Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on  Toxoplasma gondii is an obligate intracellular apicomplexan parasite that is an important pathogen of humans and animals. T. gondii causes encephalitis in immunocompromised patients, and progressive encephalitis in children infected in utero T. gondii consists of two phases: the sexual, which takes place only in felines, and the asexual, which takes place in all mammalian and avian hosts Toxoplasma infections T. gondii drugs are urgently needed. Studies on the basic biology of this organism are thus necessary for discovery of novel targets, and may also serve as a model system for the study of other apicomplexan parasites.Eukaryotic signaling pathways regulate a spectrum of intracellular activities; for example, the cAMP-dependent pathway is known to influence gene expression, apoptosis, tissue differentiation, and cellular proliferation Plasmodium falciparum, cAMP has been implicated in the differentiation of gametocytes Leishmania majorGiardia lambliaSchistosoma mansoniT. gondii, studies using cAMP analogs and cyclic nucleotide phosphodiesterase (PDE) inhibitors, which inhibit inactivation of cAMPs, implicated cAMP signaling in the asexual stage of the parasite T. gondii, whether these effects are extracellular , intracellular , or due to stimulation of both host and parasite signaling pathways remains to be determined. Moreover, although H89 is considered to be a specific inhibitor of PKA, several recent reports have suggested that H89 inhibits other kinases such as mitogen- and stress-activated protein kinase 1, p70 ribosomal protein S6 kinase or Rho-dependent protein kinase II Both cAMP and PKA have been shown to be essential signaling components in the life cycles of many eukaryotic pathogens. In in vitro kinase assay using recombinant T. gondii PKA catalytic subunit (TgPKA-C), as well as tachyzoite growth assay. The effect of PKI was weaker in TgPKA-C compared to mammalian PKA-C in the in vitro kinase assay, and PKI did not inhibit tachyzoite growth. These data support the hypothesis that the inhibitory effect of H89 on tachyzoite growth is due to TgPKA-C inhibition, since the possible inhibition of host cell PKA-C activity by PKI did not result in the inhibition of tachyzoite growth. Moreover, we generated a parasite line that expressed TgPKA-R stably, in which inhibiting the activity of parasite PKA without any effect on host cell PKA might be possible. These parasites also showed decreased growth. According to these data, TgPKA-C does indeed play an important role in the asexual stage of the T. gondii life cycle.In this study, we compared the inhibitory effects of ATP competitive chemical inhibitor H89 and mammalian natural peptide inhibitor PKI in an P. falciparum and Homo sapiens. The putative TgPKA-C (343 amino acid residues) showed 54% identity with H. sapiens PKA-Cα  and 57% identity with P. falciparum PKA-C . A multiple alignment using the ClustalW program showed that the 11 major subdomains of protein kinases (I–XI) The amino acid sequence alignment of the putative TgPKA-C , which we identified for the first time, is shown in P. falciparum and H. sapiens, is shown in H. sapiens PKA regulatory subunit Iα , 35% identity with HsPKA-RIIα (GenBank Accession Number: NP_004148), 36% identity with HsPKA-RIβ (GenBank Accession Number: NP_001158230), and 32% identity with HsPKA-RIIβ (GenBank Accession Number: NP_002727), as well as 52% identity with P. falciparum PKA-R . TgPKA-R possessed the two most conserved regions of PKA-R: the phosphate-binding cassettes (PBCs) of cAMP-binding domains A and B, specific to proteins of the PKA-R family r of 53,000 or 69,000, respectively, as detected by silver staining. These proteins reacted with antiserum containing α-GST antibody. These data indicate that we successfully purified the desired GST fusion proteins.To characterize the function of the protein, we expressed and purified TgPKA-C as a GST fusion protein using a wheat germ cell-free protein synthesis system. Purified protein was then electrophoretically separated in a denaturing gel and either silver-stained or immunoblotted with rabbit antiserum containing α-GST antibody . The purr of 69 000 was labeled  in accordance with the manufacturer's instructions. The labeled TgPKA-C band was eliminated by phosphatase treatment, indicating that TgPKA-C was labeled with [γ-32P]ATP via phosphorylation  was included as a positive control. Compared to BtPKA-C, inhibition by PKI was much weaker in TgPKA-C . However, in the experiment in which P. falciparum lysate containing cAMPs was used instead of recombinant TgPKA-C, phosphorylation of PfPKA-C markedly decreased The mechanism by which TgPKA-R, overexpressed in transgenic parasites, inhibited the activity of natural PKA-C is unclear. Two possibilities exist: TgPKA-R may act as a pseudosubstrate of TgPKA-C by inhibiting its binding to a substrate, or it may act as a cAMP sink, thereby reducing available cAMP so that PKA-C cannot be dissociated from PKA-R. When we used GST-TgPKA-C and MBP-TgPKA-R under cAMP-free conditions in an T. gondii. Several proteins have recently been reported to interact with PKA-C in apicomplexan parasites. In P. falciparum, the merozoite transmembrane protein AMA1, which is involved in the invasion of erythrocytes, has been reported to be phosphorylated by PKA T. gondii possesses a homolog of this protein (TgAMA1), TgPKA may also interact with it, thereby regulating invasion. In Trypanosoma cruzi, yeast two-hybrid experiments revealed that several proteins with known functions interact with PKA T. cruzi growth, adaptation, and differentiation. Since some of these proteins, for example, hexokinase and aquapoline, also exist in T. gondii, investigating their interaction(s) with TgPKA-C might be interesting.Further understanding of the biology of TgPKA may best be gained by investigation of the pathways and proteins with which TgPKA-C interacts. Since PKA is known to be involved in various activities of mammalian cells, many types of substrate may also be present in T. gondii RH strain and RH/ht− strain (kindly provided by Dr. X. Xuan) were used in this study. Parasites were maintained in monolayers of Vero cells cultured in Dulbecco's modified Eagle's medium (DMEM) that contained 7.5% fetal calf serum (FCS), 2 mM l-glutamine, 20 mM HEPES (pH 7.5), streptomycin, and penicillin. Host Vero cells were passaged in the same medium.Tachyzoites of T. gondii RH strain mRNA was isolated from infected Vero cells using the TRIZOL reagent  according to the manufacturer's instructions. The entire TgPKA-C open reading frame (ORF) was amplified by RT-PCR using parasite mRNA as the template and the following primers: forward, 5′-CCGGCTCGAGGATCTTTTTCGCCGTCTAAT-3′ and reverse, 5′-CGGGATCCTTACCACGACGTGAAAGGGT-3′. The PCR product was digested with XhoI/BamHI and cloned into the XhoI and BamHI sites of pEU  to express a glutathione S-transferase (GST) fusion protein. The resultant plasmid was designated pEU-GST-TgPKA-C. The entire TgPKA-R ORF was amplified from parasite mRNA by RT-PCR using the primers 5′-CGAATTCGGTAACGTTTGTGAATGCCAAAAG-3′ and 5′-GGGCTGCAGTTATTTGTCGAGGTATTTGGTGTCG-3′. Amplified fragments were digested with EcoRI/PstI and cloned into the EcoRI and PstI sites of pMal-c  in-frame with maltose-binding protein (MBP), to generate pMal-TgPKA-R. For stable expression, pMFH-TgPKA-R was generated as follows. The TgPKA-R ORF attached to the EcoRI/EcoRV restriction site was amplified from pMal-TgPKA-R using the primers 5′-CGAATTCGGGTAACGTTTGTGAATGCC-3′ and 5′-GGATATCTTTGTCGAGGTATTTGGTGTCG-3′. Amplified fragments were digested with EcoRI/EcoRV and cloned into the EcoRI and EcoRV sites of pMini.3×Flag.ht µg of pEU-GST-GFP or pEU-GST-TgPKA-C was mixed with 18 µL transcription mixture  and incubated at 37°C for 6 h. Each mRNA generated was then mixed with 10.8 µL WEPRO1240G (CellFree Sciences) and 40 ng/µL creatine kinase , then transferred to the bottom of the SUB-AMIX (CellFree Sciences) to form a bilayer and incubated at 16°C for 20 h.Protein expression using the Wheat germ cell-free protein synthesis system (CellFree Sciences) was performed as described previously µL of a 50% slurry of glutathione sepharose beads  for 16 h. The beads were then washed three times with phosphate-buffered saline (PBS)(–). Purified protein captured on the beads was separated by 10% SDS-PAGE after boiling and either silver-stained or immunoblotted  at 37°C for 2 h were pelleted and solubilized in 1% Tween 20/PBS(–) using one freeze–thaw cycle and three 10 s bursts of ultrasonication. After centrifugation , the supernatant was mixed with amylose resin beads (New England BioLabs) at 4°C for 2 h, and the beads washed five times with 1% Tween 20/PBS(–).Wheat germ extracts were mixed with 15 oblotted  with α-GµL kinase buffer  containing 5 µCi [γ-32P] ATP. Samples were then washed with TNE buffer . Reactions were performed at 30°C for 30 min in a total volume of 50 Following the kinase assays, labeled GST fusion proteins were boiled to inactivate the kinase and incubated with λ-protein phosphatase (New England Biolabs) in accordance with the manufacturer's instructions.T. gondii RH/ht− was performed as described previously µg of plasmid DNA was electroporated into 5.0×107 parasites using a GenePulser  at 2000 V, 25 µF, and 50 ohm. At 24 h after transfection, parasites were selected by exposure to 50 µg/mL mycophenolic acid and xanthine for 48 h, followed by a subsequent plaque purification. The recombinant clones stably expressing GFP or TgPKA-R-3xFlag were designated RH/GFP or RH/TgPKA-R.Transfection of the expression plasmid to 14–22  was 2 µM, and the volume of DMSO added was the same as that of H89. After incubation for 12, 24, and 36 h, the coverglass slides of RH/TgPKA-R were fixed with 4% paraformaldehyde in PBS(–) for 10 min, permeabilized by 0.1% Triton X-100 in PBS(–) for 10 min, and blocked in 3% bovine serum albumin and 5% skim milk in PBS(–). For the immunofluorescence assay (IFA), the slides were incubated with mouse α-SAG1 (tachyzoite-specific surface antigen) polyclonal antibody  at a 1∶1000 dilution for 1 h at room temperature. After reaction with the primary antibody, slides were washed three times with T-PBS [0.1% Tween 20 in PBS(–)], and primary antibody was detected with Alexa 546-conjugated α-mouse antibody (Invitrogen) at a 1∶2000 dilution for 1 h at room temperature, followed by five washes with T-PBS. The coverglass slides of RH/GFP were fixed with 4% paraformaldehyde in PBS(–) for 10 min and then washed three times with PBS(–). Each coverglass slide was dried at room temperature, mounted under a coverglass using Fluoromount-G , and observed under a fluorescence microscope . Microscopic fields were chosen randomly to count parasitophorous vacuoles up to three hundred. Vacuoles were sorted by the numbers of tachyzoites inside, and average numbers of tachyzoites per vacuole were calculated. Parasite growth was determined by the number of parasites per vacuole. The representative data of several experiments are shown in RH/TgPKA-R or RH/GFP was inoculated onto confluent Vero cell monolayers growing in four-chambered coverglass slides  at a multiplicity of infection (MOI) of 10 and allowed to invade host cells for 2 h at 37°C. Extracellular parasites were then removed by washing three times with PBS(–) and returned to culture in complete medium with or without the PKA-specific inhibitor. The final concentration of inhibitors H89 (Promega) and PKI


