2 but not HIV-MA, which binds to PS. For MuLV MA, the specificity of the interaction with PI(4,5)P2 is increased by the presence of PS.
Mutations in the polybasic region of MuLV MA inhibit its interaction with PI(4,5)P2in vitro.The N-terminal MA domain of MuLV Gag contains a polybasic region that could play a role in Gag-cell membrane binding (36, 46, 52), as previously reported for several other retroviruses, notably HIV-1 and RSV (14). Phylogenetic sequence analysis reveals the presence of several basic residues and a conserved basic cluster located at the N terminus of MuLV MA domains from exogenous (MoMuLV and Friend MuLV) or endogenous (neuro-2a-associated retrovirus [NeRV]) strains. A previous study (52) showed that mutations in this basic region of MoMuLV MA impair virus production. As PIPs are acidic lipids, the polybasic R31KR R34 region of MA is likely to be involved in the interaction. We therefore introduced substitution mutations into the unmyristoylated recombinant MA protein (Fig. 2A), changing basic residues to either neutral (mutants m1 and m2) or acidic (mutant m3) amino acids. Due to the low solubility of these mutants, the recombinant proteins (wild type and mutants) were purified under denaturing conditions (see Materials and Methods). Unfortunately, the m2 mutant aggregated and could not be analyzed. Under these conditions, the affinity of the wild-type MA protein for PC/PS/PI(4,5)P2 (75:20:5) LUVs was similar to the result reported in Fig. 1 and gave a Kd of 7.6 ± 0.7 μM (Fig. 2B and C). In contrast, the m1 and m3 mutants showed no measurable affinity (less than 35% of protein bound even at the highest lipid concentrations) (Fig. 2C). In conclusion, our results showed that the polybasic region R31KR R34 in MA is required for the interaction of MuLV MA with PI(4,5)P2/PS-containing membranes in vitro.
Overexpression of the 5ptaseIV enzyme causes a decrease in the level of MuLV production.To examine whether PI(4,5)P2 is important for MuLV particle production in cells, we did experiments aimed at modifying the PI(4,5)P2 pool in cells expressing Gag. The enzyme 5ptase IV is a phosphatase that specifically removes PIP phosphorylation at position 5, changing in particular PI(4,5)P2 into PI(4)P [but also PI(3,4,5)P3 into PI(3,4)P]. The activity of the enzyme was verified. We cotransfected 293T cells with pPH-PLC-CFP, which allows the expression of the CFP-coupled PH domain of the phospholipase C that specifically binds to PI(4,5)P2, and a vector expressing either wild-type or mutant 5PtaseIV (or LacZ as a control). The plasma membrane localization of PH-phospholipase C was observed under conditions of LacZ or 5Ptase Δ1 overexpression (Fig. 3C, left and right, respectively). This membrane localization is completely lost under conditions of 5PtaseIV overexpression (Fig. 3C, middle), which suggests that the enzyme is active in depleting PI(4,5)P2 from the plasma membrane. We then overexpressed 5ptaseIV and MuLV Gag together in 293T cells and measured viral particle production by an RT test (Fig. 3A). The reference activity (100%) was measured in the presence of a reporter control gene, YFP. In the presence of wild-type 5PtaseIV, viral production was reduced by fivefold (20% ± 4% of the control), while in the presence of the 5PtaseIV Δ1 mutant, viral production was less affected (65% ± 8% of the control). By immunoblotting, we found that the overexpression 5ptaseIV caused a decrease in viral particle production (Fig. 3B, top) without a significant modification of the intracellular level of Gag (Fig. 3B, middle) or actin (Fig. 3B, bottom), while the 5ptaseIV Δ1 mutant had little effect on viral particle release. The results showed that decreasing the intracellular PI(4,5)P2 pool partially inhibits viral production.
Mutations in the polybasic region of the Friend MuLV MA domain impair virus production.To further examine the role of the basic residues in Gag membrane binding and assembly in cells, we generated several mutants of the MA domain in a Friend MuLV Gag-Pol expression vector (pY1) (57) (Fig. 4A). Mutants were classified into two groups. The first group, already characterized in vitro, contained three mutants (m1, m2, and m3) of the polybasic region R31KRR34. In the m1 and m2 mutants, alanine or glycine residues were substituted for basic residues, while in m3, basic residues were changed to glutamic acid. The second group contained three mutants (m4, m5, and m6) of single arginine residues located in distant regions from the polybasic region (Fig. 4A).
To study the effects of the MA mutations on virus production and infectivity, we cotransfected 293T cells with plasmids expressing wild-type or mutant Gag-Pol and a murine envelope (Env) protein together with the packageable MuLV-enhanced GFP vector in order to produce retroviral vector particles that are able to achieve a single round of infection. This method allowed us to evaluate the production and the one-round replication of the MuLV retroviral vector. First, levels of Gag and Env proteins in the culture supernatant and in cell lysates were monitored 24 h posttransfection by immunoblotting using anti-capsid (CAp30) and anti-envelope (gp70) antibodies. All MA mutants allowed the synthesis of Pr65Gag (Fig. 4B, top), even though intracellular Gag levels and maturation profiles were slightly different from those of wild-type Gag, notably for mutants m1 and m3. The intracellular accumulation of Gag and Gag-derived products might be due either to a lower degradation or a higher production rate of Gag. Intracellular Env expression was equivalent for all the mutants in comparison with the wild type (Fig. 4B, top).
Only the MA mutants from the second group (m4, m5, and m6) were able to produce detectable amounts of retroviral vector particles similar to the wild-type level (Fig. 4C, bottom). These particles incorporated a normal amount of envelope protein. No particle production was detected for the m1 to m3 mutants, indicating that this MA region is essential for MuLV-based retroviral vector production.
Second, to verify if the particles produced were functional, we measured the RT activity of the cell culture supernatants and the single round of replication by transducing NIH 3T3 cells and by monitoring GFP expression by FACS analysis (Fig. 4C and see Materials and Methods for details). For the m1 and m3 mutants, only low RT activity (5.51% ± 2.54% and 6.77% ± 3.52% of the wild-type level, respectively) (Fig. 4C) and a low retroviral vector titer (4% ± 0.55% and 6.35% ± 1.98% of the wild-type level, respectively) (Fig. 4C) were monitored. The m2 mutant had no RT activity and was not infectious. The m4, m5, and m6 mutants (group 2) had no significant effect on RT activity, and vector-transducing titers were similar to the wild-type levels. We noted a good agreement between RT activity, vector titer, and CAp30 levels in the supernatants (Fig. 4), suggesting that the mutations did not cause further transducing defects in the vector particles produced.
