Diarrhea kills 2 million children worldwide each year, yet an etiological agent is not found in approximately 30–50% of cases. Picornaviral genera such as enterovirus, kobuvirus, cosavirus, parechovirus, hepatovirus, teschovirus, and cardiovirus have all been found in human and animal diarrhea. Modern technologies, especially deep sequencing, allow rapid, high-throughput screening of clinical samples such as stool for new infectious agents associated with human disease.A pool of 141 pediatric gastroenteritis samples that were previously found to be negative for known diarrheal viruses was subjected to pyrosequencing. From a total of 937,935 sequence reads, a collection of 849 reads distantly related to Aichi virus were assembled and found to comprise 75% of a novel picornavirus genome. The complete genome was subsequently cloned and found to share 52.3% nucleotide pairwise identity and 38.9% amino acid identity to Aichi virus. The low level of sequence identity suggests a novel picornavirus genus which we have designated klassevirus. Blinded screening of 751 stool specimens from both symptomatic and asymptomatic individuals revealed a second positive case of klassevirus infection, which was subsequently found to be from the index case's 11-month old twin.We report the discovery of human klassevirus 1, a member of a novel picornavirus genus, in stool from two infants from Northern California. Further characterization and epidemiological studies will be required to establish whether klasseviruses are significant causes of human infection. Picornaviruses are positive-sense ssRNA viruses consisting of eight classical genera and six new proposed genera. They share a common genomic organization with a long 5' untranslated region (UTR) (500–800 nt) containing an internal ribosome entry site (IRES), a single ORF encoding a polyprotein that is proteolytically processed, and a short 3' UTR followed by a polyA tail . Major dKobuvirus is a genus in the family Picornavirus. There are three known kobuviruses: Aichi virus, bovine kobuvirus, and porcine kobuvirus -4. All tRecently, pyrosequencing of stool samples from patients with acute flaccid paralysis from Pakistan was recently used to identify cosavirus, a new proposed picornaviral genus . In thisAs part of an ongoing investigation of pediatric gastroenteritis from Northern California, we identified 141 stool samples that were negative for viral detection by specific PCR for 7 stool viruses  and Virochip, a pan-viral microarray. 141 samples were negative by array and PCR and were subjected to two sequencing runs on a Genome Sequencer FLX without molecular bar-coding. The two sequencing runs gave 937,935 filter pass reads with an average length of 241.7 bp, ranging from 32–503 bp. Of these, 849 reads had an E-value of less than 1e-6 against the Aichi virus genome by TBLASTX. Reads that aligned to Aichi virus assembled into approximately 75% of an expected ~8 kb genome  tail. The base composition of the coding region is 17.8% A, 36.0% C, 20.7% G, and 25.5% U. The genome shares 52.3%, 49.9%, and 49.8% pairwise nucleotide identity with Aichi virus, bovine kobuvirus, and porcine kobuvirus . The latter two-thirds of the UTR comprising the IRES aligns with 68% identity to Aichi virus, the highest identity of any area of the genome. However, the first 250 nucleotides of human klassevirus 1 have only 52% pairwise identity to Aichi virus and do not align to any sequence in GenBank. The first 120 nucleotides of Aichi virus comprising stem-loops A, B, and C (SL-A/B/C) have been shown to be critical for viral replication and encapsidation and the first 50 nucleotides of all previously identified kobuvirus genomes are very highly conserved.To rule out aberrant or chimeric amplification products during the recovery of the 5' UTR, RNAse protection was used to demonstrate that the initial 250 nt of the 5' UTR is on the same strand as the subsequent 500 nt that aligns to Aichi virus  were not present in klassevirus. The 2A protein does not include any H-box/NC protein domains as is apparent in all other kobuviruses as well as some other picornaviruses and does not tblastx with significant similarity to any known sequence [The highest level of sequence identity in the coding region to other picornaviruses (Aichi virus) was found in the 2C, 3D, and VP3 genes. Cleavage sites 2A-2B, 2B-2C, 2C-3A, 3A-3B, 3B-3C, and 3C-3D were all Q-G except 3C-3D which was Q-S. The most conserved region between klassevirus and Aichi virus was in the putative nucleotide binding domain of the 2C helicase (VVYLYGPPGTGKSLLASLLA). A conserved tyrosine was identified in the third position of the 3B-VPg. The conserved 3C protease active site motif that is GXCGG in the enterovirus genus was present but changed to GLCGS, the same as in Aichi virus. The conserved KDELR, YGDD, and FLKR motifs were present in the 3D polymerase . No tand7 and 1 × 107 viral genomic copies were present per 1 mL of stool, respectively.Previously described universal kobuvirus primers failed to detect klassevirus in our collection of 751 stool samples (data not shown). New 32-fold degenerate pan-kobuvirus/klassevirus primers were designed to amplify a 200 bp amplicon from the 3D gene and used for RT-PCR screening of 751 stool specimens from symptomatic and asymptomatic individuals from Northern California under code. One additional human klassevirus 1 was detected with screening . After screening, samples origins were decoded. As it happens, the positive sample was collected from a member of the family that yielded the original sample from which klassevirus was identified. Both children were 11-month old males. Sequence recovered from the 3D gene and 5' UTR was >99% identical between the two samples. No additional kobuviruses or picornaviruses were recovered from the PCR screening using these primers, so it is not known whether these primers are, in fact, pan-kobuvirus/klassevirus primers. The Virochip (v4), a pan-viral microarray designed to detect all known viruses as well as novel viruses on the basis of sequence homology, was unable to detect the novel picornavirus genus. Quantitative PCR from samples 02394-01 and 02394-03 indicated that approximately 5 × 10This study presents the discovery and characterization of a novel picornavirus, human klassevirus 1. Klassevirus has a typical picornavirus organization with a ~700–800 bp 5' UTR, long open reading frame and, ~100 bp 3' UTR. The phylogenetic relationship of the new genus to other picornaviruses by amino amino acid sequence is shown in Figure Similar to cosavirus, this virus was identified through deep sequencing of stool, a strategy to identify novel viruses that are too divergent to be identified by other methods. Without filtering or selecting for viral particles, we were able to obtain sequence for 75% of the klassevirus genome based on TBLASTX against Aichi virus. Aligning all the pyrosequencing reads to the complete recovered genome of klassevirus indicated that 95% of the viral genome could be identified from the deep sequencing run Figure . This inUnlike previously identified kobuviruses, the first 140 nt of human klassevirus 1 is highly divergent. Published studies of Aichi virus suggests the first three stem-loop structures are required for positive and negative strand replication as well as encapsidation ,8. The tDespite the sequence divergence at the 5' end of its genome compared to known kobuviruses, human klassevirus 1 contains two stem-loops and a pseudoknot structure within the first 140 bp of its genome. Human klassevirus 1 also shares a high degree of sequence identity with Aichi virus throughout the remainder of the 5' UTR, indicating that IRES structure and function is likely preserved between the two viruses. This is especially interesting when compared to porcine kobuvirus which shares the conserved first 50 bp to the kobuvirus 5' UTR but has a hepacivirus/pestivirus-like type IV IRES ,10. The Although it remains to be determined whether human klassevirus 1 causes bona fide human infection, the data are suggestive. Screening using a newly developed PCR primer pair designed to amplify any klassevirus or kobuvirus found klassevirus only in two young children from the same family. The virus was present in relatively high copy number in both samples, suggesting that replication occurs in the gut and that human klassevirus 1 is not merely a passenger virus. However, both infants were asymptomatic at the time virus was present in their stool. The low prevalence rate is akin to that of Aichi virus, which is a rare known cause human gastroenteritis. Bovine and porcine kobuvirus, on the other hand, have both been found in healthy stool and bovine kobuvirus has been found in the serum of infected cattle . It remaFuture studies to determine a possible link to disease in humans and any unique characteristics of the viral life cycle will be required. Viral culture on human cell lines, especially those from the gastrointestinal tract, could be suggestive that the virus is competent to replicate in human cells and that humans could be a bona fide host of klassevirus. Culture would also help elucidate the importance of different secondary structures in the divergent 5' UTR as well as determine cleavage sites of the polyprotein. Further epidemiological screening and serological assays will be necessary to understand the diversity within this possible genus, the prevalence of klassevirus, and the average age of those infected. Notably, both of the cases in this study were 11 months old, which is approximately the age at which maternal antibodies decline.We have detected a new picornavirus genus in stool specimens from two twins and sequenced the viral genome. Further characterization will be required to determine the full extent to which this agent is implicated in human disease, and the spectrum of illnesses to which it may be linked.The cohort has been described previously .Stool suspensions were created by mixing 2 mL of PBS with stool (100–300 mg). 100 uL of stool/PBS mixtures were further diluted in 900 uL of PBS and extracted using the PureLink Viral RNA/DNA 96-well kit . Total RNA/DNA was randomly amplified using the round A/B protocol with 25 cycles of PCR before 454 pyrosequencing .Specific RT-PCR was done with Qiagen One-Step RT-PCR kit using 4 uL H2O, 2.5 uL 5× Buffer, 2.5 uL Q solution, 0.5 uL dNTP, 0.5 uL RT/Taq solution, 0.75 uL of F/R 10 uM primer, and 1 uL of stool total RNA. Conditions were 50C for 30 min, 95C for 15 min; 40 cycles of 95C for 30 sec, 50C for 30 sec, 72C for 1 min/kb; and final extension at 72C for 7 min. Degenerate pan-kobuvirus primers targeting the 3D region used for screening were kvF 5'-GYT TTG AYG CYA CCM TYC C-3' and kvR 5'-SGT GTT GAK GAT GGA RGT SSC-3'. Primers for genome recovery are listed in Additional file 3' RACE was done with an adapter-linked oligo-dT primer. Due to problems with secondary structure, 5' RACE was done with a combination of a 5'RACE kit (Invitrogen) and by using the reverse transcriptase activity of Tth polymerase (Promega) at 70C, TdT with 0.2 mM dATP (NEB) for 10 minutes at 37C, and the same adapter-linked oligo-dT primer.A total of 141 amplified cDNA libraries that were negative by array and PCR were cleaned via Ampure beads (Agencourt) and quantitated on the Nanodrop spectrophotometer. Aliquots of 200 ng from each sample were combined and sequenced on the Genome Sequencer FLX (Roche) using the Shotgun Sequencing protocol. Sequence analysis of Genome Sequencer FLX data was filtered against human and bacterial sequences using BLAT before unbiased BLASTn and tBLASTx (W3) searches against the BLAST nr database.Total RNA from sample 2394-03 was amplified using 40 cycles of RT-PCR with primers kv1F 5'-CCC TTT CGA CCG CCT TAT-3' and kv761R 5'-CAG CCA ACG AAC TCG AAA AT-3'. The 761 bp amplicon was gel purified and cloned using TOPO TA cloning kit (Invitrogen). TOPO TA plasmid containing the 761 bp insert was sequenced to ensure the correct sequence and insert direction. 1 ug of plasmid was linearized with HindIII and linearly amplified for 10 minutes using MaxiScript (Ambion) kit with 825 nM alpha-P32-UTP and 15 uM unlabeled UTP such that 80 bp of vector sequence flanked both side of the 5' UTR insert. The 920-bp radiolabeled probe was gel-purified on a denaturing 4% polyacrylamide gel following the RPA III kit (Ambion) protocol and quantified using scintillation counting. 80,000 cpm of probe were hybridized with ~50 ng of stool total nucleic acid or aRNA and 5 ug of yeast tRNA and digested with RNase A/T1 using the streamlined protocol from the RPA III kit. The entire sample was run on a denaturing 4% polyacrylamide gel and visualized using a phosphoimager.9 – 100 copies per reaction). Quantitative PCR was performed on a Mx3005P (Stratagene) under the same RT-PCR conditions listed above for screening PCR, with the exception of Tm 50C for 45 sec, extension at 72C for 45 sec, addition of 1× Sybr Green, and addition of melt curve analysis.In order to ascertain whether klassevirus underwent replication in the gut or was merely a passenger virus, quantitative PCR was used to determine viral titer in stool. A 134-bp amplicon was generated by RT-PCR using the same conditions above for screening PCR using primers kv3918F/kv4041R and used for standard curve generation (10ALG owns equity in Illumina, Inc.ALG carried out the initial Virochip and PCR screening, cohort maintenance, 454 sequencing, sequence analysis, full genome recovery, RT-PCR screening, RNAse protection assay, and drafted the manuscript. CR carried out initial Virochip and PCR screening, cohort maintenance, sequence recovery, and sequence analysis. CC carried out maintenance of the cohort and sequence analysis. JP and TD gathered the cohort and organized the data from the cohort. ALG, CR, CC, DG, and JD conceived of the study and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.RT-PCR Primers for Klassevirus genome recovery. Table of RT-PCR primers designed from pyrosequencing reads that were used in this study for klassevirus genome recovery and screening.Click here for file