Bombyx mori expression system. This system was confirmed by the expression of human microsomal triglyceride transfer protein (hMTP) fused with EGFP in silkworm larvae and pupae. Moreover, hMTP and human protein disulfide isomerase (hPDI) genes were inserted into two LIC vectors harboring gcLINK sequences and were combined by using the LIC through gcLINK sequences. The constructed vector was incorporated into the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid, and injected into silkworm larvae. The expressed hMTP-hPDI complex was purified from the fat bodies of silkworm larvae. This LIC vector system was applied to express the E1, E2, and E3 subunits of human α-ketoglutarate dehydrogenase (KGDH) in silkworm larvae. The expressed proteins were purified easily from fat bodies using three different affinity chromatography steps. The LIC vectors constructed as described in this report allow for the rapid expression and purification of recombinant proteins or their complexes by using the BmNPV bacmid system.A ligation independent cloning (LIC) system has been developed to facilitate the rapid and high-efficiency cloning of genes in a  Generally, to produce recombinant proteins, target genes are amplified by PCR with primers containing restriction enzyme sites. PCR products and expression vectors are digested by restriction enzymes and ligated using T4 DNA ligase. Using this protocol, the available restriction enzymes depend on the nucleotide sequences of target genes, and different enzymes should be used when many target genes are cloned in parallel.Escherichia coli is performed. Host repair enzymes yield circular plasmids.Ligation-independent cloning (LIC) has been developed as a new cloning method, which eliminates the use of restriction endonuclease digestion and ligation of PCR products E. coli expression system, although there are a few reports of using LIC in other expression systems LIC is now used for the expression of fusion protein In this study, LIC vectors that can express affinity tag-fused recombinant proteins in the N- or C-terminal were constructed for high-throughput expression and purification of recombinant proteins in a silkworm expression system. Expression and purification of human α-ketoglutarate dehydrogenase (KGDH) subunits  in silkworm larvae showed the feasibility of using LIC vectors. Moreover, an improved LIC vector, allowing the fusion of two expression plasmids into one was also constructed for expression of a protein complex based on the ‘LINK’ sequence Xba I and Kpn I was inserted into the Xba I–Kpn I site of pFastBac 1 . The constructed plasmid was named pFastEGFP-SH-LIC  or Asc I and Nar I (gcLINK2)  . The congcLINK2)  were usestructed .Sma I and incubated at 65°C for 20 min to inactivate enzyme activity. T4 DNA polymerase (2.5 U) , which has both activities of 5′-3′ polymerase and 3′-5′ exonuclease, and 2.5 mM dTTP, were added into reaction mixtures and incubated at 25°C for 30 min, followed by heating at 65°C for 20 min to inactivate T4 DNA polymerase. Because of the 3′-5′ activity of polymerase the nucleotides  are removed from 3′-ends until the first “T” residue is reached. When T4 DNA polymerase comes to this first “T”, its polymerase reaction dominates and its 3′-5′ exonuclease reaction stops. This reaction leads two specific 5′-overhangs in this LIC vector of 10 and 12 bases, respectively.Plasmid construction by LIC reaction is also described in CGAAGAGGAGCCCTACCATGATTCTTCTTGCTGTG 3′), Human MTP R (5′ GGCACCAGAGCCCCTAACCATCCGCTGGAAGT 3′), Human PDI F (5′ CGAAGAGGAGCCCTACCATGCTGCGCCGCGCTCTG 3′), and Human PDI R (5′ GGCACCAGAGCCCCTACAGCTTTCTGATCATC 3′) primer sets, respectively. The ER retention sequence (last 4 amino acids of hPDI) was removed from the intact hPDI sequence. Amplified genes were purified and treated with T4 DNA polymerase according to the protocol described above. Then, T4 DNA polymerase-treated hMTP and hPDI were mixed with T4 DNA polymerase-treated pFastgcLINK-S-LIC and pFastgcLINK-H-LIC, respectively, and heated at 65°C for 5 min. The mixtures were then cooled to room temperature for 1 h to anneal each other. Two microliters of 25 mM EDTA was added to each mixture before E. coli DH10B (Invitrogen) transformation. The constructed plasmids were named pFastgcLINK-hMTP-S and pFastgcLINK-hPDI-H. Applying the same protocol described above, pFastBac-hMTP-EGFP-SH was constructed using pFastEGFP-SH-LIC and hMTP genes amplified by PCR.hMTP and hPDI genes were amplified by PCR using Human MTP F  was ligated into the Xba I – Kpn I site in pFastbac 1. gcLINK1 and gcLINK2 sequences were inserted into the resulting plasmid (pFast-N-SH-LIC) by the same manner as described in “Construction of LIC vectors for the expression of recombinant proteins fused with affinity tag C-terminally” in Ssp I sites. Four of the Ssp I sites in this vector, except for that in the LIC sequence, were mutated by using the Quikchange Lightening Multi site-Directed Mutagenesis Kit  using the following primers . The mutated plasmid (pFMgcLINK-N-SH-LIC) was digested by Mlu I or Pvu II, and each digested plasmid was self-ligated, resulting in pFMgcLINK-N-S-LIC and pFMgcLINK-N-H-LIC, respectively. Moreover, pFMgcLINK-N-SH-LINK was digested by Mlu I and Pvu II and ligated with the FLAG tag sequence composed of 2 oligonucleotides annealed to each other . This FLAG tag sequence was phosphorylated by T4 DNA kinase before its ligation. The next step was to construct pFMgcLINK-N-F-LIC. Human E1, E2, and E3 genes, which encode E1, E2, and E3 subunits of human KGDH, were amplified by PCR. Amplified E1, E2, and E3 genes lacked their mitochondrial targeting signal-coding sequences at their N-terminus . The primers used were as follows: . Each amplified gene was inserted into pFMgcLINK-N-S-LIC, pFMgcLINK-N-H-LIC, and pFMgcLINK-N-F-LIC, respectively, using the LIC reaction. Then, pFMgcLINK-S-E1, pFMgcLINK-H-E2, and pFMgcLINK-F-E3 were constructed.A LIC cassette for the expression of recombinant proteins fused with an affinity tag in the N-terminal was amplified by PCR using primers F2 and R2, and pFast-SH-LIC, which was constructed in this study as a template . An amplE. coli BmDH10Bac CP- strain was transformed with each pFastgcLINK vector or pFMgcLINK vector containing the gene of interest. Recombinant BmNPV bacmid harboring the gene of interest was extracted from white transformants. In this study, BmNPV-CP--hMTP-EGFP, BmNPV-CP--hMTP-S/hPDI-H, BmNPV-CP--S-E1, BmNPV-CP--H-E2, and BmNPV-CP--F-E3 bacmids were constructed. Recombinant BmNPV bacmid DNA (∼10 µg) containing helper plasmid was mixed with a one-tenth volume of DMRIE-C (Invitrogen) reagent and this mixture was injected into silkworm larvae or pupae.The construction and extraction of recombinant BmNPV bacmids were performed as described in a previous report The fat bodies collected from 10 silkworm pupae were suspended in 25 mL of phosphate-buffered saline  and sonicated 3 times for 30 sec each time with 1 min intervals. After centrifugation of the resultant homogenate at 8000×g for 10 min, the collected supernatant was mixed with 0.5 mL of PBS-equilibrated TALON Metal Affinity Resin  and stirred at 4°C for 1 h. This mixture was centrifuged at 3000×g for 2 min, and the precipitated resin was washed with 120 mL of PBS. Proteins bound to resin were eluted with 2 mL of PBS containing 200 mM imidazole. This eluent was mixed and gently stirred with 0.25 mL of PBS-equilibrated Streptactin Superflow  at 4°C for 1 h. This mixture was centrifuged at 3000×g for 2 min and the supernatant was removed. After washing the affinity gel with 5 mL of PBS, hMTP-hPDI complex was eluted with 2 mL of PBS containing 5 mM desthiobiotin.The homogenate supernatant was obtained from the collected fat bodies of 5 silkworm larvae by the same manner as the purification of hMTP-hPDI complex. Supernatant was mixed with 0.5 mL of Strep-Tactin Superflow equilibrated with Tris-buffered saline  and gently stirred at 4°C for 1 h. This mixture was centrifuged at 3000×g for 2 min and the supernatant was removed. After washing the affinity gel with 120 mL of TBS, S-E1 was eluted with 2 mL of TBS containing 5 mM desthiobiotin.H-E2: Homogenate supernatant was obtained from fat bodies collected from 5 silkworm larvae by the same manner as the purification of hMTP-hPDI complex. Supernatant was mixed with 0.5 mL of TALON Metal Affinity Resin equilibrated with TBS and gently shaken at 4°C for 1 h. This mixture was centrifuged at 3000×g for 2 min and the supernatant was removed. After washing the affinity gel with 120 mL of TBS, H-E2 was eluted with 2 mL of TBS containing 200 mM imidazole.Homogenate supernatant was obtained from fat bodies collected from 5 silkworm larvae by the same manner as the purification of hMTP-hPDI complex. Supernatant was mixed with 0.5 mL of anti-FLAG M2 antibody agarose . This mixture was centrifuged at 3000×g for 2 min and the supernatant was removed. After washing the affinity gel with 120 mL of TBS, F-E3 was eluted with 2 mL of TBS containing 100 µg/mL of FLAG peptide (Sigma Aldrich Japan).Recombinant protein samples were subjected to SDS-PAGE on 10 or 12% polyacrylamide gels using the Mini-protean II system . Total proteins on SDS-PAGE gel were detected with Coomassie Brilliant Blue (CBB) R-250 or silver staining. For the specific detection of fluorescent hMTP-EGFP fusion protein on SDS-PAGE gels, samples were mixed only with sample buffer and not boiled Protein concentration was determined using Protein Assay Kit II (Bio-Rad) based on the Bradford method.Dialysis was performed in PBS overnight at 4°C using a dialysis membrane .