cdkelly's blog

This paper dealt with spider web production in terms of web mass and temperatures affect on it. They used 20 female spiders and randomly assigned them to one of their 4 temperature groups. They kept them at the temperature for six days. Fruit flies were dropped into the web with an apparatus. They created a gradient of temperatures by putting two aluminum pans on a hot plate. The pans were positioned in such a way that the entire pan wouldn’t get hot. The different temperature regions were indicated by marking on the pan, not barriers that the spider could not cross. They found that web mass was much lighter in the colder conditions.

Once the protein was produced, the team began a series of analyses to elucidate some of the features of the newly cloned Solanum dulcamara THP. They did a western blot, a gel shift assay, RNA blot analysis, and analysed the hysteresis activity. Following the completion of these analyses, they identified STHP-64 as the hydrophilic THP protein present in the November sample of Solanum dulcamara. It differed in weight slightly (3 kDa) compared to the original THP used as a template, but they attributed this to the loss of exons from the N-terminus. Functionally and structurally, the protein was extremely similar to the original and they believed that it was derived from the same gene within the plant. They observed thermal hysteresis activity for STHP-64, albeit not much. Citrate was found to increase the thermal hysteresis activity, which confirmed for them that STHP was in fact THP. The low activity was attributed to potential protein-folding issues among other things. Amino acid repeats were identified in the clone and thought to facilitate thermal hysteresis by hydrogen bonding with the ice on the plant, effectively inhibiting the further crystallization of ice. STHP-64 also demonstrated DNA-binding ability and they believed that it could act as a transcription factor for the upregulation of proteins related to pathogenesis. In conclusion, although the THP activity was low in the isolated sample from Solanum dulcamara, the THP protein was only present in the November sample. This suggests that STHP-64 is at least partially responsible for the overwintering capabilities demonstrated by Solanum dulcamara.

Overwintering plants have the ability to survive throughout the winter, and many of them contain thermal hysteresis proteins (THPs), which lower the freezing point and melting point of water. These proteins are present in fish and insects as well, but the mechanism is likely to be different. Unlike insects and fish, plant THPs allow them to survive the freezing of their extracellular water, rather than preventing freezing altogether. Solanum dulcamara is an example of an overwintering plant, and is at the center of this article. The objective of the research was to isolate and characterize a specific THP within Solanum dulcamara that allows it to survive through the winter’s harsh temperature decline.

    Researchers collected and purified samples of Solanum dulcamara RNA from leaves at two separate points in time; one sample in September and the other in November. This was done to compare the RNA present during warmer weather and colder weather; they assumed that the THP would be present in the RNA extracted from the November leaves. The extracted RNA was then used to generate a cDNA expression libraries for both samples. An antibody generated from a previously purified S. dulcamara THP was used to screen the two libraries for the presence of matching THPs. A total of 20,000 recombinants from both the September and November samples were put through the antibody screening. The November sample was found to have eight matches and like the expected, the September sample had zero. Two of these matches were then isolated and prepared for cloning. Using PCR, a clone was generated, duplicated, and integrated into E. coli as a plasmid. Finally, the protein was grown in culture and extracted.

Solanum dulcamara (Solanaceae) An invasive, perennial, semi-woody climber that can grow in a wide array of environments. It can be upwards of 13 feet tall, but generally is found to be around 3-6 feet in height. Both the leaf blades and the stems are unarmed. The shape of most of the leaves is ovate with a cordate base, arranged in a alternate pattern. These leaves are simple, with a margin that can be entire, subentire, or toothed. But, the larger leaves can have a small pair of basal lobes resulting in a hastate base, or leaflets at their base; toothed or toothed and lobed margins are associated with these larger leaves. This feature of Solanum dulcamara differentiates it from similar plants, such as Solanum nigrum or Solanum ptycanthum, because it uniquely has the aforementioned leaflets. Pubescent simple hairs or glabrous surface textures are present on all its leaves. Blueish-Purple, radially symmetrical flowers comprise the inflorescence. Solanum dulcamara can contain anywhere from 10-25 flowers, each with 5 petals and 5 tepals. Flowers contain both gynoecium and androecium. Ellipsoid to ovoid berries are present on the plant and are known to be poisonous to humans, but in fact, the entire plant is actually considered to be toxic.

It's really interesting that fusion proteins can be expressed. I assumed that Abl did something to the protein that it fused with in the cytosol, like affect its noncovalent bonding and consequently change its conformation/function. But, this implies that the fusion protein is expressed so the mutation begins at the DNA before transcription.

Does the Abl kinase act as a phosphatase? It states that one of its functions is to inhibit tyrosine kinases. Maybe it acts as a competitor for phosphorylation as a mechanism of inhibition instead?  

This is fascinating because I assumed that all SH2 domains functioned as phosphotyrosine binding partners. But in the case of Abl, it seems that the SH2 domain functions to bind to the SH3 domain and thereby maintain its autoinhibition. If the SH2 domain is not able to bind to a phosphorylated tyrosine, then its normal function will no longer work.

This facet of Abl 1b coincides with its cancerous tendencies. If the kinase activity is deregulated then the implications for the cell as a whole are not good. Cancerous cell utilize these kinds of mutations for grow and proliferate. Without normal regulation, the kinase can promote this. Therefore, it makes sense that the Abl 1b mutant is commonly a part of the cell that when mutated can lead to cancer.

Since Gab/Grb2 fulfill such a large role in the cell, it makes sense that its deregulation by Bcr-Abl results from the mutation. Gab/Grb2 bind to epidermal growth factor receptors (EGFR) which is a commonly mutated in various forms of cancer. Furthermore, because Gab2 serves a number of other functions in the cell like being the binding site for the assembly of SHP2 and potentially coordinating STAT5, its deregulation can have serious consequences for the longevity of the cell.

