ewinter's blog

In the cancer associated fibroblasts (CAFs) of epithelial ovarian cancer, miR-31 and miR-214 were downregulated, while miR-155 was upregulated (Mitra et al 2012). MicroRNAs are small noncoding RNAs that downregulate gene expression post transcriptionally by binding to mRNA. Furthermore, Mitra and colleagues found that inducing this phenotype in normal fibroblasts using miRNAs and miRNA inhibitors yielded a functional conversion of normal fibroblasts to CAFs, and the reverse experiment caused reversion of CAFs to normal fibroblasts.  These promising findings will be the basis of my treatment regarding cancer associated fibroblasts. To further explain, I tried to elucidate the mechanisms by which epigenetic changes in these specific miRNAs may cause the phenotype observed by Mitra and colleagues.

Mitra et al. did a nice experiment in which they antagonized the CAF phenotype of the three micro RNAs mentioned.  In CAFs, reversal of miR-31 and miR-214 downregulation and reversal of miR-155 upregulation caused reversion of CAFs to a normal phenotype.  Designing a treatment based off of these results seems like a logical plan.  We will use liposomal delivery to insert complementary miR-31 and miR-214 as well as a complementary strand to miR-155.  Fibroblast activated protein (FAP) and a-smooth muscle actin (aSMA) are two cell surface antigens of epithelial ovarian cancer CAFs (Mhawech-Fauceglia et. al).  We will engineer a liposome with monoclonal antibodies that can bind to these two cell surface antigens in order to target the CAFs.  

The Warburg Effect is a metabolic shift in which even in aerobic conditions, cancer cells use anaerobic respiration, thereby spending a lot more energy due to inefficiency of this pathway compared to oxidative phosphorylation.  Ginsenoside 20(S)-Rg3 is a chemical derived from the ginseng plant.  It has been shown to inhibit the Warburg Effect in ovarian cancer via two mechanisms.  The first mechanism was shown by Li et al. in 2014.  Ginsenoside 20(S)-Rg3 was shown to block STAT3.  STAT3 normally upregulates expression of hexokinase 2, but now that no longer happens.  This shift inhibits the Warburg effect.  The second mechanism was shown by Zhou et al. in 2018.   They showed that Ginsenoside 20(S)-Rg3 was a suppressor of miR-532-3p and lessened levels of DNMT3A.  These two things inhibit the Warburg Effect. 

The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) was used as a homology-based method.  We performed a Standard Nucleotide BLAST of the RZW genomic DNA using the Highly Similar Sequences (megablast) program in the Expressed Sequence Tags (EST) database for Brachypodium distachyon (taxid: 15368) in order to identify ESTs that correspond to our unknown gene.  All matches with greater than 95% Ident were saved. All of the saved ESTs were imported into the CAP3 software. CAP3 generated consensus sequences bases on the overlap between numerous ESTs. To find a full length cDNA sequence for our gene, we performed a Standard Nucleotide BLAST of the RZW genomic DNA using the Highly Similar Sequences (megablast) program in the Nucleotide Collection (nr/nt) database for Brachypodium distachyon (taxid: 15368).  We found accession number XM_003562897.4, an mRNA sequence that is predicted to code for the Brachypodium distachyon G-type lectin S-receptor-like serine/threonine-protein kinase B120 (LOC100825184). The two consensus sequences formed were aligned to the cDNA sequence using the NCBI BLAST.  To find the official identity of the RZW gene, we performed a Phytozome BLAST search of the RZW genomic DNA with Brachypodium distachyon v3.1 as the target species.  The best match to the query sequence was Bradi1g25180.1, and the predicted protein sequence was saved. The FGENESH and Phytozome predicted protein sequences were compared and the working map was updated.  From the Phytozome locus page, the functional annotation was saved as a graphic showing domains. The link to Uniprot was followed and there were 16 annotations that contained information about different domains.  

Fibroblasts are cells responsible for synthesizing the stroma of animal tissue including the extracellular matrix and collagen.  In cancer, these cells can be reprogrammed to aid in tumor growth and progression. In the cancer associated fibroblasts (CAFs) of epithelial ovarian cancer, miR-31 and miR-214 were downregulated, while miR-155 was upregulated (Mitra et al 2012).  The authors also found that mimicking this induced a functional conversion of normal fibroblasts into CAFs, and reversal caused reversion of CAFs to normal fibroblasts. When miR-214 is downregulated in CAFs, the CAFs secrete high levels of the chemokine CCL5 into the tumor microenvironment.  The authors found that miR-214 is a target of CCL5. Therefore, due to the downregulation of miR-214 in CAFs, it can no longer silence the CCL5 mRNA, so it gets translated at a much higher level. CCL5 secreted by CAFs is known to be a tumor promoting factor. It is higher and involved in cisplatin resistance because it is secreted from the CAFs and signals through STAT3/PI3K/AKT on ovarian cancer cells.