To the Editor: Chikungunya is a mosquitoborne alphavirus in the family Togaviridae. Recently, a chikungunya virus epidemic that affected thousands of persons occurred in islands in the southwestern Indian Ocean, including Mauritius and Reunion , mild thrombocytopenia (platelet count 105 × 109/L), and abnormal liver function test results . Malaria blood films and dengue serologic results were negative. A validated, in-house, generic alphavirus reverse transcription–PCR (RT-PCR) showed positive results 24 hours after collection of blood when the patient was admitted.The patient had a temperature of 39°C, bilateral conjunctivitis, tender and markedly swollen Achilles tendons, a swollen left ankle, and a maculopapular rash that involved the left forefoot and anterior portion of the shin. Initial blood examinations showed leukopenia .Chikungunya virus was identified by a heminested RT-PCR for the nonstructural protein 4 gene. First-round primers were AlphaF1  and AlphaR . Second-round primers were AlphaF2 (nt 7480–7501) (5′-TGGNTBAAYATGGAGGTIAAG-3′) and AlphaR. Sequencing of the second-round product identified the virus. A 339-bp fragment (GenBank accession no. DQ678928) had 97% identity with the African prototype strain S27 isolated in Tanzania (Tanganyika) in 1953 (AF369024) and 100% identity with viral sequences from Reunion Island in 2006 (DQ443544).Knowledge of distant epidemics aids clinical recognition of infections not commonly seen in Australia. Websites and electronic bulletins  are a conduit of information. On the basis of this case-patient, those in Australia became more aware of the chikungunya virus epidemics affecting the islands of the southwestern Indian Ocean.Laboratory diagnosis of chikungunya virus infection is usually serologic. However, alphavirus infections not endemic to Australia are unlikely to be diagnosed serologically because specific assays are generally available only for those viruses known to circulate in Australia. Because viremia of alphaviruses is brief, success of RT-PCR depends on early admission and clinical recognition of infection (Aedes, including Aedes aegypti, Ae. furcifer-taylori, Ae. luteocephalus, Ae. albopictus, and Ae. dalzieli (Aedes and Culex (Rapid establishment of a definitive diagnosis had substantial benefits that included management of the febrile patient, reduced need for further investigations, and better prognosis. Infection caused by an introduced arbovirus may have important public health implications in Australia. Because immunity in the Australian population is unlikely, consideration must be given to the potential for transmission of the virus to caregivers and the local community. Chikungunya virus is commonly spread by mosquitoes of the genera This case highlights the potential for exotic viruses to be introduced into Australia by visitors or returning travelers and the utility of molecular testing for their rapid detection. The generic nature of the RT-PCR enabled detection of an alphavirus with subsequent specific identification by sequencing. Rapid identification and differentiation in a public health setting minimized the potential for spread of the virus.


We demonstrated the feasibility of a new methodology for isolating colonocytes from feces. To reduce costs and improve the recovery rate of colonocytes from feces, we attempted to develop new immunomagnetic beads. Several sizes of magnetic beads were prepared and tagged with a monoclonal antibody against EpCAM. We made several new monoclonal antibodies against EpCAM, and each monoclonal antibody was tagged to the magnetic beads. In the simulation, the most efficient recovery of HT-29 cells was obtained using the smallest size of beads. Also, beads tagged with a monoclonal antibody with a higher affinity against EpCAM had a higher recovery rate. Similar results were obtained when the smallest size of beads with the highest-affinity monoclonal antibody was applied to clinical samples. The newly developed immunomagnetic beads may be useful for isolating colorectal cancer cells from feces, enabling the cytological or molecular biological diagnosis of CRC. Colorectal cancer (CRC) is one ofthe most common malignancies worldwide. CRC is the third leading cause of cancer-relatedmortality and the second leading cause of cancer-related incidence. Nevertheless,the survival rate of patients with CRC is high if this cancer can be diagnosedand surgically resected at an early stage . Thus, tThefecal occult blood test has been used widely as a screening test for CRC –4. HowevPreviously,we reported that cancer cells remain viable in the feces and can be isolatedfrom naturally evacuated feces using an immunomagnetic bead methods that we developed, 21. AftThese figures were relatively satisfactory, butthe numbers of cells isolated from the feces were not very high and, inaddition, commercially available immunomagnetic beads are relatively expensive.In this context, to improve the function of immunomagnetic beads for retrievingcolonocytes from naturally evacuated feces and to reduce the cost performanceof this method, we attempted to develop new immunomagnetic beads and toevaluate their usefulness.μm). Beads with each different size weretagged with commercially available antihuman EpCAM monoclonal antibody (mAb),VU-1D9   see . Brieflytion see .Immobilization of the antibody onto the beadswas carried out using a 2-step reaction. First, an antimouse IgG mAb wascoupled to the bead using the amino groups of the antibody. One hundredmicrograms of the goat antimouse IgG  were addedto 1 mL of 1 wt% bead suspension in 0.1 M borate buffer, pH 9.5, and reacted for24 hours at 37°C. After the elimination of uncoupled antibodies throughrepeated washing with TBS-T , anti-EpCAM mAb was immobilized on the bead as a second step.Twenty micrograms of commercially available anti-EpCAM mAb, VU-1D9, were addedto 1 mL of 1 wt% bead in TBS-T and mixed for 1 hour at room temperature. Afterthe reaction, uncaptured antibody was washed away with TBS-T and stored at 4°Cuntil use.2 atmosphere at 37°C, hypoxanthine-aminopterin-thymidine(HAT) medium was added to start HAT selection. Hybridoma clones were culturedfor an additional 8 days, and then the culture media were assayed for specific antibodyproduction using the ELISA method on Maxisorp microtiter plates coated with the antigen. Briefly, an aliquot of undiluted hybridoma culture supernatantwas added to the antigen coated wells. After 1 hour of incubation and a subsequentwash cycle, an appropriate dilution of peroxidase-conjugated rabbit IgG to mouse IgG reagent was added. The enzyme-linked IgG binding was detectedusing an H2O2 and o-phenylenediamine substrate solution. The colorintensity was measured automatically using a SpectraMax Plus microplatespectrophotometer .The ELISA positive hybridoma cells were cloned by limiting dilution in 96-wellculture plates and established as stable hybridoma cells.Next, we developed new antihuman EpCAM mAbs inorder to obtain an antibody with higher affinity against EpCAM. A recombinanthuman EpCAM/Fc chimera was used as an immunogen. The antigen (0.1 mg) was mixed with complete Freund'sadjuvant  and injected intraperitoneally (IP) into BALB/c mice . Subsequent injections were made using an RIBIadjuvant system, MPL + DM emulsion  every three weeks for a totalof 7 times. Three weeks later, the mice were given an intravenous (i.v.) boosterinjection of 0.1 mg of the antigen in phosphate buffered saline. Three dayslater, spleen cells from the immunized mice were fused with myeloma cells(P3X63Ag8.653) at a ratio of 7:1 in 50% polyethylene glycol 4000  in RPMI 1640 at room temperature for 1 minute. Aftercentrifugation, the cells were pelleted, washed, resuspended in RPMI 1640containing 10% NCTC 109, 20% FCS, and 50 ng/L of mouse IL-6 (R&D Systems),and plated in flat-bottomed 96-well tissue culture palates . Following overnight incubation in a humidified 5% COThe isotype of the mAb was determined using an MONO Ab-ID KIT according to the manufacturer's instructions. Ascites fluids were obtained frompristine-primed CD1-Foxn1nu mice (Charles River Japan) injected with each mAbproducing hybridoma clone. Immunoglobulin G was separately purified from eachascites fluid sample using protein G affinity chromatography .The purified IgG fractions were used for further characterization and were evaluatedfor their reactivity. The immunoglobulin concentration was determined bymeasuring the absorbance at 280 nm using an extinction coefficient of 1.38 formouse IgG.The affinities to EpCAM of the newly developed antihumanEpCAM mAbs were analyzed using flow cytometry with a BD FACSCalibur .HT-29 cells and UMUC-3 cells were used as an EpCAM positive control andnegative control, respectively. The antihuman EpCAM mAbs and nonspecific mouseIgG1 were directly labeled using Zenon mouse IgG labeling kits  according to the manufacturer's instructions.Finally, the new antihuman EpCAM mAbs were conjugatedto the optimal size of magnetic beads chosen in the present study see .5 HT-29 cells were added to the homogenized solution andfiltered through a nylon filter (pore size: 512 μm). The HT-29 cells wereretrieved using 80 μL of several immunomagnetic beads, and the mixtures wereincubated for 30 minutes under gentle rolling conditions at room temperature.The mixtures on the magnet were incubated on a shaking platform for 15 minutesat room temperature. Then, the supernatant was removed, and the retrieved cellswere counted using a NucleoCounter .Asimulation was conducted to determine the optimal bead conditions for the recoveryof HT-29 colorectal cancer cells using a previously established method . BrieflyFinally, to determine the optimal immunomagnetic beads, HT-29colorectal cancer cells were retrieved using these immunomagnetic beads, andthen the cell-bead complexes were fixed and stained using papanicolaou stain.FromMarch 2007 to July 2008, 40 patients with histologically confirmed colorectalcancer were enrolled in the present study. Nineteen of the 40 patients were enrolledin a study to analyze the optimal bead size from March 2007 to September 2007. Then,21 of the 40 patients were enrolled in a study to evaluate the quality of the antibodyfrom October 2007 to July 2008. All the patients had undergone surgicalresection of their primary cancer at the National Cancer Center Hospital, Tokyo, Japan.All patients were thoroughlyinformed of the content of the study, and provided their writtenconsent to participate inthe study. The study was approved by the Institutional Review Board of the NationalCancer Center, Japan.Before surgical resection, naturally evacuated fecal samples wereobtained from patients with colorectal cancer. Each fecal sample was dividedinto 2-gram samples for use in the evaluation of several immunomagnetic beadswith different sizes and different affinities of the EpCAM antibody. The fecalsamples were prepared as described in the simulation section. Colonocytes isolatedfrom the feces were stored at −80°C until genomic DNA extraction.GenomicDNA was extracted from colonocytes isolated from feces using an Allprep minikit according to the manufacturer's instructions.Forthe genomic DNA analysis, we targeted a consensus sequence of human Alu repeats.The sequences for the Alu primers and probe used in this study were as follows:forward primer, 5′-TAGTAGAGACGGGGTTTCACCTTG-3′; reverse primer, 5′-AGCTTGCAGTGAGCCGAGAT-3′;probe, 5′-GAGAATGGCGTGAA-3′. The reporter dye at the 5′-end of the probe wasFAM, and the quencher dye at the 3′-end was MGB.μL of a templateDNA, 10 μL of TaqMan Fast Universal PCR Master Mix , 500 nM offorward and reverse primers, and 250 nM of probe in a total reaction volume of20 μL. Real-time PCR amplification was performed using precycling heat activation at 95°Cfor 20 seconds, followed by 25 cycles of denaturation at 95°Cfor 3 seconds, and annealing/extension at 62°C for 30 seconds in an Applied Biosystems 7500 Fast Real-Time PCRSystem (Applied Biosystems). The absolute quantification of genomic DNA in eachsample was determined using a standard curve with serial dilutions (10 ng to 100 fg) of TaqMan control genomic DNA (Applied Biosystems). A negative control(without template) was run in each reaction plate.Thereaction mixture for the genomic DNA analysis consisted of 4 P < .05 were considered statistically significant.Thecell retrieval rate for each group was analyzed using a Tukey-Kramer multiplecomparisons test. Statistical differences in the cell retrieval abilities of thenew immunomagnetic beads were determined using a two-sided Mann-Whitney U test.Statistical analyses were performed using StatView Ver. 5 for Windows .Values of μm, respectively (see P = .0001) and beadsC (P < .0001)  see . Microsc001) see .