Looking back at my statistics 240 course, I realize that I remember a lot of key concepts but some of the details of calculation are a blur.  For example, I know all of the basic measures including mean, median, mode. I recall that significance in a set of data is a way to determine if an effect was actually observed. Also, confidence intervals are essentially a measure of how confident you can be that the obtained result could be repeated. A confidence interval of .9 indicates 90% confidence and .99 indicates 99% confidence. If you have a very large sample and a high confidence interval then you can more readily rely on the validity of the results. In addition, a given sample size must have a minimum of thirty participants for its results to be considered significant (n=30). All of these concepts can be related to the normal distribution. When conducting an experiment, you want to prove that your hypothesis actually generated a result. By using statistical analysis, you can provide a numeric confirmation that a difference was observed between an original condition and the experimental condition. Since it has been awhile since I took the course, I’m not currently confident with all of the calculations pertaining to these concepts. Luckily, these are things that I could open up my old notebook and refresh myself on.

 

Looking back at my statistics course, I realize that I mainly remember key concepts. For example, all of the basic measures including mean, median, mode. The idea of significance in a set of data is another element of statistics I recall. A confidence interval is essentially a measure of how confident you can be that the obtained result could be repeated. A confidence interval of .9 indicates 90% confidence and .99 indicates 99% confidence. If you have a very large sample and a high confidence interval, then you can more readily rely on the results. Also, a given sample size must have a minimum of thirty participants for results pertaining to it to be considered significant (n=30). All of these concepts can be related to the normal distribution. When conducting an experiment, you want to prove that your hypothesis actually generated a result. By using statistical analysis, you can provide a numeric confirmation that a difference was observed between an original condition and the experimental condition. 

The background and scenery of part A and part B of the replicate figure had differences that resulted from the methods as well as differences that could not be controlled. The moisture and shadow position differences between the original and the replicate were due to the weather differences and the alloted time for replication. My methods stated the weather and time of day the photos were taken. In addition, the vehicle seen in the background of part B in the replicate was due to the same reason. On the other hand, the lack of the pipe extending from the electrical box in part A of the original was a result of methods omissions. I did not put enough detail into the way that the camera was held above the web in part A and used the word horizontal to describe the camera's orientation relative to the web. The pipe visible in the original figure is a result of the camera being at a slight angle; and its absence in the replicate is due to the camera angle being more horizontal. Furthermore, I mentioned that photo for part B was taken next to the second pillar of the bike rack structure. However, I did not mention that a portion of the structure itself was present in the original image. This explains why the structure was not seen in the replicate.

The map depicted in part C of both figures showed different levels of detail. In the original figure, the map was more zoomed relative to the replicate. The resultant extra buildings and map markings on the replicate arose because I did not describe the landmarks surrounding the Student Union in my methods section. I stated that the Student Union was at the center of the map image, but did not provide further information beyond that. Therefore, the person who replicated my figure had to approximate. In addition, the replicate lacked the scale marking and the logo seen in the bottom corners of part C of the original. These markings were not mentioned once in the methods. Thus, it is not surprising that they were not featured in the replication because the person could not have know about them in the first place. The lack of cropping instructions in the methods was another reason for this omission.

In conclusion, the replicate figure was similar in many regards to the original, but exclusions from the methods section made certain components vague for the reader tasked with replication. These exclusions led to all of the discussed differences between the two figures aside from the weather. This project emphasized the importance of paying attention to details when creating a methods section so that the replication process can proceed as intended in the original work.

The three main differences between the original figure and the replicate were the sizing and orientation of the markings of the figure, the surrounding scenery, and the area encompassed by the map. This section looks at these differences in more detail and makes inferences on what portion of the methods section caused them.

The size and thickness differences in the labels, indicating lines, text, length lines, the dot on the map, and highlight circles were all caused by the same facet of the methods. Not including units of measurement with all of the dimensions in the methods led to a vagueness in the original sizes. Dimensions were meticulously included with each part of the labeling on the figure, but there were no associated units. Thus, the person tasked with the replication of the figure did not know which of the number of possible units was used in the original, causing the size differences.

As for the orientation of the figure markings, the methods described the general location, but did not go into enough detail. This caused the labels in the top left corners of parts A,B, and C to be positioned slightly past the borders of the figure and resulted in a lip at all three locations. Also stemming from the lack of detail pertaining to orientation, the “location” box in Part C was found on the left side in the replicate. I only specified that the label box was off to the side of the red dot in part C, with the connecting line at an approximate 45 degree angle. I did not say which side it was on, which led to the replicate having it on the left. The connector line made contact with the red dot and the location box in the replicate because I only said that they were connected without elaborating in the methods. I didn’t say to which part of the location box the line connected, and I didn’t mention that the line did not actually make contact with either the box or the dot in the original. All of these missing details resulted in the different orientation of the markings in part C of the replicate.

In part C of the replicate, the text covering the student union was larger in font size and displayed in a straight line. As for the red dot marking the absolute location of the web, the replicate version was larger. The label box with the word “location” inside of it was positioned of the left side of the figure. Also, the thin line connecting the box to the red indicator dot made contact and was connected to the side of the box.  

The surrounding scenery of part A and part B of the replicate was subject to different weather conditions and contained objects not seen in the original. A pipe extending from the bottom of electric box next to the web was absent from part A of the replicate figure. For both part A and part B, the background had visible moisture. A lack of shadows was observed in part B of the replicate. A vehicle is visible in the background of part A. Also, the coloration and positioning of the leaves caught in the web were different across figures. The bike rack structure shown in part B was not seen in the replicate.    

In part C of the replicate, the map encompassed a larger region of the campus. Compared to the original, part C was more congested with labels on the map itself. It showed nearly the entire campus pond as well as more of the local buildings.