Hepatitis C virus (HCV) encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors including CD81. In this study, alanine substitutions in E2 were generated within putative CD81 binding regions to define residues critical for viral entry. The effect of each mutation was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp). In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E1 and E2 proteins, CD81 binding profiles, and E1E2 association of mutants were examined.Based on these characteristics, mutants either displayed wt characteristics  or segregated into 4 distinct low infectivity (≤ 50% of wt HCVpp) mutant phenotypes: (I) CD81 binding deficient ; (II) CD81 binding competent, but lack of E1 detection on the viral particle, (despite adequate E1E2 expression in producer cell lysates and proper conformation); (III) CD81 binding competent, with adequate E1E2 expression, incorporation, association, and proper E2 conformation (i.e. no defect identified to explain the reduced infectivity observed); (IV) CD81 binding deficient due to disruption of E2 mutant protein conformation.Although most alanine substitutions within the putative CD81 binding region 1 (amino acids 474–492) displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, E1E2 association and incorporation into HCVpp suggesting that region 1 of E2 does not mediate binding to CD81. In contrast, conformationally correct E2 mutants (Y527 and W529) within the second putative CD81 binding region (amino acids 522–551) disrupted binding of E2 to CD81-GST, suggesting that region 2 is critical to CD81 binding. Likewise, all conformationally intact mutants within the third putative CD81 binding region (amino acids 612–619), except L615A, were important for E2 binding to CD81-GST. This region is highly conserved across genotypes, underlining its importance in mediating viral entry. Hepatitis C virus (HCV) is a primary causative agent of chronic hepatitis. It is a positive-strand RNA virus in the family Flaviviridae that encodes a polyprotein of approximately 3,000 amino acids. This polyprotein is cleaved into ten viral proteins including two transmembrane envelope glycoproteins, E1 and E2, which are heavily N- glycosylated in their N-terminal ectodomains. Like other Flaviviruses, the interactions of the E1 and E2 glycoproteins with cell surface receptors mediate HCV entry via receptor mediated endocytosis [Several cellular surface molecules have been implicated in HCV entry, including: CD81 -6, scavein vitro HCV infectious clone systems [The role and requirement for CD81 in HCV entry has been thoroughly characterized and documented -6,22,23. systems -32. The  systems ,34. On t systems -39. The  systems -39 and t systems ,36.Notably, the amino acid composition of these regions varies significantly between individual viral genomes because HCV undergoes rapid genetic change requiring classification into multiple, naturally occurring genotypes. Amino acid sequences between these different genotypes vary approximately 30% and even within a single genotype, differences can range from 5–10% . Thus, wIn this study, to define residues critical for viral entry, individual alanine substitutions in the three putative CD81 binding regions were generated via site-directed mutagenesis. The strategy was to target residues that are highly conserved across several strains of HCV, as retention of specific residues across genetically diverse genotypes strongly implicates those residues as being important for the interaction between HCV E2 and CD81. Although the hypervariable region II (HVR II) extends into the first putative CD81 binding region targeted (residues 474–482), residues Y474 and D481 are very highly conserved and were therefore also targeted in this study. Susceptible cell lines were challenged with HCV E1E2 pseudovirus (HCVpp) containing the individual mutations to determine the effect of each mutation on HCVpp infectivity. Additionally, producer cell expression and HCVpp incorporation of HCV E1 and E2 proteins, CD81 binding profiles, conformation and E1E2 association of mutants were also examined.in vivo, sequence alignment indicates that there is a high degree of sequence conservation within these three regions, consistent with the idea of these regions having functional importance. Among the three putative CD81-binding regions, region 3 (residues 612–619) is the most conserved, while region 1 (residues 474–492) has the greatest sequence variability, which is expected as the second hypervariable region (HVR II) extends into positions 474–482   Fig.  and Hep3LU) Fig.  cells  see Fig.  demonstrIn contrast, the majority of infectivity defective substitutions in the putative CD81 binding regions 2  and 3  demonstrate little to no CD81 binding, consistent with these regions being involved in CD81 binding. Whereas D535A, R614A and W616A all displayed a disrupted AR3A epitope in this region, indicative of more global structural aberrances Y527A, W529A, Y613A, H617A and Y618A have intact conformation and still are unable to bind CD81, defining these residues as critical for the HCV E2 and CD81 interaction. In addition, both regions 2 and 3 contain at least one group III mutation that reduces infectivity, but maintains the ability to bind CD81 . Thus, it is likely that regions 2 and 3 are also involved in other aspects of viral entry. Notably, this is in agreement with a report by Owsianka et al. , which eThe first and third putative CD81 binding domains targeted, both contain a "WHY" motif. The first "WHY" motif (487–489) has been implicated in dimerization  and the Unlike the mutations within the region 1 "WHY" motif, CD81 binding was disrupted in all three alanine substitutions within the "WHY" motif of the third region see Fig. . MutatioIn conclusion, we have determined that the second and third putative CD81 binding regions are responsible for mediating E2 binding CD81. In the second region, residues Y527, W529 and D535 are critical for CD81 binding. The third putative CD81 binding region comprises a CD81 binding region, as all alanine substitutions, aside from L615A, are unable to interact with CD81. This region is conserved across genotypes, underlining its significance. Finally, we have determined that the first putative CD81 binding region is not a CD81 binding region, as all mutations bind CD81 at wt levels.293T human embryonic kidney cells were maintained in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal calf serum with penicillin, streptomycin. Huh7 and Hep3B cells were maintained in DMEM supplemented with 10% fetal calf serum, penicillin, streptomycin and supplemented with 5 ml Hepes (1 M) (Gibco), and Nonessential amino acids (NEAA) (Gibco). The goat polyclonal antibody against hepatitis C virus (HCV) E2 and the monoclonal mouse antibody for E1 glycoproteins (GP) (genotype 1a) were obtained through ViroStat. The mouse anti-HIV p24 monoclonal antibody was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. Polyclonal rabbit glutathione-S-transferase (GST) antibody was obtained from NeoMarkers. The conformational anti-E2 AR3A antibody was provided by Dennis Burton, PhD from The Scripps Research Institute.The cDNA clone containing E1E2 from genotype 1a strain H77 in pCB6, was kindly provided by Charles Rice, PhD (Rockefeller University). All alanine substitution mutations of the HCV E2 glycoprotein were generated by site-directed mutagenesis with the Stratagene Quick-Change mutagenesis kit according to the supplier's protocols. All mutations were confirmed by DNA sequencing.--E-) into 293T producer cells. One microgram of the wt or mutant glycoprotein expression plasmid and 3 μg of pNL4-3-Luc-R--E- were used to transfect 293T cells (90% confluent) in 6-well plates with polyethylenimine (PEI). The DNA cocktail was added to 200 μl Opti-MEM media and PEI was added at 2× the volume of DNA. The mixture was incubated at room temperature for 15 min. 293T producer cells were rinsed with PBS (no Ca++/no Mg++). Eight hundred microliters of Opti-MEM was added to each well and the PEI/DNA mixture was added. After 5–6 h incubation at 37°C, the DNA cocktail was aspirated off and 3 ml cell culture media was added per well. A minimum of two wells per mutant were done at each time, for a total of 6 ml. The supernatants containing the pseudotyped viruses were collected 48 h posttransfection and filtered through a 0.45 μm-pore-size filter .Pseudotyped viruses were produced by cotransfecting DNA encoding wild-type (wt) or mutant glycoproteins with the Env-deficient HIV vector carrying a luciferase reporter gene  at 72 h post-infection (PI). The luciferase activity was measured with a luciferase assay kit (Promega) and a FB12 luminometer (Berthold detection system) according to supplier's protocol. Each sample was done in duplicate and experiments were repeated at least three times.Huh7 or Hep3B cells were seeded in 12-well plates at a density of 8 × 10++/no Mg++). The protein samples were spun down at 14 k for 10 min to clear cellular debris and transferred to fresh eppendorf tubes. SDS-PAGE loading dye was added to the protein samples, which were subsequently boiled for 5 min at 95°C, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinyl difluoride membrane (PVDF). Membranes were then probed for HCV glycoproteins E1 and E2 and actin, p24 or GST using peroxidase-conjugated secondary antibody and chemiluminescence reagent according to the supplier's protocol . To determine incorporation of E1 and E2 into the pseudotyped viruses, 4 ml of pseudotyped virus was layered onto a 1 ml cushion of 20% sucrose in PBS and centrifuged at 55,000 rpm for 45 min in a SW55Ti rotor (Beckman Coulter) at 16°C. The pelleted pseudovirions were lysed in 50 μl of 1% Triton X-100 lysis buffer and subjected to SDS-PAGE and Western Blot analysis.To determine HCV E1E2 expression and incorporation, 293T producer cells transfected with HCV E1E2/HIV plasmids as described above, were lysed in 0.5 ml of 1% Triton X-100 lysis buffer  and protease inhibitor cocktail  after harvesting virus and rinsing cells with PBS  were added and incubated at 4°C for 1 h. The slurry was spun down for 1 min at 14, 000 rpm and GSH beads were rinsed two times with 0.5% Triton X-100 lysis buffer. SDS-PAGE loading dye was added to the beads and samples were boiled at 95°C for 5 min. Slurry was spun down again and supernatant was collected for SDS-PAGE and Western Blot analysis.The CD81 clone used was kindly provided by Shoshana Levy, PhD (Stanford University). A glutathione S-transferase (GST) fusion protein containing the large extracellular loop (LEL) of human CD81 was generated as previously described . 293T pr2HPO4, 1.8 mM KH2PO4). Fifty microliters of rinsed polyA beads were then added to the cell lysate/antibody cocktail and incubated with gentle rocking at 4°C for 2 h. Beads were washed three times with 100 μl 0.5% Triton lysis buffer. SDS-PAGE loading dye was added to the beads and samples were boiled at 95°C for 5 min. Slurry was spun down and supernatant was collected for SDS-PAGE separation and Western Blot analysis with a polyclonal anti E2 antibody (Virostat).293T producer cells were transfected with 1 μg wt or a selection of CD81 binding deficient or binding competent mutant HCV E1E2 DNA constructs using PEI. After 48 h cells were lysed in 0.5% Triton X-100 lysis buffer with protease inhibitor on ice for 30 min. Cell lysates were clarified by centrifuging at 20,000 × g for 30 min at 4°C. Four-hundred microliters of clarified lysates from these cells were incubated with 1 μg of AR3A conformational antibody  with gen2HPO4, 1.8 mM KH2PO4). Fifty microliters polyA beads were then added to the cell lysate/antibody cocktail and incubated with gentle rocking at 4°C for 2 h. Beads were washed four times with 100 μl PBS with 0.2% Triton. SDS-PAGE loading dye was added to the beads and samples were boiled at 95°C for 5 min. Slurry was spun down and supernatant was collected for SDS-PAGE separation and Western Blot analysis with anti E1 antibody (Virostat).293T producer cells were transfected with 1 μg HCV E1E2 expression plasmid using PEI. Forty-eight h post-transfection cells were lysed in 4% Triton X-100 lysis buffer   with proKBR participated in the design of the study, performed the experiments and drafted the manuscript. BM set up HIV pseudotyping system. SU participated in designing experiments and drafted the manuscript. LR designed the study and participated in drafting the manuscript.


