BACKGROUND
the cold-induced angiogenic response
angiogenesis can be initiated by a variety of stimuli.
for example, angiogenesis is induced by airway smooth muscle strain in chronic asthmatics  <cit> , by exercise  <cit>  and in some species by cold exposure  <cit> .
many species are subjected to annual cycles of environmental cooling that represents a significant challenge for the cardiovascular system due to the effects of reduced oxygen diffusion and increased blood viscosity associated with low temperatures  <cit> .
one way of overcoming limitations to aerobic activity is to increase tissue capillary supply.
although the underlying process is unclear, cold-induced angiogenesis may respond to altered hormonal levels or changes in the mechanical environment of endothelial cells   <cit> .
even within the mammalian literature, it is apparent that a number of types of capillary growth exist, e.g.
pathological vs. physiological angiogenesis, albeit under the influence of a restricted set of genes  <cit> .
in order to improve the signal to noise ratio from the wealth of published data and reduce the influence of host tissue in the readout, gene expression profiles for endothelial-specific up-regulated genes have recently been identified  <cit> .
no such information exists for fish, although their use as an experimental model is increasing due to the additional interventions not possible with mammals  <cit> .
for the first time, we have used an in silico approach to identify whether orthologs between man and fish could be identified that were associated with angiogenesis, then used re-analysis of microarray data to see if these genes were differentially expressed on cold exposure and finally we determined what proportion of the up-regulated genes were angiogenic or ec-specific.
we anticipate this approach could be useful for similar studies in non-model species for which a genome sequence is not available but the human orthologs involved in the biological process are known.
RESULTS
gene assignment by ortholog identification
orthologs , as a whole, perform an equivalent function in respective genomes  <cit> .
based on this assumption, carp orthologs of known angiogenic human genes provide genes pivotal in their cold angiogenic response.
cold carp data from a study by gracey et al.
<dig>  <cit>  was to be used in this work to predict the identity of cold induced angiogenic genes.
the genes assigned to carp sequences from the original article  <cit>  were not applicable to this study as they used a blastx against multiple species databases and only looked for the best hit without using a control step.
in addition, the databases searched were from  <dig>  several years out of date, and a more stringent method of sequence contig construction was used in this work .
a problem to be overcome was how best to assign human orthologs to  <dig>  anonymous est carp sequences collected from genbank and carpbase .
many of these sequences were less than  <dig> bases and many of them contain partial or complete untranslated regions.
an obvious first step was to cluster them, using cap <dig>  <cit> , to extend the sequence length and improve the quality of any subsequent analyses.
this reduced the sequence count to a total of  <dig>  sequence contigs and singletons .
the next challenge was to identify their human orthologs.
choosing the ortholog assignment method
homologs, in the strictest sense, are characters that have been passed down following a speciation event from a common ancestor  <cit> .
a gene in two species derived from a single gene in their common ancestor is defined as an ortholog  and usually has the same or similar function between species  <cit> , i.e.
the same gene in different organisms are orthologs  <cit> .
conversely, paralogs are defined as genes derived from a single gene sequence duplication event and usually have different functions  <cit> .
orthologs and paralogs are not the only gene histories and several other events can take place, see  <cit>  for a full list.
in this work, the sole aim was to find the most likely functional human homologs of non-model transcripts and did not attempt to define or classify paralogs or any other forms of gene histories.
many bioinformatic programs and pre-computed databases of orthologs exist, a number of which are listed in a recent review  <cit> .
essentially, there are  <dig> methods of finding orthologs, tree based, graph based and a combination of the two.
tree methods use sequence alignments and phylogenetic trees for predicting evolutionary relationships, while graph based methods use pairwise sequence similarity search methods  to predict orthologs.
the review highlights the merits of each program or database and it provides the reader with a flow chart  to assist in the best choice of tool for a particular application.
after investigation of the recommended programs it was decided that none were applicable to the high-throughput analysis needed to cope with the >  <dig> k incomplete anonymous carp nucleotide sequences.
the main reasons were the inability of these programs to be queried with a high volume of data through their web servers or the databases were several years out of date.
further literature searches revealed a tool called blast on orthologous groups   <cit>  which searches a query sequence against a database of orthologous groups  as a single unit.
although this tool enabled searches against several combination method pre-computed databases including homologene  <cit> , it could only deal with one sequence at a time.
additionally, the blasto form did not permit the user to change blast filtering options which, as seen below, makes a difference to the quality and quantity of successful ortholog predictions.
ortholog assignments, rbh and filtering options
a graph based, nearest neighbour  <cit>  approach, rbh, was considered the best option since constructing multiple sequence alignments and phylogenetic trees from incomplete nucleotide sequences would be inaccurate and not amenable to high throughput.
strengthening this decision, a performance assessment of different ortholog prediction methods by altenhoff and dessimoz  <dig>  <cit>  found the rbh performed well in comparison with the other methods.
reciprocal best hit   <cit>  is a technique using blast to first search a query sequence against a genome.
if the resulting best hit matches the original query sequence from a reciprocal blast then it is an ortholog termed rbh.
a diagrammatical representation of the rbh process of carp vs. human is shown in figure  <dig>  labelled route  <dig>
an article by moreno-hagelsieb and latimer  <dig>  <cit>  tested ortholog rbh searching using different blast options.
the tests utilised bacterial genomes, neighbouring orthologs and paralogs to estimate error rates of ortholog assignments by rbh.
they found the soft filtering option -f "m s" with the addition of smith-waterman  alignments  were optimal for rbh success.
these options were tested for eukaryotes using carp vs. human rbh searches.
four different blast options were tested: soft filtering  with and without sw  and hard filtering with and without sw. the results revealed very little difference in the number of successful rbh with or without the sw algorithm against their findings of 10%.
in contrast, as with prokaryotes, soft filtering increased the number of rbhs by 2% .
table  <dig> shows the number of successful orthologs found using carp vs. human rbh for the different filtering options.
figure  <dig> shows a bar chart of the successful rbh results normalised to the default blast options .
this table shows the performance of rbh using the different blast filtering options with eukaryotic sequences.
the results from this analysis showed the soft filtering option with no smith and waterman produced the highest number of successful rbhs.
contrary to the findings of moreno-hagelsieb and latimer  <dig>  <cit> , who showed the sw algorithm improved results by 10%, in this study using sw produced  <dig> %  less successful results.
however the analyses in this work were slightly different to that of moreno-hagelsieb and latimer and are listed here:
1) they used complete bacterial genomes: a) these sequences are generated from genomic dna and are sequenced in both strands.
b) the carp sequences are generated from single pass  cdna sequencing from rna and, as such, are likely to contain more errors.
2) the protein sequences used in assessing rbh success: a) moreno-hagelsieb and latimer used full length and defined proteins from complete bacterial genomes.
b) the carp sequences were partial proteins from  <dig> frame nucleotide translations and from a eukaryotic and not a prokaryotic species.
3) the algorithm options used with blast were not exactly the same.
a) moreno-hagelsieb and latimer used the blastpgp algorithm from the ncbi  <cit>  blast, which has the smith-waterman -s t option available.
b) in this work blastall using blastx  and tblastn  were used.
the -s t option is only available for tblastn and not blastx with ncbi blastall.
note: blastpgp can only be used on protein sequences and not nucleotides.
