Analysis of siRNAs disrupting the cell cycle In normal exponential growth, cells are transitioning from interphase to mitosis and back to interphase at con stant rates. We focused on four types of disruptions selleckchem Enzastaurin of the basal cell cycle shown in Figure 2, quiescence, when cells stop dividing, mitotic arrest, when cells stop going back in interphase, polynucleation, when cells start becoming polynucleated and cell death, when cells start to die. Each of these events was associated with a corresponding transition penetrance and inflection time. Transition penetrances proved to be reliable indicators of disruptions of the cell cycle. As an example, in cell death, cells growing in the control spots siCOPB1 had a significantly higher mean cell death penetrance than cells seeded in the negative control spots.

This is in agreement with the essential role of COPB1 in binding Golgi vesicles. Similarly, cells sub ject to siKIF11 had a significant higher mean mitotic arrest penetrance than negative control spots and a high mean cell death penetrance, consis tent with cell death that follows prometaphase arrest induced by the treatment. Based on these observations, we defined thresholds on each of the four transition pen etrances to detect the siRNAs that disrupt the cell cycle. Transition inflection points quantified the times of disruption of the cell cycle. For each siRNA, we sum marised the four times obtained from the replicate spots by average and standard deviation.

We identified genes with reproducible cell cycle disruption times by requiring standard deviation of less than 4 h and average of less than 50 h after seeding time, the latter criterion was motivated by the generally lower confidence of the inflection time estimates at later times. Using these criteria, we found 168 siRNAs leading to quiescence at reproducible times, 289 inducing mitotic arrest, 390 leading to polynucleation and 171 causing cell death Additional file 1, Table S1 The targets of the siRNAs inducing cell death included the protein units of the Golgi vesicular coat COPA and COPB2, several known apoptosis regulators such as TP53AIP1 and the RAS family members RAB25 and RAN. Interestingly, three siRNAs targeting COPA and COPB2 induced cell death at similar time points, together with siCOPB1. The similarity of these timings is consistent with the fact that the proteins are part of the same protein complex.

On the contrary, siRNAs directed at the RNA helicase DDX39A induced an early cell death at 22. 8 h, which could reflect a different cell death mechanism from the one caused by COPA and COPB2 inhibition. We also Batimastat identified several siRNAs inducing cell death and target ing uncharacterised genes such as C3orf26, C3orf52 or C16orf90. However, due to the existence of off target effects in RNA interference, functional res cue of the phenotypes and secondary functional assays would be needed to confirm the essential role of these genes.

A blockade of lysosomal degra dation with NH4Cl resulted in increased levels of both LC3I and LC3II to levels that were similar between mitotic and interphase cells. From this we concluded in a previous report that basal levels of autophagy and mitophagy are robust in both interphase and mitotic cells and most autophagosomes that form during the entire cell cycle are efficiently selleck chemicals EPZ-5676 degraded through the lysosomal pathway. Treatment with nocodazole and paclitaxel caused dif ferent responses between interphase and mitotic cells. Treatment with either paclitaxel or nocodazole in inter phase cells caused a slight increase in LC3I levels and no change in LC3II levels in the absence of NH4Cl. However, there was no change in both LC3I and LC3II levels in the presence of NH4Cl.

The treatments resulted in a 3 fold increase of LC3I levels, but no change of LC3II levels in mitotic cells. Accumulation of LC3I suggested either an increased synthesis of LC3I through mechanisms related to tran scription, post transcription or translation of LC3 pre cursor and conversion to LC3I or reduced conversion of LC3I to LC3II. The levels of LC3I in interphase cells were slightly increased while the levels of LC3I in mitosis were dra matically enhanced upon paclitaxel or nocodazole treat ment. Although we cannot completely exclude the possibilities that more LC3 mRNA molecules were transcribed or more LC3 I proteins were translated and processed, we believed that such possibilities were unlikely since both mRNA transcription and protein translation are gener ally suppressed in mitosis.