Overall, these results showed that the polybasic R31KR R34 region of MA is necessary for MuLV viral particle production, while each of the isolated basic residues appears to be dispensable.
Mutations in the MA polybasic region decrease the ability of MuLV Gag to bind to cellular membranes.To assess the membrane binding ability of wild-type and mutated Gag proteins in cells, membrane flotation assays were performed on 293T cell extracts expressing wild-type Gag or either one of the mutant Gag proteins. Cells were harvested and lysed 24 h posttransfection, and the resulting PNS was loaded at the bottom of a discontinuous sucrose gradient that was submitted to equilibrium flotation centrifugation (see Materials and Methods). Fractions were collected and analyzed by Western blotting to detect MuLV Gag and cellular markers (Fig. 5). The top fractions, notably fractions 2 and 3, contained cell membrane-associated proteins (the lysosomal membrane protein Lamp2 was used as a control for a membrane binding protein), while the bottom fractions contained cytosolic proteins (the ribosomal S6 protein was used as a control) (Fig. 5A). The percentage of membrane-bound Gag was evaluated and is reported in Fig. 5B. For wild-type FrMuLV and MoMuLV Gag proteins, the majority of Gag was associated with membranes at the top of the gradient (almost 100% in fractions 2 and 3), while myr(−)Gag lost its membrane binding ability (almost 100% in fractions 7 and 8) (Fig. 5A). The polybasic-region mutants of Gag harbored a different phenotype: less than 50% of Gag remained associated with membranes (48% ± 9% for m1, 25% ± 6% for m2, and 46% ± 12% for m3), with the rest being cytosolic (group 1) (Fig. 5A and B). In contrast, the isolated basic-residue mutants harbored a wild-type-like membrane binding ability (group 2) (Fig. 5A and B) except that only 72% (±7%) of m6 Gag was associated with membranes.
These results show that the polybasic R31KR R34 region of MA is required for the stable membrane binding of MuLV Gag in cells.
Mutations in the polybasic region of MuLV MA result in intracellular Gag relocalization.Since mutations in the polybasic region of MA prevent virus production, the intracellular localization of the mutated Gag proteins was investigated by immunofluorescence microscopy in comparison with wild-type Gag. The study was performed by using both murine (NIH 3T3) and human (293T) cells, with the latter being used in order to avoid interference caused by the expression of endogenous MuLV Gag.
Figure 6 reports how MuLV Gag and mutated Gag proteins accumulated in NIH 3T3 murine cells. An unmyristoylated Gag-Pol mutant (44) was used as a control. As expected, wild-type Gag showed a punctate pattern, while myr(−)Gag was diffuse in the cytoplasm (Fig. 6). The m1, m2, and m3 mutants harbored phenotypes that were distinct from that of wild-type Gag. Mutant m1 was both diffuse and punctate in 88% of the cells (phenotype 1) (Fig. 6) and totally diffuse in 12% of them (phenotype 2). Mutant m2 was diffuse in the cytoplasm, with frequent patches observed only for NIH 3T3 cells (Fig. 6). Mutant m3 was both punctate and diffuse in all cells. The intracellular Gag localization for the single-basic-residue mutants (second group) was punctate in the cytoplasm, similarly to wild-type Gag (m4, m5, and m6) (Fig. 6). These observations suggest that the accumulation of large Gag oligomers in the cytoplasm is a prerequisite for viral particle production. Essentially similar results were obtained with 293T cells (data not shown).
Ultrastructure of MuLV mutant particles by electron microscopy.The ultrastructure and cellular localization of mutant Gag particles were analyzed by electron microscopy. Thin sections of cells expressing one mutant of each group (m3 for polybasic-region mutants and m6 for single-basic-residue mutants) were compared to wild-type Gag-Pol and myr(−)Gag-Pol. As expected, budding structures were observed mainly at the plasma membrane for wild-type Gag (Fig. 7A, right), and sometimes, particles were found in vesicles (intracellular compartments or plasma membrane diverticles) (Fig. 7A, left). Electron-dense assembled myr(−)Gag structures were found in the cytoplasm, without any apparent membrane (Fig. 7B). An intermediate phenotype was observed for the m3 mutant (polybasic-region mutant), since assembled particles were found in the cytoplasm, as for myr(−)Gag (Fig. 7C, left), or in intracellular compartments (Fig. 7C, middle and right), which could well correspond to virus-containing late endosomes or lysosomes in ultrathin sections (Fig. 7C, right). These observations were consistent with those obtained by confocal microscopy, as this Gag mutant appears either diffuse or punctate in the cytoplasm (m3) (Fig. 6). Moreover, m3 mutant Gag assembly did not take place at the plasma membrane (Fig. 7C), probably due to an improper trafficking of Gag and/or weak Gag-plasma membrane interactions (Fig. 2in vitro and Fig. 5in cellulo). Particles corresponding to the single-basic-residue m6 mutant bud at the plasma membrane and appeared to have a structure similar to that of the wild type (Fig. 7D).
This indicates that mutations of the MA polybasic region of MuLV Gag seem to prevent viral assembly and budding at the plasma membrane.
The aim of this study was to identify the viral and cellular determinants for MuLV Gag targeting to the plasma membrane. Two elements of the MA domain have historically been thought to be necessary for membrane anchoring: the myristic acid, which is required for Gag membrane binding and infectious-particle production (5, 22, 44, 52), and basic surface residues that could interact with phospholipid polar heads of the inner leaflet of the cell membrane (36). In the present study, the role of the basic residues of MuLV MA in Gag membrane binding, particle assembly, and release was examined. Two sets of mutants were designed, one group composed of three mutants in the conserved basic cluster (four basic residues in positions 31 to 34) and one group of three mutants with single substitutions of isolated basic residues exposed at the protein surface.
Our MA cosedimentation experiments with LUVs indicated that MuLV MA can interact with the biologically relevant PIP molecules [PI(4,5)P2, PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3] but not with PC (neutral) or with PS (acidic) alone (Fig. 1).
Under similar conditions, namely, using a nonmyristoylated MA protein with extruded LUVs in HEPES buffer, there is no specific interaction between HIV-1 MA and PIPs (15; data not shown). However, interactions between full-length HIV-1 Gag and PI(4,5)P2 in a solution containing LUVs and reticulocyte lysate were noted previously (12). Several factors might account for the different PIP binding properties of the MA proteins of MuLV and HIV-1. Thus, HIV-1 MA may need a cellular cofactor to bind PIPs, with this cofactor being present in the reticulocyte lysate. Our results indicate that the mechanism is different for MuLV MA. In fact, MuLV MA alone was able to specifically interact with LUVs containing PIPs in the absence of other cellular components (Fig. 1).