4) some cdnas span genes on different chromosome strands and is another way rbh results could be affected .
however, even with these differences, the results were very surprising because of other research  <cit>  and considering the -s f option is a fast approximation of the sw algorithm .
one thing to bear in mind is an e-value of 1e- <dig> and soft filtering were employed in this work, which in itself, would remove many spurious matches otherwise found using the default blast heuristic.
a further investigation into this revealed that the genes successfully found with each method did not totally overlap.
the heuristic blast alignment method predicted  <dig> functional homologs with rbh that sw did not find and the sw alignment method predicted  <dig> that failed with the blast heuristic.
the intersection of results totally concurred.
the questions to answer here were; why did one algorithm fail rbh, whilst the other method was successful?
and of these differences, which algorithm produced the more believably evolutionary related rbh successes?
to investigate this finding further and to decide on which method to use in this work, a manual inspection of sequence alignments was needed in a systematic way to avoid the problem of biased cherry picking.
this was done using a random number generator to pick  <dig> sequences to investigate ,  <dig> from each algorithm rbh success.
so the investigation was to see if the results were believable for each algorithm.
the results found that ~50%  of results suggested the default blast option found the more likely evolutionary related sequence as it found higher similar matches but at shorter alignment lengths, which is believable as sw is good at finding distant homology.
however, this was not always the case and the different sequence alignment algorithms were correct for different sequences.
as a consequence it was not possible to automate the choice without manually viewing the results.
therefore, in this work the results generated using the default blast heuristic was used for the final data.
however, it is recommended for other researchers using these methods on small datasets to use the intersection of both methods or the sw alone .
this is not a problem for small numbers of sequences but for next generation sequence data, for example, it is recommended the default heuristic be used as it will take weeks to compute sw alignments.
all but one cold induced angiogenic gene were found with both algorithms and users of this data can be confident in the ortholog assignments of these genes.
the exception was the plasminogen gene plg .
taking advantage of a closely related vertebrate
zebrafish are a model teleost organism used in the elucidation of vertebrate development, molecular, genetic, genomic and evolutionary biology  <cit> .
as such its genome has been sequenced with extensive annotation and currently in the sequence databases there are over  <dig> million ests and  <dig> thousand refseq proteins.
carp, on the other hand, currently has ~ <dig> thousand ests and no genome sequence.
it was possible to take advantage of this annotation because of the close evolutionary relationship carp has to zebrafish in that they diverged from their last common ancestor only  <dig> million years ago  <cit> .
previous groups have shown that close genome evolutionary relationships can aid ortholog identification  <cit> .
in the present study, zebrafish was also utilised as a stepping stone approach to ortholog identification.
figure  <dig>  labelled route  <dig>  shows a stepped reciprocal best hit  approach where rbh analysis is carried out twice, once between carp and zebrafish and then between zebrafish and human.
if both were successful, then a carp-human ortholog was assigned.
using the srbh analysis gained a further 8%  successfully assigned orthologs.
teleost genome duplication and srbh
although there is a discrepancy as to when it happened, there is much evidence for a genome duplication event in teleosts  <cit> .
a good example in zebrafish is the paralogs ets <dig> and etsrp which both lie next to fli1a and fli1b, respectively, but on different chromosomes.
these genes are important for vasculogenesis and angiogenesis  <cit> .
from a srbh analysis, zebrafish genes ets <dig> and etsrp best match the human gene ets <dig> and fli1a, and fli1b best matches the human fli <dig>  suggesting a genome duplication event occurred in zebrafish.
because of duplicate genes, the best and second best hits to zebrafish and carp genes were used to attain a successful srbh.
the impact of genome duplication in ortholog searching can be seen in the following example.
the zebrafish peptide np_ <dig> was the best hit to ca <dig>  however, the peptide np_ <dig> best matched human np_ <dig>  blasting np_ <dig> back against zebrafish revealed two very similar sequences, np_ <dig> and np_ <dig>  np_ <dig> was the best hit and therefore failed srbh .
this is due to genome duplication of the zebrafish genome and thus both genes in zebrafish can be considered orthologs to the same human gene.
rbh and a comparison with homologene
homologene is a database of orthologous groups defined with blast pairwise alignments and phylogenetic trees  <cit> .
since csrbh included a zebrafish and human ortholog rbh analysis, a comparison was made between homologene and this rbh step to see if the zebrafish to human ortholog assignments from this work agreed.
from the  <dig>  successful rbhs, only  <dig>  could be compared as only these had both human and zebrafish genes present in the homologene data.
this fact alone gives support to using this approach as a user supplies all genes, and as such, all are present.
from these ortholog assignments, 85% agreed with homologene orthologous groups.
a manual investigation of some of those that disagreed suggests homologene does not always perform well for a proportion of duplicated genes.
for instance, one example, the zebrafish actc1l gene best matched the human actc <dig> gene with 99% identity over the full alignment length of the gene but homologene put actc1l in the actb orthologous group  where alignment percent identity was less at 94% .
the full results of the comparison can be viewed in additional file  <dig>  in addition, ten alignments were chosen randomly and manually investigated  to see which source of data was most realistic.
the results of this suggested  <dig> out of  <dig> ortholog assignments were correct with rbh whilst homologene put the genes into different orthologous groups.
a full comparison of rbh with homologene
a full analysis was also performed that took all human proteins in the refseq database of proteins and rbh searched against the full database of zebrafish refseq proteins.
on july 22nd  <dig>  there were  <dig>  proteins in the human database.
<dig>  of these proteins resulted in a successful rbh analysis against zebrafish.
build  <dig> of homologene  contained  <dig>  human proteins.
therefore 33% of the human proteome was not represented in homologene at this time and only  <dig>  of the  <dig>  human proteins had a zebrafish homolog protein in the same orthologous group.
there were  <dig> human/zebrafish orthologs identified by homologene only and not with the rbh procedure.
in contrast, there were  <dig>  human/zebrafish orthologs found using the rbh but not with in homologene.
<dig>  human proteins were found in both homologene and in the rbh analysis.
<dig>  of these agreed, an 89% agreement.
this is very similar to the result above with only the carp data.
these findings, particularly the fact that homologene only had  <dig>  human proteins with a zebrafish ortholog and the findings of altenhoff  <cit> , give a user some further support and confidence to using a rbh ortholog assignment approach rather than phylogenetic tree methods for analysing large amounts of data from a non-model species in a quick and fairly accurate manner.
the srbh approach is endorsed by the fact other researchers have used closely related species to predict genes and sequence motifs  <cit> .
however, it should be noted that the comparisons done here with homologene only validates the rbh method but not the csrbh as, to definitively validate the csrbh approach, the carp genome and annotation need to be more complete.
in addition, the csrbh approach does not attempt to better or replace the efforts of databases such as homologene.
one reason is that it does not attempt to deal with paralogs and other sources of gene histories but only with the most functional homologs , though some attention was given to the teleost genome duplication event.
so there are examples where homologene better assigns orthologs than the methods used here.
having said that, with a non-model species not present in homologene, this approach is excellent at giving a good approximation of the human functional homologs of researchers' genes.
csrbh, combining rbh and srbh to find further orthologs
although adding the zebrafish intermediary step  improved ortholog assignment by 8%,  <dig> assignments that failed using this procedure were successfully found using a carp vs. human rbh alone .
inspection of a random sample of these found two examples of why this occurred.
blast of carp est ec <dig> vs. zebrafish best hit the gene smyhc <dig> and the reciprocal blast vs. carp found est ab <dig> as the best hit.
however, smyhc <dig> was the fifth best hit to ab <dig>  thus failing the srbh analysis .
however, this gene is successfully assigned a human ortholog from an rbh analysis .