−1 cm−1 for NADH. One unit of enzyme activity was defined as the amount of enzyme capable of catalyzing the conversion of 1 µmol of NADH to NAD+ per minute.Dihydrolipoamide dehydrogenase (DLD) activity was assayed according to Huo et al The hMTP-hPDI complex was expressed in silkworm larvae using newly constructed LIC vectors. hMTP is located in the lumen of endoplasmic reticulum (ER) as a heterodimer with human protein disulfide isomerase (hPDI) Sma I site). EGFP gene, Strep-tag II, and 6× histidine tag sequences can be removed by thrombin due to the presence of a thrombin cleavage site between the LIC site and these tag sequences. The hMTP gene could be inserted with high efficiency at the LIC site by using the LIC reaction . The LIC reaction is highly specific and the efficiency of its reaction is ∼100% --hMTP-EGFP-SH bacmid was prepared and injected into silkworms and pupae. Green fluorescence was detected in silkworm larvae and pupae , and therefore, the yield of purified hMTP-hPDI complex was low compared to the yield of human prorenin-human prorenin receptor complex. To improve the recovery of purified hMTP-hPDI complex, purification by Strep-Tactin agarose was performed after dialysis of the hMTP-hPDI complex eluent of TALON affinity chromatography against PBS. Recovery yield (10 µg/10 silkworm pupae) was constant irrespective of dialysis  in mitochondria Human KGDH is in the mitochondrial matrix and is a multi-enzyme complex in the TCA cycle. KGDH is composed of multi-copies of E1 (α-ketoglutarate dehydrogenase), E2 (dihydrolipoamide succinyltransferase), and E3 (dihydrolipoamide dehydrogenase). KGDH plays an important role in controlling the reductive potential  protein, pFMgcLINK-N-S-LIC and pFMgcLINK-N-H-LIC, were constructed in the same manner as shown in Mlu I and Pvu II sites, and pFMgcLINK-N-F-LIC was constructed, indicating that any tag can be inserted into this site instead of the 6× histidine tag and Strep-tag II. This replacement can be available to LIC vectors constructed in LIC vectors for the expression of recombinant proteins fused with an affinity tag at the N-terminus were also constructed as shown in LIC and co-expression vectors were constructed using a new gcLINK sequence and BmNPV bacmid. Using these vectors, an hMTP-hPDI co-expression vector was successfully constructed without ligation. Moreover, purification of hMTP-hPDI complex was easily performed by TALON affinity and Strep-Tactin agarose chromatography, because Strep-tag II and 6× histidine tag were fused with hMTP and hPDI, respectively. Moreover, LIC vectors for the expression of recombinant proteins fused with several tags in the N- or C-terminal end were constructed. Each human KGDH subunit was expressed in silkworm larvae and purified from its fat body. This indicates that LIC vectors constructed in this study were available for protein expression and purification in silkworms. Because these vectors facilitate high-efficiency cloning of genes and fusion with purification tags, they are applicable for high-throughput expression of proteins, co-expression of protein complexes, and simultaneous administration of gene expression in the silkworm expression system.


Fibroblast growth factors FGF-1 and FGF-2 are often upregulated in tumors, but tightly bound to heparan sulphate proteoglycans of the extracellular matrix (ECM). One mechanism of their bioactivation relies on the FGF-binding protein (FGF-BP) which, upon reversible binding to FGF-1 or -2, leads to their release from the ECM. FGF-BP increases tumorigenicity and is highly expressed in tumors like colon carcinoma. In this paper, we analyse cellular and molecular consequences of RNAi-mediated FGF-BP knockdown in colon carcinoma, and explore the therapeutic effects of the nanoparticle-mediated delivery of small interfering RNAs (siRNAs) for FGF-BP targeting.Employing stable RNAi cells, we establish a dose-dependence of cell proliferation on FGF-BP expression levels. Decreased proliferation is mirrored by alterations in cell cycle distribution and upregulation of p21, which is relevant for mediating FGF-BP effects. While inhibition of proliferation is mainly associated with reduced Akt and increased GSK3β activation, antibody array-based analyses also reveal other alterations in MAPK signalling. Additionally, we demonstrate induction of apoptosis, mediated through caspase-3/7 activation, and alterations in redox status upon FGF-BP knockdown. These effects are based on the upregulation of Bad, Bax and HIF-1α, and the downregulation of catalase. In a therapeutic FGF-BP knockdown approach based on RNAi, we employ polymer-based nanoparticles for the in vivo delivery of siRNAs into established wildtype colon carcinoma xenografts. We show that the systemic treatment of mice leads to the inhibition of tumor growth based on FGF-BP knockdown.FGF-BP is integrated in a complex network of cytoprotective effects, and represents a promising therapeutic target for RNAi-based knockdown approaches. Fibroblast growth factors (FGF) represent a large polypeptide growth factors family comprising at least 23 members. Beyond embryonic development and tissue repair in the adult, FGFs play important roles in cancer and other diseases , a 9 nucleotide hairpin loop sequence (TTCAAGAGA) and a RNA Pol III terminator sequence tract (TTTTTT), flanked by a FGF-BP "A" (mRNA 253-271):5'GCACCCAGATTAAGCAGAAATTCAAGAGATTTCTGCTTAATCTGGGTGCTTTTTT-3'FGF-BP "B" (mRNA 490-508):5'GAGACATCTGTAGATATTCCTTCAAGAGAGGAATATCTACAGATGTCTCTTTTTT-3'FGF-BP "C" (mRNA 364-382):5'GGGTTGAGTGCACTCAATTGTTCAAGAGACAATTGAGTGCACTCAACCCTTTTTT-3'Firefly Luciferase shRNA :5'GTGCGCTGCTGGTGCCAACTTCAAGAGAGTTGGCACCAGCAGCGCACTTTTTT-3'The annealed oligonucleotides were cloned into the pSingle-tTS-Vector (Clontech) and the plasmids were checked by sequencing for correct insertion. The luc3 shRNA and the empty vector were used as negative controls.2) in Iscove's modified Dulbecco's medium  supplemented with 10% fetal calf serum (FCS) unless indicated otherwise.LS174T, HCT-116 and HT29 colon carcinoma cells were obtained from the American Type Culture Collection , HCT-116 p21 -/- were obtained from Dr. Bert Vogelstein , and staStable transfections were performed in 60 mm Petri dishes with 500,000 LS174T cells and 5 μg plasmid DNA using TurboFect  or 200,000 cells  and 2 μg plasmid DNA using Fugene  as described by the manufacturers. Selection of stable integrants was started 48 hours after transfection by adding 750 μg/ml (LS174T) or 1000 μg/ml  G418 (PAA) for 2 weeks. In the case of HCT-116 p21-/- cells, 0.2 μg pcDNA 3.1/Hygro  was added to the transfection, and stable cell lines were selected in the presence of 800 μg/ml hygromycin. Clonal selection from stably mass-transfected cells was performed by clonal expansion, and stably mass transfected cells or clonal shRNA expressing cell lines were used as indicated in the experiments.The stable integration of the shRNA expression cassette was confirmed by PCR analysis of genomic DNA with vector specific primers (Sing_TetU6) using the High-Pure PCR Template Preparation Kit according to the manufacturer's protocol .t method with β-actin and GAPDH serving as reference housekeeping genes.Total RNA from tumor xenografts or cells from tissue culture was isolated using the TRI reagent  according to the manufacturer's protocol. Reverse transcription (RT) was performed using the RevertAid H Minus First Strand cDNA Kit (Fermentas) with 1 μg total RNA and random hexamer primers. Quantitative polymerase chain reaction was performed using a LightCycler (Roche) in a total volume of 10 μl per capillary containing 5 μl QuantiTect SYBR Green 2× Master Mix, 4 μl diluted cDNA (1:50) and 1 μl intron spanning qPCR primers (5 μM each) specific for FGF-BP or reference genes . The Hot Start Polymerase was activated by a 15 min pre-incubation at 95°C, followed by 55 amplification cycles at 95°C for 10 seconds, 55°C for 10 seconds and 72°C for 10 seconds. CP values obtained were between 22 and 25 for FGF-BP and between 15 and 16.5 for actin/GAPDH. A melting curve analysis was performed to verify correct PCR products and a sample containing no template was always run in parallel to control for background, which was at CP values > 50. Quantitation of gene expression was performed by the ΔΔCTo determine the RNAi-mediated downregulation of FGF-BP at the protein level, LS174T cells from cell culture were scraped and lysed in PBS/5 mM EDTA in presence of Protease Inhibitors ('lysis buffer') on ice, or tumor xenograft tissue was homogenized in liquid nitrogen, and upon addition of lysis buffer treated by sonication or three freeze-thaw cycles. After centrifugation and determination of the protein concentration in the supernatant, samples containing 100 μg protein were separated by SDS-PAGE (19% polyacrylamide containing 6 M Urea and a 10% Spacer Gel) and transferred onto a nitrocellulose membrane  by semi-dry blotting. Membranes were blocked with 5% milk powder in Tris-buffered saline + Tween  and incubated with either of the following antibodies: 1:1000 rabbit polyclonal anti-FGF-BP raised against a GST-human FGF-BP fusion protein ,29, 1:50Soft agar assays for the determination of anchorage-independent proliferation and colony formation were carried out essentially as described previously . Brieflyin vitro, a commercially available bioluminescent caspase-3/7 assay  and a fluorometric Assay based on the caspase-3 substrate Ac-DEVD-AMC (Alexis) were applied. The Caspase-Glo 3/7 assay was performed in the 96-well format as recommended by the supplier and luminescence was measured after 1 hour incubation at 27°C in the dark using a Fluostar Optima reader . To normalize for differences in cell densities, a WST-1 assay was performed in parallel on the same plate, and the results of caspase activity were adjusted to cell numbers of the different cell lines.To test for apoptosis 2, 10 mM DTT and Protease Inhibitor ) followed by sonication in an ultrasonic bath three times for 10 sec. and chilling on ice. Cellular debris was removed by centrifugation at 10,000 g for 5 min and the total protein content of the supernatant was determined by a Bio-Rad protein assay. 