This Project: This page has been made for a genomic analysis project being conducted by Biology 142 Lab Courses at Emory University in partnership with the Georgia Aquarium. The purpose of this project is to aid in the identification of possible proteins in the recently sequenced whale shark genome. This page is dedicated to the analysis done on the human TICAM2/TRAM protein.
Background Information
When pathogens are found in our bodies, our immune system is what recognizes and fights them off. These pathogens are recognized by toll-like receptor proteins, abbreviated TLRs (receptors on the outside of macrophages and dendritic cells which recognize microbes) . Recognition of these pathogens activates immune responses through signaling pathways controlled by domain containing adaptors (Verstak et al) . Adaptor proteins influence signaling pathways and cross-overs by facilitating interactions between proteins and larger signaling complexes. To ensure the appropriate signaling response, many processes rely greatly on these adaptor proteins ("Research Topics - Adaptor Proteins").
TICAM2/TRAM, an abbreviation for TRIF –related adaptor molecule or TIR containing adaptor molecule 2, is one of these adaptors ("The Toll/IL-1 Receptor Adaptor Family"). When TLR4 is activated, upon recognition of pathogens, it recruits TICAM2/TRAM to activate the production of pro-inflammatory cytokines ("Purified Anti-TICAM-2 TRAM Antibody").This process has further been supported by studies that have explored how TRAM deficient mice show defects in their ability to produce cytokine (Yammamoto et al).
The pathway within which that TRAM operates in this system is called the MyD88-independent pathway. In the signaling chain, TRAM bridges the interaction between TLR4 and TICAM1("Purified Anti-TICAM-2 TRAM Antibody").A recent study has shown that the interaction between TLR4 and TRAM is physical and that the "BB loop" regions of both the TRAM and the TLR4 domain are important for the physical interaction to occur (Ullah et al). Figure 1 below illustrates the multiple TLR signaling pathways involved in the immune response system (Liew et al).
Negative regulation of Toll-like receptor-mediated immune responses
Figure 1. Credit to Liew et al. This image shows the process of TLRs affecting nuclear activity. The process is quite complicated and takes many steps. The TRAM molecule initiates a series of chain reactions which ultimately controls and alters nuclear activity by nuclear factor kB (Liew et al).
Methods
Finding Whale shark predicted orthologs:The human protein sequence (ENSP00000415139) was used as the query in a BLAST against the predicted whale shark protein database using the Galaxy server by Georgia Aquarium. The full sequence of the best matching predicted protein, determined by the lowest e-value and alignment length, was obtained. The sequence was then used as the query in protein BLASTs against the NCBI human protein database to test possible orthology by showing a positive reciprocal match.
Predicted orthologs in other species
TICAM2/TRAM (ENSP00000415139) predicted orthologs were identified in other species by using the NCBI Blast server. The human NHEJ1 protein sequence was used as query in these searches. Protein BLASTs were performed using the human protein sequence against single species protein databases for the mouse, zebra fish, clawed frog, elephant shark, and fruit fly.
Phylogenetic tree The top whale shark predicted protein sequences,the best hit sequences (with the lowest E-value) for each non-whale shark species, and the human TRAM sequence, were used to create a multiple sequence alignment and phylogenetic tree. The source used to create the multiple sequence alignment and phylogenetic tree was ClustalW2 program.
Searching for TICAM2/TRAM in whale sharks The protein sequence for TRAM in humans was used as the query against the whale shark predicted protein database. The results of the top four hits in the BLAST are shown in Table 1 below. These 4 best hits had the lowest E-values with the lowest E-value being 3e-07. These 4 top hits were then used as queries and BLASTed against the human protein database using NCBI BLASTp.
Whale Shark ID
E-value
Alignment Length
Predicted protein length
% Identity
g24573.t1
5e -05
122
296
24.50%
g47253.t1
3e -07
32
218
40.62%
g21665.t1
1e -04
26
110
50.00%
g48010.t1
8e -06
40
244
30.00%
Table1. Human TRAM best BLASTp hits against the whale shark predicted protein database. The Galaxy server was used to BLAST the human TRAM protein sequence against the predicted whale shark protein database. The top 4 hits are displayed here with their ID, E-value, protein length, alignment length, and percent identity.
The top 4 predicted proteins from the BLAST of the whale shark predicted proteins were used as queries against the human protein database. The BLAST of these 4 predicted proteins did not return TRAM as the best hit. The results can be seen in Table 2 below.
