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Sunday, August 11, 2019

Explore Filament magazine's photos on Flickr! Filament magazine. Follow. Give Pro. Filament magazine. Followers•92 Following. Photos. In this edition of our magazine, you will find news about our service app for DTY machines, sppn.info ( MB, PDF-Datei). Filament was a quarterly erotic magazine aimed at women, published in the United Kingdom. Create a book · Download as PDF · Printable version.

Filament Magazine Pdf

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British Women's Magazines\ Filament Magazine, Time and Tide, Spare Rib, Tank Magazine, More!, Mslexia, Woman's Own, Pick Me Up, Vogue, Grazia PDF. MODELLING FOR FILAMENT MAGAZINE. Last revised May Hoping to see yourself in the pages of Filament? Read on! What sort of men are you looking. New Zealand Filament magazine stockists. How our distribution in New Zealand works. Copies of Filament arrive in New Zealand shops approximately two.

I can only assume that knee-jerk response is the product of centuries of indoctrination and not bothering to take a look around at the way the world is changing. Female sexuality and desire have not changed much through the ages, but the freedom to express it certainly has.

But that is another article altogether.


Some of us are happy being every bit as shallow in our admiration of the male form as we want to be. For years I have been personally frustrated by entertainment supposedly designed with me in mind. Television for women. Now, about Filament.

Clean color: Improving multi‐filament 3D prints

Other reviews have criticized Filament for a perceived uniformity of their models. Not so much along ethnic lines so far, their pictorials have been deliciously ethnically diverse but more with regards to body type.

I thoroughly approve, but hope that they keep their mix weighted toward the pretty boys, for reasons that are entirely civic-minded and not at all selfish. Hamilton and an article on live-action role-play. Mutants with inhibitory effects on filament formation will be highlighted in red, whereas constitutively filament-forming mutants will be highlighted in green.

DIY Filament Extruder

First, we investigated the oligomeric state of wild-type and variant Gln1 by blue native PAGE and immunoblotting. As can be seen in Figure 2C , the predominant form of Gln1 was a decamer. Importantly, however, we also identified distinct bands that corresponded to higher molecular weight forms of the enzyme.

These assemblies were predominantly detectable for the wild-type and the R23E variant.

Moreover, when compared to wild-type Gln1, the constitutively filament-forming R23E variant showed an increased number of these higher order assemblies. As a next step, we isolated His-tagged R23E Gln1 from yeast cells and investigated the purified protein by negative staining and electron microscopy.

We identified short filamentous structures, which, upon closer inspection, revealed that they were formed by a repeating unit that precisely matched the dimensions of a Gln1 decamer Figure 2—figure supplement 6. Thus, we conclude that Gln1 retains a near-native structure when it assembles into filaments.

As a next step, we performed correlative light and electron microscopy CLEM experiments on cells overexpressing the R23E variant as mCherry fusion.

The ultrastructural features of the mCherry-positive structures are shown in Figure 2D. The electron micrographs show a large number of filaments, which are laterally aligned into higher order bundles. This side-by-side bundling is consistent with the growth pattern of the filaments, which was predominately in the longitudinal direction but also included some increase in circumference over time see Video 1.

To exclude that the bundling was caused by the mCherry tag, we performed a careful ultrastructural analysis of yeast cells that expressed untagged R23E. Indeed, we could also identify fibrillar structures in the cytoplasm of R23E-expressing cells but not in control cells Figure 2—figure supplement 7. However, we noticed that the filaments were in closer contact, suggesting that the mCherry tag has some influence on the packing density.

Together, these findings indicate that Gln1 assembles into filaments by a back-to-back stacking mechanism. Once formed, these filaments can organize into higher order fibrils. Self-assembly into filaments is driven by macromolecular crowding Is Gln1 able to self-assemble or does it need additional factors?

To investigate this question, we purified wild-type and variant Gln1 from bacteria. The purified proteins were investigated by dynamic light scattering and gel exclusion chromatography for their ability to assemble into high molecular weight forms. As can be seen in Figure 3A and Figure 3—figure supplement 1 , R23E Gln1 had a strongly increased propensity to assemble into higher order forms.

This propensity was reduced in wild-type Gln1 and absent from a variant that had lost the ability to assemble into filaments in yeast cells EK. We next analyzed purified R23E Gln1 by electron microscopy Figure 3—figure supplement 2.

Our analysis revealed abundant cylindrical particles, consistent with the reported structure of Gln1 He et al.

Interestingly, these particles were organized into chains, providing further support for the proposed back-to-back assembly mechanism. However, we were unable to identify higher order structures that resembled previously observed fibrillar structures in yeast, suggesting that an important factor was missing.

Figure 3 with 3 supplements see all Download asset Open asset Self-assembly into filaments is driven by macromolecular crowding. A Equal amounts of 6xHis-tagged wild-type and variant Gln1 purified from bacteria were subjected to dynamic light scattering. Shown is the volume distribution that was derived from the intensity distribution. Note the different scales of the x axes.