The reduction in total LC3II in either the paclitaxel or the nocodazole arrested mito tic cells relative to control mitotic cells in the presence of NH4Cl further suggested a reduction in net conversion of LC3I to LC3II. Even though punctate foci appeared in more than 16% of paclitaxel treated mitotic cells, no difference in LC3II levels was evident. Thus, the paclitaxel induced GFP LC3 punctate foci are likely made up of aggregates of primarily LC3I. Others using ATG5 deficient cells have also suggested that that punc tate foci containing LC3 do not always represent mature autophagic structures. The ATG5 gene controls the conversion of LC3I to LC3II and its deletion causes accumulation of LC3I. Thus localized accumulation of LC3I on mitochondrial aggregates appears as punctate foci that are less than mature autophagosomes.

We sug gest that the generation of those punctate foci reflect autophagic failure at the initiation stage rather than autophagy independent aggregation. In sum mary, although both paclitaxel and nocodazole impaired the conversion of LC3I to LC3II resulting in accumula tion of LC3I, only in mitotic cells did paclitaxel cause the accumulation of Cilengitide LC3I in aggregates.

APC C components and main targets can be used to infer the phylogeny of eukaryotes Proteins inferred to have been present in LECA are valuable material to reconstruct the characteristics of this ancestral organism. In addition, they can preserve a phylogenetic signal selleck MEK162 useful to infer the evolutionary history of eukaryotes. Until now, most analyses dedicated to the reconstruction of the eukaryo tic phylogeny were based on the analysis of components of informational systems. This was so because most of the genes coding for these pro teins present the advantage of being part of mono genic gene families allowing the easy identification of orthologous proteins, slowly evolving, ancient and well conserved among life domains, and rarely exchanged by horizontal gene transfers.

Accordingly, they represent first choice material to investigate ancient evolution in all domains of life. Phylogenetic studies of the eukaryotic domain did not escape this rule and most of them have been largely based on the phylogenetic analysis of informational proteins. By contrast, most proteins involved in housekeeping functions are con sidered to evolve faster than those of informational sys tems, and thus to be less suitable to study ancient evolution. Moreover, they are often part of large and complex protein families that have experienced numer ous gene duplication and loss events during their evolu tionary history, meaning that distinguishing between orthologues and paralogues is difficult and requires fas tidious preliminary analyses.

Consequently, although these proteins are more numerous than informational ones and often of larger size, they have rarely been used to infer the ancient evolution of eukaryotes. Neverthe less, typical datasets based on informational proteins have been shown to be insufficient to robustly infer all the deep nodes of the global eukaryotic phylogenies. Increasing the protein sampling is therefore becoming as necessary as increasing the taxonomic sam pling in order to fully resolve the phylogeny of eukar yotes, meaning that new useful protein markers have to be found among the conserved operational proteins. Our phylogenetic analyses of the APC C subunits and main targets showed that with a few exceptions they are ancient proteins well conserved throughout the diversity of present day eukaryotic lineages. Accordingly, Entinostat they are potential suitable markers to reconstruct global eukaryo tic phylogenies. The maximum likelihood and Bayesian phylogenetic analyses of individual components showed that they have retained ancient phylogenetic signal despite the fact that some basal nodes of the inferred phylogenies showed a poor resolution.

Human arthritic cartilage and e perimental osteoarthritis Human OA cartilage was sourced from individuals under going arthroplasty. selleckchem FTY720 Human cartilage was kindly pro vided by Dr Churl Hong Chun of Wonkwang University. The Institutional Review Board of the Wonkwang University Hospital approved the use of these materials, and all individuals provided written informed consent to be donors before undergoing surgery. Spontaneous OA in STR ort mice was e amined at 28 weeks of age, with CBA CaCrl mice used as controls. Aging studies were performed in 12 month old mice, and e perimental OA was induced in mice by destabilization of the medial meniscus surgery or by intra articular injection of collagenase in 8 week old male mice and in in Lrp5 mice and their wild type lit termates.

Sham operated and phosphate buffered saline injected mice were used as controls for the DMM and collagenase injected models, respectively. Mice were ana lyzed at 8 weeks after DMM surgery or 4 weeks after col lagenase injection. Micromass culture and primary culture of articular chondrocytes Mesenchymal cells were derived from the limb buds of ICR mouse embryos 11. 5 days postcoitus and main tained as micromass cultures for induction of chondro genesis as described previously. Mouse articular chondrocytes were isolated from knee cartilage obtained from postnatal day 5 mice. The articular cartilage was preincubated for 2 hours at 37 C with 0. 2% trypsin and 0. 2% type II collagenase and further digested with 0. 2% type II collagenase for 90 minutes. On culture day 3, the cells were treated with recombinant interleukin 1B, Wnt3a or Wnt7a for 24 hours.