30-100 μg total protein was diluted in 40 μl of freshly prepared reaction buffer containing PBS/10 mM DTT. 40 μl supernatant was transferred to 96 well plates , the reaction was started by adding 10 μl substrate solution  stock solution in DMSO, dissolved in 500 μl PBS) to a final concentration of 25 μM Ac-DEVD-AMC and measured using a FLUOstar OPTIMA microplate reader . A background control containing the substrate in PBS/10 mM DTT without cell lysate was run in parallel and a time curve of fluorescence was measured at 37°C every 20 min to check for linearity. Averages of triplicates were subtracted from the background signal and values were normalized as x-fold increase in apoptosis rate relative to the corresponding values of control cells at time points indicated in the figures. Cells treated with 50 μg/ml 5-fluorouracil served as positive control.The Caspase-3 assay using Ac-DEVD-AMC was assessed in a 6-Well format at a density of 100.000 cells. At the time points indicated in the figures, cells were washed twice with PBS and scraped from the surface. Cell lysis was performed in 300 μl lysis buffer , washing, and incubating in a Streptavidin-HRP solution (1:2000). After washing again, signals were developed by incubating in a chemiluminescent substrate , and chemiluminescence was visualized by film exposure . To precisely analyze very strong as well as very weak signals within the linear range, exposure times were varied between 5 seconds and 2 minutes. Signals were scanned and quantitated by densiometry using ImageJ .The activity of a panel of mitogen-activated protein kinases (MAPKs) and other serine/threonine kinases was assessed through the determination of their relative levels of phosphorylation using the Proteome Profiler Array  according to the manufacturer's instructions. Briefly, stable FGF-BP depleted cells and control cells were seeded at 50% confluency in 6-well plates and grown for 2 days prior to lysis at 1 × 10For flow cytometry-based analysis of cell cycle distribution, the sample preparation and propidium iodide staining of nuclear DNA were performed as described previously . Brieflyin vivo was determined by treating subcutaneous tumor xenograft-bearing mice with siRNAs complexed with polyethylenimine (PEI) as described previously  end-labeled at both strands using T 4 polynucleotide kinase and γ-[32P] ATP. To remove unbound radioactivity, siRNAs were purified by microspin columns  and complexed prior to i.p. injection as described above. After 2 h, mice were sacrificed and tumors were removed for RNA preparation as described above. The total RNA was dissolved in 200 μl DEPC-treated water, and 10 μl-samples were mixed with loading buffer, heat-denatured and subjected to agarose gel electrophoresis prior to blotting and autoradiography . Quantitation was performed by phosphor imager analysis.For the radioactive determination of siRNA delivery into the tumor, 0.6 μg (0.05 nmoles) FGF-BP-specific siRNAs were -labelled siRNAs demonstrate efficient delivery of intact siRNAs into the tumors. This is true even upon systemic administration which is more relevant in a therapeutic setting than local  injection. Concomitantly, a ~30% knockdown of FGF-BP expression is observed which proved sufficient for anti-tumor effects. This is in line with previous findings in stable FGF-BP knockdown prostate carcinoma cells, which showed an already complete abrogation of tumor formation upon injection of cells with 50% reduced FGF-BP levels [The therapeutic relevance of our findings is demonstrated by our in vivo data in mice. By employing siRNA-loaded nanocarriers for a therapeutic in vivo knockdown approach in established wildtype tumor xenografts, this study goes beyond previous publications based on ex vivo generated stable knockdown cell lines that were s.c. injected and thus do not mimick a therapeutic situation . In vivoP levels , and furTaken together, the knockdown of FGF-BP exerts multiple cellular and molecular effects in colon carcinoma including the induction of apoptosis, and FGF-BP represents a promising therapeutic target, for example by RNAi-based knockdown approaches through delivery of therapeutic siRNAs.FGF-BP: Fibroblast growth factor-binding protein; PEI: polyethylenimine; RNAi: RNA interference; siRNA: small interfering RNA.The authors declare that they have no competing interests.DS generated the LS174T cell lines and analysed cellular and molecular effects of the FGF-BP knockdown ; PP generated the HCT-116 and HT29 cell lines, analysed the cellular  and molecular effects of the FGF-BP knockdown (Western blots); SH and DS performed the in vivo studies; AA conceived and coordinated the study, participated in the data analysis and wrote major parts of the paper. All authors read and approved the final manuscript.


This 0.3-kb fragment contains a 17-nucleotide difference between the A8 and 57 sequences. We previously showed that the 0.3-kb fragment influences expression levels of Env protein in both cultured cells and rat brain, but the corresponding molecular mechanisms are not well understood.A neuropathogenic variant of Friend murine leukemia virus (Fr-MLV) clone A8 induces spongiform neurodegeneration when infected into neonatal rats. Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 identified a 0.3-kb KpnI-AatII fragment containing a R-U5-5’leader sequence as an important determinant for inducing spongiosis, in addition to the luciferase (luc) gene instead of the env gene found that the vector containing the A8-0.3-kb fragment yielded a larger amount of spliced luc-mRNA and showed higher expression of luciferase when compared to the vector containing the 57-0.3-kb fragment. The amount of total transcripts from the vectors, the poly (A) tail length of their mRNAs, and the nuclear-cytoplasm distribution of luc-mRNA in transfected cells were also evaluated. The 0.3-kb fragment did not influence transcription efficiency, mRNA polyadenylation or nuclear export of luc-mRNA. Mutational analyses were carried out to determine the importance of nucleotides that differ between the A8 and 57 sequences within the 0.3-kb fragment. In particular, seven nucleotides upstream of the 5’splice site (5’ss) were found to be important in regulating the level of protein expression from spliced messages. Interestingly, these nucleotides reside within the stem-loop structure that has been speculated to limit the recognition of 5’ss.Studies with expression vectors constructed from the full-length proviral genome of Fr-MLV that incorporated the The 0.3-kb fragment containing the R-U5-5’leader sequence of Fr-MLV influences the level of protein expression from the spliced-mRNA by regulating the splicing efficiency rather than transcription, nuclear export of spliced-mRNA, or poly (A) addition to mRNA. Seven nucleotides in the 0.3-kb fragment, which reside within the stem-loop structure that has been speculated to limit recognition of the 5’ss, could pinpoint the function of this region. Proteins responsible for the constitution of the inner structures of the virion are encoded by the gag gene, which includes the matrix, capsid and nucleocapsid proteins. The pol gene encodes the enzymatic proteins, i.e. the reverse transcriptase, protease, integrase and RNase H and the env gene encodes the proteins protruding out from the viral particle surface, namely the surface (SU) and transmembrane (TM) proteins [pol gene. Only a singly spliced mRNA is usually found in simple retroviruses. Gag and Pol proteins are translated from the unspliced full-length viral mRNA, and the Env protein is translated from the spliced env-mRNA [The simple retroviruses, including MLV, are characterized by a coding structure in which the proteins . Transcrenv-mRNA . In contenv-mRNA -4.env gene of A8 as the primary determinant [env gene on the background of 57, did not exhibit neuropathogenicity. In contrast, the chimeric virus R7f, which contains a 0.3-kb fragment of A8 and the A8-env gene on the background of 57, induced spongiform neurodegeneration. It has been shown that the expression level of Env protein in both R7f-infected cultured cells and in brains of R7f-infected rats was higher than in the Rec5-infected cultured cells and brains of Rec5-infected rats [A neuropathogenic variant of Fr-MLV, clone A8, induces spongiform neurodegeneration in neonatal rats. Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 identified a 0.3-kb KpnI-AatII fragment containing the R-U5-5’leader sequence as an important determinant of neuropathogenicity, in addition to the erminant . Chimerited rats . These fgag) has specific sequences with distinct secondary structure features [Given that the 0.3-kb fragment containing the R-U5-5’leader sequence is the first untranslated region that exists in all variants of retroviral transcripts, this region dynamically impacts various stages of the viral life cycle. The R region, present at both ends of viral RNA, mediates the jump of reverse transcriptase from the 5’ site to the 3’ site during the synthesis of minus-strand DNA ,8, possifeatures ,29. Therfeatures . For exafeatures . Interesfeatures . A seconfeatures  and is afeatures -35, is ifeatures ,37. It ifeatures ,39 and afeatures ,40.luciferase (luc) gene incorporated in place of the env gene. We then examined the effects of the 0.3-kb fragment on several steps affecting protein expression levels in NIH3T3 cells. The results showed that the 0.3-kb fragment of A8 enhanced protein expression levels from the spliced mRNA through up-regulating the efficiency of splicing compared with the 0.3-kb fragment of 57, rather than through increased transcription, poly (A) addition to mRNA, or nuclear export of spliced mRNA. Furthermore, we investigated more specifically the roles of the nucleotides that differ between A8 and 57 sequences in defining the function of the 0.3-kb fragment. Lastly, we discuss the possible mechanism by which the 0.3-kb fragment participates in protein expression.In this study, to investigate the role of the 0.3-kb fragment containing the R-U5-5’leader sequence in the expression of Env protein of Fr-MLV, we constructed expression vectors having the full-length proviral genome of Fr-MLV with the env gene on the background of 57, was shown to increase Env expression about 3-fold compared to non-neuropathogenic Rec5, which contains the 57-0.3-kb fragment and the A8-env gene on the background of 57 [luc gene, in which the viral env gene was replaced with the luc gene to produce the luciferase expression vectors Rec5-L and R7f-L, respectively   but was comparable with that from Rec5-L  tail length of gapdh-mRNA  tail length of mRNA is correlated with the efficiency of translation. Therefore, to examine whether or not the 0.3-kb fragment influences polyadenylation of viral mRNA, the poly (A) tail lengths of mRNA from Rec5-L and R7f-L transfected Hela cells were compared. Total RNA