μm) of magnetic beads  showed that the affinity of clone 1-2 was 3 times higher thanthat of nonspecific mouse IgG1, while the affinities of clones B8-4 and B8-7 were500 times higher than that of nonspecific mouse IgG1 see . Theaffeads see .P < .0001) and beads F (P < .0001) , 73.5 ± 1.96, and 71.2 ± 3.39, respectively. The recovery rate using beads D was significantly lower thanthose using beads E (001) see.P = .09). However, the amount of extracted DNA using beads A was significantly higher than that using beads C (P = .02).Meanwhile, in the clinical studyexamining cell retrieval ability according to antibody affinity, the medianamount of DNA from 2-gram stool using beads D, E, and F was 0.75 ng (range 0.03–3.65), 1.66 ng(range 0.12–5.74), and 0.99 ng(range 0.08–5.67),respectively (see P = .01).Inthe clinical study examining cell retrieval ability according to bead size, themedian amount of DNA from 2-gram stool using beads A, B, and C was 1.50 ng (range 0.09–8.12), 0.58 ng(range 0–4.23), and 0.25 ng(range 0–4.62),respectively see . The amovely see . The amoThese results clarified that the smaller immunomagnetic beads and thebeads that were conjugated with higher-affinity Abs were more efficient atretrieving colonocytes from feces.CRC develops from the colorectal mucosa and therefore is in continuous contact with feces from an early stage. Previously, weproposed that colorectal cancer cells exfoliated into the feces might survivefor a considerable period in the feces, since feces do not inhibit cancerexpansion towards the colorectal lumen –22. MeanIntuitively, larger magnetic beads seem more likely to beattracted to magnetic body, compared with smaller magnetic beads, since theirlarger size corresponds to a greater number of iron particles inside the bead.Therefore, we initially thought that more cell-bead complexes using the largermagnetic beads would adhere to the magnetic body, enabling more cells to becollected. However, in a study to determine the optimal bead size, the smallerbeads adhered to the HT-29 cells more densely than the larger beads see . Thus, t5 HT-29cells, the cell retrieval rate using immunomagnetic beads tagged with a high-affinityantibody was superior to that of the beads tagged with a low-affinity antibody . We then made several mAbs againsthuman EpCAM and consequently obtained mAbs with either a high affinity or a lowaffinity to the EpCAM antigen see . Each anbody see. This rebody see.Previously, we demonstrated the feasibility of a new methodologyfor isolating colonocytes from naturally evacuated feces, followed bycytological or molecular biological analysis of the colonocytes to detectcolorectal cancer . Animprμm insize and were conjugated with a new monoclonal antibody possessing a higheraffinity to EpCAM, could effectively isolate colonocytes from feces. Thisresult is promising for the future of CRC diagnosis.We succeeded in developing suitable immunomagnetic beads for the isolationof colonocytes from feces. The new immunomagnetic beads, which were 3.0


Colorectal cancer is the second commonest cause of cancer death and the cost to primary care has not been estimated.To determine the direct primary care costs of colorectal cancer care.Retrospective case note review.Nottingham, United Kingdom.We identified people with colorectal cancer between 1995 and 1998, from computerised pathology records. Colorectal cancer related resources consumed in primary care, from hospital discharge to death, were identified from retrospective notes review. Outcome measures were costs incurred by the General Practitioner (GP) and the total cost to primary care. We used multiple linear regression to identify predictors of cost.Of 416 people identified from pathology records, the median age at primary operation of the 135 (33%) people we selected was 74.2 (IQR 14.4) years, 75 (56%) were male. The median GP cost was: Dukes A £61.0 (IQR 516.2) and Dukes D £936.2 (1196.2) p < 0.01. The geometric mean ratio found Dukes D cancers to be 10 times as costly as Dukes A. The median total cost was: Dukes A £1038.3 (IQR 5090.6) and Dukes D £1815.2 (2092.5) p = 0.06. Using multivariate analysis, Dukes stage was the most important predictor of GP costs. For total costs, the presence of a permanent stoma was the most predictive variable, followed by adjuvant therapy and advanced Dukes stage (Dukes C and D).Contrary to hospital based care costs, late stage disease (Dukes D) costs substantially more to general practice than any other stage. Stoma care products are the most costly prescribable item. Costs savings may be realised in primary care by screening detection of early stage colorectal cancers. Colorectal cancer remains the second commonest cause of cancer death in the U.K. and consumes significant resources within both primary and secondary care.We identified people with histologically proven colorectal cancer, treated at one hospital between 1995 and 1998, from computerised pathology records. People identified as deceased, had their notes reviewed by a single investigator (DM) at the local health authority. A small group of people still alive (n = 8) and registered with two GP practices, also had their notes reviewed. The study time period was from hospital discharge following excision of the primary cancer until death or the study end date (01/01/2003). We excluded those patients who died in the early post-operative period (30 days) without being discharged home, as they had consumed no community resources. Ethical approval was obtained for the study (LREC Q1110208).Our main outcomes were costs incurred by the General Practitioner (GP) and the total cost to primary care. We included resources consumed in primary care and related to colorectal cancer and excluded costs due to benign gastrointestinal symptoms (e.g. constipation). GP costs included all GP related activity (e.g. home visits) or prescribing. Total cost to primary care, included all identifiable costs related to colorectal cancer care  in addition to the GP costs. The cost of training a GP was considered when costing their time  and a 5% discount rate was chosen for costs in the main analysis. We used standard sources to calculate costs,e transformation of costs, multiple linear regression (backward variable elimination technique) was used to estimate the best predictors of primary care costs. Sensitivity analysis was performed across the three different GP costs structures, cost of stoma care (between £1000 and £2000) and through a range of discounting rates (0%–10%). Analyses were performed in Stata 7.0 .Initially we described the characteristics and cost data by Dukes stage of disease using median values, interquartile ranges and non-parametric tests where appropriate, as the cost data was non-normal. We used a non-parametric test for trendOf 416 people identified from pathology records, 127 were deceased. Within two GP practices, 8 people still alive had their notes reviewed. Overall, 22 (5%) subjects were excluded due to death in the peri-operative period. The median age at primary operation, of the 135 (33%) people included, was 74.2 (IQR 14.4) years, 75 (56%) were male and 112 (83%) of primary operations were considered curative. The median time from operation to death or end date was 2.1 years (IQR 2.4); those with a Dukes A cancer surviving longest—3.1 years (IQR 2.1); Dukes D surviving least long −0.8 years (IQR 0.9). The proportion of people dying from colorectal cancer increased across stage −11% in Dukes A and 95% in Dukes D .We found that Dukes stage was the most important determinant of GP costs in those treated for colorectal cancer, with Dukes D cancers costing approximately ten times as much as Dukes A cancers. The trend of rising cost with advancing stage seen in this study, has not been observed in hospital based care where very early and very late stage conferred least costs. In contrast, Dukes stage appeared less important than the presence of a permanent stoma when calculating total cost to primary care, probably because of the expense and consistent requirement of stoma products (£1500–£2000 per annum). However, on average, a Dukes D cancer still cost £777 more than a Dukes A cancer.Reviewing the notes of deceased patients limited our study population to a more advanced stage of disease with a reduced life expectancy but ensured an accurate end point for costs. The eight alive patients included were the result of the pilot period of this study and were therefore part of the study design. When excluded, the high proportion of permanent stomas in the small group of Dukes A cancers (50%) and their long survival period (median 3.8 years) caused the total costs of Dukes A to be higher than any other—£3043.8 (IQR 5628.1). Notwithstanding this, the cost of Dukes A to the GP remained minimal, total costs for Dukes B and C remained over £670 cheaper than Dukes D. When costs were annualised, the cost per life year showed an even greater trend of rising cost with advancing stage. As most recurrences occur within two years study time period provided up to 8 years of follow upAlthough irresectable (often advanced stage) disease is one indication for a permanent stoma, the decision is also often dependent on the site of the lesion. Regular stoma care products are required for those with permanent stomas, yet the results of GP costs indicate that this cost is rarely documented. Estimated costs were required due to the lack of documentary evidence and minimal economic data in the medical literature. We used a figure of £1500 which assumes the use of two bags per day plus additional consumables, an estimate deemed reasonable by an experienced stoma care nurse . When we varied the annual stoma cost by £1000 it made no difference to the overall conclusions of our study.Identifying actual resource use is a more robust and accurate method of economic analysis than economic modelling but it is time-consuming and expensive. Economic modelling must estimate resource consumption but in doing so, makes assumptions of use and costs. Our study reduced the number of such assumptions that had to be made.Hospital based follow up during the 1990’s would probably be considered less protocol driven in the U.K. than it is today. CEA measurements whether in hospital or primary care were not routinely used (and in certain institutions would remain debatable). USS surveillance for liver metastases has been superceded by CT Chest, Abdomen and Pelvis surveillance, although the frequency of tests (including colonoscopy) has perhaps reduced. Generally, in Nottingham at the time, follow up was hospital based under the original consultant. It was not possible to review the travel expenses aspect and a future study of the patient costs incurred would be useful.Who should perform the follow up and how intensively remain hotly debated issues but studies have shown that primary care can perform this role although the research is perhaps slightly dated.14We have only dealt with costs related to colorectal cancer but we may have underestimated the palliative care costs due to the limited documentation of District and other Nurse activity in GP records. In palliative care settings, 71.6% of patients require District nurse input with only 10% utilising GP’s and home helps.Subjects successfully operated for an early stage colorectal cancer without the creation of a stoma, had minimal ongoing primary care costs. Advanced stage disease or a permanent stoma required substantial resource allocation and despite overlooking patient and carer costs, advanced stage disease cost primary care £900 per year more than earlier stage cancers. This is a considerable cost and should be considered when apportioning resources.Ours is the first study that has looked at the primary care costs for treated colorectal cancer. From a hospital perspective, it is often considered that Dukes D confers minimal cost due to reduced life expectancy but this study has shown this to be an unreliable assumption. While hospital costs may be low, primary care costs are generally higher. Reducing the number of permanent stomas may be helped by modern surgical techniques and stapling devices, which enable very low rectal anastomoses and ileo-anal pouch formations. Identifying earlier stage disease can be achieved by screening, confirmed recently in the national FOBT pilot screening program,There are over 34,000 new cases of colorectal cancer in the U.K. per year, costing over £300 million in surgical, oncological and palliative care services. Previous hospital based studies have found Dukes A and D cancers to cost less than intermediate stage cancers but the cost to primary care has not been established and is likely to be considerable. Our study found that GP costs increased with advancing stage. Stoma care products also added significantly to primary care costs but were independent of stage. Costs savings may be realised in primary care by screening detection of early stage colorectal cancers.