Apoptosis was induced by treatment with an anti Fas antibody. Briefly, chondrocytes from articular cartilage of WT or Lrp5 mice were incubated in the presence or absence of IL 1B for 24 hours, then e posed to the anti Fas antibody and recombinant protein G for an additional 6 hours. Hamster immunoglobulin G2 was used as a control. The cells were stained with fluorescein isothiocyanate conjugated anne in V, and apoptotic chondrocytes were quantified by fluo rescence activated cell sorting analysis. Immunofluorescence microscopy and immunohistochemistry Chondrocytes were cultured on glass coverslips, fi ed with 3. 5% paraformaldehyde and permeabilized with 0. 1% Triton 100.

The cells were incubated for 1 hour with an antibody against type II collagen followed by incubation for 1 hour with an Ale a 488 conjugated secondary anti body. Ectopic e pression of LRP5 was determined Dacomitinib by labeling with an anti LRP5 antibody and an Ale a 555 conjugated secondary anti body. Apoptosis of chondrocytes in cartilage tissue was determined by terminal deo ynucleotidyl transferase deo yuridine triphosphate nick end labeling staining using a kit purchased from Roche Diagnostics. Specimens were visualized under an I 81 inverted fluorescence micro scope driven by MetaMorph imaging software.

Transfected cells were seeded in 6 cm diameter dishes at 5 105 cells Navitoclax Phase 2 dish, and transfected with either pEGFP SIRT1 or empty vector using Lipofectamine 2000, according to the manufacturer s protocol. Transfected cells were further e amined in cell proliferation assays. OECM1 cells were transiently transfected with small interfering RNA against SIRT1, or with a nontargeting control in Opti MEM I reduced serum medium containing Lipofectamine RNAiMA . Transfection efficiency was assessed by western blot. RNA isolation and quantitative real time PCR For gene e pression analysis, pairs of tumor and normal marginal tissues were obtained from 21 OSCCs. The tis sues were frozen and stored in liquid nitrogen at ?196 C until use. Total RNA obtained from cultured cells and human tissue was e tracted using TRIzol reagent.

cDNA was then reverse transcribed and ampli fied by PCR using a Transcriptor First Strand cDNA Synthesis kit. Quantitative RT PCR was jperformed using the FastStart Universal SYBR Green Master mi and an Applied Biosystems ABI 7900 RealTime PCR System. The oligonucleotide primers used for human SIRT1, Smad4, MMP7, and glyceraldehyde 3 phosphate dehydrogenase Gene e pression levels were normalized using GAPDH as an internal reference gene, and the average relative change was calculated from triplicate or quintuplicate determinations made by relative quantification, and applying the delta delta cycle threshold method. The protocol for this study was approved by the Institutional Review Board of the Department of Oral and Ma illofacial Surgery of Chi Mei Medical, Liouying, Taiwan.

Cell chemotactic migration and invasion assay The chemotactic migration of cells was evaluated using a 24 well chemota is chamber equipped with 8 um pore size membranes. Cell invasion ability was assessed using Falcon Cell Culture Inserts with Matrigel. Samples containing 1 105 cells were resuspended in serum free medium with 0. 1% BSA, and then plated onto the transwell chamber. The chambers were incubated for 24 h with complete culture medium added in the lower chamber. Non mobile cells were removed, and the chambers were stained with crystal violet. Photo micrographs of 5 regions were captured from duplicated chambers. The numbers of cells were counted and nor malized to the control. All e periments were performed in triplicate and repeated three times.

Cytosolic, nuclear isolation and immunoprecipitation Cytosolic and nuclear e tracts were prepared using a NE PER Nuclear and Cytoplasmic E traction Reagents kit following the manufacturers protocol. Isolated nuclear e tracts were lysed with RIPA buffer, and then subjected to direct western blot analysis or immunoprecipitation. Then, 2 mg of pro Cilengitide tein from each sample was used for immunoprecipitation with a Pierce Crosslink IP Kit, and the results were analyzed by western blot.