was harvested and anchored with the RVP3 primer before the first strand of cDNA was synthesized with an anti-RVP3 oligo strand. To determine the poly (A) tail length, the transcripts derived from Rec5-L and R7f-L were selectively amplified using the forward primer for viral U3 sequences of the 3’LTR and the reverse primer for the RVP3 sequence. PCR products viewed on electrophoresed gels showed no detectable differences in the smeared patterns indicating the poly (A) tail lengths of transcripts derived from R7f-L and Rec5-L  and 65% (R7f-L) of the gapdh-mRNA in the cytoplasm and about 41% (Rec5-L) and 35% (R7f-L) remaining in the nucleus . In the cells transfected with Rec5-L, 12% of n Figure . In the Further investigations were carried out to determine whether any of the nucleotides within the 0.3-kb fragment are key(s) to the observed luciferase expression effects. Using the same luciferase expression vectors, a series of point mutations was incorporated into the R7f-L 0.3-kb fragment, in which the 17 nucleotides that differ between the A8 and 57 sequences were gradually mutated into sequences of 57 from the 5’ site. The luciferase activity of these vectors was determined  R7f.567 m-L, in which only the 5th, 6th and 7th nucleotides contain the 57 sequences and (b) another vector having the exact reverse order, Rec5.567 m-L, which has only the 5th, 6th and 7th sequences retained as A8 sequences. The luciferase activity of R7f.567 m-L remained at about 95% and could not be brought down to parallel that of Rec5-L, while its exact reverse vector, Rec5.567 m-L, had a significantly increased luciferase activity (86%) that was higher than that of Rec5-L (p < 0.001).luc-mRNA expression, we mapped out the secondary structure formed by the sequence containing the 1st to 7th nucleotides of the 0.3-kb fragment of the A8 and 57 sequences. The secondary structure, as shown in Figure To explain how the 1st to 7th nucleotides might be important for Since point mutational analysis indicated the 1st to 7th nucleotides contribute to the luciferase activities of the vectors, we compared the sequences including these nucleotides in gamma retroviruses containing MLVs, Feline leukemia virus (FLV), and Gibbon ape leukemia virus (GALV)  polymerase from viral mRNA might be suppressed. The nuclear-cytoplasmic distribution of mRNA also differs between viral mRNA and gapdh-mRNA. It is generally known that nuclear export of mRNA is mediated by multiple protein factors that couple steps of nuclear pre-mRNA biogenesis to mRNA transport [gapdh-mRNA.In the luciferase expression vector system of the present study, luciferase protein is translated from spliced mRNA. When quantified in transfected cells, the amount of spliced s Figure , indicatransport  thereforNext, to investigate the roles of nucleotides that differ between A8 and 57 within the 0.3-kb fragment, we gradually mutated the 17 nucleotides that differ between them and tested their respective luciferase activities. Among the vectors investigated, only the F3-L, which carries the 1st to 7th nucleotides of 57 on the background of the A8 sequence, showed decreased luciferase activity that paralleled that of Rec5-L, which has the 57 sequence  addition to mRNA, or nuclear export of spliced-mRNA. Furthermore, seven nucleotides that apparently contribute to regulation of gene expression have been identified. Interestingly, these nucleotides reside within the stem-loop structure that has been speculated to limit recognition of the 5’ss.env gene with the luc gene [pol gene of each recombinant plasmid. R7fa-L was constructed by replacing the 57 sequences of KpnI (32) and AatII (361) with the A8 sequences in Rec5-L. R7fb-L was generated by replacing the A8 sequences of KpnI (32) and AatII (361) with the 57 sequences in R7f-L. Mutation vector F1-L was constructed by mutagenesis of R7f-L using the following forward primer: CGCCCGGGTACCCGTATTCCCAATAAAGCCTCTTGCTG; and the reverse primer: ACGGGTACCCGGGCGACTCAGTCTA. F2-L was generated by mutagenesis of F1-L using the forward primer: TCTTGCTGTTGCATCCGACTCGTGGTCTCGCTGTT; and the reverse primer: AGTCGGATGCAACAGCAAGAGGCTTTATTG. F3-L was constructed by mutagenesis of F2-L using the forward primer: TTTGGGGGCTCGTCCGGGATCTGGAGACCCTTGCCCAAGGACCACCGA; and the reverse primer: GATCCCGGACGAGCCCCCAAATGAAAGACCC. F4-L was generated by mutagenesis of F3-L using the forward primer: AAGCTGGCCAGCAATTGATCtGTGTCTGTCC; and the reverse primer: GATCAATTGCTGGCCAGCTTACCTCCCGGT. B1-L was generated by mutagenesis of R7f-L using the forward primer: ACCCGTGGTAGAACTGACGGGTTCGAGACACCCGGCCGCAA; and the reverse primer: CGTCAGTTCTACCACGGGTCCGCCAGATA. B2-L was generated by mutagenesis of B1-L using the forward primer: TTGGCCGACTAGCTCTGTACCTGGCGGACCCGTGGTGGAACTGACG; and the reverse primer TACAGAGCTAGTCGGCCAACTAGTACAGAC. B3-L was generated by mutagenesis of B2-L using the forward primer: CCATTGTCCCGTGTCTTTGATTGATTTTATGCGCCTGCGTTTGTACTAGT; and the reverse primer: TCAAAGACACGGGACAATGGACAGACACCG. R7f.5 m-L was constructed by mutagenesis of R7f-L using the forward primer: TCTTGCTGTTGCATCCGACTCGTGGTCTCGCTGTT; and the reverse primer: AGTCGGATGCAACAGCAAGAGGATTTATTG. R7f.567m-L was constructed by mutagenesis of R7f.5m-L using the forward primer: TTTGGGGGCTCGTCCGGGATCTGGAGACCCTTGCCCAAGGACCACCGA; and the reverse primer: GATCCCGGACGAGCCCCCAAATGAAAGACCC. Rec5.5m-L was constructed by mutagenesis of Rec5-L using the forward primer: TCTTGCTGTTGCATCCGACTTGTGGTCTCGCTGTT; and the reverse primer: AGTCGGATGCAACAGCAAGAGGCTTTATTG. Rec5.567m-L was constructed by mutagenesis of Rec5.5m-L using the forward primer: GGAGACCCTTGCCCAGGGACCACCGACC; and the reverse primer: AAGGGTCTCCGGATCCCGGACGAGCCC. Structures of the expression vectors were confirmed by digestion with restriction enzymes and sequence analysis. Basic recombinant DNA procedures were performed according to standard protocols [Luciferase expression vectors R7f-L and Rec5-L were constructed as described previously by replacing the viral luc gene  within irotocols .2 atmosphere. HeLa cells were grown under the same conditions as NIH3T3 except they were incubated in a 5% CO2 atmosphere.NIH3T3 cells were grown in Dulbecco’s Modified Eagle Medium – low glucose (SIGMA) supplemented with 10% fetal calf serum  and penicillin-streptomycin (GIBCO) and cells were incubated at 37°C in a 7% CO5) were plated in 24-well plates with growth medium minus penicillin and transfected the next day with 0.8 ug luciferase expression vectors, 5 ng of pRL-SV40 (Promega) using 2 ul of Lipofectamine 2000 Reagent  diluted with OPTI-MEM (Invitrogen). After 48 hours, cells were lysed and luciferase activities were measured as Relative Light Units (RLU) using a luminometer with a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The luciferase activity of each sample was normalized to that of Renilla (pRL-SV40) as an internal control.NIH3T3 cells (1 × 1020 primer and SSIII reverse transcribing kit (Invitrogen). A portion of the resulting cDNA was subjected to real-time PCR using an Applied Biosystems® 7500 Real-Time PCR System. The specific primers and probes used for detection of total mRNA at the luc region were: LucF: CGGCTTCGGCATGTTCA; LucR: TACATGAGCACGACCCGAAA: TaqMan probe: CACGCTGGGCTACTTGATCTGCGG. Spliced-mRNA was detected using s6: GGGTCTTT CATTTGGGGGCTC; s2: TGCCGCCAACGGTCTCC and the TaqMan probe: CACCACCGGGAGCTCATTTACAGGCAC. Standard curves to quantify both mRNAs derived from the luciferase expression vectors utilized vector splA8L [gapdh-mRNA was quantified as an internal control using TaqMan Rodent GAPDH Control Reagents containing primer sets and probes (Applied Biosystems). Standard curves to calculate the amount of mRNA were created using serially diluted gapdh T-easy vector. The negative control samples without the cDNA synthesis step showed undetectable amplification.RNA extraction was carried out using an RNase Mini Kit (Qiagen). RNA was treated with RNase-free DNase (Qiagen) and 2 ug of RNA were reverse transcribed using an OligodTr splA8L . In addigapdh DNA was measured as an internal control using the TaqMan Rodent GAPDH Control Reagents.Cellular genomic DNA (gDNA) was extracted using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. Real-time PCR was performed to quantify the amount of plasmid DNAs introduced into the cells. Primers and probe sets used to quantify the amount of firefly luciferase expression vector introduced were the same TaqMan primer and probe set used to detect the amount of cDNA. The amount of Nuclear and cytoplasmic fractions were obtained from cultured cells using a PARIS kit (Ambion) according to the manufacturer’s manual. As a control for the fractionation, an aliquot of total RNA from each section was electrophoresed on a 1% agarose gel in morpholinepropane-sulfonic acid (MOPS) buffer, and the cellular ribosomal RNAs were visualized by ethidium-bromide staining.gapdh-mRNA, a forward primer, Mgapdh3end (CCCTACTCTCTTGAATACCATCA), was set at the junction before the poly(A) signal and was used with the same reverse primer targeting the RV3PC-anchor sequence. The resulting PCR products were stained in ethidium bromide and electrophoresed on an 8% polyacrylamide gel to visualize the spliced mRNA. A 3% agarose gel was used to visualize gapdh-mRNA. Within the pool of reverse-transcribed cDNA, the following primers were used to detect the presence of luc-mRNA: forward primer f-597 (GGGCTCGTCCGGGATC) and reverse primer s2 (TGCCGCCAACGGTCTCC); for gapdh-mRNA, the forward and reverse primers from the Taqman Rodent GAPDH control reagents (Applied Biosystems) were used.Total RNA extracted from 24 hours post-transfected Hela-cells were ligated with RV3PC–anchor primers. Reverse transcription was then carried out using an antisense sequence of the RV3PC-anchor primer. To amplify the poly (A) tail of mRNA, a forward primer targeting the 3’end of U3 at LTR (AGCTCACAACCCCTCACTCGGC) was paired with a reverse primer targeting the RV3PC-anchor sequence. To increase the likelihood of the reverse primer binding at the poly(A) tail, ten thymines were added into the 3’end of the reverse primer sequence (CTAGCAAAATAGGCTGTCCCTTTTTTTTTT). Likewise, to detect the poly (A) tail length of the The authors declare that they have no competing interests.YS carried out real-time PCR analyses and luciferase assay experiments. NO constructed some of the vectors and carried out luciferase assays. AM determined the poly (A) tail length. STY conceived and organized the study and helped to draft the manuscript. All authors read and approved the final manuscript.