Of 731 stool specimens collected from children with diarrhea in Kathmandu, Nepal, from August 2004 through July 2005, 170 (23.3%) tested positive for rotavirus. Reverse transcription–PCR, including a revised G12-specific primer set, identified 56 (32.9%) as G2P[4] and 39 (23.0%) as G12 with P[6], P[8], or P[4]. Globally, ≈700,000 children each year die of rotavirus diarrheal disease, and most such deaths occur in developing countries . From the rotavirus-positive specimens, genomic RNA was extracted by using a QIAamp Viral RNA Mini kit , and the purified RNA preparations were used to determine G and P types by reverse transcription–PCR (RT-PCR) as described by Uchida et al  were positive for rotavirus antigen. When the distribution of rotavirus-positive cases was examined, 64% occurred in infants ages 3–23 months, whereas only 2.9% occurred in infants <3 months of age. Although samples were not collected in March and April 2005, because of political instability, rotavirus likely circulated among the children in Kathmandu throughout the year. However, a seasonal variation was found in the occurrence of rotavirus diarrhea with a clear peak in January; the rotavirus detection rates varied monthly from 7.3% to 58.6%.Of 170 rotavirus-positive specimens that underwent molecular genotyping, 4 G and 3 P genotypes were found; G2 and G12 together accounted for 62% and P[4] and P[6] together accounted for 74% of their respective genotypes . A totalThe G12 primer pair developed in this study successfully amplified 2 established G12 strains, L26 . Of 39 sG12 strains, including those in the first human cases of infection in the Philippines in 1990 , primers were designed and amplification conditions were optimized so that no cross-reactions occurred with prototype rotavirus strains carrying genotypes G1–11 and G13–14. This primer pair should become a valuable asset for the identification of G12 strains found among the nontypeable specimens in many epidemiologic studies since the primer binding sites are well conserved among G12 strains from diverse locations .


Giardia lamblia parasitizes the human small intestine to cause diarrhea and malabsorption. It undergoes differentiation from a pathogenic trophozoite form into a resistant walled cyst form. Few cyst proteins have been identified to date, including three cyst wall proteins (CWPs) and one High Cysteine Non-variant Cyst protein (HCNCp). They are highly expressed during encystation and are mainly targeted to the cyst wall.G. lamblia genome data base with the sequence of the Cryptosporidium parvum oocyst wall protein as a query and found an Epidermal Growth Factor (EGF)-like Cyst Protein (EGFCP1). Sequence analysis revealed that the EGF-like repeats of the EGFCP1 are similar to those of the tenascin family of extracellular matrix glycoproteins. EGFCP1 and HCNCp have a higher percentage of cysteine than CWPs, but EGFCP1 has no C-terminal transmembrane region found in HCNCp. Like CWPs and HCNCp, the EGFCP1 protein (but not transcript) was expressed at higher levels during encystation and it was localized to encystation-specific vesicles in encysting trophozoites. Like HCNCp, EGFCP1 was localized to the encystation-specific vesicles, cyst wall and cell body of cysts, suggesting that they may share a common trafficking pathway. Interestingly, overexpression of EGFCP1 induced cyst formation and deletion of the signal peptide from EGFCP1 reduced its protein levels and cyst formation, suggesting that EGFCP1 may help mediate cyst wall synthesis. We also found that five other putative EGFCPs have similar expression profiles and similar locations and that the cyst formation was induced upon their overexpression.To identify new cyst wall proteins, we searched the G. lamblia trophozoites into cysts. The results lead to greater understanding of parasite cyst walls and provide valuable information that helps develop ways to interrupt the G. lamblia life cycle.Our results suggest that EGFCPs may function like cyst wall proteins, involved in differentiation of  Giardia lamblia life cycle is differentiation into a cyst form (encystation) that can survive in the environment and infect a new host. Since cystic stages are key to transmission of parasites, this differentiation may be a target for interruption of the life cycle. Synthesis and assembly of the extracellular cyst wall are the major hallmarks of this important differentiation. During encystation, cyst wall structural proteins are coordinately synthesized and are mainly targeted to the cyst wall. However, only a few such proteins have been identified to date. In this study, we used a combination of bioinformatics and molecular approaches to identify new cyst structural proteins from G. lamblia and found a group of Epidermal Growth Factor (EGF)-like Repeats containing Cyst Proteins (EGFCPs). Interestingly, the levels of EGFCPs proteins increased significantly during encystation, which matches the characteristics of the Giardia cyst wall protein. Further characterization and localization studies suggest that EGFCPs may function like cyst wall proteins, involved in differentiation of G. lamblia trophozoites into cysts. Our results provide valuable information regarding the function of a new group of cyst proteins in parasite differentiation into cysts and help develop ways to interrupt the parasite life cycle.The biological goal of  Giardia lamblia is a prevalent intestinal parasite causing waterborne diarrheal disease G. lamblia trophozoite causes malabsorption and diarrhea without penetrating intestinal epithelial cells The cyst form is the infective form capable of survival under hostile environments after excretion cwp1-3 genes and a gene encoding an enzyme in the cyst wall polysaccharide biosynthetic pathway  increases with similar kinetics during encystation trans-Golgi network or Golgi equivalents of the other eukaryotic cells The three known cyst wall proteins (CWPs) have similar expression levels, architectural motifs, and biological properties. Expression of the G. lamblia trophozoites are covered by variant surface proteins (VSPs) that are cysteine-rich type I integral membrane proteins, protecting them from protease and enzyme digestion in intestine G. lamblia genome Cryptosporidium oocyst wall proteins (COWP1–9) in Cryptosporidium parvum and one COWP homolog (TgCOWP1) in Toxoplasma gondiiC. parvum development A group of oocyst membrane proteins have been identified recently, including nine http://www.giardiadb.org/giardiadb/) G. lamblia genome. Sequence analysis revealed that the EGF-like repeats of the EGFCPs are similar to those of the tenascin family glycoproteins. Tenascins are extracellular matrix glycoproteins that are highly expressed during embryonic cell development and reappear during wound repair, nervous regeneration and tumorigenesis G. lamblia genome data base (http://www.giardiadb.org/giardiadb/) To identify new cyst protein, we used the type I domain sequences of COWP8 to screen the G. lamblia genome data base (G. lamblia WB (ATCC 30957), clone C6, were cultured in modified TYI-S33 medium Trophozoites of G. lamblia genome data base (http://www.giardiadb.org/giardiadb/) AY465056) XM_001704009). The EGFCP1 coding region with 360 nt of 5′- flanking regions was cloned and the nucleotide sequence was determined. The egfcp1 gene sequence in the data base was correct. To isolate the cDNA of the egfcp1 gene, we performed RT-PCR with egfcp1-specific primers using total RNA from G. lamblia. For RT-PCR, 5 µg of DNase-treated total RNA from vegetative and 24 h encysting cells was mixed with oligo (dT)12–18 and Superscript II RNase H- reverse transcriptase (Invitrogen). Synthesized cDNA was used as a template in subsequent PCR with primers EGFCP1F (CACCATGATAGCCGCGGCCTTT) and EGFCP1R (CACACATCTACCATCGCG). Genomic and RT-PCR products were cloned into pGEM-T easy vector (Promega) and sequenced .The G. lamblia cell line at the indicated differentiation stages using TRIzol reagent (Invitrogen). For RT-PCR, 5 µg of DNase-treated total RNA from vegetative and 24 h encysting cells was mixed with oligo (dT)12–18 and Superscript II RNase H- reverse transcriptase (Life Technologies). Synthesized cDNA (∼20 ng) was used as a template in subsequent PCR with 20–45 cycles (depending on the genes and primers). Semi-quantitative RT-PCR analysis of total egfcp1 , egfcp1-ha, endogenous egfcp1, cwp1 (U09330), ran (U02589), glycyl t-RNA synthetase , egfcp2 , egfcp4 , egfcp5 , and egfcp6  gene expression was performed using primers egfcp1F and egfcp1R; egfcp13F and egfcp1HAR; egfcp1HAF and egfcp13R; cwp1F and cwp1R; ranF and ranR; glycylrealF and glycylrealR; egfcp2realF and egfcp2realR; egfcp3realF and egfcp3R; egfcp4realF and egfcp4R; egfcp5realF and egfcp5R; and egfcp6realF and egfcp6R, respectively. For quantitative real time PCR, SYBR Green PCR master mixture was used (Applied Biosystems). PCR was performed using an Applied Biosystems PRISMTM 7900 Sequence Detection System (Applied Biosystems). Specific primers were designed for detection of the total egfcp1, egfcp1-ha, endogenous egfcp1, cwp1, ran, glycyl t-RNA synthetase, egfcp2, egfcp3, egfcp4, egfcp5, and egfcp6 genes: egfcp1realF and egfcp1realR; egfcp1HAF and egfcp1HAR; egfcp1HAF and egfcp13R; cwp1realF and cwp1realR; ranrealF and ranrealR; glycylrealF and glycylrealR; egfcp2realF and egfcp2realR; egfcp3realF and egfcp3realR; egfcp4realF and egfcp4realR; egfcp5realF and egfcp5realR; egfcp6realF and egfcp6realR. Two independently generated stably transfected lines were made from each construct and each of these cell lines was assayed three separate times. The results are expressed as relative expression level over control. Student's t-tests were used to determine statistical significance of differences between samples.Total RNA was extracted from All constructs were verified by DNA sequencing with BigDye Terminator 3.1 DNA Sequencing kit and an Applied Biosystems 3100 DNA Analyser (Applied Biosystems). Plasmid 5′Δ5N-Pac was a gift from Dr. Steven Singer and Dr. Theodore Nash egfcp1 gene was amplified using oligonucleotides egfcp1F and egfcp1R. The product was cloned into the expression vector pET Directional TOPO (Invitrogen) in frame with the C-terminal His and V5 tag to generate plasmid pEGFCP1. The pEGFCP1 plasmid was freshly transformed into Escherichia coli BL21 (DE3) pLysE . An overnight pre-culture was used to start a 250-ml culture. E. coli cells were growth to an A600 of 0.5, and then induced with 1 mM isopropyl-D-thiogalactopyranoside (Promega) for 4 h. Bacteria were harvested by centrifugation and sonicated in 10 ml of buffer A  containing 10 mM imidazole and protease inhibitor mixture (Sigma). The samples were centrifuged, and the supernatant was mixed with 1 ml of 50% slurry of nickel-nitrilotriacetic acid superflow (Qiagen). The resin was washed with buffer A containing 20 mM imidazole and eluted with buffer A containing 250 mM imidazole. Fractions containing EGFCP1 were pooled, dialyzed in 25 mM HEPES pH 7.9, 20 mM KCl, and 15% glycerol, and stored at −70°C. Protein purity and concentration were estimated by Coomassie Blue and silver staining compared with serum albumin. EGFCP1 was purified to apparent homogeneity (>95%).The genomic E. coli and used to generate rabbit polyclonal antibodies through a commercial