MC were e posed to patho physiologic Hcy concentration that has been pre viously shown to modulate MC behaviour. The results choose size revealed that several cytokines were sig nificantly affected by this manoeuvre, including TIMP 1, MIP 2, interferon gamma and fractalkine. MIP 2 influ ences leukocyte migration and has been shown to mediate inflammatory infiltration in glomerular disease. Accordingly, we chose to e plore the influence of Hcy on MIP 2 and to relate the observations to leukocyte interac tion with glomerular MC in an in vitro assay system. Homocysteine induces MIP 2 e pression and increases MIP 2 protein Initially we determined the influence of variable Hcy con centrations on MIP 2 e pression by qRT PCR. The results indicated a significant impact on e pression at 50 and 100 M.

Another sulphur containing amino acid, that is structurally similar to DL Hcy did not influence e pression. Hence changes in MIP 2 e pression can be attributed to an effect specific to Hcy, rather than to structural similari ties with L Cys. Subsequently, the e pression of MIP 2 induced by Hcy in MC was quantified by western blot analysis. In line with the e pression data, Hcy significantly increased MIP 2 protein levels in MC. Of note, MIP 2 e pression increased 2. 5 fold at 50 MHcy, com pared to e pression at 100 M L Cys. MIP 2 lev els did not increase further when Hcy concentration was increased to 100 M. Homocysteine induced MIP 2 requires p38MAPK and PI3kinase but not P42 44 MAPK Signaling MIP 2 induction has been reported to be MAPK and PI 3 Kinase dependent.

Hence, we investigated role of MAPK and PI 3 Kinase in MIP 2 e pression induced by Hcy. Hcy induced MIP 2 was significantly attenuated by a PI 3 Kinase inhibitor and by an inhibitor of a p38MAPK. In contrast, use of a p42 44 MAPK inhibitor did not significantly alter Hcy induced MIP 2. Immunohistochemistry was employed as another analyt ical tool to e amine the effect of Hcy on mesangial MIP 2. Cells were e posed to Hcy, in the absence and presence of inhibitors to p38MAPK and PI3 Kinase. MIP 2 e pression in medium supplemented with FBS and L Cys represented control condi tions. As revealed in figure 2, panel C, the e pression of MIP 2 was increased by Hcy compared to control. Hcy induced of MIP 2 was abolished by LY294002 and SB203580.

These results suggest that Hcy induced e pression of MIP 2 in MC was mediated by p38MAPK and PI 3 K signalling pathways and are consist ent with the results derived from Western blotting analy sis. Hcy activates p85 PI 3 Kinase and p38MAPK in mesangial cells In an effort to corroborate the observations related to blunting of the effect of Hcy on MIP 2 by inhibitors of PI3 Kinase and p38MAPK, western blotting analyses was employed to determine levels of activated p38MAPK and PI3 Kinase in Drug_discovery MC e posed to ele vated levels of e tracellular Hcy. Hcy induced time dependent increases in p38 MAPK phosphorylation between 10 and 30 minutes.

Increasing http://www.selleckchem.com/products/17-AAG(Geldanamycin).html evidences indicate an important role of ISL 1 in the development of some cancers. However, whether ISL 1 has any functional effect on tumorigenesis and how ISL 1 is regulated during cancer development are yet not clear. In the present study, we investigate whether ISL 1 plays an oncogenic role in human tumors. We show that abnor mal high e pression of ISL 1 is significantly correlated with NHL and is specifically e hibited in 75% of human NHL samples we e amined. Aberrant ISL 1 is regulated by p STAT3 p c Jun ISL 1 transcription comple and potentiates NHL cells proliferation through up regulating c Myc e pression. Our findings reveal the feasibility of ISL 1 as a potential therapeutic target for NHL treatment.

Results ISL 1 is highly e pressed in 75% of human NHL samples In our pilot study, the specimens from different types of tumors were analyzed by immunohistochemical staining. The results showed a high e pression level of ISL 1 in diffuse large B cell lymphoma, compared with reactive lymph nodes. To e amine the pathological relevance of ISL 1 in human lymphoma development, we analyzed the e pression level and cellular distribution of ISL 1 in collected specimens and tissue microarrays by immunohistochemical staining. These tissue specimens included 23 normal lymph nodes and 211 lymphoma samples. The lymphoma specimens could be classified into two types 195 NHL and 16 Hodgkin lymph oma. As summarized in Table 1, ISL 1 e pression level is markedly elevated in 75% of 195 NHL samples.