ID
Length
Name/Description
E-Value
g24573.t1
268
ribonuclease P protein subunit p30 isoform b [Homo sapiens]
7.8
g47253.t1
858
toll-like receptor 5 precursor [Homo sapiens]
7e-76
g21665.t1
201
PREDICTED: uncharacterized protein LOC105373256 [Homo sapiens]
1.1
g48010.t1
784
toll-like receptor 2 precursor [Homo sapiens]
1e-28
Table 2. Top predicted whale shark protein BLASTp hits against human protein database. The top results of each BLAST are shown here with their protein lengths, name/descriptions, and E-values.
Because none of the BLASTS returned TRAM as a best hit using the predicted whale shark proteins as the queries,we were not confident that either of the two best predicted whale shark proteins and the human TRAM are orthologs. However, given the significant E-values of 7e-76 and 1e-26 with the toll-like receptor 5 precursor (TLR5 precursor) and toll-like receptor 2 precursor (TLR2 precursor), we believed that the two proteins may share the same protein domain. This belief was further supported by the similar functionality TLR proteins and TRAM have in the initiation of immune responses against pathogens. The protein domains of the top two whale shark predicted proteins ( g48010.t1 and g47253.t1) are shown in Figure 2.
Protein Domains As shown in Figure 2, all three (Human TRAM protein and the two predicted whale shark proteins) proteins contained a TIR_2 super-family domain. This shows that the domain of the human TRAM protein is conserved in the two top predicted whale shark proteins. The TIR,or Toll/interleukin-1 (IL-1) receptor, domain is a special type of domain which is found only in the signaling TLR system, toll-like receptor proteins (O’ Neill and Bowie). The TIR domain is key to the function of TLRs in triggering innate immune responses to pathogens (O’Neill and Bowie). Additionally, the conservation of sequence among TIR domains in various proteins (adaptors, receptors, etc) is only 20-30%, and the size of the domain also varies, giving rise to a great amount of diversity in the domain (Xu et al). Thus, it is likely that the two predicted whale shark proteins, although not matched with a human TRAM protein, are somehow involved in an innate immune system response in whale sharks similar to that of humans.
Human TRAM
g47253.t1 whale shark
CD search result summary
g48010.t1 whale shark
CD search result summary
Figure 2. Protein domains of 2 best whale shark predicted proteins and Human TRAM protein. The protein domains were obtained using the NCBI server to search the protein sequences for domains.
Orthologs The human TRAM protein sequence was used as query in NCBI BLASTs against the databases for other species. A TIR domain-containing adaptor molecule 2 ortholog was found in mice as the BLAST returned TRAM as the best hit. A possibly ortholog was found in zebrafish as the BLAST returned an alternate form of TRAM as the best hit.Although there were no orthologs found in the other species, results showed that the TIR_2 superfamily protein domain was conserved in all the species that were examined. All species BLASTed against, aside from the fruit fly, had best hits of proteins related to or in the same immune response system as TRAM.
Species
Name
Length
Accession
E-value
Mouse
TIR domain-containing adaptor molecule 2 [ mus musculus]
232
NP_775570.1
1e-124
Zebra fish
TIR domain-containing adaptor molecule 1 [danio rerio]
PREDICTED: TIR domain-containing adapter molecule 1-like [Xenopus (Silurana) tropicalis]
559
XP_004911105.1
1e-19
Table 3. Best hits with human TRAM protein against multiple species. The human TRAM sequence was used as the query in NCBI BLASTs against the databases of individual species. The Species, Name ,Length, Accession number, and E-value of the best hit from each search is shown. None of the searches resulted in TRAM protein as the best hit.
Phylogenetic Tree and Alignment Data A multiple sequence alignment was conducted using the ClustalW2 program with the best hit protein sequences of the individual species above. Alignment results showed low homology( many gaps in alignment) in the earlier sections of the sequence. There were high levels of homology in the section of the sequences that contained the TIR protein domains. The alignment of the sequences where the protein domain is located can be seen below in Figure 3.
Figure 3. Multiple sequence alignment of individual species. The alignment of the species was found by entering the best hit FASTA sequences for all the relevant species using the ClustlW2 program.