The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic γ subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 Å resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 Å resolution was consistent with a proteolysed activated form of PhK that had lost the α subunits and possibly the γ subunits.Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 Å resolution structure of PhK heterotetramer (αβγδ) PhK phosphorylates inactive glycogen phosphorylase b (GPb) on a single serine, Ser14, converting it to active phosphorylase a (GPa). Active phosphorylase catalyzes the phosphorolysis of glycogen to release glucose-1-phosphate. All eukaryotic protein kinases share a common catalytic core, which catalyzes the transfer of the γ-phosphate from a nucleoside triphosphate donor to the side-chain hydroxyl of a serine, threonine, or tyrosine in substrate proteins, but each kinase recognizes only specific substrates and each is regulated by a distinct mechanism.Protein phosphorylation and dephosphorylation reactions are major control mechanisms in eukaryotic cells. Skeletal muscle phosphorylase kinase (PhK) was the first protein kinase to be recognized by function and to be purified over five decades ago . PhK is 4 and a total molecular weight (MW) of 1.3 × 106. The α  and β  homologous subunits are regulatory inhibitory subunits. Autophosphorylation or phosphorylation of these subunits by cyclic adenosine-monophosphatase-dependent kinase in response to hormonal stimulation leads to activation of the kinase. ADP binds to the β subunit with high affinity and also stimulates activity. Neuronal stimulation is effected by Ca2+ ions, which bind to the intrinsic calmodulin (δ) subunit , thus coupling muscle contraction with energy production. PhK differs from most other calmodulin-regulated enzymes in that the δ-calmodulin subunit is tightly associated in the holoenzyme in the absence of calcium. The γ subunit (44.7 kDa) is the catalytic subunit. It is composed of a kinase domain (residues 20–276) and a regulatory calmodulin-binding autoinhibitory domain (residues 298–396). PhK is one of several kinases that do not require phosphorylation on the kinase activation segment for activity. When isolated, the kinase domain is constitutively active. It has a glutamate in place of the more typical phosphorylatable serine, threonine, or tyrosine in the activation segment  or residues 40–477 (β subunit) share sequence similarities with family 15 glycosyl hydrolases , whereas 833–854 .The activation of PhK is complex, with several different layers of control. In vitro pH is a further regulator. Inactive PhK assayed in the presence of calcium exhibits low activity at pH 6.8. Activity is markedly increased at pH 8.2, but still requires the presence of calcium. Activation of PhK by phosphorylation or limited proteolysis results in an increase in activity at pH 6.8 but little change in activity at pH 8.2 .2+, Mg2+ at pH 8.2 showing that GPb bound toward the end of the lobes. At the same time, In order to understand the structural basis of the mechanisms involved in control of PhK through protein-protein interactions and by phosphorylation, a structure of the whole enzyme is required. Intact PhK has been difficult to crystallize for X-ray studies, but it has proved amenable to single-particle analysis by electron microscopy. We previously determined the three-dimensional (3D) structure of PhK at 22 Å resolution using negative stain electron microscopy and the random conical tilt method . The 222t residues 1–298), whose kinase domain structure has been solved by X-ray crystallography . Km values for ATP showed a 2-fold difference between the PhK holoenzyme and PhKγt . The kinetic parameters suggest that the activity of the kinase, when incorporated into the holoenzyme, has slightly different properties that make it a more efficient enzyme.The PhK was shown to be functionally active at pH 8.2 in the presence of calcium against GPb substrate with kubstrate A. PhK hoComparison between specific activities for the calcium-activated states of PhK at pH 8.2 (5.2 μmol/min/nmol) and at pH 6.8 (0.65 μmol/min/nmol) B shows t6 Da) as calculated with a protein partial specific volume of 0.84 Da/Å3. A gallery of the class averages of PhK and the corresponding reprojections of the 3D map is shown in PhK was prepared under conditions  where it is known to be in the active conformation as indicated from the kinetic studies. PhK was imaged in cryoconditions. A range of recognizable particle shapes could be distinguished in the electron micrographs as illustrated in The comparison with our previous model obtained from negatively stained PhK at 22 Å resolution is shown in The high-resolution  cryomodel of active PhK is consistent with the broad features noted in the lower-resolution  cryomodel of inactive PhK , but theWe have raised polyclonal antibodies against the γ kinase subunit peptide with the sequence (TAEEALAHPFFQQY residues 277–290). Immunoblot assays of the binding of the anti-γ antibody to denatured PhK show that these antibodies recognize the γ subunit A. Wilkint (residues 1–298)   into thes 1–298) , and eac3D model C. In thit were converted to SPIDER format and filtered to a resolution comparable to the PhK 3D cryo-EM volume. The characteristic bilobal shape of PhKγt is shown in two orientations: front and side views , formed principally from β sheet, is connected to a larger C-terminal lobe (residues 110–292), formed mainly from α helices. The two lobes are joined by a hinge region around residues Lys108 and Gly109. The atomic models of PhKγde views D, middlet X-ray structure had been determined with a substrate analog peptide bound. The substrate peptide is shown in red in the magnified view of the site  and GPb dimer (MW 1.94 × 105) is not easy. The PhK/GPb complex was analyzed by gel filtration on a Superose 6 column, but the results were inconclusive. The Km for the PhK phosphorylation of GPb is 9 μM  correspond to 1 PhK (αβγδ):0.8 GPb dimer. Depending on the value of Kd, saturation of PhK with GPb could have been between 1% and 80%. The appearance of the particles on the grids showed all particles with an increase in size, indicating that some were GPb bound, compared with the native PhK particles.The PhK/GPb complex was formed in solution before deposition on the electron microscope grid. Divalent cations Mgt 8-fold . Quantit is 9 μM . For the is 9 μM . Hence, The 3D structure was calculated with 18,704 defocus-corrected particles and refined to 18 Å resolution as estimated from the 0.5 FSC cut-off. The final reconstruction is shown in 2GPB; A difference map was calculated to localize more precisely the additional density arising from GPb bound to PhK. The PhK map was first filtered to 18 Å, the same resolution as for the 3D model PhK/GPb. Then the PhK and PhK/GPb maps were normalized and their difference computed. The extra density 4 or possibly (βδ)4 PhK particles. The SDS gel in After this work was completed, we analyzed the PhK preparation by analytical size exclusion chromatography  followedComparison of Figures The apparent difference in molecular mass between the large and small particles estimated from the volumes of the 3D EM maps is ∼750 kDa. The loss of four α subunits would correspond to a loss of 4 × 138 kDa = 552 kDa. The loss of the four γ subunits would correspond to a further loss of 4 × 44.7 kDa = 179 kDa. The loss of both the α and γ subunits would correspond to 730 kDa, roughly in agreement with the difference observed from the EM maps. However, the threshold level used to display the EM maps is partly empirical and quantitative estimates of molecular mass are approximate.4β4γ4δ4 stoichiometry and a corresponding molecular mass of 1.3 × 106Da. Cross-linking and immunoelectron microscopy from the work of Carlson and colleagues have provided information for the possible subunit arrangement in the PhK holoenzyme. These results are summarized in 2 tetramer comprising each lobe of the “butterfly.” The lobes are connected by bridges that appear to be formed, at least in large part, by the β subunits. The important role for the β subunit in forming the quaternary structure of PhK is supported by various observations. (i) An epitope in the β subunit (residues 708–815) is localized to an interior position on the lobes near the interconnecting cross-bridges (The PhK holoenzyme has an α-bridges . (ii) Se-bridges . (iii) A-bridges . The αγδ-bridges . (iv) Th-bridges . (v) Cro-bridges . The posThe α subunit is the most susceptible of the four subunits to proteolysis by a number of proteases . It is pt, the γ subunit interacts with the other subunits in a way that is consistent with the cross-linking experiments. The C-terminal region of the γ subunit (residues 298–396) acts as an autoinhibitor of catalytic activity. In the presence of calcium, it is proposed that the interaction of the C-terminal region with the calmodulin-δ-subunit Ca2+ sensor serves to relieve inhibition. The interaction most likely involves the PhK calmodulin-binding region residues 343–366 (termed PhK5)   and posse bridge C and 3D.e bridge .In our proposed model, only one GPb monomer from the dimer is firmly bound to PhK in the PhK/GPb complex. The other monomer apparently projects into the solution and is not visualized upon averaging. This arrangement would lead to the formation of GPab hybrids in which one subunit is phosphorylated and the other is not, at least transiently. There is supportive evidence (reviewed in m for substrate GPb of 8–9 μM (m (1.8 mM) for a peptide substrate from GPb  observed in our activity measurements. The α and β regulatory subunits act as inhibitors of γ catalytic activity. Alleviation of inhibition occurs through phosphorylation, or by raising pH from 6.8 to 8.2 (which might mimic the increase in negative charge resulting from phosphorylation), or by proteolysis . The α sNonactivated PhK from fast-twitch skeletal muscle of female New Zealand White rabbits was purified by the modified method of t were measured by monitoring the conversion of GPb to GPa  for 2 hr. Five microliters PhK at about 1 mg/ml was placed on a 400-mesh copper grid coated with a thin holey-carbon film. The grid was blotted, vitrified by freeze plunging in liquid ethane, and transferred at liquid nitrogen temperature into the microscope using a side-entry nitrogen-cooled Gatan 626 cryoholder. All images were taken on a JEM-2100F (JEOL) using an acceleration voltage of 200 kV at a magnification of 50,000× with a defocus from −2.8 to −4.8 μm .For cryo-EM, PhK was activated and glycerol or sucrose removed for optimal high-contrast conditions in vitrified ice by dialysis against 100 mM NaCl, 0.3 mM CaClFor the PhK/GPb complex, PhK (2.22 mg/ml) was incubated with GPb (1.08 mg/ml) overnight at 4°C at a molar ratio of 1 PhK (αβγδ) for 0.8 GPb dimers. Five microliters was deposited on a holey-carbon film grid, and the grid was washed by contact with a drop of dialysis buffer for 30 s, blotted, vitrified, and examined in the electron microscope as described above.2, and HEPES 50 mM (pH 8.2), was incubated with 4-fold molar excess of γ-antibodies for 90 min at room temperature to allow PhK-γ-subunit-antibody complex formation. The specimen was negatively stained using 2% uranyl acetate.Micrographs of the negatively stained immunolabeled PhK were obtained with a Philips CM120 (FEI) at 100 kV. PhK, in a buffer of 200 mM NaCl, 0.5 mM CaCl2) on Kodak SO 163 films.Images were recorded under low-dose conditions (10 electrons per AParticles were chosen via automated particle picking  and datat (PDB 2PHK) into the cryo-EM map and the fitting of the atomic X-ray structure of GPb to the PhK/GPb map are described in Fitting of the atomic X-ray structure of the truncated kinase domain PhKγ