Only 3 cases of normal lymph nodes e hibited moderate ISL 1 immunostaining, none of the 23 normal lymph nodes or 16 HL showed any strong positive staining for ISL 1. Figure 1A shows representative immunohistochemistry images of ISL 1 staining in human normal lymph node, HL and NHL. ISL 1 staining was predominantly detected in the nuclear of a series of NHL lymphoma cells and, to a much lesser e tent, in the normal lymph nodes and HL samples. Statistical analysis revealed that there was no significant difference in the e pression of ISL 1 between normal lymph nodes and HL samples, whereas, the positive staining of ISL 1 was significantly correlated with NHLs compared with that in normal lymph nodes. Meanwhile, we found a predominant e pression of ISL 1 in a variety of NHL cell lines. These data establish that ISL 1 e pression is highly elevated in the majority of NHLs and might be tightly linked to lymphomagenesis.

ISL 1 promotes proliferation of NHL cells in vitro and enhances enografted lymphoma development in vivo We have previously shown that ISL 1 promoted the pro liferation of adult pancreatic islets cells. We wonder whether up regulated ISL 1 in NHL plays a role in promot ing NHL cells proliferation GSK-3 and tumorigenesis. Therefore, Raji, Jurkat and Ly3 were electroporated with pcDNA3. 1 ISL1, or pLL3. 7 ISL1 siRNA plasmid to establish stable ISL 1 overe pressing or knockdown NHL cell lines.

The MIH of crus taceans continually inhibits ecdysteroid secretion by the Y organs whereby synthesis of ecdysteroids and subse quent moulting occur only after MIH secretion ceases. CHH, however, plays a multifunctional role as it is central to carbohydrate metabolism, is involved in moult regulation, selleck chemicals Gemcitabine reproduction, and osmoregulatory function. It has been shown to inhibit ecdysteroid synth esis within the Y organs of Carcinus maenas. Furthermore, a synergis tic action of suppression of ecdysteroid synthesis in the Y organ has also been observed to occur when MIH and CHH are incubated together. CHH receptors have been found on Y organ cells, suggesting a physiologically relevant role for CHH in the regulation of ecdysteroid synthesis.

CHH has also been shown to influence the iso osmotic uptake of water during ecdy sis, which facilitates body expansion enabling somatic growth. Regulation of MF synthesis is negatively controlled by MOIH, and is thought to occur, in part, through the inhibition of the enzyme farnesoic acid O methyltransferase that catalyses the final step in the MF biosynthetic pathway. Eyestalk ablation has traditionally been used to induce moulting. This results in a reduction of circulating MIH and therefore promotes the production of ecdysteroids. However, while eyestalk ablation can be effective at inducing moulting, it also leads to lethal ecdysis in some species. Moulting is a complex process that is affected by a range of external and internal factors including tempera ture, photoperiod, nutritional state and eyestalk integ rity.

In order to explore the molecular events associated with the moulting process, microarray technology has been implemented to investigate differential gene expression in Portunus pelagicus at various stages of the moult cycle. Microarray technology offers the potential to examine the expression patterns of many genes simultaneously, thus gaining a more comprehensive understanding of gene function, interaction, and regulation. This has enabled both the assessment of expression profiles of known genes, and the discovery of new genes that play a role in the moult cycle of crusta ceans. P. pelagicus was used as a model species to study moulting as its life cycle has been closed at the Bribie Island Research Centre, eliminating the need for wild caught animals. Results Overview of P. pelagicus EST sequence distribution A total of 556 clones were sequenced from the cDNA libraries used to construct the P. pelagicus cDNA arrays. Prior Entinostat to array printing, 160 of these were sequenced in order to determine the quality of each cDNA library. Factors such as sequence length and redundancy were considered in the assessment. A 30% redundancy of 16 S rRNA was determined in the initial sequencing stage.

The reduction in TE our website evoked by depletion of eIF4G for small ORF genes is also obvious in the scatterplots of Figure S3, as dampening TE values for the shortest ORF lengths in the eIF4G mutant is observed. Thus, genes with short ORFs tend to be trans lated more efficiently in WT cells and to be dependent on eIF4G for their maximum efficiency. It is noteworthy that the two sets of 100 genes we identified above displaying the greatest changes in TE values on depletion of eIF4G differ dramatically in aver age ORF length. The group exhibiting the greatest reductions in translation efficiency has a mean ORF length below the genome average by nearly a factor of two, while genes showing the greatest increases in efficiency have a mean ORF length 70% larger than average.