To the Editor: Genotypic drug resistance testing before initiation of first-line antiretroviral therapy (ART) is recommended to detect drug-resistant viruses and to avoid treatment failure caused by preexisting drug-resistant viruses  and high HIV-1 viral load of 980,000 copies/mL plasma, we started directly observed ART with tenofovir and emtricitabine plus nevirapine. Genotypic resistance testing showed no evidence of resistance. Within the first 4 weeks of ART, the viral load decreased to 540; however, 4 weeks later it increased to 15,000, and then 12 days later to 71,000 HIV-1 RNA copies/mL . ResistaWe hypothesized that preexisting drug-resistant HIV-1 minority variants might have caused this early treatment failure. Thus, we performed clonal analysis of the RT gene before and during ART. At baseline, 17/222 clones (7.7%) carried the K65R mutation, synonymous to ≈51,000 HIV-1 RNA copies. Later, the K65R mutation was comprised in all clones. Further preexisting drug-resistance mutations have been detected in single, K65 wild-type, separate clones: K70R, V106A, and V108I. However, neither the K103N nor the M184V mutation was detected in any of those 222 clones, but both mutations were rapidly selected during early treatment failure.Thus, the presence of the K65R mutation in a substantial fraction of the virus population and the rapid acquisition of the K103N and M184V mutations led eventually to early treatment failure. We cannot formally rule out that K103N mutants were present before ART at a very low frequency <0.5% and that K65R/K103N double mutants were potentially selected. The M184V mutation was acquired later because only 12.5% of K65R/K103N viruses carried the M184V mutation at the first time point during treatment failure.Because of the considerable absolute number of less replication-competent K65R-harboring viruses in the patient, we assume that this variant has been transmitted. The additional presence of isolated, nonnucleoside RT inhibitor (NNRTI)/nucleoside RT inhibitor (NRTI) resistance mutations is another indication for the transmission of drug-resistant viruses, although they and the K65R mutation were not found in the same viral genomes. Presumably, the index patient was treated with nevirapine, stavudine, and lamivudine, the commonly prescribed first-line ART in resource-limited settings (The prevalence of K65R-harboring drug-resistant HIV-1 minority variants is not negligible in treatment-naive patients. We have shown that 2.7% of HIV-1–infected patients, mainly those infected with HIV-1 subtype B, harbor the K65R mutation as a minority variant (Immigration from resource-limited settings is increasing, and data suggest that minority variants harboring the K65R mutation are quite prevalent in those infected with HIV-1 subtype C, who are treatment naive (


Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen we uncover Kermit, previously implicated in G protein and PCP signaling, as a novel binding partner of Go. Through pull-down and genetic interaction studies, we find that Kermit interacts with Go and another PCP component Vang, known to undergo intracellular relocalization during PCP establishment. We further demonstrate that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI. Our results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment.In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue – the phenomenon known as planar cell polarity (PCP). In  Drosophila where mutations in PCP genes affect the uniform arrangement of ommatidia in eyes, anterior-posterior organization of sensory cuticle bristles, and proximo-distal orientation of wing hairs . While 78% of the control adult MS1096-Gal4, UAS-Gαo/X flies had folded wings, this number decreased to 22% in the MS1096-Gal4, UAS-Gαo/X; kermit[SH0225]/+ flies. Independent repetition of the cross confirmed this finding; statistical analysis revealed that the result is highly significant .Overexpression of pal case  -tagged recombinant protein after bacterial expression. Recombinant Gαo was also purified and immobilized on CNBr-Sepharose – procedure leading to active Gαo competent to interact with guanine nucleotides and partner proteins  and kermit[ex 31] were homozygous viable without any discernible phenotypes [kermit[SH0225] allele  animals were the fz[H51]/[P21] transheterozygotes [37]. The kermit alleles were SH0225kermit [ex31kermit [29]. To screen for Gαo-interacting mutations, the UAS-Gαo transgene on the first chromosome /+ progeny flies were analyzed in two independent crosses. All crosses were performed at 25°C. Cells producing multiple hairs were counted in the dorsal region framed by the veins 3 and 4 and the intervein 1. To monitor Vang localization in pupal wings 30h after puparium formation, Act-Stbm-YFP flies were used as described [13].kermit cDNA (clone LP09416) was obtained from Drosophila Genomics Resource Center (DGRC) and subcloned into pMAL-c2X (NEB) by EcoRI and SalI after amplification using the oligonucleotides: forward: CAATCCGAATTCATCATGCCGCTCTTCAC, reverse: GGTATGGTCGACCAATTACTTGGGACTGG. Recombinant Kermit was purified according to the manufacturer’s instruction, along with MBP expressed by the parental pMAL-c2X plasmid.kermit was subcloned into pUAST-attb [GGTATGCTCGAGCAATTACTTGGGACTGG. The φχ-22A line [UAS-kermit transformant on the second chromosome.For fly transformation, AST-attb  by EcoRI22A line  was usedGCAGGTGGTACCATGTCCTGCACCGTTTCCG, reverse: GGACATGTCGACCTAAGTTGGACTATCCG. Human RGS19 was obtained from ImaGenes and subsequently cloned into pQE-30, using the oligonucleotides: forward:CG5036 cDNA (clone LD40005) was obtained from DGRC and subcloned into pQE-30 plasmid (Qiagen) by KpnI and SalI, primers used were: forward: CTCCGCGGTACCATGCCCACCCCGCATG, reverseGCTGGGGTCGACCTAGGCCTCGGAGGAG. Both plasmids along with pQE32-Gαo were used to express recombinant proteins purified as described [6-Rab5, and GST were produced as described previously [escribed . MBP-Fz,eviously .6-Gαo, GST, MBP, MBP-Kermit, His6-CG5036, and His6-Rab5  for 30min at RT. Soluble proteins were added in equimolar amounts , or rabbit anti-Gαo/i3  antibodies.The following proteins were covalently linked on CNBr-activated Sepharose  according to manufacturer’s instructions: Hisis6-Rab5 . Immobil amounts . Drosophescribed . Bacteriescribed ,15. Afte


The expression of fluorescent protein (FP) genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs  appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues.35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER)-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed.The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here. Aequorea victoria, fluorescent proteins (FPs) have become an increasingly powerful tool for use in molecular biology [ZmUbi1) or cauliflower mosaic virus (CaMV) 35S promoter [Since the discovery and isolation of the green fluorescent protein (GFP) from the Pacific jellyfish  biology -3. The l biology . In plan biology ,6. Additpromoter ,8. Whilepromoter ,10, endopromoter -13, rootpromoter ,15 and vpromoter ,17. FPs promoter  and can promoter .Although other FPs have been added to the color palette during the past 12 years, GFP has remained the most commonly used FP in these studies; GFPs generally form monomers in physiologically-relevant concentrations overexpressed in the cytosol, whereas native coral-derived FPs (yellow through far red) tend to autotetramerize. Nevertheless coral-derived FPs have gained wider use in recent years in transgenic plant studies simply because many of them are brighter, owing partially to their longer wavelengths. The longer wavelengths needed to excite RFPs also have much lower levels of autofluorescence in mature green tissue as compared to UV or blue light, which is used to visualize GFP [Discosoma sp. was the first coral-derived FP to be used in plants as a reporter gene [Discosoma sp. are constantly being discovered and have recently been exploited to produce novel FPs, potentially resulting in improved FP reporter genes for plant biotechnology [DsRed, derived from coral ter gene ,21, and ter gene  along witer gene . Furtherchnology . Anotherchnology . EosFP iNicotiana benthamiana using agroinfiltration and stable transgenic Arabidopsis thaliana and tobacco (Nicotiana tabacum) plants were assayed using epifluorescence and confocal microscopy, and spectrofluorescence measurements.The aim of this study was to survey a sample of promising FPs that have seldom been used in plants to compare their performance as reporter genes. We also set out to improve them for use in applications where whole-plant fluorescence is of paramount importance . We evaluated and modified the following FPs : DsRed2 563, 582), tdTomato  mOrange  and pporRFP . Whereas many of these proteins are often called red fluorescent proteins (RFPs), as Shaner et al. [, 582, tdNicotiana benthamiana was designed to rapidly screen the expression vectors for functionality, but to also assay gross comparisons of the effect of ER-targeting, e.g., adding a signal peptide fusion to the N-terminus and an HDEL ER retention signal to the C-terminus that successfully improved whole plant fluorescence for GFP [The agroinfiltration experiment in  for GFP . We were for GFP ,18. With1 transgenic tobacco (Nicotiana tabacum cv. Xanthi) lines were selected for analysis and, therefore, microscopy and fluorospectroscopy analysis of these lines yield a realistic variation with regards to the range of fluorescence in these eudicot species. There was a wide range of fluorescent intensity for the different transgenic lines for each OFP construct, with up to an eight-fold difference from the highest to the lowest expressing line  and switchgrass (Panicum virgatum) as a transgenic reporter gene [Whereas agroinfiltration is useful as a first-pass gross screen for cassette functionality, stable transformation more closely mimics the range of expression levels seen when gene constructs are integrated in variable insertion sites. Stable expression was required in order to quantitatively compare the expression levels of the different fluorescent protein encoding genes in similar tissues. Transformants (up to 10 events) of the most fluorescent Te Figure . Howevere Figure . Also, poperties , which eoperties . While tter gene ,28. Indeter gene . We chosArabidopsis thaliana most commonly used codons as a model, pporRFPmut was stably transformed into tobacco and transgenic T0 lines were screened for fluorescence. Ten transgenic lines were selected and carried through to the T1 progeny as above, where they were directly