Heterorhabditis bacteriophora and its symbiotic bacterium, Photorhabdus luminescens, are important biological control agents of insect pests. This nematode-bacterium-insect association represents an emerging tripartite model for research on mutualistic and parasitic symbioses. Elucidation of mechanisms underlying these biological processes may serve as a foundation for improving the biological control potential of the nematode-bacterium complex. This large-scale expressed sequence tag (EST) analysis effort enables gene discovery and development of microsatellite markers. These ESTs will also aid in the annotation of the upcoming complete genome sequence of H. bacteriophora.The entomopathogenic nematode H. bacteriophora TTO1 strain. Cluster analysis revealed the presence of 3,051 contigs and 7,835 singletons, representing 10,886 distinct EST sequences. About 72% of the distinct EST sequences had significant matches  to proteins in GenBank's non-redundant (nr) and Wormpep190 databases. We have identified 12 ESTs corresponding to 8 genes potentially involved in RNA interference, 22 ESTs corresponding to 14 genes potentially involved in dauer-related processes, and 51 ESTs corresponding to 27 genes potentially involved in defense and stress responses. Comparison to ESTs and proteins of free-living nematodes led to the identification of 554 parasitic nematode-specific ESTs in H. bacteriophora, among which are those encoding F-box-like/WD-repeat protein theromacin, Bax inhibitor-1-like protein, and PAZ domain containing protein. Gene Ontology terms were assigned to 6,685 of the 10,886 ESTs. A total of 168 microsatellite loci were identified with primers designable for 141 loci.A total of 31,485 high quality ESTs were generated from cDNA libraries of the adult H. bacteriophora cDNA libraries. BLAST searches revealed ESTs potentially involved in parasitism, RNA interference, defense responses, stress responses, and dauer-related processes. The putative microsatellite markers identified in H. bacteriophora ESTs will enable genetic mapping and population genetic studies. These genomic resources provide the material base necessary for genome annotation, microarray development, and in-depth gene functional analysis.A total of 10,886 distinct EST sequences were identified from adult  Heterorhabditis bacteriophora, and its mutualistic bacterium, Photorhabdus luminescens, are important biological control agents of insect pests  – [GenBank:EG025806], [GenBank:ES408468] – [GenBank:ES414355], [GenBank:ES738967] – [GenBank:ES744677], [GenBank:EX006911] – [GenBank:EX015306], [GenBank:EX910019] – [GenBank:EX916843], and [GenBank:FF678120] – [GenBank:FF681586]. The removal of vector and short length (20 bp or less) sequences resulted in 31,485 high-quality ESTs with an average length of 531 bp. The cumulative length of all high-quality EST sequences was 16,713,919 bases.A total of 31,586 ESTs from adult H. bacteriophora assembled contigs) with lengths ranging from 60 to 2,856 bp and a mean of 743 bp , with the final assembly generated by Vector NTI Advance™ 10. The final assembly contained 3,051 contigs generated from 23,650 ESTs and 7,835 singletons  had significant matches  to 4,460 C. elegans proteins in Wormpep190 database. Based on sequence similarity results, 12 H. bacteriophora ESTs were identified to be involved in RNA interference (RNAi) pathway .The similarity search against s Figure . Of the ay Table . The curH. bacteriophora ESTs to all nematode EST sequences from GenBank  matched to ESTs from clade V parasitic nematodes and the remaining 78 ESTs (14%) matched to ESTs from parasitic nematodes in other clades. Among these parasitic nematode-specific ESTs, 142 had matches to proteins of other organisms, enabling putative function assignment. Among these ESTs are those encoding F-box-like/WD-repeat protein, theromacin, Bax inhibitor-1-like protein, and PAZ domain containing protein, which represent interesting targets for in-depth functional analysis. The remaining 412 had no matches to any protein in the current databases, and are thus considered novel sequences.In order to identify parasitic nematode-specific ESTs, a comparison of s Figure  of whichH. bacteriophora distinct ESTs). The GO assignment included 1,117 Biological Process terms assigned 13,438 times to 4,653 distinct ESTs, 244 Cellular Component terms assigned 2,454 times to 1,778 distinct ESTs, and 669 Molecular Function terms assigned 4,035 times to 3,190 distinct ESTs. "Embryonic development ending in birth or egg hatching" (40.9%) was the most dominant term out of the 4,653 distinct ESTs assigned to Biological Process GO category, followed by "nematode larval development" (28.3%), "positive regulation of growth rate" (27.6%), "reproduction" (24.7%), and "growth" . Biological Process term associations of H. bacteriophora distinct ESTs that may present potential interests included: (i) 164 with "determination of adult life span"; (ii) 22 with "defense response"; (iii) 22 with dauer-related biological processes, including "dauer larval development", "dauer entry", and "dauer exit"; and (iv) 37 with stress-related biological processes. Protein binding (53.8%) was the most dominant term among the 3,190 H. bacteriophora ESTs annotated to the Molecular Function category, followed by identical protein binding (2.6%) and cytochrome-c oxidase activity (2.5%). Among the 1,778 H. bacteriophora ESTs annotated to Cellular Component category, 221 were nuclear and 218 were in cytoplasm. The GO assignment for each H. bacteriophora EST is given in additional file H. bacteriophora distinct ESTs.Gene Ontology (GO) terms were assigned to 6,685 distinct ESTs with BLASTx search against the April 2008 release of GO database , tri-nucleotide (46.3%), tetra-nucleotide (2.7%), penta-nucleotide (0.5%), and hexa-nucleotide (0.5%) (Table H. bacteriophora distinct ESTs). The primers designed for the 141 microsatellite loci are potentially useful for genetic linkage mapping and population genetic studies.In total, we identified 168 microsatellite loci from 157 H. bacteriophora distinct ESTs showed 71.9%  matches to proteins from GenBank's nr database. The remaining 28.1% H. bacteriophora distinct ESTs represented novel genes yet to be assigned a function, demonstrating enormous novel gene discovery potential of this EST study. Among H. bacteriophora distinct ESTs having matches to proteins of other organisms in GenBank's nr database, a vast majority (95.9%) matched nematode proteins. About 71%  H. bacteriophora distinct ESTs match to 4,460 proteins from Wormpep190 that contains 23,771 extensively curated C. elegans proteins. H. bacteriophora homologs in C. elegans represent 18.8% of proteins in C. elegans. This finding suggests that H. bacteriophora and C. elegans have vastly different in proteomes, which may be explained in part by free-living versus parasitic life styles.This work produced a total of 31,485 high quality ESTs representing 10,886 distinct sequences. Sequence similarity searches of H. bacteriophora during its life cycle. None of these ESTs matched to genes or proteins of P. luminescens subsp. laumondii TTO1, the natural symbiont of H. bacteriophora TTO1 [H. bacteriophora ESTs are consistent with our previous observations [Interestingly, 26 distinct ESTs (0.3%) matched to proteins from various prokaryotic organisms  and BAX inhibitor-1-like protein in wasp Nasonia vitripennis (GenBank accession number XP_001605379 ). BAX inhibitor-1 is a member of Bcl-2 family that suppresses programmed cell death [C. elegans, C. briggsae, C. japonica, C. remanei, and Pristioncus pacifiucs (Wormbase). However, these genes have very low sequence similarities to H. bacteriophora Contig1066. BAX inhibitor-1 is involved in preventing endoplasmic reticulum stress-related programmed cell death in Arabidopsis [Two other parasitic nematode-specific ESTs are Contig2528 and Contig1066. Contig2528 encodes a homolog of theromacin in the segmented worm ssulatum . Theromassulatum . H. bactbidopsis  and humabidopsis .H. bacteriophora distinct ESTs involved in different biological processes. Here we discuss the genes involved in some biological processes of interest in detail. A number of ESTs related to defense responses and stress responses were identified in these H. bacteriophora distinct ESTs  homolog that is essential for DAF-16-mediated defense response to pathogenic bacteria and increased resistance to oxidative and UV-induced damage [C. elegans homologs exhibit a "pathogen susceptibility increased" phenotype when silenced by RNAi [GO assignments based on sequence similarity searches aid identification of d damage . EST ES4d damage . Another by RNAi . HoweverH. bacteriophora distinct ESTs involved in stress response encode a homolog to C. elegans catalase CTL-2 that likely is involved in protecting cells from reactive oxygen species as an antioxidant enzyme [C. elegans homolog showed an "organism stress response abnormal" phenotype when silenced with RNAi [H. bacteriophora. Once the functions of these genes are determined, they can be potentially used for genetic manipulations of entomopathogenic nematodes. ESTs involved in dauer larval development, dauer entry, and dauer exit were also identified  and another EST (Contig211) encodes a homolog of C. elegans VIG-1 (45% similarity at the amino acid level). TSN-1  containing 5 staphylococcal/micrococcal nuclease domains and a tudor domain is a RISC component in C. elegans, Drosophila and mammals [C. elegans was shown to have nuclease activity and therefore thought to contribute to RNA degradation in RNAi [vig-1 gene was also shown to be a component of RISC [Brugia malayi (57% similarity at the amino acid level). Another EST (FF679415) encodes a putative homolog to Drosophila and human Drosha [C. elegans. These findings suggest that H. bacteriophora may have structurally different RNAi pathway components than its relative, C. elegans.We have identified genes encoding RNAi induced silencing complex (RISC) components. One EST (EX014403) encodes a homolog of  mammals . The pur in RNAi . The pro of RISC . We did n Drosha  rather trde-4 [gfl-1 and zfp-1 [H. bacteriophora TTO1 that was shown to be necessary for systemic RNAi [sid-1 gene has been found in H. bacteriophora GPS11 [H. bacteriophora TTO1 is sequenced.Other RNAi related genes we were able to identify are those encoding homologs of SMG-2, SMG-5, RDE-4, GFL-1, and ZFP-1. SMG-2 and SMG-5 are involved in nonsense-medicated mRNA decay (NMD) where eukaryotic mRNAs with premature stop codons are selectively and rapidly degraded ,31. The rde-4 , gfl-1 and zfp-1  were shomic RNAi . HoweverH. bacteriophora distinct ESTs, of which we were able to design primers for 141 based on the flanking sequences. These microsatellite markers may be useful for genetic mapping, linkage analysis, and population genetic studies. In a separate effort, microsatellite loci with 2- or 3-bp repeat units were selected for microsatellite marker development, along with the microsatellite loci enriched from genomic DNA of H. bacteriophora [This EST project also enabled the development of genetic markers. We have identified 168 microsatellite loci from riophora . Eight pH. bacteriophora ESTs representing 10,886 distinct sequences. Among these, 7,828 (71.9%) ESTs matched to proteins in GenBank's nr database. The vast majority (95.9%) of the best matches was to nematode proteins, a small portion (0.3%) to prokaryotic proteins and the remaining 3.8% to other