Unexpectedly, the affinity of PI(4,5)P2 for MuLV MA increased dramatically (fourfold) upon the addition of PS (Fig. 1). In contrast, there was no such effect of PS on the other PIPs, suggesting that PI(4,5)P2 is the major MA-interacting PIP in cells. In fact, all membrane layers containing PI(4,5)P2 also contain PS, especially in the internal layer of the plasma membrane (32). A PS potentiation of the interaction between PI(4,5)P2 and a cellular protein was also previously described for Myo1c, a protein of the myosin superfamily (26). The alternative mechanism, that is, an initial binding of PS but not of PI(4,5)P2 alone and the stabilization of the interaction by PI(4,5)P2, was also described for the protein kinase Cα (PKCα)-C2 domain (33). This cooperation between PS and PIPs appears to be a general feature for protein-membrane interactions. In the case of the PKCα-C2 domain, the binding of PS stabilizes the protein in an orientation that favors the interaction with PI(4,5)P2. Similarly, in the case of MA, the interaction with PI(4,5)P2 could trigger a conformational change or stabilize a specific orientation of the protein, allowing the interaction with PS. This model is supported by the fact that in other retroviruses, MA binding to PI(4,5)P2 causes large structural modifications. Nuclear magnetic resonance data show that HIV-1 MA binding triggers the "myristyl switch" (48), and in vitro assembly data suggest that it may induce a profound reorganization in Gag oligomer formation (trimers instead of dimers) (1, 18). In the case of HIV-2, the first MA helix is destabilized upon PI(4,5)P2 binding but is not sufficient to trigger the myristyl switch (49). EIAV MA also shows motions upon PI(4,5)P2 binding, with possible consequences on trimerization (11). PI(4,5)P2 binding could then be a key step in retroviral assembly, causing structural modifications that promote myristate exposure, multimerization, and PS binding.
The main difference between our results and previous observations of Myoc1 or PKCα-C2 is that for MuLV-MA, PS is providing the specificity for PI(4,5)P2 binding.
The affinities of the interaction between MA and PI(4,5)P2 alone for HIV-1, HIV-2, and EIAV were measured by nuclear magnetic resonance [with soluble species of truncated PI(4,5)P2]. The Kd values were all quite high, between 100 and 300 μM (i.e., low affinities) (11, 48, 49). In the present study, the Kd was lower (Kd = 22.5 ± 1.3 μM), indicating a high affinity of the interaction between MuLV MA and PI(4,5)P2. This affinity may suggest the specific targeting and binding of MuLV Gag to the plasma membrane. In addition, the Kd obtained in the presence of PS, 5.4 ± 1.7 μM, enhances it. The affinity enhancement by PS may therefore also exist for other retroviruses. Another study was carried out on RSV matrix (16), but the authors reported no affinity of MA for PIPs. Those experiments, however, were done in a simpler system, and, as for HIV-1, the interaction with PIPs may necessitate more complex environments.
Highly consistent with our data, an analysis of retroviral particle lipidome was recently carried out (8). The authors showed that PI(4,5)P2 and PS were the most enriched phospholipids in the particles for HIV-1 and MuLV, suggesting that these lipids are required for retrovirus assembly and most probably for Gag targeting and budding at the plasma membrane. A decrease in the level of viral production under conditions of 5PtaseIV overexpression was also reported, in agreement with our observations for MuLV Gag showing that the depletion of the intracellular PI(4,5)P2 pool upon 5PtaseIV phosphatase overexpression decreased levels of MuLV release (Fig. 3).
Our data showed that the polybasic-region mutants partially lose the capacity to bind biomimetic membranes (Fig. 2) and cellular membranes (Fig. 5 and 6), indicating that the polybasic region R31KRR34 is involved in MuLV Gag membrane binding. The data also showed that multiple substitutions in this region impaired particle release and the localization of Gag (Fig. 4, 6, and 7), in agreement with previous work on MoMuLV Gag (52). Part of wild-type Gag traffics through the late endosomal/lysosomal pathway, as previously shown (3, 27, 51), while part of Gag is located at the plasma membrane (Fig. 7), but mutants were diffuse in the cytoplasm (Fig. 5 and 6) or eventually accumulated in intracellular compartments (Fig. 6 and 7), suggesting that the polybasic region of MuLV MA was not required for Gag targeting to internal compartments. By electron microscopy, the assembly of these mutants was observed either in the cytoplasm or in internal compartments, as opposed to wild-type Gag, which buds mainly at the plasma membrane (Fig. 7). This favors the notion that the MuLV MA basic cluster is most probably a signal for Gag targeting to and assembling at the plasma membrane. These results suggest that the polybasic region in MA not only is necessary for the stabilization of Gag membrane binding, in synergy with the anchoring of the myristate, but also contributes to the "choice" of the viral assembly site.
In contrast, the isolated basic-residue mutants harbored mostly wild-type-like phenotypes and have no major role in particle assembly.
We propose a scheme for MuLV Gag targeting and assembly, notably at the level of MA-cell membrane interactions. Our results favor the fact that MuLV Gag, having a high affinity for PI(4,5)P2/PS, is directly addressed at the plasma membrane but could also bind endosomal membranes since MA is able to interact with all PIPs. The virions could then be produced by budding at the plasma membrane or at internal membranes, as suggested previously for HIV-1 (21, 23, 37, 41).
In conclusion, it appears that the MA-PIP interaction is an obligatory step for Gag membrane targeting and binding and, thus, for efficient retroviral assembly at the plasma membrane. Even though no classical PIP binding cleft has been observed for the MuLV MA domain, comparisons between several retroviral MA domains (36) allow the identification of a conserved polybasic region in the N-terminal domain of Gag. This MA basic cluster is most probably the PIP binding site of Gag and represents a potential target for new antiviral drug design.
We acknowledge the PLATIM IFR128 microscope platform at ENS Lyon (France), Mike Spielt in the M. Summers laboratory for providing us the recombinant HIV-1 MA protein, E. Freed for providing us pcDNA4TOMyc5ptaseIV and the Δ1 mutant, W. Mothes for providing us plasmid pPH-PLC-CFP, and A. Rein for providing us the anti-CAp30 rabbit antibody. We gladly thank Jorge Vera for his helpful suggestions concerning the purification of MA proteins under denaturing conditions.
This work was supported by INSERM and CNRS (grant RISC AO 2008 to D.M.). We gratefully acknowledge partial support from the NIH (grant AI30917 to M.F.S.). E.H.-P. is a fellowship receiver of the French Government.
- Received 3 June 2009.
- Accepted 1 October 2009.
- Copyright © 2010 American Society for Microbiology