Using the alignment data, a phylogenetic tree was then made and can be seen below in Figure 3. From this tree, the similarity between the human and mouse sequences can be seen clearly, as they are grouped together. The two whale shark predicted proteins are also grouped together in the tree, showing their sequence similarity. The whale shark predicted proteins are grouped the furthest away from the human TRAM protein, further suggesting that there is no whale shark homolog to the human TRAM protein (Figure 4).
Figure 4. Phylogenetic tree of TRAM best hits. The best hits from BLAST searches of protein databases for individual species and the two best whale shark predicted proteins were used to create a phylogenetic tree. The program used to create the tree was ClustalW2.
Conclusion Although we were not able to strongly identify a predicted TRAM ortholog in whale sharks, we found strong evidence of the conservation of the TIR signaling protein domain. The conservation of this domain is supported by the high levels of alignment of the protein sequences in the region where the domain is found. The lack of a human TRAM ortholog in elephant sharks, which are closely related to whale sharks further shows a lack of orthologs of the human TRAM protein in whale sharks.
However, innate immune response systems play huge role in the survival of any organism. Thus, it is important that all species have some form of innate response to fight pathogens. Given the diversity of proteins involved in the TLR system, of which there are similar systems in a variety of species, it is highly likely that the whale shark has a system that uses similar proteins and processes. The results we found suggest that although there is no TRAM ortholog in whale sharks, there may be orthologs of other proteins which are involved in the innate immune response system in humans such as the toll-like receptor proteins. This concept is supported by the conservation of the TIR domain found in multiple species and the whale shark predicted proteins. Further research needs to be done to determine whether whale sharks do have orthologs of the proteins involved in the human TLR system and whether they have similar immune response processes.
References
Liew, Foo Y, et al. "Negative regulation of Toll-like receptor-mediated immune responses." Nature reviews. Immunology 5.6 (2005):446-458.
O'Neill, Luke A. J., and Andrew G. Bowie. "The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling." Nature reviews. Immunology 7.5 (2007):353-364.
Ullah, M O, et al. "Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling." Protein expression and purification 106(2015):31-40.
Verstak, B, et al. "The TLR signaling adaptor TRAM interacts with TRAF6 to mediate activation of the inflammatory response by TLR4." Journal of leukocyte biology 96.3 (2014):427-436.
Yamamoto, Masahiro, et al. "TRAM is specifically involved in the Toll-like receptor 4–mediated MyD88-independent signaling pathway." Nature Immunology 4.11 (2003):1144-1150.
This Project:
This page has been made for a genomic analysis project being conducted by Biology 142 Lab Courses at Emory University in partnership with the Georgia Aquarium. The purpose of this project is to aid in the identification of possible proteins in the recently sequenced whale shark genome. This page is dedicated to the analysis done on the human TICAM2/TRAM protein.
Background Information
When pathogens are found in our bodies, our immune system is what recognizes and fights them off. These pathogens are recognized by toll-like receptor proteins, abbreviated TLRs (receptors on the outside of macrophages and dendritic cells which recognize microbes) . Recognition of these pathogens activates immune responses through signaling pathways controlled by domain containing adaptors (Verstak et al) . Adaptor proteins influence signaling pathways and cross-overs by facilitating interactions between proteins and larger signaling complexes. To ensure the appropriate signaling response, many processes rely greatly on these adaptor proteins ("Research Topics - Adaptor Proteins").
TICAM2/TRAM, an abbreviation for TRIF –related adaptor molecule or TIR containing adaptor molecule 2, is one of these adaptors ("The Toll/IL-1 Receptor Adaptor Family"). When TLR4 is activated, upon recognition of pathogens, it recruits TICAM2/TRAM to activate the production of pro-inflammatory cytokines ("Purified Anti-TICAM-2 TRAM Antibody").This process has further been supported by studies that have explored how TRAM deficient mice show defects in their ability to produce cytokine (Yammamoto et al).
The pathway within which that TRAM operates in this system is called the MyD88-independent pathway. In the signaling chain, TRAM bridges the interaction between TLR4 and TICAM1("Purified Anti-TICAM-2 TRAM Antibody").A recent study has shown that the interaction between TLR4 and TRAM is physical and that the "BB loop" regions of both the TRAM and the TLR4 domain are important for the physical interaction to occur (Ullah et al). Figure 1 below illustrates the multiple TLR signaling pathways involved in the immune response system (Liew et al).