eukaryotic proteins. GO terms were assigned to 6,685 H. bacteriophora distinct ESTs. "Embryonic development ending in birth or egg hatching" and "protein binding" were the most dominant terms in the categories of Biological Process and Molecular Function, respectively. This EST collection offers unprecedented opportunities for research on this unique nematode-bacterium symbiotic complex. The comparison of ESTs of H. bacteriophora TTO1 with those of AHPNs, FLNs, and PPNs resulted in the identification of 554 parasitic nematode-specific ESTs. These ESTs should be valuable for future research related to insect parasitism by these nematodes. We were able to identify a small number of ESTs involved in RNAi, among which is an EST encoding a Drosha homolog, suggesting structurally different RNAi pathway components from those in C. elegans. In addition, we have identified 157 microsatellite loci which may prove valuable once their polymorphisms are tested and validated. Overall, novel, parasitic nematode-specific, and C. elegans homologous genes have been identified in this EST study, greatly facilitating genome annotation, gene functional analysis, population genetic studies, and microarray development.We have generated 31,485 high quality Heterorhabditis bacteriophora TTO1-M31e strain propagated on a lawn of Photorhabdus luminescens bacterium. Poly(A) RNA was used for cDNA library construction with two different strategies. The first group of libraries were constructed using the CloneMiner™ cDNA Library Construction Kit (Invitrogen) following the manufacturer's instructions. Briefly, 2 μg single-stranded mRNA was converted into double stranded cDNA containing attB sequences on each end. Through site-specific recombination, attB-flanked cDNA was cloned into the attP-containing pDONR222 vector. The second group of libraries were constructed using SMART technology with modifications. Briefly, the double stranded cDNA was synthesized with SMART oligos from poly(A) RNA with SuperScript® III First-Strand Synthesis System (Invitrogen) and Advantage® High Fidelity 2 PCR kit (Clontech). The double-stranded cDNA was normalized with duplex-specific nuclease (Evrogen) and then was nebulized, end repaired with End Repair Kit (Lucigen), size separated, and ligated into pSMART hinc II Vector System (Lucigen). The cloning and sequencing of both pDONR222 and pSMART libraries were not directional, leading to the production of ESTs from both 5' and 3' ends. The sequences were generated by ABI 3730 machines from the cDNA libraries using and deposited in GenBank dbEST.Total RNA and poly(A) RNA were isolated from adult hermaphrodites of the isogenic line of EST sequences in FASTA format were downloaded from GenBank dbEST. The sequences were processed by removing vector sequences with Vector NTI Advance™ 10 program. The processed EST sequences were assembled into contigs (contiguous sequences) using the ContigExpress module embedded in Vector NTI Advance™ 10 (Invitrogen). These stringent parameters of assembly (overlap length cutoff of 40 and overlap percent identity cutoff of 95%) were used to assure proper assembly. The distinct EST sequences, including the contig consensus sequences and the singleton sequences, were searched against GenBank's nr database and Wormpep190 databases in a local Linux workstation using the BLASTx algorithm . The E vH. bacteriophora ESTs to ESTs of FLNs, "-l fln.gi" was included in the command with fln.gi containing all GI numbers of EST entries from free-living nematodes. The BLAST outputs were parsed with in-house developed perl scripts to extract match information. ESTs with no significant matches to ESTs of FLNs were extracted and further searched against GenBank nr and Wormpep190 databases using BLASTx algorithm. EST entries with no significant matches to proteins of FLNs were designated parasitic nematode-specific ESTs, which were further characterized. The cutoff value of BLAST searches was 1e-5.All nematode EST sequences were downloaded from GenBank dbEST to a local Linux workstation and formatted as a database for tBLASTx searches. Gene index (GI) numbers of all nematode EST sequences were extracted and grouped according to the categories of AHPNs, PPNs, and FLNs. The tBLASTx searches were performed in a local Linux workstation against the complete nematode EST database with the -l option enabled to restrict the database search to the list of GI's of the targeted group . For exaH. bacteriophora ESTs were searched using the BLASTx algorithm against the annotated sequences of FASTA format in the April 2008 release of GO database. The BLAST output was parsed and terms assigned with the assistance of in-house developed perl scripts accessing the MySQL database of "mygo" in a local Linux workstation. The distribution of GO terms in each of the main ontology categories, Biological Process, Cellular Component, and Molecular Function [H. bacteriophora distinct ESTs assigned in a single GO category was considered as 100% [For assignment of Gene Ontology terms, the distinct Function , was exa as 100% ,38.H. bacteriophora distinct ESTs were searched for microsatellite loci using msatfinder v. 2.0.9 [The set of 10,886 v. 2.0.9  in a locv. 2.0.9  in a locXB conducted vector sequence removal, assembly and analysis of EST sequences and prepared the manuscript. TAC provided the poly(A) RNA for cDNA library construction. RKW, SC, and JS led groups at the Genome Center at Washington University School of Medicine in St. Louis, MO that conducted cDNA library construction, sequencing, and data deposition. PSG, BJA, TAC, SC, RG, SAH, JS, and PWS initiated the study and obtained funding. PSG supervised the study and assisted in manuscript preparation. All authors read, edited, and approved the final manuscript.ContigComponents. Components of H. bacteriophora assembled contigs. The table includes the components of H. bacteriophora assembled contigs, including contig names and their comma-delimited components in GenBank accession numbers.Click here for filedefense_stress_response_genes. H. bacteriophora distinct ESTs similar to C. elegans genes involved in defense and stress responses. This table contains H. bacteriophora distinct ESTs matching to C. elegans genes that are involved in defense and stress responses. Information of each H. bacteriophora distinct EST includes matching C. elegans gene, its description, E value of the BLASTx search, alignment, and percentage similarity.Click here for fileNematodeTaxa. Nematode species that ESTs used in the analysis came from. The table includes the nematode species in the categories of AHPNs, FLNs, and PPNs, the clade they belong to, and their corresponding number of ESTs in GenBank when the analysis was done.Click here for filepanESTs. Summary of BLASTx identification of parasitic nematode-specific H. bacteriophora ESTs. The table describes the best BLASTx matches to GenBank's nr database of the 554 parasitic nematode-specific H. bacteriophora ESTs, including distinct EST ID, length, and annotation of the top BLASTx hit, including accession number, length, description, E value, bit score, frame, query_start, query_end, hit_start, hit_end, positives, and identities.Click here for fileGOassignment. Summary of GO assignment of H. bacteriophora distinct ESTs. The table describes the GO assignment for each of 10,886 H. bacteriophora distinct ESTs, of which 4,201 have "no GO assignment". The GO terms separated by semicolons include accession numbers, term descriptions, and term types.Click here for filemsat. Summary of microsatellite loci identified in H. bacteriophora distinct ESTs. The table describes the microsatellite loci identified in H. bacteriophora distinct ESTs, including the ID and length of H. bacteriophora distinct EST ID, the start and stop coordinates and motif units of the microsatellite loci, GC contents of the distinct ESTs and the flanking sequences, primer sequence, length, and Tm of the left and right primers, and the size of the amplification product from these primers. The asterisk (*) marking H. bacteriophora distinct EST IDs denotes that the primers designed for these loci amplify other adjacent loci too.Click here for file


Peste des petits ruminants virus , which causes a severe disease in sheep and goats, has only recently been officially declared to be present in Turkey. We carried out a study to determine the prevalence, distribution, and host range of PPRV in Turkey. A total of 1,607 animals, reared in 18 different locations, were monitored for the presence of antibodies to PPRV and the related virus of large ruminants, Rinderpest virus (RPV). Only two farms had animals that were free of antibody responses to either disease. Prevalence for PPRV infection varied (range 0.87%–82.6%) and was higher in sheep (29.2%) than in goats (20%). The overall antibody responses to PPRV and RPV were 22.4% and 6.28%, respectively. Two PPRVs of lineage 4, which comprises many other PPRVs whose origins are in the Middle East, the Arabian Peninsula, and southern Asia, were isolated from Turkish sheep. Peste des petits ruminants virus (PPRV) is a morbillivirus that primarily infects sheep and goats. The virus is present in Africa (Rinderpest virus (RPV), Canine distemper virus, and human measles virus n Africa –3, the Mn Africa ,7 and isPPRV infection has only recently been officially reported in Turkey, in September 1999 ,17, but Domestic ruminant species  from throughout Turkey were examined for virus-specific antibodies. The sampling procedure depended on the presence of suspected infection and focused on two groups of animals. The first included 193 sheep that local authorities reported as having clinical signs of PPRV infection. These animals were examined, blood samples were collected, and any animals with signs of disease were sampled by swabbing for virus isolation. Cattle grazing with sheep or goats were also sampled to monitor for antibodies to the two viruses. The second group consisted of 1,414 animals randomly selected for serologic screening for PPRV and RPV antibodies from herds near the flocks of sheep and goats in which PPRV-like infection was reported. The numbers of serum samples collected from sheep, goats, and cattle were 884, 209, and 321, respectively.Competitive enzyme-linked immunosorbent assays were performed as described in the manual of Peste des Petits Ruminants enzyme-linked immunosorbent assay (ELISA) kit A total of 328 field samples, including heparinized blood, organ (lung), and swab specimens, were cultured to obtain virus isolates. The processed samples were spread onto Vero cells seeded in rolling culture tubes. The cells were grown in Dulbecco’s modified Eagle medium enriched with 5% fetal bovine serum as a regular culture medium. The cell culture media were changed every 2 days and the inoculated cells observed for 12–14 days. The positive culture tubes were frozen at -80°C when the cytopathic effect (CPE) was 90%, and virus stocks were prepared from the positive samples.EcoRI at 37°C for 1 hour. Samples were then analyzed on 1.7% agarose gels to determine the cleavage patterns of the amplicons. DNA products obtained with PPR F1b and F2d primers were sequenced by using a T7 polymerase-based commercial kit  with 35SdATP as the radiolabel.Detection of PPRV RNA by reverse transcription-polymerase chain reaction (RT-PCR) was performed as described Morbillivirus genus, and the phylogenetic tree was generated with the DNADIST and FITCH programs of the PHYLIP 3.73 software Sequence data were analyzed with the GCG  package. The nucleic acid sequences obtained from PCR products were aligned with known sequences