ewinter's blog

The Bcl-2 protein family consists of proteins that contain at least one evolutionarily conserved BH domain out of the four that exist (BH1, BH2, BH3, BH4).  Within this family, there exists pro-apoptotic proteins and anti-apoptotic proteins that work together to govern the fate of the cell.  Anti-apoptotic proteins conserve all four BH domains, and include Bcl-2, Bcl-XL, Bcl-W, Mcl-1, and A1.  Pro-apoptotic proteins can be subdivided into two groups, those with multiple BH domains such as Bax and Bak, and those with only the BH3 domain such as Bid, Bim, Bad, PUMA, and NOXA. 

TP53 is the gene on chromosome 17 that encodes the p53 tumor suppressor protein.  Mutations in TP53 are present in 96% of high-grade serous ovarian cancer.  Many types of mutations in p53 have been characterized.  These include single base substitutions leading to missense or nonsense mutations and single base deletions or insertions leading to frameshift mutations or in-frame deletions or insertions.  80% of these mutations occur in the DNA binding domain, encoded by amino acid residues 102 to 292, leading to a loss of the ability to bind DNA and act as a transcription factor. Given the rate at which TP53 is mutated in HGSOC, it comes at no surprise that it is a hot target for therapy.  However, the wide range of TP53 mutations found in HGSOC present a major difficulty in TP53 targeted therapy because treatment must be specific for the mutation present. 

At first glance, the replica of the original diagram is quite accurate.  Upon further inspection, there are some noticeable differences.  The font of the text is different, so it was likely not specified.  The letters are also misplaces, for example, the C touches the bottom of the figure, whereas the B does not.  This likely means that it was done by hand.  There is a noticeable difference in leaf color in box A.  In this same box, there is a leaf from another plant overhanging the frame that is in differing orientations.  These combined observations lead me to believe that the side from which to photograph the plant was not specified.  For boxes B and C, the picture is noticeably closer to the flower in the second figure.  This could be the result of a lack of specificity, but could also be the result of measurement inaccuracies.  

This interspecific interaction is between a juniper and a forsythia.  The forsythia is surrounded by juniper, and it is clear that they are sharing the same soil.  The location of this interspecific interaction is on the west side of the Life Science Laboratories (LSL) at UMass Amherst.  Upon exiting the Integrated Science Building on the second floor, a right turn was taken, the doors to the LSL were passed, and the interspecific interaction was spotted directly in front of the single glass window that is between the LSL entrance doors and the windows looking into the laboratories. Three pictures were taken at 4 pm on a sunny day with snow covering the ground.  One picture, which included both organisms, was taken from the sidewalk, on the side farthest from the plants.   One was a top-down view of the juniper from approximately 1 foot above the plants. One was a picture of the forsythia with buds that were approximately 5 inches from the camera lens in focus, with the tan wall of the LSL in the background (no windows or juniper).  

Targeted drug delivery would be performed by creating 3 antibody binding domains for antigens CA125, KASH5, and HSF1 on a liposome. To test for the presence of these antigens in the HGSOC, we would perform an enzyme linked immunosorbent assay (ELISA) for autoantibody (AAb) reactivity.  Autoantibodies for these three antigens will be present in the blood, so ELISA will would tell us if the antibodies for these particular antigens were present. To test for treatment progression, periodic ELISA assays will be performed.  If the autoantibody levels in the blood remain high, more treatment is needed.  A possible indication of cancer evolution and/or metastasis is if less than three autoantibodies remain high.  In this event, a new liposome with only the respective two monoclonal antibodies will need to be engineered.

The idea of engineering a liposome with three monoclonal antibodies attached to one liposome presents a daunting challenge.  Additionally, the binding affinity between each monoclonal antibody to its antigen must be considered carefully. It is important that the interaction between each antibody and antigen is relatively weak.  If one is too strong, the liposome will bind and inject its contents to healthy cells that merely express the antigen in normal levels, leading to cell death. To prevent metastasis, it will be possible to engineer liposomes with the respective antibodies for the three surface antigens as needed.  However, if these tumors mutate and/or metastasize and lose the overexpression of CA125, KASH5, or HSF1, assaying the new tumor cells and designing a new treatment in a timely manner will likely be an insurmountable task.   

The idea of engineering a liposome with three monoclonal antibodies attached one liposome presents a daunting challenge.  Additionally, the binding affinity between each monoclonal antibody to its antigen must be considered carefully. It is important that the interaction between each antibody and antigen is relatively weak.  If one is too strong, the liposome will bind and inject its contents to healthy cells that merely express the antigen in normal levels, leading to cell death.

The predicted amino acid sequence from the RZW gene of Brachypodium distachyon, was given by two different programs.  FGENESH provides an ab initio method, while Phytozome compares the RZW gene to known expressed tag sequences (ESTs) in B. distachyon.  The predictions differed in one area.  The FGENESH predicted protein had a string of four amino acids: KSLQ, in the middle, while Phytozome predicted that instead of these four, there was a chain of 109 amino acids.  All amino acids before and after this noted difference were the same. Furthermore, two contiguous sequences were made from the bank of ESTs. These sequences had a gap of a few hundred base pairs in between them; there was a part of the gene that was unaccounted for by ESTs.  This may help to explain the difference in predicted protein sequence. Either way, the Phytozome predicted protein sequence is trusted more, because it analyzes cDNA libraries, as opposed to just known trends in mRNA splicing.

Two methods were used to arrive at our final predicted protein sequence for the RZW gene of Brachypodium distachyon.  The first method was an ab initio method.  The program FGENESH was used.  FGENESH analyzes genomic DNA and comes up with the most likely protein sequence, based on common characteristics of introns and exons.  For example, introns start with the nucleotides GT and end with AG. The program also recognizes the start codon, ATG, and the stop codons: TAG, TAA, and TGA.  The second method used a program called Phytozome. Phytozome is a DNA library, so it compares the genomic sequence of the RZW gene to known DNA sequences in B. distachyon.  It compares the genomic DNA to expressed sequence tags (ESTs), which are overlapping sequences derived from cDNA clones.  Because Phytozome compares the RZW gene to known cDNA sequences, its predicted protein sequence is more trustworthy than that of FGENESH.  

Figure 1.  Volvox in its natural form.  The multicellular organism is composed of up to 50,000 cells that adhere to each other, forming a sphere.  Each colony is composed of two differentiated cell lines: a large number of somatic cells with flagella and a smaller number of germ cells lacking in soma that lie in the interior.  Photo by Specious Reasons, available at https://www.flickr.com/photos/28594931@N03/20108157326/.