Figure 1. Credit to Liew et al. This image shows the process of TLRs affecting nuclear activity. The process is quite complicated and takes many steps. The TRAM molecule initiates a series of chain reactions which ultimately controls and alters nuclear activity by nuclear factor kB (Liew et al).
Methods
Finding Whale shark predicted orthologs:The human protein sequence (ENSP00000415139) was used as the query in a BLAST against the predicted whale shark protein database using the Galaxy server by Georgia Aquarium. The full sequence of the best matching predicted protein, determined by the lowest e-value and alignment length, was obtained. The sequence was then used as the query in protein BLASTs against the NCBI human protein database to test possible orthology by showing a positive reciprocal match.
Predicted orthologs in other species
TICAM2/TRAM (ENSP00000415139) predicted orthologs were identified in other species by using the NCBI Blast server. The human NHEJ1 protein sequence was used as query in these searches. Protein BLASTs were performed using the human protein sequence against single species protein databases for the mouse, zebra fish, clawed frog, elephant shark, and fruit fly.
Phylogenetic tree
The top whale shark predicted protein sequences,the best hit sequences (with the lowest E-value) for each non-whale shark species, and the human TRAM sequence, were used to create a multiple sequence alignment and phylogenetic tree. The source used to create the multiple sequence alignment and phylogenetic tree was ClustalW2 program.
Searching for TICAM2/TRAM in whale sharks
The protein sequence for TRAM in humans was used as the query against the whale shark predicted protein database. The results of the top four hits in the BLAST are shown in Table 1 below. These 4 best hits had the lowest E-values with the lowest E-value being 3e-07. These 4 top hits were then used as queries and BLASTed against the human protein database using NCBI BLASTp.
The top 4 predicted proteins from the BLAST of the whale shark predicted proteins were used as queries against the human protein database. The BLAST of these 4 predicted proteins did not return TRAM as the best hit. The results can be seen in Table 2 below.
Because none of the BLASTS returned TRAM as a best hit using the predicted whale shark proteins as the queries,we were not confident that either of the two best predicted whale shark proteins and the human TRAM are orthologs. However, given the significant E-values of 7e-76 and 1e-26 with the toll-like receptor 5 precursor (TLR5 precursor) and toll-like receptor 2 precursor (TLR2 precursor), we believed that the two proteins may share the same protein domain. This belief was further supported by the similar functionality TLR proteins and TRAM have in the initiation of immune responses against pathogens. The protein domains of the top two whale shark predicted proteins ( g48010.t1 and g47253.t1) are shown in Figure 2.
Protein Domains
As shown in Figure 2, all three (Human TRAM protein and the two predicted whale shark proteins) proteins contained a TIR_2 super-family domain. This shows that the domain of the human TRAM protein is conserved in the two top predicted whale shark proteins. The TIR,or Toll/interleukin-1 (IL-1) receptor, domain is a special type of domain which is found only in the signaling TLR system, toll-like receptor proteins (O’ Neill and Bowie). The TIR domain is key to the function of TLRs in triggering innate immune responses to pathogens (O’Neill and Bowie). Additionally, the conservation of sequence among TIR domains in various proteins (adaptors, receptors, etc) is only 20-30%, and the size of the domain also varies, giving rise to a great amount of diversity in the domain (Xu et al). Thus, it is likely that the two predicted whale shark proteins, although not matched with a human TRAM protein, are somehow involved in an innate immune system response in whale sharks similar to that of humans.
Human TRAM
g47253.t1 whale shark
g48010.t1 whale shark
Figure 2. Protein domains of 2 best whale shark predicted proteins and Human TRAM protein. The protein domains were obtained using the NCBI server to search the protein sequences for domains.
Orthologs
The human TRAM protein sequence was used as query in NCBI BLASTs against the databases for other species. A TIR domain-containing adaptor molecule 2 ortholog was found in mice as the BLAST returned TRAM as the best hit. A possibly ortholog was found in zebrafish as the BLAST returned an alternate form of TRAM as the best hit.Although there were no orthologs found in the other species, results showed that the TIR_2 superfamily protein domain was conserved in all the species that were examined. All species BLASTed against, aside from the fruit fly, had best hits of proteins related to or in the same immune response system as TRAM.