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Me on a camel
Written By: Gary on April 27, 2006 One Comment
Originally uploaded by garylapointe.
So we went on a camel ride yesterday!
We rode them up a dune (15 minutes) and then back down across the Sahara desert (and hour?) to a village where we had a tour.
It wasn't as hard as I thought it'd be!


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Thomas & Friends are an ever popular brand with toddlers and young children and in the evolving world of gender neutrality Mattel has responded to feedback from parents and created a range of apparel for little girls to go with the infant and boys range. Available at Myer since mid-October the brand new range of Thomas & Friends apparel is sure to excite the Thomas … [Read more...]
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We all know adults who are very difficult to buy for at Christmas, people who have all they need and want that just leave you scratching your head for great gift ideas. Flaschengeist have something perfect for those hard to buy for people at Christmas with an extensive range of cocktail flavours, gourmet olive oils, delicious dukkahs and artisan wines. The best thing … [Read more...]
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We often give gifts which are impersonal because we are time poor or stuck for ideas, but choosing something a little more sentimental doesn't need to be difficult. Thinking about something based on their personality type or interest is the first step in ensuring a heartfelt gift. Something homemade is always going to be special, because we all know it is the thought and … [Read more...]


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Team Members
Albert Coppens
Former trader (NL)
Albert was born in the Netherlands; his trading journey began in 2007 but he has been interested in the financial markets and trading since the age of 15. In (insert year) Albert Completed a Bachelor of Business Administration and worked for four years in the financial industry. He started the 240trade company as a dream to help and develop traders of all levels from around the globe.
These days Albert is not only trading, teaching and working hard to reach his goals, he also shares and develops trading strategies which have been proven to stand the test of time. He is responsible for most of the education and webinars within the 240trade community but he does also get assistance from his team Mark Bleakley and Karl Rollon.
Albert is mostly a technical trader who specialises in Advanced Fibonacci concepts in his approaches to the market. He has developed multiple strategies which also include but are not limited to Pivot Point, W.D. Gann, Fundamental and Wolf Wave analyses. These are just some of the technical tools and trading methodologies Albert adopts in his trading, there are many more.
Albert loves to help people and has mentored hundreds of traders over the years. His mission together with his team (Mark Bleakley and Karl Rollon) is to build 240trade into a caring community of experienced and knowledgeable traders who support each other to achieve their financial, trading and personal goals. His promise to you is to deliver quality trading education, including live and pre-recorded webinars, trade ideas, written materials and to work with you closely to build you into a top trader from the ground up.
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Former trader (AU)
Mark was born and bred in the frontier town of Darwin, Australia. He has also lived and worked in Canberra, Australia's capital city as a professional musician and music teacher, he has completed a Bachelor of Music and Diploma of Teaching and Learning. Mark's trading career began 11 years ago after he was introduced to forex, stock, futures and options trading by a friend. These days he mainly trades Forex and Stock Indices. During his trading career he has been trained one on one by some top traders, including three former bankers. He continues to work with his mentors to improve all the time. He is a big believer in the power of patience and hard work.
Some of the methodologies he has been taught and uses include Richard Demille Wyckoff's approaches, in particular building on the work of Tom Williams (the former syndicate trader who revealed a lot to the retail trading public before he passed) Tom invented Volume Spread Analysis based on the work of Wyckoff. Mark has also studied and uses W.D. Gann's famous Fibonacci Fan lines, chart patterns, some candlestick formations (the back tested high converting ones only) and fundamental factors that move the market. He really enjoys helping people grow as traders and over the last 6 months he has worked with and mentored over 50 traders. Some of these traders are now being presented with opportunities to work for a fund and commence their professional trading careers.
Karl Rollon
Sales and marketing (THA)
Despite being fairly new to trading, Karl has had success in a short period of time in his trading career. He first started trading last October, 2018 and was introduced to the markets by a former student. A highly committed family man and an Educator by profession, Karl studied a Bachelor of Science and Commerce, Majoring in Accountancy, as a starter to find his chosen career.
He found his passion in sharing the joy of learning to his students and has completed TEFL and TESOL certificates and furthermore has more than a decade of experience in the education industry. Being fond of numbers, he enjoys teaching Mathematics and also English as a Second Language. After seven years working in international schools, he chose to serve the less privileged for a cause. He is originally from the Philippines and his family are presently living and working in Thailand and have been there for more than a decade now. Karl loves to spend quality time with his one and only wife and three kids.
A committed life-long learner and currently establishing his foundation and knowledge in best trading practices through access to quality materials and teachings from his industry experienced mentors. Technical analysis is one of his main passions and also encompassing technical analysis into a practical strategy that combines price action analysis on higher time frames. With exemplary growth in such a short period of time, some of the approaches that have been successful for Karl include: candlestick patterns, volume spread analysis, recognizing pin bars, inside bars, engulfing bars, Guppy moving averages, identifying trends and drawing dynamic trend lines, major pivot points (mainly Monthly and Yearly Pivot Points being the strongest areas of supply and demand), Fibonacci Retracements, Fibonacci Projections and Ichimoku Kinko Hyo