The ab initio program FGENESH predicted that the mRNA is 2229 nucleotides spanning 7 exons (Fig. 1-A).  The homology-based program Phytozome predicted that the mRNA is 3193 nucleotides spanning 9 exons (Fig. 1-B).  FGENESH predicted that the protein is 742 amino acids long (Fig. 2-A). Phytozome predicted the protein is 847 amino acids long (Fig. 2-B).  In both gene maps, exons 1, 2, and 3 cover the exact same regions.  Exons 4 and 5 in the Phytozome prediction are predicted introns in the FGENESH output.  Exon 6 of the Phytozome output begins 14 bases after the beginning of exon 4 of the FGENESH output and they end at the same place.  Phytozome predicted exons 5, 6, and 7 are identical to FGENESH predicted exons 7, 8, and 9 respectively.  In the FGENESH and Phytozome predicted protein sequences, amino acid positions 1-437 are exactly homologous.  FGENESH positions 442-742 are exactly homologous to Phytozome positions 547-847. The discrepancy between the two peptide is that the FGENESH amino acid contains ‘KSLQ’ at positions 438-441, which is replaced in the Phytozome output by a set of 109 amino acids occupying positions 438-546.  Nowhere does this sequence of 109 amino acids contain ‘KSLQ.’

We next narrowed our literature search in pursuit of gene function of our specific gene.  A search of Bradi1g25180 did not yield any results, so we switched our focus to homologous proteins.  Bradi1g25180 was searched on www.arabidopsis.org in order to find homologous proteins.  There were twenty homologs identified, but searches for all but one yielded no papers about specific function.  The arabidopsis thaliana gene AT4g21390 (gene B120), also known as T6K22.120 according to Uniprot, is homologous to Bradi1g25180 according to www.arabidopsis.org.  A search for “AT4g21390” yielded nothing, but a search for “T6K22.120” yielded a paper in which the gene was hypothesized to be the Arabidopsis thaliana counterpart to the S-locus receptor kinase (SRK) when compared to SRK in Arabidopsis lyrata (Kusaba et al. 2001).  However, the authors ultimately concluded that the homologous counterpart was actually the adjacent ARK3 gene, at position 110, and that gene B120 is related to the BRLK gene in Brassica oleracea.

We next narrowed our literature search for gene function.  A search of Bradi1g25180 did not yield any results, so we switched our focus to homologous proteins.  Bradi1g25180 was searched on www.arabidopsis.org in order to find homologous proteins.  There were twenty homologs that came up, but searches for them yielded no papers about function.  Notable homologs that yielded no results about function include the following: two arabidopsis thaliana genes (AT4g21390 and AT1g61610), two Glycine max genes (GLYMA13g32242 and GLYMA15g07090), one Oryza sativa gene (OSJNBB0041B22), and one Sorghum bicolor gene (SB02G035940).  

The B120 gene in Arabidopsis thaliana is AT4g21390, which is also known as T6K22.120 according to Uniprot.  This gene is one of three genes hypothesized to potentially be the Arabidopsis thaliana counterpart to the S-locus receptor kinase (SRK) when compared to SRK in Arabidopsis lyrata (Kusaba et al. 2001).  SRK is the female S-locus transmembrane protein expressed in the stigma. If it binds to the SRC protein expressed on pollen, the plant undergoes the self-incompatibility response and prevents seed formation.  

The ab initio protein prediction and the homology based protein prediction had a substitution that involved 4 amino acids becoming 140.  Ab initio methods are purely based on theoretical trends of transcription and translation, while homology based methods compare the reference sequence to known sequences.  I believe the homology-based protein prediction is more accurate. In both DNA and protein level homology searches, the “G-type lectin S-receptor-like serine/threonine-protein kinase” terminology was repeated in results with high levels of homology.  This makes me confident to say that the Bradi1g25180.1 gene encodes a G-type lectin receptor kinase. However, the functions of many of these G-type lectin receptor kinases are not well characterized. Sun et al implicated an Arabadopsis thaliana G-type lectin receptor kinase called GsSRK to salt stress.  They showed that is is upregulated when a plant is under salt stress, and also showed that its overexpression leads to increased salt tolerance. Unfortunately, they did not perform a knockout of this gene. To my knowledge, there are no papers that have studied a knockout mutation of a G-type lectin receptor kinase in any organism.  

The vaccine that the scientists have developed should be used to save the German Shepherd.  The German Shepherd is above all known for its intelligence, and is also loyal, confident, and courageous (American Kennel Club).  These dogs have the intelligence to adapt to any situation. They are docile and obedient yet can be aggressive when necessary. Their superior intelligence makes them comparatively easy to train.  Due to these character traits, it comes as no surprise that they are one of the top service dogs (Hayner, 2019). Service dogs are used for a wide range of things. To name a few, they assist the blind and the deaf, and they are often used as police dogs.

It is well established that German Shepherds have characteristics that make them a good service dog.  The heritability of these characteristics must be taken into consideration. In their study, Wilsson and Sundgren noted a high heritability on average for numerous behavioral characteristics in German Shepherds.  Their findings are important because they give us confidence that the descendents of the German Shepherds we save will be able to maintain their integral role in our communities. German shepherds improve the quality of life for many people and play integral roles in keeping our communities safe.  The extinction of the German Shepherd would leave big shoes to fill. Although with proper training, other dogs theoretically would be up to the tasks, it makes sense to save the German Shepherd, as they are the gold standard for service dogs.

Sources:

“German Shepherd Dog Dog Breed Information.”  American Kennel Club.

https://www.akc.org/dog-breeds/german-shepherd-dog/

Hayner, LM. (2019).  Let’s Meet Some of the Best Service Dog Breeds.  Dogster.

https://www.dogster.com/lifestyle/best-service-dog-breeds

Wilsson and Sundgren (1996). The use of a behavior test for selection of dogs for service and breeding.  II. Heritability for tested parameters and effect of selection based on service dog characteristics. Elsevier.  53 (2-3): 235-241.

https://www.sciencedirect.com/science/article/pii/S0168159196011756