vendor .Recombinant EGFCP1 protein was purified from pac gene were selected and maintained with 54 µg/ml puromycin. Western blots were probed with anti-AU1  or anti-HA monoclonal antibody , and detected with horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG  and enhanced chemiluminescence . Western blots were also probed with anti-EGFCP1 antibody (1/10000), anti-CWP1 antibody (1/10000) Cells transfected with pN series plasmid were selected with G418 as described previously G. lamblia cells. Four ml of the collected medium was incubated with 40 µl of anti-HA antibody conjugated to beads (Bethyl Laboratories Inc.). The beads were washed three times with 500 µl of luciferase lysis buffer (Promega). Finally the beads were then resuspended in sample buffer and analyzed by Western blot and probed with anti-HA monoclonal antibody .5′Δ5N-Pac, pPTEGFCP1▵sp, and pPTEGFCP1–6 cell lines were cultured in growth medium or encystation medium for 24 h. The cultured media were collected and centrifuged at 800×g for 10 min and then passed through a 0.22 µm syringe filter to eliminate 6 cells/coverslip) and then fixed and stained Stably transfected cells were harvested after 24 h in growth or encystation medium under drug selection, washed in phosphate-buffered saline, and attached to glass coverslips (2×10G. lamblia genome data base (http://www.giardiadb.org/giardiadb/) AY465056) XM_001704009)  . A typic1704009)  [18], . WeΔ5N-Pac) . As a coΔ5N-Pac) .We further tried to understand whether EGFCP1 can be released into medium. The EGFCP1 overexpressing cell line (pPTEGFCP1) was cultured in growth or encystation medium for 24 h. Both culture media were then immunoprecipitated with anti-HA antibody and then subjected to Western blot analysis. Intact EGFCP1  was detected in the encystation medium, whereas there was no evidence for EGFCP1 release into the normal growth medium . We did egfcp2  gene is controlled by its own promoter and contains an HA epitope tag at its C terminus , 114815 (EGFCP3), 16477 (EGFCP4), 16322 (EGFCP5) and 8687 (EGFCP6) . RT-PCR terminus  and stabdetected , which idetected . When thdetected . The HA-p<0.05) relative to the control cell line which expresses only the puromycin selection marker (5′▵5N-Pac)  on cyst formation. We found that the cyst number in the EGFCP2  overexpressing cell line increased by ∼1.3-2.0-fold (▵5N-Pac)  and 6E, We further tried to understand whether EGFCP2–6 can be released into medium. The EGFCP2–6 overexpressing cell lines (pPTEGFCP2–6) were cultured in growth or encystation medium for 24 h. Both culture media were then immunoprecipitated with anti-HA antibody and then subjected to Western blot analysis. Intact EGFCP5  was detected in the encystation medium , whereasImmunofluorescence assays showed that the HA-tagged EGFCP2–6 were localized to the ER in vegetative trophozoites (data not shown). During encystation, the HA-tagged EGFCP2–6 were localized to the ESVs in encysting trophozoites . CWP1 waG. lamblia can form a resistant cyst wall composed of proteins and polysaccharides to protect it during infection G. lamblia genome data base. EGFCP1 protein (but not transcript) was expressed at higher levels during encystation and it formed high molecular weight disulfide bonded complexes that were detected in non-reducing gels. We also found that EGFCP1 was localized to the ER during vegetative growth and to the ESVs of encysting trophozoites . CWP1, CWP3, HCNCp and EGFCP1 have acidic pI (3.59–4.72), but CWP2 has a pI of 8.13. HCNCp has an integral membrane domain, but CWP1–3 and EGFCP1 have no transmembrane domains. The CWP1–3, HCNCp, and EGFCP1 protein levels increased greatly during encystation and they could target to the ESVs of encysting trophozoites Toxoplasma gondiihttp://smart.embl-heidelberg.de) (data not shown). The similar localization of EGFCP1 and HCNCp in the ESVs, cyst wall and cell body of cysts The EGF repeats of EGFCP1 are similar to those of the tenascin family of extracellular matrix glycoproteins Ascaris suum to protect the parasites from host digestive enzymes EGFCP1 has two putative TIL cysteine rich domains (Pfam: PF01826) . One is G. lamblia genome data base using the type I domain of COWP8 as a query sequence. This search detected three other open reading frames, including 114161, 14331, and 17328 (data not shown). Open reading frames 14331 and 17328 were annotated as VSP and HCMp, respectively. Open reading frame 114161 was described as a cysteine-rich protein with a C-terminal transmembrane region To date, only COWP1 and COWP8 have been localized to the oocyst wall which are of interest G. lamblia genome data base with the amino acid sequences of the EGFCP1, including open reading frames 113038 (EGFCP2), 114815 (EGFCP3), 16477 (EGFCP4), 16322 (EGFCP5) and 8687 (EGFCP6). They had high expectation values (from 6.6e−149 to 5.0e−64) to EGFCP1. We found that these putative EGFCPs have similar characteristics with EGFCP1: i) they were expressed at higher levels during encystation (except that the expression of EGFCP2 was too low to be detected); ii) they formed high molecular weight disulfide bonded complexes that were detected in non-reducing gels (except that the expression of EGFCP2 was too low to be detected); iii) they were localized to the ER during vegetative growth and partly to the ESVs of encysting trophozoites and to the cyst wall and cell body of cysts; iv) Overexpression of these five putative EGFCPs induced the cyst formation. Like EGFCP1, EGFCP5 can also be released into the medium during encystation. We did not detect the release of EGFCP2, 3, 4, 6 into the medium, possibly because they are too little to be detected. All the six putative EGFCPs (including EGFCP1) have acidic pIs, signal peptides  −73 to 4.1e−10) to EGFCP1 (data not shown). Among these open reading frames, two have been annotated as VSPs. One have been annotated as HCNCp. Twenty-four have been annotated as HCMps. Six have been annotated as cysteine-rich proteins but not classified as HCMps (they are too short and/or have <20 CxC and/or CxxC) http://www.cbs.dtu.dk/services/TMHMM/) (data not shown). Finally, there are six open reading frames without transmembrane domains as predicted by TMHMM  and TUBBR (GGCGGGATCCTTTTATTTCCGCCCGTCCAG) generated a 0.3-kb product that was digested with NheI and BamHI. Another PCR with primers egfcp1BF (GGCGGAGATCTATGATAGCCGCGGCCTTTCTC) and egfcp1MR (GGCGACGCGTCACACATCTACCATCGC) generated a 1.7-kb PCR product that was digested with BglII and MluI and cloned into NheI/MluI digested pPop2NHA NheI/BamHI fragment. The resulting plasmid, pPTEGFCP1, contained the egfcp1 gene controlled by α2-tubulin promoter with an HA tag fused at its C-terminus. For constructing pNEGFCP1, we integrated the AU1 tag sequence into the pNLop2-1 vector CTAGCAAAAACGCGTGATACGTATCGATACATCTAAG) and AUR (AATTCTTAGATGTATCGATACGTATCACGCGTTTTTG) were phosphorylated and annealed. The double-strand DNA fragment was ligated into NheI/EcoRI-digested and dephosphorylated pNLop2-1 to produce pNAU. The egfcp1 gene and its 360-bp 5′-flanking region was amplified with oligonucleotides egfcp1NF (GGCGGCTAGCTCAACAGAGGAGAACGA) and egfcp1MR, digested with NheI/MluI, and ligated in place of the NheI/MluI-excised luciferase gene in pNAU. The resulting plasmid, pNEGFCP1, contained the egfcp1 gene controlled by its own promoter with an AU1 tag fused at its C-terminus. For constructing pPTEGFCP1Δsp, a PCR with oligonucleotides egfcp1dBF (GGGCGAGATCTATGGATGGCGAGGTGGATGTAAAC), egfcp1MR generated a 1.5-kb product was digested with BglII and MluI, and cloned into BamHI/MluI digested pPTEGFCP1. The resulting plasmid, pPTEGFCP1Δsp, contains an egfcp1 gene lacking the coding sequence for the predicted signal peptide sequence . For constructing pPEGFCP2 , a PCR with oligonucleotides EGFCP2NF (GGGCGGCTAGCGAGGAGCTCTCTGCGAAAGAC) and EGFCP2MR (GGGCGACGCGTATTGCCATAGAAACAAGCTCC) (or EGFCP4NF (GGGCGGCTAGCTGCTGGTTTGAAGGAGGCGCT) and EGFCP4MR (GGGCGACGCGTCGCTGCTCTTTCACAATATCC), EGFCP5NF (GGGCGGCTAGCTGGTCTTAAAGCGGCTGATGG) and EGFCP5MR (GGGCGACGCGTAAGTGTTTTTACATTACAGTG), EGFCP6NF (GGGCGGCTAGCTGACCTTGGGGGCAACGGGCG) and EGFCP6MR (GGGCGACGCGTCTGGGAGCTGCTGACTGCACA)) generated a 1.7-kb product was digested with NheI and MluI, and cloned into NheI/MluI digested pPTEGFCP1. The resulting plasmid, pPEGFCP2, pPEGFCP4, pPEGFCP5, or pPEGFCP6, contains the egfcp2, egfcp4, egfcp5, or egfcp6 gene controlled by its own promoter with an HA tag fused at its C-terminus. For constructing pPEGFCP3, a PCR with oligonucleotides EGFCP3NF (GGGCGGCTAGCTTAGAGTAAATTTAAGGGACT) and EGFCP3XR (GGGCGTCTAGACTATACCCATACGATGTTCCAGATTACGCTATCGCTATAGA AACAGGCTCC) generated a 1.7-kb product was digested with NheI and XbaI, and cloned into NheI/XbaI digested pPop2N egfcp3 gene controlled by its own promoter with an HA tag fused at its C-terminus.Schematic representation of plasmid construction. For constructing pPTEGFCP1, a PCR with oligonucleotides TUBNF ((0.03 MB PDF)Click here for additional data file.Figure S2Comparison of TSA417, HCNCp and EGFCP1. (A) Summary of characteristics of TSA417, HCNCp and EGFCP1. TSA417 is a representative of VSPs. (B) Comparison of cysteines of EGFCPs. The number of cysteine-containing motifs from Cx0C to Cx20C is shown. The number zero is not shown.(0.01 MB PDF)Click here for additional data file.Figure S3http://smart.embl-heidelberg.de) in Alignment of the amino acid sequences of the EGF or EGF-like repeats of EGFCP1. The EGF or EGF-like repeats predicted by SMART analysis ((0.06 MB PDF)Click here for additional data file.Figure S4Ancylostoma caninum anti-coagulant precursors (AcSP6 and AcASP5), A. ceylanicum Ascaris-type serine protease inhibitor (Acl), and Apis mellifera (honeybee) chymotrypsin inhibitor (AMCI) . All alignments were carried out using Clustal W 1.83 Alignment of the amino acid sequences of the two putative TIL domains of EGFCP1 and (0.03 MB PDF)Click here for additional data file.Figure S5E. coli using nickel affinity chromatography under native conditions. Purified EGFCP1 protein was analyzed by SDS-PAGE and Coomassie Blue staining. (B) EGFCP1 protein levels in different stages. The wild-type non-transfected WB cells were cultured in growth (Veg) or encystation medium (Enc) for 2, 5, 9, and 24 h and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-EGFCP1 antibody. A representative full-length result is shown  Det(0.11 MB PDF)Click here for additional data file.Figure S6http://smart.embl-heidelberg.de). Two to eleven EGF or EGF-like repeats are present in these open reading frames. Thirty-four to one hundred and forty-six cysteines are present in these open reading frames. They have no transmembrane domains as predicted by TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). Some have acidic pIs and signal peptides (black boxes) as predicted by Signal P Six open reading frames with some characteristics of EGFCPs. Amino acid sequences with similarity to that of EGFCP1 were found in six other open reading frames including 92495, 16833, 14573, 10330, 113268 and 103983.The number and location of the EGF or EGF-like repeats in EGFCPs are predicted by SMART analysis ((0.05 MB PDF)Click here for additional data file.Table S1Primers used for semi-quantitative RT-PCR and quantitative real time PCR.(0.01 MB PDF)Click here for additional data file.


Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% of these strains clustered in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection).Multilocus sequence typing of 169 non-O157 enterohemorrhagic  Escherichia coli (EHEC) strains are a highly pathogenic subgroup of Shiga toxin–producing E. coli (STEC) that cause severe human diseases, including bloody diarrhea and hemolytic uremic syndrome (HUS) , the current standard for phylogenetic analyses of bacteria, indicated extensive allelic variations and homolog recombinations in pathogenic lineages and demonstrated repeated and independent evolution of pathogenic strains (E. coli (HUSEC) (www.ehec.org).A recent phylogenetic analysis of E. coli by API 20 E  and serotyped  as outgroup . The isolation was achieved by using previously described procedures (utgroup  .– (249 were non–sorbitol-fermenting EHEC O157:H7/H– and 106 were sorbitol-fermenting EHEC O157:H–), and 169 (32.3%) belonged to 34 non-O157 serotypes. Because the phylogeny of E. coli O157:H7/ H- has been extensively studied and is well established  belonged to serotypes O157:H7/HMLST analysis of 169 non-O157 EHEC isolates distinguished 29 different sequence types (STs), which clustered into 10 CCs and 12 singletons . The pre– . The nonmotile strains within these serogroups shared the H antigen–encoding fliC gene with the motile strains that expressed the respective H antigen , O145:H28/H– , O111:H8/H– , and O103:H2/H antigen . These 8 antigen .– (fliCH11), which was the most common non–O157 EHEC associated with HUS and clustered into 4 STs as single-locus variants (–), none of which could be successfully serotyped, was matched by its ST to an O typeable strain, indicating a recent conversion from the smooth to the rough strain form.The most frequent serotypes including O26:H11/H–, O103:H2/H–, O111:H8/H–, and O145:H28/H– clustered into the 5 most prevalent STs . Howevervariants . Each of– (fliCH8) were ST16 (CC29). In contrast, the EHEC O111:H10 isolate with ST43 (CC10) shared none of the 7 MLST loci with the O111:H8/H– strains, indicating that EHEC O111 causing HUS originate from 2 different clonal sources. Similar differences were observed between EHEC O145:H25 (ST342)/O145:H– (fliCH25) (ST659) and O145:H28 (ST32). Whereas ST659 is a single-locus variant of ST342, both allelic profiles differ in all loci from ST32.The relationships among members of the different STs and CCs are demonstrated in the stx genotypes and the presence of the eae gene  isolated from HUS patients and all 31 STs identified within these serotypes . The streae gene . PhenotyE. coli O157 serologically is even more important. This exclusion was not always possible because patients’ serum for the investigation of immunoglobulin M anti-O157 lipopolysaccharide antibodies is frequently not available. However, at least some of these strains might represent emerging clones in the human population, such as O111:H10 (www.ehec.org).Most (81.1%) of the non-O157 EHEC clustered into 3 CCs and belonged to a limited number of serotypes. These strains were recovered independently from different regions in Germany over an 11-year period. For the remaining strains, epidemiologic support is not as strong, and the clonal analysis demonstrated that their chromosomal backgrounds are highly divergent from those of CC29, CC32, and CC20. In 14 STs, we have only 1 isolate. In these cases, excluding concurrent or recent infection by Escherichia coli strains and Shigella dysenteriae M1354  as an outgroup generated from allelic profiles based on the eBURST algorithm (12). Each ST is represented by a circle named with its ST, and the corresponding serotypes are given . Black lines connecting pairs of STs indicate that they share 6 (thick lines) or 5 (thin) alleles. Gray lines connecting pairs of STs of increasing line length indicate that the STs share <4 alleles. In addition, the STs and, if applicable, the connecting lines of a clonal complex are shaded in gray.Minimum spanning tree of hemolytic uremic syndrome–associated enterohemorrhagic


N-glycosylations that have been found to be associated with an antigenic change of the human H1N1 virus during the early epidemic of this virus, 2009 H1N1 HA still retains unique three-codon motifs, some of which became N-glycosylation sites via a single nucleotide mutation in the human H1N1 virus. We thus hypothesize that the 2009 H1N1 HA antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in humans. Indeed, amino acid substitutions predicted here are occurring in the recent 2009 H1N1 variants. The present study suggests that antibodies elicited by natural infection with the 1918 pandemic or its early descendant viruses play a role in specific immunity against 2009 H1N1, and provides an insight into future likely antigenic changes in the evolutionary process of 2009 H1N1 in the human population.The pandemic influenza virus (2009 H1N1) was recently introduced into the human population. The hemagglutinin (HA) gene of 2009 H1N1 is derived from “classical swine H1N1” virus, which likely shares a common ancestor with the human H1N1 virus that caused the pandemic in 1918, whose descendant viruses are still circulating in the human population with highly altered antigenicity of HA. However, information on the structural basis to compare the HA antigenicity among 2009 H1N1, the 1918 pandemic, and seasonal human H1N1 viruses has been lacking. By homology modeling of the HA structure, here we show that HAs of 2009 H1N1 and the 1918 pandemic virus share a significant number of amino acid residues in known antigenic sites, suggesting the existence of common epitopes for neutralizing antibodies cross-reactive to both HAs. It was noted that the early human H1N1 viruses isolated in the 1930s–1940s still harbored some of the original epitopes that are also found in 2009 H1N1. Interestingly, while 2009 H1N1 HA lacks the multiple  In April 2009, pandemic (H1N1) 2009 influenza virus (2009 H1N1) was first found in patients with febrile respiratory illness in the United States and Mexico, and has spread rapidly across the world by human-to-human transmission. On the 11th of June 2009, the World Health Organization declared a global pandemic of 2009 H1N1 infection. H1N1 influenza virus caused a pandemic in 1918 (1918 H1N1) The 2009 H1N1 resulted from genetic reassortment between the recently circulating swine H1 viruses in North America and the avian-like swine viruses in Europe In this study, we generated three-dimensional (3D) structures of the HA molecules of 1918 H1N1, its descendent, recent seasonal H1N1 viruses, and 2009 H1N1, and compared their antigenic structures to look for evidence for the existence of shared epitopes for neutralizing antibodies. Since the 2009 H1N1 HA antigenic sites will be targeted by antibody-mediated selection pressure in humans in the near future, we further discuss possible directions of antigenic changes in the evolutionary process of this pandemic virus.It is known that the H1 HA molecules have four distinct antigenic sites: Sa, Sb, Ca, and Cb We then constructed 3D structures of the representative strains of seasonal H1 viruses that had been isolated since 1934, and tracked the amino acid substitutions on their HA molecules . We confN-glycosylation sites  in the globular head region of HA It is well-documented that antigenic changes of HA occasionally result in the acquisition of carbohydrate side chains on the HA molecule N-glycosylation site at the same position in the globular head region of HA . Of these, the Cand1 sites with positions at 177 and 179 had actually become potential N-glycosylation sites in human H1N1 viruses, although these two sites did not exist concurrently Cand1 sites are still present on the surface of CA2009 HA, suggesting the likelihood of additional N-glycosylation at these sites during future antigenic changes of 2009 H1N1 HA.Interestingly, CA2009 also has a single potential on of HA , despited CA2009 . We founIn this paper, we employed 3D structures constructed by a homology modeling method to map amino acid residues on the antigenic sites of HA. When compared to the presentation of simple primary sequences, the 3D presentation has following advantages: (a) There are several amino acid residues that are buried beneath the surface of the HA molecule, even if they are included in the antigenic sites described by the primary amino acid sequences. Since such amino acid residues do not directly contribute to the interaction with antibodies, the surface structures of antigenic sites that are accessible for antibodies can be compared more precisely in the presentation by 3D models than by the primary amino acid sequence. (b) An epitope likely consists of multiple amino acid residues belonging to different antigenic regions presented by the primary amino acid sequence. Such conformational epitopes can be illustrated only by the 3D presentation. (c) One of the purposes of this study is to provide a structural basis to confirm antigenic similarity between the 1918 H1N1 and the pandemic 2009 H1N1 viruses. For this purpose, we employed a homology modeling method rather than simply mapping on the existing crystal structure (e.g. 1918 H1N1 HA), since this method is generally used to generate a 3D structure of a protein molecule if there is no available crystal structure of the target protein Our analysis indicated that 2009 H1N1 had undergone less significant antigenic changes of HA in the pig population than human H1N1 virus since their emergence in the early 1900s. The Centers for Disease Control and Prevention reported that vaccination with recent (2005–2009) human H1N1 viruses was unlikely to provide protection against 2009 H1N1 This virus will soon be subjected to complex immunological selection pressure by the antibody response that will be induced in the human population by vaccination and/or natural infection with homologous viruses, and pre-existing immunity cross-reactive to the early descendants of 1918 H1N1. In the present study, we showed that the antigenic structure of 2009 H1N1 HA might still be similar, at least in part, to that of the 1918 H1N1 HA. We speculate that the 2009 H1N1 HA antigenic sites involving the conserved amino acids will soon be targeted by neutralizing antibodies in humans. Thus, it is of interest to monitor whether these antigenic sites of 2009 H1N1 will undergo similar patterns of amino acid substitutions to those seen in seasonal H1N1 viruses during its epidemic period . Intereshttp://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).Nucleotide sequences for HA genes of SC1918 (AF117241), BR2007 (CY030230), CA2009 (FJ966082), A/Puerto Rico/8/1934/Mount Sinai (AF389118), A/Bellamy/1942 (CY009276), A/Albany/4836/1950 (CY021701), A/USSR/90/1977 (DQ508897), A/Singapore/6/1986 (CY020477), A/Texas/36/1991 (AY289927), and A/Hong Kong/1035/1998 (AF386777) MODELLER 9v6 N-glycosylation sites and candidate codons (Cand1) in HA sequences. The programs are available upon request.Custom-made programs were developed with the Ruby language and used for investigating the numbers of potential Table S1(0.04 MB PDF)Click here for additional data file.Figure S1Amino acid substitutions of seasonal human H1N1 virus HAs shown in close-up views of each antigenic site. The strains used in this analysis are corresponding to those shown in (1.02 MB PDF)Click here for additional data file.File S1PDB file of the homology model of H1 HA  after energy minimizations.(0.20 MB ZIP)Click here for additional data file.File S2PDB file of the homology model of H1 HA (A/Bellamy/1942) after energy minimizations.(0.20 MB ZIP)Click here for additional data file.File S3PDB file of the homology model of H1 HA (A/Albany/4836/1950) after energy minimizations.(0.20 MB ZIP)Click here for additional data file.File S4PDB file of the homology model of H1 HA (A/USSR/90/1977) after energy minimizations.(0.20 MB ZIP)Click here for additional data file.File S5PDB file of the homology model of H1 HA (A/Singapore/6/1986) after energy minimizations.(0.20 MB ZIP)Click here for additional data file.File S6PDB file of the homology model of H1 HA (A/Texas/36/1991) after energy minimizations.(0.20 MB ZIP)Click here for additional data file.File S7PDB file of the homology model of H1 HA (A/Hong Kong/1035/1998) after energy minimizations.(0.20 MB ZIP)Click here for additional data file.File S8PDB file of the homology model of H1 HA (A/Brisbane/59/2007) after energy minimizations.(0.20 MB ZIP)Click here for additional data file.