text: HomeAccommodation The Golden Heart Birdlip Gloucestershire The Golden Heart is a delightful country pub with rooms at Birdlip deep in the Gloucestershire Cotswolds. Situated in the heart of the Cotswolds and in a beautiful location between Brimpsfield and Cowley near Cirencester, our centuries-old pub sits alongside the A417 and is easily accessible from the M4 and M5.Why stop by? Good Food, Award Winning & Exotic Meats, Vegetarian, Vegan & Gluten Free options available, Locally Sourced Produce, Traditional Cotswold Building, Cosy, Intimate Dining Rooms, Traditional Ales, Local Ciders, Bed & Breakfast, Outside Catering, Open Fire, Large Garden & Patio, Ample Parking.Dining. At the Golden Heart, we are committed to providing the best locally-sourced produce and we regularly support National and Local livestock shows and markets. Exotic meats have long been a feature of the Golden Heart's extensive menu – whether you opt for the healthy option of a low-fat ostrich casserole or kangaroo steak or the more adventurous crocodile, zebra and rattlesnake mixed grill.Stay. Our en-suite accommodation costs from £45 for a double room per night, £58.00 for bed & breakfast (£30 for a single or £38 for bed & breakfast), and all rooms are situated at the rear of the premises. Breakfast is included in the price of the room and is freshly cooked to your own specifications and required time! Tweet Please quote 'Let's Stay' Contact Details Birdlipnear CirencesterGloucestershire T: 01242 870261 Email


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My third week of Stitches n Scraps KAL! I finished the three pieces, connected them with the final rows of ribbing, and seamed the bottom. The middle gaps are where the handle and spout will stick out.
It's looking adorable, but I haven't found the perfect buttons, yet. I'll be scrounging around in my button jars for just the right ones.
Next week you'll see a (hopefully) finished and blocked cozy. :)


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- Home
- Afghan Vintage Kuchi Old Style Dresses
- Crochet Dress Balochi Dress In Turquoise Light Blue Color
Product Description
You have seen above retro fashion Balochi Dress, made of good quality fabric. This costume is beautified with hand embroidery work, which is also known as Crochet work or needle work. These dresses resemble very old Balochistan tribal fashion artwork and prepared in few months. These dresses are known as Balochistan women formal costume, but also loved around the globe as the crochet work is very unique and attractive.
- Main theme color of the dress is turquoise or light blue.
- The package contains only single shirt.