even more compelling was the situation with est au <dig> .
it aligns with the human protein np_ <dig> as the best hit and the alignment was highly similar with a percent identity of 91% over  <dig> amino acids.
the same est best aligned to the zebrafish protein np_ <dig> with 37% identity over  <dig> amino acids.
hence, the est is significantly more similar to the human protein rather than the zebrafish.
the human protein np_ <dig> was blast searched against zebrafish to see if the full-length protein found a likely ortholog in zebrafish.
the best hit to the human protein was np_ <dig>  the same as found by searching est au <dig>  therefore, there is evidence that either some genes do not exist in zebrafish and do so in carp or are not currently found in zebrafish as the genome is not yet completely sequenced or annotated.
another piece of evidence is, if a simple blast search is performed against the multiple species nr database of proteins at the ncbi  <cit> , 24% of carp sequences preferentially match a human protein, whilst 30% match a zebrafish protein.
this supports the use of a conditional stepped reciprocal best hit  approach, which uses whichever species is a closer match to the query and performs the appropriate ortholog prediction route .
in addition, for any that fail, a rescue was be performed by searching through the other route.
csrbh outperformed all other methods and produced 3% and 11% more successful ortholog assignments than the srbh and rbh, respectively.
in total  <dig>  contigs were assigned an ortholog, totalling  <dig>  non-redundant genes .
see table  <dig> and figure  <dig> for the number of successful ortholog assignments for all the different analysis methods used in this work.
we propose that csrbh should be the method of choice for researchers working with non-model species which have no sequenced genome and partially sequenced genes.
this table displays the number of human protein orthologs found using csrbh for the  <dig>  cdna carp contiguous sequences.
<dig>  total none redundant genes were found and  <dig> of them were predicted as angiogenesis genes.
from the re-analysis,  <dig> genes were up-regulated and 24% of them were predicted as angiogenesis genes.
a weakness with this approach is that it does not determine an orthologs absolute gene history and it will not report to a user whether the ortholog found was derived from a gene duplication event or from a speciation.
that said, these methods will enable a user to quickly find the most likely functional homolog of a gene.
prediction of angiogenic genes
established angiogenic genes
previous groups have developed complex data mining methods using decision trees to identify genes of interest  <cit> .
in this study,  <dig> simple strategies were combined to generate as a complete a set of angiogenic genes as possible.
first the cardiovascular and angiogenesis groups at birmingham university used their knowledge to produce a list of angiogenic genes that included those from the recent literature  <cit>  and the sabiosciences gearray® human angiogenesis microarray  <cit> .
a total of  <dig> angiogenesis related genes were predicted this way and a list of  <dig> genes were contained in the successful csrbh results .
however, it is possible many other genes have an undiscovered role in angiogenesis and, therefore, three further methods were employed to predict additional angiogenic genes.
putative angiogenic genes
the second and third approaches used perl programs to scan article abstracts from pubmed  <cit>  and gene ontology processes from amigo  <cit>  using the following angiogenic related keywords as baits: 'angiogenic, angiogenesis, neovascularization, neovascularisation, vasculogenesis, hypoxia, endoth vegf'.
this predicted a further  <dig> angiogenic genes .
a fourth method used gene expression of ecs.
during the activation of angiogenesis, ecs become activated and switch on several signalling pathways that cause proliferation, migration and invasion  <cit> .
this angiogenic phenotype leads to an up-regulation of ec genes.
therefore, genes up-regulated in the endothelial transcriptome could be angiogenic.
the endothelium transcriptome has been extensively defined  <cit>  and genes up-regulated in ecs were compared with the human/carp orthologs to identify additional putative angiogenic genes;  <dig> were found .
total angiogenic genes
combining the results from the four data mining methods gave a total of  <dig> non-redundant predicted angiogenic genes .
we postulate this gave us a comprehensive set and the best chance of discovering cold-induced angiogenic genes from the carp data.
however, it should be noted, and the authors fully acknowledge, that not every gene predicted here is a genuine angiogenesis gene and there will be some false positives.
very few bioinformatic predictions are 100% foolproof and this study is aimed at guiding the bench scientist into making more informed decisions before going into the laboratory.
the number of different methods that predicted a gene angiogenic now ranks the genes.
for example, the gene hif1a is a well-known angiogenic gene and was predicted as such by all four methods.
column i entitled "number of methods" in additional file  <dig> gives the number of methods predicting a gene angiogenic and is ranked in a descending order.
conversely, genes that are not so well known to be angiogenic genes are only found by one or two methods.
if this approach is employed and genes selected that were predicted by more than one method, reduces the high percentage of angiogenic genes from 25% down to  <dig>  this is reduced further if you only take genes with  <dig> and/or  <dig> methods.
but given the full list, a researcher can decide based on the presence of functional domains or personal knowledge which ones are of interest to investigate with full knowledge that the human functional homolog was only loosely associated with the biological process of interest.
re-analysis of gene expression data
three established methods of measuring gene expression used in biological research are microarrays, cdna and serial analysis of gene expression  libraries.
to investigate numerous pathologies many institutes around the world have generated, analysed and deposited large amounts of expression data into public repositories  <cit> .
examples of such databases are the gene expression omnibus , cancer genome anatomy project , national center for biotechnology information  and european bioinformatics institute  arrayexpress.
it has been shown that data mining of relevant data sets can successfully lead to the identification of biologically interesting targets  <cit> .
cold response differentially expressed genes
carp is an ectotherm with a body temperature similar to the water it is immersed in and can adapt to a range of temperatures .
a recent study investigated physiological adaptation of carp exposed to gradually lower temperatures  <cit> .
microarray analysis measured the change in gene expression, in several tissues, at several cooling stages from  <dig> to 10°c vs. a control temperature of 30°c.
overall,  <dig> transcriptional regulatory, rna splicing and translation control genes were found to be up-regulated in all tissues.
interestingly, some tissue-specific affects were also seen, e.g.
in the glycolytic pathway of brain and lipid metabolism of liver.
this data set proved appropriate for re-analysis to predict cold-induced angiogenic genes in skeletal and heart muscle tissues.
persistently up-regulated genes
two strategies were applied to the data.
the first utilised the data without intervention for heart and muscle tissues, employing a threshold score > =  <dig>  the total number of up-regulated cdnas for heart was  <dig>   <dig> non-redundant genes, and for muscle  <dig> cdnas,  <dig> genes.
any cdnas with significant contradicting expression were removed.
for both heart and muscle combined there were  <dig> non-redundant genes,  <dig> of which were predicted angiogenic genes .
this table conveys the numerical breakdown of results for the differential cdna expression due to cold temperatures on the carp array.
<dig> were the total number of putative cold induced angiogenic genes.
initial cooling, fold ratio subtraction and microarray re-analysis
it is thought angiogenesis is switched on in the initial stages of cooling, before the animal has become acclimatised to a particular temperature.
this has been shown in rats subjected to cooling to 4°c which led to a  <dig>  fold increase in vegf expression for  <dig> to  <dig> hours and returned to the basal level at  <dig> hours  <cit> .
therefore, the microarray data was analysed between the first and last time point at each target temperature.
first, ratios were calculated between each of the cooling time points vs. a control group .
analysing heart and muscle separately and combining the results gave  <dig> unambiguous cdnas with a ratio subtraction > =  <dig> and this represented  <dig> non-redundant genes.