The goal of the present study was to assess the effect of genetic variability at the APOA5/A4/C3/A1 cluster locus on the risk of metabolic syndrome.APOA5 Ser19Trp, APOA5 -12,238T>C, APOA4 Thr347Ser, APOC3 -482C>T and APOC3 3238C>G (SstI) polymorphisms were analyzed in a representative population sample of 3138 men and women from France, including 932 individuals with metabolic syndrome and 2206 without metabolic syndrome, as defined by the NCEP criteria.The p = 0.03) for APOA5 Trp19 carriers, 0.81 [0.69–0.95] (p = 0.01) for APOA5 -12,238C carriers and 0.84 [0.70–0.99] (p = 0.04) for APOA4 Ser347 carriers. Adjustment for plasma triglycerides,  abolished these associations and suggests that triglyceride levels explain the association with metabolic syndrome. There was no association between the APOC3 -482C>T or APOC3 3238C>G polymorphisms and metabolic syndrome. The decreased risk of metabolic syndrome observed in APOA5 -12,238C and APOA4 Ser347 carriers merely reflected the fact that the APOA5 Trp19 allele was in negative linkage disequilibrium with the common alleles of APOA5 -12,238T>C and APOA4 Thr347Ser polymorphisms.Compared with homozygotes for the common allele, the odds ratio (OR) [95% CI] for metabolic syndrome was 1.30 [1.03–1.66] (APOA5 Trp19 allele increased susceptibility to metabolic syndrome via its impact on plasma triglyceride levels.The  Metabolic syndrome is a complex disease characterized by the ustering of several metabolic disorders ,2. ExcesAPOA5 gene is located on chromosome 11q23 and codes for a 369 amino-acid protein secreted by the liver  and for women . Educational level was assessed by counting the number of years of schooling and classifying the values into 3 categories: primary, secondary/technical and university. Anthropometric measurements included body weight , waist girth  and were performed on subjects in light clothing without shoes. Body mass index was calculated according to the Quetelet equation.Diabetic subjects were identified by fasting glycemia ≥126 mg/dl or antidiabetic treatment. Abdominal obesity was defined by waist girth ≥102 cm in men and ≥88 cm in women. Metabolic syndrome was defined  polymorphism was assessed using the following probes: APOC3-F1: 5'-GAAGGTCGGAGTCAACGGATTACCTGCCTATCCATCCTGCC-3', APOC3-F2: 5'-GAAGGTGACCAAGTTCATGCTACCTGCCTATCCATCCTGCG-3' and APOC3-R: 5'-GCACTGAGAATACTGTCCCTTT-3'. In the sample, 3.5% was undetermined. The APOC3 -482C>T (rs2854117) polymorphism was also assessed in an Amplifluor® system using the following probes: APOC3-F1: 5'-GAAGGTGACCAAGTTCATGCTAGTAAAGGCACAGAAGACCG-3', APOC3-F2: 5'-GAAGGTCGGAGTCAACGGATTGAGTAAAGGCACAGAAGACCA-3' and APOC3-R: 5'-TGAAACCCAGAGATGGAGGT-3'. In the sample, 3.8% was undetermined. The APOA4 Thr347Ser polymorphism was assessed as previously described  for metabolic syndrome were similar in magnitude (data not shown).Table  p = 0.02 and APOAGTACC haplotype  was associated with a 33% greater risk of metabolic syndrome (p < 0.005). The other haplotype  was associated with a 26% reduction in metabolic syndrome risk (p = 0.03). Since the APOC3 and APOA5 SNPs are in different haplotype blocks, the association with metabolic syndrome was tested for the two blocks independently. Using only the SNPs of APO5  and APOA4 Thr347Ser, we found 4 possible haplotypes with a frequency of above 1%. When compared with the common haplotype , the GTA (estimated frequency 0.05) and the CCT (estimated frequency 0.19) haplotypes were respectively associated with a 33% greater risk (p < 0.02) and a 17% lower risk (p = 0.034) of metabolic syndrome. Both associations disappeared after adjustment for triglyceride levels. In contrast, haplotype analyses with the two APOC3 SNPs did not reveal any significant association with metabolic syndrome.Haplotype analyses using the 5 SNPs were performed to assess the relationship with metabolic syndrome. The overall association between the haplotypes and metabolic syndrome was statistically significant (p = 0.011). With respect to the most common haplotype (CTACC), 2 haplotypes were significantly associated with metabolic syndrome. The APOA5 Trp19 allele was associated with an increased risk of metabolic syndrome and that this association could possibly be explained by an increase in plasma triglyceride levels. Furthermore, the APOA5 -12,238C and APOA4 Ser347 alleles were associated with a lower risk of metabolic syndrome – possibly due to their negative linkage disequilibrium with the APOA5 Trp19 allele. In contrast, the APOC3 -482C>T and 3238C>G SNPs did not appear to be related to metabolic syndrome in this French sample.The goal of the present study was to assess the contribution of SNPs in the APOA5/A4/C3/A1 cluster to the risk of metabolic syndrome. Our results showed that the APOA5 Trp19 allele only or both the APOC3 -482T and APOC3 3238G alleles were found to be independently associated with elevated triglycerides. These results are consistent with earlier studies in different population samples that reported similar linkage disequilibrium patterns and associations with triglycerides . In addmulation  and a suAPOA5 Trp19 allele increased the odds ratio for metabolic syndrome, whereas the APOA5 -12,238C and APOA4 Ser347 alleles decreased it. Other studies have reported associations between APOA5 SNPs and metabolic syndrome [APOA5 -12,238C and APOA4 Ser347 alleles merely reflected the negative linkage disequilibrium existing between these alleles and the APOA5 Trp19 allele. Several lines of evidence suggest that the association with metabolic syndrome is mediated by triglycerides. Firstly, the APOA5 Trp19 allele is associated strongly and independently with triglyceride levels but far less so with blood pressure. Secondly, adjustment for triglycerides (but not for other metabolic syndrome-related criteria) abolished the associations between APOA5 or APOA4 SNPs and the risk of metabolic syndrome. Overall, this suggests that APOA5 and APOA4 genetic variability affected susceptibility to metabolic syndrome.The APOC3 -482C>T and APOC3 3238C>G SNPs did not significantly affect the risk of metabolic syndrome in this sample of French origin, despite significant association with triglyceride level. These SNPs do not belong to the same haplotype as the APOA5 Ser19Trp SNP and have an independent effect on plasma triglycerides, which may explain the difference vis-à-vis APOA5. Furthermore and in contrast to the APOA5 Ser19Trp SNP, the APOC3 SNPs were not associated with another component of metabolic syndrome (such as blood pressure) which could explain this difference. Lastly, the prevalence of hypertriglyceridemia was higher in carriers of the APOA5 Trp19 allele (35%) than in carriers of the APOC3 -482T (23%) or APOC3 3238G (22%) alleles and thus increased the probability of fulfilling the definition of metabolic syndrome.In contrast, the APOA5 SNPs and metabolic syndrome [This study has both strengths and limitations. It was performed on a representative sample of the French population and therefore avoided potential selection bias for patients and controls. There were more than 900 subjects with metabolic syndrome, which yields enough statistical power to detect an OR of 1.3 for an allele frequency of 10% at α = 0.05 and β = 90%. Despite this large number of subjects with metabolic syndrome, the statistical power of the study was still not sufficient to account for possible type 1 error due to multiple testing. Therefore, corrections for multiple testing were not applied in the present analysis. In our sample, subjects without metabolic syndrome were, on average, 5 years younger than those with metabolic syndrome. Since metabolic syndrome prevalence increases with age, this may possibly confound (underestimate) the odds ratio associated with the SNPs. However, all our analyses were adjusted for age, in order to account for a possible impact of the latter parameter on the association. Since only a few SNPs per gene were studied, we cannot completely rule out the possibility that other SNPs in different gene loci or specific haplotypes can also contribute to genetic susceptibility to metabolic syndrome. Indeed, other studies have reported associations between syndrome -12. LastAPOA5 Trp19 allele carriers.Over the past decades, genetic variability in the APOA5/A4/C3/A1 cluster has been associated (to varying extents) with variability in plasma lipid, apolipoprotein and lipoprotein levels and with an increased risk of cardiovascular disease. The results of the present study further suggest that the association of SNPs in this cluster with metabolic syndrome may be explained by the propensity of plasma triglycerides to increase in The authors declare that they have no competing interests.DC, AW, PD, JBR, DA, AB, JF and PA participated in the design and coordination of the study. JD and AM carried out the molecular genetic studies, performed the statistical analyses, wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Surveillance of amantadine and oseltamivir resistance among influenza viruses was begun in Hong Kong in 2006. In 2008, while both A/Brisbane/59/2007-like and A/Hong Kong/2652/2006-like viruses (H1N1) were cocirculating, we detected amantadine and oseltamivir resistance among A/Hong Kong/2652/2006-like viruses (H1N1), caused by genetic reassortment or spontaneous mutation. A high rate of oseltamivir resistance in seasonal influenza virus A (H1N1) infection was reported in Europe in the winter of 2007–08  of 697 isolates tested were resistant to oseltamivir. They all carried the H274Y mutation in the NA gene and showed an ≈1,000-fold reduction in susceptibility to oseltamivir . Analysis of the M2 genes showed that 3 (3.4%) of 87 oseltamivir-resistant isolates also carried the S31N mutation associated with amantadine resistance. These 3 viruses were isolated from 2 infants and an elderly woman in different hospitals during 2008 . To eliminate the possibility of mixed strains, we designed a real-time PCR using single nucleotide polymorphisms to detect antimicrobial drug–susceptible strains (with S31 and H274) and drug resistant strains (with S31N and H274Y) in the samples (method available on request). Results showed that the 3 isolates did not possess S31 and H274.To further study the genetic mechanism of emergence of antiviral drug resistance among subtype H1N1 viruses, we performed nucleotide sequencing on a partial segment of the hemagglutinin (HA) gene on 84 (97%) of 87 oseltamivir-resistant and 37 (5%) of 697 oseltamivir-susceptible viruses isolated in our laboratory in 2008. PCR and DNA sequencing of the HA gene were carried out by using the primers H1–1 and H1–2, which flank a fragment of 612 nt of the HA segment .Of 37 oseltamivir-susceptible viruses, analysis of the HA, NA, and M2 genes showed that 20 were susceptible to amantadine and similar to the clade 2B A/Brisbane/59/2007 virus (GenBank accession no. CY030232) , incubated the cultures at 33°C, and examined the cytopathic effect (CPE) daily for 7 days. By observing the timing of CPE appearance and relative progressive rate of CPE, we found no substantial difference in growth efficiency and similar replication patterns for the 4 different types of subtype H1N1 strains.Reassortment of gene segments likely occurred among clade 2B and clade 2C subtype H1N1 viruses while they were cocirculating in Hong Kong. Clade 2B A/Brisbane/59/2007-like viruses also were cocirculating with clade 2C A/Hong Kong /2652/2006-like viruses in about the same proportion during the 2008 influenza season. We identified 3 clade 2B A/Brisbane/59/2007-like viruses (isolated from 1 outbreak) that were oseltamivir-sensitive in their HA and NA genes but carried an amantadine-resistant M2 gene that was similar to clade 2C A/Hong Kong/2652/2006 virus. We also identified 3 virus isolates that were resistant to both amantadine and oseltamivir. The HA, NA, and M2 gene sequences of the 3 oseltamivir- and amantadine-resistant subtype H1N1 isolates were deposited in GenBank (accession nos. FJ227684–FJ227689 and FJ581446–FJ581448).All 3 isolates were clade 2C A/Hong Kong/2652/2006-like viruses, but they acquired oseltamivir resistance through spontaneous mutation or reassortment with a clade 2B A/Brisbane/59/2007-like virus. Gene sequencing results confirmed that no epidemiologic link existed among the 3 patients infected with these isolates.Monitoring of antiviral resistance among influenza isolates showed that resistance pattern changed among subtype H1N1 viruses when different lineages were introduced. While oseltamivir resistance appears to be largely confined to clade 2B viruses and amantadine resistance to clade 2C viruses, oseltamivir- and amantadine-resistant viruses due to spontaneous mutation or reassortment were detectable when clade 2B and 2C viruses were cocirculating. If antiviral resistance markers are combined with sequence data of HA, NA, and M genes, systematic monitoring would make it possible to track the spread of influenza viruses globally and to clarify the underlying mechanism for the spread of such resistance.