Phylogenetic Tree and Alignment Data
A multiple sequence alignment was conducted using the ClustalW2 program with the best hit protein sequences of the individual species above. Alignment results showed low homology( many gaps in alignment) in the earlier sections of the sequence. There were high levels of homology in the section of the sequences that contained the TIR protein domains. The alignment of the sequences where the protein domain is located can be seen below in Figure 3.
<span style="color: #000000; font-family: monospace; font-size: 12px;">Human -----------------------------------------------------------E Mouse -----------------------------------------------------------E ElephantShark -----------------------------------------------------------A ZebraFish ---------------------------------------------------------LLS ClawedFrog ---------------------------------------------------------YTV g48010.t1 ------------------------------------------------------------ Fruitfly LNQPPKLDYIPILVAILTAFIFVMICISLVFIFRQEMRVWCHSRFGVRLFYNAQKDVDKN g47253.t1 ----------------------------------------------------------KA Human AEEEVFLKFVILHAEDDTDEALRVQNLLQDDFGIKPGIIFAEMP-CGRQHLQNLDDAVNG Mouse QDEEEFLKFVILHAEDDTDEALRVQDLLQNDFGIRPGIVFAEMP-CGRLHLQNLDDAVNG ElephantShark DNEDTFYKFVILHAQNDTDEALRLLNLLENKFQVIPGRLFNDMP-AGNQQLQNLSDAVNG ZebraFish DIDETFYAFVILHEAEDADEAQRLREKLEGIISANGATFSEDFAQAGRSTLRCIEDAIDN ClawedFrog PDDSLFFNFVVLHVREDSEVARRVCDVLQSLGAGNGTTFCEGFEIPGSNPLTCIQEAVEN g48010.t1 VCNDGFVAFCATAP-----VIFVLMVTVVVYQKGKWSFHYGYYLLRAWIHENKLQKNSTK Fruitfly EREKLFDAFVSYSSKDELFVNEELAPMLEMGEHRYKLCLHQRDFPVGGYLPETIVQAIDS g47253.t1 YKFDAYLCFSSNDIEWVTNSLLQYLDSQFNEKNKFQICFEDRDFIPGEDHITNIRDAIWS . : * . : . Human SAWTILLLTENFLRDTWCNFQFYTSLMNSVNRQHKYNSVIPMRPLNNPLPRERTPFALQT Mouse SAWTILLLTENFLRDTWCNFQFYTSLMNSVSRQHKYNSVIPMRPLNSPLPRERTPLALQT ElephantShark SAWTILLLTEDFLSEAWCKFQSYITLMNSLNKQHKYNTVIPVRILNNPLPHKMTPLVIQA ZebraFish SAFVLLLLTQNFK-SNQSMTTTDSAIVNSLEHHHKLNSVIPLLPRENRLSRKNIPLVLQT ClawedFrog SAYIILLMTEHFE-TRWAEFQSNAVLMNSINDENRTGCVIPFLPETNRLPIKKMPLALKS g48010.t1 GYKFDAFVSYNSKDEAWVFDQLMNNLENEGPPFHQLCFHHRDFEVGKPIVENIVDAIYKS Fruitfly SRRTIMVVSENFIKSEWCRFEFKSAHQSVLRDRRRRLIVIVLGEVPQKELDPDLRLYLKT g47253.t1 SKKTVCIVTRQFLKDGWCVEAFNIAQSRLFHELREVMVVLVVGKLP-------------- . .:: . .. Human INALEEESRGFPTQVERIFQESVYKTQQTIWKETRNMVQRQFIA---------------- Mouse INALEEESQGFSTQVERIFRESVFERQQSIWKETRSVSQKQFIA---------------- ElephantShark INALEEGNPAFDRQVNLTFQDSTYEQQRTIWHRERKLKQQGISAFNGNKTNKSRAEEG-- ZebraFish KVPLDESNRTFERKALKALSQDAVEKQRKIWMKKQTLKKLEEEYKRRQEENFINANLRLE ClawedFrog LIPLDELSPVFRRAVCNTFKQEKILSQKQHWERQQQKKKERQRMLELQRSEQANDSGQYL g48010.t1 RKTICIISKNYLQSEWCSMEMQVALYRLFDEHSDVLILVFLEEIPGHVLSSYHHLRKL-- Fruitfly NTYLQWGDKLFWQKLRFALPDVSSSQRSNVAGQSCHVPINHASYHHHHHVHQQAMPLPHS g47253.t1 ---------DYQLMKYQPIRAYIQSRQYMRWPEDPQDH---------------------- : : : Human ------------------------------------------------------------ Mouse ------------------------------------------------------------ ElephantShark ------------------------------------------------------------ ZebraFish REKLARLKLESESPHNNMFHSSSAQPPSCGPMLQQHDSWPQKPSYIHIENAQNIMIGNHS ClawedFrog MHMCANEGNVNLSAQYMYQHP------------------IPYAPVIHINNAENIQIGNHN g48010.t1 ------------------------------------------------------------ Fruitfly VHHHQQQFMLPPPPQQPGSFRRQPSLHQQQQQQQQIRGNNNTTQQQQQQQAALLMGGGSV g47253.t1 ------------------------------------------------------------</span>Figure 3. Multiple sequence alignment of individual species. The alignment of the species was found by entering the best hit FASTA sequences for all the relevant species using the ClustlW2 program.Using the alignment data, a phylogenetic tree was then made and can be seen below in Figure 3. From this tree, the similarity between the human and mouse sequences can be seen clearly, as they are grouped together. The two whale shark predicted proteins are also grouped together in the tree, showing their sequence similarity. The whale shark predicted proteins are grouped the furthest away from the human TRAM protein, further suggesting that there is no whale shark homolog to the human TRAM protein (Figure 4).