<dig> of these genes were predicted angiogenic, see table  <dig> and additional file  <dig>  column e for both heart and muscle sheets.
to further discover genes switched on at initial cooling, microarray analysis using limma from the bioconductor r package  <cit>  was used to fit a linear model to the arrays .
an adjusted p-value < =  <dig>  was employed as a significance threshold.
<dig> and  <dig> unambiguous cdnas were found to be up-regulated in heart and skeletal muscle respectively .
combined, this represented  <dig> non-redundant genes, of which  <dig> were predicted to be angiogenic .
differential gene expression on initial cooling: overlap with ratio subtraction and limma analyses
both these methods are seeking to find genes up-regulated at the first time point on immediate cooling of fish vs. fish that have acclimatised to the temperature.
the results should corroborate each other.
dealing the heart tissue first, there were  <dig> cdnas that reached a p-value < =  <dig>  and  <dig> cdnas that had a ratio subtraction > =  <dig> .
these cdnas did not overlap but the ratio subtraction results, though not significant, for the limma cdnas were positive.
so they concurred but not with significance for the ratio subtraction.
muscle, in contrast, gave better corroboration between ratio and limma analyses.
there were  <dig> cdnas with limma adjusted p-value < =  <dig> .
there were  <dig> total ratio subtractions that reached significance .
of these,  <dig> agreed with significant limma results.
the results were encouraging.
in general, the number of genes predicted by a method are always a balance between false positives and false negatives.
in this case  <dig> cdnas are in common in total between the "ratio" and the "limma" methods for muscle.
however, differences between the two methods are expected as the first is based on a hard cut-off on the fold change, the second is based on a conservative cut-off on a p-value after multiple test correction .
the reason for using the different methods is: if  <dig> biological replicates were present than the second would be the ideal, however, here only different temperatures were used as replicates and as such, there were no real biological replicates.
therefore, the comparison between the methods might be more informative.
in the results genes that are predicted by  <dig> method and not contradicted by the others are used: this is equivalent to lowering the multiple test correction threshold in one method when results agree with the other.
total cold induced genes
<dig> cdna sequences were unambiguously up-regulated for all three methods, representing  <dig> genes and  <dig> of these were identified as angiogenesis-related genes .
summary of analyses
the combined results of this analysis are summarised in figure  <dig> and are as follows: from the  <dig>  contigs searched,  <dig>  were successfully assigned an ortholog, which amounted to  <dig>  non-redundant gene orthologs.
<dig> of the orthologs were predicted to be angiogenesis genes and  <dig> of the predicted orthologs were found to be induced by cold temperature.
finally, cross-referencing these genes found  <dig> cold induced angiogenic genes .
biologically relevant genes
the aim of this study was to develop a bioinformatic method of predicting genes of biological interest in a non-model species.
although many putative angiogenesis genes were found, some key angiogenic genes were not present on the carp array.
for instance, vascular endothelial growth factor  is a major activator of angiogenesis  <cit>  and it would have been interesting to see the effect of cold exposure on this gene.
nevertheless, table  <dig> displays  <dig> out of the  <dig> angiogenesis-related genes up-regulated by low temperature.
on investigation of these genes , there is encouraging evidence that these methods were successful.
from the  <dig> putative angiogenic genes up regulated due to cold temperatures,  <dig> genes considered biologically interesting in terms of angiogenesis are listed.
the four highlighted in bold are reviewed in the text.
mitogen-activated protein kinase  <dig>
mapk <dig> is part of the mapk-signalling pathway, which is the interface to a myriad of cellular processes like differentiation, proliferation and development  <cit> .
it is also activated by two upstream kinases raf and mek, which are themselves activated by growth factor receptors  <cit> .
upon activation, mapk <dig> is phosphorylated and translocates to the nucleus where it phosphorylates targets such as c-fos, c-jun, c-myc and tal- <dig> and initiates transcription of many genes  <cit> .
during angiogenesis mapk <dig> is activated in ecs and induces ec proliferation, critical to the establishment of new vessels  <cit> , abrogates apoptosis and promotes the transcription of vegf, itself a key activator of angiogenesis  <cit> .
it is possible this is done via the hypoxia-inducible factor  <dig> alpha  gene as mapk <dig> can activate hif1a and this transcription factor is known to mediate vegf expression  <cit> .
the involvement of hif1a in cold-induced angiogenesis fits well as reduced oxygen diffusion and increased blood viscosity can lead to tissue hypoxia.
interestingly, the mapk-pathway regulates expression of mmp <dig>  <cit>  which also was predicted as a cold-induced angiogenic gene in this study.
this finding is supported by the fact that inhibition of mek, an upstream activator of mapk <dig>  curtails the expression of mmp <dig>  <cit>  and that the mapk-pathway induces transcription of mmp <dig> via the upstream promoter site ap- <dig>  <cit> .
matrix metallopeptidase  <dig>
mmp <dig> is a member of a family of zinc containing endopeptidases  and plays a major role in angiogenesis  <cit> .
mmp <dig> is synthesised as an inactive zymogen  that is activated by proteolysis  <cit> .
during angiogenesis, mmp <dig> is secreted by endothelial cells to degrade the extracellular matrix, thus allowing ecs to migrate and form new blood vessels  <cit> .
coupled to this are the facts that mmp <dig> inhibition significantly decreased cerebral ec migration and proliferation  <cit>  and that a positive correlation with micro vessel density and mmp <dig> expression exists  <cit> .
mmp <dig> is also known to release vegf from extracellular matrix  stores  <cit> .
ras homolog gene family, member a
another cold-induced angiogenic gene that was predicted in this study was rhoa, which is also known to induce mmp <dig> expression  <cit> .
rhoa is a member of the rho-family gtpases which are part of the ras superfamily  <cit> .
it has been shown that dominant-negative rhoa in ecs impairs tube formation and sprouting in vitro  <cit> .
this is replicated in vivo where dominant-negative n19rhoa impairs vessel assembly and dominant-active v14rhoa stimulates ecs to form vessels  <cit> .
rhoa has also been shown to induce cytoskeleton re-organisation, enhance migration and increase angiogenic capacity  <cit> .
vegf increases rhoa activity by enhancing its recruitment to the membrane and mediating the rhoa/rho pathway during angiogenesis  <cit> .
over expression of dominant-active rhoa leads to increased tyrosine phosphorylation of vegfr <dig>  which is the key inducer of angiogenesis  <cit> .
again, related to hypoxia and low oxygen diffusion at low temperatures, rhoa is up-regulated in hypoxia and has been shown to be required for the accumulation of hif1a, which induces vegf expression  <cit> .
granulin
some of the cold-induced genes have not been widely studied as angiogenic factors but have nevertheless been implicated in the process.
these include granulin and nucleolin.
granulin is not a direct acting angiogenic factor but has been shown to stimulate vegf expression in breast carcinoma cells  <cit> .
granulin has also been shown by yeast two hybrid assays to bind the hiv tat protein that is actively secreted by hiv infected cells and acts as an ec growth and angiogenic factor  <cit> .
in addition, granulin has been shown to be expressed within proliferating ovarian carcinomas blood vessels and interact with perlecan.
therefore, it is thought to regulate tumour angiogenesis and influence cancer growth  <cit> .
finally, an anti-granulin antibody has been shown to inhibit tumour angiogenesis in human hepatomas implanted into athymic mice  <cit> .
nucleolin
nucleolin is one of the major proteins of the nucleolus but is also expressed on the cell surface where it binds a variety of ligands involved in several cell processes.
expression of nucleolin is particularly high on the surface of ecs in angiogenic blood vessels  <cit> .
inhibition of nucleolin in ecs prevents capillary tube formation and retards ec migration  <cit> .
it is of interest that mmp <dig>  also a predicted cold-responsive angiogenic gene in this study, is associated with nucleolin in angiogenesis.
mimicking hypoxia led to a 3-fold increase in mmp <dig> protein levels, due to enhanced translational efficiency caused by nucleolin binding to the 3' utr of mmp <dig>  <cit> .
these data showed that mmp <dig> expression during angiogenesis can be post-transcriptionally regulated by nucleolin.
lastly, antibodies to nucleolin have been shown to suppress tumour growth and angiogenesis  <cit> .