GPR110 is an orphan G protein-coupled receptor--a receptor without a known ligand, a known signaling pathway, or a known function. Despite the lack of information, one can assume that orphan receptors have important biological roles. In a retroviral insertion mutagenesis screen in the mouse, we identified GPR110 as an oncogene. This prompted us to study the potential isoforms that can be gleaned from known GPR110 transcripts, and the expression of these isoforms in normal and transformed human tissues.Various epitope-tagged isoforms of GPR110 were expressed in cell lines and assayed by western blotting to determine cleavage, surface localization, and secretion patterns. GPR110 transcript and protein levels were measured in lung and prostate cancer cell lines and clinical samples, respectively, by quantitative PCR and immunohistochemistry.We found four potential splice variants of GPR110. Of these variants, we confirmed three as being expressed as proteins on the cell surface. Isoform 1 is the canonical form, with a molecular mass of about 100 kD. Isoforms 2 and 3 are truncated products of isoform 1, and are 25 and 23 kD, respectively. These truncated isoforms lack the seven-span transmembrane domain characteristic of GPR proteins and thus are not likely to be membrane anchored; indeed, isoform 2 can be secreted. Compared with the median gene expression of ~200 selected genes, GPR110 expression was low in most tissues. However, it had higher than average gene expression in normal kidney tissue and in prostate tissues originating from older donors. Although identified as an oncogene in murine T lymphomas, GPR110 is greatly overexpressed in human lung and prostate cancers. As detected by immunohistochemistry, GPR110 was overexpressed in 20 of 27 (74%) lung adenocarcinoma tissue cores and in 17 of 29 (59%) prostate adenocarcinoma tissue cores. Additionally, staining with a GPR110 antibody enabled us to differentiate between benign prostate hyperplasia and potential incipient malignancy.Our work suggests a role for GPR110 in tumor physiology and supports it as a potential therapeutic candidate and disease marker for both lung and prostate cancer. GPCRs are seven transmembrane receptors that vary extensively in their biological functions. Upon ligand binding, these receptors transduce a signal via a G protein. This fact has been used extensively in pharmacology to select inhibitors of biological pathways. A large fraction of all drugs currently on the market target GPCRs. Drugs targeting members of this integral membrane protein superfamily represent the core of modern medicine .There are many so-called orphan receptors--receptors without a known ligand, a known signaling pathway, or a known function. Despite the lack of information, one can assume that orphan receptors have important biological roles. One of these orphan receptors is GPR110, about which little is known other than its gene structure and potential isoforms that can be inferred from published transcript data. In a large murine retroviral mutagenesis screen, we identified GPR110 as an oncogene.The GPR110 protein contains two protein domains where cleavage can potentially occur: the SEA domain and the GPS domain. Self-cleavage has been reported for the SEA domain in human MUC1  and in rKpnI and XbaI restriction sites to make constructs pcDNA/Iso1 and pcDNA/Iso2. Sequences of these clones matched published RefSeq sequences on NCBI. GPR110 isoform 3 with no stop codon was amplified from pcDNA/Iso1 using the primers 110_F_Kpn and reverse primer 5'-CCCTCTAGACCGAAATTGGGTGACC-3'. A version of isoform 1 with no stop codon was amplified from the pcDNA/Iso1 construct using primer 110_F_Kpn and reverse primer 5'-CCCTCTAGATTCATTTGAGACAAACTGAG-3'. Isoforms 1-3 containing no stop codons were then cloned into a version of pcDNA3.1(+) containing the HA epitope between restriction sites XbaI and ApaIon the pcDNA3.1(+) vector creating constructs Iso1-HA, Iso2-HA, and Iso3-HA.GPR110 isoforms 1 and 2 were amplified from PC-3 cDNA using a set of primers designed to their common 5' UTR and their respective 3' UTR regions. Forward primer 5'-CACCAGTCACAGACTATGC-3' and reverse primer 5'-ACCCGATCGAATACTGAGC-3'  and reverse primer 5'-CAGGGGAATCTCTTGAACCCG-3' . Products from the first PCR reactions were used as templates in a nested PCR with the following primers: forward primer 5'-TTCGGTACCACCATGAAAGTTGGAGTGC-3' (110_F_Kpn), reverse primer 5'-CCCTCTAGATTATTCATTTGAGACAAACTG-3'  and reverse primer 5'-CCTTCTAGAGATTGTGCCATTGCACTC-3' . The PCR products were then cloned into pcDNA3.1(+) (Invitrogen) using Three additional HA-tagged versions of isoform 1 were made using pcDNA/Iso1 as a template with the QuikChangeII Site-Directed Mutagenesis Kit (Stratagene). The following primers were used for the three constructs: HA466: 5'-TTAGAATTATCAGAGCAAAGTACCCATACGATGTTCCAGATTACGCTACCACAGACTGCAACAG-3' and 5'-CTGTTGCAGTCTGTGGTAGCGTAATCTGGAACATCGTATGGGTACTTTGCTCTGATAATTCTAA-3'; HA1036: 5'-CCTGCAGCAGTGGCTACCCATACGATGTTCCAGATTACGCTAGGGGAAACATCACAGC-3' and 5'-GCTGTGATGTTTCCCCTAGCGTAATCTGGAACATCGTATGGGTAGCCACTGCTGCAGG-3'; and HA1393: 5'-GTCTTACTGCGGGAAGAAAAGTACCCATACGATGTTCCAGATTATGCCAGCTCACG-3' and 5'-CGTGAGCTGGCATAATCTGGAACATCGTATGGGTACTTTTCTTCCCGCAGTAAGAC-3'. Construct HA466 has a HA tag located N-terminal to the SEA domain whereas HA1036 and HA1393 contain tags located between the SEA and GPS domains.5 cells per well. Each well was transfected with 4 μg of DNA and 10 μl of Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were incubated in lysis buffer . Cell lysates were run on 4-12% Bis-Tris gels in NuPAGE MOPS buffer (Invitrogen) and transferred to nitrocellulose membranes. Blots were blocked in 5% nonfat dry milk (wt/vol) in TBST  and followed by incubation with primary antibody . After washing with TBST, blots were incubated with an anti-mouse Ig secondary antibody (Southern Biotech #1010-04), and developed with 1 Step NBT/BCIP (Pierce). For deglycosylation reactions, samples denatured in protein loading buffer were treated with 500 units of PNGase F (NEB) in 1× G7 Reaction Buffer and 1% NP40 for 1.5 hours at 37°C.All cell lines were seeded in 6-well plates at a cell density of 4 × 10For cell surface detection, two 10 cm dishes of HEK293 were transfected with Lipofectamine 2000 for each construct according to manufacturer's instructions. Approximately 24 hr post transfection, surface proteins were isolated using the Cell Surface Protein Isolation Kit (Pierce). Purification fractions were assayed by immunoblotting as described above using an anti-GAPDH antibody (Ambion), an anti-β1 integrin antibody , and HA.11 (Covance).Immunoprecipitation of media samples was done using Protein-G agarose (Invitrogen). 200 μl of Protein-G agarose (50% slurry) was washed in lysis buffer and incubated with 5 μg of HA.11 antibody for 1 hr at 4°C. The conjugated beads were washed 3 times with lysis buffer to remove any excess antibody. 15 μl aliquots of beads were incubated with 1 mL of media from transfections of various GPR110 isoforms overnight at 4°C with rocking. Beads were washed in lysis buffer and then boiled for 10 min in SDS Loading Buffer. Samples were then assayed by immunoblotting as described above.Cell lines HEK293T/17, HeLa, PC-3, LNCaP, DU145, A549, NCI-H460, and NCI-H23 were obtained from the American Type Culture Collection. With the exception of LNCaP, all human cell lines used are part of the NCI-60 panel of reference cell lines, for which extensive expression analysis and significant chemical compound screening assays have been done. In addition, these cell lines have been used in xenograft cancer models. All cultures were grown in media supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin. HEK293T/17 cells were maintained in DMEM (Cellgro); PC-3 and A549 in F12K (Hyclone) with 2 mM glutamine; LNCaP, NCI-H460, and NCI-H23 in RPMI with 2 mM glutamine (Hyclone) and supplemented with 10 mM HEPES, 4.5 g/L glucose, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate; DU145 and HeLa in MEM/EBSS (Hyclone) with 2 mM glutamine, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, and 0.1 mM nonessential amino acids.The FirstChoice Human Total RNA Survey Panel (Ambion) was used to screen GPR110 expression in normal human tissues. The Human Lung Cancer TissueScan Real Time Expression Panel (Origene) containing cDNA from 40 lung tumor and 8 normal lung samples was used to screen GPR110 expression in lung tumors. Additional human lung adenocarcinoma RNAs were from Asterand, and normal lung RNAs from Ambion and Biochain. RNA was extracted from frozen mouse spleen and thymus tumor samples and from human cell lines with the RNeasy Mini Kit (Qiagen). Mouse RNA samples were treated with rDNase (Ambion) prior to reverse transcription. For all cDNA synthesis, 500 ng of RNA was reverse transcribed using the SuperScript First-Strand Synthesis System III (Invitrogen).Ct-ΔΔ) were calculated using normal mouse spleen cDNA. GUSB  was used as an endogenous control for all human samples. ABI Taqman probe Hs00228100_m1 (spanning exons 2 and 3) was used to detect human GPR110. The following SYBR primers were used to detect the three GPR110 isoforms in GPR110 positive cell lines: Isoform 1 - 5'-CTTTCTGTCATCATTCGGCAAAAC-3' and 5'-TTGGTTACTGAGGCTGAATTAAGG-3', Isoform 2 - 5'-GAATTCATCTTCTGCTATATACTCC-3' and 5'-CCAACTTGGGCGACAAGAGTGAC-3', Isoform 3 - 5'-CAACAACCTCAGCCAGAGTGT-3' and 5'-ATGCCCTTCTAAGACCATTGTGT-3', and endogenous control GUSB - 5'-CACTGAAGAGTACCAGAAAAGTC-3' and 5'-TCTCTGCCGAGTGAAGATCC-3'.Quantitative PCR (qPCR) was done on the Stratagene MX3000P. All cDNA samples were assayed in triplicates except the Origene Lung Expression Panels, which were run in duplicates for each gene probe. Mouse tumor samples were assayed with the Mouse ACTB-VIC/MGB probe (ABI) as an endogenous control and GPR110 probe Mm00505409_m1 (ABI). Tumor samples containing no integration sites in the GPR110 locus were used as control tumors. Relative expression values  were processed according to standard procedures. LS-A2021 and LS-A2019 , or control rabbit IgG antibodies (Invitrogen) were incubated with the arrays for 1 hour at room temperature. For detection of bound antibody, the arrays were then processed using the SuperPicture kit (Invitrogen) according to the manufacturer's instructions.The genomic locations of the proviral integrations were determined using the splinkerette-based PCR method . This meNot only is the ligand(s) of GPR110 unknown, but also very little is known about the putative transcripts that direct protein synthesis. To date, all published research on GPR110 has dealt with its sequence identification and analysis -9. PubliUsing cDNAs from various primary prostate and lung tumors, we searched for novel PCR products in the extracellular domain of GPR110. A summary of GPR110 isoforms is shown in Figure Sea urchin sperm protein, Enterokinase, and Agrin. Self-cleavage was reported for this domain in human MUC1 [Of the three isoforms, isoform 1 must be the canonical form, with a predicted molecular mass of about 100 kD. Isoforms 2 and 3--truncated products of isoform 1--are 25 and 23 kD, respectively. These isoforms lack the seven-span transmembrane domain and thus are not likely to be membrane anchored. However, the presence of a signal peptide suggests isoforms 2 and 3 may be potentially secreted. Figure man MUC1 . GPR110 We constructed C-terminal HA-tagged versions of