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India's repeated charge that Pakistan has been providing shelter to terrorist and D-Company boss Dawood Ibrahim was vindicated when close aide Farooq Takla was arrested from Delhi airport last week. He has revealed to investigation agencies the details of his residence, safe house and his security contingent which is managed by Pakistan Rangers.
Dawood aide Farooq Takla reveals Dawood Ibrahim's new safe house in Bara Anda island near Karachi. This island is protected by Pakistan coast guard commandos. The Island has secret escape route, that takes Dawood to Dubai in less than six hours.
India's No 1 enemy Dawood Ibrahim tracked down on island near Pakistan's Karachi
Sources have told that Takla confessed to Pakistan Rangers providing security cover to Dawood and his family in and around his bungalow in Karachi's posh Clifton locality. He then goes on to elaborate on how the dreaded terrorist accused of masterminding the 1993 Bombay blasts – is whisked away to a safe house each time a foreign VVIP arrives in the country or when Indian government increases international pressure on Pakistan.
India's number 1 enemy Dawood Ibrahim tracked down to an island near Karachi.
MEGA TIMES NOW EXCLUSIVE| India's number 1 enemy Dawood Ibrahim tracked down to an island near Karachi. LISTEN IN as @Aruneel_S gives us more details pic.twitter.com/420th7exXc
— TIMES NOW (@TimesNow) March 14, 2018
Pakistan has provided the underworld don with a luxurious safe house at an island near Karachi which is guarded by the Pakistani coast guard. The safe house also has an escape route mapped out which is made for Ibrahim to get out safely and travel to Dubai in a few hours if need be. Farooq Takla, who was arrested by the Indian authorities last week and a close aide of Dawood, was responsible for looking after the arrangements of his master whenever he was in Dubai. Takla is now being questioned by the Central Bureau of Investigation (CBI) at their headquarters.
The safe house too is strategically located in an area called 'Anda Group of Islands' and none except Dawood and his wife are permitted in and around the area. Officials in the Pakistani establishment use a special frequency to speak with the Don. And while Pakistan Rangers deploy security personnel here as well, Pakistan's Coast Guard conducts regular patrols to not just ensure safety for Dawood but also a safe passage to Dubai if and when required.
Takla has further revealed that the Pakistani establishment is capable of ferrying Dawood to Dubai's safety in less than six hours in the face of threats and that he was responsible for handling matters for Dawood once he reached UAE. He has confessed that he not only received Dawood in Dubai once under such a scenario but also transported him to other locations here.
He said Dawood faced threats from elements within Pakistan and that a local gang and Chotta Rajan's gang made separate attempts on his life between 2000 and 2005.
UPA Government approved Farooq Takla passport renewal!
This segment of Zee News brings to you information about big revelation on Farooq Takla, a close aide of gangster Dawood Ibrahim. According to sources, Farooq Takla's passport was renewed in 2011 and at that time P. Chidambaram was Home Minister of the country. India's number one enemy and a gangster Dawood Ibrahim's close aide was allowed to renew his passport by the UPA Government. Watch this video for more information.
Takla, meanwhile in Dubai, used to drive taxis to evade authorities and has two sons there – one has completed his post-graduation and the other is studying commerce. He added that it was always his wish breathe his last in India where his ailing mother lives with his brother. And while Takla's arrest does shed light on the modus operandi of D-Company boss, it is learnt that Takla has also claimed Indian authorities would never be able to bring Dawood back to India.
Dawood Ibrahim says he wants to come back to India and breathe his last here. Why being loyal suddenly? Indian govt should not adhere to his demands and allow him to die and show his love and loyalty to his country Pakistan. No one's missing him here in India!
#DawoodIbrahim says he wants to come back to India and breathe his last here. Why being loyal suddenly? Indian govt should not adhere to his demands and allow him to die and show his love and loyalty to his country https://t.co/eeIJhqWYFZ one's missing him here in india!
— Savitha (@SavithaKartha) March 9, 2018
Under the Congress raj Dawood Ibrahim did bomb blast and left the country and they did nothing to catch him.. He talks to news reporters but Congress never did anything to put pressure on him, never said he is in Pakistan… Now the same Congress is pouncing on BJP for Nirav Modi scam.
Source: Zee News


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Make It Funky Holiday Party
Presented By
Buy Ticketspurplereindeer.eventbrite.com
When the light turns green, you go.
When the light turns red, you stop.
But what do you do when the light turns blue with orange and lavender spots?
You go to the Purple Reindeer Holiday Party. Not quite Thanksgiving, not quite Christmas, you seize the chance to double down on holiday cheer.
Make It Funky DJs in the house.
It's a holiday party, dress to impress.
*Wearing/rolling/pouring purple optional
RSVP for free entry before 10pm:
0 Comments
Who's Going


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Were you charged the wrong amount? Did you not receive a deduction you were expecting?
Just inform us about the incorrect charge using the contact form at the bottom of this page. Please include the amount of the excess charge or missing credit, and a quick explanation of why this is incorrect. Our team will check your claim and add a credit to your Medici Living account if there was an error.


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Today was beautiful. The sun was out and the temperature was in the low to mid 60's. That's what I call perfect garden weather! I didn't have much time to garden but needed to do a little something and was able to spend about 30 minutes on a small project. Back in the fall I used my black tarp technique to cover an existing bed and the results were great!
The black tarp technique helps to kill off vegetation underneath it. It triggers seed germination, growth, then kills the plants off leaving a nearly weed free environment. I learned this technique after reading The Market Gardener (Amazon Aff Link) and it really works. It took me less than 30 minutes today to move the tarp to a new location in the garden, rake the dead weeds out, level the soil, and sow new seeds! It's really very simple. I didn't till the ground so I did not disturb the soil ecosystem in any significant way.
In a couple weeks I can do the same to the new section of ground I covered and continue my way around the garden. The black tarp technique works really well with succession planting and really makes it easier to get the garden started. The tarp could easily fit on either a raised bed or an in ground garden. You can read more about using a black tarp in the garden here.
What plants did I plant today?
Today I planted seeds for cilantro, spinach, lettuce, kale, mustard, and dill in the garden. We're expecting rain tonight so these seeds should get a good watering in to trigger germination.
Cilantro loves starting in cooler weather and tends to bolt when it gets hot so now is the perfect time to get the seeds in the garden. The others are semi frost tolerant too and should do alright in the garden at this point in Tennessee!
What's next in the garden?
- This week I'll be sowing seeds for peppers, tomatoes, eggplant, basil, and a few other warm season plants. Some of them will be ornamental.
- In a couple weeks we'll be planting potatoes and another succession of greens.
We're in zone 6b-7 of Middle TN so make sure you calibrate to your zone before deciding to plant on my schedule! We also have an additional issue to factor in with a hillside that blocks late afternoon sunlight. That's great for the summer, we stay cooler, but we're in a frost pocket which we have to plan around.