Conclusion
Although we were not able to strongly identify a predicted TRAM ortholog in whale sharks, we found strong evidence of the conservation of the TIR signaling protein domain. The conservation of this domain is supported by the high levels of alignment of the protein sequences in the region where the domain is found. The lack of a human TRAM ortholog in elephant sharks, which are closely related to whale sharks further shows a lack of orthologs of the human TRAM protein in whale sharks.
However, innate immune response systems play huge role in the survival of any organism. Thus, it is important that all species have some form of innate response to fight pathogens. Given the diversity of proteins involved in the TLR system, of which there are similar systems in a variety of species, it is highly likely that the whale shark has a system that uses similar proteins and processes. The results we found suggest that although there is no TRAM ortholog in whale sharks, there may be orthologs of other proteins which are involved in the innate immune response system in humans such as the toll-like receptor proteins. This concept is supported by the conservation of the TIR domain found in multiple species and the whale shark predicted proteins. Further research needs to be done to determine whether whale sharks do have orthologs of the proteins involved in the human TLR system and whether they have similar immune response processes.
References
Liew, Foo Y, et al. "Negative regulation of Toll-like receptor-mediated immune responses." Nature reviews. Immunology 5.6 (2005):446-458.
O'Neill, Luke A. J., and Andrew G. Bowie. "The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling." Nature reviews. Immunology 7.5 (2007):353-364.
"Purified Anti-TICAM-2 TRAM Antibody". Biolegend- The Path to Legendary Discovery. Web. 30 Mar. 2015. <http://www.biolegend.com/purified-anti-ticam-2-tram-antibody-9432.html>.
"Research Topics - Adaptor Proteins." R&D Systems.Web. 31 Mar. 2015. <http://www.rndsystems.com/molecule_group.aspx?g=1066>.
"The Toll/IL-1 Receptor Adaptor Family". Invivogen-Innovation Within Reach. Web. 29 Mar. 2015. <http://www.invivogen.com/review-toll-il1-adaptor-family>.
Ullah, M O, et al. "Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling." Protein expression and purification 106(2015):31-40.
Verstak, B, et al. "The TLR signaling adaptor TRAM interacts with TRAF6 to mediate activation of the inflammatory response by TLR4." Journal of leukocyte biology 96.3 (2014):427-436.
Yamamoto, Masahiro, et al. "TRAM is specifically involved in the Toll-like receptor 4–mediated MyD88-independent signaling pathway." Nature Immunology 4.11 (2003):1144-1150.
"Multiple Sequence Alignment - CLUSTALW." Multiple Sequence Alignment - CLUSTALW. N.p., n.d. Web. 14 Apr. 2015. <__http://www.genome.jp/tools/clustalw/__>.
"Basic Local Alignment Search Tool." BLAST:. N.p., n.d. Web. 14 Apr. 2015. <__http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome__>.
"Galaxy / Whale Shark." Galaxy / Whale Shark. N.p., n.d. Web. 14 Apr. 2015. <__http://whaleshark.georgiaaquarium.org/__>.
Xu, Y. et al. (2000) Nature 408(6808), 111–115