from representatives of the Animals with clinical signs of PPRV were detected in 11 provinces . In many, while only 1 sheep in Konya was found to have seroconverted, probably following natural infection with RPV before 1999. The overall occurrence of PPRV infection in cattle was 15.57% , and approximately 27% of cattle were antibody positive for RPV, indicating previous exposure to the virus either by natural infection or, most probably, by vaccination, since all cattle in the study were >6 months of age.A total of 1,607 animals from 18 farms were sampled for antibodies to PPRV and RPV . Only twThe study showed no substantial relationship between the occurrence of PPRV infection and geographic location. Although the main portal of entry of the disease is thought to be in the southeastern part of Anatolia, distribution of the prevalence values did not show a clear pattern across the country, and the disease was detected in varying percentages in almost every region studied .EcoRI recognition sequence site in the amplified genome region of the isolates. While the PPR vaccine strain (Nigeria 75/1) produced, as expected, two fragments of 202 bp and 246 bp on cleavage with EcoRI, isolates Sakarya 1 and Sakarya 2 were not digested by this restriction enzyme (data not shown). Partial sequencing of the F protein–coding region of the two PPRV isolates showed them to be identical (GenBank accession number AF384687). The Turkey 2000 sequence was then aligned with the sequences of other PPRV isolates from around the world. A total of 328 samples were spread onto Vero (African Green Monkey Kidney) cells. Two nasal swab samples (Sakarya 1 and Sakarya 2), from sheep in Sakarya Province, showed CPE on Vero cells. The CPE was observed on day 3 after inoculation and was initially characterized by the formation of rounded cells; later, syncytia developed. RT-PCR was performed on cell culture supernatants after the first passage on the Vero cells. The expected amplification product of 448 bp was observed by using RNA prepared from culture supernatants from only the two samples (data not shown). Restriction fragment length polymorphism analysis of the RT-PCR products indicated nucleotide substitutions in the We investigated the prevalence, host range, and distribution of PPRV in small private farms in Turkey. We also demonstrated the presence of the disease by observing animals in the field and by isolating virus from clinical specimens. This wide-ranging survey is the first to be carried out on this disease in Turkey. PPRV infection has only recently been officially declared to be present in Turkey in the Elazig Province in eastern Anatolia ,17. Our The prevalence of the disease was as high as 28.5% in sheep and goats reared in small private flocks, and the disease was found in almost every region across Turkey. Occurrence of infection did not vary substantially by geographic locations of the livestock tested. Although the presence of PPRV infection in Turkey has been reported before ,19, the . This finding may indicate that in some cases no interference occurs. Another possible reason is the cross-reactivity of the PPRV assay for antibodies to RPV, as noted by Anderson et al. Cattle act as dead-end hosts for PPRV and show no clinical signs of infection. Nevertheless, they develop a humoral immune response to PPRV that protects them against natural or experimental challenge with virulent RPV Only two PPRVs were isolated—from nasal swab samples of two sheep from Sakarya Province. The reason for the poor success in isolating virus could be the nature of the samples. In previous studies, virus isolations were made from spleen, mesenteric lymph nodes ,26, or iOur use of molecular epidemiologic techniques provided data that suggest cross-border transmission into Turkey of PPRV infection that is actively circulating in neighboring countries. The viruses we isolated are PPRV lineage 4, which includes viruses whose origins are in the Middle East, Arabia, and south Asia


Engineered Cre and the mutated ligand-binding domain of estrogen receptor fusion recombinase (CreERT) allow temporal control of Cre activity.Cre/T, which show 4-hydroxytamoxifen (4-OHT)-inducible and spontaneous (4-OHT-independent) Cre activities, referred to Tg(BK5-CreERT)I and Tg(BK5-CreERT)S, respectively. The transgenic construct is driven by the bovine Keratin 5 promoter, which is active in the basal epithelial lineage of stratified and pseudo-stratified epithelium across multiple organs. Despite the difference in 4-OHT dependency, the Tg(BK5-CreERT)I and Tg(BK5-CreERT)S mouse lines shared similar Cre-mediated recombination among various organs, except for unique mammary epithelial Cre activity in Tg(BK5-CreERT)S females.In this study, we have generated two distinct transgenic mouse lines expressing CreERThese two new transgenic mouse lines for the analysis of basal epithelial function and for the genetic modification have been created allowing the identification of these cell lineages and analysis of their differentiation during embryogenesis, during perinatal development and in adult mice. Howeveiewed in ,2) offerstradiol ,4. Thus,loxP recombination activity through female germ-line only, which potentially confines the breeding strategy available for the analysis of tissue-specific gene ablation, that is in generalized germ-line deleted strains [loxP-mediated recombination in utero or at adult stage; these have proved to be powerful genetic resources and have mostly concentrated on the analysis of epidermal development and disease [T driven by the bovine K5 promoter on an inbred (C57BL/6J) background in this report.Keratin 5 (K5) is a member of type II keratins and expresses with its type I keratin partner keratin 14 (K14) in the basal layer of stratified squamous epithelium (SSC) . Utilizi strains ,14. As a disease -19. To sBK5-CreERT transgenic plasmid , a 1.8-kb of EcoRI-digested/Klenow filled-in of Cre-ERT fusion gene derived from pCre-ER(T) plasmid (kindly provided by Dr. Richard R. Behringer with an agreement of Dr. Pierre Chambon), a 0.5-kb SV40 polyadenylation signal (pA) and 2 copies of the ~1.2-kb HS4 insulator sequence from the 5' region of the chicken β-globin locus . This 5'-HS4 served as a barrier element that protects genes from any chromosomal position effect, which has been thought to result in histone modifications via USF proteins [NotI/SalI-digested transgene was eluted and separated from the pBluscript vector backbone for pronuclear microinjection [The d Figure  was creaproteins ,21. The njection .Tg(BK5-CreERT)I and Tg(BK5-CreERT)S mice bearing the same transgene, BK5-CreERT 26Sortm1Sor; the Jackson Laboratory) on a C57BL/6;129 mix background are referred to as R26R mice in this report. The Tg(BK5-CreERT)I/+;R26R/+ and Tg(BK5-CreERT)S/+;R26R/+ bigenic mice were generated by crossing R26R/R26R females with Tg(BK5-CreERT)I/+ and Tg(BK5-CreERT)S/+ males, respectively. Occasionally, the offspring of R26R/R26R males and Tg(BK5-CreERT)S/+ females were generated and are referred to as R26R;Tg(BK5-CreERT)S, as they are the progeny of Tg(BK5-CreERT)S/+ females; these were used for the analysis of spontaneous Cre activity through the female germ-line. The genotyping protocol for the R26R allele was according to the methodology for PCR genotyping presented on the web site of the Jackson Laboratory. Genotyping of the Tg(BK5-CreERT)I and Tg(BK5-CreERT)S mice was determined by a 300-bp PCR product amplified by a forward primer (5'-GGACATGTTCAGGGATCGCCAGGCG-3') and a reverse primer (5'-CGACGATGAAGCATGTTTAGCTG-3'). The PCR conditions were 5 min at 94°C, 28 cycles for 1 min at 94°C, 1 min at 64°C, 1 min at 72°C and a final 2 min extension at 72°C. All animals were housed in microisolator cages (up to 5 mice per cage) using specific pathogen free husbandry. All experiments with mice were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at National Yang-Ming University.The T Figure  were gen4-hydroxytamoxifen  was dissolved in dimethyl sulfoxide  at a concentration of 25 mg/ml. The 4-OHT solution was emulsified in sunflower seed oil (Sigma) by vortex followed by mixing on a rotator for 4~6 hours. The mice were injected intraperitoneally with three doses of 4-OHT (4~5 mg/kg body weight) every other day.The detailed experimental procedures had been described previously . After XBK5-CreERT transgenic founders were obtained. Each transgenic founder was backcrossed to C57BL/6J for expansion of the individual transgenic line. All transgenic founders were fertile and transmitted BK5-CreERT transgenes to their progeny. To examine the Cre recombinase activity, BK5-CreERT mice were bred with ROSA26 Cre Reporter (R26R) mice [Tg(BK5-CreERT);R26R bigenic mice; this was followed by intraperitoneal 4-OHT injection. Among these four transgenic mouse lines, three lines exhibited 4-OHT induced Cre recombinase activity. We selected an efficient and tightly controlled 4-OHT inducible BK5-CreERT transgenic mouse line, referred to here as Tg(BK5-CreERT)I, to be the representative line for further study.Through pronuclear microinjection, four 6R) mice  to generTg(BK5-CreERT)I;R26R bigenic mice was assessed in terms of reporter (LacZ) gene expression S. The detailed mechanism that triggers this spontaneous Cre activity remained unclear. X-gal stained tissues of Tg(BK5-CreERT)S;R26R bigenic mice revealed that Cre activity initiated reporter LacZ gene expression started at around embryonic day (E) 14.5 S;R26R mice was undetectable at birth S mice might not exactly match that of endogenous K5 expression during organogenesis. Furthermore, in the urogenital system of the Tg(BK5-CreERT)S;R26R females at P7, the transitional epithelia lining of the bladder and the stratified epithelia lining of sinus vagina (SV) markedly exhibited β-galactosidase activity, whereas very few X-gal positively stained epithelial cells in the Müllerian vagina (MV) could be detected S transgenic mice exhibited female germ-line Cre activity, we obtained offspring S) from a cross of a R26R/R26R male and a Tg(BK5-CreERT)S/+ female and analyzed spontaneous β-galactiosidase expression compared to of control R26R mice. Our results showed that the X-gal positively stained patterns remained specific to the K5-expressing cell types as shown by examples of skin and trachea S skin, the β-galactosidase activity showed tissue/lineage-specific expression in the epidermal, hair follicles and sebaceous cells S trachea, intense X-gal-stained cells were found in the basal cells of the pseudo-stratified epithelium S mouse line does not behave as a female germ-line deleter as described in earlier reports [Tg(BK5-CreERT)S mice enabled us to bypass the female germ-line deletion effect and to use this line for Cre-mediate recombination in a tissue-specific manner during embryonic and early postnatal development.Previously Hafner et al and Ramirez et al. independently demonstrated that K5-Cre and K14-Cre express in female oocytes and have suggested their applicability as female germ-line deleter strains ,14. To da Figure  &3g. In s Figure , suggest reports ,14. The Tg(BK5-CreERT)S transgenic mice, but not in our Tg(BK5-CreERT)I transgenic mice, Cre-mediated recombination in the mammary gland was detected and X-gal-stained cells were observed in both the luminal epithelium and the basal epithelial cells of the mammary glands at P7 (data not shown), in virgins S;R26R anterior prostate (AP) lobes S and Tg(BK5-CreERT)I, which exhibit spontaneous and 4-OHT-inducible Cre activity driven by a bovine K5 promoter, respectively. For analysis of Cre/loxP-mediated recombination at the embryonic and early postnatal stages using Tg(BK5-CreERT)S mouse line, a breeding scheme that considers whether the male or the female is carrying the BK5-CreERT transgene is not necessary because there was no female germ-line Cre-mediated recombination in our Tg(BK5-CreERT)S females. The 4OHT-induced Tg(BK5-CreERT)I mouse line will enable us to perform conditional genetics in temporally and spatially controlled manner.Taken together, we reported two new transgenic mouse lines, These genetic resources generated in this study will not only help us to understand the biology of the skin, but will also support studies of the basal epithelial lineages of pseudo-stratified, stratified and transitional epithelia as they appear in the thymic medulla, the esophagus, the forestomach, the trachea, the bronchus, the bladder, the cervicovagina, etc. Moreover, glandular organs such as prostate glands, which are composed of K5-expressing basal epithelium, can also be a target organ using these new transgenic mouse lines; this will allow the genetic fine-dissection of basal epithelial function in terms of epithelial identity and differentiation.The authors declare that they have no competing interests.CCL performed the experiments and analyzed the data. LRY and JLC participated in the data interpretation. TFT contributed the materials for the initial design of transgenic construct. CMC performed the experiments, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.