compared to the native pporRFP gene for expression of fluorescence. Surprisingly, the overall fluorescence level of the codon-altered pporRFPmut was significantly lower than that of the native pporRFP  and 1.7 × 105 (± 0.5 × 105), respectively. We are curious as to why plant codon optimization failed, since it has increased expression and accumulation of other proteins synthesized in plants, most notably Bacillus thuringiensis endotoxins [As a result of this high fluorescence using the native pporRFP gene, we modified the nucleotide sequence with the intention of optimizing the codons for plants to further enhance the fluorescent expression levels. Utilizing P Figure . The avedotoxins . One spedotoxins . Lower e5 (± 0.6 × 105) and 3.2 × 105 ). Furthermore, the distributions of fluorescence between the two gene variants have very little overlap  vs. leaves (tobacco)) , so we caution against making definitive conclusions regarding direct OFP expression in tobacco vs. Arabidopsis. We did, however, observe a similar trend of dramatic enhancement for ER-targeting of mOrange that we observed in tdTomato. When stably expressed in Arabidopsis, we observed a 2-fold enhancement of fluorescence in ER-targeted mOrange compared with the non-targeted counterpart (6.5 x 105 (± 2.7 × 105) and 3.3 x 105 . Additionally, the ER-targeted tdTomato resulted in more than a 4-fold increase in fluorescence as compared to the non-targeted tdTomato (10.8 × 105 (± 3.5 × 105) and 2.4 x 105 . For recombinant proteins that have been shown to be relatively stable when synthesized in the cytosol, ER-targeting and retention has also shown a three-fold increase [We overexpressed the entire collection of genes (except for pporRFP-ER) in s Figure . Again, s Figure ,4, and 5s Figure . We obseincrease . ER-targincrease -38. The in vivo reporter genes of choice for plant transformation. Endoplasmic reticulum targeting allows the accumulation of greater OFP monomers than non-targeting in select OFPs. TdTomato-ER is the most brightly fluorescing FP marker gene ever characterized in transgenic plants, followed by mOrange-ER. These OFP variants will be especially valuable in quantifying inducible expression in plant organs.OFPs have emerged as the real-time ATGGCTCTTTCAAAGCAAAGTGG-3′ and reverse primer 5′- TTAGTGATGGTGATGGTGATGGG-3′. mOrange and tdTomato containing vectors were obtained from the laboratory of Roger Tsien . mOrange CDS was amplified from pRSET-mOrange using the forward primer 5′- ATGGTGAGCAAGGGCGAGGAGAATA-3′ and reverse primer 5′ TTACTTGTACAGCTCGTCCATGC-3′. The tdTomato CDS was amplified from pRSET-tdTomato using the forward primer 5′- ATGGTGAGCAAGGGCGAGGAGGT-3′ and reverse primer 5′-TTACTTGTACAGCTCGTCCATGC -3′. The resulting products were cloned into the entry vector pCR8/GW-TOPO (Invitrogen). DsRed2 coding sequence (Clontech) was recombined into the entry vector pDONR/Zeo from pET160/GW-DsRed2 using BP Clonase (Invitrogen). The N-terminal signal polypeptide sequence (MKTNLFLFLIFSLLLSLSSAEF) and C-terminal ER-retention polypeptide sequence (HDEL) were added to coding sequences through assembly and amplification PCR as described by Richardson et al [ATGAAAACTAATCTTTTCTTGTTTCTTATCTTTTCACTTCTTTTGAGCTTAAGCTCTGCAG-3′) and 3′ assembly primer 3′ER20 (5′- TTACAACTCGTCATGCTTGTACAGCTCGTCCATGCCG-3′) were used in conjunction with sequence specific assembly primers in assembly PCR from a template of fluorescent protein coding sequence described above. Sequence specific assembly primers were as follows: mOrangeER (5′- GGCCATGTTATTCTCCTCGCCCTTGCTCACGAACTCTGCAGAGCTTAAGCTCAAAAGAA-3′), tdTomatoER (5′- CTCTTTGATGACCTCCTCGCCCTTGCTCACGAACTCTGCAGAGCTTAAGCTCAAAAG-3′), DsRed2ER (5′- GATGACGTTCTCGGAGGAGGCGAACTCTGCAGAGCTTAAGCTCAAAAGAA). A mutagenized ppor sequence was created using the GeMS program [Arabiopsis thaliana codon usage as a template, and a codon cutoff frequency of 0.2. Full length mutatgenized ppor product was assembled from partially overlapping 60-mer oligos designed via the program Gene Design [The coding region of pporRFP was amplified from the vector pGem-T-gbr15 using the forward primer 5′-on et al . Common  program , utilizie Design . All proAgrobacterium tumefaciens GV3850. See the Additional file http://plantsciences.utk.edu/stewart.htm) as follows: mMDC32-DsRed2, mMDC32-tdTomato, mMDC32-mOrange, mMDC32-pporRFP, mMDC32-pporRFP-mut, mMDC32-DsRed2-ER, mMDC32-tdTomato-ER, mMDC32-mOrange-ER, and mMDC32-pporRFP-mut-ER.OFP-containing entry vectors were recombined into the plant binary destination vector pMDC32 [Nicotiana benthamiana was performed as described by Liu et al. [Arabidopsis Col1 ecotype was performed using the floral dip method [Arabidopsis plants were grown in growth chambers and allowed to self-fertilize. Spectrofluorometry analysis was completed on Arabidopsis T2 generation seeds, screened on MSA media containing 50 mg/L hygromycin, resistant plants were transferred to potting media and grown in growth chambers . Plants were 9-week-old rosettes when spectrofluometry was performed. Self-fertilized tobacco plants were grown in the greenhouse . Tobacco plants for spectrofluorometry analysis were started as T1 segregating seeds grown in potting media, screened for fluorescent protein expression using microscopy, transplanted to individual pots and grown to six-weed old stage under greenhouse conditions.Agroinfiltration of u et al.  Stable tu et al.  method. p method . ArabidoEpifluorescent microscopy of plants was performed using the tdTomato filter set: 535/30 nm excitation and 600/50 nm band pass emission or the mOrange filter set: 535/30 excitation and 585/40 nm band pass emission filter . Confocal microscopy images were produced using a Leica TCS SP2 microscope , which allows for adjustable bandwidths for the detected fluorescence. The samples were excited with a 543 nm HeNe laser and fluorescence emission was collected from 555–604 nm for mOrange, 570–620 nm for DsRed and tdTomato, and 590 – 610 nm for pporRFP. Chlorophyll autofluorescence was checked for each sample by exciting the sample with 488 nm light from an argon ion laser and collecting emission from 650–750 nm. When chlorophyll autofluorescence was imaged along with the fluorescent protein, images were collected using sequential scanning to prevent bleedthrough fluorescence. Fluorescence measurements  and ER retention signals (3′). Click here for file


We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX  with a Kd value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation  Despite all advances in molecular medicine, the treatment of metastatic disease is still an unresolved problem, thus the mortality of cancer is still high. In 2013, about 1,600,000 incidences of cancer and 580,000 cancer deaths were registered alone in the United States During haematogeneous metastasis formation tumor cells have to adhere to and transmigrate the endothelium at the target site of the future metastasis. In doing so, they mimic the behavior of normal leukocytes as they use the selectin-mediated migratory pathway of leucocytes in inflammation in vitro process called Systematic Evolution of Ligands by EXponential Enrichment (SELEX) by amplifying DNA or RNA oligonucleotides based on their affinity for a target molecule Aptamers are DNA or RNA oligonucleotides with defined three-dimensional structures displaying high affinity and specificity for target molecules. With respect to their binding characteristics, they are comparable to antibodies. The advantages of aptamers over antibodies are (i) their simple and affordable synthesis, (ii) the possibility for their long-term storage at room temperature and thus (iii) their stability during shipping, (iv) the diverse possibilities of conjugating to other reagents and (v) their lack of immunogenicity We started to select DNA aptamers aiming at their medical application in inhibiting tumor metastasis. We succeeded in selecting an aptamer with high affinity for E- and P-selectin and tested the binding to its target molecules via filter retention assays (FRA). To assess the aptamer's ability to block the interaction of cancer and leukemia cells with selectins, we used laminar flow assays under physiological shear stress conditions, employing human colorectal cancer cell line HT29, human chronic eosinophilic leukemia cell line EOL-1, and primary human pulmonary microvascular endothelial cells (HPMECs). The selected DNA aptamers inhibited the interaction of cancer cells with the endothelium by blocking their selectin-mediated adhesion. Thus the selected aptamers provide an attractive alternative to existing comparable reagents.The initial DNA library used for SELEX was purchased from Metabion. It consisted of a randomized region of 50 nucleotides flanked by constant regions at the 5′- as well as 3′-end  to allow for PCR amplification. Further oligonucleotides were purchased from Invitrogen.2 in phosphate buffered saline (PBS), pH 7.5) including 1 mg bovine serum albumin (BSA)/mL as previously described For biotinylation reaction, 50 μg recombinant human E-selectin/IgG-Fc-chimeras  were incubated with threefold molar excess of sulfo-NHS-LC-biotin (Thermo Scientific) as described in detail in the manufactureŕs manual. Finally, the biotinylated rh E-selectin was immobilized on 5 mg streptavidin-coated magnetic beads  and suspended in selection buffer (3 mM MgClStreptavidin-coated magnetic beads (5 mg) were washed five times with 500 μL washing buffer (1 mg BSA/mL PBS), resuspended in 500 μL PBS containing 0.9 mM biotin, and incubated for 30 minutes at room temperature. The biotin-streptavidin-coated magnetic beads were washed five times with 500 μL washing buffer, resuspended in 1.5 mL PBS including 1.25 mg BSA/mL and were subsequently used for pre-selection.in vitro selection of E-selectin specific DNA aptamers, we used the SELEX procedure (50-CATGCTTATTCTTGTCTCCC-3′) were incubated with 30 nmol pre-selection beads for 30 minutes at room temperature. Streptavidin-binding DNA was removed via magnetic separation. The first round of selection was initiated by incubation of pre-selected DNA library with 50 pmol target beads for 30 minutes at room temperature. After removing of unbound DNA by washing with 200 μL selection buffer, bound DNA was eluted in 55 μL water by heating the mixture to 80°C for 3 minutes. Eluted DNA was amplified via PCR using 1 μM 5′-biotinylated reverse primer (5′-GGGAGACAAGAATAAGCATG-3′), 1 μM forward primer (5′-GCCTGTTGTGfAGCCTCCTAAC-3′), 1.5 mM MgCl2, 200 μM dNTPs in 1×PCR buffer B and 0.05 U FIREPol DNA Polymerase per μL (the latter two purchased from Solis