Images were acquired from harvested, spheroplasted, and lysed cells. Images were taken at the same intensity settings. The inset is the corresponding DIC image. D Gln1-mCherry was purified from yeast and incubated in a phosphate-citrate buffer of pH 7 with or without a crowding agent for 1 hr. Samples were analyzed by fluorescence microscopy and images were taken at the same intensity settings. A first hint came from our attempts to purify filaments from yeast cells.

When we lysed filament-containing yeast, the filaments were unstable Figure 3B. To identify components that are necessary for filament integrity, we performed experiments with modified lysis buffers.

One obvious difference to yeast cytoplasm was that the lysis buffer lacked a high background concentration of macromolecules. We therefore tested the influence of macromolecular crowders on filament stability. To further investigate the role of macromolecular crowding, we purified mCherry-tagged R23E from yeast cells and incubated it for 1 hr in the presence or absence of a crowder. The samples were subsequently analyzed by fluorescence microscopy for the formation of higher order structures.

Intriguingly, we found that R23E Gln1 formed filamentous structures in the presence of a crowder but not in its absence Figure 3D. Importantly, similar structures could not be detected when we replaced R23E with the assembly-incompetent variant P83R.

Further inspection of in vitro formed Gln1 filaments revealed structures of varying thickness Figure 3—figure supplement 3. This suggests that in vitro reconstituted filaments are able to assemble into higher order bundles, as in yeast cells.

Thus, we conclude that the assembly of Gln1 is strongly dependent on macromolecular crowding but independent of other cellular components. A drop in intracellular pH triggers filament formation Wild-type Gln1 only formed filaments in energy-depleted cells, in contrast to the R23E variant, which assembled also in dividing cells.

This raised questions about the trigger for assembly in starved yeast.

Again experiments with lysates of R23E-expressing cells were revealing. These experiments showed that the stability of R23E filaments could not only be increased by crowders but also by acidifying the lysis buffer.

A cell lysate prepared with a lysis buffer of pH 5 contained abundant filaments, while the filaments began to fall apart when the lysis buffer was adjusted to pH 6, and they were absent from lysates adjusted to pH 7 or 8 Figure 4A. This raised the possibility that intracellular pH changes are the trigger for filament formation.

Figure 4 with 2 supplements see all Download asset Open asset A drop in intracellular pH triggers filament formation. Images were acquired immediately after lysis. Note that the lysis buffer did not contain a crowder.

Classic filament LEDbulbs

B Yeast cells expressing mCherry-tagged Gln1 were washed twice with water and resuspended in a glucose-containing buffer of the indicated pHs with or without the proton carrier 2,4-dinitrophenol DNP.

Images were taken 1 hr after addition of the buffer. C Yeast cells expressing Gln1-mCherry were washed twice with water and resuspended in phosphate buffers of different pHs to induce starvation. The buffers contained proton carriers and energy inhibitors for rapid equilibration of inside and outside pH.

D Gln1-mCherry was purified from yeast and incubated in an acidic or basic buffer containing a crowding agent for 1 hr. Samples were analyzed by fluorescence microscopy. This is because yeast cells have to continuously expend energy to maintain the proton gradient across the plasma membrane usually yeast media are acidic. To test whether pH changes are sufficient to trigger the assembly of Gln1, we artificially acidified the cytoplasm by adding the protonophore 2,4-dinitrophenol DNP to dividing yeast.

Strikingly, acidified yeast abundantly formed filamentous structures, despite the presence of glucose Figure 4B. Next, we tested whether a neutral or basic outside pH could prevent the formation of filaments in energy-depleted yeast.

Indeed, starved yeast maintained in a buffer of pH 7 contained shorter and smaller filaments than yeast in a buffer of pH 5 Figure 4C. Moreover, filaments were largely absent when the buffer was adjusted to pH 8. Intracellular pH changes could directly or indirectly affect the assembly of Gln1.

To differentiate between these two possibilities, we purified mCherry-tagged Gln1 from yeast and incubated it for 1 hr in a buffer of pH 5 or 8. Afterwards, the samples were examined by fluorescence microscopy for the formation of higher order structures.

Indeed, Gln1 formed filamentous structures in the acidic buffer but not in the control buffer with a basic pH Figure 4D. Importantly, this effect was specific, because the assembly-deficient variant P83R did not assemble into higher order structures in an acidic buffer. To follow pH-induced assembly over time, we exposed bacterially purified Gln1 to an acidic buffer and monitored the formation of higher order structures by dynamic light scattering.

DIY Filament Extruder

As can be seen in Figure 4—figure supplement 1 , Gln1 progressively assembled into higher order structures, whereas a mutant version of Gln1 EK did not.Adding a physical mechanism increases cost and print time as extruders travel to a cleaning station.

Thus, we conclude that Gln1 assembly is directly regulated by protons and that the trigger for filament formation in yeast cells is a starvation-induced drop in the intracellular pH. Curiously, however, the crystal structure of yeast Gln1 also revealed a new back-to-back association between two decamers Figure 2A. Step 6: Now to the machining.