CONCLUSIONS
we provide evidence in this study that genes of biological interest in a non-model species, without a sequenced genome, can be discovered by combining re-analysis with a fast and efficient method of finding orthologs between fish and mammals.
with the advent of next generation sequencing we envisage this approach will be useful to researchers doing similar studies in other organisms or for other biological processes.
programs using the methods from this work can be downloaded from  and can be used to run these analyses locally.

 the cold-induced angiogenic response angiogenesis can be initiated by a variety of stimuli. for example, angiogenesis is induced by airway smooth muscle strain in chronic asthmatics, by exercise and in some species by cold exposure. many species are subjected to annual cycles of environmental cooling that represents a significant challenge for the cardiovascular system due to the effects of reduced oxygen diffusion and increased blood viscosity associated with low temperatures. one way of overcoming limitations to aerobic activity is to increase tissue capillary supply. although the underlying process is unclear, cold-induced angiogenesis may respond to altered hormonal levels or changes in the mechanical environment of endothelial cells. even within the mammalian literature, it is apparent that a number of types of capillary growth exist, e.g. pathological vs. physiological angiogenesis, albeit under the influence of a restricted set of genes. in order to improve the signal to noise ratio from the wealth of published data and reduce the influence of host tissue in the readout, gene expression profiles for endothelial-specific up-regulated genes have recently been identified. no such information exists for fish, although their use as an experimental model is increasing due to the additional interventions not possible with mammals. for the first time, we have used an in silico approach to identify whether orthologs between man and fish could be identified that were associated with angiogenesis, then used re-analysis of microarray data to see if these genes were differentially expressed on cold exposure and finally we determined what proportion of the up-regulated genes were angiogenic or ec-specific. we anticipate this approach could be useful for similar studies in non-model species for which a genome sequence is not available but the human orthologs involved in the biological process are known.  gene assignment by ortholog identification orthologs, as a whole, perform an equivalent function in respective genomes. based on this assumption, carp orthologs of known angiogenic human genes provide genes pivotal in their cold angiogenic response. cold carp data from a study by gracey et al. was to be used in this work to predict the identity of cold induced angiogenic genes. the genes assigned to carp sequences from the original article were not applicable to this study as they used a blastx against multiple species databases and only looked for the best hit without using a control step. in addition, the databases searched were from several years out of date, and a more stringent method of sequence contig construction was used in this work. a problem to be overcome was how best to assign human orthologs to anonymous est carp sequences collected from genbank and carpbase. many of these sequences were less than bases and many of them contain partial or complete untranslated regions. an obvious first step was to cluster them, using cap, to extend the sequence length and improve the quality of any subsequent analyses. this reduced the sequence count to a total of sequence contigs and singletons. the next challenge was to identify their human orthologs. choosing the ortholog assignment method homologs, in the strictest sense, are characters that have been passed down following a speciation event from a common ancestor. a gene in two species derived from a single gene in their common ancestor is defined as an ortholog and usually has the same or similar function between species, i.e. the same gene in different organisms are orthologs. conversely, paralogs are defined as genes derived from a single gene sequence duplication event and usually have different functions. orthologs and paralogs are not the only gene histories and several other events can take place, see for a full list. in this work, the sole aim was to find the most likely functional human homologs of non-model transcripts and did not attempt to define or classify paralogs or any other forms of gene histories. many bioinformatic programs and pre-computed databases of orthologs exist, a number of which are listed in a recent review. essentially, there are methods of finding orthologs, tree based, graph based and a combination of the two. tree methods use sequence alignments and phylogenetic trees for predicting evolutionary relationships, while graph based methods use pairwise sequence similarity search methods to predict orthologs. the review highlights the merits of each program or database and it provides the reader with a flow chart to assist in the best choice of tool for a particular application. after investigation of the recommended programs it was decided that none were applicable to the high-throughput analysis needed to cope with the > k incomplete anonymous carp nucleotide sequences. the main reasons were the inability of these programs to be queried with a high volume of data through their web servers or the databases were several years out of date. further literature searches revealed a tool called blast on orthologous groups which searches a query sequence against a database of orthologous groups as a single unit. although this tool enabled searches against several combination method pre-computed databases including homologene, it could only deal with one sequence at a time. additionally, the blasto form did not permit the user to change blast filtering options which, as seen below, makes a difference to the quality and quantity of successful ortholog predictions. ortholog assignments, rbh and filtering options a graph based, nearest neighbour approach, rbh, was considered the best option since constructing multiple sequence alignments and phylogenetic trees from incomplete nucleotide sequences would be inaccurate and not amenable to high throughput. strengthening this decision, a performance assessment of different ortholog prediction methods by altenhoff and dessimoz found the rbh performed well in comparison with the other methods. reciprocal best hit is a technique using blast to first search a query sequence against a genome. if the resulting best hit matches the original query sequence from a reciprocal blast then it is an ortholog termed rbh. a diagrammatical representation of the rbh process of carp vs. human is shown in figure labelled route an article by moreno-hagelsieb and latimer tested ortholog rbh searching using different blast options. the tests utilised bacterial genomes, neighbouring orthologs and paralogs to estimate error rates of ortholog assignments by rbh. they found the soft filtering option -f "m s" with the addition of smith-waterman alignments were optimal for rbh success. these options were tested for eukaryotes using carp vs. human rbh searches. four different blast options were tested: soft filtering with and without sw and hard filtering with and without sw. the results revealed very little difference in the number of successful rbh with or without the sw algorithm against their findings of 10%. in contrast, as with prokaryotes, soft filtering increased the number of rbhs by 2%. table shows the number of successful orthologs found using carp vs. human rbh for the different filtering options. figure shows a bar chart of the successful rbh results normalised to the default blast options. this table shows the performance of rbh using the different blast filtering options with eukaryotic sequences. the results from this analysis showed the soft filtering option with no smith and waterman produced the highest number of successful rbhs. contrary to the findings of moreno-hagelsieb and latimer, who showed the sw algorithm improved results by 10%, in this study using sw produced % less successful results. however the analyses in this work were slightly different to that of moreno-hagelsieb and latimer and are listed here: 1) they used complete bacterial genomes: a) these sequences are generated from genomic dna and are sequenced in both strands. b) the carp sequences are generated from single pass cdna sequencing from rna and, as such, are likely to contain more errors. 2) the protein sequences used in assessing rbh success: a) moreno-hagelsieb and latimer used full length and defined proteins from complete bacterial genomes. b) the carp sequences were partial proteins from frame nucleotide translations and from a eukaryotic and not a prokaryotic species. 3) the algorithm options used with blast were not exactly the same. a) moreno-hagelsieb and latimer used the blastpgp algorithm from the ncbi blast, which has the smith-waterman -s t option available. b) in this work blastall using blastx and tblastn were used. the -s t option is only available for tblastn and not blastx with ncbi blastall. note: blastpgp can only be used on protein sequences and not nucleotides. 