BioDyne). Separation of single-stranded DNA aptamers from double-stranded PCR products was done by streptavidin-coated magnetic beads. Therefore, PCR products were diluted 1∶2 in 2×B&W buffer , added to streptavidin-coated magnetic beads and subsequently washed twice with 1×B&W buffer. After incubation for 15 min at RT the supernatants were removed and beads were re-suspended in 150 mM NaOH following incubation for another 10 minutes at room temperature. The combined supernatants, containing the single-stranded DNA, were neutralized using 100 mM HCl and utilized as start pool for a next selection round. After incubation of DNA single strands with target beads the amounts of washing steps were raised from round to round increasing stringency.For rocedure  includinIsolation of selected aptamers was accomplished by molecular T-A-cloning. Therefore, the vector pUC19-T  was restricted with XcmI (Thermo Scientific) to generate 3′ thymidine overhangs 32P] adenosine-5′-triphosphte  using T4 polynucleotide kinase (Thermo Scientific) and purified via gel extraction followed by isopropanol precipitation. For binding assays, constant amounts of radioactive DNA (<1 nM) was incubated with increasing amounts of corresponding proteins (0–1 μM) for 30 minutes at room temperature in selection buffer. Afterwards protein-aptamer complexes were filtrated  through a pre-equilibrated (15 minutes in 0.4 M KOH) nitrocellulose membrane (Whatman) for 5 minutes in demineralized water at room temperature and then washed in selection buffer. After filtration, the nitrocellulose membrane was dried and exposed for 3 hours to a phosphor imaging screen (Bio-Rad). For quantification we used the One site-Specific binding model (Quantity One software).Filter retention assays (FRA) were used to determine dissociation constants of the aptamer selectin interactions. Therefore, DNA was radioactively labeled with [<$>\raster="rg1"<$>-32P] adenosine-5′-triphosphte as described above and incubated with 100 μL full medium at 37°C. After definite time points (0–720 min), 10 μL of each sample were frozen in liquid nitrogen and subsequently analyzed via 10% denaturing gel electrophoresis.DNA aptamer was radioactively labeled with [?-2. For all assays, HT29 cells were cultivated until 80% confluency; EOL-1 cells were grown in suspension. Primary human pulmonary microvascular endothelial cells (HPMECs) were obtained from Promocell, cultured in endothelial cell growth medium MV supplemented with the corresponding supplement mix as provided by the supplier, and 100 μg penicillin/mL and 100 μg streptomycin/mL. HPMECs were sub-cultured using a specific detach kit (PromoCell) according to the manufacturer’s instructions. All experiments with primary cells were performed during the first six passages.The human colorectal cancer cell line HT29 (purchased from the European Cell Culture Collection) and the human chronic eosinophilic leukemia cell line EOL-1 (from DSMZ) were maintained in RPMI-1640 supplemented with 2 mM L-glutamine, 10% fetal calf serum (FCS), 100 μg penicillin/mL and 100 μg streptomycin/mL (latter reagents were purchased from PAA) at 37°C in a humidified atmosphere of 5% CO2+ and Mg2+  for 30 min at 37°C. In parallel as a control, chambers were coated likewise with 0.02 mg/mL IgG-Fc (R&D Systems). Slides were washed with 50 μL DPBS and incubated with or without aptamer (0.15 mg aptamer/mL DPBS+) for 30 minutes at room temperature. IBIDI microslides capillaries were washed again with 50 μL DPBS+ and then perfused with 1×105 HT29 tumor cells per mL at a laminar flow rate of 8.5 mL/h We analyzed the aptamer-mediated inhibition of selectin ligand interaction under laminar flow conditions representing endothelial shear stress found in post-capillary venules of metastatic organs such as lungs 5 HT29 tumor cells per mL as described above (control without aptamer).To determine the inhibitory potential of our aptamer on shear-resistant tumor cell adhesion towards human pulmonary endothelium, IBIDI treat microslides VI were coated with confluent HPMEC monolayers, which remained untreated or were stimulated with recombinant human (rh) TNFα (Peprotech) for 4 h prior to the flow adhesion assay (10 ng/mL). The stimulated HPMECs were incubated with 0.15 mg aptamer/mL medium for 30 minutes at 37°C and then perfused with 1×10For all statistical analyses Graphpad Prism 6  was used. The values are given as mean ± standard deviation. The One site-Specific binding model (Graphpad Prism 6) was used for calculating the dissociation constants. The two-tailed unpaired t-test was used for comparisons between two groups. Significant differences between two means with p<0.05 are marked with *, very significant differences (p<0.01) with ** and extremely significant differences (p<0.001) with ***.Kd) of 170±65 nM. Sequence analyses of 50 single oligonucleotides derived from this pool revealed no sequence similarities between these oligonucleotides. However, investigating several of these molecules by filter retention assays, we identified the rh E-selectin specific DNA aptamer SDA (5′-GCCTGTTGTGAGCCTCCTAACGATTTGGATTTGGGGTGGAGGGTATGGTTTGTGCTGGCGTTCTCATTTCCCATGCTTATTCTTGTCTCCC-3′) with a Kd value of 98±16 nM  but not at all to streptavidin. Finally, control DNA (5'-GCCTGTTGTGAGCCTCCTAACGAGGAGTGGGCTAAAGGTATGTTGTGGGTTTGGTTCCATGCTTATTCTTGTCTCCC-3'), which differed from SDA in the randomized region, did neither bind to rh E- nor rh P-selectin (Due to sequence similarities between E- and P-selectins selectin .All attempts to minimize the 91 nucleotide long aptamer failed despite applying Mfold P<0.001 vs. E-selectin).We investigated whether the SDA was able to interfere with the adhesion of selectin ligand presenting tumor cells to E- and P-selectins under physiological shear stress conditions. Therefore, rh-selectins were immobilized on a laminar flow micro-chamber and the capillaries were perfused either with HT29 cells in case of human E-selectin or with EOL-1 cells in case of human P-selectin. Adhesive events were recorded. In absence of SDA, HT29 cells adhered on E-selectin-coated chambers with 11.89±4.9 events per minute . To examP<0.05 vs. P-selectin). Upon incubation with control DNA, flow adhesion of EOL-1 cells to P-selectin remained unchanged with 13.00±3.7 events per minute (P<0.01 vs. SDA).To examine if SDA also inhibits EOL-1 cell adhesion to human P-selectin, we immobilized human P-selectin on the chamber’s surface. In the laminar flow assay, unaffected EOL-1 cell adhesion to human P-selectin resulted in 10.42±3.7 events per minute . This vaX and sLeA. First, non-stimulated HPMECs were coated on a micro-chamber. Adherence of selectin ligand-presenting HT29 cells was determined to be 1.50±1.3 cells per minute (−rh TNFα). After E-selectin production was induced by treatment with rh TNFα for 4 h prior to the flow adhesion experiments, the number of HT29 cells adhering to HPMECs increased to 23.17±12.7 events per minute . To investigate the influence of SDA on this cell-cell interaction, rh TNFα-stimulated HPMECs were incubated either with SDA or control DNA. The following laminar flow assay with HT29 cells showed that SDA reduced HT29 adhesion on E-selectin presenting HPMECs significantly to 45% . In contrast, control DNA did not show any significant effect  and HT29 cells. Non-stimulated HPMECs do not present E-selectin at their surface. Upon TNFα-stimulation, HPMECs produce E-selectin and present it on their cell surface allowing for the interaction with HT29 that carry the E-selectin ligands sLeMetastasis formation is the main clinical hurdle in cancer therapy in vitro selection for DNA aptamers binding to E-selectin and identified an aptamer, named Selectin DNA Aptamer (SDA) that comprises high affinities for both, human E- as well as for human P-selectin. For SDA a dissociation constant of approximately 100 nM was determined, which documents the high affinity of the bimolecular interaction between the aptamer and the selectin.We performed an X and sLeA, respectively In vitro binding assays showed an almost similar affinity of SDA for recombinant human P- and E-selectin. Assays with recombinant murine selectins showed that SDA retained affinity for murine selectin as well which was also not unexpected due to the sequence analogy between human and murine selectins (data not shown). We did not prove the possible binding affinity of SDA for L-selectin, because of its lacking importance in the metastasis process. Furthermore L-selectin interacts with other ligands than E- or P-selectin.Due to amino acid sequence similarities between E- and P-selectin and the same affinity for sLein situ or even in vivo applications in coming investigations. This case and the demonstrated stability of the SDA are encouraging features for future successful in vivo studies.As mentioned above, nucleic acids in general and RNA in particular are not remarkably stable in serum due to the presence of various nucleases in vivo studies.As SDA is able to inhibit the adhesion to E- as well as P-selectin, we hypothesized that this aptamer interferes with the lectin domains of the selectins, as those are responsible for the carbohydrate binding in vivo application, since it can only be dealt with synthetic PBS omitting any nucleases To the best of our knowledge, only one thio-modified DNA aptamer (ESTA-1) has been described capable in binding and blocking E-selectin efficiently Compared to the already existing modified aptamers, we have selected a stable non-modified DNA aptamer with inhibitory capacity on selectin-ligand interactions between vascular endothelium and tumor cells by reducing the initial and rate limiting step during transendothelial migration of cancer cells.In addition to oligonucleotide antagonists, alternative selectin targeting inhibitors are presented by a substance class named glycomimetics. These are modified oligosaccharides which mimic natural carbohydrates comprising the capability to bind the mimicking carbohydrate ligand. One glycomimetic that blocks E-, P-, and L-selectin is GMI-1070 dK values of about 100 nM. Its ability to inhibit the interaction of human cancer and leukemia cells to selectins even in a flow chamber simulating the blood flow in physiological environment highlights SDA as a promising tool for further invivo studies. The generation of mice harboring corresponding human selectins for those invivo assays is one important next step and subject of current experiments.In summary, we have selected a non-modified 91 nucleotide long DNA aptamer, SDA, which shows high affinity for human E- and P-selectin with