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Alexa Discord Community
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Become part of a growing community of Alexa Developers. The VoiceTV community is all about bringing talented developers, marketers, clients and agencies into a single hub for you to learn, network and improve.
By joining you can:
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• Access to student-only channel when purchasing any Alexa Course


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Demille Flannel (Purple)
$ 42.00
This variant is currently sold out
"Demille" Flannel - Premium blended plaid fabric in a classic collared button up silhouette, highlighted by a custom all over paint splatter, spray and brush marks, complete with rear neck cuban and branding details. Fits true to size for a relaxed fit. Size up for an oversized fit.


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A NOTE TO READERS: The following article is a drastically rewritten and updated version of an article prepared by Bob Mason six years ago. This article is to be published soon in the Winter 2018 … [Read more...]
Naming A Trust As IRA Beneficiary: Great Idea? Bad Idea?
This is the most advanced topic I have written for my public readers. But it can be an important topic, so hang in there. According to conventional wisdom you should always name your spouse as … [Read more...]
Trusts and Trucks – A Trusts Primer
Lately I have written a number of difficult posts that relate to trusts. A reader asked me why I hadn't written anything that simply explains what a trust is. I was surprised to see that I hadn't, so … [Read more...]
Should Special Needs Trust Beneficiaries Eat Their Pets?
Updated April 14, 2017 Recent Question: Bob, is it OK for the trustee of a special needs trust to purchase a $2,000 pure bred spaniel for the trust beneficiary, Edwina? Recent Answer: Only if … [Read more...]
Sole Benefit Trusts and Special Needs Trusts
A Sole Benefit Trust is an often neglected yet important type of Special Needs Trust that can be useful in planning for certain categories of individuals with disabilities. For example, a "Sole … [Read more...]


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Been Injured in a Trucking Accident? Our Indiana Truck Accident Attorneys Can Help
If you drive on the freeways, you may notice tire debris on the roadways. Rubber is typically an indication that a tire blowout occurred. Tire blowouts are not uncommon for truckers to experience on the roadways and can result in serious accidents.
Big rig accidents can cause fatal injuries due to their massive size and weight. According to the Insurance Institute for Highway Safety (IIHS), "A total of 4,102 people died in large truck crashes in 2017."
If you or a loved one were involved in an automobile accident with a truck and sustained injuries, contact our Indiana truck accident attorneys. At the Ken Nunn Law Office, we believe injured victims deserve justice for any injuries and property damage caused by the careless actions of a truck driver.
Main Causes of Tire Blowouts
When you are behind the wheel of your motor vehicle, it can be scary and intimidating to travel alongside a semi-truck. What can become even more frightening is when the commercial vehicle has a tire blowout. A tire blowout can happen for several reasons including:
- Potholes – Potholes can produce serious injuries to any driver no matter the size of their tires. The initial impact of the tire and the hole can cause the tire to puncture or slip open. When a tire has any type of damage, it has the potential to deflate and release air rapidly, which can lead to a blowout.
- Poor Maintenance – The overall maintenance of a truck plays a vital role in how safe the truck functions and operates. However, in some cases, trucking companies fail to comply with safety procedures or requirements and do not check the maintenance of their trucks often enough. When a trucking company fails to maintain their trucks, they place their drivers and other motorists in severe danger.
- Overloaded Cargo – In some cases, the tires placed on commercial vehicles can be punctured due to heavy cargo loads.
- Defective Tires – When defective tires are placed on commercial vehicles, they can fail to function according to their capability and result in an accident.
- Inadequate Tire Inflation – Sometimes tires have too much or not enough air. Without proper air pressure, a tire can blowout.
- Braking Too Often – Tires tend to wear down faster when truck drivers brake excessively. The tires tread depth diminishes and causes the tires to become unsafe.
Steps to Take after a Truck Crash
The moments immediately following a trucking accident are imperative if you plan to file a lawsuit in the future. It's important to contact the police and emergency responders right after the collision. The police will review the scene of the accident and determine what factors contributed to the crash.
If you are able, make sure to take photos and witness statements, which can be used as evidence to help build your case. If the truck driver was at fault for the accident, it's best for you to contact an Indiana truck accident attorney before making any decisions about what legal steps to take next.
Can I File a Lawsuit after a Tractor-Trailer Accident?
Filing a lawsuit is not always a straightforward process and, without legal guidance, you may be left with countless questions. When you file a lawsuit, you can seek compensation for any injuries and property damage you suffered as a result of the collision.
Seeking compensation after a truck accident can be difficult since these large corporations have extensive insurance policies. The reason why they carry such large policies is that they are fully aware that trucking accidents can lead to devastating and permanent injuries and, in some cases, death. Although trucking companies have large policies, it doesn't mean they won't attempt to offer you a low settlement.
The insurance company's ultimate goal is to make a profit, which means they will strive to resolve a lawsuit by paying as little as possible. This could leave you with a settlement that is not comparable to the injuries you sustained. In other words, the settlement amount offered may not compensate for all the medical expenses and treatment required for you to recover.
Compensation can include medical bills, lost wages, pain and suffering, out of pocket expenses, property damage, and even the wrongful death of a loved one.
Is the Operator Held Legally Responsible?
In order to pursue a lawsuit, you must prove that the truck driver acted negligently. However, in some cases, that can be difficult to conclude. In these types of cases, liable parties can vary and can include the trucking company, the manufacturer, and the owner of the commercial truck. These are just some of the potential parties who may be found at fault. It's in your best interest to solicit legal advice in order to hold the correct party or parties responsible.
Contact Our Trusted Legal Team
If you or someone you know has fallen victim to an accident involving a tire blowout from a large truck, our Indiana truck accident attorneys can help you navigate the complex legal system.
Our team has handled numerous truck accident cases, and we have the knowledge and experience to seek justice on your behalf. We know you may feel stressed and afraid about the outcome of your case, however, when you have our trusted legal team on your side, you can feel confident that your case will be handled with compassion and determination. Our law firm has dedicated our practice to helping innocent people seek the justice they rightfully deserve, which is why many people have trusted us with their cases.
Truck accidents in Indiana happen more often than people know. If you're involved in a trucking accident that wasn't your fault, you need our legal knowledge. Call our office today at (800) 225-5536 or fill out our free consultation form.