4) some cdnas span genes on different chromosome strands and is another way rbh results could be affected. however, even with these differences, the results were very surprising because of other research and considering the -s f option is a fast approximation of the sw algorithm. one thing to bear in mind is an e-value of 1e- and soft filtering were employed in this work, which in itself, would remove many spurious matches otherwise found using the default blast heuristic. a further investigation into this revealed that the genes successfully found with each method did not totally overlap. the heuristic blast alignment method predicted functional homologs with rbh that sw did not find and the sw alignment method predicted that failed with the blast heuristic. the intersection of results totally concurred. the questions to answer here were; why did one algorithm fail rbh, whilst the other method was successful? and of these differences, which algorithm produced the more believably evolutionary related rbh successes? to investigate this finding further and to decide on which method to use in this work, a manual inspection of sequence alignments was needed in a systematic way to avoid the problem of biased cherry picking. this was done using a random number generator to pick sequences to investigate, from each algorithm rbh success. so the investigation was to see if the results were believable for each algorithm. the results found that ~50% of results suggested the default blast option found the more likely evolutionary related sequence as it found higher similar matches but at shorter alignment lengths, which is believable as sw is good at finding distant homology. however, this was not always the case and the different sequence alignment algorithms were correct for different sequences. as a consequence it was not possible to automate the choice without manually viewing the results. therefore, in this work the results generated using the default blast heuristic was used for the final data. however, it is recommended for other researchers using these methods on small datasets to use the intersection of both methods or the sw alone. this is not a problem for small numbers of sequences but for next generation sequence data, for example, it is recommended the default heuristic be used as it will take weeks to compute sw alignments. all but one cold induced angiogenic gene were found with both algorithms and users of this data can be confident in the ortholog assignments of these genes. the exception was the plasminogen gene plg. taking advantage of a closely related vertebrate zebrafish are a model teleost organism used in the elucidation of vertebrate development, molecular, genetic, genomic and evolutionary biology. as such its genome has been sequenced with extensive annotation and currently in the sequence databases there are over million ests and thousand refseq proteins. carp, on the other hand, currently has ~ thousand ests and no genome sequence. it was possible to take advantage of this annotation because of the close evolutionary relationship carp has to zebrafish in that they diverged from their last common ancestor only million years ago. previous groups have shown that close genome evolutionary relationships can aid ortholog identification. in the present study, zebrafish was also utilised as a stepping stone approach to ortholog identification. figure labelled route shows a stepped reciprocal best hit approach where rbh analysis is carried out twice, once between carp and zebrafish and then between zebrafish and human. if both were successful, then a carp-human ortholog was assigned. using the srbh analysis gained a further 8% successfully assigned orthologs. teleost genome duplication and srbh although there is a discrepancy as to when it happened, there is much evidence for a genome duplication event in teleosts. a good example in zebrafish is the paralogs ets and etsrp which both lie next to fli1a and fli1b, respectively, but on different chromosomes. these genes are important for vasculogenesis and angiogenesis. from a srbh analysis, zebrafish genes ets and etsrp best match the human gene ets and fli1a, and fli1b best matches the human fli suggesting a genome duplication event occurred in zebrafish. because of duplicate genes, the best and second best hits to zebrafish and carp genes were used to attain a successful srbh. the impact of genome duplication in ortholog searching can be seen in the following example. the zebrafish peptide np_ was the best hit to ca however, the peptide np_ best matched human np_ blasting np_ back against zebrafish revealed two very similar sequences, np_ and np_ np_ was the best hit and therefore failed srbh. this is due to genome duplication of the zebrafish genome and thus both genes in zebrafish can be considered orthologs to the same human gene. rbh and a comparison with homologene homologene is a database of orthologous groups defined with blast pairwise alignments and phylogenetic trees. since csrbh included a zebrafish and human ortholog rbh analysis, a comparison was made between homologene and this rbh step to see if the zebrafish to human ortholog assignments from this work agreed. from the successful rbhs, only could be compared as only these had both human and zebrafish genes present in the homologene data. this fact alone gives support to using this approach as a user supplies all genes, and as such, all are present. from these ortholog assignments, 85% agreed with homologene orthologous groups. a manual investigation of some of those that disagreed suggests homologene does not always perform well for a proportion of duplicated genes. for instance, one example, the zebrafish actc1l gene best matched the human actc gene with 99% identity over the full alignment length of the gene but homologene put actc1l in the actb orthologous group where alignment percent identity was less at 94%. the full results of the comparison can be viewed in additional file in addition, ten alignments were chosen randomly and manually investigated to see which source of data was most realistic. the results of this suggested out of ortholog assignments were correct with rbh whilst homologene put the genes into different orthologous groups. a full comparison of rbh with homologene a full analysis was also performed that took all human proteins in the refseq database of proteins and rbh searched against the full database of zebrafish refseq proteins. on july 22nd there were proteins in the human database. of these proteins resulted in a successful rbh analysis against zebrafish. build of homologene contained human proteins. therefore 33% of the human proteome was not represented in homologene at this time and only of the human proteins had a zebrafish homolog protein in the same orthologous group. there were human/zebrafish orthologs identified by homologene only and not with the rbh procedure. in contrast, there were human/zebrafish orthologs found using the rbh but not with in homologene. human proteins were found in both homologene and in the rbh analysis. of these agreed, an 89% agreement. this is very similar to the result above with only the carp data. these findings, particularly the fact that homologene only had human proteins with a zebrafish ortholog and the findings of altenhoff, give a user some further support and confidence to using a rbh ortholog assignment approach rather than phylogenetic tree methods for analysing large amounts of data from a non-model species in a quick and fairly accurate manner. the srbh approach is endorsed by the fact other researchers have used closely related species to predict genes and sequence motifs. however, it should be noted that the comparisons done here with homologene only validates the rbh method but not the csrbh as, to definitively validate the csrbh approach, the carp genome and annotation need to be more complete. in addition, the csrbh approach does not attempt to better or replace the efforts of databases such as homologene. one reason is that it does not attempt to deal with paralogs and other sources of gene histories but only with the most functional homologs, though some attention was given to the teleost genome duplication event. so there are examples where homologene better assigns orthologs than the methods used here. having said that, with a non-model species not present in homologene, this approach is excellent at giving a good approximation of the human functional homologs of researchers' genes. csrbh, combining rbh and srbh to find further orthologs although adding the zebrafish intermediary step improved ortholog assignment by 8%, assignments that failed using this procedure were successfully found using a carp vs. human rbh alone. inspection of a random sample of these found two examples of why this occurred. blast of carp est ec vs. zebrafish best hit the gene smyhc and the reciprocal blast vs. carp found est ab as the best hit. however, smyhc was the fifth best hit to ab thus failing the srbh analysis. however, this gene is successfully assigned a human ortholog from an rbh analysis. even more compelling was the situation with est au. it aligns with the human protein np_ as the best hit and the alignment was highly similar with a percent identity of 91% over amino acids. the same est best aligned to the zebrafish protein np_ with 37% identity over amino acids. hence, the est is significantly more similar to the human protein rather than the zebrafish. the human protein np_ was blast searched against zebrafish to see if the full-length protein found a likely ortholog in zebrafish. the best hit to the human protein was np_ the same as found by searching est au therefore, there is evidence that either some genes do not exist in zebrafish and do so in carp or are not currently found in zebrafish as the genome is not yet completely sequenced or annotated. another piece of evidence is, if a simple blast search is performed against the multiple species nr database of proteins at the ncbi, 24% of carp sequences