the three isoforms to determine their expression and possible secretion . To aid in the detection of potential cleavage sites, we made three additional HA-tagged versions of isoform 1 through site-directed mutagenesis: one construct with an HA tag located N terminal to the SEA domain (HA466), and two others with tags located between the SEA and GPS domains (HA1036 and HA1393) Figure . To checBecause GPR110 contains 19 predicted N-linked glycosylation sites, the bands running at 100 and 80 kD fall short of the expected size. However, the banding pattern of the tagged GPR110 proteins does not indicate the presence of another cleavage site. To estimate the sizes of the non-glycosylated protein, cell lysates from transfected HEK cells were treated with the glycosidase PNGaseF. This treatment reduced the four bands of isoform 2 to a single band of the expected molecular weight of 26 kD (molecular weight plus tag), and isoform 3 to ~25 kD (MW plus tag) Figure . Thus, bAs a member of the GPCR family, GPR110 isoform 1 is expected to be present at the cell surface, but because isoforms 2 and 3 both lack the seven transmembrane domain, they may not be. To determine whether the isoforms reach the cell surface, we transiently transfected HEK cells with constructs encoding isoforms 1, 2 and 3. We then labeled all cell surface proteins with biotin using a crosslinking reagent, lysed the cells, and purified the proteins using avidin-linked agarose beads. Fractions from the cell surface isolation procedure, lysate  using an anti-HA antibody (HA.11) linked to protein-G agarose. Immunoprecipitation led to a 40-fold enrichment of GPR110 over the concentration in the culture media. Cell lysates (L) and the IP fraction of the media (M) were assayed by western blot for the GPR110 isoforms using the HA.11 antibody Figure . In the Having identified isoforms that are encoded by the GPR110 gene, we determined the expression of GPR110 in 15 normal human tissues by quantitative PCR. For this we used a Taqman probe spanning exons 2 and 3 (ExJ2-3) of GPR110, which measures both isoforms 1 and 2; and GUSB, a moderately expressed gene, as an endogenous control gene. The relative expression of GPR110, as compared to the median gene expression of ~200 selected genes within each tissue, was low in most tissues, except in kidney and prostate Figure . BecauseIn a large retroviral insertion screen in mice -14, we hHaving identified GPR110 as an oncogene, we screened various tumor samples for its overexpression. We assayed three human lung cancer cell lines  by qPCR for GPR110 using the ExJ2-3 Taqman probe, and EGFR, a gene frequently mutated or amplified in lung cancer. All three lines express EGFR at levels equal to GUSB (data not shown). Although GPR110 is only moderately expressed in A549 and weakly, if at all, expressed in H460 and H23 Figure , we deciTo confirm our data obtained with qPCR, we performed immunohistochemistry on primary lung cancer tissue cores. Currently there are no commercially available monoclonal antibodies for GPR110. The polyclonal peptide antibodies LS-A2021 and LS-A2019 work well in staining on formalin-fixed paraffin-embedded tissue sections Figure  that werBecause the normal tissue screen indicated that GPR110 may be elevated in prostate tissue with increased age and thus hyperplasia, we also measured GPR110 expression in three commonly studied prostate lines, PC-3, LNCaP, and DU145. In PC-3, a prostate adenocarcinoma cell line, GPR110 expression was high Figure ; LNCaP tAs before with the lung cancer tissue cores, we also performed immunohistochemistry on prostate tissues. On these, we hypothesized that GPR110 expression may enable us to differentiate between benign prostate hyperplasia (BPH) and potential incipient malignancy. On BPH cores, 94% (33/35) were positive for PSA Figure , arrows;In a large retroviral mutagenesis screen, we identified Gpr110 as an oncogene. As an orphan G protein-coupled receptor, this protein has not been subject to experimental study. We identified three isoforms of GPR110 at the protein level by their production in multiple human cell lines. Isoform 1 is cleaved within the SEA domain and isoform 2 is secreted into the culture medium. However, all isoforms are glycosylated, and all are present on the cell surface. At the transcript level, GPR110 is overexpressed in some lung tumors as well as highly expressed in a commonly used prostate cancer cell line. In lung and prostate tumor tissue cores, GPR110 protein is overexpressed, and it may differentiate between benign prostate hyperplasia and prostate carcinoma. This work supports GPR110 as a potential therapeutic candidate and disease marker for both lung and prostate cancer.The authors declare the following financial competing interests: Picobella, LLC is the organization financing the publication of this manuscript and applying for patents relating to the content of this manuscript. AL, BW, LL, and NC have each received a salary from Picobella, LLC, within the last five years. BW and MW hold shares of Picobella, LLC. The authors declare that there are no non-financial competing interests.AL, BW, GB, NC, and LL did the experimental work; AL, BW and MW planned the study, and AL and MW wrote the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/40/prepub


All complexes were characterized by several techniques using elemental analysis , FT-IR, electronic spectra, and molar conductance measurements. The elemental analysis data suggest the stoichiometry to be 1:1 [M:L] ratio formation. The molar conductance measurements reveal the presence of 1:1 electrolytic nature complexes. These new complexes showed excellent antitumor activity against two kinds of cancer cells that are K562 (human chronic myeloid leukemia) cells and Jurkat (human T lymphocyte carcinoma) cells.(6-(cyclohexylamino)-1,3-dimethyl-5(2-pyridyl)furopyrimidine-2,4-dione) abbreviated as CDP was synthesized and characterized. Ti(IV), Zn(II), Fe(III), and Pd(II) metal complexes of this ligand are prepared by the reaction of salts of Ti(IV), Zn(II), Fe(III), and Pd(II) with CDP in acetonitrile. Characterization of the ligand and its complexes was made by microanalyses, FT-IR,  Nitrogen-containing ligands such as Schiffbases and their metal complexes played an important role in the development of coordination chemistry, resulting in an enormous number of publications,ranging from pure synthetic work to physicochemical  and biocPyrimidines representa very interesting class of compounds because of their wide applications inpharmaceutical, phytosanitary, analytical, and industrial aspects, for example,as antibacterial, fungicide , anti-inIt has long been known that metal ions involve inbiological processes of life and have been subject of interest. The modes ofaction of these metal ions are often complex but are believed to involvebonding to the heteroatoms of the heterocyclic residues of biologicalmolecules, that is, proteins, enzymes, nucleic acids, and so forth .From these points of view, it is interesting to studydifferent types of transition metal complexes of these biologically activeligands. In this paper, the synthesis, characterization, and antitumorproperties of a number of the first row transition metal complexes with one ofthe above ligands have been studied.N,N′-dimethylbarbituricacid, 2-pyridinecarbaldehyde, titanium(VI) tetra fluoride, zinc(II) acetatedihydrate, iron(III) chloride hexahydrate, and palladium chlorides were Merck chemicals  and were used without further purification. Organicsolvents were reagent grade. Electronic spectra were recorded by Camspec UV-Visiblespectrophotometer model Wpa bio Wave S2 100. The IR spectra were recorded usingFT-IR Bruker Tensor 27 spectrometer. 1H-NMR and 13C-NMRwere recorded on a Bruker AVANCE DRX 500 spectrometer. All the chemical shiftsare quoted in ppm using the high-frequency positive convention; 1Hand 13C-NMR spectra were referenced to external SiMe4.The percent composition of elements was obtained from the MicroanalyticalLaboratories, Department of Chemistry, OIRC, Tehran.2, with RPMI-1640 Medium HEPESModification with L-glutamine and 25 mM HEPES  supplementedwith 10% heat-inactivated fetal bovine serum (FBS) , 2.7% sodium bicarbonate, and500 mg/L ampicillin.The human chronic myeloid leukemia—K562 cell line—and the human Tlymphocyte carcinoma-Jurkat cell line, used for treatment with thedrugs, were provided. K562 and Jurkat cells were grown at 37°C in anatmosphere containing 5% CON, N′-dimethylbarbituricacid  and 2-pyridinecarbaldehyde  in DMF (3 mL) in ascrew-capped vial was added cyclohexylisocyanide  via a syringe and was shaken for 1 minute. The reaction mixture was then kept for about 30 minutesat room temperature (25°C) and the completion of reaction was confirmedby TLC (EtOAc-hexane 1:2). Then, the resulting crystals were filtered and washed with diethyl ether(20 mL) to yield as light pink crystals. The dried product thusobtained showed a single spot on TLC and was pure enough for all analyticalpurposes  for M = Ti(IV), Zn(II), Fe(III), and Pd(II).All complexes are quite stable and could be stored without any appreciablechange. All complexes were characterized by several techniques using elementalanalyze , FT-IR, electronic spectra, and molar conductancemeasurements. The elemental analysis data suggest the stoichiometry to be 1:1[M:L] ratio formation. The molar conductance measurements reveal the presenceof 1:1 electrolytic nature complexes. The complexes Ti(C19H20N4O3)F4, Zn(C19H20N4O3)(OAC)2,Fe(C19H20N4O3)Cl3, and Pd(C19H20N4O3)Cl2 complexeshave been tested against two human cancer cell lines: K562 and Jurkat. The IC50 cytotoxicity values of the complexes were compared to those found for thestarting organic bases as well as for some of the anticancer agents usednowadays, that are cisplatin and oxaplatin compounds [CDPligand and TiCH20N4O3F4The general method used for testing on antitumorproperties of these compounds is the standard testing method that has beenpreviously described in greater detail.2 atmosphere and 100% humidity, the tested compounds in the concentration ranges of0.1–250 μM for CDP, of 0.1–150 μM for Ti(C19H20N4O3)F4, of 0.1–100 μM for Zn(C19H20N4O3)(OAC)2, of 0.1–200 μM for Fe(C19H20N4O3)Cl3, and of 0.1–97 μM for Pd(C19H20N4O3)Cl2 were added.The incubation lasted for 72 hours and at the end of this period IC90 and IC50 of the dead cells andlive cells were measured by trypan blue. The mechanism by which these complexes act asantitumor agents is apoptosis. IC90 and IC50 values thatare the compounds concentrations lethal for 90% and 50% of the tumor cells weredetermined both in control and in compounds concentrations lethal for both incompounds-treated cultures. The compounds were first dissolved in DMSO and thenfiltrated. The corresponding 50% and 90% inhibitory doses (IC50 and IC90)values are shown in After preincubationlasting for 12 hours at 37°C in a 5% COIt is clearfrom the above discussion that Ti(IV), Zn(II), Fe(III) and Pd(II) complexesand CDP ligand offer a new outlook for chemotherapy. The resultsof antitumor activity show that the metal complexes exhibit antitumorproperties and it is important to note that they show enhanced inhibitoryactivity compared to the parent ligand. Themechanism by which these complexes act as antitumor agents is apoptosis. It hasalso been proposed that concentration plays a vital role in increasing thedegree of inhabitation.