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There's something to be said for learning to take a compliment, and a lot of us are really bad at it. When a friend, coworker, or boss praises us for a job well done, we'll brush it off and say something like, "eh, it's no big deal." If you're trying to improve your compliment-accepting skills, here's how to respond instead.
Photo by Thank you for visiting my page.
There are a number of reasons you might not be great at taking a compliment. Maybe you're shy and it makes you feel uncomfortable. Maybe you don't want to come across as boastful. Whatever the reason, there's a good case to be made for learning to accept praise. For one, sometimes deflecting a compliment comes across as condescending. It makes the giver feel uncomfortable. Also, deflecting a compliment can diminish your value, as The Art of Manliness puts it. Politely accepting praise usually comes across as more professional than simply shrugging it off.
You probably know your default, go-to reply for rejecting a compliment. For many people, it's "no big deal." For me, it's one of two reactions: 1) I say something negative about myself instead, or 2) I ignore the compliment altogether and instead praise the giver. Pinpoint your default reply, then replace it with something better.
For example, next time you've worked hard on a project, your boss compliments your ability to deliver under pressure, and you're tempted to tell him or her, "it wasn't a big deal," consider the fact that you might be deflecting. Instead, try to replace this gut reaction with a simple "thank you." And when someone thanks you, The Muse suggests embracing "you're welcome" instead of shrugging off gratitude for hard work or even for a favour:
The funny thing is, "You're welcome" is so easy, but also so overlooked. If you listen to yourself the next 10 times someone thanks you, I'd bet your response is more along the lines of "sure," "no problem," or "any time."
So, practice saying, "you're welcome" in daily life (e.g., after someone thanks you for holding the door open). Then, you'll be able to say it much more readily when a co-worker thanks you for helping him out with a big project.
Both are obvious replies, and you might not be a fan of "you're welcome" for simple favours — to each their own — the point here is: learning to take a compliment can be surprisingly beneficial. By replacing your go-to reply with a more accepting phrase, it's an easy enough skill to learn.
You Need to Stop Responding to Praise With "It's No Big Deal" — Here's How [The Muse]


text: You need some dental work, but you're afraid to get it. You think you can live with the throbbing pain in your mouth if it means you'll never have to see the inside of a dental operatory again. It's trading one type of pain for another, as you see it, and you'll take your ...Read More


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He then pointing the gun to her chest, forced her to her knees and ordered her to open the cash register. When he began pulling cash from the register Anna screamed.
That scream told her husband, John that something was definitely wrong.
John grabbed his gun and came out from the back. The armed robber fired once at him. John returned fire with three shots, one of which found the mark and killed the scumbag.
If my husband hadn't shot him, he would have been shot," Anna said.
John had to agree. "I didn't want it to come to that," he said. "But we had to defend ourselves."


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Sometimes a girl has to get her pink on! And that's just what little munchkin Milania is doing in her perfectly styled nursery. Designed by her interior designing mama, the room has everything a little princess could want. The chandelier and fabric hanging gorgeously highlight the room. A monogramed chevron wall frames the little one's crib and all the pops of pink bring the nursery full circle. So, put on your princess hat and jump into the dreamy world of little Milania.
Photography: KB Digital Designs
10 Comments
Tina says:
Love this room! Just gorgeous. Where did you get the mirror?
casey says:
how do you do the ceiling
Tracie Patterson says:
Ceiling looks like white tulle
Tracie Patterson says:
Try etsy
Anonymous says:
Can you tell us how you did the draped fabric on the ceiling?
Stay at Home Mama says:
I'm wanting to do fabric on my ceiling like this but can't seem to find the right fabric…what kind is this?
Anonymous says:
Did anyone find out where the material for bedding or the colors on wall came from or the names?
LauraUNF99 says:
I LOVE everything about this room for my daughter's playroom!! we need details from paint colors to where you got those wall sayings, etc. PLEASE SHARE 🙂
Brittany says:
Do you know what paint color was used on the walls? I LOVE IT!!!
Sara says:
do you know where they got the bedding? i love it!
Comments are closed.