preferentially match a human protein, whilst 30% match a zebrafish protein. this supports the use of a conditional stepped reciprocal best hit approach, which uses whichever species is a closer match to the query and performs the appropriate ortholog prediction route. in addition, for any that fail, a rescue was be performed by searching through the other route. csrbh outperformed all other methods and produced 3% and 11% more successful ortholog assignments than the srbh and rbh, respectively. in total contigs were assigned an ortholog, totalling non-redundant genes. see table and figure for the number of successful ortholog assignments for all the different analysis methods used in this work. we propose that csrbh should be the method of choice for researchers working with non-model species which have no sequenced genome and partially sequenced genes. this table displays the number of human protein orthologs found using csrbh for the cdna carp contiguous sequences. total none redundant genes were found and of them were predicted as angiogenesis genes. from the re-analysis, genes were up-regulated and 24% of them were predicted as angiogenesis genes. a weakness with this approach is that it does not determine an orthologs absolute gene history and it will not report to a user whether the ortholog found was derived from a gene duplication event or from a speciation. that said, these methods will enable a user to quickly find the most likely functional homolog of a gene. prediction of angiogenic genes established angiogenic genes previous groups have developed complex data mining methods using decision trees to identify genes of interest. in this study, simple strategies were combined to generate as a complete a set of angiogenic genes as possible. first the cardiovascular and angiogenesis groups at birmingham university used their knowledge to produce a list of angiogenic genes that included those from the recent literature and the sabiosciences gearray® human angiogenesis microarray. a total of angiogenesis related genes were predicted this way and a list of genes were contained in the successful csrbh results. however, it is possible many other genes have an undiscovered role in angiogenesis and, therefore, three further methods were employed to predict additional angiogenic genes. putative angiogenic genes the second and third approaches used perl programs to scan article abstracts from pubmed and gene ontology processes from amigo using the following angiogenic related keywords as baits:'angiogenic, angiogenesis, neovascularization, neovascularisation, vasculogenesis, hypoxia, endoth vegf'. this predicted a further angiogenic genes. a fourth method used gene expression of ecs. during the activation of angiogenesis, ecs become activated and switch on several signalling pathways that cause proliferation, migration and invasion. this angiogenic phenotype leads to an up-regulation of ec genes. therefore, genes up-regulated in the endothelial transcriptome could be angiogenic. the endothelium transcriptome has been extensively defined and genes up-regulated in ecs were compared with the human/carp orthologs to identify additional putative angiogenic genes; were found. total angiogenic genes combining the results from the four data mining methods gave a total of non-redundant predicted angiogenic genes. we postulate this gave us a comprehensive set and the best chance of discovering cold-induced angiogenic genes from the carp data. however, it should be noted, and the authors fully acknowledge, that not every gene predicted here is a genuine angiogenesis gene and there will be some false positives. very few bioinformatic predictions are 100% foolproof and this study is aimed at guiding the bench scientist into making more informed decisions before going into the laboratory. the number of different methods that predicted a gene angiogenic now ranks the genes. for example, the gene hif1a is a well-known angiogenic gene and was predicted as such by all four methods. column i entitled "number of methods" in additional file gives the number of methods predicting a gene angiogenic and is ranked in a descending order. conversely, genes that are not so well known to be angiogenic genes are only found by one or two methods. if this approach is employed and genes selected that were predicted by more than one method, reduces the high percentage of angiogenic genes from 25% down to this is reduced further if you only take genes with and/or methods. but given the full list, a researcher can decide based on the presence of functional domains or personal knowledge which ones are of interest to investigate with full knowledge that the human functional homolog was only loosely associated with the biological process of interest. re-analysis of gene expression data three established methods of measuring gene expression used in biological research are microarrays, cdna and serial analysis of gene expression libraries. to investigate numerous pathologies many institutes around the world have generated, analysed and deposited large amounts of expression data into public repositories. examples of such databases are the gene expression omnibus, cancer genome anatomy project, national center for biotechnology information and european bioinformatics institute arrayexpress. it has been shown that data mining of relevant data sets can successfully lead to the identification of biologically interesting targets. cold response differentially expressed genes carp is an ectotherm with a body temperature similar to the water it is immersed in and can adapt to a range of temperatures. a recent study investigated physiological adaptation of carp exposed to gradually lower temperatures. microarray analysis measured the change in gene expression, in several tissues, at several cooling stages from to 10°c vs. a control temperature of 30°c. overall, transcriptional regulatory, rna splicing and translation control genes were found to be up-regulated in all tissues. interestingly, some tissue-specific affects were also seen, e.g. in the glycolytic pathway of brain and lipid metabolism of liver. this data set proved appropriate for re-analysis to predict cold-induced angiogenic genes in skeletal and heart muscle tissues. persistently up-regulated genes two strategies were applied to the data. the first utilised the data without intervention for heart and muscle tissues, employing a threshold score > = the total number of up-regulated cdnas for heart was non-redundant genes, and for muscle cdnas, genes. any cdnas with significant contradicting expression were removed. for both heart and muscle combined there were non-redundant genes, of which were predicted angiogenic genes. this table conveys the numerical breakdown of results for the differential cdna expression due to cold temperatures on the carp array. were the total number of putative cold induced angiogenic genes. initial cooling, fold ratio subtraction and microarray re-analysis it is thought angiogenesis is switched on in the initial stages of cooling, before the animal has become acclimatised to a particular temperature. this has been shown in rats subjected to cooling to 4°c which led to a fold increase in vegf expression for to hours and returned to the basal level at hours. therefore, the microarray data was analysed between the first and last time point at each target temperature. first, ratios were calculated between each of the cooling time points vs. a control group. analysing heart and muscle separately and combining the results gave unambiguous cdnas with a ratio subtraction > = and this represented non-redundant genes. of these genes were predicted angiogenic, see table and additional file column e for both heart and muscle sheets. to further discover genes switched on at initial cooling, microarray analysis using limma from the bioconductor r package was used to fit a linear model to the arrays. an adjusted p-value < = was employed as a significance threshold. and unambiguous cdnas were found to be up-regulated in heart and skeletal muscle respectively. combined, this represented non-redundant genes, of which were predicted to be angiogenic. differential gene expression on initial cooling: overlap with ratio subtraction and limma analyses both these methods are seeking to find genes up-regulated at the first time point on immediate cooling of fish vs. fish that have acclimatised to the temperature. the results should corroborate each other. dealing the heart tissue first, there were cdnas that reached a p-value < = and cdnas that had a ratio subtraction > =. these cdnas did not overlap but the ratio subtraction results, though not significant, for the limma cdnas were positive. so they concurred but not with significance for the ratio subtraction. muscle, in contrast, gave better corroboration between ratio and limma analyses. there were cdnas with limma adjusted p-value < =. there were total ratio subtractions that reached significance. of these, agreed with significant limma results. the results were encouraging. in general, the number of genes predicted by a method are always a balance between false positives and false negatives. in this case cdnas are in common in total between the "ratio" and the "limma" methods for muscle. however, differences between the two methods are expected as the first is based on a hard cut-off on the fold change, the second is based on a conservative cut-off on a p-value after multiple test correction. the reason for using the different methods is: if biological replicates were present than the second would be the ideal, however, here only different temperatures were used as replicates and as