Widespread deaths of American Crows (Corvus brachyrhynchos)were associated with the 1999 outbreak of West Nile (WN) virus in the New York City region. We compared six organs from 20 crow carcasses as targets for WN virus detection. Half the carcasses had at least one positive test result for WN virus infection. The brain was the most sensitive test organ; it was the only positive organ for three of the positive crows. The sensitivity of crow organs as targets for WN virus detection makes crow death useful for WN virus surveillance.


In Finland like in many other countries, employers are legally obliged to organize occupational health services (OHS) for their employees. Because employers bear the costs of OHS it could be that in spite of the legal requirement OHS expenditure is more determined by economic performance of the company than by law. Therefore, we explored whether economic performance was associated with the companies' expenditure on occupational health services.We used a prospective design to predict expenditure on OHS in 2001 by a company's economic performance in 1999. Data were provided by Statistics Finland and expressed by key indicators for profitability, solidity and liquidity and by the Social Insurance Institution as employers' reimbursement applications for OHS costs. The data could be linked at the company level. Regression analysis was used to study associations adjusted for various confounders.Nineteen percent of the companies (N = 6 155) did not apply for reimbursement of OHS costs in 2001. The profitability of the company represented by operating margin in 1999 and adjusted for type of industry was not significantly related to the company's probability to apply for reimbursement of the costs in 2001 . Profitability measured as operating profit in 1999 and adjusted for type of industry was not significantly related to costs for curative medical services  nor to OHS cost of prevention in 2001 .We did not find a relation between the company's economic performance and expenditure on OHS in Finland. We suppose that this is due to legislation obliging employers to provide OHS and the reimbursement system both being strong incentives for employers. Occupational health services (OHS) are regarded as a fundamental right of every worker. In order to reach the highest attainable standard of health, workers' health at work should be protected ,2. ThereFiler and Golbe  have desIn making decisions on OHS investments the company is balancing the costs and benefits of OHS. Preventive services are supposed to lead to lower occupational accidents and diseases, lower sickness absence and disability pensions which all improve the economic performance of the company -7. CuratInvestments in safety and health compete with other investments in the company. While companies make decisions on resource allocation the economic situation of the company might affect OHS differently than other input decisions. Acquiring outside funding for OHS investments will be difficult and therefore investment decisions on OHS are dependent on the liquidity of the company (cash flows). Cash flows indicate if there is internal funding available in general, also for investments in OHS.Filer and Golbe  studied There is some evidence to support these assumptions but there are only few studies. Nickell and Nicolitsas found in their study that declining company finances lead to lower pay and to lower safety levels as indicated by abolishment of "restrictive practices" such as restrictions to hours of work, manning ratios on machines, and inflexibility of working practices . AbolishThe small amount of research in this field may be due to a lack of data. In Finland, however, it is possible to study the relationship between economic performance and OHS expenditure because employers are entitled to reimbursement for about half of the costs of preventive and curative medical occupational health services. Based on the reimbursement claims, the National Social Insurance Institution keeps an employer-based register of the contents and costs of OHS. In addition, Statistics Finland keeps a register with the firms' annual financial statements. These financial statements allow the calculation of key ratios that measure a company's economic performance. Firm specific identification codes made it possible to combine the information in both registers at the firm level.Based on these administrative sources, we studied if key ratios for a company's economic performance were associated with the OHS expenditure two years later.It has been obligatory for employers to organize preventive occupational health services for their employees since 1979. According to a population survey conducted in 2006, two out of three employees had attended an occupational health examination in the past three years, and around half of them had had occupational health personnel assessing their workplace in the past three years. Although organizing curative medical services is voluntary for employers, over 90% of employees can obtain GP level services from their OHS unit. Around half of the primary care level GP visits of these employees take place within OHS . A more Finland introduced public health insurance to reimburse the costs of curative medical care in the private sector in 1964. Since then employers have got reimbursement for the costs of OHS. Employers first pay all costs of OHS and apply for reimbursement within six months after closing their accounts. The share of the costs reimbursed has varied during the over 40 years of reimbursement. In 2001 and still nowadays, the reimbursement is 60% for preventive and 50% for curative medical services. So, despite of the reimbursement the firms will always bear a considerable part of the cost themselves.In a prospective design, data for economic performance of companies from 1999 were used to predict their expenditure on OHS in 2001.Statistics Finland collects the financial statements of all Finnish firms from the tax authorities. The register also contains data such as the number of persons employed by the company, number of blue- and white-collar workers, year of establishment, registered office, and industry.The Social Insurance Institution (SII) registers employers' reimbursement applications for OHS. This register contains data on service mix and costs. We chose to use registers from the years 1999 and 2001. In 1999, the renewed reimbursement system where the costs for prevention and curative medical services are reported separately had been in force for four years. As this project was launched, 2001 was the last year for which all reimbursement applications had been processed. Companies apply for reimbursement within six months of closing their accounts. After this, it takes over a year to process all the applications at the SII.The Social Insurance Institution register was merged with the Statistics Finland data at the Statistics Finland by using firm-specific identification codes. To protect the privacy of the companies we had at our disposal only the merged unidentifiable data.Firms are a heterogeneous group, e.g. differences in the legal construction affect the regulations about bookkeeping. To be sure that the firms in the study would employ personnel and would therefore be obliged to provide OHS we had to restrict our sample Figure . For thi- were not companies- had not been in business continually through 1999-2001- had financial statements of insufficient quality as assessed by Statistics Finland - had a turnover of less than €50,000 per annum- had less than ten employees in 2001- had values in either 1% tail of any key ratio. - had very high values of prevention costs  and curative medical service costs  in 2001. This indicates that expenditure has been beyond the normal limits because of exceptional circumstances.This resulted in 6,155 firms that had valid data for both economic performance and OHS expenditure and that could be included in the analysis.Key ratios for profitability are calculated from the profit and loss account by deducting costs from turnover and dividing this margin by turnover. The difference between the ratios results from the variation in costs deducted. The three first key ratios for solidity proportionate the annual profit to different capital titles. The two other key ratios for solidity, relative indebtedness and equity ratio, give an idea about the accumulated wealth of the company. Key ratios for liquidity indicate how large a share of its debts the company could pay with its liquid assets. Definitions for key ratios can be found in the appendix 1 . It can be used for testing the functional form of a model, especially to detect nonlinearities and omitted variables . The tesWe assumed that industry, the proportion of blue collar workers, age of the company, geographical region and grade of urbanisation of the area could all be related to the economic performance and to the expenditure of OHS. These variables were therefore introduced as confounders in all models.We used the STATA software packages for the analysis.The companies included in the study were a representative sample in terms of region and the type of the municipality. The size distribution of the companies in the study naturally differed from that of all Finnish companies as those with less than ten employees were excluded. For the same reason of exclusion of small firms, the industry distribution of the included companies differed from that of all Finnish companies. 'Real estate, renting, and business activities' and 'financial intermediation' were underrepresented and 'mining and quarrying plus manufacturing' slightly overrepresented.  except for relative indebtedness and quick ratio which were lower in the companies included in the study table . The corThe average turnover of the companies in the study was about €24 million, and the average age of a company was 18 years.Of the companies included in the study, 19% had not applied for reimbursement in 2001. Among the companies who had applied for reimbursement, the costs were the highest for the companies' own OHS units and lowest for municipal health centres  of the companies included in the study.Click here for file