Conserved neutralizing epitopes are considered to be a key role for eliciting broadly neutralizing antibody (NAb). Previously, two conserved neutralizing epitopes of HIV-1 CRF01_AE envelope were identified at amino acid 93-112 of the C1 (C1E) and at 218-239 of the C2 (C2E) regions. To access the potency of antibody directed against conserved epitopes, a monoclonal antibody (MAb) specific to the C2E region was developed and characterized.The immunogenicity of two epitopes was examined by immunizing BALB/c mice with the matching synthetic peptides. One MAb, C2EB5, directed against peptide C2E, was generated by conventional methods, while C1E1 and C1E2 peptides induced slight antibody response in mice. The neutralizing activity of MAb C2EB5 was examined using a peripheral blood mononuclear cell (PBMC) based method and various HIV-1 subtypes including A, B, C, D, and CRF01_AE; C2EB5 was compared with other known neutralizing MAbs  and with sCD4. The exposure of the C2 epitope on native virus was investigated using virus capture by these MAbs.The MAb C2EB5 demonstrated cross-neutralization against various HIV-1 subtypes. The overall potency of MAb C2EB5 against 5 subtypes was ranked in the following order: subtype C> CRF01_AE> subtype D> subtype A> subtype B. The epitope exposure for MAb C2EB5 was also correlated with the neutralization properties of each subtype.This study demonstrates the cross-clade neutralizing activity of a MAb directed against an epitope located in the C2 region of the HIV-1 env and highlights differences in the exposure of antigenic epitopes on the surface of various HIV-1 subtypes. The epitope for this newly identified neutralizing MAb made against a subtype CRF01_AE peptide is particularly exposed in subtype C viral isolates. The great variability HIV-1 antigenic epitopes has been considered to be a major mechanism used by the virus to evade the host immune response. To elicit broadly neutralizing antibody (NAb) against HIV-1, one or more conserved epitopes should be recognized to overcome the extensive antigenic diversity. However, there are few conserved epitopes on the envelope protein that are accessible for specific antibody binding and neutralization. These epitopes have been hidden either by glycosylation or conformational masking . The majThree monoclonal antibodies  directed against gp120 and three MAbs against gp41  have been extensively described in their neutralizing activities. Of the anti-gp120 MAbs, 2G12 recognizes a unique epitope in a carbohydrate-rich region on the outer domain involving the C3-V4 region , whereasgag) and E (env), is an important HIV-1 recombinant form prevalent in Asia. Since we demonstrated some conserved neutralizable epitopes, which are located on amino acids 93-112 (C1 region) and 218-239 (C2 region) of HIV-1 CRF01_AE primary isolates in previous study [The emergence of circulating recombinant forms (CRFs) has been recognized and it is thought that they will make the HIV-1 epidemic more complex. This may have serious issues for the future of antiretroviral therapy and vaccine development. At least 32 circulating recombinant forms have been reported in HIV-1 group M . CRF01_ATwo human MAbs (4E10 and 447-52D) and sCD4 were kindly gifted from the National Institute for Biological Standards and Control  whereas MAb C2EB5 was produced in this study . The MAb50) for each virus stock, both primary isolates and TCLA strains, was titrated on PHA-stimulated PBMCs and the value of TCID50 was calculated by Spearman-Karber method.Five HIV-1 CRF01_AE primary isolates were obtained from National HIV Repository and Bioinformatic Center (Thailand) (NHRBC). These viruses with prefix MENO were collected from HIV-1 seropositive cases from the northern part of Thailand through National serosurveillance in the year 2002, including MENO12 (AY243187), MENO23 (AY243194), MENO24 (AY243195), MENO31 (AY243202) and MENO43 (AY243213). HIV-1 TCLA strains including, 2 subtype A (92RW009 and VI191), 4 subtype B , 2 subtype C (92BR025 and DU174), 1 subtype D (92UG024) and 2 CRF01_AE (NP1525 and NPO3) were obtained from National Institute for Biological Standards and Control . These viruses were thawed from liquid nitrogen and co-cultivated with PHA-stimulated donor PBMCs in IL-2 medium. The viral multiplications were followed up by measuring p24 level . The value of 50% tissue culture infectious dose (TCIDenv nucleotide sequences (C2-V4) obtained from 43 HIV-1 CRF01_AE primary isolates  [NMVEQMQEDVIS whereas amino acid sequence of peptide C1E2 is different from C1E1 at position 101, where an N residue is changed to a K, as underlined in Table The designation of peptides corresponding C1 (C1E1 and C1E2) and C2 regions (C2E) has been described previously . The pepF322214) . The amiSix to eight week old female BALB/c mice  were immunized with synthetic peptides by intraperitoneal injection protocols. Two groups of mice (2 mice/group) were primed with 100 μg peptide/100 μl complete Freund's adjuvant . Two weeks later, the first group was intraperitoneally boosted with 100 μg peptide/100 μl incomplete Freund's adjuvant (IFA)  whereas the second group was boosted with 200 μg peptide/100 μl IFA. For control groups, the mice were immunized with normal saline instead of peptide utilized the same preparation of peptide immunizations. All mice were bled and sacrificed after boosting 3 days, and then all sera was kept frozen. The spleenocytes were separated immediately to hybridize with myeloma cells Ag8.653 by using 41.3% polyethylene glycol  as fusion reagent [2SO4. The absorbance was measured at wavelength 450 nm. The cutoff is defined as the mean value of absorption of serum samples from mice immunized with normal saline or that of fresh culture medium.The flat-bottom 96-well plates were coated with 100 μl of peptide  for overnight at 4°C. Following washing steps, the plates were blocked with blocking buffer  for 1 h. The plates were washed again before incubating with 100 μl of sera from BALB/c mice (for detect Ab response in BALB/c mice) or 100 μl of culture supernatants (for detect Ab production in hybridoma cell culture) for 1 h at 37°C. After washing steps, 100 μl of HRP-conjugated goat-anti mouse IgG  was added and the plates were allowed to incubate for 1 h at 37°C. Then, 100 μl of TMB substrate  was added and the reaction was stopped with 100 μl of 1 M H50) was pre-incubated with equal volume of serially diluted MAb or sCD4 at 37°C for 1 h. After that, 75 μl of PHA-stimulated PBMCs (1.34 × 106 cells/ml) was added and allowed to incubate at 37°C, 5% CO2 for 18 h. The infected cells were then washed twice and re-suspended in 400 μl of IL-2 medium before transferring 200 μl of cell suspension into duplicated well of round-bottom 96-well plate. The replication of the virus in supernatant was followed up by measuring p24 antigen on day 4. For virus control, it was performed by incubating the virus supernatant with HIV seronegative sera. To calculate percent neutralizing activity, p24 level of virus control was subtracted with p24 level of virus containing each dilution of MAb or sCD4 before being divided by p24 level of virus control and then multiplied by 100. The toxicity of MAb C2EB5 on PBMCs was assayed by adding various concentration of C2EB5 into 50,000 PBMCs (106 cells/ml) overnight. The viable PBMCs were counted after washing and stained with vital dye (typan blue).Neutralization test was assayed by a method based on PBMC infection and reduction of p24 gag protein in culture fluids, as described previously . BrieflyThis procedure was modified from the protocol, which has been described previously ,22. BrieAll statistical analyses were performed on non-parametric analysis by program SPSS version 1.5. The association between antigenic exposures and HIV-1 subtypes were determined by Wilcoxon Signed-Rank test. The difference of antigenic epitope exposure of MAb C2EB5 and neutralizing activity was determined by Mann-Whitney U test.Previously, we have investigated the activity of epitopes located at amino acids 93-112 of the C1 and 218-239 of the C2 regions as conserved neutralizable epitopes in HIV-1 CRF01_AE primary isolates by using peptides from C1E1, C1E2 and C2E to inhibit neutralizing activities of sera from HIV-1 CRF01_AE infected LTNPs [50 ± SD 32.00 ± 6.92, > 50, 24.94 ± 21.11, 29.78 and 21.81 ± 6.71 μg/ml, respectively  HIV-1 strain was selected for further study and named MAb C2EB5. This MAb C2EB5 did not show any cross-reaction with peptides C1E1 and C1E2 by the ELISA method. The neutralizing activity of MAb C2EB5 was investigated against 14 isolates of HIV-1 from various subtypes including, subtype A; 92RW009 and VI191, subtype B; MN, IIIB, QH0692, and SF162, subtype C; 92BR025 and DU174, subtype D; 92UG024 and CRF01_AE; NPO3, CM244, NP1525, MENO23, MENO43. HIV-1 isolates used in this neutralization study were selected based on C2 amino acid similarity (CRF01_AE) and difference (other subtypes) to explore the cross-reactivity of the monoclonal antibody (Table _AE HIV-1p < 0.05). This observation was correlated with low neutralizing activity of the C2EB5 MAb against HIV-1 subtype B isolates. HIV-1 subtype C demonstrated high antigenic epitope exposure to MAb C2EB5, which also correlated well with the neutralizing activity of the C2EB5 MAb against subtype C isolates. However, this comparison is based upon analysis of a small number of samples investigated in this study.The MAb C2EB5 was also characterized by performing virus capture to assess the exposure of the antigenic epitopes on the virion; data for C2EB5 was compared with capture data for the 4E10 and 447-52D MAbs and for sCD4. We initially assessed MAb binding to native, intact viruses by a virus binding ELISA . We locaThe antigenic diversity of HIV-1, particularly within the Env glycoprotein, is a major tool used by the virus as an immune evasion strategy, and this poses a major obstacle for the development of an effective HIV-1 vaccine. Therefore, a major focus of vaccine developers has been the difficulty in the elicitation of a broadly neutralizing antibody response. This effort has been directed towards a limited number of conserved epitopes on the envelope glycoprotein of HIV-1 primary isolates. Previously, we attempted to define these conserved neutralizable epitopes in CRF01_AE primary isolates from Thailand. We defined locations of various conserved epitopes and utilized these data to design synthetic peptides. We found that synthetic peptides representing amino acids 93-112 (C1E1 and C1E2) of C1 and 218-239 (C2E) of C2 regions could absorb NAbs in sera collected from Thai long-term non-progressors (LTNPs). Presence of these NAbs in sera of these subjects implies that these amino acids are associated with particular properties of neutralizable epitopes. Recently, these data were re-examined by creating a monoclonal antibody directed against peptide C2E to investigate its neutralization property.in vitro due to their lacking of conformational structure, combined epitopes, and allelic representations [Unfortunately, conserved neutralizable epitopes appear to be poorly immunogenic and Abs against them are rarely produced in infected subjects ,24. The ntations -27. Indentations ,24. The ntations ,29, it wThe reason that MAb C2EB5 was able to neutralize HIV-1 subtype C comparable to CRF01_AE may be due to the homology within the C2 amino acids (218-239) for these 2 subtypes (except at only one position at 227, Table This is the first such study utilizing amino acid sequences of HIV-1 CRF01_AE primary isolates to design MAb. This MAb, in addition to neutralizing CRF01_AE, also cross-neutralizes other subtypes, particularly subtype C, which accounts for the largest population of HIV-1 infection in the world. As described above, high concentration of MAb C2EB5 was required to neutralize subtype B. However, it is our hope that MAbs directed against conserved regions are an alternative way to develop an effective vaccine against HIV. Accordingly, these data may facilitate our understanding of essential characteristics to design an immunogen to induce broadly neutralizing antibodies; this information may assist in the development of an effective HIV-1 vaccine.50: 50% tissue culture infectious dose; TCLA: T-cell line adapted.CRF: circulating recombinant form; gp: glycoprotein; HRP: horse radish peroxidase; IFA: incomplete Freund's adjuvant; IL-2: interleukin-2; LTNP: long-term non-progressor; MAb: monoclonal antibody; NAb: neutralizing antibody; PBMC: peripheral blood mononuclear cell; PHA: phytohemagglutinin; sCD4: soluble CD4; SD: standard deviation; TCIDThe authors declare that they have no competing interests.AS participated in the design of the study, determined immunogenicities, performed MAb, investigated neutralizing activities, analyzed data and drafted the manuscript. JP participated in determining immunogenicities and performing MAb. WK and SS participated in the design of this study and were responsible for data analysis. NT prepared virus primary isolates and TCLA strains. RS conceived of the study, participated in the design of this study, analyzed data and drafted the manuscript. All authors have read and approved the final manuscript.