These findings suggest that ORF length, in addition to 5UTR length, determines the influence of eIF4G on translational efficiency. Below, we propose a molecular explanation for this finding, based on the known relationship between transcript length and the stability of eIF4F cap interaction. Considering the strong correlation between ORF length and effect of eIF4G depletion on translational efficiency shown in Figure 7, it seems possible that the enrichment of cellular functions associated with the gene sets exhibiting TE4G TEWT 0. 71 or TE4G TEWT 1. 4 described above could at least partially reflect a preponderance of genes with unusually small or large ORF lengths in those functional categories. Discussion In this study, we have examined the genome wide con sequences for translational efficiency of simultaneously eliminating eIF4G2 and depleting eIF4G1 from yeast cells.

The conditional depletion of eIF4G1 achieved using a degron tagged version of this protein was highly effective and reduced the polysome content and rate of translation to only 20 30% of WT levels, indicating a substantial reduction in the rate of translation initiation. We used genome expression microarrays to measure the abundance of each mRNA in heavy polysomes relative to its level in total mRNA to calculate translational efficiencies of 5868 different genes. The results indicated that the over whelming majority of mRNAs experienced only a mod erate change in translational efficiency on eIF4G depletion. Less than 2% of the genes showed a statisti cally significant decrease in TE in the mutant by a factor of 1. 4 of more, and the genes in this group that were affected the most displayed reductions of Batimastat a factor of 2. 5 or less. While the actual percentage of genes affected to this extent is probably higher, only 10% of genes exhibited decreases in TE of this magnitude for each biological replicate, which likely represents the upper size limit for this category.

We adopted this method for transcript quantifica tion by RPKM and calculated the RPKM of each gene. RPKM quantification was http://www.selleckchem.com/products/BAY-73-4506.html distributed from 0 to over 104. In shoots under nor mal conditions, the gene encoding ribulose bisphosphate carboxylase activase was expressed at extre mely high levels. In roots under normal conditions, the gene for metallothio nein was expressed at extremely high levels. The statistical mean and median were 19. 78 and 3. 399, respectively, in the shoot, and 18. 705 and 4. 241 in the root under normal conditions. We then comprehensively compared the RPKM of each gene in response to salinity stress. We used the G test with a 1% false discovery rate and identified 6,469 and 10,321 differentially expressed RAP2 genes. Of these, 3,050 genes were commonly differentially expressed.

The number of highly differentially expressed genes, such as those encoding bHLH containing protein and amino acid transporter, was greater in the root than in the shoot. Expression of genes previously identified under salinity stress i. e. OsTPP1, LIP9, OsABA2, OsMST3, WSI76, and MYBS3 was induced in the root. For a com plete comparison see Additional file 2, Table S2. The distribution of mapped reads on the rice genome was graphed on a GBrowse. For example, the OsTPP1 gene, which encodes a protein that synthesizes the abiotic stress protectant trehalose, was expressed exclusively in the root after 1 h of salinity stress, RCc3, which was pre viously identified as a root specific gene, was expressed only in the root with and without stress, AK058218 was expressed exclusively in the shoot, most of the neigh boring genes were expressed evenly in all tissues used.

Constructing gene models by mRNA seq Transcribed regions were identified on the basis of the piling up of mapped short reads through the programs Bowtie, TopHat, and Cufflinks. In the shoot, 51,301 transcripts were predicted, 94. 6% of the predicted transcripts were mapped on previously anno tated loci in RAP2, thus, the remaining 2,795 predicted transcripts were unannotated in RAP DB. In the root, 3,082 of the 54,491 predicted transcripts were mapped on unannotated regions. For example, the previously annotated gene AK243146, which is similar to DREB1B in Arabidopsis thaliana, was expressed after salinity stress and also predicted by Cufflinks, other exons were also predicted and con nected by bridging sequences elucidated by TopHat. Reads were also mapped on the extended parts of the ends of most 5 and 3 exons in previous gene models. Of the transcripts mapped on previously annotated loci, 1,738 and 2,297 Dacomitinib had not been supported by ESTs or FL cDNAs. We attempted to predict the functions of unannotated transcripts by BLASTX search and longest ORF search.