such, there were no real biological replicates. therefore, the comparison between the methods might be more informative. in the results genes that are predicted by method and not contradicted by the others are used: this is equivalent to lowering the multiple test correction threshold in one method when results agree with the other. total cold induced genes cdna sequences were unambiguously up-regulated for all three methods, representing genes and of these were identified as angiogenesis-related genes. summary of analyses the combined results of this analysis are summarised in figure and are as follows: from the contigs searched, were successfully assigned an ortholog, which amounted to non-redundant gene orthologs. of the orthologs were predicted to be angiogenesis genes and of the predicted orthologs were found to be induced by cold temperature. finally, cross-referencing these genes found cold induced angiogenic genes. biologically relevant genes the aim of this study was to develop a bioinformatic method of predicting genes of biological interest in a non-model species. although many putative angiogenesis genes were found, some key angiogenic genes were not present on the carp array. for instance, vascular endothelial growth factor is a major activator of angiogenesis and it would have been interesting to see the effect of cold exposure on this gene. nevertheless, table displays out of the angiogenesis-related genes up-regulated by low temperature. on investigation of these genes, there is encouraging evidence that these methods were successful. from the putative angiogenic genes up regulated due to cold temperatures, genes considered biologically interesting in terms of angiogenesis are listed. the four highlighted in bold are reviewed in the text. mitogen-activated protein kinase mapk is part of the mapk-signalling pathway, which is the interface to a myriad of cellular processes like differentiation, proliferation and development. it is also activated by two upstream kinases raf and mek, which are themselves activated by growth factor receptors. upon activation, mapk is phosphorylated and translocates to the nucleus where it phosphorylates targets such as c-fos, c-jun, c-myc and tal- and initiates transcription of many genes. during angiogenesis mapk is activated in ecs and induces ec proliferation, critical to the establishment of new vessels, abrogates apoptosis and promotes the transcription of vegf, itself a key activator of angiogenesis. it is possible this is done via the hypoxia-inducible factor alpha gene as mapk can activate hif1a and this transcription factor is known to mediate vegf expression. the involvement of hif1a in cold-induced angiogenesis fits well as reduced oxygen diffusion and increased blood viscosity can lead to tissue hypoxia. interestingly, the mapk-pathway regulates expression of mmp which also was predicted as a cold-induced angiogenic gene in this study. this finding is supported by the fact that inhibition of mek, an upstream activator of mapk curtails the expression of mmp and that the mapk-pathway induces transcription of mmp via the upstream promoter site ap-. matrix metallopeptidase mmp is a member of a family of zinc containing endopeptidases and plays a major role in angiogenesis. mmp is synthesised as an inactive zymogen that is activated by proteolysis. during angiogenesis, mmp is secreted by endothelial cells to degrade the extracellular matrix, thus allowing ecs to migrate and form new blood vessels. coupled to this are the facts that mmp inhibition significantly decreased cerebral ec migration and proliferation and that a positive correlation with micro vessel density and mmp expression exists. mmp is also known to release vegf from extracellular matrix stores. ras homolog gene family, member a another cold-induced angiogenic gene that was predicted in this study was rhoa, which is also known to induce mmp expression. rhoa is a member of the rho-family gtpases which are part of the ras superfamily. it has been shown that dominant-negative rhoa in ecs impairs tube formation and sprouting in vitro. this is replicated in vivo where dominant-negative n19rhoa impairs vessel assembly and dominant-active v14rhoa stimulates ecs to form vessels. rhoa has also been shown to induce cytoskeleton re-organisation, enhance migration and increase angiogenic capacity. vegf increases rhoa activity by enhancing its recruitment to the membrane and mediating the rhoa/rho pathway during angiogenesis. over expression of dominant-active rhoa leads to increased tyrosine phosphorylation of vegfr which is the key inducer of angiogenesis. again, related to hypoxia and low oxygen diffusion at low temperatures, rhoa is up-regulated in hypoxia and has been shown to be required for the accumulation of hif1a, which induces vegf expression. granulin some of the cold-induced genes have not been widely studied as angiogenic factors but have nevertheless been implicated in the process. these include granulin and nucleolin. granulin is not a direct acting angiogenic factor but has been shown to stimulate vegf expression in breast carcinoma cells. granulin has also been shown by yeast two hybrid assays to bind the hiv tat protein that is actively secreted by hiv infected cells and acts as an ec growth and angiogenic factor. in addition, granulin has been shown to be expressed within proliferating ovarian carcinomas blood vessels and interact with perlecan. therefore, it is thought to regulate tumour angiogenesis and influence cancer growth. finally, an anti-granulin antibody has been shown to inhibit tumour angiogenesis in human hepatomas implanted into athymic mice. nucleolin nucleolin is one of the major proteins of the nucleolus but is also expressed on the cell surface where it binds a variety of ligands involved in several cell processes. expression of nucleolin is particularly high on the surface of ecs in angiogenic blood vessels. inhibition of nucleolin in ecs prevents capillary tube formation and retards ec migration. it is of interest that mmp also a predicted cold-responsive angiogenic gene in this study, is associated with nucleolin in angiogenesis. mimicking hypoxia led to a 3-fold increase in mmp protein levels, due to enhanced translational efficiency caused by nucleolin binding to the 3' utr of mmp. these data showed that mmp expression during angiogenesis can be post-transcriptionally regulated by nucleolin. lastly, antibodies to nucleolin have been shown to suppress tumour growth and angiogenesis.  we provide evidence in this study that genes of biological interest in a non-model species, without a sequenced genome, can be discovered by combining re-analysis with a fast and efficient method of finding orthologs between fish and mammals. with the advent of next generation sequencing we envisage this approach will be useful to researchers doing similar studies in other organisms or for other biological processes. programs using the methods from this work can be downloaded from and can be used to run these analyses locally. 
the csrbh approach outperformed all other methods and attained carp to human functional homolog relationships for contiguous sequences. finally, re-analyses of cold carp microarray data identified carp genes up-regulated in response to low temperatures in heart and muscle. we show that csrbh functional homologs relationships and re-analyses of gene expression data can be combined in a non-model species to predict genes of biological interest before a genome sequence is fully available. for example, some species increase their vascular network to minimise the effects of reduced oxygen diffusion and increased blood viscosity associated with low temperatures. the total number of up-regulated differentially expressed genes was and of them were putatively angiogenic. in this work, carp sequences were collected and built into contigs.
 physiological processes occur in many species for which there is yet no sequenced genome and for which we would like to identify the genetic basis. for example, some species increase their vascular network to minimise the effects of reduced oxygen diffusion and increased blood viscosity associated with low temperatures. since many angiogenic and endothelial genes have been discovered in man, functional homolog relationships between carp, zebrafish and human were used to predict the genetic basis of cold-induced angiogenesis in cyprinus carpio. in this work, carp sequences were collected and built into contigs. human-carp functional homolog relationships were derived via zebrafish using a new conditional stepped reciprocal best hit protocol. data sources including publications, gene ontology and cdna libraries were then used to predict the identity of known or potential angiogenic genes. finally, re-analyses of cold carp microarray data identified carp genes up-regulated in response to low temperatures in heart and muscle.  the csrbh approach outperformed all other methods and attained carp to human functional homolog relationships for contiguous sequences. this represented non-redundant genes and of them were predicted to have a role in angiogenesis. the total number of up-regulated differentially expressed genes was and of them were putatively angiogenic. of these, genes representing the functional homologs ncl, rhoa, mmp grn and mapk are angiogenesis-related genes expressed in response to low temperature.  we show that csrbh functional homologs relationships and re-analyses of gene expression data can be combined in a non-model species to predict genes of biological interest before a genome sequence is fully available. programs to run these analyses locally are available from. 